KR20060092218A - Method for linking sequences of interest - Google Patents

Abstract

Multiplex overlap-extension RT-PCR provides an efficient method of linking two or more nucleotide sequences encoding for domains or subunits of a heteromeric protein, in a single reaction. Especially, the linkage of variable region encoding sequences from e.g. immunoglobulins, T cell receptors or B cell receptors is eased with the method of the present invention. This allows for a more efficient way of generating libraries of variable region encoding sequences. The capability to perform the multiplex overlap-extension RT- PCR using template derived from an isolated single cell enables the generation of cognate pair libraries in a high-throughput format.

Description

Methods of Linking Sequences of Interest {METHOD FOR LINKING SEQUENCES OF INTEREST}

The present invention relates to complex molecular amplification methods capable of binding the nucleotide sequence of interest in connection with amplification, in particular polymerase chain reaction (complex PCR). This method is particularly advantageous for generating cognate pair libraries and combinatorial libraries of variable region encoding sequences from immunoglobulins, T cell receptors or B cell receptors.

Antigen binding proteins involved in the immune response exist in mammals as many polyclonal repertoires that exhibit a wide variety of binding specificities. This difference is created by rearrangement of the gene sequence encoding the variable region of the binding protein. Such variable region binding proteins include soluble and membrane bound forms of B cell receptors (also known as immunoglobulins or antibodies) and membrane bound T cell receptors (TcRs). With respect to immunoglobulins, their affinity is subsequently enhanced through the so-called affinity maturation process involving the cycle of body hypermutation of variable genes, following the recognition of antigen by the B cell antigen receptor.

Notably, immunoglobulins or fragments thereof such as Fab fragments, Fv fragments and single chain Fv (scFv) molecules were cloned and recombinantly expressed. However, all other variable region binding proteins can in principle also be cloned and expressed using the same concept of antibodies.

Known methods for isolating antibodies with the desired binding specificities include generating hybridomas from the immunized host and screening for specific clones or generating combinatorial expression libraries in Escherichia coli consisting of immunoglobulin variable domains, which are subsequently Very often it involves hatching using techniques such as phage display, for example.

The main limitation in using hybridoma technology to produce therapeutic antibodies is the absence of human lymphomas suitable as fusion partners for human B lymphocytes. It is well known that heterohybriomas (ie fusion of human B cells with mouse lymphomas) are unstable and therefore rarely elicit cell lines suitable for production purposes. Human B cells that proliferate through infection with the Epstein-Barr virus exhibit a similar instability challenge. The absence of a firm cellular methodology of preparing human antibodies for treatment can be offset by more recent advantages in molecular biology.

The use of combinatorial libraries and phage display can produce many repertoires of antibody clones with potential differences of more than 10 ¹⁰ . Screening for binding to specific targets from this repertoire can be performed to create sub-libraries. The sub-library can be used to generate polyclonal or monoclonal antibodies. The variable region encoding sequences constituting the library (eg, immunoglobulin heavy chain variable region and light chain variable region encoding sequences) can be amplified from lymphocytes, plasma cells, hybridomas or any other immunoglobulin expressing individual of cells. Current techniques for generating combinatorial libraries include the isolated isolation of variable region encoding sequences from cell individuals. Thus, for example, inherent pairings of immunoglobulin heavy chain variable region and light chain variable region encoding sequences will be lost. Rather, in a combinatorial library, the sequences will be randomly paired and only unique combinations of these variable sequences will occur by chance. Thus, in order to isolate the variable region encoding sequences that are responsible for the desired binding specificities, a significant amount of screening is required. This is usually done in conjunction with methods for hatching clones that exhibit the desired specificity, such as ribosome display or phage display. Even in that case, the variability achieved will not be large enough to isolate variable region encoding sequence pairs that result in high affinity binding proteins similar to those found in the original cells. In addition, incubation processes commonly used to screen combinatorial libraries introduce strong bias, e.g., efficient folding, slow off-rate, or other system-dependent parameters that further reduce the diversity of the library, for example in E. coli. do. In addition, clones derived from such combinatorial libraries tend to produce more binding proteins that are cross-reactive to self antigens, as opposed to their own pairs (hereafter referred to as cognate pairs), It does not undergo negative selection in vivo for its antigen, such as for B and T lymphocyte receptors during certain stages of development of B and T lymphocyte receptors. Therefore, cloning unique pairs of variable region encoding sequences is the preferred approach. Moreover, the frequency of clones exhibiting the desired binding specificity is greater than in conventional combinatorial libraries, especially when the starting material cells are derived from donors with high frequency of cells encoding specific binding pairs, such as immune or immunized donors. It is expected to be significantly higher in the library of cognate pairs. This does not require that the size of the cognate pair library be as large as the combinatorial library; Cognate pair library sizes of 10 ⁴ to 10 ⁵ clones or even as few as 10 ² to 10 ³ clones derived from donors with an ongoing relevant immune response are highly desired to obtain binding proteins that exhibit a wide range of desired binding specificities. Will be enough.

In order to generate cognate pair libraries, binding of variable region encoding sequences derived from the same cell is required. Currently, two different approaches are disclosed that can achieve cognate pairing of variable region encoding sequences.

In-cell PCR is an approach to intracellularly bind heavy chain variable region and light chain variable region encoding sequences from immunoglobulins after the cell population has been fixed and permeated. Such binding can be performed by overlap-extension RT-PCR (WO 93/03151) or by recombination (Chapal, N. et al., 1997 BioTechniques 23, 518-524). The amplification processes disclosed in this document include: i) reverse transcription using constant region primers to generate immunoglobulin cDNA, ii) PCR amplification of heavy and light chain variable region encoding sequences using primer sets containing overlap-extension design or recombinant sites, iii) if a recombinant approach is chosen, a three or four step process consisting of binding by recombination, iv) nested PCR of the product creating restriction sites for cloning. Because the cells have permeated, there is a significant risk that the amplification product will leak out of the cells to introduce scrambling of the heavy and light chain variable region encoding sequences and cause a loss of cognate pairing. Thus, the procedure includes a washing step after each reaction, which annoys the process and reduces the efficiency of the reaction.

More generally, intracellular PCR is known to be inefficient, resulting in low sensitivity. Thus, intracellular PCR binding techniques are not widely used at all, and in fact original research has no reliable reproducibility in a way that can be used to verify that binding actually occurs in cells. However, this is of great importance for disrupting the cognate pairs by avoiding scrambling of the heavy chain variable region and light chain variable region encoding sequences.

Different intracellular approaches are disclosed in WO 01/92291. This approach is based on RNA trans-splicing and achieves binding of V _H and V _L encoding mRNAs in cells. This approach requires the presence of DNA constructs that induce trans-splicing in cells.

Single-cell PCR is a different approach to achieving cognate pairing of heavy chain variable region and light chain variable region encoding sequences. Coronella, J. A. et al. 2000 Nucleic Acids Res. 28, E85; Wang, X. et al. 2000 J. Immunol. Methods 20, 217-225. In that document, individuals of immunoglobulins expressing cells are distributed by diluting to a density of one cell per reaction during the cloning process, thereby eliminating scrambling of the heavy chain variable region and light chain variable region encoding sequences. Basically, the disclosed process contains i) reverse transcription using oligo-dT-, random hexamer- or constant region primers to generate cDNA, ii) restriction sites for fractionation and cloning of cDNA products into several tubes. Performing PCR amplification on individual variable chain encoding sequences (separated tubes) using primer sets, iii) nested PCR (optional) of the product creating restriction sites for cloning and iv) heavy chain variable regions from isolated tubes And a three or four step procedure consisting of binding them (basically a multistep process) by cloning the light chain variable region encoding sequences into a suitable vector.

In humans there are two types of light chains: lambda (λ) and kappa (κ). This means that three or more separate PCR reactions should be performed and analyzed using cDNA generated from every single cell and the appropriate fragments should be cloned into a single vector to achieve cognate pairing. Thus, the disclosed single-cell PCR approach requires a large number of manipulations to generate libraries of cognate pairs. Although cognate pair libraries do not have to be as large as combinatorial libraries to obtain binding proteins exhibiting a wide variety of binding specificities, the generation of libraries of, for example, 10 ⁴ to 10 ⁵ clones by the single-cell PCR approach described Still annoying work. In addition, a large number of manipulations greatly increase the risk of contamination and human error.

In order to obtain a high affinity binding protein corresponding to the affinity generally observed during the immune response, cognate pairing of variable region sequences associated with amplification of the variable region sequences is highly preferred. In order to produce a wide variety of libraries, it is essential to have cloning techniques that can be suitable for high throughput formats and to minimize the risk of contamination and scrambling.

There is also a desire to reduce the number of cloning steps that can produce combinatorial libraries suitable for high throughput formats.

The present invention utilizes complex molecular amplification methods, such as complex overlap-extension RT-PCR or complex RT-PCR, followed by ligation or recombination to bind two or more nucleotide sequences of interest, such as variable region encoding sequences. Provide an efficient way of combining. This method can be applied to single cells, thereby cloning cognate pairs in a high throughput format.

Description of the Drawings

1 is a diagram illustrating different types of overlap-extension tails. The thick line is the gene-specific portion of the primer and the normal line represents the overlapping tail. Vertical bars indicate complementary areas. Primers promote binding of two nucleotide sequences of interest. 1 (I) shows two types of type I overlap-extension tails in which only the extension tails are fully or partially overlapped; 1 (II) shows a type II overlap-extension tail where a portion of the 5′-nucleotide of the first primer extension tail is complementary to the gene-specific portion of the neighboring primers; 1 (III) shows the type III overlap-extension tail in which the full overlap-extension tail is complementary to the gene-specific region of the neighboring primers.

2 shows a schematic overview of a complex overlap-extension primer mix that can be applied to the binding of immunoglobulin variable region encoding sequences. The cDNA encoding light chain (LC) and heavy chain variable region (V _H ) to be bound are shown as tubes in which their sense strands 5 'and 3' ends and amplified products of expected size are indicated. The set of complex overlap-extension primers used to amplify the encoding sequences is indicated by arrows. The protruding bent arrow tail on the 5 'side, indicated by a dash, represents the cloning tail. Duplicate-extension tails are shown in bold. Restriction sites present in the tail are named in relation to the tail. The total number of primers in the complex overlap-extension primer mix is 16, which is on the outer primer comprising one C _κ and one C _H1 primer and on the overlapping stretch primer comprising six V _L and eight V _H primers. Distributed. The C _H1 primers are annealed at the 5 'end of heavy chain constant domain 1. The product resulting from the complex overlap-extension RT-PCR is expected to be approximately 1070 bp, which constitutes the whole kappa light chain and variable genes that make up the constant region and bind genes and variable genes (C _κ + J _L + V _L ), It constitutes a heavy chain variable region consisting of various segments and binding genes (V _H + D + J _H ). 5 'and 3' indicate the direction of the open reading frame. Since the annealing position of the C _H1 primer is close to the heavy chain J-region, only a small portion of the C _H1 region encoding sequence is amplified by the complex overlap-extension primer mix.

3 is a series of diagrams showing products of different binding directions that can be obtained as the primer has a binding tail. Filled areas indicate overlapping areas. 5 'and 3' indicate the direction of the open reading frame. 3A is a diagram showing the head-to-head orientation of the product. 3B is a diagram illustrating tail-to-tail orientation. FIG. 3C is a diagram first showing head-to-tail orientation with light chain encoding sequences. FIG. FIG. 3D is a diagram showing head-to-tail orientation with a heavy chain encoding sequence first.

4 is a schematic representation of an immunoglobulin expression vector pLL113 when the encoding sequence is in head-to-tail orientation. The vector includes the following elements: bla = promoter that allows expression of ampicillin resistance gene. Amp = gene encoding ampicillin resistance. pUC ori = pUC replicator. AdMLP = Adenovirus major late promoter. Human IgGl = sequence encoding the immunoglobulin isotype G1 termination chain. hGH pA = human growth hormone poly A signal sequence. bGH poly A = bovine growth hormone poly A sequence. Human kappa LC = sequence encoding the immunoglobulin kappa light chain. FRT = Flp recognition target site. Hygromycin = gene encoding hygromycin resistance. SV40 poly A = monkey virus 40 poly A signal sequence.

5A is an electrophoretic gel showing the results of a two step complex overlap-extension RT-PCR followed by semi-nested PCR. Amplification products are derived from cDNA isolated from single CHO Flp-In pLL113 cells. Lanes 1-12 are sample lanes and arrows represent 1076 bp correct nested complex-redundant-extension RT-PCR product; M1 is a 100 bp ladder. W is water used as a negative control template. C is a positive cDNA control template derived from cell line HB-8501. In separate panels, the same lanes W, C and 100 bp ladders are shown less clearly for analyzing individual DNA fragments. 5B is a sketch of the gel shown in FIG. 5A, showing related fragments from the gel.

FIG. 6 shows a series of photos and graphs of electrophoretic gels showing the results from a single step complex overlap-extension RT-PCR reaction without further PCR amplification. In each panel, M1 is a 100 bp ladder and M2 is a 500 bp ladder. 6A is an electrophoretic gel showing amplification products derived from lysates corresponding to 100, 10, 1 or 0 cells. Arrows indicate overlap-extension products. FIG. 6B is a sketch of the gel of FIG. 6A. FIG. 6C is an electrophoretic gel demonstrating the presence of overlap-extension products of 100 and 1 cell lanes in FIG. 6A. FIG. 6D is an electrophoretic gel showing restriction enzyme cleavage of the overlap-extension product from one cell lane of FIG. 6C with NheI and NcoI, respectively.

FIG. 7 is an electrophoretic gel showing the results of single-step complex overlap-extension RT-PCR followed by semi-nested PCR amplification. M1 is a 100 bp ladder and M2 is a 500 bp ladder. Results from complex overlap-extension primer mixtures containing C _H1 , C _H2 , C _H3 , C _H4 or C _H5 as outer primers in complex overlap-extension RT-PCR reactions. The reaction was performed on cell lysates corresponding to 100, 10, 1 or 0 cells. The size of the overlap-extension product is indicated by the arrow.

FIG. 8 is an electrophoretic gel showing results from semi-nested PCR amplification using single-step complex overlap-extension RT-PCR followed by human B lymphocytes hatched as template. M1 is a 100 bp ladder. Lanes 5 and 6 show bands of the expected size for the overlap-extension product.

9A is a schematic diagram of a mammalian expression vector (Em465 / 01P582 / Em465 / 01P581) used to generate IgGl-lambda expressing cell lines when the encoding sequence is in head-to-head orientation. The vector includes the following elements: Amp = gene encoding ampicillin resistance. pUC ori = pUC replicator. AdMLP = Adenovirus major late promoter. EFP = extension factor promoter. AP leader = alkaline phosphatase leader sequence. VH = heavy chain variable region encoding sequence. IgG1 HC = sequence encoding the immunoglobulin isotype G1 heavy chain constant region. rBG poly A = rabbit beta-globin poly A signal sequence. bGH poly A = bovine growth hormone poly A sequence. IgK leader = sequence encoding the murine kappa leader. IgL (1b or 1c) = sequence encoding the immunoglobulin lambda light chain family 1b or 1c. FRT = Flp recognition target site. Hygromycin = gene encoding hygromycin resistance. SV40 poly A = monkey virus 40 poly A signal sequence. 9B and 9C are ethidium bromide stained agarose gels loaded with PCR products isolated from cell lines CHO Flp-In / Em464 / 01P581 and CHO Flp-In / Em464 / 01P582, respectively. Lanes 1 to 4 correspond to total RNA template concentrations of 50 pg, 5 pg, 0.5 pg or 0 pg used for the complex overlap-extension RT-PCR reaction. M is a 100 bp ladder (New England Biolabs, New England, USA). Arrows indicate overlap-extension PCR products.

10 is a flowchart showing the steps applied to generate a cognate antibody expression library in which polyclonal or monoclonal antibodies can be expressed.

FIG. 11 is a schematic diagram of an E. coli vector, JSK301, used to generate a library of Fab vectors by inserting into the vector a double-extension fragment comprising cognate variable region encoding sequences at the indicated NotI / XhoI restriction sites. Vectors include the following elements: Amp and Amp pro = ampicillin resistance gene and promoter thereof. pUC19 ori = the replica. Human CH1 = sequence encoding human immunoglobulin gamma 1 heavy chain domain 1. Stuffer = unrelated sequence insert that is cleaved upon insertion of a duplicate-extension fragment. tac P and lac Z = bacterial promoters that can be excised at NheI and AscI restriction sites.

12 is a schematic diagram showing generation of cognate Fab expression vector library. Step I describes the insertion of cognate pairs (VH ₁ -VL ₁ to VHx-VLx) of variable region encoding sequences into E. coli vector JSK301 by XhoI-NotI digestion. Step II describes the insertion of bacterial promoters and leader cassettes ( pel B leader-P tac -promoter to induce expression of VHx and P lac promoter- pel B leader to induce expression of VLx) by AscI-NheI digestion. .

FIG. 13 illustrates the binding and further PCR amplification of the α-, β- and γ-subunits that make up the G-protein using single-step complex overlap-extension RT-PCR. The size of the individual coding regions as well as the size of the bound products are shown. Restriction sites introduced by primer tail during amplification are indicated in the final product.

14 (A) PBMC purified from donor blood; Point coordinates of analytical FACS staining of (B) magnetically labeled unlabeled CD19 negative cell fractions and (C) magnetically sorted CD19 + cell fractions. The scattered coordinates, CD19 / CD38 coordinates, and CD38 / CD45 markers for each fraction are shown.

FIG. 15 shows the CD19 + fraction from FIG. 9C, which was stored in liquid nitrogen, thawed and stained with anti-CD19, anti-CD38 and anti-CD45. The point coordinates corresponding to FIG. 9C are shown.

16 depicts the gate used on CD19 + cell fractions for sorting. Interstitial gates and fluorescent gates based on CD38 and CD45 were used to classify CD38 hi, CD45 intermediate (CD45in) cells.

FIG. 17 is an electrophoretic gel showing successful complex overlap-extension RT-PCR response to donor TT03 (column A, wells 1-12 from eight 96 well plates). Samples were applied to the agarose gel in two rows (A and B) with 48 samples each. The expected size of the overlap-extension fragment is approximately 1070 bp. Presumed overlap-extension fragments are indicated by arrows.

18 shows ELISA analysis of periplasmic extract from plate G060. ELISA plates were coated with goat-anti-human kappa and the captured Fab fragments were detected with HRP-conjugated gt-anti-human Fab-specific antibodies.

19 shows ELISA analysis of periplasmic extract from plate G060. ELISA plates were coated with 10 μg / ml of ovalbumin (Sigma A-5503) and the captured Fab fragments were detected with HRP-conjugated gt-anti-human Fab-specific antibodies.

20 depicts ELISA analysis of periplasmic extract from plate G060. ELISA plates were coated with tetanus toxoid and the captured Fab fragments were detected with HRP-conjugated gt-anti-human Fab-specific antibodies.

21 depicts a one step competitive ELISA assay of periplasmic extract from plate G060. ELISA plates were coated with tetanus toxoid (TT) and soluble TT was added to each well at 10 ⁻⁷ M to compete with immobilized TT for binding of Fab fragments from bacterial supernatant. Captured Fab fragments were detected with HRP-conjugated gt-anti-human Fab-specific antibodies.

FIG. 22 shows alignment of variable heavy chain protein sequences from TT antigen-binding clones from plate G060. The degree of sequence homology is shown in different shades; 100%, 80% and 60% are shown in black, gray and light gray, respectively. CDR1 is located at alignment positions 34-41. CDR2 is located at alignment positions 55-73. CDR3 is located at alignment positions 107-127. Immature stop codons are marked with asterisks. The alignment is divided into eight separate figures (a-h) divided into two rows from left to right with FIGS. 22A-D in the top row and FIGS. 22E-H in the bottom row.

FIG. 23 shows alignment of variable light chain protein sequences from TT antigen-binding clones from plate G060. The degree of sequence homology is shown in different shades; 100%, 80% and 60% are shown in black, gray and light gray, respectively. CDR1 is located at alignment positions 26-42. CDR2 is located at alignment positions 58-64. CDR3 is located at alignment positions 97-106. Immature stop codons are marked with asterisks. The alignment is divided into eight separate figures (a-h) divided into two rows from left to right with FIGS. 23A-D in the top row and FIGS. 23E-H in the bottom row.

24 depicts a competitive ELSIA assay for determining the apparent affinity of clones selected from plate G060. Soluble TT dilutions at concentrations of 100 nM to 25 pM (4 fold dilution) were added to Fab fragments to compete for binding of Fab-fragments with immobilized TT. The degree of reaction is expressed as the ratio of binding observed at a given soluble TT concentration to binding when no soluble TT is added to the reactant.

FIG. 25 depicts dual phylogenetic point matrix number coordinates showing intragene and intergene correlation of antibody heavy and light chain variable domain sequences. Three phylogenetic V _H and V _L sequences are paired in a dot matrix to represent the actual pairing of a particular V gene. A) TT binding clone obtained from combinatorial library using phage display. B) TT binding clones obtained from a library of cognate pairs using the present invention.

Description of the invention

The present invention provides methods for amplification and binding of two or more discontinuous nucleotide sequences of interest, which can make cloning of the sequences suitable for high throughput formats. This is basically achieved by reducing the number of steps needed to amplify and bind the cloned sequence.

One aspect of the present invention involves amplifying a nucleotide sequence of interest by complex molecular amplification using a template derived from an isolated single cell, an individual of homologous cells, or an individual of genetically diverse cells, and subsequently combining the amplified sequences. To a plurality of discontinuous nucleotide sequences. When the template is an isolated single cell or an individual of homologous cells, the binding results in a nucleic acid segment comprising the nucleotide sequence of interest linked to each other in a cognate fashion. When the template is an individual of genetically diverse cells, the binding results in a library of segments, ie a so-called combinatorial library, each comprising a nucleic acid sequence of interest that is joined in any manner.

In one embodiment of the invention, the complex molecular amplification method is preferably complex PCR amplification followed by a reverse transcription step. In a preferred embodiment, reverse transcription, amplification and binding are performed in a single step using complex overlap-extension RT-PCR or alternatively in two steps using complex RT-PCR followed by ligation or recombination. do.

A further embodiment of the invention relates to the generation of cognate pair library comprising linked variable region encoding sequences, in particular heavy chain variable region and light chain encoding sequences or T cell receptor (TcR) alpha chain encoding sequences and beta chain encoding sequences. . This method yields a lymphocyte-containing cell fraction from one or more suitable donors and optionally determines B lymphocytes or T lymphocytes, depending on whether a particular lymphocyte entity from this fraction, eg, immunoglobulin or variable region encoding sequence from TcR, is desired. Hatching. Lymphocyte-containing cell fractions or hatched cell fractions are dispensed into containers arranged to yield one cell in each container. Aligned single cells are subjected to reverse transcription (RT) steps or alternative cDNA production processes using nucleic acids derived from individuals of single cells as templates. Following the RT step, pairs of variable region encoding sequences generated from each cell are amplified and conjugated to molecules according to one of the methods of the present invention.

The cloning methods disclosed herein eliminate the cumbersome and inefficient cloning approach and further reduce the risk of contamination and loss of diversity during multiple cloning steps.

Another aspect of the invention is directed to a library of cognate pairs produced by complex molecular amplification and binding methods. Initial libraries of cognate pairs (parent libraries) generated by the methods of the present invention can be screened to generate cognate pair sub-libraries encoding target-specific binding protein variable domains or full-length binding proteins.

In a further embodiment of the invention, the libraries and sub-libraries of the invention can be used to express recombinant monoclonal or polyclonal proteins, wherein the inherent binding affinity and specificity present in the donor is preserved. .

Justice

The term "cognate pair" describes a pair of contemplated native non-contiguous nucleic acids contained within or derived from a single cell. In a preferred embodiment, the cognate pair comprises two variable region encoding sequences that encode together for a binding protein variable domain and whose gene sequences are derived from the same cell. Thus, when expressed as complete binding proteins or as stable fragments thereof, they preserve the binding affinity and specificity of the binding proteins originally expressed from these cells. A cognate pair consists of, for example, an antibody variable heavy chain encoding sequence associated with variable light chain encoding from the same cell, or a T cell receptor α chain encoding sequence associated with a β chain encoding sequence from the same cell. A library of cognate pairs is a collection of such cognate pairs.

The term "heat-initiating polymerase" describes a polymerase that is inert or has very low activity at the temperature used in reverse transcription. Such polymerases must be activated at high temperatures (90-95 ° C.) which exhibit functionality. This is advantageous, for example, in single-stage RT-PCR processes because it interferes with the polymerase interference in reverse transcriptase reactions.

The term "homogeneous population of cells" describes a population of genetically identical cells. Specifically, a homogenous population of cells derived by clonal extension of isolated single cells is contemplated by the present invention.

The term "isolated single cell" describes a cell that is physically separated from the population of cells corresponding to "single cell in a single container". When distributing a population of cells individually in multiple containers, a population of isolated single cells is obtained. As specified in the section entitled "Template Source", the proportion of containers with a single cell need not necessarily be 100% to call it a population of single cells.

Terms derived from “bind” or “bind” in connection with amplification describe a combination with a single fragment of an amplified nucleic acid sequence encoding the nucleic acid sequence under consideration. With respect to cognate pairs, fragments comprise nucleic acid sequences encoding variable domains derived from the same cell, eg, antibody heavy chain variable regions associated with antibody light chain variable region encoding sequences. Coupling can be accomplished simultaneously or as a step immediately after amplification. It does not require the form or functionality of the fragment and the fragment can be linear, circular, single stranded or double stranded.

Binding is not necessarily permanent, one of the nucleic acid sequences contemplated can be separated from the fragment if desired, and one of the variable region encoding sequences can be separated from, eg, cognate pair fragments. However, unless the original variable regions composed of cognate pairs are mixed with other variable regions, they are considered cognate pairs even if they are not combined with a single fragment. The bond is preferably a nucleotide phosphodiester bond. However, the bonds can also be obtained by different chemical crosslinking processes.

The term "complex molecular amplification" describes the simultaneous amplification of two or more target sequences in the same reaction. Suitable amplification methods include polymerase chain reaction (PCR) (US4,683,202), ligase chain reaction (LCR) (Wu and Wallace, 1989, Genomics 4, 560-9), strand displacement amplification (SDA) technology (Walker et al. , 1992, Nucl.Acids Res. 20, 1691-6), self-suspending sequence replication (Guatelli et al., 1990, Proc. Nat. Acad. Sci, USA, 87, 1874-8) and nucleic acid sequence sequence amplification (NASBA) ) (Compton J., 1991, Nature 350, 91-2). The latter two amplification methods include isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DAN (dsDNA).

The term “complex PCR” is a modification of PCR, in which two or more target sequences are modified in the same reaction with more than one primer set, eg, one primer set and kappa chain variable modified for amplification of heavy chain variable regions in the same PCR reaction. It describes amplification at the same time by including one primer set modified for amplification of the region. In addition, primer sets modified for amplification of lambda chain variable regions can be combined with these primer sets.

The term “complex RT-PCR” describes a complex PCR reaction, which is processed by a reverse transcription (RT) step. Complex RT-PCR can be performed as a two-step process with separate RT steps prior to complex PCR, or as a single-step process when all components for both RT and complex PCR are combined in a single tube.

The terms "complex overlap-extension PCR" and "complex overlap-extension RT-PCR" are used where complex PCR or complex RT-PCR is performed using a complex overlap-extension primer mix to amplify the target sequence, thereby simultaneously Means amplification and binding.

The term "multiple containers" describes any object (collection of objects) capable of physically separating a single cell from a population of cells. It may be a tube, multiwell plate (eg, 96-well, 384-well, microtiter plate or other multiwell plate), array, microarray, microchip, gel, or gel matrix. Preferably the object is one applicable to PCR amplification.

As used herein, the term “polyclonal protein” or “polyclonality” refers to a protein composition that comprises a protein molecule that is different but homogeneous, preferably a protein molecule selected from an immunoglobulin superfamily. Thus, each protein molecule is homologous to other molecules of the composition and also contains one or more stretches of variable polypeptide sequences, which are characterized by different amino acid sequences between individual members of the polyclonal protein. . Known examples of such polyclonal proteins include antibody or immunoglobulin molecules, T cell receptors and B cell receptors. Polyclonal proteins can be composed of defined subsets of protein molecules, which are conventional such as polyclonal antibodies that exhibit binding activity of a portion to a desired target, eg, binding specificity to a desired target antigen. Defined by the features.

As used herein, the term “individuals of genetically different cells” refers to a cell individual when the individual cells in the individual differ from one another at the genomic level. Individuals of such genetically different cells are, for example, individuals of cells derived from a donor, or fragments of B lymphocytes or T lymphocytes containing such cells, eg, cell fragments.

The term “primer set” is used interchangeably with the term “primer pair” to describe two or more primers capable of amplifying together the amplification of the nucleotide sequence under consideration (eg, one member of a cognate pair). Primer sets of the present invention can be designed to amplify a family of nucleotide sequences containing variable region encoding sequences. Examples of different families are antibody kappa light chains, lambda light chains, heavy chain variable regions, and α, β, γ or δ T cell receptor variable regions. Primer sets for amplification of a family of nucleotide sequences containing variable region encoding sequences often consist of multiple primers when several primers are degenerate primers.

The term "sequence of sequence" refers to the degree of equality between nucleic acid sequences for the shortest length of two sequences, expressed as a percentage. This can be calculated as (N _ref -N _dif ) × 100 / N _ref , where N _ref is the number of shorter residues in the sequence, and N _dif is at N _ref, where the longest match between the two sequences is optimally arranged. The total number of mismatched balances. Here, the DNA sequence AGTCAGTC is the sequence T A A TCA A TC GG (N _dif = 2 and N _ref = 8) (underlining indicates optimal alignment, and the darker portions represent two mismatched residues other than 8). And 75% sequence identity.

The term "randomly" or "randomly" related to binding refers to the binding of nucleotide sequences that are not derived from the same cell but are reversely bound among individuals of genetically different cells. If the nucleotide sequence under consideration is a variable region encoding sequence, this will result in a combinatorial library of bound sequences. On the other hand, when encoding for heteromeric proteins in which the nucleotides considered are not different, randomly bound sequences will appear similar in sequence from a single cell.

The term “template derived from an isolated single cell” relating to reverse transcription relates to nucleic acid in such isolated cells. The nucleic acid may be in the form of, for example, RNA, mRNA, DNA or genetic DNA. The nucleic acid may be isolated from the cell or present in the remaining contents of the cell, in which case the cell will be in an inactive or fused form.

Amplification and Bonding Process

One feature of the invention is the modification of PCR in which two or more target sequences are amplified simultaneously in the same tube by including in the same reaction one or more sets of primers, e.g., all the primers needed to amplify the variable region encoding sequences. To reduce the number of tubes needed to amplify the nucleotide sequence of interest. In general, this method is known as a multiplex polymerase chain reaction (complex PCR).

Complex PCR (Complex RT-PCR), preceded by complex PCR amplification and reverse transcription, is well known in the field of diagnostics for quantitative assays of mRNA levels and for the identification of viruses, bacteria and parasites, for example, analysis of mutations, deletions and polymorphisms in DNA. (Markoulatos, P. et al. 2002. J. Clin. Lab. Anal. 16, 47-51). However, using a complex primer mix consisting of four or more primers constituting a V _κ and / or V _λ primer set with a V _H primer set, the immunoglobulin light chain variable region encoding sequence is identical to the immunoglobulin heavy chain variable region encoding sequence. Examples of amplification in are rare (Chapal, N. et al. 1997. BioTechniques 23, 518-524; Liu, AH et al. 1992. Proc. Natl. Acad. Sci. USA 89, 7610-7614; Embleton, MJ et al. 1992. Nucleic Acids Res. 20, 3831-3837). The reason for this may be that the primer set adapted for amplification of the sequences encoding the variable domains of the antigen binding protein is generally composed of multiple degenerate primers to obtain the diversity of these variable region encoding sequences. Thus, the complexity of the PCR reaction is greatly increased when performing complex PCR amplification on variable region encoding sequences.

Another feature of the invention is that two or more target sequences amplified by complex PCR are tightly coupled to the amplification process. In particular, cognate pairs of variable region encoding sequences are bound by this process.

One embodiment of the invention utilizes that the complex primer mix can be designed to work in an overlap-extension PCR procedure, resulting in simultaneous amplification and binding of the nucleotide sequence of interest. This complex overlap-extension PCR technique serves to reduce the number of reactions required to isolate and bind the nucleotide sequence of interest, especially cognate pairs of bound variable regions.

Other embodiments of the invention apply binding by ligation or recombination as an alternative to binding by complex overlap-extension PCR. In this procedure, the binding is not performed simultaneously with the complex PCR amplification but as a step immediately after the amplification. However, binding can still be performed in the same tube in which the complex PCR is performed.

In order to perform complex overlap-extension PCR, it is necessary to have two or more primer sets (complex primer mix) in which at least one primer of each set is equipped with a overlap-extension tail. The overlap-extension tail allows binding of the product produced by each primer set during amplification. This primer mix is referred to as a composite overlap-extension primer mix. Complex overlap-extension PCR differs from conventional overlap-extension PCR in that the sequences to be bound are generated simultaneously in the same tube, thereby providing direct binding of the target sequence during amplification without any intermediate purification. In addition, conventional overlap-extension PCR requires a separate binding PCR reaction using either an outer primer set or a nested primer set to generate the bound product (Horton, RM et al. 1989. Gene 77 , 61-68). This additional amplification step is optional in the complex overlap-extension PCR of the present invention.

Another feature of the present invention is the reverse transcription (RT) step, which precedes complex PCR or complex overlap-renal PCR amplification, using templates derived from individuals of isolated single cells or isogenic cells.

Another feature of the present invention is the use of nucleotide sequences derived from individuals of isolated single cells or allogeneic cells as templates for complex PCR amplification. Preferably, RNA from a single cell is reverse transcribed into cDNA before complex PCR. For amplification of some nucleic acid sequences of interest, genomic DNA can be used as an alternative to mRNA. By using an isolated single cell or an individual of homologous cells derived by clonal proliferation of an isolated single cell as a template, the nucleotide sequence encoding the heteromeric protein of interest is derived from various cells in the individual of the cell. It is possible to prevent mixing with the sequence. This is important if one wishes to obtain the original composition of the sequence of interest. In particular, when generating cognate pairs of variable region encoding sequences, it is an important feature to use an isolated single cell or an individual of homologous cells as a template.

Complex overlap-extension PCR is a rarely used technique. WO 99/16904 describes the binding of exons from genomic sequences in a single reaction to generate cDNA without the use of reverse transcription. The described method constructed a complex overlap-extension primer mix, using one primer set (consisting of two primers) per exon to be bound. Each individual primer set could be overlapped with an adjacent primer set by complementary overlap-extension tails. cDNA was generated from a template of genomic DNA by performing nested PCR, which is described as an essential step following a duplicate-extension PCR reaction using a complex overlap-extension primer mix.

The production of cDNAs from exons of genomic DNA described in WO 99/16904 is a different field from the cloning of sequences encoding heteromeric proteins. First, heteromeric proteins are generally expressed from various genes, but the exon binding described in WO 99/16904 relates to the binding of exons from a single gene. In addition, the present invention facilitates the generation of libraries of bound nucleic acid sequences of interest, especially libraries of cognate pairs of variable regions and combinatorial libraries, which combine a series of exons from a single gene to produce a single non-variable cDNA. The height is a completely different case. In addition, the present invention uses nucleic acids derived from single cells, preferably nucleic acids in the form of RNA that do not need to be isolated from residual cell content before they can be used as templates.

Few publications describe complex overlap-extension RT-PCRs with respect to binding of variable region encoding sequences.

The simplest form of complex overlap-extension RT-PCR has been described for the isolation of scFv encoding sequences from hybridoma cell lines (Thirion, S. et al. 1996. Eur. J. Cancer Prev. 5, 507-511 and Mullinax, RL et al. 1992. BioTechniques 12, 864-869). The method described by Thirion and Mullinzx used a separate binding step followed by reverse transcription of mRNA by oligo-dT primers to total RNA extracted from hybridoma cell lines. The binding step was performed with a total of four primers, each constituting two primer pairs for the amplification of the heavy chain variable region and light chain variable region encoding sequences. The V _L forward primer and the V _H or C _H reverse primer contained complementary overlap-extension tails, thereby allowing simultaneous amplification and binding of the heavy chain variable region and light chain variable region encoding sequences. This method did not use nested PCR to increase the sensitivity of the binding method.

Other examples of complex overlap-extension RT-PCRs involving the binding of variable region encoding sequences are described in the aforementioned WO 93/03151, which refers to heavy chain variable regions originating from the same cells without the need to isolate single cells prior to cloning and Methods of cloning light chain variable region encoding sequences are provided. The method described in WO 93/03151 required a wash between the RT step and the complex overlap-extension PCR step. In addition, one of the specific purposes caused by WO 93/03151 has been to solve the problem of isolating single cells to obtain cognate pairs of variable region encoding sequences.

None of these known complex overlap-extension RT-PCR techniques have been developed to function on templates derived from isolated single cells. No method was able to carry out the RT-PCR reaction as a single step.

One embodiment of the invention includes the binding of a plurality of discontinuous nucleotide sequences of interest. Such methods include amplifying a nucleotide sequence of interest in a complex PCR or complex RT-PCR amplification procedure using a template derived from an individual of an isolated single cell or allogeneic cell and binding the amplified nucleotide sequence of interest. In addition, this method includes any step of performing further amplification of the bound product.

Another embodiment of the invention includes a method of generating a library of cognate pairs comprising linked variable region encoding sequences. Such methods include providing a lymphocyte containing cell fraction from the donor, which cell fraction is optionally concentrated for a particular lymphocyte individual therefrom. In addition, individuals of isolated single cells are obtained by individually distributing cells from lymphocyte containing cell fractions or concentrated cell fractions into multiple containers. Complex molecular amplification (complex RT-PCR amplification) of variable region encoding sequences contained within an individual of isolated single cells is performed and pairs of variable region encoding sequences within an individual of isolated single cells (each pair is a single cell Derived from). In addition, this technique involves two arbitrary steps, in which the individual isolated single cells of a single cell individual are propagated to a population of allogeneic cells prior to performing complex RT-PCR amplification to allow for a variety of allogeneic cells. Obtain multiple containers with individuals (with one individual homologous cell in one container). The second optional step involves performing further amplification of the bound variable region encoding sequence.

In a preferred embodiment of the invention, each member of the library of cognate pairs consisting of immunoglobulin light chain variable region encoding sequences is linked to an immunoglobulin heavy chain variable region encoding sequence originating from the same cell or to a beta chain variable region. A sequence encoding a T cell receptor binding domain consisting of a gamma chain variable region joined with a chain variable region or a delta chain variable region, where the bound variable region originates from the same cell.

Complex RT-PCR amplification of the present invention is a two step process in which reverse transcription (RT) is performed separately from complex PCR amplification (or another complex molecular amplification) or RT and complex PCR amplification steps are performed with the same primers in one tube. It may be carried out as a single step process to be performed.

Reverse transcription (RT) is performed with enzymes containing reverse transcriptase activity to generate cDNA from total RNA, mRNA or target specific RNA from isolated single cells. Primers that can be used for reverse transcription are, for example, oligo-dT primers, random hexamers, random decamers, other random primers, or primers specific for the nucleotide sequence of interest.

The two-step combined RT-PCR amplification procedure allows the cDNA produced in the RT step to be distributed to one or more vessels to allow for storage of the template fractions before amplification proceeds. In addition, the distribution of cDNA into one or more tubes enables the performance of one or more complex PCR amplifications of nucleic acids derived from the same template. Although this increases the number of separate reactions, this opens up the possibility of reducing the complexity of the complex primer mix if this should be desired. These two step methods amplify and bind the heavy chain variable region and kappa light chain variable region encoding sequences, for example, in one tube, and amplify and bind the heavy chain variable region and lambda light chain variable region encoding sequences in different tubes using the same template. Can be applied to Single cells typically only express one of the light chains. However, it is often easier to run these reactions simultaneously instead of waiting for the results of one reaction before carrying out another reaction. In addition, amplification of both kappa and lambda functions as an internal negative control, since only kappa or lambda is expected to be amplified from a single cell.

In a single step complex RT-PCR procedure, reverse transcription and complex PCR amplification are performed in the same vessel. First all the ingredients needed to perform both reverse transcription and complex PCR in a single step are added into the vessel and the reaction is carried out. In general, it is not necessary to add additional components when the reaction is initiated. The advantage of single step complex RT-PCR amplification is that it further reduces to the number of steps needed to generate the linked nucleotide sequences of the present invention. This is particularly useful when performing complex RT-PCR on an array of single cells where the same reaction needs to be performed in multiple vessels. Single step complex RT-PCR is performed by using the reverse primers present in the complex primer mix required for the complex PCR amplification also as primers for reverse transcription. Generally, the compositions required for single-step complex RT-PCR include nucleic acid templates, enzymes with reverse transcriptase activity, enzymes with DNA polymerase activity, deoxynucleoside triphosphate mixes (dATP, dCTP, dGTP and dTTP). DNTP mix) and complex primer mix. The nucleic acid template is preferably whole RNA or mRNA derived from an isolated single cell in purified form as a lysate of the cell or in a form still present in the intact cell. In general, the exact composition of the reaction mixture requires some optimization for each complex primer mixture to be used in the present invention. This applies for both two-step and single-step combined RT-PCR procedures.

In another embodiment of the invention, it may be suitable to use genomic DNA instead of RNA as a template. In this case, the reverse transcription step is omitted and the remaining steps of the present invention are performed as described throughout this specification.

For some single stage complex RT-PCR reactions, it may be advantageous to add additional components during the reaction. For example, the polymerase is added following the RT step. Other components can be, for example, dNTP mixtures or complex primer mixes, possibly with different primer compositions. This can be considered as a one-tube composite RT-PCR, because this also limits the number of tubes needed to obtain the desired bound product.

The nucleotide sequences of interest amplified by the complex RT-PCR can be linked to each other by several methods using various complex primer mixes, for example by complex overlap-extension RT-PCR, ligation or recombination. Preferably, the complex RT-PCR amplification and binding process is a single step or two steps. However, the binding process can also be performed as a multistep process using, for example, a stuffer fragment to bind the nucleic acid sequence of interest with PCR, ligation or recombination. Such stuffer fragments may contain cis-elements, promoter elements or related coding sequences or recognition sequences. In a preferred embodiment, the binding process is carried out in the same vessel as the complex RT-PCR amplification.

In one embodiment of the present invention, the binding of multiple discontinuous nucleotide sequences of interest is performed with complex PCR amplification using a complex overlap-extension primer mix. This results in amplification and binding of the target sequence together. In general, the compositions required for complex overlap-extension PCR include nucleic acid templates, enzymes with DNA polymerase activity, deoxynucleoside triphosphate mixes (dNTP mixes including dATP, dCTP, dGTP and dTTP) and complex overlap- Elongation primer mix.

In certain embodiments of the invention, the binding of a plurality of discontinuous nucleotide sequences of interest is performed by complex overlap-extension RT-PCR using templates derived from individuals of isolated single cells or allogeneic cells. This method also includes any step of performing further molecular amplification of the bound product. Preferably, the complex overlap-extension RT-PCR is performed as a single stage / 1-tube reaction.

The complex overlap-extension primer mixes of the present invention can be used to amplify and bind two or more variable region encoding sequences, eg, to amplify and bind sequences from an immunoglobulin heavy chain variable region family and a kappa or lambda light chain variable region family, or T cells. Two or more primer sets capable of priming amplification and binding of sequences from the receptor family α, β, γ or δ.

In another embodiment of the invention, a plurality of nucleotide sequences of interest amplified by complex RT-PCR are joined by ligation. To achieve this, the complex primer mix used in the complex RT-PCR is designed such that the amplified target sequence is cleaved by a suitable restriction enzyme and covalent binding by DNA ligation can be performed (primer design is called " primer mixture " And design ". Following complex RT-PCR amplification using this complex primer mix, the restriction enzymes necessary to form compatible ends of the target sequence are added to the mixture with ligase. Purification of the PCR product is not necessary before this step, but purification can be performed. The reaction temperature for concomitant digestion and ligation is about 0 to 40 ° C. However, if the polymerase from the complex PCR reaction is still present in the mixture, an incubation temperature below room temperature is preferred, with a temperature of 4-16 ° C. more preferred.

In another embodiment of the present invention, multiple nucleotide sequences of interest amplified by complex RT-PCR are recombinantly bound. In this method, the amplified target sequences can be bound using the same recombination site. Thereafter, binding can be performed by adding a recombinase to promote recombination. Some suitable recombinase systems include Flp recombinase with various FRT sites, Cre recombinase with various lox sites, integrase Φ31, and β-recombinase-six, which perform recombination between attP and attB sites. six) There is a Gin-gix system as well as a system. Recombination is illustrated for two nucleotide sequences (V _H bound with V _L ) (Chapal, N. et al. 1997 Bio Techniques 23, 518-524; incorporated herein by reference).

In a preferred embodiment of the invention, the nucleotide sequence of interest comprises a variable region encoding sequence and the binding results in a cognate pair of the variable region encoding sequence. Such cognate pairs may comprise one or more constant region encoding sequences in addition to the variable region.

In a more preferred embodiment of the invention, the nucleotide sequence of interest comprises an immunoglobulin variable region encoding sequence and the binding results in a cognate pair of light chain variable region and heavy chain variable region encoding sequences. Such cognate pairs may comprise one or more constant region encoding sequences in addition to the variable region. Such cognate pairs can also be isolated from template derived from lymphocyte containing cell fractions, eg, B-lymphocyte family cells enriched from whole blood, mononuclear cells or leukocytes.

In a preferred embodiment of the invention, the nucleotide sequence of interest comprises a TcR variable region encoding sequence and the binding is an cognate pair of an α chain variable region and β chain variable region encoding sequence or a γ chain variable region and δ chain variable region encoding sequence. Creates. Such cognate pairs may comprise one or more constant region encoding sequences in addition to the variable region. Such cognate pairs can also be isolated from templates derived from lymphocyte containing cell fractions, eg, T-lymphocyte family cells enriched from whole blood, mononuclear cells or leukocytes.

Another aspect of the invention is the use of complex RT-PCR using individuals from genetically diverse cells as template sources. The majority of heteromeric protein encoding sequences do not vary from cell to cell as is the case with variable region encoding sequences from binding proteins. Thus, when using the present invention for the cloning of such non-variable heteromeric protein encoding sequences, there is no need to perform initial isolation of single cells.

In this embodiment of the invention, the plurality of discontinuous nucleotide sequences of interest perform complex RT-PCR amplification of the nucleotide sequences of interest using templates derived from individuals of genetically diverse cells, and the amplified nucleotides of interest It is randomly bound by a method comprising joining sequences. In addition, this method includes any step of performing further amplification of the bound product. As in the single cell method, binding can be performed using complex overlap-extension primer mixes for amplification, or by ligation or recombination. Preferably, the template derived from the individual of the cell is not strictly contained within the cell. The individual of the cell can for example be lysed.

Applying a random binding process to an individual in a cell expressing a variant binding protein can simply generate a combinatorial library of variable region encoding sequences. Preferably, the individual of the cell constitutes a cell expressing a variable region binding protein, for example B lymphocytes, T lymphocytes, hybridoma cells, plasma cells or mixtures of these cells.

In the above-mentioned embodiments the individual of the cell can be permeated or lysed, for example without further purification, or the template nucleic acid can be isolated from the cell by standard procedures. Single-step combined RT-PCR procedures are preferred. However, a two-step procedure may also be used in the embodiments.

The present invention also provides a combinatorial library consisting of linked pairs of immunoglobulin light chain variable region and heavy chain variable region encoding sequences.

An efficient method for increasing the specificity, sensitivity, and yield of the complex RT-PCR binding process is further molecular amplification of the bound nucleotide sequence obtained from the complex RT-PCR followed by binding or recombination by extension or complex overlap-extension. The binding using RT-PCR is performed. Preferably such further amplification is performed by PCR amplification using primer mixes adapted to amplify the bound nucleic acid sequences of interest. The primer mix used may be the outer primer of the complex primer mix or the complex overlap-extension primer mix, which is the whole product annealed to the outermost 5 'and 3' ends of the sense strand of the bound variable region encoding sequence. It means a primer that enables the amplification of. The outer primer may also be described as a primer in a complex overlap-extension primer mixture that does not contain duplicate extension tails. In addition, nested or semi-nested primer sets can be used for further amplification of bound nucleotide sequences. Such nested PCR functions in particular not only to increase the amount of bound product but also to increase the specificity of the method. For the present invention, semi-nested PCR (described in the title entitled Primer Mix and Design ) is also considered to function as nested PCR. Thus, although this is desired, it is not necessary to perform further PCR amplification of the bound product from the complex overlap-extension RT-PCR or the bound product by ligation or recombination in the present invention.

Further amplification is carried out, for example, by agarose gel electrophoresis of the bound product, and excision of fragments corresponding to the expected size of the bound variable region encoding sequence, thereby fractional or whole complex overlap-extension RT-PCR. The reaction product, ligation product or recombinant product, or fractions of either of these products can be carried out directly or using partially purified bound products from any of these reactions. In the case of products bound by complex overlap-extension RT-PCR, further amplification is preferably carried out directly on the fragments from the complex overlap-extension RT-PCR reaction, which is an individual that is not bound in the first reaction. This is because it assists in binding the target sequence.

Sequence of interest

The nucleotide sequence of interest of the present invention can be selected from sequences encoding various subunits or domains that, when expressed, form a protein or part of a protein. Such proteins consisting of two or more non-identical subunits are known as heteromeric proteins. Heteromeric proteins are common in all kinds of species. Some of the classes to which these proteins belong are, for example, enzymes, inhibitors, structural proteins, toxins, channel proteins, G-proteins, receptor proteins, immunoglobulin superfamily proteins, transport proteins and the like. The nucleotide sequences encoding such heteromeric proteins are discontinuous, meaning that they originate, for example, from different genes or from different mRNA molecules. However, the term discontinuous, as used herein, may also refer to a nucleotide sequence that encodes domains of the same protein, where the domains are separated by nucleotide sequences of interest.

In one embodiment of the invention, the nucleotide sequence of interest contains a variable region encoding sequence from an immunoglobulin superfamily, such as an immunoglobulin (antibody), B cell receptor and T cell receptor (TcR's). In particular, variable region encoding sequences from immunoglobulins are of interest. This variable region encoding sequence is a combination of Fab's, Fv's, scFv's and fragments of the variable region encoding sequence as well as complementarity determining regions (CDR's) that bind genes or V-genes or combinations thereof, as well as full length antibodies. Contains water. In general, the present invention can be applied using any combination of variable region encoding sequences and fragments thereof. The present invention illustrates the binding of the variable domains of the entire light and heavy chains. However, the present invention also allows the binding of only the variable regions of the heavy and light chains to generate Fv or svFv encoding sequences, or the entire light and heavy chain variable region + constant domain CH to generate Fab, Fab 'or F (ab) ₂ Enables the combination of ₁ + hinged areas. It is also possible to add any region of the heavy chain constant region domain to the variable heavy chain to generate a truncated antibody encoding sequence or full length antibody encoding sequence.

In another embodiment of the invention, the variable region encoding sequence comprises one type of immunoglobulin light chain (kappa or lambda) encoding sequence and one immunoglobulin heavy chain variable region encoding sequence.

Variable region encoding sequences derived from T cell receptors (TcR's) are also of interest. Such TcR encoding sequences are encoding sequences for full-length alpha and beta chains or gamma and delta chains, as well as soluble TcR's or just the variable domains of these chains or their single chain fusion proteins (eg, single chain αβ or single chain γδ). It includes.

Mold source

One feature of the present invention is the ability to link nucleotide sequences derived from isolated single cells, isogenic cell individuals or genetically different cell individuals not separated into a single tube. The cells used in the present invention can be, for example, bacteria, yeast, fungi, insect cells, plant cells or mammalian cells or fractions of such cells. Blood cells derived from mammals are an example of a fraction of cells that can be used in the present invention.

A preferred feature of the invention is the use of isolated single cells or homologous genotype cell individuals as template sources, as it prevents the mixing of nucleic acid sequences of interest, in particular sequences encoding various regions. This is important if, for example, one wishes to obtain the original pairs of sequences encoding the various regions.

Another preferred feature of the invention is to obtain a single cell or a single cell individual from lymphocytes such as B lymphocytes, T lymphocytes, plasma cells and / or cell fractions comprising various stages of development of such cell lineages. Other individuals of cells expressing binding proteins from an immunoglobulin superfamily can also be used to obtain single cells. Cell lines, such as hybridoma cells, cell lines of the B lymphocytes or T lymphocyte lineages, or virus immortalized cell lines or donor derived cells that participate in an immune response, are also applicable to the present invention. Donor-derived lymphocyte-containing cell fractions can be obtained from these cells from many natural tissues or liquids, such as blood, bone marrow, lymph nodes, spleen tissue, tonsil tissue, or from tumor infiltration or from inflammatory tissue infiltration. Suitable cell donors for the present invention can be selected from vertebrates, all of which have an acquired immune system. The donor may be naive or hyperimmune with respect to the desired target. For the isolation of antigen binding proteins with binding specificities for the desired targets, hyperimmunized donors are preferred. Such a hyperimmune donor can be a donor immunized with a target or fragment of a target, or is a recovering patient, or an unhealthy individual who has a natural immune response against a target, that is, an autoimmune patient, a cancer patient, an infectious disease , For example HIV patients, A, B, or hepatitis C patients, SARS patients and the like, or patients with chronic diseases.

When using recombinant proteins for treatment, they are preferably derived from sequences having species identity with the individual to be treated (ie, human sequences for the treatment of humans). First, recombinant proteins derived from foreign sequences (ie, non-humans) will be recognized by the immune system leading to an immune response involving polyclonal anti-protein antibodies. Such anti-protein antibodies can occupy active sites and block drug action, which will accelerate drug clearance and potentially induce adverse reactions such as hypersensitivity upon repeated exposure.

However, immunogenicity can also be observed when the recombinant protein is derived from a sequence having species identity. Such immunogenicity can be induced, for example, by post-immune modifications which may differ from those seen in vivo. Combinatorial libraries of sequences encoding various regions can also cause immunogenicity because they consist of random pairs of various region encoding sequences generated in vitro. The rules governing the formation of antibody heavy and light chain encoding sequence pairs (or T cell receptors) in vivo are not fully understood. Thus, this follows that some of the pairs formed in vitro can be recognized as foreign by the immune system, even if both sequences that make up the pair are completely human. Binding proteins obtained from a cognate library on the other hand do not produce such abnormal combinations, and consequently they have less potential immunogenicity than binding proteins from combinatorial libraries. This does not mean that the products from the combinatorial library are inadequate for treatment, but only need to be monitored to a greater extent with respect to the aforementioned side effects.

For use in the present invention, the cell donor should preferably be the same species as the species to be treated with the product obtainable from the linked nucleotide sequences of the present invention. Preferably, the cell donor is a livestock, pet, human or transgenic animal. Transgenic animals with human immunoglobulin loci are described in US Pat. No. 6,111,166 and Nature Biotechnology, 2002, 20: 889-893, Kuroiwa, Y. et al. Such transgenic animals can produce human immunoglobulins. Thus, fully human antibodies to a particular target can be produced by conventional immunization techniques of such transgenic animals. This allows the generation of libraries encoding binding proteins that do not have a natural human antibody response or have specificity for more different targets, such as limited human antigens. Such transgenic animals can likewise be developed to produce human T cell receptors.

In a further embodiment of the invention, the lymphocyte-containing cell fraction consists of whole blood, bone marrow, monocytes, or leukocytes obtained from a donor. Monocytes can be isolated from blood, bone marrow, lymph nodes, spleen, infiltration around cancer cells and inflammatory infiltration. Monocytes can be separated by density centrifugation techniques, such as Ficoll gradients. If monocytes are separated from a sample consisting of tissue, the tissue must disintegrate before gradient centrifugation is performed. Disintegration can be performed, for example, by mechanical methods such as grinding, electroporation and / or by chemical methods such as enzymatic treatment. Isolation of leukocytes can be performed directly from the donor using leukopheresis. Raw preparations containing lymphocytes, for example bone marrow or tissue, can also be used in the present invention. Such preparations will have to disintegrate, for example, to facilitate single cell distribution as described above.

A further feature of the invention is the enrichment of lymphocyte-containing cell fractions, ie whole blood, monocytes, white blood cells or bone marrow, for certain lymphocyte individuals such as cells from B lymphocytes or T lymphocyte lineages. Enrichment of B lymphocytes can be performed using magnetic bead cell separation or fluorescence activated cell sorting (FACS), for example using lineage-specific cell surface marker proteins such as CD19 or other B cell lineage-specific markers. Enrichment of T lymphocytes can be performed using cell surface markers such as, for example, CD3 or other T cell lineage-specific markers.

A preferred feature of the present invention is the further sorting of concentrated B lymphocytes to obtain plasma cells prior to dispensing the cells individually into a plurality of tubes. Isolation of plasma cells is generally performed by FACS sorting using surface markers such as CD38, possibly in combination with CD45. Other plasma cell-specific surface markers or combinations thereof may be used, for example, CD138, CD20, CD21, CD40, CD9, HLA-DR, or CD61L, and the correct selection of markers is dependent on plasma cell sources, namely tonsils, Depends on blood or bone marrow Plasma cells may also be obtained from non-concentrated lymphocyte-containing cell individuals obtained from any one of these sources. Plasma cells isolated from blood are sometimes called early plasma cells or plasmablasts. Such cells in the present invention are also called plasma cells even if they are CD19 positive in contrast to the plasma cells present in the bone marrow. Plasma cells are desired for the isolation of immunoglobulin encoding sequence pairs of the same origin, which results in higher frequency of these cells producing antigen-specific antibodies that reflect acquired immunity to the desired antigen, most of which are somatic cells. This is because they have undergone sex hypervariation and thus encode high affinity antibodies. In addition, the mRNA levels of the plasma cells are elevated compared to the remaining B lymphocyte individuals and thus the reverse transcription process is more efficient when using single plasma cells. As an alternative to plasma cell isolation, memory B cells can be isolated from lymphocyte containing cell fractions using cell surface markers such as CD22.

An alternative feature of the invention is the selection of concentrated B lymphocytes for antigen specificity prior to dispensing the cells in multiple tubes. Isolation of antigen-specific B lymphocytes is performed by contacting the enriched B lymphocytes with the desired antigen or antigens to enable the antigen to bind to surface exposed immunoglobulins and then separating the binder. This is for example coating magnetic beads with the desired antigen or antigens and subsequently magnetic bead cell sorting, FACS, coating the column with antigen and subsequently affinity chromatography, filter screening assays or known in the art. Can be done by other methods. Plasma cells as well as B lymphocytes, unconcentrated monocytes, white blood cells, whole blood, bone marrow, or tissue preparations can be isolated for antigen specificity if desired.

Another feature of the invention is the sorting of concentrated T lymphocytes (eg CD3 positive cells) using surface markers CD45R0 and / or CD27 to obtain a fraction of memory T cells. T lymphocytes can also be selected for MHC-antigen specificity using MHC-peptide complexes. See Callan, M.F. et al., 1998, J. Exp. Med. 187, 1395-1402; Novak, E.J. et al., 1999, J. Clin. Invest 104, R63-R67].

A further feature of the invention is the immortalization of any of the cell fractions isolated (ie B lymphocytes, plasma cells, memory cells or T lymphocytes) as described above. Immortalization can be performed, for example, with the Epstein Bar virus prior to cell distribution (Traggiai, E., et al., 2004, Nat Med 10, 871-875). Alternatively, isolated single cells can be immortalized and expanded prior to reverse transcription (Traggiai, E., et al., Nat Med, 2004, Aug, 10 (8): 871-5).

A further feature of the invention is that in order to obtain an isolated single cell individual, the desired cell population (e.g., cell lines of hybridoma cells, B lymphocytes or T lymphocyte lineages, whole blood cells, bone marrow cells, monocytes, Leukocytes, B lymphocytes, plasma cells, antigen-specific B lymphocytes, memory B cells, T lymphocytes, antigen / MHC-specific T lymphocytes, or memory T cells). This isolation of a single cell means physical separation of the cells from the cell individual in such a way that the single tube contains a single cell, or is loaded in such a way that a microarray, chip or gel matrix produces a single cell. Cells can be distributed directly into multiple tubes, such as an array of single tubes, by limiting dilution. The single tubes used in the present invention are preferably those applicable to PCR (ie, PCR tubes and 96 well or 384 well PCR plates, or larger tube arrays). However, other tubes may also be used. When distributing a single cell into multiple single tubes (ie 384 well plates), a single cell individual is obtained. Such dispensing can be done, for example, by distributing the volume into a single tube containing, on average, a cell concentration of 1, 0.5, or 0.3 cells to obtain a tube containing on average one cell or less. Since distribution of cells by limiting dilution is a statistical issue, the fraction of the tube will be empty and the major fraction will contain a single cell and the minor fraction will contain two or more cells. If two or more cells are present in the tube, some shuffling of the sequences encoding the various regions may occur in the cells present in the tube. However, since this is an incident, it will not affect the overall utility of the present invention. In addition, combinations of various region encoding sequences that do not have the desired binding affinity and specificity will most likely not be selected and thus will be removed during the screening process. Thus, incidental events such as shuffle would not affect the final library of the present invention much.

An alternative is to distribute the cells by limiting dilution, for example using a cell sorter such as a FACS instrument or a robot that accurately distributes one cell into one tube. These alternatives are less efficient and they are more efficient to obtain a constant distribution of one cell into one tube.

Concentration, sorting, and isolation processes as described above are performed to keep the majority of cells intact. Disruption of cells during enrichment and sorting can result in intermingling of various region encoding sequences. However, this is not expected to be a problem since the frequency of fracture is expected to be low. Washing the cells and possible RNAse treatment prior to dispensing into a single tube will remove any RNA that leaks during the process.

In addition, given the above description of a method of distributing cells to obtain a single cell individual among individuals of a single tube, it is not to be interpreted as an absolutely necessary feature to contain a single cell per tube. Rather, this indicates that the majority of the tubes contain a single cell, ie the number of tubes with two or more cells is less than 25% or even better than 10% of the total amount of cells distributed.

A further feature of the present invention is the performance of reverse transcription using templates derived from cells individually distributed in multiple tubes.

For the purpose of reverse transcription (RT), according to the present invention, nucleic acid in one cell to serve as a template source for RT is derived from a single cell even though they are not necessarily isolated from the remaining contents of this single cell. Is considered.

If a single cell is finally distributed into their single tube, the single cell may be expanded to obtain homogenous genotype cells prior to reverse transcription. This process yields more mRNA to be used as a template, which can be important when rare targets are amplified or linked. However, the cells must remain genetically identical with respect to the target gene during expansion. Individuals of the isolated cells or homologous genotype cells can remain intact or lysed unless the template for reverse transcription is degraded. Preferentially, cells are lysed to facilitate subsequent reverse transcription and PCR amplification.

In a different embodiment of the present invention, the complex overlap-extension RT-PCR method disclosed or the complex RT-PCR followed by ligation or recombination are also not separated into a single tube but all remain together as pools of cells. It can be used in templates derived from genetically diverse individuals. This approach will not require the distribution of single cells. However, the cells that can be used for this approach are the same as those described for the single cell approach, for example individuals of sorted B lymphocytes or T lymphocytes (pools). If one-step complex RT-PCR or ligation or recombination followed by ligation or recombination is performed on cells of such individuals, it is desirable to lyse the cells prior to the reaction and, if desired, the entire RNA or mRNA can be isolated from lysate.

The sensitivity of the one-stage complex redundant-extension RT-PCR of the present invention makes it possible to use very small amounts of template. As shown in Examples 2 and 3, one-step complex overlap-extension RT-PCR can be performed on the amount of template corresponding to the lysate of a single cell.

Primer mixtures and designs

Primer mixtures of the invention comprise at least four primers that form a primer set, two by two, which can amplify at least two different target sequences of interest. A mixture of two or more such primer sets constitutes a composite primer mixture. Preferably the complex mixture is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 primer sets (primer pairs). In particular, for amplification of various region encoding sequences, individual primers constitute two or more several primers in a composite primer mixture. Preferably, the individual primer sets are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 45, 50, 60, 70 , 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280 or 300 primers. Preferably the total number of primers in the composite primer mixture is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 45, 50, 60, 70 , 80, 90, 100, 125, 150 or 200, and at most 225, 250, 275, 300, 325, 350, 375, or 400 primers.

All of the primers of the invention comprise a gene specific region, preferably all primers additionally comprise a primer tail at the 5 'end of the primer, ie a 5' non-fused to the 3'-end of the gene-specific primer portion. Coding sequence. Such primer tails are approximately 6-50 nucleotides, but can also be longer if desired. Upon amplification, the primer tail is added to the target sequence.

Primer tails of the invention are, for example, cloning tails and linking tails, for example tails adapted for ligation by ligation, recombination or overlap-stranded tails.

Cloning tails are 6 to 20 nucleotides and contain restriction sites and / or recombinant sites, which are useful for inserting linked products into appropriate vectors.

In order to enable ligation by ligation, the primer set of the composite primer mixture is placed in one part of the first primer set (forward or reverse primer (s)) at the linking tail of one part of the second primer set upon cleavage. It is designed to be compatible with the restriction position. For linkage of two or more target sequences, the second portion of the second primer set is provided with a restriction position that is compatible with the restriction region located in one portion of the third primer set upon cleavage. This second restriction position located in the second primer set is not compatible with that of the first primer set. A significant number of target sequences can be linked by designing primer sets in this manner. Restriction sites that should appear or appear less frequently in the target sequence should be selected. It is also desirable that the compatible restriction positions are not the same so that the ligation positions will have cleavage-resistance for the particular restriction enzyme used. This reaction regulates the direction of linking target sequence 1 with target sequence 2 because the linkage between the same target sequences is cleavable by restriction enzymes. Suitable pairs of restriction sites are, for example, SpeI and XbaI (alternatively NheI or AvrII may replace one or both of them), NcoI and BspHI, EcoRI and MfeI or PstI and Nsil. For ligation, SpeI can be located, for example, at target sequence 1, XbaI can be located at target sequence 2, NcoI can be located at the other end of target sequence 2, and BspHI is located at target sequence 3 You can do it. To further simplify the process, it is advantageous if restriction enzymes are applied in the same buffer. To enable linkage by recombination, primer sets of complex primer mixtures can be designed as illustrated, for example, in Chapal, 1997, BioTechniques 23, 518-524, which is incorporated herein by reference. Is incorporated.

To enable linkage of the nucleotide sequence of interest in the same step as the complex PCR amplification, a tail adapted for overlap-extension PCR is added to at least one primer of each primer set of the complex primer mixture to add the complex overlap-extension primer mixture. Create Redundant-extension tails are typically longer and 8 to 75 nucleotides, and may allow restriction or recombinant positions to be allowed for subsequent insertion of promoters, ribosomal binding regions, termination sequences, or linker sequences such as scFvs. It may contain. Duplicate-extension tails may also contain stop codons if desired. In general, there are three types of overlap-extension tails, as shown in FIG. 1. In type I, the overlap-extension tails in the two primer sets overlap with each other alone. The nucleotides of both overlap-extension tails do not necessarily need to be complementary to each other. In one aspect of the invention, complementary nucleotides represent 60-85% of the overlap-extension tail. In type II, duplicate kidney tails, 4-6 of the 5 'nucleotide, are complementary to the gene-specific regions of adjacent target sequences. In type III overlap-extension tail the full overlap is complementary to adjacent target sequences. Type I and II overlap-extension tails are preferred when regulatory elements and the like are inserted between linked target sequences later. Type II overlap-extension tails are preferred when the target sequence is linked by a linker defined as shown with the scFv. Type III overlap-extension tails are preferred when the target sequences are linked in-frame to each other.

The design of the overlap-extension tails depends on sequence properties such as length, relative GC content (GC%), presence of restriction sites, palindrome, melting point, and gene-specific portions to which they are coupled. The length of the overlap-extension tail is 8 to 75 nucleotides, preferably they are 15 to 40 nucleotides. More preferably they are 22 to 28 nucleotides. The use of very long overlap-extension tails (50 to 75 nucleotides) may favor the ligation of the product produced by each primer set. However, the ratio between the length of the overlap-extension tail and the gene-specific region will probably need to be adjusted when using very long overlap-extension tails. GC% preference depends on the length of the overlap-extension tail. Since shorter tails have shorter areas when they are complementary, they need higher GC% to intensify the interaction than longer tails. Other principles of primer design should likewise be followed, ie primer dimerization and hairpin formation should be minimized. They should not participate in false priming. In addition, Taq DNA polymerase often adds adenosine (A) at the 3 'end of the newly synthesized DNA strand, which allows the redundant-extension tail to accommodate the addition of the 3'-non-type A, resulting in a redundant-extension tail design. Can be accommodated in

The selection of primers having binding tails, eg, overlap-extension tails, which are tails suitable for binding by ligation or recombinantly binding, define the binding sequence and direction of the target sequence. It is not essential to the present invention whether the binding tail is provided with a forward primer (s) or reverse primer (s) of the primer set, or possibly both forward and reverse primers. However, this requires some consideration in any way, because the order and orientation of the target sequences in the final product can be controlled, for example, for insertion of regulatory elements such as promoters and terminal sequences, or in-frame of individual target sequences. This may be related to (in-frame) binding.

For the binding of the two nucleotide sequences of interest, the binding tail can be added to either the reverse primer (s) or forward primer (s) of each primer set used for PCR amplification of each target sequence. The present invention illustrates the addition of tails suitable for binding by overlap-extension tail and ligation to each primer set of V _H and V _L forward primers (eg, FIGS. 2 and 9 respectively). This results in the binding direction (head-to-head and bidirectional) of the product being 5'-5 '. However, it is natural that binding tails can also be added to each set of reverse primer (s) (eg C _κ and / or C _{λ in} the first set, and C _H or J _H in the second set). This results in the binding direction (tail-to-tail and bidirectional) of the product being 3'-3 '. The third option is the reverse primer (s) of the first primer set (eg C _κ and / or C _λ primers) and the forward primer (s) of the second primer set (eg V _H primer (s) ), Or vice versa. This results in a 3'-5 'orientation (head-to-tail, and single direction). Figure 3 shows the possible directions that can be generated depending on which primer of each primer set is provided with a binding tail.

When binding more than two nucleotide sequences of interest, some primer sets are forward primers and one primer is complementary to the tail of the preceding primer set, and the other tail is complementary to one of the primers of the subsequent primer set. It is necessary to have a binding tail for both reverse primers. This theory is believed to be the case for all primer sets that amplify a target sequence that must be bound between two different target sequences.

The design of gene specific primer moieties will generally have to comply with known primer design regulations such as minimizing primer dimerization, hairpin formation and nonspecific annealing. In addition, as many 3 'bases, as many G or C nucleotides should be avoided as possible. The melting point (Tm) of the gene specific region of the primer set should preferably be the same as each other or +/- 5 ° C. In the present invention, Tm values of 45 to 75 ° C. are preferred, and Tm values of about 60 ° C. are suitable for most applications. Advantageously, the starting primer design can be assisted by a computer program developed for this task. However, primer design generally requires laboratory testing and routine optimization. This can be done, for example, by analyzing the sequencing of amplification products obtained using size, restriction enzyme fragment length polymorphism (RFLP) and primer sets. The use of degenerate sites in primers is useful when amplifying sequences with variable regions or when investigating new family members belonging to a particular class of protein. The number of degenerate sites may also require optimization.

The present invention includes an improved set of primers that can be used together in a highly complex format. The primer set described by de Haard, HJ et al. 1999. J. Biol. Chem. 274, 18218-18230 was used as the starting point, and the 3 'end of the primer was used to reduce nonspecific interactions. Modifications were made by trimming and adding tails suitable for binding by overlap-extension tail or ligation.

One of the features of the present invention lies in a primer mixture consisting of two or more primer sets capable of priming amplification of two or more nucleotide sequences of interest and facilitating their binding. Primer mixtures of the invention are, for example, from heteromeric proteins belonging to the class of enzymes, inhibitors, structural proteins, toxins, channel proteins, G-proteins, receptor proteins, immunoglobulin superfamily proteins, transport proteins and the like. Amplification of two or more subunits or domains can be primed.

Another feature of the invention includes a primer set comprising one or more primer set members of each primer set comprising a redundant-extension tail capable of hybridizing to the overlap-extension tail of the primer set members of the second primer set. It is a complex overlap-extension primer mix.

The overlap-extension tail can immediately bind the nucleotide of interest during complex overlap-extension PCR amplification by having each individual product resulting from a primer set having a tail complementary to the adjacent product. However, this does not mean that such binding must occur during the first PCR amplification. Depending on the reaction setup, the majority of actual binding can be performed during further amplification (complex PCR amplification) by the outer primer of the first PCR amplification.

Another advantage of the present invention is that it is a primer set designed to amplify a family of nucleotide sequences containing variable region coding sequences. Examples of such families include kappa light chains (eg human VKI-VI) from immunoglobulins, lambda light chains (eg human VL1-10), variable heavy chains (eg human VH1-7, And VH1-15) of mice, and α, β, γ, or δ TCR variable regions. Primer sets for amplification of nucleotide sequence families containing variable region coding sequences often include multiple primers, several of which may be degenerate primers. Amplification of a family of immunoglobulin light chain variable region coding sequences can include, for example, constant region kappa primers (C _κ primer (s)) and / or lambda primers (C _λ primer (s)) (reverse primers) or many such primers. Together with the kappa _chain (V _Lκ primer (s)) or kappa leader sequence (V _LκL primer (s)) and / or lambda chains (V _Lλ primer (s)) or lambda leader sequence (V _LλL primers) S)) (forward primer) using a primer set consisting of multiple primers complementary to the 5 'end. Alternatively, light chain binding region primers (J _Lκ and / or J _Lλ primer (s)) can be used as reverse primers instead of constant region primers. Alternatively, the forward primer is annealed in the UTR region preceding the leader sequence of the variable light chain. Equally, families of immunoglobulin heavy chain variable region coding sequences can be amplified with either primer set using various primer combinations. For example, with multiple heavy chain binding region primers (J _H primer (s)) or heavy chain constant region primer (s) (reverse primers), heavy chain variable region (V _H primer (s)) or leader sequences of such regions (V _HL primer (s)) (forward primers) can be amplified with a primer set consisting of multiple primers complementary to the 5 'end. C _H primers may be isotype specific, in theory any C _H primer may be used (e.g., C _H1 , C _H2 , C _H3 or C _H4 ), and primers capable of forming full-length heavy chains may also be used. Can be. Alternatively, the forward primer is annealed in the UTR region preceding the leader sequence of the variable heavy chain.

The use of forward primers annealed in the leader sequence instead of the 5 'end of the variable region is particularly useful when cross-hybridization is observed for the variable region primers. Since mutations due to cross-hybridization will be removed from the final protein, the leader sequence is removed during intracellular protein processing. Example 9 describes the design of antibody variable heavy and kappa light chain leader primers. An advantage over previous antibody leader primers is that the site of priming is located at the 3'-terminus (C-terminus) of the leader coding sequence, because it is a bacterium in a manner that allows functional leader sequences in both bacteria and eukaryotic systems. This is because it allows for shuttleting of the amplified sequence between the expression vector and the eukaryotic expression vector. It goes without saying that the design system described in Example 9 can be easily applied to antibody lambda light chains, TcR, α, β, γ, or δ chains.

One feature of the present invention is a primer annealed at the 3 'end of the leader coding sequence preceding the variable region coding sequence, and their use for amplification of the variable region coding sequence.

A preferred feature of the invention is that at least 90% sequence identity (preferably 95%) with the gene specific region of SEQ ID NOs 86 to 92 corresponding to the primer annealed at the C-terminus of the heavy chain leader coding sequence (V _HL primer) Use of primers). The gene specific sequence of SEQ ID NO corresponds to the base number 18 for the 3 'end of the sequence (see Table 11).

Another preferred feature of the invention is at least 90% sequence identity (preferably with the gene specific region of SEQ ID NOs 93 to 98 corresponding to the primer annealed at the C-terminus of the kappa light chain leader coding sequence (V _LκL primer) Use at least 95% identity). The gene specific sequence of SEQ ID NO corresponds to the base number 25 for the 3 'end of the sequence (see Table 11).

In one embodiment of the invention, the complex overlap-extension primer mix used in the complex overlap-extension PCR and possibly the reverse transcription step is a) one or more C _L complementary to the sense strand of the immunoglobulin light chain region coding sequence. Or J _L primer; b) at least one V _L 5 ′ primer or V _L leader primer complementary to the antisense strand of the immunoglobulin light chain variable region coding sequence and capable of forming a primer set with the primer (s) in a); c) at least one C _H or J _H primer complementary to the sense strand of an immunoglobulin constant heavy chain domain coding sequence or heavy chain binding region; And d) one or more V _H 5 ′ primers or V _H leader primers complementary to the antisense strand of the immunoglobulin heavy chain variable region coding sequence and capable of forming a primer set with the primer (s) in c).

Primer sets of the present invention, for example, V _Lκ + C _κ , V _Lλ + C _λ , V _Lκ + J _Lκ , V _Lλ + J _Lλ , V _LκL + C _κ , which can amplify the variable region coding target sequence V _LλL + C _λ , V _LκL + J _Lκ , V _LλL + J _Lλ , V _H + J _H , V _H + C _H , V _HL + J _H , or V _HL + C _H , or a combination thereof. .

Further embodiments include C _L (J _L ) primer (s) and V _L (V _LL ) primer (s) suitable for amplifying a sequence comprising a kappa light chain variable region or a lambda light chain variable region.

Preferred embodiments of the invention are C _L (J _L ) primer (s) and V _L (V _LL ) primer (s) suitable for amplifying both kappa and lambda light chain variable region coding sequences.

Even more preferred embodiments of the invention are omnidirectional primers for light chain amplified V _LL primers having at least 90% sequence identity (preferably at least 95% identity) with the gene specific regions of SEQ IDs 93-98, heavy chains The forward primer for amplification is a V _HL primer having at least 90% sequence identity (preferably at least 95% identity) with the gene specific region of SEQ IDs 86-92.

Another embodiment of the invention has the immunoglobulin V _L / V _LL and V _H / V _HL primer binding tails, preferably in the form of complementary overlap-extension tails. This results in variable region coding sequences that are bound in head-to-head form. For binding of the variable region coding sequence in head-to-head form, preferably in the form of complementary overlap-extension tails, the C _L / J _L and V _H / V _HL primers contain a binding tail, or V _L / V _LL and C _H / J _H primers contain binding tails. For binding the variable region coding sequence in tail-to-tail form, the C _L / J _L and C _H / J _H primers contain the binding tail, preferably in the form of complementary overlap-extension tail (FIG. 3). ).

Preferentially, the composite primer mix comprising the composite overlap-extension primer mix of the present invention comprises two primer sets. Thus, the composite primer mix includes four or more different primers. In another aspect of the invention, the composite primer mix comprises four or more different primers. The complex primer mix of the present invention is used for amplification of target sequences in a single vessel. For example, kappa, lambda and heavy chain variable regions are all amplified in the same vessel. One complex primer mix of the present invention consists of 16 different degenerate primers distributed as follows: 8 V _H primers, one C _H1 primer, six V _Lκ primers, and one C _κ primer (FIG. 2). ). Another set consists of 19 degenerate primers distributed as follows: 8 V _H primers, 4 J _H primers, 6 V _Lκ primers, and one C _κ primer. The third set consists of 22 degenerate primers distributed as follows: 8 V _H primers, one C _H1 primer, 11 V _Lλ primers, and two C _λ primers. The fourth set consists of 27 degenerate primers distributed as follows: 8 V _H primers, one C _H1 primer, six V _Lκ primers, one C _κ primer, 11 V _Lλ primers, and 2 C _λ primers.

The invention also includes primers for further PCR amplification of the bound product obtained by ligation or recombination following complex RT-PCR or by complex overlap-extension RT-PCR. Such further PCR amplification can be performed using a primer mix suitable for amplifying the bound target sequence. This primer mix is a complex primer mix or a complex overlap-extension primer, meaning a primer that can be annealed to the outermost 5 'and 3' ends of the sense strand of the bound nucleotide sequence to amplify the entire bound product. It may include the outer primer of the mix. Examples of primers that can be used as the outermost primers in the present invention are C _κ / J _κ and / or C _λ / J _λ primers that form primer sets with J _H or C _H primers. This process generally serves to increase the amount of binding product obtained from ligation or recombination followed by complex RT-PCR, or from complex overlap-extension RT-PCR.

Alternatively, a set of nested primers can be used for further amplification of bound nucleotide sequences compared to the outer primers used in the main complex RT-PCR or complex overlap-extension RT-PCR reactions. The design of the nested primers is generally for the gene specific primers described above, except that it primes 3 'for the annealing position of the outer primer used in the composite RT-PCR or the complex overlap-extension RT-PCR. Stick to the same design rules as Therefore, the product formed from nested PCR is shorter in length than the bound product obtained by ligation or recombination following complex RT-PCR or by complex overlap-extension RT-PCR. In addition to increasing the amount of bound product, nested PCR further serves to increase the overall specificity, particularly of the complex overlap-extension RT-PCR technique. However, it should be noted that not all of the previously described complex primer mix / complex overlap-extension primers are suitable for combination with nested primer sets when performing further amplification. In this case, the outer primer of the complex primer mix / complex overlap-extension primer mix can be used for further amplification, or semi-nested PCR can be used as described later.

In one embodiment of the invention, a mixture of J _L and J _H primers is used as nested primers for further amplification of bound immunoglobulin variable region coding sequences.

In addition, the nested primer set of the present invention is characterized by the reverse (or forward) outer primer (s) from the first composite primer mix / complex overlap-extension primer mix and the first composite primer mix / complex overlap-extension primer mix. And a second nested primer that primes 3 'for the annealing position of the forward (or reverse) outer primer (s). The use of such primer sets for further PCR amplification is generally known as semi-nested PCR. Semi-nested PCR can be applied, for example, when it is difficult to design nested primers (V and J primers) for any specific region, such as a variable region sequence, because such primers are complementary. This is because annealing must be performed in a typical crystal region (CDR). In addition, semi-nested PCR can be used, for example for cloning purposes, if it is desired to retain either end of the intact bound sequence.

One of the features of the present invention is to maintain a constant light chain coding sequence that remains intact during further amplification reactions. The C _L (reverse) primer (s) used for further amplification is only slightly modified compared to the outer primer (s) used for the main reaction (complex RT-PCR or complex overlap-extension RT-PCR reaction). This modification involves the addition of a few bases to the 3 'end of the C _L primer (s) used for main amplification. In addition, different cloning tails can be added to the nested C _L primer (s). The forward primer (s) used for further amplification are fully nested compared to the constant heavy chain specific outer primer (s) used for the main reaction. The combination use of the outer primer (s) and slightly modified nested C _L primer (s) in the main reaction with the fully nested omnidirectional primer (s) in further amplification resulted in a fully nested primer set. This results in an increase in specificity comparable to the specificity achievable by the nested PCR used.

In a preferred embodiment of the present invention, nested PCR is performed with J _H primer (s) and modified C _L primer (s), wherein two to ten gene-specific base pairs are combined primer mix / complex overlap- It is added at the 3 'end as compared to the first C _L primer (s) of the stretch primer mix.

Optimization of Complex Overlap-kidney PCR

Parameters for multiple overlap-extension PCR steps in both two- and single-step procedures can be optimized for several parameters (see, for example, Henegariu, O. et al. 1997. BioTechniques 23, 504-511; Markoulatos, P. et al. 2002. J. Clin. Lab. Anal. 16, 46-51. Although the ratio between the outer primer and the inner primer is less important for this reaction, the same optimization parameters generally apply for complex RT-PCR.

a. Primer concentration

The concentration of primers with overlap-extension tails (eg V _H and V _L primers) is preferably lower than the concentration of outer primers (eg J _H and kappa primers) without overlap-extension tails.

If one of the target sequences is amplified with low efficiency compared to others, for example as a result of higher GC%, it may be possible to equalize the amplification efficiency. This can be done by using a high concentration of primer sets that mediate amplification with low efficiency, or by lowering the concentration of other primer sets. For example, for the heavy chain variable region, the coding sequence tends to have a higher GC%, resulting in lower amplification efficiency than the light chain variable region. This is also true for using V _L primers at lower concentrations compared to V _H primers.

In addition, when using multiple primers, the overall concentration of the primer may be a problem. The upper limit is determined experimentally by titration experiments. For “AmpliTaq Gold” from Applied biosystems, the upper limit has been found to be 1.1 μM total oligonucleotide concentration, but for other systems it may be as high as 2.4 μM. The upper limit of this total oligonucleotide concentration affects the maximum concentration of the individual primers. If the individual primer concentrations are too low, poor PCR sensitivity can result.

The quality of oligonucleotide primers has been found to be important for complex overlap-extension PCR. HPLC purified oligonucleotides yielded the best results.

b. PCR Cycling Conditions

Preferably, the cycling conditions are as follows:

denaturalization: 10-30 seconds 94 ℃ Annealing: 30-60 seconds 50-70 ℃ Less than about 5 ° C below the Tm of the primer kidney: 1 minute x EPL 65-72 ℃ EPL is the expected product length in kb. Cycles 30-80 Final height 10 minutes One 65-72 ℃

For single step complex overlap-extension RT-PCR, the following steps were introduced into the cycling program prior to the outlined amplification cycling.

Reverse transcription 30 minutes 42-60 ℃ These conditions are also used when separate reverse transcription is performed. Polymerase Activation 10-15 minutes 95 ℃ High temperature starting polymerase may be advantageous for single stage RT-PCR. Activation according to the manufacturer.

It is possible to optimize for all these parameters. In particular, the annealing temperature is important. Therefore, every individual primer set at the start that will constitute the final primer mix must be tested separately to ascertain the optimum annealing temperature and time, as well as elongation and denaturation time. This will provide a good idea for the means by which these parameters can be optimized for complex overlap-extension primer mixes.

For example, problems with poor PCR sensitivity due to low primer concentrations or low template concentrations can be overcome by using multiple thermal cycles. Many thermal cycles consist of 35 to 80 cycles, preferably about 40 cycles. In addition, longer elongation times can improve the complex overlap-extension PCR process. Longer elongation times consist of 1.5-5 minutes x EPL compared to standard 1 minute elongation.

c. Use of adjuvant

Complex PCR reactions can be significantly improved using PCR additives such as DMSO, glycerol, formamide, or betaine, which facilitate template denaturation by relaxing DNA.

d. dNTP and MgCl ₂

Deoxynucleoside triphosphate (dNTP) quality and concentration are important for complex overlap-extension PCR. The best dNTP concentration is between 200 and 400 μM for each dNTP (dATP, dCTP, dGTP and dTTP) and at higher concentrations amplification is quickly suppressed. Low dNTP concentrations (100 μM for each dNTP) are sufficient to achieve PCR amplification. dNTP stocks are sensitive to thawing / freezing cycles. After 3 to 5 such cycles, complex PCR often does not work well. To avoid this problem, small aliquots of dNTP are prepared and kept frozen at -20 ° C.

Optimization of the Mg ²⁺ concentration is important because most DNA polymerases are magnesium dependent enzymes. In addition to DNA polymerase, template DNA primers and dNTPs bind to Mg ²⁺ . Therefore, the optimal Mg ²⁺ concentration will depend on the dNTP concentration, template DNA and sample buffer composition. If the primer and / or template DNA buffer contains a chelator such as EDTA or EGTA, the apparent Mg ²⁺ optimal concentration may be changed. Excess Mg ²⁺ concentrations stabilize DNA double strands and inhibit complete denaturation of DNA, which reduces yield. Excess Mg ²⁺ may also stabilize the spurious annealing of the primer against incorrect template sites, thus reducing specificity. On the other hand, insufficient Mg ²⁺ concentration reduces the amount of product.

A good balance between dNTP and MgCl ₂ is about 200-400 μM dNTP (each) for 1.5-3 mM MgCl ₂ .

e. PCR buffer concentration

In general, KCl based buffers are sufficient for complex overlap-extension PCR, while buffers based on other components such as (NH ₄ ) ₂ SO ₄ , MgSO ₄ , Tris-HCl, or combinations thereof are subject to complex overlap-extension PCR. Can be optimized to work. Primer pairs involved in the amplification of longer products are more effective at lower salt concentrations (eg, 20-50 mM KCl), while primer pairs involved in the amplification of shorter products have higher salt concentrations (eg, 80). To 100 mM KCl). By raising the buffer concentration twice, instead of once, the efficiency of the complex reaction can be improved.

f. DNA polymerase

The present invention is exemplified by Taq polymerase. Alternatively, other types of heat resistant DNA polymerases can be used including, for example, Pfu, Phusion, Pwo, Tgo, Tth, Vent, Deep-vent. Polymerases with or without 3′-5 ′ exonuclease activity may be used alone or in combination with others.

Vectors and libraries

Binding of the nucleotide sequences of interest according to the present invention results in a nucleotide segment comprising the desired nucleotide sequence bound. In addition, such libraries of bound nucleic acid sequences of interest are produced by the methods of the invention, in particular the libraries of variable region encoding sequences.

One feature of the present invention is the insertion of a segment containing a linked nucleotide sequence of interest produced by a method of the invention or a library of linked nucleotide sequences of interest into a suitable vector. The library may be a combinatorial library or library of cognate pairs of variable region encoding sequences.

The restriction sites generated by the outer primers, nested primers or semi-nested primers are preferably designed to match the appropriate restriction sites of the selection vector. If a recombinant site suitable for one of the semi-nested, nested primers or external primers is provided and the selection vector contains one, the bound nucleic acid sequence of interest can also be inserted into the vector by the synthesis.

Basically, there is no limitation to the vector which can be used as a carrier of the product produced by one of the complex RT-PCR-binding methods of the present invention. The selection vector can be, for example, a vector suitable for amplification and expression in cells, including bacteria, yeast, other fungi, insect cells, plant cells, or mammalian cells. Such vectors can be used to facilitate additional cloning steps, shuttle between vector systems, display of the inserted product into the vector, express the inserted product and / or integrate the host cell into the genome.

Cloning and shuttle vectors are preferably bacterial vectors. However, other forms of vector can also be applied to the cloning and shuttle processes.

The display vector can be, for example, a phage vector or phagemid vector derived from fd, M13, or fl filamentous bacteriophage. Such vectors can facilitate the display of proteins, including, for example, binding proteins or fragments thereof, on the surface of filamentous bacteriophages. Display vectors suitable for display on ribosomes, DNA, yeast cells or mammalian cells are known in the art. These include, for example, vectors or viral vectors encoding chimeric proteins.

Expression vectors exist for all mentioned species and the vector selected is entirely dependent on the protein being expressed. Some expression vectors may additionally be integrated into the genome of the host cell by random integration, or site specific integration, using appropriate recombinant sites. Expression vectors are designed to provide additional encoding sequences, which further encode a larger protein, eg, full length, when the bound product is inserted in-frame into the additional encoding sequence when incorporated into the appropriate host cell. Enable expression of monoclonal antibodies. Such in-frame insertion can also promote expression of chimeric proteins that promote display on the surface of filamentous bacteriophages or cells. In a bacteriophage display system, the linked nucleotide sequence of interest can be inserted in-frame into a sequence encoding a coat protein such as pIII or pVIII (Barbas, CF et al. 1991. Proc. Natl. Acad. Sci USA 88, 7978-7982; Kang, AS et al. 1991. Proc. Natl. Acad. Sci. USA 88,4363-4366).

In one embodiment of the invention, each segment of the linked nucleotide sequence of interest consists of an immunoglobulin heavy chain variable region encoding sequence associated with a light chain variable region encoding sequence. Preferably these binding sequences are inserted into a vector containing sequences encoding one or more immunoglobulin constant domains. Insertion is engineered such that the bound heavy chain variable region and / or light chain variable region encoding sequences are inserted in-frame into the constant region encoding sequence. Such insertion can produce, for example, an expression vector encoding a Fab expression vector, full length antibody expression vector or fragment of full length antibody. Preferably such vectors are expression vectors suitable for screening (e.g., E. coli, phagemid or mammalian vectors) and the constant region heavy chain encoding sequences are human immunoglobulins, ie IgGI, IgG2, It is selected from IgG3, IgG4, IgM, IgAl, IgA2, IgD, or IgE to allow expression of Fab or full length recombinant antibody. In addition to the constant heavy chain encoding sequence, the vector may also contain a constant light chain encoding sequence selected from human lambda or kappa chains. This is appropriate when the linked nucleotide sequence encodes only immunoglobulin variable region encoding sequences (Fv's).

In another aspect of the invention, the individual segments of the bound nucleotide sequence consist of a γ chain variable region encoding sequence associated with a TcR a chain variable region encoding sequence or a δ chain variable region encoding sequence associated with a β chain variable region encoding sequence. Preferably, these joined sequences are inserted into a vector containing a sequence encoding one or more TcR constant domains. Such insertions are engineered such that the inserted and linked variable region encoding sequences are in frame at the corresponding TcR constant region encoding sequences. In further embodiments, such a vector is a chimeric expression vector comprising a sequence that encodes a leucine zipper in-frame to a TcR constant region. It has been demonstrated that such constructs increase the stability of soluble TcR's (Willcox, B. E. et al. 1999. Protein Sci 8,2418-2423).

Libraries of cognate pairs of the invention can be introduced into a vector by two different methods. In the first method, single cognate pairs are inserted individually into a suitable vector. Such vector libraries can be kept separate or pooled. In a second method, all race pairs are pooled prior to vector insertion, followed by bulk insertion into a suitable vector to produce a pooled vector library (illustrated in FIG. 12). Libraries of such vectors contain a wide variety of pairs of variable region encoding sequences.

One aspect of the invention is a library of cognate pairs of linked variable region encoding sequences. Preferably, the individual cognate pairs of the library comprise immunoglobulin light chain variable region encoding sequences associated with heavy chain variable region encoding sequences.

Another preferred library of cognate pairs includes linked TcR region encoding sequences, wherein each individual TcR region encoding sequence is an alpha chain variable region encoding sequence and / or a delta chain variable region encoding sequence associated with a beta chain variable region encoding sequence. And a TcR gamma chain variable region encoding sequence associated with the.

Embodiments of the invention are sub-libraries of cognate pairs of linked variable region encoding sequences that encode for the desired binding specificities derived for a particular target. Preferably, such cognate pairs comprise a linked immunoglobulin light chain and heavy chain variable region encoding sequence, a TcR alpha chain variable region and a beta chain variable region encoding sequence and / or a TcR gamma chain variable region and a delta chain variable region encoding sequence. Include.

A further embodiment is a sub-library selected from the parent library of cognate pairs of variable region encoding sequences as described throughout the present invention.

Preferred embodiments of the invention are libraries or sub-libraries that encode for cognate pairs of full length immunoglobulins selected from human immunoglobulins, i.e., IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM. .

Another preferred feature of the invention is a library or sub-library that encodes for soluble and stable cognate pairs of TcR.

A feature of the present invention is the diversity of libraries consisting of at least 5, 10, 20, 50, 100, 1000, 10 ⁴ , 10 ⁵ or 10 ⁶ different cognate pairs.

In a further embodiment of the invention, a library of cognate pairs of bound variable region encoding sequences can be obtained by a method comprising the steps described herein. Such a library is also called a parent library.

Screening and Selection

The parent library of bound variable region encoding sequence pairs isolated from the donor, using one of the methods of the present invention, is expected to exhibit a variety of binding proteins, some of which may be irrelevant, that is, in particular combinatorial libraries It does not bind to the required target for. Thus, the present invention encompasses enrichment and screening for sub-libraries encoding a subset of the diversity of binding specificities induced for a particular target.

For libraries of cognate pairs, the diversity of the library is expected to represent the diversity present in the donor material with only a few randomly coupled variable regions. Thus, the incubation step may not be necessary before screening for target specific binding affinity in a library of cognate pairs.

In a further embodiment of the invention, a method of generating a library of bound variable region encoding sequence pairs generates a sub-library by selecting a subset of bound variable region sequence pairs encoding binding proteins having the desired target specificity. Further comprises. Such selection of bound variable region encoding sequences is also referred to as a library of target specific cognate pairs.

Preferred embodiments of the invention are libraries of target-specific cognate pairs of variable region encoding sequences delivered to mammalian expression vectors.

Immunological assays are generally suitable for the selection of target-specific immunoglobulin variable region encoding sequences. Such assays are known in the art and include, for example, ELISPOTS, ELISA, membrane assays (eg, western blots), arrays on filters, or FACS. Such assays can be performed in a direct manner using polypeptides generated from immunoglobulin variable immunorecording encoding sequences. Immunoassays can also be performed with or after hatching methods such as phage display, ribosomal display, bacterial surface display, yeast display, eukaryotic virus display, RNA display or covalent display (FitzGerald, K., 2000. Drug Discov Today 5,253-258). As shown in FIG. 10, both the cognate Fab expression library and the cognate full length antibody expression library can be subjected to screening to generate a sub-library of positive clones. Such screening assays and incubation processes are also suitable for combinatorial libraries of bound variable regions or Fv or scFv fragments. In a preferred embodiment of the invention, the selection of target-specific cognate pairs or combination pairs of variable region encoding sequences is performed by using a high throughput screening assay. The high power screening assay can be, but is not limited to, an ELISA assay performed with a semi-automated or fully automated device. Such assays can also be membrane assays, where bacteria can be picked and grided by a robot onto appropriate membranes on agar plates that produce arrays of colonies expressing antibody binding molecules. Molecules are secreted through the membrane onto a second lower antigen-coated membrane that can be developed separately and used to identify clones that secrete antigen binding molecules to the desired target (de Wildt, RM, et al. 2000. Nat. Biotechnol 18, 989-994).

If the co-pair or combination pair of antigen-binding clones is selected by appropriate techniques, it is possible to perform further analysis by DNA sequencing of bound immunoglobulin light chain variable region and heavy chain variable region encoding sequences. . First, such DNA sequencing will provide information on library diversity, such as germ cell origin, family distribution and maturation within CDR regions. Such analysis allows the selection of clones that exhibit a wide variety of variances, resulting in repeated clones. Second, DNA sequencing will reveal variations introduced during the isolation process.

When analyzing variable region encoding sequences, there are three types of mutations to consider when testing whether mutations are allowed: i) The most frequent form of variation is from cross-priming within the family, where V Gene primers are primed with error subsets in one particular V gene family. The change introduced is mainly the substitution of natural codons at one specific position. Due to the high sequence homology within the V gene family, these changes are generally considered to be conservative and acceptable changes; ii) less frequent variations are induced by inter-family cross-priming (eg, VH3 family primers prime VH1 family encoding sequences) and sometimes lead to more significant structural changes without natural counterparts. Such changes can potently affect the immunogenicity of the variable regions by creating new epitopes. Such changes can be readily identified and subsequently repeated by using standard molecular biological techniques or clones can be excluded from the library; iii) Errors generated by Taq DNA polymerase are most readily identified in the constant region encoding sequence and can be easily removed. However, Taq derived variations will also be present in the variable region encoding sequences, where they are indistinguishable from natural somatic mutations, which are the result of random variations in the variable region encoding sequences. Given that mutations are non-systemic and only affect certain pairs in a unique way, it seems reasonable to ignore such changes.

Sequence analysis can also be used to identify the extent of scrambling in cognate pair libraries as illustrated in Table 20 for VH group H4.

As described in Example 9, the presence of the mutations described in i) and ii) is characterized by expression libraries when using primers that anneal in the leader sequence of the variable region encoding sequence instead of primers that anneal in the 5 'region of the variable region. Can be overcome.

In a further embodiment of the invention, the target specific and possible sequenced pairs of sub-libraries of the bound immunoglobulin light chain variable region and heavy chain variable region encoding sequences can be delivered to a mammalian expression vector. Such delivery may be performed into any of the vectors described in the paragraphs above to allow expression of full length recombinant antibodies. If screening is performed with a mammalian cognate full-length antibody expression library, such delivery may not be necessary.

In another embodiment of the invention, the maternal library is generated from lymphocyte-containing cell fractions that are hatched against T lymphocytes. The pairs of bound variable region encoding sequences that make up the parent library encode a subset of pairs of bound variable region sequences consisting of alpha and beta and / or gamma and delta chains that encode a binding protein with the desired target specificity, It can be chosen to generate a sub-library of cognate pairs or combination pairs. Antigen-specific T cell receptors are subsequently subjected to standard techniques such as staining with tetrameric MHC-peptide complexes (eg, Callan, MF et al. 1998. J. Exp. Med. 187, 1395-1402; Novak, EJ et al. 1999. J. Clin. Invest 104, R63-R67) for measuring cell responses in IL-2 release form or transfected by more ideal methods such as yeast or retroviral display technology. It can be identified from the pool of.

Host Cells and Expression

Libraries of the invention can be delivered to vectors suitable for the expression and production of proteins encoded from the bound nucleic acid sequences of interest, in particular variable region containing binding proteins or fragments thereof. Such vectors are described in the Vectors and Libraries section and are provided for example in the expression of full length antigens, Fab fragments, Fv fragments, scFv, membrane bound or soluble TcRs or TcR fragments of selected species.

One feature of the invention is the introduction of a cognate pair of linked variable region encoding sequences or a library or sub-library of vectors of a single clone encoding a cognate pair of linked variable region encoding sequences for amplification and / or expression into a host cell. It is. The host cell can be selected from bacteria, yeast, other fungi, insect cells, plant cells or mammalian cells. For expression, mammalian cells such as Chinese hamster ovary (CHO) cells, COS cells, BHK cells, myeloma cells (eg Sp2 / 0 cells, NSO), NIH 3T3, fibroblasts or immortalized human cells such as , HeLa cells, HEK 293 cells, or PER. C6 is preferred.

The introduction of the vector into a host cell includes many transformations or trans known to those of skill in the art, including calcium phosphate precipitation, electrophoresis, microinjection, liposome fusion, RBC ghost fusion, protoplast fusion, and viral injection. It can be performed by the specification method. Methods of producing monoclonal full length antibodies, Fab fragments, Fv fragments and scFv fragments are known.

The production of recombinant polyclonal antibodies used for processing is a brand new field. Recombinant polyclonal production techniques are described in PCT application WO 2004/061104. In summary, this technique involves the production of cell collections suitable as manufacturing cell lines. The description of libraries of cognate pairs is described below, but may also apply to combinatorial libraries. Individual cells in the cell collection can express a distinct number of recombinant polyclonal binding proteins, eg, from a library of cognate pairs. In order for the individual cells to express a single cognate pair rather than several cognate pairs of polyclonal binding proteins, a nucleic acid sequence encoding the cognate pair is introduced at a single site specific site in the genome of each individual cell. This is an important feature of the cell collection, because it prevents scrambling of the heavy and light chains expressed from each cell, but with the exception of small differences in the variable regions of the individual cognate pairs, Because it creates. This feature will enable unbiased growth of the cell collection over the time period required for production. To ensure single site-specific integration, a host cell line with only one integration site should be used, such cell line, for example, an Invitrogen CHO Flp-In cell containing a single FRT site (Invitrogen's CHO Flp). -In cell). Suitable vectors for these cell lines contain the corresponding FRT site and are introduced into the genome by using Flp synthase. There are several known recombinant enzymes, eg, phageΦC31 integrase, which can be used with Cre, beta-recombinase, Gin, Pin, PinB, PinD, R / RS, lambda integrase, or corresponding recombinant sites. do. In addition, suitable vectors contain selection markers that allow the selection of site-specific integration.

Production of polyclonal producing cell lines and production of recombinant polyclonal proteins from such cell lines can be obtained by several different transfection and preparation techniques.

One method is to use a library of vectors mixed together into a single composition to transfect host cell lines into a single integration site per cell. This method is called bulk transfection or bulk phase transfection. In general, the vector and host cell design described above will allow polyclonal cell lines to be obtained upon appropriate selection that allow unbiased growth. Frozen stocks of polyclonal cell lines will be produced before initiation of recombinant polyclonal protein production.

Another method is to use a library of vectors divided into fractions containing about 5-50 individual vectors of the library in the composition for transfection. Preferably, the fraction of the library consists of 10 to 20 individual vectors. Each composition is then transfected into an aliquot of the host cell. This method is called anti-bulk transfection. The number of aliquots transfected depends on the size of the library and the number of individual vectors in each fraction. If the library consists of, for example, 100 distinct cognate pairs that are divided into fractions containing 20 distinct members in the composition, an aliquot of 5 host cells constitutes a distinct fraction of the original library. It needs to be transfected with the library composition. Aliquots of host cells are selected for site-specific integration. Preferably, distinct aliquots are selected separately. However, they may be pooled before selection. Aliquots will be analyzed for their clonal diversity and only aliquots of sufficient diversity will be used to generate polyclonal cognate pair library stocks. In order to obtain the required polyclonal cell lines for preparation, aliquots can be mixed before producing the frozen stock, immediately after they are recovered from the stock, or after a short proliferation and adaptation time. Optionally, the cell aliquots are maintained separately throughout the production process, and the polyclonal protein composition is constructed by combining the product of each aliquot rather than the cell aliquot before production.

The third method is a high power method in which host cells are separately transfected by using individual vectors that make up a library of cognate pairs. This method is called individual transfection. Individually transfected host cells are preferably selected separately for site specific integration. Individual cell clones generated at selection can be analyzed for proliferation time, and clones with similar growth rates are used to generate polyclonal cognate pair library stocks. These individual cell clones can be mixed to produce the required polyclonal cell lines immediately after they are recovered from the feedstock, shortly after their proliferation and adaptation time, before producing the feedstock. This method can eliminate any possible residual sequence bias during transfection, integration and selection. In addition, individually transfected host cells are mixed before selection is performed, and such mixing will allow control of sequence bias due to transfection.

A common feature in the manufacturing techniques outlined above is that all individual cognate pairs that make up the recombinant polyclonal protein can be produced at one time or in a limited number of bioreactors. The only difference is the step of selecting to produce a collection of cells that make up the polyclonal producing cell line.

One embodiment of the invention is an individual of a host cell comprising a cognate library or sub-library of binding pairs of variable region encoding sequences.

In a further embodiment, the individual of the host cell constitutes the lymphocytes, using the ligation of the present invention or binding by recombinant or multimeric overlap-extension RT-PCR techniques to bind cognate pairs followed by complex RT-PCR amplification. Libraries obtained from individuals of isolated single cells are included.

Another embodiment of the invention is an individual of a host cell comprising a combinatorial library or sub-library of linked pairs of variable region encoding sequences.

The individual of the host cell according to the present invention will include various individuals of the cell corresponding to the diversity of the library into which the cell is transformed / transfected. Preferably, each cell of the cell individual constitutes only one cognate pair of the entire library of cognate pairs and the individual members of the cognate pair library are at least 50%, more preferred, of the total number of individual members expressed from the individual of the host cell. Preferably at least 25%, most preferably at least 10%.

In a preferred embodiment of the invention, the individual of the host cell is a mammalian cell.

Individuals of the host cell as described above can be used for the expression of recombinant polyclonal binding proteins, because the individual cells of the individual constitute different variable region encoding sequences of different diversity.

One embodiment of the invention is a recombinant polyclonal protein expressed from an individual in a host cell comprising a library of vectors encoding various cognate pairs of linked variable region encoding sequences, wherein the library is in a method of the invention. Can be obtained by Typically, recombinant polyclonal proteins of the invention consist of at least 2,5, 10, 20,50, 100, 1000, 10 ⁴ , 10 ^5, or 10 ⁶ proteins of different cognate pairs.

Preferred embodiments of the invention are recombinant polyclonal immunoglobulins expressed from an individual in a host cell comprising a library of vectors encoding various cognate pairs of heavy chain variable region and light chain variable region encoding sequences.

Another preferred embodiment of the invention is a library of vectors encoding various cognate pairs of TcR alpha chain variable region and / or TcR gamma chain variable region coupled with a beta light chain variable region encoding sequence. Recombinant polyclonal TcR expressed from an individual of a host cell comprising a.

Another embodiment of the invention is a host cell suitable for the production of monoclonal proteins. In particular, monoclonal antibodies consist of cognate pairs of light chain variable regions with heavy chain variable regions, or monoclonal TcRs consist of alpha variable regions with beta variable regions or cognate pairs of delta variable regions with gamma variable regions. Preferably such monoclonal producing cell line is not a hybridoma cell line.

Monoclonal antibodies or TcRs can be generated by adding the following steps to the method of binding the various discontinuous nucleotide sequences of interest; a) inserting the bound nucleic acid sequence into a vector; b) introducing said vector into a host cell; c) culturing said host cell under conditions suitable for expression; d) to obtain the protein product expressed from the vector inserted into the host cell. Preferably, the vectors introduced into the host cell encode individual cognate pairs of variable region encoding sequences.

Application of the present invention

One of the major applications of the present invention is in variable region encoding sequences, in particular immunoglobulin and light chain variable region encoding sequences or TcR alpha and beta chains or gamma and delta chain variable region encodings, by high efficiency methods for the generation of cognate pair libraries. A combination of cognate pairs of sequences. In addition to the generation of cognate pair libraries, the binding by the ligation or recombinant or complex overlap-extension RT-PCR technique of the present invention followed by the combination RT-PCR can be achieved from individuals of genetically different cells, individuals of such cells in the generation of combinatorial libraries Cell lysates, or RNA purified from an individual of such cells. Libraries, sub-libraries, or monoclonals from such libraries facilitate the expression of polyclonal or monoclonal proteins. In particular monoclonal or polyclonal antibodies can be obtained from the libraries of the invention.

The use of recombinant monoclonal antibodies in diagnosis, treatment and prevention is well known. Recombinant monoclonal and polyclonal antibodies raised by the present invention will have the same application as antibody products generated by the techniques present. In particular, pharmaceutical compositions comprising polyclonal recombinant immunoglobulins as active ingredients in combination with one or more pharmaceutically acceptable excipients may be produced by the present invention. More preferably, the pharmaceutical composition is comprised of polyclonal recombinant immunoglobulins consisting of cognate pairs of variable region encoding sequences. Pharmaceutical compositions of such polyclonal recombinant immunoglobulins can be used as medicaments. The polyclonal recombinant immunoglobulins of the composition can be specific or responsive to predetermined disease targets, so that the composition can be used for cancer, infections, inflammatory diseases, allergies, asthma and other in mammals such as humans, livestock or pets. It can be used to treat, alleviate or prevent diseases such as respiratory disease, autoimmune disease, immunological dysfunction, cardiovascular disease, central nervous system disease, metabolic and endocrine diseases, transplant rejection, or pregnancy that is not desired.

The present invention has further applications that cannot be obtained with conventional monoclonal combinatorial antibody techniques. In situations where the protective antigen is rarely or completely unknown, such as causing infectious disease, it is impossible to screen for monoclonal antibodies that provide protection against the disease.

However, in the present invention, antibody-expressing cells are obtained directly from a donor with an established protective antibody response, eg, a patient in the recovery phase, and using a starting material from each of them to cognate in the immunoglobulin and light chain variable region encoding sequences. You will be able to generate a pair of libraries.

For example, under circumstances where viruses are known but protective antigens are not known, it may be possible to generate sub-libraries of cognate antibody gene pairs with broad reactivity to antigen structure on the virus. If recombinant polyclonal antibodies are produced from this sub-library, they will contain almost protective antibodies.

In situations where antigens are not fully known, recombinant polyclonal antibodies generated from cognate pair libraries, such as patients in the recovery phase, can be used in the same way that hyperimmune immunoglobulins are used today. This is because the recombinant polyclonal antibodies produced are cognate pairs that closely resemble the antibody immune response of convalescent patients.

Another application of the techniques for joining cognate pairs of variable regions described herein is for diagnostic and analytical purposes. When administering a medicament to a patient, there is always a possibility of an immune response induced against the medicament. Such immunogenicity can be assessed by conventional techniques, eg, drug-specific binding assays with serum or plasma derived from an individual treated with a medicament. Alternatively, the methods of the invention can be used to minimize the immune response of short patients after administration of a medicament by separating cognate pairs of variable heavy and light chain encoding sequences. Antibodies expressed from such libraries can then screen for reactivity with the agent or component of the agent. This method is particularly useful when the presence of the medicament in plasma or serum interferes with conventional methods. Such agents are, for example, antibodies. In conventional methods, identification of anti-drug immune responses (eg, anti-gene, anti-structural, or anti-Fc immune responses) in connection with the treatment of the antibody results in the complete removal of the antibody from the blood from the treatment. May be performed until In the present invention, the sequences encoding such anti-drug antibodies can be isolated and analyzed from the individual treated with the medicament within two weeks. Thus, this would be an alternative method of evaluating whether generic drugs and specifically antibody based drugs cause an immune response.

Another application of the present invention is the validation and comparison of vaccine and vaccination programs. This is particularly useful during vaccine development, since in addition to comparing the antibody binding affinity and flow of serum titers, it will be possible to assess and compare the sequence diversity of antibody responses generated in response to novel candidate vaccines. The invention can also be used to analyze, monitor and compare antibody responses in the supervision of vaccine efficacy in a subject.

The present invention has also found application beyond the field of variable region containing binding proteins. Those skilled in the art have only a problem of applying the techniques described herein to join two or more transcribed nucleotide sequences encoding heteromeric proteins in addition to binding proteins. The binding of sequences encoding for a domain or subunit from a heteromeric protein is advantageous for the separation of such protein encoding sequences, since it can significantly reduce several steps. In addition, splice modifications, mutations, or novel family members of these proteins can be isolated using only one complex primer mix / complex overlap-extension primer mix.

The binding of sequences encoding the distal domain of a single protein is also possible. This technique facilitates the study of the importance of specific domains in multidomain proteins, as it facilitates the removal of intermediate domains.

Generation of chimeric or coupled proteins is also a region to which the present invention may be applied. Even if the protein to which the binding can be made is a chimeric line between different origins, for example human and murine proteins, the present invention can be used by mixing cells prior to reverse transcription. Such cell mixtures may consist of individuals of cells from each species or single cells from each species.

Example 1: 2-Step Complex Redundant-Elongated RT-PCR

In this example, reverse transcription (RT) was performed using a template derived from isolated single cells, and the produced cDNA was used as a template for more than one complex overlap-extension PCR.

a. cell

IgG1-kappa expression Chinese hamster ovary (CHO) cell lines were generated using Flp-In technology (Invitrogen, Carlsbad, CA, USA).

IgG-kappa mammalian expression plasmid vector pLL113 (FIG. 4) was constructed based on the Flp-In expression vector, pcDNA5 / FRT. CHO-Flp-In cells were prepared using Lipofectamin 2000 (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions, pOG44 conferring transient expression of pLL113 and Flp recombinase containing the antibody encoding gene. Simultaneous infection. Transformants were selected and demonstrated for generation of IgG-kappa by immunoassay. Selected cell lines were designated CHO Flp-In pLL113 and maintained in Hams F-12 medium fed with 2 mM L-glutamine, 10% FCS and 900 Hygromycin B (floating medium).

CHO Flp-In pLL113 cells were harvested using trypsin and washed three times with flotation medium. After the last wash, cells were resuspended in the original volume of flotation medium. Cell concentration was measured using the Casy-1 system (Schaerfe System GmbH, Reutlingen, Germany) and diluted to a concentration of 1 cell / 5 μl in flotation medium.

b. Reverse transcription

CHO Flp-In pLL113 cell suspension containing an average of one cell was dispersed in a single well of a 96-well PCR plate (Thermo-Fast 96, skirted AB-0800, ABgene, Epsom, Surrey, UK).

cDNA was synthesized from distributed cells using a reverse transcriptase (RT) step in the Qiagen One-Step RT-PCR protocol (Qiagen OneStep RT-PCR Kit, Cat # 210210, Hilden, Germany). .

Each well contained the following reagents in a total volume of 20 μl:

1 x one-step RT-PCR buffer,

DNTP's with a final concentration of 1 mM

5 pmol oligo poly-dT (18),

26 U RNase initiator (RNasin, Promega, Madison USA, cat.No N2111),

2 μl one step RT-PCR enzyme mix, and

5 μl CHO Flp-In pLL113 cell suspension.

The RT reaction was carried out by incubating the reaction mix at 55 ° C. for 30 minutes. The reverse transcriptase was then inactivated by incubating the reaction mixture at 94 ° C. for 10 minutes.

c. Compound Overlap-Kidney PCR

A fragment of the cDNA product generated in step b) was used as a template for complex overlap-extension PCR. The reaction was carried out in 96-well plates.

Each well contained the following reagents in a total volume of 40 μl:

1 x one step RT-PCR buffer,

DNTP's with a final concentration of 500 μM

Complex overlap-extension primer mix at concentrations as described in Table 1,

26 U RNase initiator (RNasin, Promega, Madison USA, cat.No N2111),

2 μl one step RT-PCR enzyme mix, and

1 μl cDNA template (derived from single cell (step b)).

The complex overlap-extension primer mix used included the primers shown in Table 1 below.

Table 1

The reaction was carried out using the 96-well MWG Primus HT thermocycler (MWG Biotech AG, Ebersberg, Germany) under the following cycle conditions:

Denaturation 30 sec 95 ° C .: 50 cycles,

Firing 30 seconds 50 ° C .: 50 cycles,

Extension 5 min 72 ° C .: 50 cycles,

Last extension 10 minutes 72 ° C .: 50 cycles

d. Nested PCR

Nested PCR was performed using the complex overlap-extension product as a template. The reaction was carried out in 96-well plates.

Each well contained 50 μl final volume containing the following reagents:

1 x Bio Taq Buffer,

DNTP's with a final concentration of 400 μM

2 mM MgCl ₂ ,

Nested PCR primer mix,

1.25 U BIOTAQ DNA polymerase (Cat. No BIO-21040, Bioline, UK.), And

1 μl complex overlap-extension PCR product (step c).

The primers used are shown in Table 2 below.

TABLE 2

The reaction was carried out using the 96-well MWG Primus HT gene amplifier (MWG Biotech AG, Ebersberg, Germany) under the following cycle conditions:

Denaturation 30 seconds 95 ℃: 25 cycles,

Firing 30 seconds 50 ° C: 25 cycles each

Extension 5 minutes 72 ° C .: 25 cycles each,

Last extension 10 minutes 72 ℃: 25 cycles each

Nested PCR products were analyzed by 1% agarose gel electrophoresis using ethidium bromide for detection (FIG. 5). The expected size of the overlap-extension product was 1076 bp. This product was observed in lanes 1, 5, 6, 7, 8 and 12 indicated by arrows (FIG. 5A). In the described experiments, negative controls were contaminated so that similar contaminants were present in the sample. 5B shows a fragment of FIG. 5A associated with this experiment.

This experiment shows one method for performing two-step complex overlap-extension PCR using a template derived from a single cell. It also shows that approximately 20 complex overlap-extension PCR's can be performed using cDNAs generated from isolated single cells.

Example 2: Single-Stage Complex Redundant-Elongated RT-PCR

In this example, reverse transcription and complex overlap-extension PCR were performed in one step using templates from lysed cells, at concentrations corresponding to 100, 10 or 1 cells.

a. cell

Anti-Human Product Modified Dulbecco's Medium of Isocobe Obtaining Human Hybrid Tumor Cell Line HB-8501 Producing IgG1-kappa Antibody from the American Type Culture Collection and Containing 10% Fetal Bovine Serum (Vitacell, Kiev , Ukrain, cat. No 30-2005). Prior to performing the complex overlap-extension RT-PCR, cells were harvested, counted and frozen at −80 ° C. in culture medium at a concentration of 200 cells / μl.

b. Single Phase Complex Redundant-Eight RT-PCR

The Qiagen One-Step RT-PCR kit (Qiagen cat. No 210212, Hilden, Germany) was used for the complex overlap-extension RT-PCR, essentially following the manufacturer's advice. Prior to addition to the PCR tubes, lysates were thawed and diluted in H ₂ O to yield lysate concentrations corresponding to 100, 10 and 1 cells per 5 μl.

Each PCR tube was 50 μl in total volume and contained the following reagents:

1 x one-step RT-PCR buffer,

DNTP's with a final concentration of 400 μM

Complex overlap-extension primer mix at concentrations as described in Table 3,

2 μl one-step RT-PCR enzyme mix,

50 U RNase initiator (RNasin, Promega, Madison USA, cat.No N2515) and

5 μl diluted cell lysate.

The complex overlap-extension primer mixes used are shown in Table 3 below.

TABLE 3

The reaction was carried out using the following cycle conditions:

Reverse Transcription: 30 min 55 ℃

Polymerase activity: 15 min 95 ° C, inactivate reverse transcriptase and activate Taq polymerase.

PCR reactions:

Denaturation 30 seconds 94 ℃: 50 cycles each

Firing 30 seconds 44 ℃: 50 cycles each

Extension 3 min 72 ℃: 50 cycles each

Final extension 10 minutes 72 ° C .: 50 cycles each.

Ten microliters of reaction product were analyzed using 1.5% agarose gel electrophoresis using ethidium bromide for detection (FIG. 6A).

The expected size of the fragment (exact size depends on the length of the variable region) is:

V _H : 410 bp,

LC: 680 bp

Overlap-extension fragment: 1070 bp.

Separate DNA fragments with mobility corresponding to heavy chain variable region (V _H ) and light chain variable and constant region (LC) lengths are shown for all dilutions of cell lysates. Less strenuous fragments with mobility corresponding to the putative overlap-extension fragments appeared from 100 and 10 cells in lysate. Duplicate-extension fragments of one cell lysate sample, although originally shown on the gel, are difficult to recognize (see arrows in the photograph shown in FIG. 6A and sketches in FIG. 6B).

c. Identification of duplicate-extension fragments

From the experiments reproduced as described, the presence of overlap-extension bands was demonstrated. Regions around 1070 bp were cut from lanes corresponding to 1 cell and lanes corresponding to 100 cells in an agarose gel, using Qiaex II (Qiagen cat. No 20051, Hilden, Germany) Purified and eluted in 20 μl of water.

1 microliter of eluate was estimated according to the manufacturer's instructions for overlapping-extension fragments (J _H primers corresponding to SEQ ID NOs 32 to 35 and C _LK primers corresponding to SEQ ID NO 17; the concentration of each primer was 0.5 PCR (Biotaq kit, Bioline, UK cat.NO BIO-21040) was performed. The cycle parameters are as follows:

Denaturation 30 seconds 95 ℃: 30 cycles each

Firing 30 seconds 55 ℃: 30 cycles each

Extension 1 min 72 ° C .: 30 cycles each.

Ten microliters of reaction product each were analyzed using 1% agarose gel electrophoresis using ethidium bromide for detection. Several fragments, including both 1 and 100 cells, showed fragments with the mobility of the expected overlap-extension fragments (arrows in FIG. 6C). 1 kb fragments from gel lanes corresponding to one cell were cleaved from the gel and purified using QIAX II as described above. Purified fragments were digested with restriction enzymes Nhel and Ncol (individually) and reaction products were analyzed using 1% agarose gel electrophoresis using ethidium bromide for detection (FIG. 6D). Restriction sites were present in the overlap between VH and LC, and the expected sizes after digestion were approximately 410 and 680 bp in length, respectively (FIG. 2). NheI digestion was partial because large fragments existed in their original size.

Example 3: Combined Single-Step Complex Over-Elongation RT-PCR and Nested PCR

In this example, reverse transcription and complex overlap-extension PCR reactions were performed in a single step and then using semi-nested PCR amplification with templates from lysed cells at concentrations corresponding to 100, 10 or 1 cells. .

a. Single-Stage Complex Redundant-Elongated RT-PCR

Complex overlap-extension RT-PCR using HB-8501 cell lysate was performed as described in Example 2 using a complex overlap-extension primer mix comprising the primers shown in Table 4 below.

Table 4

However, using only one of the C _H1-5 primers for each complex overlap-extension primer mix, it was confirmed that five different complex overlap-extension primer mixes were obtained.

The remaining parameters are as described for the complex overlap-extension RT-PCR reaction of Example 2 with varying firing temperatures to 50 ° C. The reaction was carried out using lysates corresponding to 100, 10, 1 and 0 cells, each heavy chain constant region reverse primer (C _H1 , C _H2 , C _H3 , C _H4 , C corresponding to SEQ ID NOs 49-53, respectively) _H5 ).

b. Semi-Nested PCR

One microliter of complex overlap-extension RT-PCR reaction product was performed by semi-nested PCR (Biotaq kit, Bioline, UK cat. NO BIO-21040) as essentially suggested by the manufacturer. The total volume of reaction was 50 μl. The primers used are shown in Table 5 below.

Table 5

Cycle conditions are as follows:

Denaturation 30 seconds 95 ℃: 25 cycles,

Firing 30 seconds 50 ° C: 25 cycles each

Extension 1.5 minutes 72 degrees Celsius

Final extension 5 minutes 72 ° C.

Each reaction of 10 microliters was analyzed using 1.5% agarose gel electrophoresis using ethidium bromide for detection (FIG. 7).

The putative overlap-extension fragments from the semi-nested PCR corresponding to lysates from one cell and the CH4 primers were used for the first reaction (see arrows in FIG. 7) were cut from agarose gels and the QIAEX II kit Purification using (Qiagen cat. No. 20051, Hilden, Germany) and pCR2.1 using Invitrogen TOPO TA cleaning kit (Invitrogen cat.No 45-0641, Carlsbad, CA, USA) -Inserted into TOPO. Inserts of eight clones were listed and seven of them appeared to be composed of heavy chain variable regions bound to the light chain variable and constant regions by the expected overlap regions.

In summary, the reactivity of the combined complex overlap-extension RT-PCR and semi-nested PCR reactions was satisfactory, with 4 of 5 constant region primers producing significant amounts of overlap-extension products from lysates corresponding to single cells. Can be amplified.

Example 4 Combined Single-Step Complex Redundant-Kidney RT-PCR and Nested RCR Using Concentrated Human B Lymphocytes as Template Source

In this example, a semi-nested amplification PCR was performed after a single step, using a single human B lymphocyte isolated from a reverse transcription and complex overlap-extension PCR reaction as a template source.

a. B-cell isolation

Male human donors were inoculated with tetanus toxoid. 120 ml blood samples were collected from the donor 6 days after inoculation and peripheral blood mononuclear cells (PBMC's) were isolated using Lymphoprep (Axis-Shield, Oslo Norway, prod. No 1001967) according to the manufacturer's instructions. . CD19-positive cell individuals were concentrated using magnetic bead cell sorting. PBMC's were stained with FITC-conjugated anti-CD19 antibodies (Becton Dickinson, NJ, USA, cat. No 345776). Magnetic bead cell sorting and column purification using anti-FITC-conjugated magnetic microbeads were performed according to manufacturer's instructions (Miltenyi Biotec, Gladbach, Germany, cat. No 130-042-401). Cells were diluted to a concentration of 200 cells per ml in PBS containing 2 nM EDTA and 0.5% BSA. Five microliters of diluted cells were dispensed into PCR tubes to yield approximately single cells per tube. The tube was stored at -80 ° C until use.

b. Compound Redundant-kidney RT-PCR and Semi-Nested PCR

Conditions for complex overlap-extension RT-PCR and semi-nested PCR are as described in Example 3. However, the reaction was performed only with the complex overlap-extension primer mix consisting of primer C _H3 corresponding to SEQ ID NO: 51. Sixteen samples from the combined complex overlap-extension RT-PCR and semi-nested PCR reactions were performed with 1% agarose gel electrophoresis using ethidium bromide for detection, respectively, 10 μl of semi-nested PCR reaction Analyze by doing (Fig. 8).

As shown in FIG. 8, two of the 16 lanes (lanes 5 and 6) contained fragments of expected mobility (approximately 1 kb). Lane 2 also contained fragments that were less strong at the expected mobility. 1 kb fragments of lanes 5 and 6 were cut from agarose gels, purified using Qiagen II kit (Qiagen cat. No 20051), invitrogen TOPO TA cloning kit (Invitrogen cat.No 45-0641, Carlsbad, CA, USA) was used to insert pCR2.1-TOPO. Two clones from each isolated fragment had inserts of the correct size. Restriction enzyme digesters (individually, NcoI and NheI) exhibited fragments of expected sizes (410 and 680 bp) indicating the correct binding between the heavy chain variable region and the light chain variable and constant region encoding sequences.

Two clones from the fragments of lane 5 were arranged and found to match, indicating that the bound V _H and LC are cognate pairs.

Example 5: V _λ Combined single-step complex overlap-extension RT-PCR and nested PCR with specific primers

In this example, reverse transcription and complex overlap-extension PCR reactions were performed in a single step using _Vλ -specific primers followed by a half nested PCR reaction. Total RNA purified from two different cell line expressing lambda gene families 1b and 1e in combination with the same heavy chain variable region was used as template.

a. cell

Two IgGl-lambda expressing Chinese hamster ovary (CHO) cell lines were generated using Flp-In technology (Invitrogen, Carlsbad, CA, USA).

IgGl-lambda mammalian expression vectors pEm465 / 01P581 and pEm465 / 01P582 (FIG. 9A), representing two lambda gene families, were constructed based on the Flp-In expression vector, pcDNA5 / FRT. CHO-Flp-In cells were simultaneously concomitant with the above-described plasmid containing pOG44 conferring transient expression of the antibody encoding gene and Flp recombinase using Fuzen 6 (Fugene 6, Roche, Mannheim, Germany) according to the manufacturer's instructions. Infected. Transformants were selected for the inserts. The selected cell lines were named CHO Flp-In / Em464 / 01P581 & CHO Flp-In / Em464 / 01P582 and were supplied with 2 mM L-glutamine, 10% FCS and 900 μg / ml hygromycin B in a Ham's F-12 medium ( Flotation medium).

Cell lines were harvested using trypsin and washed three times in flotation medium. Approximately 10 ⁷ cells were used for total RNA purification using the Nucleo Spin L kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer's instructions. Final RNA concentration was determined by OD ₂₆₀ measurement.

b. Single-Stage Complex Redundant-Elongated RT-PCR

Example 3 Using a complex overlap-extension RT-PCR using 50 pg, 5 pg or 0.5 pg total RNA as a template, essentially a complex overlap-extension primer including the primers shown in Table 6 and the cycle conditions specified below It was performed as follows.

Reverse Transcription: 30 min 55 ℃

Polymerase activity: inactivates 95 ° C reverse transcriptase and activates Taq polymerase

PCR reactions:

Denaturation 30 seconds 95 ℃: 35 cycles each,

Firing 30 seconds 50 ° C: 35 cycles each

Elongation 5 minutes 72 ℃: 35 cycles each,

Final elongation 10 min 72 ° C.

Table 6

c. Semi-Nested PCR

One microliter of complex overlap-extension RT-PCR reaction product was performed by semi-nested PCR (Biotaq kit, Bioline, UK cat. No BIO-21040) essentially as suggested by the manufacturer. The total volume of reaction was 20 μl. The primers used are shown in Table 7 below.

TABLE 7

Cycle conditions are as follows:

Denaturation 30 seconds 95 ℃: 25 cycles,

Firing 30 seconds 50 ° C: 25 cycles each

Elongation 1.5 minutes 72 ℃: 25 cycles each,

Final elongation 5 min 72 ° C.

Ten microliters of nested product were analyzed by 1.5% agarose gel electrophoresis using ethidium bromide for detection (FIGS. 9B and 9C). The arrow points to the overlap-extension product with the expected migration properties of approximately 1 kb.

This experiment describes that the lambda complex overlap-extension primer mix shown in Table 6 is applicable to single-step complex overlap-extension RT-PCR independently of the template used. In addition, certain overlap-extension PCR products were generated in 0.5 pg total RNA. This sensitivity suggests that cognate linked heavy chain variable region and light chain variable region encoding sequences can be amplified from a single cell.

Example 6: Generation of sub-libraries of tetanus-specific cognate pairs of antibody encoding sequences

In this example, the steps outlined in the process diagram described in FIG. 10 are illustrated using tetanus toxoid (TT) as the target antigen.

a. Donor

Donors vaccinated with the tetanus vaccine were augmented with tetanus vaccine (Statens Serum Institut, Denmark). Six days after tetanus vaccine enhancement approximately 200 ml of blood sample was flowed from the donor into a tube containing anticoagulant.

Donors were generally required to be in a healthy state with no known or chronic infection. They have never had an autoimmune disease or have been injected with any immunosuppressant and have not been vaccinated in the last three months. In addition, upon increasing the TT-vaccine, the donor must not have had any serious infections in the last month.

b. Preparation of Peripheral Blood Mononuclear Cells (PBMC)

PBMCs were isolated from blood samples using Limpopripe (Axis-Shield PoC AS, Norway, prod. No 1001967) at the manufacturer's suggestion. In summary, blood was diluted 1: 1 in PBS and these suspensions were layered on lymphpoprim at a ratio of 2: 1. The vial was centrifuged at 800 g, 25 ° C. for 20 minutes and a white mesophase band was collected. Cells were washed in PBS containing 2 mM EDTA.

c. Enrichment of B Cells

The B-cell lineage of PBMC's was concentrated by magnetic bead cell sorting using the following procedure.

Isolated PBMCs were stained with anti-CD19-FITC (Becton Dickenson, NJ, USA cat. No 345776). All steps were performed at 4 ° C. in the dark. Staining was performed with 10 μl anti-CD19-FITC per 1 × 10 ⁶ cells in a volume of 100 μl per 1 × 10 ⁶ cells using M-buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA). This will stain the B-cell lineage of PBMCs. The cells were incubated for 20 minutes and then washed twice with M-buffer. Wherein -CD19-FITC stained cells to 1 × 10 ⁶ wherein the 1 × 10 ⁶ cells in a volume of 100 per 10 ㎕ ㎕ M- buffer per cell -FITC magnetic beads (Miltenyi Biotec, Gladbach, Germany, cat. No 130-042 -401), magnetically labeled with anti-FITC conjugated microbeads. Incubation was performed for 15 minutes and then washed once with M-buffer. Cells were resuspended in degassed M-buffer.

 The MACS LS column (Miltenyi Biotec, Gladbach, Germany, cat.No 130-042-401) was pretreated with degassed M-buffer according to the manufacturer's instructions. A suspension of cells stained with anti-CD19-FITC and labeled with anti-FITC-magnetic beads was injected into the column and passed through. Stained and labeled cells (CD19 +) will be maintained in the magnetic field around the column, while unstained cells (CD19−) will pass through the column. The column was washed with degassed M-buffer. Magnetic field was removed and CD19 + cells were collected.

d. Classification of Plasma Cells

Eluates from the MACS column were centrifuged and resuspended in FACS buffer (PBS, pH 7.2, 2% BSA) at a concentration of 1 × 10 ⁶ cells / 60 μl FACS buffer. Anti-CD19-FITC (Becton Dickenson, NJ, USA, cat.No 345776) (10 μl / 10 ⁶ cells), anti-CD38-PE (Becton Dickenson, NJ, USA, cat.No 555460) (10 μl / 10 ⁶ cells) and anti-CD45-PerCP (Becton Dickenson, NJ, USA, cat. No 345809) (20 μl / 10 ⁶ cells) was added. Incubation was performed for 20 min at 4 ° C. in the dark, then washed twice and resuspended in FACS buffer.

Cells were sorted by fluorescence activated cell sorting (FACS) using the following gating parameters:

1. Scattering and lateral scattering to avoid dead cells having lymphocytes and monocytes, including plasma blaster and plasma cells, which may aggregate or be granulocytes, and cells with very high side scatter.

2. Cells expressing CD19 positive and increasing levels of CD38 (CD38 ^hi ). PBMCs were hatched on the MACS column for CD19 expression, but this would basically be the gate to CD38 as this would reveal any contaminants.

3. CD45 positive cells. All lymphocytes express CD45. However, plasma cells down-regulate their CD45 expression compared to previous lymphocyte differentiation stages. Thus, separate individuals of cells corresponding to plasma cells can be obtained upon gating for CD45.

FACS sorted cells were collected directly as single cells in single wells of 96 well plates containing 5 μl of PBS buffer supplemented with 5 U RNase inhibitors per well (Rnasin, Promega, Madison USA, cat. No N2515). At this point, cells can be frozen for subsequent RT-PCR or immediately applied to RT-PCR.

e. Binding of cognate pairs of immunoglobulin variable region encoding sequences

Multiple overlap-extension RT-PCR techniques were applied to single cells to achieve cognate binding of the anti-tetanus toxoid heavy chain variable region and light chain variable region encoding sequences.

e-1. Single-Stage Complex Redundant-Elongated RT-PCR

In essence the Qiagen One-Step RT-PCR kit (Qiagen cat. No 210212, Hilden, Germany) was used for the complex overlap-extension RT-PCR as recommended by the manufacturer. Frozen 96-well plates containing single cells per well were removed from the freezer and 15 μl of RT-PCR reaction mixture was added to each sample (single cell) immediately after the ice crystals disappeared from the wells.

The RT-PCR reaction mixture contains a total of 20 μl volume of the following reagents:

1 x one-step RT-PCR buffer,

DNTP's with a final concentration of 400 μM,

Complex overlap-extension primer mixes of the concentrations described in Table 8,

0.8 μl one-step RT-PCR enzyme mix, and

20 U RNase inhibitors (RNasin, Promega, Madison USA, cat. No N2515).

The composition of the composite overlap-extension primer mix is shown in Table 8.

Table 8

The cycle conditions are as follows:

Reverse transcription: 30 minutes 55 ℃

Polymerase activation: inactivate 95 ° C reverse transcriptase for 15 minutes

Activate Taq polymerase.

PCR reactions:

Denaturation 30 seconds 94 ℃: 35 cycles

Annealing 30 sec 52 ° C .: 35 cycles

Extension 5 min 72 ° C: 35 cycles

Last extension 10 minutes 72 ° C .: 35 cycles

e-2. Additional amplification

1 μl of complex overlap-extension RT-PCR reaction product from each sample was treated with semi-nested PCR (Biotaq kit, Bioline, UK cat.No BIO-21040) as essentially suggested by the manufacturer using 96 well plates It was. The total volume of each reaction is 50 μl, which contains the final concentration of 1 × Biotaq buffer, 200 μM dNTP's (each), 2 mM MgCl ₂ , 1.25 U Bio Taq polymerase and the primers described in Table 9.

The cycle conditions are as follows:

Denaturation 30 seconds 95 ℃: 30 cycles

Annealing 30 sec 55 ° C .: 30 cycles

Extension 1.5 min 72 ° C .: 30 cycles

Last extension 5 minutes 72 ° C .: 30 cycles

Table 9

10 μl of a limited set of samples were analyzed by 1.5% agarose gel electrophoresis with ethidium bromide for visualization, demonstrating the success of the complex overlap-extension RT-PCR.

Expected fragment size (exact size depends on the length of the variable region):

V _H : ~ 410 bp

LC: ~ 680 bp

Redundant-height fragment: ~ 1070 bp

2 μl of all samples derived from the same donor and run in 96 well plates were combined in a single tube. The pooled sample was digested with XhoI and NotI. Digested overlap-extension fragments were purified by preparative 1% agarose gel electrophoresis; Double-extension fragments were excised from agarose gels and purified using the Qiaex II kit (Qiagen cat. No 20051, Hilden, Germany). If not desired, there is no need to pool cognate pairs at this stage. In that case each individual reactant will be restriction cleaved and the product is individually cloned into the vector described below.

f. Cognate Fab expression library

A library of Fab vectors is generated by inserting pools of XhoI / NotI digested overlap-extension fragments into the E. coli vector JSK301 (FIG. 11) by ligation. E. coli (TOP10) was transformed by electrophoresis using a library of Fab vectors and transformants were selected on 2 × YT agar containing 100 μg / ml carbenicillin. Plasmid DNA was prepared from colonies obtained directly on agar plates. Plasmid preparations were derived from the minimum number of colonies per donor to maintain diversity, which corresponds to three times the total number of initially sorted single plasma cells. Fab expression libraries were generated by inserting a leader cassette and a prokaryotic promoter derived from phh3 (Den, W. et al. 1999. J. Immunol. Methods 222, 45-57) into the resulting plasmid preparation, which incorporated the plasmid preparation into AscI and This is accomplished by digestion with NheI and subsequent ligation. The library generation method is outlined in FIG. E. coli (TG1) was transformed by electrophoresis using the resulting Fab expression library and transformants were selected on 2 × YT agar containing 100 μg / ml carbenicillin. Den et al. J Immunol Methods. Individual donors were kept separate during the process disclosed in 1999 Jan 1; 222 (1-2): 45-57. However, they can be combined at any stage desired.

g. Screening of Clones

Fab express clones were screened for TT antigen-binding by antigen-specific ELISA assay.

g-1. Master Plate Generation and Fab Expression

Individual selected colonies of TG1 cells, each containing a cognate Fab expression vector from the library generated in step (f), were placed in a single well of a 96 well plate containing 2 × YT / 100 μg / mL Amp / 1% glucose. Picked. Colonies were grown overnight at 37 ° C. with gentle shaking. The plate is referred to as the master plate, to which glycerol was added to a final concentration of 15% and then stored at -80 ° C.

Prior to storing the master plates, one or more 384 well plates containing 2 × YT / 100 μg / mL Amp / 1% glucose were used to inoculate using a 96-pin replicator. The plate was sealed and shaken at 37 ° C. for 2-3 hours.

Fab expression was induced by adding an appropriate volume of 2 × YT / 100 μg / mL Amp / 0.2 mM IPTC to achieve a final IPTG concentration of 0.1 mM. The plate was sealed and shaken at 30 ° C. overnight. The following day, Fab containing the supernatants were analyzed by ELISA for TT antigen-binding specificity.

g-2. ELISA analysis

384 well ELISA plates (Nunc, Roskilde, Denmark, cat. No 265196) were coated overnight at 4 ° C. with tetanus toxoid (TT) antigen diluted to a final concentration of 1 μg / ml in 25 μl volume PBS per well. Excess binding sites in the wells were blocked for 1 hour at room temperature by adding 2% M-PBS-T (2% Scheme Milk Powder in PBS, 0.05% Tween 20). Wells were washed twice with PBS-T (PBS, 0.05% Tween 20).

Fab containing bacterial supernatant from g-1 was diluted 1: 2 in 2% M-PBS-T and transferred to duplicate ELISA wells. Incubation was performed for 1 hour at room temperature. Wells were washed four times with PBS-T. A 1: 10.000 dilution of goat-anti-human Fab / HRP (Sigma, St. Louis, MO, USA, cat. No A0293) in 2% M-PBS-T was added to the wells. Incubation was performed for 1 hour at room temperature. Wells were washed four times with PBS-T. TMB plus (KemEnTec, Copenhagen, Denmark, cat. No 4390L) substrate was added and incubation was performed for 5-15 minutes. The reaction was stopped by adding an equal volume of 1 MH ₂ SO ₄ . The extent of the reaction was read at 450 nm in an ELISA reader (Multiscan Ascent, Labsystems, Franklin, USA).

Subsequently, the original bacterial clones corresponding to the TT antigen-binding clones were recovered from the original master plate. Plasmid DNA was prepared from isolated antigen positive Fab clones to generate cognate Fab expression sub-library of TT antigen-binding clones.

Clones were also analyzed by anti-light chain ELISA assay to obtain a correlation between the number of antigen-binding clones and the number of Fab expressing clones. In addition, the assay provides information on kappa and lambda light chain representation in clones.

h. Cognate antibody expression library

To facilitate expression of full-length human antibodies, cognate variable regions from bacterial Fab expression vectors must be transferred to mammalian expression vectors containing constant domains from one of the human immunoglobulin isotypes. This movement can be done per clone or as a whole. How to perform a full move is described below.

The entire move is performed in two stages. First, a plasmid preparation of individually isolated antigen-binding Fab clones is pooled. By digesting the pooled Fab expression sub-library with AscI and NheI and inserting the mammalian promoter and leader cassette by subsequent ligation, the leader containing the prokaryotic promoter and cassette was replaced with a human alkaline phosphatase leader sequence (AP Leader), human extension factor 1-alpha promoter (EFP), adenovirus major late promoter (AdMLP), mammalian promoter and leader cassette consisting of IgK leader sequences. E. coli (TOP10) was transformed by electrophoresis using the resulting promoter exchanged Fab expression library and transformants were selected on 2 × YT agar containing 100 μg / ml carbenicillin. Plasmid DNA was prepared from colonies obtained directly on agar plates. To maintain diversity, plasmid preparations were derived from the minimum number of colonies per donor, which is approximately three times the total number of clones of the original library.

Second, cognate variable regions from promoter exchanged Fab expression vectors were isolated by digesting the plasmid preparation with XhoI and NotI. Isolated fragments were inserted into mammalian expression vectors by ligation resulting in cognate antibody expression libraries. The vector used was essentially identical to FIG. 9A, except that the IgL in this case corresponds to the kappa light chain encoding sequence. The mammalian expression vector used is based on a site-specific Flp-In system (Invitrogen Corporation, Carlsbad, CA, USA, Cat No K6010-01). E. coli (TOP10) was transformed by electrophoresis using the resulting cognate antibody expression library and transformants were selected on 2 × YT agar containing 100 μg / ml carbenicillin. Plasmid DNA was prepared from colonies obtained directly on agar plates. To maintain diversity, plasmid preparations were re-derived from the minimum number of colonies per donor, which is approximately three times the total number of clones in the original library. The resulting plasmid preparation of the cognate antibody expression library was used to transfect mammalian host cells, such as CHO cells from Invitrogen Flip-In System (Invitrogen Corporation, Carlsbad, CA, USA, Cat No K6010-01). Recombinase-mediated integration can produce stable mammalian expression cells. The cell line can be used to generate recombinant polyclonal antibodies.

Example 7: Screening by High Density Membrane Assay

Fab expression clones from Example 6f were screened in a high density membrane assay.

a. Master plate formation

The master plate was formed as described in step (g-1) of Example 6.

b. PVDF membrane manufacturing

PVDF membranes (Amersham, Uppsala, Sweden, cat. NoRPN202OF) were coated overnight at 4 ° C. according to product instructions using TT antigen diluted to a final concentration of 1 μg / ml in PBS. Excess binding sites on the membranes were blocked for 1 hour in 2% M-PBS (2% skim milk powder in PBS). The membrane was rinsed three times with PBS. The membrane was immersed in 2 x YT for several minutes.

c. Clone consolidation

Clone immobilization to 384 well plates was performed by transferring clones from the master plate to one or more 384 well plates containing 20 μl of 2 × YT per well using a 96-pin replicator.

Using a 384 pin replicator, the bacteria were transferred to one or more double nylon membranes (Amersham, Uppsala, Sweden, cat.No RPN2020B) placed on a 2 × YT / Carb / 1% glucose agar plate. Plates were incubated at 37 ° C. for 4-6 hours.

d. Fab induction

The PVDF membrane prepared in step b was placed on a 2 x YT / Carb / 0.1 mM IPTG agar plate. Nylon membranes with growing bacterial colonies were placed on top of PVDF membranes (one nylon membrane per PVDF membrane) on agar plates containing IPTG. IPTG containing agar plates were incubated overnight at 30 ° C. to promote IPTG inducible Fab expression from colonies. Fab molecules diffuse through the nylon membrane into the PVDF membrane, where TT-antigen binding Fabs will be retained on the membrane.

e. detection

The nylon membrane was removed and the PVDF membrane was washed twice with PBS-T (PBS, 0.05% Tween 20) for 5 minutes. The membrane was incubated with 2% M-PBS for 30 minutes. The PVDF membrane was washed twice with PBS-T for 5 minutes, followed by goat-anti-human IgG / HRP diluted 1: 10.000 in 2% M-PBS (Sigma, St. Louis, MO, USA, cat.No A0293 ) Was incubated for 1 hour. The PVDF membrane was washed three times with PBS-T for 5 minutes. Finally, the PVDF membrane was incubated for 5 minutes using a SuperSignal West Femto chemiluminiscent substrate (Pierce, Rockford, II, USA, cat. No 34095) according to the product instructions. The excess substrate was removed and the PVDF membrane was placed under a CCD camera for the detection of chemiluminescent signals generated at the position of the PVDF membrane where the Fab fragment bound to the TT-antigen.

Positive TT-antigen binding clones will appear as dots on the image. The original bacterial clones were then recovered in turn from the original master plate. Plasmid DNA was prepared from isolated antigen positive Fab clones to produce cognate Fab expression sub-library of TT-antigen binding clones.

f. Correlation Between Antigen Binding Clones and Fab Expression Clones

The nylon membrane removed in step e was placed in a second series of PVDF membranes coated with an anti-light chain antibody or anti-Fab antibody according to the process of step b. Thereafter, steps d and e were repeated.

Fab positive clones will then appear as dots in the image and there will be a correlation between the number of TT-antigen binding clones and the number of Fab expressing clones.

Example 8 Exemplary Examples Describes Separation of Sequences Encoding Heteromeric Proteins

This example illustrates that the trimer guanine nucleotide binding protein (G-protein) can be separated in a single step using the complex overlap-extension RT-PCR technique of the present invention. The G-protein subunit family is large and alone in humans, with about 16 different alpha subunits, 5 beta subunits and 12 gamma subunits. For this example, subunit GαS (gene bank accession number X04408), subunit Gβ1 (gene bank accession number AF501822) and subunit Gγ1 (gene bank accession number BC029367) were selected. The binding process is illustrated in FIG. 13, where step 1 is a complex overlap-extension RT-PCR step and step 2 is an additional amplification step of the bound product.

a. cell

Individuals of human liver cells were obtained from surgical secretion samples. Liver cells were digested and lysed to separate total RNA from lysate using the RNA L kit (Macherey-Nagel, Duren, Germany, cat. No 740 962.20) according to the product instructions.

b. Complex Redundant-Elongated RT-PCR

The Qiagen One Step RT-PCR kit (Qiagen cat. No 210212, Hilden, Germany) was used for essentially complex overlap-extension RT-PCR according to product recommendations.

Each PCR tube contains the following reagents in a total volume of 50 μl:

1 x One Step RT-PCR Buffer,

DNTP at a final concentration of 400 μM each,

Complex overlap-extension primer mix at concentrations as shown in Table 10,

2 μl one step RT-PCR enzyme mix,

1 U / μl RNase inhibitor (RNasin, Promega, Madison USA, cat.No N2515), and

50 ng total RNA.

The complex overlap-extension primer mix used included the primers shown in Table 10 below.

Table 10

Uppercase sequences correspond to gene specific regions.

Cycling conditions are as follows:

Reverse transcription: 30 minutes 42 ℃

Polymerase Activation: Inactivate 95 min reverse transcriptase and activate Taq polymerase

PCR reactions:

c. Amplification PCR

1 μl of complex overlap-extension RT-PCR reaction product was subjected to additional PCR amplification steps (Biotaq kit, Bioline, UK cat. No BIO-21040) essentially as suggested by the manufacturer. Primers used are α1 and γ2 shown in Table 10 corresponding to SEQ ID NOs 80 and 85. The total volume of reaction is 50 μl.

Cycling conditions are as follows:

5 μl of reaction product was analyzed by 1% agarose gel electrophoresis using ethidium bromide for detection. The size of the product is expected to be 2215 bp. However, there may be additional unbound fragments of the subunits. The expected size of these fragments is 1228 bp for Gα, 1064 bp for Gβ, and 262 bp for Gγ.

d. Cloning

The remaining reaction product was subjected to 1% agarose gel electrophoresis with ethidium bromide for detection, 2215 bp duplicate-extension fragments were cut out of the agarose gel and the QiaexII kit (Qiagen) , Hilden, Germany, cat.No 20051), and inserted into pCR2.1-TOPO using the Invitrogen TOPO TA cloning kit (Invitrogen cat.No 45-0641, Carlsbad, Calif., USA).

In addition, promoter sequences or ribosomal binding sites can be inserted upstream of each subunit encoding sequence to facilitate their expression from the vector. Restriction sites present in the overlap-extension sequence can be applied for such insertion.

e. General consideration

As described above, the binding of the sequences encoding the subunits constituting the heteromeric protein can be performed for most heteromeric proteins in which the sequences of the individual subunits are known. In addition, when using multiple overlap-extension primer mixtures capable of amplifying several members of a particular family, for example all G-proteins α-, β- and γ-subunits, which cell types the family members are expressed by Without first analyzing, it is possible to identify and bind any combination of subunits from cell types. For example, the above example amplifies and links all 16 Gα subunits, 5 Gβ subunits, and 12 Gγ subunits, thereby identifying which combination of subunits are expressed in each cell and simultaneously sub This can be done using a template derived from one isolated liver cell, using a complex overlap-extension primer mixture capable of isolating the encoding sequence responsible for the combination of units. In addition, the use of denatured primers can help to identify new members of a particular family.

When binding subunits of heteromeric proteins using the complex overlap-extension RT-PCR technique of the present invention, the total length of the bound product must be taken into account, which limits the number of base pairs that the DNA polymerase can amplify. Because of this. Deep Vent polymerase from New England Biolabs, Mass., USA, is one of the DNA polymerases capable of producing very long primer stretches up to 14,000 bp in length. Theoretically, 14,000 bp can encode a protein with an average weight of 510 kDa. Thus, it is possible to separate the encoding sequences for very large heteromeric proteins using the methods of the present invention. Elongation capacity can be further increased, possibly by using a mixture of DNA polymerases.

Example 9 Design of Leading Primer

In this example, a complex set of primers was designed in which the location of priming is maintained within sequences encoding the leading regions of the human antibody heavy and kappa light chain families.

When using primers that anneal to the N-terminal encoding sequence of the variable region family, some cross hybridization can be observed. Primers of one particular gene family sequence can be hybridized to cDNA of another gene family, which in many cases generates new sequences. Proteins generated from such sequences are potentially immunogenic when used in therapy. In general, the new sequence can be corrected by PCR or removed from the library.

An alternative solution is to position the priming in the sequence encoding the leader peptide region of the antibody variable region. Hybrid sequences generated as a result of cross-priming can be removed during intracellular processing of the antibody, where the peptide lines containing the potential immunogenic sequence will be cleaved and therefore will not be present in the released antibody product. The previous set of leading primers for antibody cloning is located at the 5 'end of the leading encoding sequence allowing direct eukaryotic expression (Campbell M.J. et al. 1992 Mol lmmunol. 29, 193-203). Unfortunately, eukaryotic leading peptides are not suitable for prokaryotic processing.

The ease of manipulation of nucleic acid sequences in bacteria makes it attractive for developing cloning systems that enable sequence shuttle between vectors to bacterial vectors and other host oils. Thus, a complex primer set was designed in the present invention in which the location of priming is maintained at a location within the leader region encoding sequence, which allows for functional expression of the antibody or antibody fragment in both eukaryotic and prokaryotic expression systems.

The sequences required for signal peptidase cleavage in the C-terminal region of any leading peptide appear to be very similar for prokaryotic and eukaryotic systems (Nielsen H. et al. 1997 Protein Eng. 10, 1-6. ). Moreover, mutations in the C-terminal region of the lead peptide have little effect on the conformation of the N-terminal region, and vice versa. Thus, it is expected that the C-terminal sequence can be shifted between species without losing function.

The basic concept of primer design in this example is to place the site of priming in the last six codons of the C-terminal region of the lead peptide. The remaining portion of the leading encoding sequence, which is upstream of the priming (about 50 nucleotides), must be supplied by an appropriate expression vector that matches the host species. For example, a vector suitable for expression in gram negative bacteria can be designed with partial or full length PelB leader sequences. For expression of antibodies in eukaryotic cells, vectors with partially native antibody heavy chain leader encoding sequences and partially variable kappa chain leader encoding sequences will be suitable. Regardless of the expression system, the last six amino acids in the leader will be derived from the endogenous antibody leader encoding sequence contained in the nucleic acid fragment inserted into the vector.

The following primer sequences were designed for use in complex overlap-extension RT-PCR (Table 11). However, the overlapping tails (lower case) can be easily redesigned to act on binding by ligation or recombination methods.

Table 11

M = A / T, R = A / G, Y = C / T, S = G / C. Uppercase sequences correspond to gene specific regions.

It should be noted that the restriction sites in the primer design are slightly modified compared to the primers described in the examples above. Thus, the overlapping sequence between the heavy and light chain variable region encoding sequences comprises NotI and NheI restriction sites and the constant kappa primer (C _Lκ ), The existing NotI site was replaced with AscI site. Of course the latter modification should be observed when designing primers for further PCR amplification. Moreover, the NotI site in the cloning vector shown in FIG. 11 should be changed to the AscI site, and the AscI site of the promoter unit should be changed to the NotI site.

Functionality for freshness split was tested in silico using SignalP provided by CBS of Danish Technical University.

Chimeric leader peptides tested for variable heavy chains include a nucleic acid sequence consisting of a truncated PelB sequence, a C-terminal NotI site (SEQ ID NO 100: ATG AAA TAT CTT CTA CCA ACA GCG GCA GCT GGA TTA TTG GCG GCC GCC), And natural V in connection with the NotI site _HL  It is encoded by the gene specific portion of one of SEQ ID NOs 86 to 92 which encodes six C-terminal amino acids from a family member. All seven sequences showed signal peptide cleavage in Gram negative bacteria using the SignalP program.

Chimeric leader peptides tested for the variable light chains include a nucleic acid sequence consisting of a truncated PelB sequence, a C-terminal NheI site (SEQ ID NO 101: ATG AAA TAT TTG CTA CCA ACA GCG GCA GCT GGA TTA TTG TTA CTA GCG) And the gene specific portion of one of SEQ ID NOs 93 to 98, which encodes six C-terminal amino acids from the native V _Lκ family member with respect to the NheI site. All six sequences showed signal peptide cleavage in Gram negative bacteria using the SinalP program.

The sequences of SEQ ID NOs 100 and 101 are suitable for constructs of bacterial expression vectors similar to those shown in FIG. 11, wherein Asc I shown in FIG. 11 is substituted with SEQ ID NO 100 and the NheI site is SEQ ID NO 101 Is substituted. In addition, the NotI site shown in FIG. 11 needs to be substituted for AscI site.

Mammalian expression vectors (FIG. 9) can likewise be redesigned to allow expression in mammalian cells after moving the variable region encoding fragments from the just described bacterial vector.

Example 10: Binding by Single Tube Complex RT-PCR and Ligation

This example illustrates that cognate pairs comprising bound heavy chain variable region and light chain variable region encoding sequences can be generated in a single tube reaction using ligation instead of overlap-extension PCR to effect binding.

a. Composite RT-PCR

Cell lines generated in-house expressing Fab molecules with specificity for tetanus toxoid were dispensed at limit dilution to obtain single cells in a single PCR tube.

In addition to single cells, each PCR tube contains the following reagents in a total volume of 20 μl:

1 x Fusion HF Buffer

DNTP at a final concentration of 200 μM each

Complex primer mixes of concentrations as described in Table 12

0.8 U Fusion Polymerase (FinnZymes Cat.No F-530-L)

1 μl Sensiscript reverse transcriptase (Qiagen Cat. No 205213)

20 U RNase Inhibitor

Table 12

W = A / T, R = A / G, S = G / C. Uppercase sequences correspond to gene specific regions.

Cycling conditions are as follows:

b. Binding by ligation

In order to effect binding by ligation, restriction enzymes and ligase were added directly to the complex RT-PCR reaction product. Alternatively, the complex RT-PCR can be purified prior to the addition to remove the mixture from the polymerase.

The following reagents were added to each tube in a total volume of 40 μl:

1 x NEBⅡ buffer

1 mM ATP

50 U Xba I (New England Biolabs Cat.No R0145L)

25 U Spe I (New England Biolabs Cat.No R0133S)

100 μg / ml BSA

400 U T4 DNA ligase (New England Biolabs Cat.No M0202S)

The reaction was incubated at 16 ° C. overnight.

Bound heavy and light chain variable region encoding sequences were purified from the reaction by gel electrophoresis and ablation of a band of about 1000 bp.

In an alternative version of the method, the complex RT-PCR reaction is performed using C _H primers instead of J _H primers in the complex primer set. The primer may be equipped with a cloning tail to insert the heavy chain variable region in-frame into the expression vector, or may be subjected to semi-nested PCR using the primers of Table 5.

Example 11 Generation of Recombinant Polyclonal Immunoglobulins Having Specificity for Tetanus Toxoid

In this example, results from a single donor (TT03) immunized with tetanus toxoid (TT) were used to illustrate the steps shown in the flowchart of FIG. 10.

a. Donor

Eight donors previously immunized with tetanus vaccine were boosted with tetanus vaccine (Statens Serum Institut, Denmark). Donors were designated by the numbers TT01 to TT08. Six days after boosting with tetanus vaccine, about 200 ml of blood sample was taken from the donor and injected into a tube containing anticoagulant.

The donor is healthy without chronic infection, autoimmune disease, or immunosuppressive agents and has not received any vaccination within the last three months.

a-1. Donor Characteristics Monitoring

Preliminary blood collection of the donor was performed at the time of immunization. After 14 days, additional blood collection was performed to determine serum titers. All donors responded with increased TT-titer. In addition, ELISPOT analysis was performed to determine the frequency of TT-specific plasma cells. Various cell fragments can be used for ELISPOT, such as most blood samples from day 6, PBMC fragments, self sorted fragments, or FACS sorted fragments. ELISPOT can be used to assess donor material or to identify optimal responders, which can be run through a classification step and multiple overlap-extension RT-PCR. For donor TT03, ELISPOT was performed using CD19 + cell fragments (see section c). The ELISPOT assay was performed with minor modifications as described in Lakew, M. et al. 1997. J. Immunol.Methods 203, 193-198. The frequency of TT specific plasma cells in PBMC fragments was calculated to 0.021%.

b. Preparation of Peripheral Blood Mononuclear Cells (PBMC)

PBMCs were isolated from blood samples using Lymphoprep (Axis-Shield PoC AS, Norway, prod. No 1001967) according to product recommendations. In short, the blood was diluted 1: 1 in PBS and the suspension was stratified on lymphoprep in a 2: 1 ratio. The vial was centrifuged at 800 g at 25 ° C. for 20 minutes and a white mid layer band was collected. Cells were washed with PBS containing 2 mM EDTA.

From 200 ml of whole blood drawn from donor TT03, about 2 × 10 ⁸ PBMCs were obtained.

c. Enrichment of B Cells

The B-cell lineage (CD19 + cells) of PBMCs was concentrated by magnetic bead cell sorting using the following method.

Isolated PBMCs were stained using anti-CD19-FITC (Becton Dickenson, NJ, USA, cat. No 345776). All steps were performed at 4 ° C. in the dark. Staining with 10 μl of anti-CD19-FITC per 1 × 10 ⁶ cells at a volume of 100 μl per 1 × 10 ⁶ cells using M-buffer (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA) Was performed. This will stain the B-cell lineage of PBMCs. Cells were incubated for 20 minutes and then washed twice with M-buffer. Wherein -CD19-FITC 1 x 10 ⁶ cells per section of 10 ㎕ the stained cells in the M- buffer solution of 1 x 10 ⁶ 100 ㎕ volume per cell -FITC magnetic beads (Miltenyi Biotec, Gladbach, Germany, cat. No 130 -042-401) magnetically labeled with anti-FITC-conjugated microbeads. After 15 minutes of incubation, a single wash step with M-buffer was performed. The cells were resuspended in degassed M-buffer solution.

The MACS LS column (Miltenyi Biotec, Gladbach, Germany, cat. No 130-042-401) was pretreated with degassed M-buffer in accordance with product instructions. Cell suspensions stained with anti-CD19-FITC and labeled with anti-FITC-magnetic beads were applied to the column and allowed to pass. Unstained cells (CD19 ÷) pass through the column while stained and labeled cells (CD19 +) remain in the magnetic field around the column. The column was washed with degassed M-buffer. The magnetic field was removed and CD19 + cells were harvested.

Analytical staining of starting material (PBMC), unlabeled fragments from TT03 and CD19 labeled fragments was performed using anti-CD19-FITC, anti-CD38-PE, and anti-CD45-PerCP (FIG. 14). This indicates that magnetic cell sorting produces two separate fragments compared to PBMC fragments. CD19 negative cells are shown in panel B and CD19 positive cells are shown in panel C. 11% of the cells in the PBMC fragment were CD19 + and the range of CD19 positive cell purification was 99.5% of R1 (scatter gare in FIG. 14C) for TT03.

As shown in FIG. 14C, anti-CD38 / anti-CD45 plots showed distinct individuals of CD38hi, CD45in cells (R2) corresponding to 1.1% of R1. The subject contained plasma cells and was harvested during later sorting steps. This indicates that 0.12% of cells in the PBMC fragment correspond to plasma cells.

CD19 positive cell fragments were frozen in FCS (Invitrogen, Cat. No. 16000-044) + 10% DMSO (Sigma, Cat. No. D2650) for subsequent sorting. The assay as shown in FIG. 14 was performed on MACS purified cells that were frozen to ensure that the cells were intact (FIG. 15). The staining pattern shown in FIG. 15 was slightly the same as that shown in FIG. Cells harvested by sorting are a subset of R1 and R2. This corresponds to about 1.1% of MACS purified cells.

d. Classification of Plasma Cells

Frozen eluate from the MACS column was thawed, centrifuged and resuspended in FACS buffer (PBS, pH 7.2, 2% BSA) at 1 × 10 ⁶ cells / 60 μl FACS buffer concentration. Anti-CD19-FITC (Becton Dickenson, NJ, USA, cat.No 345776) (10 μl / 10 ⁶ cells), anti-CD38-PE (Becton Dickenson, NJ, USA, cat.No 555460) (10 μl / 10 ⁶ cells) and anti-CD45-PerCP (Becton Dickenson, NJ, USA, cat.No 345809) (20 μl / 10 ⁶ cells) were added and the mixture was incubated for 20 min in 4 ° C. Washes and resuspension in FACS buffer were performed.

Cells were sorted by fluorescence activated cell sorting (FACS) using the following gating parameters:

1. Forward scattering and lateral scattering to retain lymphocytes and monocytes, including plasma blaster and plasma cells, and to avoid lethal cells and cells with very high side scatter, which may be coagulum or granulocytes.

2. Cells expressing CD19 positive and increased levels of CD38 (CD38hi). This is basically the only gate for CD38 because PBMC is abundant on the MACS column for CD19 expression, but it exposes any contaminants.

3. CD45 intermediate positive cells. All lymphocytes express CD45. However, plasma cells subregulate their CD45 expression compared to previous lymphocyte differentiation stages. Thus, individual individuals of cells corresponding to plasma cells can be obtained when gated against CD45.

FACS sorted cells from donor TT03 were collected as a subset of two gates P2 and P1 (FIGS. 16A and 16B, respectively). These cells were CD38hi (FL2-A), CD45in (FL3-A) and CD19 +, and CD19 + was due to MACS purification. Conveniently, cells are collected in bulk, counted, and then 10% FCS (Invitrogen Cat. No. 16000-044), 100 units / ml penicillin-streptomycin (Invitrogen, cat. No. 15410-122), 2 mM L Diluted with RPMI (Invitrogen, Cat No. 21875-034) containing glutamine (cat No. 25030-024). The cells were then distributed into 50 96 well PCR plates with one cell per well in 5 μl medium. The plate was frozen immediately after sealing and then stored for subsequent RT-PCR analysis.

e. Binding of cognate pairs of immunoglobulin variable region encoding sequences

Multiple overlap-extension RT-PCR techniques were applied to single cells obtained from donor TT03 to achieve homologous binding of the transcribed anti-Tetanus Toxoid heavy chain variable region and light chain variable region encoding sequences.

e-1. Single Phase Complex Redundant-Elongated RT-PCR

The Qiagen One-Step RT-PCT Kit (Qiagen cat. No. 210212, Hilden, Germany) was used for complex overlap-extension RT-PCR essentially according to the manufacturer's recommendations. Fifty frozen 96 well PCR plates containing approximately one cell per well were removed from the freezer, and when ice crystals were removed from the wells, 15 μl of RT-PCR reaction mixture was added immediately to each well.

The RT-PCR reaction mixture contained the following reagents in a total volume of 20 μl:

1 × One Step RT-PCR Buffer

DNTP at a final concentration of 400 μM each

Complex overlap-extension primer mixes at the concentrations listed in Table 13

0.8 μl one-step RT-PCR enzyme mix

20 U RNase Inhibitor (RNasin, Promega, Madison USA, cat.No N2515)

The composition of the composite overlap-extension primer mix is shown in Table 13 below.

Table 13

Cycling conditions are as follows:

Reverse Transfer: 30 ℃ 55 ℃

Polymerase Activation: Inactivates reverse transcription at 95 ° C. for 15 minutes and activates Taq polymerase.

PCR reactions:

Denaturation 30 seconds 94 degrees Celsius

Annealing 30 sec 52 ℃

Extended 5 minutes 72 ℃

Final extension 10 min 72 ° C

The denaturation, annealing and expansion are carried out in 35 cycles.

e-2. Additional amplification

In 1 μl of the complex overlap-extension RT-PCR reaction product from each sample, semi-nested PCR as essentially suggested by the manufacturer using a 96 well PCR plate (ABgene, cat. No. AB-0800). (Biotaq kit, Bioline, UK cat.No BIO-21040) was performed. The total volume of each reaction was 50 μl, which contained a final concentration of 1 × Biotaq buffer, 200 μM dNTP (each), 2 mM MgCl ₂ , 1.25 U Bio Taq polymerase and the primers shown in Table 14 below.

Table 14

Cycling conditions are as follows:

Denaturation 30 seconds 95 degrees Celsius

Annealing 30 sec 55 ℃

Extended 1.5 minutes 72 ℃

Final extension 5 minutes 72 ℃

The denaturation, annealing and expansion are carried out in 30 cycles.

Column A from each plate, 10 μl of samples from wells 1 to 12 were analyzed by 1.5% agar gel electrophoresis using ethidium bromide to verify that the complex overlap-extension RT-PCR was successful. . The expected size of the overlap-expansion fragment was approximately 1070 bp (the exact size depends on the length of the variable region). 17 shows samples from eight 96 well plates. The average number of successful overlap-expansion RT-PCR fragments from 50 96 well plates was estimated at 8 per plate. Thus, the total number of successful overlap-extension RT-PCR fragments was estimated to be approximately 400.

10 [mu] l from all reactions carried out running in 96 well plates and originating from the same donor were solidified in a single tube. An aliquot consisting of 200 μl of pooled PCR product was subsequently subjected to the preparation procedure using QIAquick PCR purification kit, using 60 μl of buffer EB for elution (Qiagen cat. No. 28106, Hilden, Germany). Purified. Purified pools of overlapping PCR products were digested with XhoI and NotI and subsequently purified by preparative 1% agar gel electrophoresis; The overlap-extension fragments were cut from the agar gel and purified using the Qiaex II kit (Qiagen cat. No 20051, Hilden, Germany).

f. Homogenous Fab Expression Library

Fab expression libraries were generated by a two step ligation process. First, the pool of digested overlap-expansion fragments described above was ligated with XhoI / NotI digested E. coli vector JSK301 (FIG. 11). The ligation reaction was subsequently transformed with electrostatic E. coli cells (XL-1-blue electroporation qualified, Stratagen, cat. No. 200228, La Jolla, USA) according to the manufacturer's instructions. Transformed E. coli cells were plated onto 2 × YT agar containing 100 μg / ml carbenicillin. Biomass corresponding to approximately 10 ¹⁰ to 10 ¹¹ cells originating from more than five independent colony numbers based on the total number of overlapping PCR products was obtained from the Qiagen plasmid preparation maxi kit (Qiagen cat. No. 12163, Hilden, Germany). Used as starting material for plasmid preparation. To allow Fab expression of the cloned homologous linked VH and VL encoding sequences, the prokaryotic promoter and leader cassette were inserted in the second ligation step. The bidirectional promoter fragment used (SEQ ID NO 321) was extracted from the parent JSK301 by AscI / NheI digestion. Purified pools of plasmids were digested in the same manner with AscI / NheI restriction endonuclease and gel purified as previously described. Purified fragments are subsequently ligated and transformed into electrical E. coli cells (TGI, Stratagene, cat. No. 200123, La Jolla, USA) and contain 100 μg / ml carbenicillin and 1% glycos Were plated on 2 × YT agar. The Fab expression library generation process is outlined in FIG. 12.

g. Screening of Clones

Fab expressing clones were screened for TT antigen-binding by antigen-specific ELISA assay.

g-1. Master Plate Generation and Fab Expression

A single well of a 96 well plate containing 2 × YT / 100 μg / ml Carb / 1% glucose containing individual selected colonies of TG1 cells, each providing homologous Fab expression vectors from the library produced as described in section (f). Picked into wells. The plate was incubated overnight at 37 ° C. with gentle shaking. Four 96 well plates were solidified into either 384 well plates containing 2 × YT / 100 μg / ml Carb / 1% glucose using a 96-pin replicator. These 384 well plates were incubated overnight at 37 ° C. These plates were called master plates and stored at −80 ° C. after adding glycol to them at a final concentration of 15%.

Masterplates were used for inoculation of 384 deepwell plates containing 2 × YT / 100 μg / ml Carb / 0.1% glucose using a 96-pin replicator. Plates were sealed and incubated at 37 ° C. for 2-3 hours with shaking.

Fab expression was induced by adding an equal volume of 2 × YT / 100 μg / ml Carb / 0.2mM IPTG to a final IPTG concentration of 0.1 mM. Plates were sealed and incubated overnight at 30 ° C. with shaking. The following day, Fab containing supernatants were analyzed for TT antigen-binding specificity by ELISA.

g-2. ELISA test

384 well ELISA plates (Corning Inc., Corning, NY, USA, cat.No. 3700) were coated overnight at 4 ° C. using tetanus toxoid (TT) antigen, and in 1 volume of PBS in a volume of 25 μl per well Dilution to a final concentration of μg / ml. Excess binding sites in the wells were blocked for 1 hour at room temperature by adding 2% M-PBS-T (2% skim milk powder in PBS, 0.05% Tween 20). Wells were washed twice with PBS-T (PBS, 0.05% Tween 20).

Fab-containing bacterial supernatants from section (g-1) were diluted 1: 2 in 2% M-PBS-T and transferred to replicated ELISA wells. Incubation was performed at room temperature for 1 hour. These cells were washed four times with PBS-T. Chlorine-anti-human Fab / HRP (Sigma, St. Louis, MO, USA, cat. No A0293) diluted 1: 10,000 in 2% M-PBS-T was added to the wells. Incubate at room temperature for 1 hour. Wells were washed four times with PBS-T. TMB plus (KemEnTec, Copenhagen, Denmark, cat. No 4390L) substrate was added and incubated for 5-15 minutes. The reaction was stopped by adding an equal volume of 1M H ₂ SO ₄ and analyzed at 450 nm using a spectrometer (Multiscan Ascent, Labsystems, Franklin, USA).

Origin bacterial clones corresponding to TT antigen-binding clones can subsequently be removed from the origin master plate. Plasmid DNA can be prepared from isolated antigen positive Fab clones, generating a homologous Fab expression sub-library of TT antigen-binding clones.

To obtain an association between the number of antigen binding clones and the number of Fab expressing clones, a subset of clones was further analyzed by anti-kappa ELISA assay. Anti-kappa assays are generally used, except that the wells are coated with a 1: 1000 dilution of goat anti-human kappa antibody (Caltag, Califonia, USA, Cat. No H16000) using carbonate buffer at pH 9.6. Performed with TT-test.

g-3. Screening Results

Clones from four 384 well plates were screened for reactivity with anti-kappa Abs and TT using an ELISA assay following the procedure described in g-2. The results obtained are summarized in Tables 15-19 below. Anti-kappa ELISA results reveal the expression of Fab fragments in a given clone, and TT ELISA results show the function of Fab fragments.

Table 15: ELISA screening, anti-kappa coatings (total 1440 clones)

Of the 1440 single clones analyzed, 482 clones or 34% showed anti-kappa reactivity at levels above 2x background reactivity. 395 clones or 27% of the clones exhibited a reactivity greater than 3x background reactivity, and the like.

The same clones were analyzed for TT-antigen reactivity by ELISA and the results are shown in Table 16 below:

Table 16: ELISA screening, TT coating (total 1440 clones)

From this table, it was confirmed that the 9.0% clone showed reactivity with TT on the 2x background (defined as the signal obtained by the reactivity of the Fab fragment with unrelated specificity). 104 clones reacted on 3x background (7.2%) and the like.

Of the Fab positive clones (48 clones in total), approximately 27% (130/482) of the clones showed reactivity with TT at the 2 × background level. These levels do not change significantly at other background levels.

Six 384 well plates were screened for clones with TT reactivity. The number of clones causing reactivity with the antigen is shown in Table 17 below. No anti-kappa ELISA was performed using these plates.

Table 17: ELISA screening, TT coating (total 2160 clones)

The percentage of TT positive clones in plates G054-G059 was compared to that identified in plates G050-G053 (Table 16).

Results from all clones screened for TT (Tables 16 and 17) are summarized in Table 18 below.

Table 18: All screened clones, TT reactive clones

In sum, a total of 339 clones showed reactivity with TT on at least 2 × background level (Table 18). This corresponds to 9.4% of all clones screened.

All positive clones showing reactivity with TT on a 2x background were inoculated into 96 well plates from the master plate as described previously. The following day, bacteria were collected by centrifugation at 4000 rpm for 15 minutes, and the pellet was resuspended in 0.8 mM EDTA, 0.4 x PBS, 0.8 M NaCl and incubated on ice for 15 minutes. Peripheral cytoplasmic extracts were collected by centrifugation and the reactivity of the clones was further analyzed. Here, from one plate (G060), reactivity with anti-kappa (FIG. 18), egg albumin (unrelated antigen) (FIG. 19), TT (FIG. 20), and 10 ⁻⁷ M concentration in solution The results for the single step competition assay (FIG. 21) using TT-antigen of are confirmed. This ELISA assay was performed on the same periplasmic extract at the same dilution.

Most clones expressed Fab fragment (90/96) (FIG. 13) and none of the clones reacted with egg albumin (FIG. 19). Reactivity with immunized TT was completely inhibited or reduced by TT in solution (FIG. 21), indicating that the clone specifically reacted with TT. Clone reactivity on plate G060 is summarized in Table 19 below.

Table 19: Summary for G060 Plates (96 clones total)

h. Diversity Analysis and Clone Approval

을 서열화하고, 경쇄 엔코딩 서열을 프라이머 LSN-LCP를 이용하여, P tac 프로모터로 어닐링되는

을 서열화하였다. 서열 데이터를 벡터 NTI 소프트웨어(Informax, Frederick, MD, USA)를 이용하여 분석하였다. 중쇄의 생성된 서열 데이터를 위쪽 AscI 제한 부위의 한 염기쌍 5'로 트리밍시킨 직후, 아래쪽 XhoI 제한 부위의 염기쌍 3'로 트리밍하였다. 경쇄 서열 데이터를 상류 NheI 제한 부의의 제 2 5' 염기쌍, 및 가변 쇄 C-말단 라이신을 엔코딩하는 최후 코돈 "AAA"의 염기쌍 3'으로 트리밍하였다. 트리밍 처리는 각 DNA 서열의 번역과 같은 추가 분석을 촉진시키도록 수행되었다.Sequence analysis was performed on plasmids isolated from 47 clones (from plate G060) from the homologous TT antigen-binding Fab expression sub-library. Variable heavy chain encoding sequences are annealed to the P tac promoter using primers LSN-HCP

, And the light chain encoding sequence is annealed to the P tac promoter using primers LSN-LCP.

Was sequenced. Sequence data were analyzed using vector NTI software (Informax, Frederick, MD, USA). The resulting sequence data of the heavy chain was trimmed to base pair 3 ′ of the bottom XhoI restriction site immediately after trimming to one base pair 5 ′ of the upper AscI restriction site. The light chain sequence data was trimmed with a second 5 'base pair of upstream NheI restriction and a base pair 3' of the last codon "AAA" encoding the variable chain C-terminal lysine. Trimming treatments were performed to facilitate further analysis, such as translation of each DNA sequence.

Variable heavy and light chain encoding sequences were analyzed for germline gene utilization by comparing these sequences with the V-base germline variable region sequence database (MRC Center for Protein Engineering, Cambridge, UK). Accordingly, closely related wiring alleles are derived from 12 different variable heavy chain wiring alleles belonging to the VH-family of VH1 to VH5, and V originating from eight different variable kappa light chain wiring alleles belonging to VKI, VKIII and VKIV. -Was determined for each sequence representing the gene repertoire (Figure 20).

Variable gene sequences were also translated into aligned protein sequences using AlignX software (Informax, Frederick, MD, USA) as shown in FIGS. 22 and 23. Based on protein sequence alignment, the variable chain sequences could be divided into groups named V (D) J rearrangement, as appropriate, H for variable heavy chain sequences and L and other unique numbers for variable kappa chain sequences. . In each rearrangement group, sequences were sorted according to mature genotype (mature type in Table 20 below) within CDR 1, 2 and 3 regions indicated in low alphabetical order. Seven of the clones had premature stop codons, six of which were tan mutations (TGA) (clone ID g060: b12, d08, f06, c12, f03, c04) and one clone was an opal mutation (TGA) (Clone ID g060: h12). These codons were most easily inhibited by E. coli resulting in functional Fab fragments. Four of these clones were components of the group containing overlapping pseudo clones. Since additional codons were a single component of these groups, they would have to be analyzed to replace the remaining three clones with premature stop codons. Alternatively, the sequence could be modified by standard molecular biology techniques such as PCR, replacing the stop codons with appropriate codons.

47 analyzed V-region sequences could be divided into 20 unique V (D) J rearrangement groups (“groups” named in Table 20 below) for both variable heavy and variable kappa genes. Four of the rearrangement groups could be further divided into 1 to 4 mature forms (a to d), resulting in 27 unique antibody encoding sequences (Table 12). In general, specific heavy chain rearrangement groups are combined with specific light chain rearrangement groups (eg, H1 and L1, H12 and L24, etc.). In addition, the mature forms match the variable heavy chain pairs with the variable light encoding sequences (eg, matching H4c and L13). The stringency in matching these mature and rearranged groups means homogeneous matching of the variable region encoding sequences.

However, the heavy chain rearrangement group H4 is exceptional. H4 pairs with two different light chains L28 and L13. L13 is unique for H4, including mature forms a, b, and c that match the L13 mature type. L28, on the other hand, was found to pair with a single component in heavy chain group H2. Two of the seven H4a heavy chain sequences are paired with heavy chain L13a, and five of the seven H4a heavy chain sequences are paired with L28a. In sum, from this confirmation, a complex overlap-renal RT-PCR case can be proposed when genotype combinations H2-L28 and H4a-L13a and plasma cells producing two TT-specific antibodies are present in a single well. have. Very rare intermingling in light and heavy chain gene pairing, such as for H2 and H4a and L28, was expected that the experiment was based on the dilution limit of plasma cells.

The sequence identity among the individual cognate pairs of variable heavy and variable light encoding sequences from the same group and mature is at least 90%, preferably at least 95%. For example, take g060g03 and g060a01 from clones g060g03 corresponding to group H1 mature, nucleotide SEQ ID pair 168: 215, wherein the variable heavy encoding sequence is in SEQ ID NO 168 and the variable light chain encoding sequence is SEQ ID Corresponds to NO215. Clone g060a01 corresponds to nucleotide SEQ ID pair 133: 180. If SEQ ID NO 168 is aligned with SEQ ID NO 133 (variable heavy chain), 4/369 bases are not identical, and 8/327 are not identical for variable light chains (SEQ ID NOs 215 and 180), which is Corresponds to 98.3% sequence identity between these two cognate pairs (g060g03 and g060a01). However, when looking at sequence identity among different groups, sequence identity is not expected to be high because polyclonal sub-libraries and diversity are required. In this particular embodiment, the lowest identity among cognate pairs is approximately 40% (eg, g060b11 and g060h11 have 39.5% sequence homology).

One embodiment of the invention is a sub-library of cognate pairs of immunoglobulin heavy chain variable region and light chain variable region encoding sequences, wherein the immunoglobulins obtainable from the library can react or bind to tetanus toxin.

A further embodiment of the invention is a cognate pair of immunoglobulin heavy chain variable region and light chain variable region encoding sequences comprising an individual cognate pair having at least about 90% sequence identity and one individual SEQ ID pair selected from the group Is a sub-library of:

Table 20:

15881YY00

15881YY00

i. Apparent affinity

Competition assays were set up to determine the apparent affinity or IC ₅₀ of clones selected from plate G060.

Briefly, Fab fragments were expressed in 50 ml culture as follows: 50 ml 2 × YT / 100 μg / ml Carb / 0.1% glucose was added to 0.5 ml overnight and shaken at 37 ° C. for about 2 hours. IPTG was added to a final concentration of 0.1 mM and shaking continued at 30 ° C. overnight. The following day, the bacteria were collected by centrifugation at 4000 rpm for 15 minutes, and the pellet was resuspended in 1 ml 0.8 mM EDTA, 0.4 x PBS, 0.8 M NaCl and incubated for 15 minutes on ice. Periplasmic extracts were collected by centrifugation and stored at -20 ° C.

Competition assays were performed as follows: Periplasmic extract was added to a series of tubes at the appropriate dilution predicted by titration. As a positive control, phage display induced Fab fragment mp584 derived from human hybridoma cell line HB8501 expressing anti-TT antibody was used. Soluble TT was added to the first tube at a concentration of 100 nM and subsequently diluted in a four-fold step in the next tube to prepare a total of seven TT dilutions (from 100 nM to 25 pM). The reaction was incubated at room temperature for about 45 minutes. Samples were transferred to ELISA plates coated at 1 μg / ml with TT and blocked as described above. Plates were incubated for 1 hour at room temperature, then washed 4 × with PBS-T, goat-anti-human Fab / HRP was added at a 1: 10.000 dilution and incubated for 1 hour. TMB plus substrate (KemEnTech, Denmark, cat. No. 4390A) was added and incubation was performed for about 10 minutes and the reaction was stopped with 1M H ₂ SO ₄ . Plates were read at 450 nm. Data is plotted in FIG. 24.

The apparent affinity of the clones analyzed is shown in Table 21.

Table 21

Clone Apparent affinity g060a10 0.50 nM g060b06 0.60 nM g060b12 1.2 nM g060c04 0.55 nM g060d04 1.1 nM g060f01 1.2 nM g060g04 0.9 nM g060g07 0.35nM g060h11 3.0 nM mp584 (pos.cont.) 6.0 nM

As shown in Table 21, Fab fragments have an apparent affinity in the low nanomolar or high picomolar range. In addition, all cognate paired Fab fragments exhibit higher apparent affinity than phage display induced Fab fragments.

j. summary

In donor TT03, the frequency of TT specific plasma cells in the peripheral blood monocyte fraction was calculated to be 0.022%. About 400 cognate pairs were generated from TT03. 3600 clones obtained from cells transformed with the cognate pair library were screened by ELISA, of which 339 clones showed TT reactivity in ELISA screening. 47 of these clones were analyzed for clone diversity. Of these 47 clones, 27 were found to be similar to unique non-scrambled variable region coding sequences. Three of these clones contained premature stop codons that needed correction before transfer to a mammalian expression vector. Transfer to the mammalian expression vector is described in Example 1 section h. Apparent affinity for selected clones was measured in the low nanomolar to high picomolar range.

k. View

Tetanus toxin is one of the most toxic substances known to have a lethal dose of several nanograms. This toxin is produced by Clostridium tetani , a soil bacterium that also exists in the digestive tract of up to 25% of humans. Although the disease is virtually eliminated in the western world by the tetanus immunity program, 100 to 200 cases are still observed in most western countries each year, with a case mortality rate of 50%. In developing countries, the number of cases is significantly higher. Bacterial growth in contaminated penetrating wounds can lead to toxin release and ultimately lead to stiffness, spasms, respiratory arrest and death.

Hyperimmune immunoglobulin products isolated from human blood donors with high antibody response titers to tetanus toxins can be used to prevent tetanus, or can be used with active immunity if early onset of established tetanus treatment. However, due to the lack of human products, horse hyperimmune anti-tetanus toxins are used in developing countries. Recombinant monoclonal or polyclonal antibodies against tetanus toxin are likely to replace hyperimmunoglobulin products for therapeutic and / or prophylactic use. Recombinant monoclonal antibodies generated from conventional hybridoma techniques have been described as effective against TT [Chin, J, et al. 2003. Biologicals 31, 45-53. Interestingly, a synergistic effect was observed when mixing two monoclonal antibodies. Thus, recombinant polyclonal anti-tetanus toxin antibodies capable of reacting with or binding to tetanus toxins can potentially be very effective in the treatment or prophylactic protection of patients at risk for tetanus outbreaks.

One embodiment of the invention is a recombinant polyclonal immunoglobulin or fragment thereof capable of reacting or binding to tetanus toxin.

Preferred embodiments of the invention are recombinant polyclonal immunoglobulins capable of reacting or binding to tetanus toxin consisting of cognate pairs of immunoglobulin heavy chain variable regions and light chain variable regions.

Another embodiment of the invention is a recombinant polyclonal immunoglobulin capable of reacting or binding to tetanus toxin obtained by the method according to the invention.

Other embodiments of the invention include SEQ ID NOs: 229: 276, 262: 309, 240: 287, 245: 292, 267: 314, 246: 293, 258: 305, 242: 289, 231: 278, 263: 310, 232: 279, 237: 284, 255: 302, 260: 307, 251: 298, 233: 280, 228: 275, 244: 291, 250: 297, 252: 299, 264: 311, 272: 319, 235: Recombinant polyclonal capable of reacting or binding to tetanus toxins comprising individual cognate pairs of immunoglobulin heavy chain variable regions and light chain variable regions having at least 90% sequence identity with one individual sequence number pair selected from 282 or 238: 285 Immunoglobulin.

Another embodiment of the invention is a pharmaceutical composition comprising a recombinant polyclonal antibody capable of reacting or binding to tetanus toxin as an active ingredient for the treatment or prevention of tetanus. Preferably, the recombinant polyclonal antibody is combined with a pharmaceutically acceptable excipient.

A further embodiment of the invention is the use of a recombinant polyclonal immunoglobulin capable of reacting or binding to tetanus toxin as a medicament for the treatment or prophylactic protection of a patient at risk of developing tetanus.

A further embodiment is a method of preventing or treating a patient at risk of developing tetanus by administering to a patient in need thereof a composition comprising a recombinant polyclonal antibody capable of reacting or binding to tetanus toxin.

Example 12 Comparison of Results from Two Donors

In the examples below, the results obtained from donor TT08 are compared with the results obtained from donor TT03 in Example 11. The results are summarized in Table 22.

TT03 TT08 TT specific plasma cells (ELISPOT) in PBMC fraction 0.021% 0.011% CD19 + cells (FACS) in PBMC fraction 11% 5% Plasma cells (FACS CD38hi, CD45in) in PBMC fraction 0.12% 0.08% Predicted number of linked V _H and V _L sequences 400 400 Number of clones screened for Fab expression 1400 2000 Number of Fab positive clones (2x background) 482 558 Number of clones screened for TT-specificity 3600 3400 Number of TT-specific clones (2x background) 339 188 Number of sequences analyzed from TT-specific clone 47 102 VH germline allele 12 (VH1-5) NA VL reproductive allele 8 (VKI, III, IV) NA Number of unique cognate pairs 27 40 Apparent affinity range 0.35-3.0nM NA

NA = not analyzed

This clearly demonstrated that a library of similar nature could be isolated from two different donors immunized with TT.

The reason for the higher number of unique cognate pairs in the library obtained from donor TT08 may be due to the higher number of sequences analyzed.

Example 13: Comparison of combinatorial phage display libraries generated from the same donor with libraries of cognate pairs obtained from Example 11

In this example, combinatorial phage display libraries were prepared from the same donor used previously to prepare cognate pair libraries, comparing library diversity, affinity, and specificity between cognate pair libraries and combinatorial libraries.

a. Combination Phage Display Library Manufacturing

Phage display libraries were prepared from the CD19 + fraction of cells obtained from donor TT03 (same as the cell fraction obtained in Example 11c).

Total RNA was prepared from about 5 × 10 ⁶ CD19 ⁺ cells using the NucleoSpin RNA L kit (Machery-Nagel cat. No. 740 962.20). CDNA was subsequently synthesized in an oligo (dT) primed reaction using ThermoScript reverse transcriptase (Invitrogen cat. No. 11146-016).

HotMasterTaq DNA polymerase (Eppendorf cat. No. 0032 002.692) and de Haard et al. (J. Biol. Chem. 274, 18218-18230; 1999), and V _H and kappa chains were PCR amplified essentially using primers modified at the 5 'end with respect to restriction enzyme recognition sequences.

The kappa and V _H PCR products were serially inserted into an Em351 phage display vector (modified from phh3 described in Den, W. et al. 1999 J. Immunol. Methods 222, 45-57) to prepare a combinatorial phage display library. Generated.

The final library is TG1. Electroporation into E. coli strain (Stratagene). The size of the combinatorial library contained 3 × 10 ⁶ independent clones with high insertion frequency.

b. Panning Combination Library

Fab display phage particles were prepared according to standard methods (eg Antibody Engineering, A Practical Approach 1996, ed. McCafferty, Hoogenboom and Chiswell).

Panning was performed on tetanus toxin (TT; SSI batch no. 89-2), diluted to 1 μg / ml in PBS and fixed in Maxisorp immunotubes (Nuc. Cat. No. 444202). After a one hour incubation period and several washing steps, bound phage particles were eluted using 100 mM TEA (triethylamine).

Phage particle eluate was neutralized and used to infect exponentially growing TG1 cells, from which the Fab display phage particles with high TT specificity were obtained. According to the general method outlined above, a second panning using eluted phage was performed.

In parallel, according to the method, three pannings were performed on the C-fragment of tetanus toxin molecule (Sigma cat. No. T3694).

Single colonies were screened for binding to C-fragment and tetanus toxin from the unselected library and from the two sets of panning described above after each meeting panning.

c. Comparison of Specificity and Affinity of Combination Phage Particle Clones and Cognate Pair Clones

Single colonies were taken from the unselected library and after each round of panning (for intact tetanus toxin (TT) and tetanus toxin C-fragments, respectively). Fab display phage particles were analyzed for reactivity by ELISA assay. The number of Fab positive clones showing TT and / or C-fragment reactivity of at least 2 × and at least 4 × of background reactivity is shown in Tables 23-25 below. All results are shown as the number of specific clones / number of Fab positive clones.

Table 23: Clones selected for TT and analyzed by TT-specific ELISA

Increase 2x background 4x background Unchecked clone - 13 / approximately 5000 After the first panning 61/99 59/93 After the second panning 165/169 163/166

Table 24: Clones selected for TT and analyzed by C-fragment-specific ELISA

Increase 2x background 4x background Unchecked clone 0/13 0/13 After the first panning 0/85 0/85 After the second panning 0/85 0/85

Table 25: Clones selected for C-fragment and analyzed by C-fragment-specific ELISA

Increase 2x background 4x background After the first panning 1/35 0/35 After the second panning 12/47 9/47 After the third panning 64/126 26/126

Panning of the phage display library for TT has been shown to increase the number of TT specific clones when the number of pannings is increased, and only a few clones have been identified in clones that are not selected. Clones from the unselected library or after two pannings of the library for TT were shown to be inactive with the tetanus toxin C-fragment. In order to obtain Fab fragments with C-fragment specificity from the combinatorial phage display library, this library had to be panned specifically for C-fragment.

For comparison, C-fragment ELISA was performed on 13 TT-specific clones obtained from Example 11, of which 7 showed reactivity greater than 2 × background. Thus, Fab fragments specific for tetanus toxin C-fragment could be expressed from a library of cognate pairs obtained from TT-immunized individuals.

This clearly shows the drawbacks of using panning to identify clones that are specific for a particular antigen. If important antigen fragments or epitopes are not known, they can be released during panning, resulting in a less efficient product.

In addition, the apparent affinity of the combination clones was measured as described in the assay of Example 11i. Ten of the combined clones obtained after two pannings for TT were analyzed and found to have an apparent affinity of 1 to 15 nM.

Four of the nine clones (Table 21) analyzed from the cognate pair library for comparison showed affinity of the picomolar range.

This indicates that pairing the first selected variable region by the donor immune system, together with overbody mutations that the pair received as a pair, potentially leads to higher apparent binding affinity than a random combination of these variable regions. .

d. Sequence Comparison of TT Specific Combination Phage Particle Clones and Cognate Pair Clones

The sequence data generated from the two libraries is so large that raw sequences cannot be directly compared. To visualize the differences between V _H and V _L sequence pairs in phage display libraries and cognate pair libraries, phylogenies were generated for the V _H and V _L sequences and the pairs were illustrated by dot matrix. Pairing of phylogenetic information in the dot matrix (FIG. 25) showed a very different distribution profile of the V _H and V _L sequence pairs in both libraries. Dispersive aspects of the V _H and V _L sequence pairs in the phage display library (FIG. 25A) showed little phylogenetic relationship between the V _H and V _L genes consistent with the formation of random pairs of V genes in this library. In contrast, the V _H and V _L sequences from a library of cognate pairs exhibit clustering patterns indicating V gene coevolution as predicted for cognate pairs. In addition, gene diversity is greater for V genes in cognate pair libraries compared to V genes from combinatorial libraries, indicating that the method of separating V genes of cognate pairs has less variation.

Example 14 Combination of Single Step Complex Over-Kidney RT-PCR and Nested PCR Using T Cells as Template Source

In this example, we describe how single step complex overlap-renal RT-PCR can be performed on T lymphocytes derived from human donors.

a. Obtaining Lymphocyte-Containing Cell Fractions

Blood samples were obtained from human donors treated with the antigen of interest, for example, by immunization, natural infection, malignant tumor, autoimmune response or other disease. Peripheral blood monocytes (PBMC) were isolated using Lymphoprep (Axis-Shield, Oslo Norway, prod. No 1001967) according to the manufacturer's instructions.

In this example, the PBMC fraction was further stimulated to generate antigen specific T cells. However, complex overlap-extension RT-PCR can also be performed directly from PBMC fractions on single cells or on cell fractions containing large amounts of T cells.

b. Generation of Antigen Specific T Cells

PBMC cell fractions are resuspended in a suitable culture medium containing related cytokines such as IL2. In addition, the desired antigen is added to the culture, where the antigen is presented to T cells by antigen presenting cells (APC) present in the PBMC fraction. Alternatively, APC feed cells pretreated to present the desired antigen can be added to the PBMC fraction. Such APC feeder cells are, for example, APCs exposed to the antigen of interest in the form of peptides, proteins, or other molecules, or cells transfected to express and present microbially infected APCs or antigens, or for example primary tissues or cell lines. APC may be co-cultured with other antigenic cells, such as cancer cells. Many different forms of APC feed cells are known in the literature, including transformed cell lines, B cell lines, dendritic cells, and the like.

PBMC fractions are incubated for about 3-5 weeks, during which new antigen presenting cells are added with cytokines, eg, weekly. As a result, T lymphocytes proliferated, activated, and matured. At the end of the culture period, the cell culture is dominated by antigen specific T cells. Whether such cells are CD4 + or CD8 + depends on the disease, antigen, APC cells, and cytokine mixture used during the stimulation period.

Antigen specificity can be tested using, for example, CTL assays, proliferation assays and MHC tetramers loaded with the desired antigen [Altman, J. D., et al. 1996. Science 274, 94-96.

Antigen-specific T cells can be distributed in PCR tubes by limiting dilution or by using FACS to determine single cell pr. Obtain a container. The vessel can be stored at -80 ° C until use.

b. Single Phase Complex Redundant-Eight RT-PCR

Complex duplicate-extension RT-PCR was performed using the Qiagen One-Step RT-PCR kit (Qiagen cat. No 210212, Hilden, Germany) essentially according to the manufacturer's recommendations. Before adding the PCR reaction mixture to the PCR tube, the cells were thawed.

PCR reaction mixtures and cycling conditions were initially set as described in Example 11 e-1. However, certain optimal amounts can be predicted for new primer sets.

The complex overlap-extension primer mixtures used included the primers shown in Table 26d.

Table 26

c. Semi-Nested PCR

Semi-nested PCR is similarly suggested to perform as described in Example 11 e-2, and some optimization can be expected.

Primers used are shown in Table 27.

Table 27

Capitalized sequences correspond to gene specific regions.

To demonstrate the success of the complex overlap-extension RT-PCR, a portion of the sample from the semi-nested PCR reaction was treated with 1% agarose gel electrophoresis of 10 μl from each semi-nested PCR reaction. And analyzed using ethidium bromide for detection. The expected size of the overlap-extension fragment is about 850 bp (the exact size depends on the length of the variable region).

About 10 microliters from all reactions performed from the same donor were combined in one tube. An aliquot of the pooled PCR product was subsequently purified using a QIAquick PCR Purification Kit (Qiagen cat. No. 28106, Hilden, Germany) according to the manufacturer's method. Purified pools of overlapping PCR products can be digested with appropriate restriction enzymes, purified and inserted into the appropriate vector.

In this experiment, the constant region primers (SEQ ID NOS: 376 and 377) used in the semi-nested PCR were designed to subclones the semi-nested PCR product into a suitable vector. The design of the Cβ primers depends on the change of SER to MET at position 21 in the chain, whereby the NsiI site can be introduced into the nucleic acid sequence:

This change should be relatively safe because SER21 is exposed on the β chain of the loop structure at the proximal end of the domain. Thus, such relative conservative changes will not interfere with the overall domain structure.

The design of the Cα primers depends on the change in nucleotide corresponding to positions 15-17 of the α chain constant region peptide, so that the SacI site can be introduced into the nucleic acid sequence:

Thus, a suitable vector will contain the remainder of the constant region of the TCR α and β chains and will contain the appropriate modification in the restriction site. Such changes can be carried out using standard PCR and subcloning techniques.

d. Additional considerations

Large amounts of variable region primers can potentially interact with inhibitors during complex overlap-extension PCR amplification. To avoid this, the variable region primers can be divided into subsets and used separately in suitable combinations.

Another embodiment

All publications and patent applications cited herein are hereby incorporated by reference as if each individual document were specifically and individually indicated to be incorporated herein. Although the foregoing invention has been described in detail in the manner of illustration and example for clarity of understanding, it will be apparent to those skilled in the art in view of the teachings of the present invention that specific changes and modifications can be made without departing from the spirit or scope of the appended claims. Will be obvious to you.

                         SEQUENCE LISTING <110> Symphogen A / S   <120> METHOD FOR LINKING SEQUENCES OF INTEREST <130> 15881PCT00 <160> 377 <170> PatentIn version 3.3 <210> 1 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 1 agcctatact attgattagg cgcgcccagr tgcagctggt gcart 45 <210> 2 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 2 agcctatact attgattagg cgcgccsagg tccagctggt rcagt 45 <210> 3 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 3 agcctatact attgattagg cgcgcccagr tcaccttgaa ggagt 45 <210> 4 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 4 agcctatact attgattagg cgcgccsagg tgcagctggt ggag 44 <210> 5 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 5 agcctatact attgattagg cgcgcccagg tgcagctaca gcagt 45 <210> 6 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 6 agcctatact attgattagg cgcgcccags tgcagctgca ggagt 45 <210> 7 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 7 agcctatact attgattagg cgcgccgarg tgcagctggt gcagt 45 <210> 8 <211> 46 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 8 agcctatact attgattagg cgcgcccagg tacagctgca gcagtc 46 <210> 9 <211> 20 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 9 gacsgatggg cccttggtgg 20 <210> 10 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 10 cgcctaatca atagtatagg ctagccgaca tccagwtgac ccagtct 47 <210> 11 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 11 cgcctaatca atagtatagg ctagccgatg ttgtgatgac tcagtct 47 <210> 12 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 12 cgcctaatca atagtatagg ctagccgaaa ttgtgwtgac rcagtct 47 <210> 13 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 13 cgcctaatca atagtatagg ctagccgata ttgtgatgac ccacact 47 <210> 14 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 14 cgcctaatca atagtatagg ctagccgaaa cgacactcac gcagt 45 <210> 15 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 15 cgcctaatca atagtatagg ctagccgaaa ttgtgctgac tcagtct 47 <210> 16 <211> 38 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 16 atatatatgc ggccgcttat taacactctc ccctgttg 38 <210> 17 <211> 26 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 17 atattctcga gacggtgacc agggtg 26 <210> 18 <211> 25 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 18 atattctcga gacggtgacc attgt 25 <210> 19 <211> 27 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 19 atattctcga gacggtgacc agggttc 27 <210> 20 <211> 25 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 20 atattctcga gacggtgacc gtggt 25 <210> 21 <211> 51 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 21 accgcctcca ccggcggccg cttattaaca ctctcccctg ttgaagctct t 51 <210> 22 <211> 43 <212> DNA <213> Artificial <220> <223> primer sequence <400> 22 gctagcatta ttattaccat ggcccagrtg cagctggtgc art 43 <210> 23 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 23 gctagcatta ttattaccat ggccsaggtc cagctggtrc agt 43 <210> 24 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 24 gctagcatta ttattaccat ggcccagrtc accttgaagg agt 43 <210> 25 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 25 gctagcatta ttattaccat ggccsaggtg cagctggtgg ag 42 <210> 26 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 26 tattggcgcg ccatggccsa ggtgcagctg gtggag 36 <210> 27 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 27 gctagcatta ttattaccat ggcccaggtg cagctacagc agt 43 <210> 28 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 28 gctagcatta ttattaccat ggcccagstg cagctgcagg agt 43 <210> 29 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 29 gctagcatta ttattaccat ggccgargtg cagctggtgc agt 43 <210> 30 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 30 gctagcatta ttattaccat ggcccaggta cagctgcagc agtc 44 <210> 31 <211> 26 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 31 atattctcga gacggtgacc agggtg 26 <210> 32 <211> 27 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 32 atattctcga gacggtgacc attgtcc 27 <210> 33 <211> 27 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 33 atattctcga gacggtgacc agggttc 27 <210> 34 <211> 27 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 34 atattctcga gacggtgacc gtggtcc 27 <210> 35 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 35 ccatggtaat aataatgcta gccgacatcc agwtgaccca gtct 44 <210> 36 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 36 ccatggtaat aataatgcta gccgatgttg tgatgactca gtct 44 <210> 37 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 37 ccatggtaat aataatgcta gccgaaattg tgwtgacrca gtct 44 <210> 38 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 38 ccatggtaat aataatgcta gccgatattg tgatgaccca cact 44 <210> 39 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 39 ccatggtaat aataatgcta gccgaaacga cactcacgca gt 42 <210> 40 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 40 ccatggtaat aataatgcta gccgaaattg tgctgactca gtct 44 <210> 41 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 41 tattggcgcg ccatggccca grtgcagctg gtgcart 37 <210> 42 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 42 tattggcgcg ccatggccsa ggtccagctg gtrcagt 37 <210> 43 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 43 tattggcgcg ccatggccca grtcaccttg aaggagt 37 <210> 44 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 44 tattggcgcg ccatggccsa ggtgcagctg gtggag 36 <210> 45 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 45 tattggcgcg ccatggccca ggtgcagcta cagcag 36 <210> 46 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 46 tattggcgcg ccatggccca gstgcagctg caggagt 37 <210> 47 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 47 tattggcgcg ccatggccga rgtgcagctg gtgcagt 37 <210> 48 <211> 38 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 48 tattggcgcg ccatggccca ggtacagctg cagcagtc 38 <210> 49 <211> 23 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 49 aagtagtcct tgaccaggca gcc 23 <210> 50 <211> 20 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 50 tgagttccac gacaccgtca 20 <210> 51 <211> 20 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 51 agaggtgctc ttggaggagg 20 <210> 52 <211> 21 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 52 agttttgtca caagatttgg g 21 <210> 53 <211> 23 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 53 gttgcagatg taggtctggg tgc 23 <210> 54 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 54 gccatggcgc gccaatagct agccgacatc cagwtgaccc agtct 45 <210> 55 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 55 gccatggcgc gccaatagct agccgatgtt gtgatgactc agtct 45 <210> 56 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 56 gccatggcgc gccaatagct agccgaaatt gtgwtgacrc agtct 45 <210> 57 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 57 gccatggcgc gccaatagct agccgatatt gtgatgaccc acact 45 <210> 58 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 58 gccatggcgc gccaatagct agccgaaacg acactcacgc agt 43 <210> 59 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 59 gccatggcgc gccaatagct agccgaaatt gtgctgactc agtct 45 <210> 60 <211> 40 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 60 atatatatgc ggccgcttat taacactctc ccctgttgaa 40 <210> 61 <211> 30 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 61 ggaggcgctc gagacggtga ccagggtgcc 30 <210> 62 <211> 30 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 62 ggaggcgctc gagacggtga ccattgtccc 30 <210> 63 <211> 30 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 63 ggaggcgctc gagacggtga ccagggttcc 30 <210> 64 <211> 30 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 64 ggaggcgctc gagacggtga ccgtggtccc 30 <210> 65 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 65 cgcctaatca atagtatagg ctagcccagt ctgtgctgac tcagcca 47 <210> 66 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 66 cgcctaatca atagtatagg ctagcccagt ctgtgytgac gcagc 45 <210> 67 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 67 cgcctaatca atagtatagg ctagcccagt ctgtcgtgac gcagc 45 <210> 68 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 68 cgcctaatca atagtatagg ctagcccart ctgccctgac tcagcct 47 <210> 69 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 69 cgcctaatca atagtatagg ctagcctcct atgwgctgac tcagcca 47 <210> 70 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 70 cgcctaatca atagtatagg ctagcctctt ctgagctgac tcaggac 47 <210> 71 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 71 cgcctaatca atagtatagg ctagcccacg ttatactgac tcaaccg 47 <210> 72 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 72 cgcctaatca atagtatagg ctagcccagg ctgtgctgac tcagc 45 <210> 73 <211> 49 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 73 cgcctaatca atagtatagg ctagccaatt ttatgctgac tcagcccca 49 <210> 74 <211> 46 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 74 cgcctaatca atagtatagg ctagcccagr ctgtggtgac ycagga 46 <210> 75 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 75 cgcctaatca atagtatagg ctagcccwgc ctgtgctgac tcagc 45 <210> 76 <211> 31 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 76 gccgcttatt atgaacattc tgtaggggcc a 31 <210> 77 <211> 31 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 77 gccgcttatt aagagcattc tgcaggggcc a 31 <210> 78 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 78 atatatatgc ggccgcttat tatgaacatt ctgtaggggc cactg 45 <210> 79 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 79 atatatatgc ggccgcttat taagagcatt ctgcaggggc cactg 45 <210> 80 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 80 atatatatgc ggccgcttaa tgggctgcct cgggaa 36 <210> 81 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 81 gccatggcgc gccaatagct agccttagag cagctcgtac tgacgaa 47 <210> 82 <211> 41 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 82 tattggcgcg ccatggccat gagtgagctt gaccagttac g 41 <210> 83 <211> 47 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 83 ggtacctaat aataatcgat cgcttagttc cagatcttga ggaagct 47 <210> 84 <211> 48 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 84 cgatcgatta ttattaggta ccccatgcca gtaatcaata ttgaggac 48 <210> 85 <211> 38 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 85 ggaggcgctc gagttatgaa atcacacagc ctcctttg 38 <210> 86 <211> 34 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (34) <223> gene-specific region <400> 86 tattcccagc ggccgccgcc acaggtgccc actc 34 <210> 87 <211> 34 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (34) <223> gene-specific region <400> 87 tattcccagc ggccgcccct tcmtgggtct tgtc 34 <210> 88 <211> 35 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (35) <223> gene-specific region <400> 88 tattcccagc ggccgcctta raaggtgtcc agtgt 35 <210> 89 <211> 32 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (32) <223> gene-specific region <400> 89 tattcccagc ggccgccccc agatgggtcc tg 32 <210> 90 <211> 34 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (34) <223> gene-specific region <400> 90 tattcccagc ggccgccctc caaggagtct gttc 34 <210> 91 <211> 35 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (35) <223> gene-specific region <400> 91 tattcccagc ggccgcccca tggggtgtcc tgtca 35 <210> 92 <211> 33 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (18) .. (33) <223> gene-specific region <400> 92 tattcccagc ggccgccgca acaggtgccc act 33 <210> 93 <211> 41 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (41) <223> gene-specific region <400> 93 ggcggccgct gggaatagct agcgytccca ggtgccagat g 41 <210> 94 <211> 40 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (40) <223> gene-specific region <400> 94 ggcggccgct gggaatagct agcggtccct ggatccagtg 40 <210> 95 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (42) <223> gene-specific region <400> 95 ggcggccgct gggaatagct agcgctccca gataccaccg ga 42 <210> 96 <211> 39 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (39) <223> gene-specific region <400> 96 ggcggccgct gggaatagct agcgatctct ggtgcctac 39 <210> 97 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (42) <400> 97 ggcggccgct gggaatagct agcgatctct gataccaggg ca 42 <210> 98 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <220> <221> misc_feature (222) (25) .. (42) <223> gene-specific region <400> 98 ggcggccgct gggaatagct agcggttcca gcctccaggg gt 42 <210> 99 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 99 atatatatgg cgcgccttaa cactctcccc tgttgaa 37 <210> 100 <211> 48 <212> DNA <213> Artificial <220> <223> Vector sequence <400> 100 atgaaatatc ttctaccaac agcggcagct ggattattgg cggccgcc 48 <210> 101 <211> 48 <212> DNA <213> Artificial <220> <223> Vector sequence <400> 101 atgaaatatt tgctaccaac agcggcagct ggattattgt tactagcg 48 <210> 102 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 102 tgttgttcta gatgaaggcg cgcccagrtg cagctggtgc art 43 <210> 103 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 103 tgttgttcta gatgaaggcg cgccsaggtc cagctggtrc agt 43 <210> 104 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 104 tgttgttcta gatgaaggcg cgcccagrtc accttgaagg agt 43 <210> 105 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 105 tgttgttcta gatgaaggcg cgccsaggtg cagctggtgg ag 42 <210> 106 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 106 tgttgttcta gatgaaggcg cgcccaggtg cagctacagc agt 43 <210> 107 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 107 tgttgttcta gatgaaggcg cgcccagstg cagctgcagg agt 43 <210> 108 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 108 tgttgttcta gatgaaggcg cgccgargtg cagctggtgc agt 43 <210> 109 <211> 44 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 109 tgttgttcta gatgaaggcg cgcccaggta cagctgcagc agtc 44 <210> 110 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 110 aacaacacta gtgataggct agccgacatc cagwtgaccc agtct 45 <210> 111 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 111 aacaacacta gtgataggct agccgatgtt gtgatgactc agtct 45 <210> 112 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 112 aacaacacta gtgataggct agccgaaatt gtgwtgacrc agtct 45 <210> 113 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 113 aacaacacta gtgataggct agccgatatt gtgatgaccc acact 45 <210> 114 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 114 aacaacacta gtgataggct agccgaaacg acactcacgc agt 43 <210> 115 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 115 aacaacacta gtgataggct agccgaaatt gtgctgactc agtct 45 <210> 116 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 116 tattcccatg gcgcgcccag rtgcagctgg tgcart 36 <210> 117 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 117 tattcccatg gcgcgccsag gtccagctgg trcagt 36 <210> 118 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 118 tattcccatg gcgcgcccag rtcaccttga aggagt 36 <210> 119 <211> 35 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 119 tattcccatg gcgcgccsag gtgcagctgg tggag 35 <210> 120 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 120 tattcccatg gcgcgcccag gtgcagctac agcagt 36 <210> 121 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 121 tattcccatg gcgcgcccag stgcagctgc aggagt 36 <210> 122 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 122 tattcccatg gcgcgccgar gtgcagctgg tgcagt 36 <210> 123 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 123 tattcccatg gcgcgcccag gtacagctgc agcagtc 37 <210> 124 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 124 ggcgcgccat gggaatagct agccgacatc cagwtgaccc agtct 45 <210> 125 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 125 ggcgcgccat gggaatagct agccgatgtt gtgatgactc agtct 45 <210> 126 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 126 ggcgcgccat gggaatagct agccgaaatt gtgwtgacrc agtct 45 <210> 127 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 127 ggcgcgccat gggaatagct agccgatatt gtgatgaccc acact 45 <210> 128 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 128 ggcgcgccat gggaatagct agccgaaacg acactcacgc agt 43 <210> 129 <211> 45 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 129 ggcgcgccat gggaatagct agccgaaatt gtgctgactc agtct 45 <210> 130 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 130 atatatatgc ggccgcttaa cactctcccc tgttgaa 37 <210> 131 <211> 20 <212> DNA <213> Artificial <220> <223> Vector Sequence <400> 131 aggaaacagg agatatacat 20 <210> 132 <211> 20 <212> DNA <213> Artificial <220> <223> Vector sequence <400> 132 tcgccaagga gacagtcata 20 <210> 133 <211> 369 <212> DNA <213> Homo Sapiens <133> 133 gggcgcgccc aggtgcagct ggtgcaatct ggacctgaag tgaagaagcc tggggcctca 60 gtgagggtct cctgcaaggc ctctggttac tcctttaaga actatggtat ccactgggtg 120 cgacaggccc ctggacaggg gcttgagtgg atggggtgga tcagcgctga caatggtgac 180 acaaccactg cactgaacct ccggggcaga gtctccatga ccacagacac atccacaaac 240 acagtctaca tggaggtgaa gagcctaaga tctgacgaca cggccatata tttctgtgcg 300 cgagattttt atagtgggag ttaccggtcc tttgactgct ggggccaggg caccctggtc 360 accgtctcg 369 <210> 134 <211> 378 <212> DNA <213> Homo Sapiens <400> 134 gggcgcgccc aggtgcagct acagcagtct gggggaggcc tggtcaagcc tggggggtcc 60 ctgagactct cctgtgcagc ctctggattc accttcagta gctatagcat gaactgggtc 120 cgccaggctc cagggaaggg gctggagtgg gtctcatcca ttagtagtag tagtagttac 180 atatactacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaagaac 240 tcactgtatc tgcaaatgaa cagcctgaga gccgaggaca cggctgtgta ttactgtgcg 300 agacgccctg gttgggctgc tactcgggcg gcgggtgctt ttgatatctg gggccaaggg 360 acaatggtca ccgtctcg 378 <210> 135 <211> 357 <212> DNA <213> Homo Sapiens <400> 135 gggcgcgccc aggtgcagct ggtgcaatct ggacctgaac tgaagaagcc tggggcctca 60 gtgaggatct cctgcaaggc ctctgagaaa tgtgatatcc actgggtgcg acaggcccct 120 ggacaggggc ttgagtggat gggatggatc agcgctgacg atggtgggac aaccactgcg 180 ctgaacctcc ggggcagagt ctccatgacc acagacagag ccacaaacac agtatacatg 240 gaactgaaga gcctaagatc tgacgacacg gccatttatt tctgtgcgcg agatttttat 300 agtgggactt accggtcctt tgactactgg ggccagggaa ccctggtcac cgtctcg 357 <210> 136 <211> 396 <212> DNA <213> Homo Sapiens <400> 136 gggcgcgcca aggtgcagct ggtggagtct gggggaggcg tggtccagcc tgggacgtcc 60 ctcagactct cctgtgtagt ctctggattc accttcagaa cctatggtat gaactgggtc 120 cgccaggctc caggcaaggg gctggagtgg ctggcatttc tatcatctga tgggagcgat 180 gaattctacg cggactccgt gaagggccga ttcaccgtct ccagggacaa ttccaagagc 240 actctctttc tgaaaatgaa cagtctgaga cctgatgaca cggctgtcta ttactgtgcg 300 agggatcgtg gggcccaaat tacacttttt ggagcccctc ttataaggcc ttcgtccttt 360 gactcctggg gccagggcac cctggtcacc gtctcg 396 <210> 137 <211> 362 <212> DNA <213> Homo Sapiens <400> 137 gggcgcgccc aggtgcagct gcaggagtct ggacctgagg tgaagaagcc tggggcctca 60 gtgaaggtgt cctgcaaggc ttctggctac acgtttaaca gttatggaat cgcctgggtg 120 cgacaggtcc ctggacaagg gcttgagtgg atgggatgga tcagccctta cagtggtcac 180 acaaactatg cagagaaggt ccagggcaga gtcaccatga ccacagacac cgccacgagc 240 acagcctgca tggagctgac gagcctgaga tctgacgaca cggccgttta tttctgtgcg 300 agagactaca gtagtccgta ccactttgac tactggggcc agggcacctg gtcaccgtct 360 cg 362 <210> 138 <211> 366 <212> DNA <213> Homo Sapiens <400> 138 gggcgcgccc agatcacctt gaaggagtct ggtcctacgc tggtgaaacc cacacagacc 60 ctcacgctga cctgcacctt gtctgggttc tcactctaca ctactggagt gggtgtgggc 120 tggatccgtc agcccccagg aaaggccctg gagtggctgg cacgcattta ttgggatgat 180 gatgagcgct acaacccgtc tctgaagagc aggctcacca tcaccaagga cgcctccaaa 240 aaccaggtgg tccttaaaat gaccaacatg gaccctgtgg acacagccac atattactgt 300 gcccggacca tgggcgtcgt tcttccattt gactactggg gccagggaac cctggtcacc 360 gtctcg 366 <139> <211> 351 <212> DNA <213> Homo Sapiens <400> 139 gggcgcgcca aggtgcagct ggtggagtct gggggaggcc tggtcaagcc gggggggtcc 60 ctgagactct cctgtgtagt ttctgggttc cccctcaata gatacaccat gaactgggtc 120 cgccaggctc cagggaaggg gctggagtgg ctctcgtcca ttagtagtac tagttcttac 180 atatactacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaagaat 240 tctctgtttc tgcagatgaa cagcctgaga gccgacgaca cggctctcta tttctgtgcg 300 agtggcaata ctcatgacta ctggggccag ggaaccctgg tcaccgtctc g 351 <210> 140 <211> 378 <212> DNA <213> Homo Sapiens <400> 140 gggcgcgccc aggtgcagct ggtgcagtct ggggctgagg tgaagaagct tgggacgtca 60 gtgagggtct cctgcaagac tcctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cggaagttcc agggcagaat cacggttacc gcggacaaat ccacgaacac agtgtacatg 240 gatttgagca acctggcatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cgatatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 141 <211> 396 <212> DNA <213> Homo Sapiens <400> 141 gggcgcgccc aggtgcagct tcaggagtct gggggaggcg tggtccagcc tgggacgtcc 60 ctcagactct cctgtgtagt ctctggattc accttcagaa cctatggtat gaactgggtc 120 cgccaggctc caggcaaggt gctggagtgg ctggcatttg tatcatctga tgggagcgat 180 gaattctacg cggactccgt gaagggccga ttcaccgtct ccagggacaa ttccaagagc 240 actctctttc tgaaaatgaa cagtctgaga gctgatgaca cggctgtcta ttactgtgcg 300 agagatcgtg gggcccaaat tacacttttt ggagcccctc ttataaggcc ttcgtccttt 360 gactcctggg gccagggaac cctggtcacc gtctcg 396 <210> 142 <211> 378 <212> DNA <213> Homo Sapiens <400> 142 gggcgcgccg aggtgcagct ggtggagtct ggggctgagg tgaagaagcc tgggacctca 60 gtgagggtct cctgcaagac ttctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccagct ttgggacaac aagctacgca 180 cagaagttcc agggcagagt cacgattacc gcggacaaag ccacgaacac agtgtacatg 240 gatttgagcg acctgacatc tgaggacacg gccatatatt actgtgcgaa agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cgctatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 143 <211> 366 <212> DNA <213> Homo Sapiens <400> 143 gggcgcgccc aggtgcagct gcaggagtct ggtcctacgc tggtgaaacc cacacagacc 60 ctcacgctga cctgctcgtt ctctggtttc tcactcggca ctactggagt caatgtgggc 120 tggatccgtc agcccccggg aaaggccctg gagtggcttg cactcatttc ttgggatggt 180 ggtaagcact acagcccatc tctgaactcc aggatcaccc tcactaagga cgcctccaga 240 gagcaggtgg tggtccctac aatgaccaac atggaccctg cggacacagg cagatattat 300 tgtgcacgta tagtggggac tcacggcttt gactactggg gccagggaac cctggtcacc 360 gtctcg 366 <210> 144 <211> 393 <212> DNA <213> Homo Sapiens <400> 144 gggcgcgcca aggtgcagct ggtggagtct ggagctgagg tgaagaagcc tggggccaca 60 gtcagggtct cctgtaaggc ttctggttac aggtttaacg actattgtat cagctgggtg 120 cgacaggccc ctggacaagg gcttgagtgg atggggtgga tcaacggtaa caatgctgac 180 acattctatg caccgaagct ccagggcaga gtcaccatga gcacagacac atccacgagc 240 acagcctaca tggagctgag gaacttgaga tcggacgaca cggccgttta tttctgtgcg 300 cgagatcgag gacgtattac tctttttggc gaagttattt taagggcggg atggttcgac 360 tcctggggcc agggcaccct ggtcaccgtc tcg 393 <210> 145 <211> 372 <212> DNA <213> Homo Sapiens <400> 145 gggcgcgccc aggtgcagct ggtgcagtct gggggaggcc tggtcaagcc gggggggtcc 60 ctgagactct cctgtgcagc ctctggattc tcctttagta ataataacat gaattgggtc 120 cgccagactc caggaaaggg actggagtgg gtcgcatcta ttagttttgg aagtcattac 180 atatcctacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaggaat 240 gcagtttatc tgcagatgaa cagcctgaga gtcgaggaca cggctgtcta ttactgcacg 300 agatgcaggg gcggaactcg tacctattat tacatggacg tctggggcaa aggcaccctg 360 gtcaccgtct cg 372 <210> 146 <211> 372 <212> DNA <213> Homo Sapiens <400> 146 gggcgcgcca aggtgcagct ggtggagtct gggggaggcc tggtcaagcc aggggggtcc 60 ctgagactct cctgtgcagc ctctggatcc tcctttagta ataataacat gaattgggtc 120 cgccagactc caggaaaggg actggagtgg gtcgcatcca ttagttttgg aagtcattac 180 atatcctacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaggaat 240 gcagtttatc tgcagatgaa cagcctgaga gtcgaggaca cggctgtcta ttactgcacg 300 agatgcaggg gcggaactcg tacctattat tacatggacg tctggggcaa aggcaccctg 360 gtcaccgtct cg 372 <210> 147 <211> 390 <212> DNA <213> Homo Sapiens <400> 147 gggcgcgccc agatgcagct ggtgcaatct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgccagtc ttctggaggc ccccccaaaa gttatactct cagctgggtg 120 cggcaggccc ctggacaagg ccctgagtgg atgggcggaa tcattctaat ctttggccca 180 ccaaactacg cccagaagtt ccaggacaga ctcacgatca ccgcggacaa gtccaccaac 240 acagtctaca tggagttaag tagcctgaga tctgatgaca cggccatgta ctactgtgtg 300 acagcccccg acgacactgg cactatatta gctcgtcaca accgttacta ctttgactcc 360 tggggccagg gcaccctggt caccgtctcg 390 <210> 148 <211> 390 <212> DNA <213> Homo Sapiens <400> 148 gggcgcgcca aggtgcagct ggtgcagtct ggagcagagg tgaagaagcc cggggagtct 60 ctgaaaatct cctgtcaggc ttctggatac ggctttaccg tctactggat cggctgggtg 120 cgccagccgc ccgggaaagg cctggagtgg ctgggtatca tctatcctgg tgactctgat 180 accagataca atccgtcctt ccaaggccag gtcaccatct cagccgacaa gtccgtcagc 240 accacctacc tgcagtggag cagcctgaag gcctcggaca ccgccattta ctactgtgcg 300 agacatctgg actcatacga tgttttcact ggttataatt tggggggcta catggacgtc 360 tggggcaagg gaaccctggt caccgtctcg 390 <210> 149 <211> 351 <212> DNA <213> Homo Sapiens <400> 149 gggcgcgcca aagtgtagct ggtgcagtct gggggaggcc tggtcaagcc tggggggtcc 60 ctgagactct cctgtgtagt ctctggattc cccctcaata gatacatcat gaactgggtc 120 cgccagactc cagggaaggg gctggagtgg ctctcgtcca ttagtagtac cagttcttac 180 atatactacg cagactcagc gaagggccga ttcaccatct ccagagacaa cgccaagaat 240 tctctgtttc tgcagatgaa cagcctgaga gccgaggaca caggtctcta ttactgtgcg 300 agtggcaata ctcatgacta ttggggccag ggcaccctgg tcaccgtctc g 351 <210> 150 <211> 378 <212> DNA <213> Homo Sapiens <400> 150 gggcgcgccc agatgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacctca 60 gtgagggtct cctgcaagac ttctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cagaagttcc agggcagagt cacgattacc gcggacaaat ccacgaacac agtgtatatg 240 gatttgagca acctgacatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cggtatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 151 <211> 372 <212> DNA <213> Homo Sapiens <400> 151 gggcgcgcca aggtgcagct ggtggagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaaggc ctctggaggc agcttcagca cctatgctat cacctgggtg 120 cgccaggccc ctggacaggg gcttgaatgg atgggaggga tcatccctat ctttgcttca 180 agagactacg cacagaagtt tcagggcaga gtcacagtca ccgcggacga atccacgagg 240 acagtgtaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca 300 agagtgctgg gaggtacaag gctctactac gctctgaacg tctggggcca agggacaatg 360 gtcaccgtct cg 372 <210> 152 <211> 372 <212> DNA <213> Homo Sapiens <400> 152 gggcgcgcca aggtgcagct ggtggagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaagac atctggaggc agtttcagca catactctat cacctgggtg 120 cgccaggccc ctggacaggg gcttgagtgg atgggaggga tcaaccctat cttcgctaca 180 agagactacg cacagaagtt ccagggcaga gtcacgatca ccgcggacga atccacgagg 240 acagtctaca tggagttgag gaacttgaga tctgaggaca cggccgtgta ttattgtgca 300 agagtgttcg gaggaacaag actctactac gccctgaacg tctggggcca aggcaccctg 360 gtcaccgtct cg 372 <210> 153 <211> 378 <212> DNA <213> Homo Sapiens <400> 153 gggcgcgccg aagtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacgtca 60 gtgagggtct cctgcaagac tcctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cggaagttcc agggcagaat cacggttacc gcggacaaat ccacgaacac agtgtacatg 240 gatttgagca acctggcatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cgatatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 154 <211> 372 <212> DNA <213> Homo Sapiens <400> 154 gggcgcgcca aggtgcagct ggtggagtct gggggaggcc tggtcaagcc gggggggtcc 60 ctgagactct cctgtgcagc ctctggattc tcctttagta ataataacat gaattgggtc 120 cgccagaccc caggaaaggg actggagtgg gtcgcatcca ttagttttgg aagtcattac 180 atatcctacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaggaat 240 gcagtttatc tgcagatgaa cagcctgaga gtcgaggaca cggctgtcta ttactgcacg 300 agatgcaggg gcggaactcg tacctattat tacatggacg tctggggcaa agggacaatg 360 gtcaccgtct cg 372 <210> 155 <211> 390 <212> DNA <213> Homo Sapiens <400> 155 gggcgcgcca aggtgcagct ggtgcagtct ggagcagagg tgaagaagcc cggggagtct 60 ctgaaaatct cctgtcaggc ttctggatac ggctttaccg tctactggat cggctgggtg 120 cgccagccgc ccgggaaagg cctggagtgg ctgggtatca tctatcctgg tgactctgat 180 accagataca atccgtcctt ccaaggccag gtcaccatct cagccgacaa gtccgtcagc 240 accacctacc tgcagtggag cagcctgaag gcctcggaca ccgccattta ctactgtgcg 300 agacatctgg actcatacga tgttttcact ggttataatt tggggggcta catggacgtc 360 tggggcaagg gaaccctggt caccgtctcg 390 <210> 156 <211> 378 <212> DNA <213> Homo Sapiens <400> 156 gggcgcgccc aggtgcagct ggtggagtct ggggctgaag tgaagaagcc tgggtcctca 60 gtgaaggtct cctgcaagtc ttctggaggc tatgttttca gctgggtgcg acaggcccct 120 gggcaaggac tagagtggat gggagggatc atctccaact ttcgcacggc agagtacgca 180 cggaagttcc agggtagagt caccatgacc gcggacacat ccacgaacac aatctacatg 240 gagctgacca gcctgacatc tgaagacacg gccgtatatt tctgtgtgag cgccccccga 300 gacacgtcga ctatagcagc tcgttttaat cgatacttct ttgacacctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 157 <211> 378 <212> DNA <213> Homo Sapiens <400> 157 gggcgcgccc aggtgcagct acagcagtcg gggggaggcg tggtccagcc tgggaggtcc 60 ctgagactct cctgtgctgc ctctggattc gccttcagag actatgccat gcactgggtc 120 cgccaggccc caggcaaggg gctggagtgg atgggagtta tctcatttaa tggagatcag 180 atattttacg cagactccat gaagggccgc ttcaccatct ccagagagaa ctccaagaac 240 acgctgcatc tgcgcatgaa cagcctgaga cctgaggaca cggctgtcta ttactgtgcg 300 agagcccgac ttcttttttg tagcggtggt aggtgcgaca tggactcttg gggccaggga 360 accctggtca ccgtctcg 378 <210> 158 <211> 378 <212> DNA <213> Homo Sapiens <400> 158 gggcgcgcca aagtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacctca 60 gtgagggtct cctgcaagac ttctggaggc tatgttttca gttgggtgcg acaggcccct 120 ggacacgggc ctgaatggat gggagggatc atcaccaact ttgggacagc aacctacgca 180 cagaagttcc agggcagagt ctcgattacc gcggacacat ccacgaacac attttacatg 240 gatttgaaca atctgaaatc tgacgacacg gccgtttatt actgtgcgag cgccccccga 300 gacacgtcga ctatagcggc tcggtttaat cggtacttct ttgacttctg gggcccgggc 360 accctggtca ccgtctcg 378 <210> 159 <211> 348 <212> DNA <213> Homo Sapiens <400> 159 gggcgcgcct aggtgcagct ggtgcagtct gggggaggct tggtaaaacc tggggggtcc 60 cttagactct cctgtgcagc ctctggattc actttcagta acgcctggat gagctgggtc 120 cgccaggctc cagggaaggg gctggagtgg gttggccatg ttaaaagcat gactgatggt 180 gggacaacag actacgctgc acccgtgaaa ggcagattca ccatctcaag agatgattca 240 gaaaacacgc tgtatctgca aatgaacagc ctgaagaccg acgacacagc cgtgtattac 300 tgtaccactc atgactactg gggccagggc accctggtca ccgtctcg 348 <210> 160 <211> 378 <212> DNA <213> Homo Sapiens <400> 160 gggcgcgcca aagtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacgtca 60 gtgagggtct cctgcaagac tcctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cggaagttcc agggcagaat cacggttacc gcggacaaat ccacgaacac agtgtacatg 240 gatttgagca acctggcatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cgatatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 161 <211> 393 <212> DNA <213> Homo Sapiens <400> 161 gggcgcgccc aggtgcagct ggtggagtct ggggctgaga tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgccaggc ctctggaggc accttcagca actatggcat caattgggtg 120 cgacacgccc ctggacaagg gcttgagtgg atgggaggaa tcgtccctat ctatggtcca 180 ccgaagtacg cacagaagtt ccagggcaga gtcacgatta ctgcggacac gtccacgagc 240 acagcctaca tggagctgag cagcctgagc tctgatgaca cggccgtgta ttactgtgcg 300 cgactttccc ggactgtgtt tggctcgggg acttatcgtc cgggctacta ctacatggac 360 gtctggggca tagggaccac ggtcaccgtc tcg 393 <210> 162 <211> 372 <212> DNA <213> Homo Sapiens <400> 162 gggcgcgcct aggtgcagct acagcagtct gggggaggcc tggtcaagcc gggggggtcc 60 ctgagactct cctgtgcagc ctctggattc tcctttagta ataataacat gaattgggtc 120 cgccagactc caggaaaggg actggagtgg gtcgcatcca ttagttttgg aagtcattac 180 atatcctacg cagactcagt gaagggccga ttcaccatct ccagagacaa cgccaggaat 240 gcagtttatc tgcagatgaa cagcctgaga gtcgaggaca cggctgtcta ttactgcacg 300 agatgcaggg gcggaactcg tacctattat tacatggacg tctggggcaa aggcaccctg 360 gtcaccgtct cg 372 <210> 163 <211> 378 <212> DNA <213> Homo Sapiens <400> 163 gggcgcgcca aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacctca 60 gtgagggtct cctgcaagac ttctggaggc tatgttttca gctgggtgcg acaggcccct 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cagaagttcc agggcagagt cacgattacc gcggacaaat ccacgaacac agtgtacatg 240 gatttgagca acctgacatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cggtatttct ttgactcctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 164 <211> 372 <212> DNA <213> Homo Sapiens <400> 164 gggcgcgccg aagtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaagac tgaaggaggc accttcagca cctatgttat cagttggatg 120 cgacaggccc ctggacaagg gcttgagtgg atgggaggga tcgtccccat ctttaacaca 180 ccaaactacg ctcagaaatt ccagggcaga gtcacaatta ccgcggacag atccacgagc 240 acagcctaca tggagctgag gagcctggga tctgaggaca cggccgtcta ttactgtgcg 300 agggtagtgg gaggtacaag gtcctactac gctttgggct tctggggcca ggggaccacg 360 gtcaccgtct cg 372 <210> 165 <211> 366 <212> DNA <213> Homo Sapiens <400> 165 gggcgcgccc aggtcacctt gaaggagtct ggtcctacgc tggtgaaacc cacacagacc 60 ctcacgctga cctgcacgtt ctctggtttc tcactcggca ctactggagt caatgtgggc 120 tggatccgtc agcccccagg aaaggccctg gagtggcttg cactcatttc ttggggtggt 180 ggtaagcact acagcccatc tctgaactcc aggatcaccc tcactaagga cgcctccaga 240 gagcaggtgg tggtccttac aatggccaac atggaccctg tggacacagg cagatattat 300 tgtgcacgta tagtggggac tcacggcttt gactactggg gccagggcac cctggtcacc 360 gtctcg 366 <210> 166 <211> 372 <212> DNA <213> Homo Sapiens <400> 166 gggcgcgccg aggtccagct ggtgcagtct gggggaggtg tggtgcggcc tggggggtcc 60 ctgagactct cgtgtgcagg ctctggattc acctttgatg aatacgctat gagctgggtc 120 cgccaagctc cagggaaggg gctggagtgg gtcgctttta ttaattggaa tggtgatagc 180 acatattatg cagactctgt gaagggccga ttcaccgtct ccagaaccaa cgccaagaac 240 tccctgtatc tgcaaatgaa cagtctgaga gccgaggaca cggccttcta ttactgtgcg 300 agagaccccc gcactaaact ggggatgtcc tattttgact attggggcca gggcaccctg 360 gtcaccgtct cg 372 <210> 167 <211> 390 <212> DNA <213> Homo Sapiens <400> 167 gggcgcgccg aagtgcagct ggtgcagtct ggagcagagg tgaagaagcc cggggagtct 60 ctgaaaatct cctgtcaggc ttctggatac ggctttaccg tctactggat cggctgggtg 120 cgccagctgc ccgggaaagg cctggagtgg ctgggtatca tctatcctgg tgactctgat 180 accagataca atccgtcctt ccaaggccag gtcaccatct cagccgacaa gtccgtcagc 240 accacctacc tgcagtggag cagcctgaag gcctcggaca ccgccattta ctactgtgcg 300 agacatctgg actcatacga tgttttcact ggttataatt tggggggcta catggacgtc 360 tggggcaagg ggacaatggt caccgtctcg 390 <210> 168 <211> 369 <212> DNA <213> Homo Sapiens <400> 168 gggcgcgccg aggtgcagct ggtgcagtct ggacctgaag tgaagaagcc tggggcctca 60 gtgagggtct cctgcaaggc ctctggttac tcccttaaga actatggtat ccactgggtg 120 cgacaggccc ctggacaggg gcttgagtgg atggggtgga tcagcgctga caatggtgac 180 acaaccactg cactgaacct ccggggcaga gtctccatga ccacagacac atccacaaac 240 acagtctaca tggaggtgaa gagcctaaga tctgacgaca cggccatata tttctgtgcg 300 cgagattttt atagtgggag ttaccggtcc tttgactact ggggccaggg caccctggtc 360 accgtctcg 369 <210> 169 <211> 378 <212> DNA <213> Homo Sapiens <400> 169 gggcgcgccg aagtgcagct ggtgcagtcg ggcccaggac tggtgaagcc ttcggagacc 60 ctgtccctca catgcgctgt ctctggtgcc tccgtcagcg gtggtgatta ctactggagc 120 tggatccggc agcccccagg gaaggcactg gagtggattg ggtatatcta ttacataggg 180 agcaccaact acaatccctc tctcaagagt cgactctccc tatcagtaga cacggccaag 240 agccagttct ccctgaagtt gagctctgtg accgctgcgg acacggccat ttatttctgt 300 gcgagagcac gtcggacgta tagtggctac gactccgcct ttgactactg gggccaggga 360 accctggtca ccgtctcg 378 <210> 170 <211> 372 <212> DNA <213> Homo Sapiens <400> 170 gggcgcgccc aggtgcagct gcagcagtcg ggcccaggac tggtgaagcc ctcggagacc 60 ctgcccctca cctgcactgt ctctggtggc tccttcagta cctactactg gagctggatc 120 cggcagtccc cagagaaggg actggagtgg attggatata tccaaaacag tgtgaacacc 180 aactacaacc cctccctcaa gagtcgtgtc atcatttcag tggacacgtc caacaaccag 240 ttctccctga agctgaggtc tgtgaccgct gcggacacgg ccgtatatta ctgtgcgaga 300 gtaagcggct ggggcccaag gggaggcatc tactttgact actggggcca gggaaccctg 360 gtcaccgtct cg 372 <210> 171 <211> 378 <212> DNA <213> Homo Sapiens <400> 171 gggcgcgcca aggtgcagct ggtgcagtct ggggctgaag tgaagaagcc tgggtcctca 60 gtgaaggtct cctgcaagtc ttctggaggc tatgttttca gctgggtgcg acaggcccct 120 gggcaaggac tagagtggat gggagggatc atctccaact ttcacacggc agagtacgca 180 cagaagttcc agggtagagt caccatgacc gcggacacat ccacgaacac aatctacatg 240 gagctgacca gcctgacatc tgaagacacg gccgtatatt tctgcgtgag cgccccccga 300 gacacgtcga ctatagcagc tcgttttaat cgatacttct ttgacacctg gggccagggc 360 accctggtca ccgtctcg 378 <210> 172 <211> 378 <212> DNA <213> Homo Sapiens <400> 172 gggcgcgccg aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggacgtca 60 gtgagggtct cccgcaagac tcctggaggc tatgttttca gctgggtgcg acaggcccca 120 ggacaagggc ctgagtggat gggagggatc atcaccaact ttgggacaac aaactacgca 180 cggaagttcc agggcagaat cacggttacc gcggacaaat ccacgaacac agtgtacatg 240 gatttgagca acctggcatc tgaggacacg gccgtgtatt actgtgcgag agccccccga 300 ggcacgtcga ctatagcagc tcgttttaat cgatatttct ttgactcctg gagccagggc 360 accctggtca ccgtctcg 378 <210> 173 <211> 372 <212> DNA <213> Homo Sapiens <400> 173 gggcgcgccg aggtgcagct ggtggagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaaggc ctctggaggc agcttcagca cctatatttt cacctgggtg 120 cgccaggccc ctggacaggg gcttgagtgg atgggaggga tcaatcctat ctttgctaca 180 agagactacc caaagaagtt ccagggcaga gtcacgatca ccgcggacga atccacgagg 240 actgtctata tggagttgag cagcctcaca tctgaggaca cggccgtcta ttactgtgca 300 agagtgttgg gaggtacaag gctctactac gctctgaacg tctggggcca agggaccacg 360 gtcaccgtct cg 372 <210> 174 <211> 372 <212> DNA <213> Homo Sapiens <400> 174 gggcgcgccg aagtgcagct ggtgcagtct gggggaggtg tggtgcggcc tggggggtcc 60 ctgagactct cgtgtgcagg ctctggattc acctttgatg aatacgccat gagctgggtc 120 cgccaagctc cagggaaggg gctgaagtgg gtcgctttta ttaattggaa tggtgatagc 180 acatattatg cagactctgt gaagggccga ttcaccgtct ccagatccaa cgccaagaac 240 tccctgtatc tgcaaatgaa cagtctgaga gccgaggaca cggccttcta ttgctgtgcg 300 agagaccccc gcactaaact ggggatgtcc tattttgact attggggcca gggcaccctg 360 gtcaccgtct cg 372 <175> 175 <211> 372 <212> DNA <213> Homo Sapiens <400> 175 gggcgcgccg aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaaggc ctctggaggc agcttcagca cctatgctat cacctgggtg 120 cgccaggccc ctggacaggg gcttgaatgg atgggaggga tcatccctat ctttgcttca 180 agagactacg cacagaagtt tcagggcaga gtcacaatca ccgcggacga atccacgagg 240 acagtgtaca tggagccgag gagcctgaga tctgaagaca cggccgtgta ttactgtgca 300 agagtgctgg gaggtacaag gctctactac gctctgaacg tctggggcca aggcaccctg 360 gtcaccgtct cg 372 <210> 176 <211> 378 <212> DNA <213> Homo Sapiens <400> 176 gggcgcgccg aggtgcagct ggtgcagtcg ggcccaggac tggtgaagcc ttcggagacc 60 ctgtccctca catgcgctgt ctctggtgcc tccgtcagcg gtggtgatta ctactggagc 120 tggatccggc agcccccagg gaaggcactg gagtggattg ggtatatcta ttacataggg 180 agcaccaact acaatccctc tctcaagagt cgactctccc tatcagtaga cacggccaag 240 agccagttct ccctgaagtt gagctctgtg accgctgcgg acacggccac ttatttctgt 300 gcgagagcac gtcggacgta tagtggctac gactccgcct ttgactactg gggccaggga 360 accctggtca ccgtctcg 378 <210> 177 <211> 372 <212> DNA <213> Homo Sapiens <400> 177 gggcgcgccg aggtgcagct ggtggagtct ggggctgagg tgaagaagcc tgggtcctcg 60 gtgaaggtct cctgcaaggc ctctggaggc agcttcagca cctatgctat cacctgggtg 120 cgccaggccc ctggacaggg gcttgaatgg atgggaggga tcatccctat ctttgcttca 180 agagactacg cacagaagtt tcagggcaga gtcacaatca ccgcggacga atccacgagg 240 acagtgtaca tggagctgag cagcctgaga tctgacgaca cggccgtgta ttactgtgca 300 agagtgctgg gaggtacaag gctctactac gctctgaacg tctggggcca agggaccacg 360 gtcaccgtct cg 372 <210> 178 <211> 369 <212> DNA <213> Homo Sapiens <400> 178 gggcgcgccc aggtcacctt gaaggagtct ggtcctacag tggtgaaacc cacacagacc 60 ctcacgctga cctgtagcct ctctgggttc gcactcggca ctaccggagt ggctgtgggc 120 tggatccgtc agcccccagg aaaggccctg gaatggcttg gactcatcga ttggaatgat 180 gataggcgct acagaccctc tctgaagacc agactcacca tcacccagga catgtccagg 240 aaccaggtgg tccttagact gaccaacttg gacccactgg acacaggcac atatttttgt 300 gcacgttcag tagtgccggc gactagggcc tttgacttct ggggccaggg caccctggtc 360 accgtctcg 369 <210> 179 <211> 366 <212> DNA <213> Homo Sapiens <400> 179 gggcgcgccc aggtcacctt gaaggagtct ggtcctacgc tggtgaaacc cacacagacc 60 ctcacgctga cctgcacctt gtctgggttc tcactctaca ctactggagt gggtgtgggc 120 tggatccgtc agcccccagg aaaggccctg gagtgactgg cacgcattta ttgggatgat 180 gatgagcgct acaacccgtc tctgaagagc aggctcacca tcaccaagga cgcctccaaa 240 aaccaggtgg tccttaaaat gaccaacatg gaccctgtgg acacagccac atattactgt 300 gcccggacca tgggcgtcgt tcttccattt gactactggg gccagggaac cctggtcacc 360 gtctcg 366 <210> 180 <211> 327 <212> DNA <213> Homo Sapiens <400> 180 ctagccgaaa ttgtgctgac tcagtctcca tcctccctgt ctgcatctct aggagacaga 60 gtcaccatca cttgccgggc aagtcagcac attagcaatt atttaaattg gtatcagcag 120 aaacctggga aagcccctaa actcctgatc tgtgctgcat ccagtttgca aagtggggtc 180 ccatcaaggt tcactggcag tggatctggg gcagattaca ccctcaccat cagcagtctg 240 caacctgaag attttgcaac ttactactgt caacagagtt acagtacctc gtggacgttc 300 ggccaaggga ccacggtgga aatcaaa 327 <210> 181 <211> 327 <212> DNA <213> Homo Sapiens <400> 181 ctagccgaaa ttgtgttgac acagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt atttagcctg gtatcagcag 120 aaaccaggga aagttcctaa gctcctgatc tatgctgcat ccactttgca atcaggggtc 180 ccatctcggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagcctg 240 cagcctgaag atgttgcaac ttattactgt caaaagtata acagtgcccc gtacactttt 300 ggccagggga ccaagctgga gatcaaa 327 <210> 182 <211> 327 <212> DNA <213> Homo Sapiens <400> 182 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctct aggagacaga 60 gtcagcatca cttgccgggc aagtcaacat attagcaatt atataaattg gtatcagcag 120 aaacctggga aagcccctaa actcctgatc tatgctgctt ccactttgca aagtggggtc 180 ccatcaaggt tcactggcag tggatctggg gcagattaca ctctcaccat caccagtctg 240 caacctgaag attttgcaac ttactactgt caacagagtt acggtacctc gtggacgttc 300 ggccaaggga ccacggtgga aatcaaa 327 <210> 183 <211> 327 <212> DNA <213> Homo Sapiens <400> 183 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctgt gggagacaga 60 gtcaccatca cttgccaggc gagtcaagac attaccaact atttaaattg gtatcagcag 120 aaaccaggga aagcccctaa gctcctgatc tacgatgcat ccaatttgca accaggggtc 180 ccatcaaggt tcagtggaag tggatctgtg acagatttta ctttcaccat cagcagcctg 240 cggcctgaag atattgcaac atattactgt caacagtatg atggtccagt gcttactttc 300 ggcggaggga ccaaggtaga gatcaaa 327 <210> 184 <211> 327 <212> DNA <213> Homo Sapiens <400> 184 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcaccatct cttgccgggc aagtcagccc attagcacct atttaaattg gtatcagcag 120 aaaccaggga aagcccctaa gatcctgatc tttggtgcat ctcgtttgca aagtggtgtc 180 ccatcaaggt tcagcggcag tggatctggg acagatttca gtctcaccat caccagtctc 240 caacctgaag attttgcaac ttacatctgt caacaaagca agagtccccc gtacaatttt 300 ggccggggga ccaagctgga gatcaaa 327 <210> 185 <211> 330 <212> DNA <213> Homo Sapiens <400> 185 ctagccgaaa ttgtgttgac acagtctcct tccaccctgt ctgcatctgt aggcgacaga 60 gtcaccatca cttgccgggc cagtcagagt attggtagct ggttggcctg gtatcaacag 120 aaaccaggga aagcccctaa actcctgatc tataaggcgt ctactttaca aagtgagacc 180 ccttcaaggt tccgcggcag tggatctggg accgaattca ctctcaccat cagcagcctg 240 cagcctgatg attttgcaac ttactactgc caacagttta atagtttttc tccgtggacg 300 ttcggccaag ggaccaaggt ggaattcaaa 330 <210> 186 <211> 327 <212> DNA <213> Homo Sapiens <400> 186 ctagccgatg ttgtgatgac tcagtctcca tcgtccctgt ctgcatctgt tggtgacaga 60 gtcaccatca cttgccgggc aagtcagagc attaacatta atttaaattg gtttcagcag 120 aaacccggga aagcccctaa cctactgatc tattctgcat ccactttgca aactggggtc 180 ccctcaaggt tcagtggaag tggatctggg acagagttca ctctcaccat cagcagtcta 240 caacctgaag attttgcaac ttactactgt caacagattt acagtcatgt taggacgttc 300 ggccaaggga ccaaggtgga gatcaaa 327 <210> 187 <211> 345 <212> DNA <213> Homo Sapiens <400> 187 ctagccgatg ttgtgatgac tcagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt cttttgtaca gctccaacaa taagaattac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgttcgg ccaagggacc aaggtggaaa tcaaa 345 <210> 188 <211> 327 <212> DNA <213> Homo Sapiens <400> 188 ctagccgaaa ttgtgttgac acagtctcca tcctccctgt ctgcatctgt gggagacaga 60 atcaccatca cttgccaggc gagtcaagac attaccaact atttaaattg gtatcagcag 120 aaaccaggga aagcccctaa gctcctgatc tacgatgcat ccaatttgca accaggggtc 180 ccatcaaggt tcagtggaag tagatctggg acagatttta ctttcaccat cagcagcctg 240 cggcctgaag atattgcaac atattactgt caacagtatg atggtccagt gcttactttc 300 ggcggaggga ccaaggtaga gatcaaa 327 <210> 189 <211> 345 <212> DNA <213> Homo Sapiens <400> 189 ctagccgaaa ttgtgttgac acagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt cttttataca gctccaacaa taagaactac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcacc 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgttcgg ccaagggacc aaggtggaaa tcaaa 345 <210> 190 <211> 330 <212> DNA <213> Homo Sapiens <400> 190 ctagccgaaa ttgtgttgac acagtctcca tccttcctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc cagtcagggc attgggaatt ttttagcctg gtatcagcaa 120 aaaccaggga aagcccctaa gctcctaatc tctggtgcat ccactttgca aagttgggtc 180 ccatcaaggt tcagcggccg tggatctggg accgaattca ccctcacaat cagcagcctg 240 cagcctgaag attttgcaac ttattactgt caacagctta atcgttaccc tccatacact 300 tttggccagg ggaccaagct ggagatcaaa 330 <210> 191 <211> 327 <212> DNA <213> Homo Sapiens <400> 191 ctagccgaaa ttgtgatgac gcagtctcca tcctccctgt ctgcatctgt gggagacaga 60 gtcaccatca cttgccaggc gagtcaggac attagcaaat atttaaattg gtatcagcag 120 aaaccaggga gagcccctaa actcctgatc tacgaagcat ccaatttgga gacaggggtc 180 ccaccaaggt tcagtggaag tggatctggg acacatttta ctttcaccat caccggcctg 240 cagcctgaag atattgcaac atattactgt caacagtgtg atagtctgcc tccggtcttt 300 ggccagggga ccaagctgga agtcaaa 327 <210> 192 <211> 330 <212> DNA <213> Homo Sapiens <400> 192 ctagccgaaa ttgtgctgac tcagtctcca ggcaccctgg ctttgtctcc aggtgataga 60 gccaccctct cctgcggggc cagtcagagc gtgttcggcg acttcttagc ctggtaccaa 120 cacaagcctg gccaggctcc caggctcctc atctatggtg cttccaccag ggccactggc 180 atcccagaca ggttcagtgg cagtggagct gggacagact tcactctcac catcagcaga 240 ctggagcctg aggattttgc agtttatttc tgtcagtagt atggtgactc agtgttcacg 300 ttcggccaag ggaccaagtt gggaatcaaa 330 <210> 193 <211> 330 <212> DNA <213> Homo Sapiens <400> 193 ctagccgaaa ttgtgatgac acagtctcca ggcaccctgg ctttgtctcc aggtgataga 60 gccaccctct cctgcggggc cagtcagagc gtgttcggcg acttcttagc ctggtaccaa 120 cacaagcctg gccaggctcc caggctcctc atctatggtg cttccaccag ggccactggc 180 atcccagaca ggttcagtgg cagtggagct gggacagact tcactctcac catcagcaga 240 ctggagcctg aggattttgc agtttatttc tgtcagcagt atggtgactc agtgttcacg 300 ttcggccaag ggaccaagtt ggaaatcaaa 330 <210> 194 <211> 330 <212> DNA <213> Homo Sapiens <400> 194 ctagccgaaa ttgtgttgac acagtctcca ggcaccctgt ctttgtctcc agggcaaaga 60 gccaccctct cctgcagggc cagtcagagt gttaacagag actacctagc ctggtaccag 120 cagaaacctg gctaggctcc caggctcctc atctatggtg catccagcag ggccactggc 180 atcccagaca ggttcagtgg cagtgggtct ggaacagact tcactctcac catcagcaga 240 ctggagcgag acgattttgc agtgtatttc tgtcaccagt atggtagctc acctaacact 300 tttggccagg ggaccaagct ggagatcaaa 330 <210> 195 <211> 330 <212> DNA <213> Homo Sapiens <400> 195 ctagccgaaa cgacactcac gcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcagagc gttagcacca actacttagc ctggtaccga 120 cagaaacctg gccaggctcc caggctcctc atccatggtg catccagccg ggccactggc 180 atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtatttc tgtcagcagt atggtagctc acctcagacg 300 ttcggccaag ggaccaggtt ggaaatcaaa 330 <210> 196 <211> 327 <212> DNA <213> Homo Sapiens <400> 196 ctagccgaaa ttgtgctgac tcagtctcca tcctccctgt ctgcatctgt tggtgacaga 60 gtcaccacca cttgccggac aagtcagagc attttcatta atttaaattg gtttcagcag 120 aaacccggga aagcccctaa actcctgatc tattctgcat ccactttgca aactggggtc 180 ccctcaaggt tcagtggcag tggatctggg acagagttca ctctcaccat cagcagtcta 240 caacctgaag attttgcaac ttactactgt caacagattt acagtcaggt taggacgttc 300 ggccaaggga ccaaggtgga gatcaaa 327 <210> 197 <211> 330 <212> DNA <213> Homo Sapiens <400> 197 ctagccgaca tccagatgac ccagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcagagt gttaacagcg acaacttagc ctggtaccag 120 cagaaacctg gccaggctcc caggctcctc atgtctggtg caaccagtag ggccactgac 180 gtcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtattac tgtcaccagt atggtagctc agataacact 300 tttggccagg ggaccaagct ggagatcaaa 330 <210> 198 <211> 330 <212> DNA <213> Homo Sapiens <400> 198 ctagccgatg ttgtgatgac tcagtttcct tcctctctgt ctgcatctgt gggagacaga 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt ttttagcctg gtatcagcag 120 aaaccaggga aagttcctga gctccttatc tatggtgcat ccactttgca atcaggggtc 180 ccatctcggt tcagtggcag tggatctggg gcagagttca cactcaccat caacagcctg 240 cagcctgaag atgttgcgac ttattactgt caaaagtatg acagtggcct gagattcact 300 ttcggccctg ggaccaaagt ggatatcaaa 330 <210> 199 <211> 330 <212> DNA <213> Homo Sapiens <400> 199 ctagccgatg ttgtgatgac tcagtctcct tcctctctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt ttttagcctg gcatcagcag 120 aaaccagggc aagttcctaa actcctgatc tatggtgcat ccactttgca atcaggggtc 180 ccatctcgct tcagtggcag tggatctggc acagatttca ctctcaccat caacggcctg 240 cagcctgaag atgttgcaac ttattactgt caaaagtatg acagtggcct gatattcact 300 ttcggccctg ggaccagagt ggagatcaaa 330 <210> 200 <211> 345 <212> DNA <213> Homo Sapiens <400> 200 ctagccgatg ttgtgatgac tcagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt cttttataca gctccaacaa taagaactac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgctcgg ccaagggacc aaggtggaaa tcaaa 345 <210> 201 <211> 330 <212> DNA <213> Homo Sapiens <400> 201 ctagccgatg ttgtgatgac ttagtctcca ggcaccctgg ctttgtctcc aggtgataga 60 gccaccctct cctgcggggc cagtcagagc gtgttcggcg acttcttagc ctggtaccaa 120 cacaagcctg gccaggctcc caggctcctc atctatgttg cttccaccag ggccactggc 180 atcccagaca ggttcagtgg cagtggagct gggacagact tcactctcac catcagcaga 240 ctggagcctg aggattttgc agtttatttc tgtcagcagt atggtgactc agtgttcacg 300 ttcggccaag ggaccaagtt ggaaatcaga 330 <210> 202 <211> 330 <212> DNA <213> Homo Sapiens <400> 202 ctagccgaaa cgacactcac gcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcagagc gttagcacca actacttagc ctggtaccga 120 cagaaacctg gccaggctcc caggctcctc atccatggtg catccagccg ggccactggc 180 atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtatttc tgtcagcagt atggtagctc acctcagacg 300 ttcggccaag ggaccaggtt ggaaatcaaa 330 <210> 203 <211> 330 <212> DNA <213> Homo Sapiens <400> 203 ctagccgaaa ttgtgctgac tcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcggagt gtaaacagca acaacttagc ctggtatcag 120 cagaaacctg accaggctcc caggctcctc atgtatggtg catccagtag ggccactggc 180 atcccagaca ggttcactgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtattac tgtcatcagt atggtgcctc agataacact 300 tttggccagg ggaccaagct ggagatcaag 330 <210> 204 <211> 327 <212> DNA <213> Homo Sapiens <400> 204 ctagccgaaa ttgtgatgac acagtctcca tccttcctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc cagtcaggac attatcactt atttagcctg gtatcaacaa 120 aaaccaggga aagcccctga ggtcctgatc tttggtgcgt ccactttgca aagtggggtc 180 ccatcaagat tcagcggcag tggatctggg accgaattca ctctcactat ctccagcctg 240 cagcctgaag attttgcaac ttattactgt caacagatta ttcgttaccc tcgcactttc 300 ggccaaggga ccagggtgga aatcaaa 327 <210> 205 <211> 330 <212> DNA <213> Homo Sapiens <400> 205 ctagccgatg ttgtgatgac tcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgccgggc cagtcagagt gttaacagca acaacttagc ctggtaccag 120 cagaaacctg gccaggctcc caggctcctc gtctctggtg caaccaatag ggccactgac 180 atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtattac tgtcaccagt atggtagctc agagaacact 300 tttggccagg ggaccaagct ggagatcaga 330 <206> 206 <211> 327 <212> DNA <213> Homo Sapiens <400> 206 ctagccgaaa ttgtgatgac gcagtctcca cccttcctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc cagtcagggc ttaagcactt atttagcctg gtatcaggta 120 aaaccaggga aagcccctaa gctcctgatc tatgctgcat ccactttgca aagtggggtc 180 ccatcaaggt tcagcggcag tggatctggg acagaattca ctctcacaat caacagcctg 240 cagcctgaag attttgcaac ttattactgt caacaacttg atacttaccc tctcactctc 300 ggcggaggga ccaaggtgga gatcaaa 327 <210> 207 <211> 345 <212> DNA <213> Homo Sapiens <400> 207 ctagccgaaa ttgtgttgac acagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt cttttataca gctccaacaa taagaactac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgttcgg ccaagggacc aaggtggaaa tcaaa 345 <210> 208 <211> 345 <212> DNA <213> Homo Sapiens <400> 208 ctagccgaca tccagatgac ccagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt cttttataca gctccaacaa taagaactac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgttcgg ccaagggacc agggtggaaa tcaaa 345 <210> 209 <211> 330 <212> DNA <213> Homo Sapiens <400> 209 ctagccgaaa ttgtgatgac acagtctcca ggcaccctgg ctttgtctcc aggtgataga 60 gccaccctct cctgcggggc cagtcagagc gtgttcggcg acttcttagc ctggtaccaa 120 cacaagcctg gccaggctcc caggcccctc atctatggtg cttccaccag ggccactggc 180 atcccagaca ggttcagtgg cagtggagct gggacagact tcactctcac catcagcaga 240 ctggagcctg aggattttgc agtttatttc tgtcagcagt atggtgactc agtgttcacg 300 ttcggccaag ggaccaagtt ggaaatcaaa 330 <210> 210 <211> 330 <212> DNA <213> Homo Sapiens <400> 210 ctagccgatg ttgtgatgac tcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcagagt gttaacagcg acaacttagc ctggtaccag 120 cagaaacctg gccaggctcc caggctcctc atgtctggtg caaccagtag ggccactgac 180 atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtattac tgtcaccagt atggtagctc agataacact 300 tttggccagg ggaccaagct ggagatcaaa 330 <210> 211 <211> 330 <212> DNA <213> Homo Sapiens <400> 211 ctagccgaca tccagttgac ccagtctcca tcctccctgt ctgcatctgt tggagacagc 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt ttttagcctg gtatcagaag 120 aaaccgggaa aagttcctag gctcctgatc tatggtgcat ccactttgca atcaggggtc 180 ccatctcggt tcagtggcag tggatctggg accgatttca ctctcaccat cagcagcctg 240 cagcctgaag atgttgcgac ttattactgt caggagtata gcaatgccct gatattcact 300 ttcggccctg ggaccaaagt tcatatcaaa 330 <210> 212 <211> 330 <212> DNA <213> Homo Sapiens <400> 212 ctagccgaaa ttgtgatgac acagtctcca tccttcctgt ctgcgtctgt aggagacaga 60 gtcaccatca cttgccgggc cagtcagggc attgggaatt ttttagcctg gtatcagcaa 120 aaaccaggga aagcccctaa gctcctgatc tctggtgcat ccactttgca aagttgggtc 180 ccatcaaggt tcagcggccg tggatctggg accgaattca ctctcacaat cagcagcctg 240 cagcctgaag attttgcaac ttattactgt caacagctta atcgttaccc tccatacact 300 tttggccagg ggaccaagct ggagatcaaa 330 <210> 213 <211> 327 <212> DNA <213> Homo Sapiens <400> 213 ctagccgaaa ttgtgatgac acagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc aagtcagacc attagcaacc atttaaattg gtatcagcag 120 aaaccaggga gagcccctaa gctcctgatc tatgttgggt ccagtctgca aagtggggtc 180 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagtggtctg 240 caacctgaag attttgcaac ttactactgt caacagagtt acagtccctc gtacactttt 300 ggccagggga ccaaggtgga gatcaaa 327 <210> 214 <211> 330 <212> DNA <213> Homo Sapiens <400> 214 ctagccgaca tccagttgac ccagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcagagc gttagcacca actacttagc ctggtaccga 120 cagaaacctg gccaggctcc caggctcctc atccatggtg catccagccg ggccactggc 180 atcccagaca ggttcagtgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtatttc tgtcagcagt atggtagctc acctcagacg 300 ttcggccaag ggaccaggtt ggaaatcaaa 330 <210> 215 <211> 327 <212> DNA <213> Homo Sapiens <400> 215 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctct aggagacaga 60 gtcaccatca cttgccgggc aagtcagcat attagcaatt atttaaattg gtatcagcag 120 aaacctggga aagcccctaa actcctgatc tatgctgcat ccagtttgca aagtggggtc 180 ccatcaaggt tcactggcag tggatctggg gcagattaca ctctcaccat cagcagtctg 240 caacctgaag attttgcaac ttactactgt caacagagtt acagcacctc gtggacgttc 300 ggccaaggga ccacggtggg aatcaaa 327 <210> 216 <211> 327 <212> DNA <213> Homo Sapiens <400> 216 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcagcatca cttgccgggc aagtcggagc gttaagacct atttaaattg gtatcagcag 120 aaaccaggga aagcccctaa gctcctggtc tatggttcgt ccagtttgga aagtggggtc 180 ccatcaagat tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 240 caacctgagg attttgcaac ttactactgt caacagagtt tcggtacccc ctacactttt 300 ggccagggga ccaagctgga caccaaa 327 <210> 217 <211> 327 <212> DNA <213> Homo Sapiens <400> 217 ctagccgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aagtgacaga 60 gtcaccatta cttgccgggc aagtcagagc attaacaagt tcttaaactg gtatcagcag 120 aaaccaggga aagcccctca gctcctcatc tatgctgcaa ccaatttgca gagtggggtc 180 ccatcaaggt tcagtggcag tggatctggg acagacttca ctctcaccat cagcagtctg 240 caaactgaag attttgccac ttactactgt caacagagtt acgatatgcc tcggacgttc 300 ggccaaggga ccaaggtgga gatcaaa 327 <210> 218 <211> 330 <212> DNA <213> Homo Sapiens <400> 218 ctagccgaaa ttgtgctgac tcagtctcca ggcaccctgt ctttgtctcc aggggaaaga 60 gccaccctct cctgcagggc cagtcggagt gtatacagca acaacttagc ctggtatcag 120 cagaaacctg accaggctcc caggctcctc atgtatggtg catccagtag ggccactggc 180 atcccagaca ggttcactgg cagtgggtct gggacagact tcactctcac catcagcaga 240 ctggagcctg aagattttgc agtgtattac tgtcatcagt atggtgcctc agataacact 300 tttggccagg ggaccaagct ggagatcaag 330 <210> 219 <211> 345 <212> DNA <213> Homo Sapiens <400> 219 ctagccgaca tccagatgac ccagtctcca gactccctgg ctgtgtctct gggcgagaag 60 gccaccatca actgcaagtc cagccagagt cttttataca gctccaacaa taagaactac 120 ttagcttggt accagcagaa accaggacag cctcctaagt tgctcattta ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agtactcctc cgatgttcgg ccaagggacc aaggtggaaa tcaaa 345 <210> 220 <211> 330 <212> DNA <213> Homo Sapiens <400> 220 ctagccgaaa ttgtgatgac gcagtctcct tcctctctgt ctgcatctgt aggagacaga 60 gtcaccatct cttgccgggc gagtcagggc attagcaatt ttttagcctg gtttcagcag 120 aaaccaggtc aagttcctaa gctcctgatc tatgttgcat ccactttgca atcaggggtc 180 ccatctcggt tcagtggcag tggatctggg acagatttca gtctcaccat caacggcctg 240 cagcctgagg atgttgcaac ttattactgt caaaggtatg acagtggcct gatattcact 300 ttcggccctg ggaccaaagt ggagatcaaa 330 <210> 221 <211> 327 <212> DNA <213> Homo Sapiens <400> 221 ctagccgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc aagtcagacc attagcaacc atttaaattg gtatcagcag 120 aaaccaggga gagcccctaa gctcctgatc tatgttgggt ccagtctgcg aagtggggtc 180 ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagtggtctg 240 caacctgaag attttgcaac ttactactgt caacagagtt acagtccctc gtacactttt 300 ggccagggga ccaaggtgga gatcaaa 327 <210> 222 <211> 330 <212> DNA <213> Homo Sapiens <400> 222 ctagccgaaa ttgtgttgac acagtctcct tcctctctgt ctgcatctgt gggagacaga 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt ttttagcctg gtatcagcag 120 aaaccaggga aagttcctga gctccttatc tatggcgcat ccactttgca atcgggggtc 180 ccatctcggt tcagtggcag tggatctggg acagatttca ctctcaccat caacagcctg 240 cagcctgaag atgttgcaac ttattactgt caaaagtatg acagtggcct gagattcact 300 ttcggccctg gggccaaagt ggatatcaaa 330 <210> 223 <211> 327 <212> DNA <213> Homo Sapiens <400> 223 ctagccgatg ttgtgatgac tcagtctcca tcctccctgt ctgcatctgt aggagacaga 60 gtcagcatca cttgccgggc aagtcggagc gttaagacct atttaaattg gtatcagcag 120 aaaccaggga aagcccctaa gctcctggtc tatggttcgt ccagtttgga aagtggggtc 180 ccatcaagat tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 240 caacctgagg attttgcaac ttactgctgt caacagagtt tcggtacccc ctacactttt 300 ggccagggga ccaagctgga catcaaa 327 <210> 224 <211> 330 <212> DNA <213> Homo Sapiens <400> 224 ctagccgaca tccagttgac ccagtctcct tcctctctgt ctgcatctgt gggagacaga 60 gtcaccatca cttgccgggc gagtcagggc attagcaatt ttttagcctg gtatcagcag 120 aaaccaggga aagttcctga gctccttatc tatggtgcat ccaccttgca atcaggggtc 180 ccatctcggt tcagtggcag tggatctggg acagagttca ctctcaccat caacagcctg 240 cagcctgaag atgttgcgac ttattactgt caaaagtatg atagtggcct gagattcact 300 ttcggccctg ggaccaaagt ggatatcaaa 330 <210> 225 <211> 327 <212> DNA <213> Homo Sapiens <400> 225 ctagccgaca tccagatgac ccagtcttct tccaccctgt ctgcatctgt aggagacaga 60 gtcaccatca cttgccgggc cagtcagaat attaatatct ggttggcctg gtatcagcag 120 aaagcaggga aagcccccaa actcctgatc tataaggcgt ctactttaga aaggggggtc 180 ccctcaaggt tcagcggcag tggatctggg acagagttca ctctcaccat caccggcctg 240 cgccctgacg atttcggaag ttattattgc caacactatg atggtaattc actaacgttc 300 ggccaaggga ccagggtgga tatccaa 327 <210> 226 <211> 330 <212> DNA <213> Homo Sapiens <400> 226 ctagccgaca tccagttgac ccagtctcct tccaccctgt ctgcatctgt aggcgacaga 60 gtcaccatca cttgccgggc cagtcagagt attggtagct ggttggcctg gtatcaacag 120 aaaccaggga aagcccctaa actcctgatc tataaggcgt ctactttaca aagtgagacc 180 ccttcaaggt tccgcggcag tggatctggg accgaattca ctctcaccat cagcagcctg 240 cagcctgatg attttgcaac ttactactgc caacagttta atagtttttc tccgtggacg 300 ttcggccaag ggaccaaggc ggaattcaaa 330 <210> 227 <211> 123 <212> PRT <213> homo sapiens <400> 227 Gly Arg Ala Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys 1 5 10 15 Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe             20 25 30 Lys Asn Tyr Gly Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Trp Ile Ser Ala Asp Asn Gly Asp Thr Thr Thr Ala     50 55 60 Leu Asn Leu Arg Gly Arg Val Ser Met Thr Thr Asp Thr Ser Thr Asn 65 70 75 80 Thr Val Tyr Met Glu Val Lys Ser Leu Arg Ser Asp Asp Thr Ala Ile                 85 90 95 Tyr Phe Cys Ala Arg Asp Phe Tyr Ser Gly Ser Tyr Arg Ser Phe Asp             100 105 110 Cys Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 228 <211> 126 <212> PRT <213> homo sapiens <400> 228 Gly Arg Ala Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe             20 25 30 Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Arg Pro Gly Trp Ala Ala Thr Arg Ala Ala Gly             100 105 110 Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 125 <210> 229 <211> 119 <212> PRT <213> homo sapiens <400> 229 Gly Arg Ala Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 1 5 10 15 Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Glu Lys Cys Asp             20 25 30 Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly         35 40 45 Trp Ile Ser Ala Asp Asp Gly Gly Thr Thr Thr Ala Leu Asn Leu Arg     50 55 60 Gly Arg Val Ser Met Thr Thr Asp Arg Ala Thr Asn Thr Val Tyr Met 65 70 75 80 Glu Leu Lys Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Phe Cys Ala                 85 90 95 Arg Asp Phe Tyr Ser Gly Thr Tyr Arg Ser Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser         115 <210> 230 <211> 132 <212> PRT <213> homo sapiens <400> 230 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 1 5 10 15 Pro Gly Thr Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe             20 25 30 Arg Thr Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Ala Phe Leu Ser Ser Asp Gly Ser Asp Glu Phe Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Ser 65 70 75 80 Thr Leu Phe Leu Lys Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Gly Ala Gln Ile Thr Leu Phe Gly Ala             100 105 110 Pro Leu Ile Arg Pro Ser Ser Phe Asp Ser Trp Gly Gln Gly Thr Leu         115 120 125 Val Thr Val Ser     130 <210> 231 <211> 121 <212> PRT <213> homo sapiens <400> 231 Gly Arg Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Val Lys Lys 1 5 10 15 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe             20 25 30 Asn Ser Tyr Gly Ile Ala Trp Val Arg Gln Val Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Trp Ile Ser Pro Tyr Ser Gly His Thr Asn Tyr Ala     50 55 60 Glu Lys Val Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ala Thr Ser 65 70 75 80 Thr Ala Cys Met Glu Leu Thr Ser Leu Arg Ser Asp Asp Thr Ala Val                 85 90 95 Tyr Phe Cys Ala Arg Asp Tyr Ser Ser Pro Tyr His Phe Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Trp Ser Pro Ser Arg         115 120 <210> 232 <211> 122 <212> PRT <213> homo sapiens <400> 232 Gly Arg Ala Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys 1 5 10 15 Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu             20 25 30 Tyr Thr Thr Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Leu Ala Arg Ile Tyr Trp Asp Asp Asp Glu Arg Tyr     50 55 60 Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys 65 70 75 80 Asn Gln Val Val Leu Lys Met Thr Asn Met Asp Pro Val Asp Thr Ala                 85 90 95 Thr Tyr Tyr Cys Ala Arg Thr Met Gly Val Val Leu Pro Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 233 <211> 117 <212> PRT <213> homo sapiens <400> 233 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Pro Leu             20 25 30 Asn Arg Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Ser Ser Ile Ser Ser Thr Ser Ser Tyr Ile Tyr Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Leu                 85 90 95 Tyr Phe Cys Ala Ser Gly Asn Thr His Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser         115 <210> 234 <211> 126 <212> PRT <213> homo sapiens <400> 234 Gly Arg Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Leu Gly Thr Ser Val Arg Val Ser Cys Lys Thr Pro Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Arg Lys Phe Gln     50 55 60 Gly Arg Ile Thr Val Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Ala Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 235 <211> 132 <212> PRT <213> homo sapiens <400> 235 Gly Arg Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln 1 5 10 15 Pro Gly Thr Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe             20 25 30 Arg Thr Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Val Leu         35 40 45 Glu Trp Leu Ala Phe Val Ser Ser Asp Gly Ser Asp Glu Phe Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Ser 65 70 75 80 Thr Leu Phe Leu Lys Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Asp Arg Gly Ala Gln Ile Thr Leu Phe Gly Ala             100 105 110 Pro Leu Ile Arg Pro Ser Ser Phe Asp Ser Trp Gly Gln Gly Thr Leu         115 120 125 Val Thr Val Ser     130 <210> 236 <211> 126 <212> PRT <213> homo sapiens <400> 236 Gly Arg Ala Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Ser Phe Gly Thr Thr Ser Tyr Ala Gln Lys Phe Gln     50 55 60 Gly Arg Val Thr Ile Thr Ala Asp Lys Ala Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asp Leu Thr Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala                 85 90 95 Lys Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 237 <211> 122 <212> PRT <213> homo sapiens <400> 237 Gly Arg Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Thr Leu Val Lys 1 5 10 15 Pro Thr Gln Thr Leu Thr Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu             20 25 30 Gly Thr Thr Gly Val Asn Val Gly Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Leu Ala Leu Ile Ser Trp Asp Gly Gly Lys His Tyr     50 55 60 Ser Pro Ser Leu Asn Ser Arg Ile Thr Leu Thr Lys Asp Ala Ser Arg 65 70 75 80 Glu Gln Val Val Val Pro Thr Met Thr Asn Met Asp Pro Ala Asp Thr                 85 90 95 Gly Arg Tyr Tyr Cys Ala Arg Ile Val Gly Thr His Gly Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 238 <211> 131 <212> PRT <213> homo sapiens <400> 238 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ala Thr Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe             20 25 30 Asn Asp Tyr Cys Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Trp Ile Asn Gly Asn Asn Ala Asp Thr Phe Tyr Ala     50 55 60 Pro Lys Leu Gln Gly Arg Val Thr Met Ser Thr Asp Thr Ser Thr Ser 65 70 75 80 Thr Ala Tyr Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val                 85 90 95 Tyr Phe Cys Ala Arg Asp Arg Gly Arg Ile Thr Leu Phe Gly Glu Val             100 105 110 Ile Leu Arg Ala Gly Trp Phe Asp Ser Trp Gly Gln Gly Thr Leu Val         115 120 125 Thr val ser     130 <210> 239 <211> 124 <212> PRT <213> homo sapiens <400> 239 Gly Arg Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe             20 25 30 Ser Asn Asn Asn Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ala Ser Ile Ser Phe Gly Ser His Tyr Ile Ser Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 65 70 75 80 Ala Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Thr Arg Cys Arg Gly Gly Thr Arg Thr Tyr Tyr Tyr Met             100 105 110 Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser         115 120 <210> 240 <211> 124 <212> PRT <213> homo sapiens <400> 240 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Phe             20 25 30 Ser Asn Asn Asn Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ala Ser Ile Ser Phe Gly Ser His Tyr Ile Ser Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 65 70 75 80 Ala Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Thr Arg Cys Arg Gly Gly Thr Arg Thr Tyr Tyr Tyr Met             100 105 110 Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser         115 120 <210> 241 <211> 130 <212> PRT <213> homo sapiens <400> 241 Gly Arg Ala Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Gln Ser Ser Gly Gly Pro Pro             20 25 30 Lys Ser Tyr Thr Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro         35 40 45 Glu Trp Met Gly Gly Ile Ile Leu Ile Phe Gly Pro Pro Asn Tyr Ala     50 55 60 Gln Lys Phe Gln Asp Arg Leu Thr Ile Thr Ala Asp Lys Ser Thr Asn 65 70 75 80 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Met                 85 90 95 Tyr Tyr Cys Val Thr Ala Pro Asp Asp Thr Gly Thr Ile Leu Ala Arg             100 105 110 His Asn Arg Tyr Tyr Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr         115 120 125 Val Ser     130 <210> 242 <211> 130 <212> PRT <213> homo sapiens <400> 242 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ala Ser Gly Tyr Gly Phe             20 25 30 Thr Val Tyr Trp Ile Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Asn     50 55 60 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Ser 65 70 75 80 Thr Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile                 85 90 95 Tyr Tyr Cys Ala Arg His Leu Asp Ser Tyr Asp Val Phe Thr Gly Tyr             100 105 110 Asn Leu Gly Gly Tyr Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr         115 120 125 Val Ser     130 <210> 243 <211> 117 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (6) .. (6) <223> Xaa corresponds to a stop codon <400> 243 Gly Arg Ala Lys Val Xaa Leu Val Gln Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Pro Leu             20 25 30 Asn Arg Tyr Ile Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Ser Ser Ile Ser Ser Thr Ser Ser Tyr Ile Tyr Tyr Ala     50 55 60 Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 65 70 75 80 Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu                 85 90 95 Tyr Tyr Cys Ala Ser Gly Asn Thr His Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser         115 <210> 244 <211> 126 <212> PRT <213> homo sapiens <400> 244 Gly Arg Ala Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe Gln     50 55 60 Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 245 <211> 124 <212> PRT <213> homo sapiens <400> 245 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Ala Ser Arg Asp Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Val Thr Ala Asp Glu Ser Thr Arg 65 70 75 80 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Leu Gly Gly Thr Arg Leu Tyr Tyr Ala Leu             100 105 110 Asn Val Trp Gly Gln Gly Thr Met Val Thr Val Ser         115 120 <210> 246 <211> 124 <212> PRT <213> homo sapiens <400> 246 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Ser Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Asn Pro Ile Phe Ala Thr Arg Asp Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Arg 65 70 75 80 Thr Val Tyr Met Glu Leu Arg Asn Leu Arg Ser Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Phe Gly Gly Thr Arg Leu Tyr Tyr Ala Leu             100 105 110 Asn Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 247 <211> 126 <212> PRT <213> homo sapiens <400> 247 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Pro Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Arg Lys Phe Gln     50 55 60 Gly Arg Ile Thr Val Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Ala Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 248 <211> 124 <212> PRT <213> homo sapiens <400> 248 Gly Arg Ala Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe             20 25 30 Ser Asn Asn Asn Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ala Ser Ile Ser Phe Gly Ser His Tyr Ile Ser Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 65 70 75 80 Ala Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Thr Arg Cys Arg Gly Gly Thr Arg Thr Tyr Tyr Tyr Met             100 105 110 Asp Val Trp Gly Lys Gly Thr Met Val Thr Val Ser         115 120 <210> 249 <211> 130 <212> PRT <213> homo sapiens <400> 249 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ala Ser Gly Tyr Gly Phe             20 25 30 Thr Val Tyr Trp Ile Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Asn     50 55 60 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Ser 65 70 75 80 Thr Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile                 85 90 95 Tyr Tyr Cys Ala Arg His Leu Asp Ser Tyr Asp Val Phe Thr Gly Tyr             100 105 110 Asn Leu Gly Gly Tyr Met Asp Val Trp Gly Lys Gly Thr Leu Val Thr         115 120 125 Val Ser     130 <210> 250 <211> 126 <212> PRT <213> homo sapiens <400> 250 Gly Arg Ala Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ser Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly         35 40 45 Gly Ile Ile Ser Asn Phe Arg Thr Ala Glu Tyr Ala Arg Lys Phe Gln     50 55 60 Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asn Thr Ile Tyr Met 65 70 75 80 Glu Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Val                 85 90 95 Ser Ala Pro Arg Asp Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 251 <211> 126 <212> PRT <213> homo sapiens <400> 251 Gly Arg Ala Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Val Val Gln 1 5 10 15 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe             20 25 30 Arg Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Met Gly Val Ile Ser Phe Asn Gly Asp Gln Ile Phe Tyr Ala     50 55 60 Asp Ser Met Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ser Lys Asn 65 70 75 80 Thr Leu His Leu Arg Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Ala Arg Leu Leu Phe Cys Ser Gly Gly Arg Cys             100 105 110 Asp Met Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 252 <211> 126 <212> PRT <213> homo sapiens <400> 252 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly His Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Ala Thr Tyr Ala Gln Lys Phe Gln     50 55 60 Gly Arg Val Ser Ile Thr Ala Asp Thr Ser Thr Asn Thr Phe Tyr Met 65 70 75 80 Asp Leu Asn Asn Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Ser Ala Pro Arg Asp Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 253 <211> 116 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa corresponds to a stop codon <400> 253 Gly Arg Ala Xaa Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe             20 25 30 Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Gly His Val Lys Ser Met Thr Asp Gly Gly Thr Thr Asp     50 55 60 Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 65 70 75 80 Glu Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Asp Asp Thr                 85 90 95 Ala Val Tyr Tyr Cys Thr Thr His Asp Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser         115 <210> 254 <211> 126 <212> PRT <213> homo sapiens <400> 254 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Pro Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Arg Lys Phe Gln     50 55 60 Gly Arg Ile Thr Val Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Ala Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 255 <211> 131 <212> PRT <213> homo sapiens <400> 255 Gly Arg Ala Gln Val Gln Leu Val Glu Ser Gly Ala Glu Met Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Gln Ala Ser Gly Gly Thr Phe             20 25 30 Ser Asn Tyr Gly Ile Asn Trp Val Arg His Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Val Pro Ile Tyr Gly Pro Pro Lys Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 65 70 75 80 Thr Ala Tyr Met Glu Leu Ser Ser Leu Ser Ser Asp Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Leu Ser Arg Thr Val Phe Gly Ser Gly Thr Tyr             100 105 110 Arg Pro Gly Tyr Tyr Tyr Met Asp Val Trp Gly Ile Gly Thr Thr Val         115 120 125 Thr val ser     130 <210> 256 <211> 124 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa corresponds to a stop codon <400> 256 Gly Arg Ala Xaa Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe             20 25 30 Ser Asn Asn Asn Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ala Ser Ile Ser Phe Gly Ser His Tyr Ile Ser Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 65 70 75 80 Ala Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Thr Arg Cys Arg Gly Gly Thr Arg Thr Tyr Tyr Tyr Met             100 105 110 Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser         115 120 <210> 257 <211> 126 <212> PRT <213> homo sapiens <400> 257 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe Gln     50 55 60 Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 258 <211> 124 <212> PRT <213> homo sapiens <400> 258 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Glu Gly Gly Thr Phe             20 25 30 Ser Thr Tyr Val Ile Ser Trp Met Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Val Pro Ile Phe Asn Thr Pro Asn Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Arg Ser Thr Ser 65 70 75 80 Thr Ala Tyr Met Glu Leu Arg Ser Leu Gly Ser Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Val Gly Gly Thr Arg Ser Tyr Tyr Ala Leu             100 105 110 Gly Phe Trp Gly Gln Gly Thr Thr Val Val Val Val Ser         115 120 <210> 259 <211> 122 <212> PRT <213> homo sapiens <400> 259 Gly Arg Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys 1 5 10 15 Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu             20 25 30 Gly Thr Thr Gly Val Asn Val Gly Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Leu Ala Leu Ile Ser Trp Gly Gly Gly Lys His Tyr     50 55 60 Ser Pro Ser Leu Asn Ser Arg Ile Thr Leu Thr Lys Asp Ala Ser Arg 65 70 75 80 Glu Gln Val Val Val Leu Thr Met Ala Asn Met Asp Pro Val Asp Thr                 85 90 95 Gly Arg Tyr Tyr Cys Ala Arg Ile Val Gly Thr His Gly Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 260 <211> 124 <212> PRT <213> homo sapiens <400> 260 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe             20 25 30 Asp Glu Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Glu Trp Val Ala Phe Ile Asn Trp Asn Gly Asp Ser Thr Tyr Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Thr Asn Ala Lys Asn 65 70 75 80 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Phe                 85 90 95 Tyr Tyr Cys Ala Arg Asp Pro Arg Thr Lys Leu Gly Met Ser Tyr Phe             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 261 <211> 130 <212> PRT <213> homo sapiens <400> 261 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Glu Ser Leu Lys Ile Ser Cys Gln Ala Ser Gly Tyr Gly Phe             20 25 30 Thr Val Tyr Trp Ile Gly Trp Val Arg Gln Leu Pro Gly Lys Gly Leu         35 40 45 Glu Trp Leu Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Asn     50 55 60 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Val Ser 65 70 75 80 Thr Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile                 85 90 95 Tyr Tyr Cys Ala Arg His Leu Asp Ser Tyr Asp Val Phe Thr Gly Tyr             100 105 110 Asn Leu Gly Gly Tyr Met Asp Val Trp Gly Lys Gly Thr Met Val Thr         115 120 125 Val Ser     130 <210> 262 <211> 123 <212> PRT <213> homo sapiens <400> 262 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys 1 5 10 15 Pro Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Ser Leu             20 25 30 Lys Asn Tyr Gly Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Trp Ile Ser Ala Asp Asn Gly Asp Thr Thr Thr Ala     50 55 60 Leu Asn Leu Arg Gly Arg Val Ser Met Thr Thr Asp Thr Ser Thr Asn 65 70 75 80 Thr Val Tyr Met Glu Val Lys Ser Leu Arg Ser Asp Asp Thr Ala Ile                 85 90 95 Tyr Phe Cys Ala Arg Asp Phe Tyr Ser Gly Ser Tyr Arg Ser Phe Asp             100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 263 <211> 126 <212> PRT <213> homo sapiens <400> 263 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ala Ser Val             20 25 30 Ser Gly Gly Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ile Gly Ser Thr Asn Tyr     50 55 60 Asn Pro Ser Leu Lys Ser Arg Leu Ser Leu Ser Val Asp Thr Ala Lys 65 70 75 80 Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala                 85 90 95 Ile Tyr Phe Cys Ala Arg Ala Arg Arg Thr Tyr Ser Gly Tyr Asp Ser             100 105 110 Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 264 <211> 124 <212> PRT <213> homo sapiens <400> 264 Gly Arg Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Glu Thr Leu Pro Leu Thr Cys Thr Val Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu         35 40 45 Glu Trp Ile Gly Tyr Ile Gln Asn Ser Val Asn Thr Asn Tyr Asn Pro     50 55 60 Ser Leu Lys Ser Arg Val Ile Ile Ser Val Asp Thr Ser Asn Asn Gln 65 70 75 80 Phe Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Ala Arg Val Ser Gly Trp Gly Pro Arg Gly Gly Ile Tyr Phe             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 265 <211> 126 <212> PRT <213> homo sapiens <400> 265 Gly Arg Ala Lys Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ser Ser Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly         35 40 45 Gly Ile Ile Ser Asn Phe His Thr Ala Glu Tyr Ala Gln Lys Phe Gln     50 55 60 Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asn Thr Ile Tyr Met 65 70 75 80 Glu Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Val                 85 90 95 Ser Ala Pro Arg Asp Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Thr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 266 <211> 126 <212> PRT <213> homo sapiens <400> 266 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Thr Ser Val Arg Val Ser Arg Lys Thr Pro Gly Gly Tyr Val             20 25 30 Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met Gly         35 40 45 Gly Ile Ile Thr Asn Phe Gly Thr Thr Asn Tyr Ala Arg Lys Phe Gln     50 55 60 Gly Arg Ile Thr Val Thr Ala Asp Lys Ser Thr Asn Thr Val Tyr Met 65 70 75 80 Asp Leu Ser Asn Leu Ala Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ala Pro Arg Gly Thr Ser Thr Ile Ala Ala Arg Phe Asn Arg Tyr             100 105 110 Phe Phe Asp Ser Trp Ser Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 267 <211> 124 <212> PRT <213> homo sapiens <400> 267 Gly Arg Ala Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Ile Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Asn Pro Ile Phe Ala Thr Arg Asp Tyr Pro     50 55 60 Lys Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Arg 65 70 75 80 Thr Val Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Leu Gly Gly Thr Arg Leu Tyr Tyr Ala Leu             100 105 110 Asn Val Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser         115 120 <210> 268 <211> 124 <212> PRT <213> homo sapiens <400> 268 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe             20 25 30 Asp Glu Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu         35 40 45 Lys Trp Val Ala Phe Ile Asn Trp Asn Gly Asp Ser Thr Tyr Tyr Ala     50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Ser Asn Ala Lys Asn 65 70 75 80 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Phe                 85 90 95 Tyr Cys Cys Ala Arg Asp Pro Arg Thr Lys Leu Gly Met Ser Tyr Phe             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 269 <211> 124 <212> PRT <213> homo sapiens <400> 269 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Ala Ser Arg Asp Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Arg 65 70 75 80 Thr Val Tyr Met Glu Pro Arg Ser Leu Arg Ser Glu Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Leu Gly Gly Thr Arg Leu Tyr Tyr Ala Leu             100 105 110 Asn Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 270 <211> 126 <212> PRT <213> homo sapiens <400> 270 Gly Arg Ala Glu Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys 1 5 10 15 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ala Ser Val             20 25 30 Ser Gly Gly Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ile Gly Ser Thr Asn Tyr     50 55 60 Asn Pro Ser Leu Lys Ser Arg Leu Ser Leu Ser Val Asp Thr Ala Lys 65 70 75 80 Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala                 85 90 95 Thr Tyr Phe Cys Ala Arg Ala Arg Arg Thr Tyr Ser Gly Tyr Asp Ser             100 105 110 Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 125 <210> 271 <211> 124 <212> PRT <213> homo sapiens <400> 271 Gly Arg Ala Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys 1 5 10 15 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe             20 25 30 Ser Thr Tyr Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu         35 40 45 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Ala Ser Arg Asp Tyr Ala     50 55 60 Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Arg 65 70 75 80 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val                 85 90 95 Tyr Tyr Cys Ala Arg Val Leu Gly Gly Thr Arg Leu Tyr Tyr Ala Leu             100 105 110 Asn Val Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser         115 120 <210> 272 <211> 123 <212> PRT <213> homo sapiens <400> 272 Gly Arg Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Val Val Lys 1 5 10 15 Pro Thr Gln Thr Leu Thr Leu Thr Cys Ser Leu Ser Gly Phe Ala Leu             20 25 30 Gly Thr Thr Gly Val Ala Val Gly Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Trp Leu Gly Leu Ile Asp Trp Asn Asp Asp Arg Arg Tyr     50 55 60 Arg Pro Ser Leu Lys Thr Arg Leu Thr Ile Thr Gln Asp Met Ser Arg 65 70 75 80 Asn Gln Val Val Leu Arg Leu Thr Asn Leu Asp Pro Leu Asp Thr Gly                 85 90 95 Thr Tyr Phe Cys Ala Arg Ser Val Val Pro Ala Thr Arg Ala Phe Asp             100 105 110 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 273 <211> 122 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (52) .. (52) <223> Xaa corresponds to a stop codon <400> 273 Gly Arg Ala Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys 1 5 10 15 Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu             20 25 30 Tyr Thr Thr Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys         35 40 45 Ala Leu Glu Xaa Leu Ala Arg Ile Tyr Trp Asp Asp Asp Glu Arg Tyr     50 55 60 Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys 65 70 75 80 Asn Gln Val Val Leu Lys Met Thr Asn Met Asp Pro Val Asp Thr Ala                 85 90 95 Thr Tyr Tyr Cys Ala Arg Thr Met Gly Val Val Leu Pro Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 274 <211> 109 <212> PRT <213> homo sapiens <400> 274 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Ser             20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Cys Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Thr Gly Ser Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr                 85 90 95 Ser Trp Thr Phe Gly Gln Gly Thr Thr Val Glu Ile Lys             100 105 <210> 275 <211> 109 <212> PRT <213> homo sapiens <400> 275 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu         35 40 45 Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala                 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 276 <211> 109 <212> PRT <213> homo sapiens <400> 276 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln His Ile Ser             20 25 30 Asn Tyr Ile Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Thr Gly Ser Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Thr Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Gly Thr                 85 90 95 Ser Trp Thr Phe Gly Gln Gly Thr Thr Val Glu Ile Lys             100 105 <210> 277 <211> 109 <212> PRT <213> homo sapiens <400> 277 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr             20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Asp Ala Ser Asn Leu Gln Pro Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Arg Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Gly Pro                 85 90 95 Val Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 278 <211> 109 <212> PRT <213> homo sapiens <400> 278 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Pro Ile Ser             20 25 30 Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ile         35 40 45 Leu Ile Phe Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Thr Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Ile Cys Gln Gln Ser Lys Ser Pro                 85 90 95 Pro Tyr Asn Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 279 <211> 110 <212> PRT <213> homo sapiens <400> 279 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly             20 25 30 Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Gln Ser Glu Thr Pro Ser Arg Phe     50 55 60 Arg Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe                 85 90 95 Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Phe Lys             100 105 110 <210> 280 <211> 109 <212> PRT <213> homo sapiens <400> 280 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn             20 25 30 Ile Asn Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu         35 40 45 Leu Ile Tyr Ser Ala Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser His                 85 90 95 Val Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 281 <211> 115 <212> PRT <213> homo sapiens <400> 281 Leu Ala Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu yle lys         115 <210> 282 <211> 109 <212> PRT <213> homo sapiens <400> 282 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Ile Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr             20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Asp Ala Ser Asn Leu Gln Pro Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu 65 70 75 80 Arg Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Gly Pro                 85 90 95 Val Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 283 <211> 115 <212> PRT <213> homo sapiens <400> 283 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu yle lys         115 <210> 284 <211> 110 <212> PRT <213> homo sapiens <400> 284 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Ser Gly Ala Ser Thr Leu Gln Ser Trp Val Pro Ser Arg Phe     50 55 60 Ser Gly Arg Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Arg Tyr                 85 90 95 Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 285 <211> 109 <212> PRT <213> homo sapiens <400> 285 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser             20 25 30 Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Glu Ala Ser Asn Leu Glu Thr Gly Val Pro Pro Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr His Phe Thr Phe Thr Ile Thr Gly Leu 65 70 75 80 Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Cys Asp Ser Leu                 85 90 95 Pro Pro Val Phe Gly Gln Gly Thr Lys Leu Glu Val Lys             100 105 <210> 286 <211> 110 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (93) .. (93) <223> Xaa corresponds to a stop codon <400> 286 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ala Leu Ser 1 5 10 15 Pro Gly Asp Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Phe             20 25 30 Gly Asp Phe Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Xaa Tyr Gly Asp                 85 90 95 Ser Val Phe Thr Phe Gly Gln Gly Thr Lys Leu Gly Ile Lys             100 105 110 <210> 287 <211> 110 <212> PRT <213> homo sapiens <400> 287 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ala Leu Ser 1 5 10 15 Pro Gly Asp Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Phe             20 25 30 Gly Asp Phe Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Asp                 85 90 95 Ser Val Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 288 <211> 110 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (45) .. (45) <223> Xaa corresponds to a stop codon <400> 288 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Gln Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn             20 25 30 Arg Asp Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Xaa Ala Pro Arg         35 40 45 Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Arg Asp Asp Phe Ala Val Tyr Phe Cys His Gln Tyr Gly Ser                 85 90 95 Ser Pro Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 289 <211> 110 <212> PRT <213> homo sapiens <400> 289 Leu Ala Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser             20 25 30 Thr Asn Tyr Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile His Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Ser                 85 90 95 Ser Pro Gln Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 110 <210> 290 <211> 109 <212> PRT <213> homo sapiens <400> 290 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Thr Thr Cys Arg Thr Ser Gln Ser Ile Phe             20 25 30 Ile Asn Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Ser Ala Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Gln                 85 90 95 Val Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 291 <211> 110 <212> PRT <213> homo sapiens <400> 291 Leu Ala Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn             20 25 30 Ser Asp Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Met Ser Gly Ala Thr Ser Arg Ala Thr Asp Val Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser                 85 90 95 Ser Asp Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 292 <211> 110 <212> PRT <213> homo sapiens <400> 292 Leu Ala Asp Val Val Met Thr Gln Phe Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Glu Leu         35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Asn Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Gly                 85 90 95 Leu Arg Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys             100 105 110 <210> 293 <211> 110 <212> PRT <213> homo sapiens <400> 293 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp His Gln Gln Lys Pro Gly Gln Val Pro Lys Leu         35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Gly                 85 90 95 Leu Ile Phe Thr Phe Gly Pro Gly Thr Arg Val Glu Ile Lys             100 105 110 <210> 294 <211> 115 <212> PRT <213> homo sapiens <400> 294 Leu Ala Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Leu Gly Gln Gly Thr Lys Val             100 105 110 Glu yle lys         115 <210> 295 <211> 110 <212> PRT <213> homo sapiens <220> <221> MISC_FEATURE (222) (8) .. (8) <223> Xaa corresponds to a stop codon <400> 295 Leu Ala Asp Val Val Met Thr Xaa Ser Pro Gly Thr Leu Ala Leu Ser 1 5 10 15 Pro Gly Asp Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Phe             20 25 30 Gly Asp Phe Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile Tyr Val Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Asp                 85 90 95 Ser Val Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Arg             100 105 110 <210> 296 <211> 110 <212> PRT <213> homo sapiens <400> 296 Leu Ala Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser             20 25 30 Thr Asn Tyr Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile His Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Ser                 85 90 95 Ser Pro Gln Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 110 <210> 297 <211> 110 <212> PRT <213> homo sapiens <400> 297 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Asn             20 25 30 Ser Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Arg         35 40 45 Leu Leu Met Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ala                 85 90 95 Ser Asp Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 298 <211> 109 <212> PRT <213> homo sapiens <400> 298 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ile             20 25 30 Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Val         35 40 45 Leu Ile Phe Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Ile Arg Tyr                 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Arg Val Glu Ile Lys             100 105 <210> 299 <211> 110 <212> PRT <213> homo sapiens <400> 299 Leu Ala Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn             20 25 30 Ser Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Val Ser Gly Ala Thr Asn Arg Ala Thr Asp Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser                 85 90 95 Ser Glu Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Arg             100 105 110 <210> 300 <211> 109 <212> PRT <213> homo sapiens <400> 300 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Pro Phe Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Leu Ser             20 25 30 Thr Tyr Leu Ala Trp Tyr Gln Val Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Thr Tyr                 85 90 95 Pro Leu Thr Leu Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 301 <211> 115 <212> PRT <213> homo sapiens <400> 301 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu yle lys         115 <210> 302 <211> 115 <212> PRT <213> homo sapiens <400> 302 Leu Ala Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Phe Gly Gln Gly Thr Arg Val             100 105 110 Glu yle lys         115 <210> 303 <211> 110 <212> PRT <213> homo sapiens <400> 303 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ala Leu Ser 1 5 10 15 Pro Gly Asp Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Phe             20 25 30 Gly Asp Phe Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg         35 40 45 Pro Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Asp                 85 90 95 Ser Val Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 304 <211> 110 <212> PRT <213> homo sapiens <400> 304 Leu Ala Asp Val Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asn             20 25 30 Ser Asp Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Met Ser Gly Ala Thr Ser Arg Ala Thr Asp Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser                 85 90 95 Ser Asp Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 305 <211> 110 <212> PRT <213> homo sapiens <400> 305 Leu Ala Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Ser Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Lys Lys Pro Gly Lys Val Pro Arg Leu         35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Glu Tyr Ser Asn Ala                 85 90 95 Leu Ile Phe Thr Phe Gly Pro Gly Thr Lys Val His Ile Lys             100 105 110 <210> 306 <211> 110 <212> PRT <213> homo sapiens <400> 306 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Gly             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Ser Gly Ala Ser Thr Leu Gln Ser Trp Val Pro Ser Arg Phe     50 55 60 Ser Gly Arg Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Arg Tyr                 85 90 95 Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 307 <211> 109 <212> PRT <213> homo sapiens <400> 307 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser             20 25 30 Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Val Gly Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Pro                 85 90 95 Ser Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 308 <211> 110 <212> PRT <213> homo sapiens <400> 308 Leu Ala Asp Ile Gln Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser             20 25 30 Thr Asn Tyr Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg         35 40 45 Leu Leu Ile His Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Ser                 85 90 95 Ser Pro Gln Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 110 <210> 309 <211> 109 <212> PRT <213> homo sapiens <400> 309 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Ser             20 25 30 Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Thr Gly Ser Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr                 85 90 95 Ser Trp Thr Phe Gly Gln Gly Thr Thr Val Gly Ile Lys             100 105 <210> 310 <211> 109 <212> PRT <213> homo sapiens <400> 310 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Arg Ser Val Lys             20 25 30 Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Val Tyr Gly Ser Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Phe Gly Thr                 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Asp Thr Lys             100 105 <210> 311 <211> 109 <212> PRT <213> homo sapiens <400> 311 Leu Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Ser Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn             20 25 30 Lys Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu         35 40 45 Leu Ile Tyr Ala Ala Thr Asn Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Thr Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Met                 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 312 <211> 110 <212> PRT <213> homo sapiens <400> 312 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser 1 5 10 15 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Val Tyr             20 25 30 Ser Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Arg         35 40 45 Leu Leu Met Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg     50 55 60 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ala                 85 90 95 Ser Asp Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 313 <211> 115 <212> PRT <213> homo sapiens <400> 313 Leu Ala Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Lys Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu             20 25 30 Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro         35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser     50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys                 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Pro Met Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu yle lys         115 <210> 314 <211> 110 <212> PRT <213> homo sapiens <400> 314 Leu Ala Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Val Pro Lys Leu         35 40 45 Leu Ile Tyr Val Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Asn Gly Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asp Ser Gly                 85 90 95 Leu Ile Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 315 <211> 109 <212> PRT <213> homo sapiens <400> 315 Leu Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser             20 25 30 Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Val Gly Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Pro                 85 90 95 Ser Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 316 <211> 110 <212> PRT <213> homo sapiens <400> 316 Leu Ala Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Glu Leu         35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Gly                 85 90 95 Leu Arg Phe Thr Phe Gly Pro Gly Ala Lys Val Asp Ile Lys             100 105 110 <210> 317 <211> 109 <212> PRT <213> homo sapiens <400> 317 Leu Ala Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Arg Ser Val Lys             20 25 30 Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Val Tyr Gly Ser Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Cys Cys Gln Gln Ser Phe Gly Thr                 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys             100 105 <210> 318 <211> 110 <212> PRT <213> homo sapiens <400> 318 Leu Ala Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser             20 25 30 Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Glu Leu         35 40 45 Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Gly                 85 90 95 Leu Arg Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys             100 105 110 <210> 319 <211> 109 <212> PRT <213> homo sapiens <400> 319 Leu Ala Asp Ile Gln Met Thr Gln Ser Ser Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn             20 25 30 Ile Trp Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Glu Arg Gly Val Pro Ser Arg Phe     50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Thr Gly Leu 65 70 75 80 Arg Pro Asp Asp Phe Gly Ser Tyr Tyr Cys Gln His Tyr Asp Gly Asn                 85 90 95 Ser Leu Thr Phe Gly Gln Gly Thr Arg Val Asp Ile Gln             100 105 <210> 320 <211> 110 <212> PRT <213> homo sapiens <400> 320 Leu Ala Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly             20 25 30 Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu         35 40 45 Leu Ile Tyr Lys Ala Ser Thr Leu Gln Ser Glu Thr Pro Ser Arg Phe     50 55 60 Arg Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe                 85 90 95 Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Ala Glu Phe Lys             100 105 110 <210> 321 <211> 402 <212> DNA <213> Artificial <220> <223> Vector sequence <400> 321 gctagcgctg gttgggcagc gagtaataac aatccagcgg ctgccgtagg caataggtat 60 ttcattatga ctgtctcctt ggcgactagc tagtttagaa ttaattcgtg aaattgttat 120 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 180 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagatctta 240 ctccccatcc ccctgttgac aattaatcat cggctcgtat gatgtgtgga attgtgagcg 300 gataacaatt tcacacagga aacaggagat atacatatga aatacctgct gccgaccgct 360 gctgctggtc tgctgctcct cgctgcccag ccggggcgcg cc 402 <210> 322 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 322 tattggcgcg ccatggccgc ccagtctgtg acccagc 37 <210> 323 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 323 tattggcgcg ccatggccgc ccagaagata actcaa 36 <210> 324 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 324 tattggcgcg ccatggccga tgctaagacc acccag 36 <210> 325 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 325 tattggcgcg ccatggccgc ccagacagtc actcag 36 <210> 326 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 326 tattggcgcg ccatggccaa acaggaggtg acacaga 37 <210> 327 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 327 tattggcgcg ccatggccgg agactcggtt acccaga 37 <210> 328 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 328 tattggcgcg ccatggccgg agattcagtg acccaga 37 <210> 329 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 329 tattggcgcg ccatggccag caattcagtc aagcaga 37 <210> 330 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 330 tattggcgcg ccatggccgg acaaaacatt gaccag 36 <210> 331 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 331 tattggcgcg ccatggccaa aaatgaagtg gagcaga 37 <210> 332 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 332 tattggcgcg ccatggccga agaccaggtg acgcaga 37 <210> 333 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 333 tattggcgcg ccatggccgg aatacaagtg gagcaga 37 <210> 334 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 334 tattggcgcg ccatggccct acatacactg gagcaga 37 <210> 335 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 335 tattggcgcg ccatggccga agacaaggtg gtacaaa 37 <210> 336 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 336 tattggcgcg ccatggccga aaaccaggtg gagcaca 37 <210> 337 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 337 tattggcgcg ccatggccca ggagaatgtg gagcag 36 <210> 338 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 338 tattggcgcg ccatggccgg agagagtgtg gggctg 36 <210> 339 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 339 tattggcgcg ccatggccgg agaggatgtg gagcaga 37 <210> 340 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 340 tattggcgcg ccatggccag ccaaaagata gaacaga 37 <210> 341 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 341 tattggcgcg ccatggccag tcaacaggga gaagag 36 <210> 342 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 342 tattggcgcg ccatggccca acaaccagtg cagagt 36 <210> 343 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 343 tattggcgcg ccatggccag ccaagaactg gagcaga 37 <210> 344 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 344 tattggcgcg ccatggccgg tcaacagctg aatcaga 37 <210> 345 <211> 37 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 345 tattggcgcg ccatggccac ccagctgctg gagcaga 37 <210> 346 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 346 tattggcgcg ccatggccca gcagcaggtg aaacaa 36 <210> 347 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 347 tattggcgcg ccatggccat actgaacgtg gaacaa 36 <210> 348 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 348 tattggcgcg ccatggccga ccagcaagtt aagcaa 36 <210> 349 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 349 tattggcgcg ccatggccgg acaacaggta atgcaa 36 <210> 350 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 350 tattggcgcg ccatggccaa ggaccaagtg tttcag 36 <210> 351 <211> 21 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 351 taggcagaca gacttgtcac t 21 <210> 352 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 352 gccatggcgc gccaatagct agccggtgaa gaagtcgccc aga 43 <210> 353 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 353 gccatggcgc gccaatagct agccgatgcc atggtcatcc aga 43 <210> 354 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 354 gccatggcgc gccaatagct agccgaagcc caagtgaccc aga 43 <210> 355 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 355 gccatggcgc gccaatagct agcccatgcc aaagtcacac aga 43 <210> 356 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 356 gccatggcgc gccaatagct agccgacaca gccgtttccc aga 43 <210> 357 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 357 gccatggcgc gccaatagct agccgaaacg ggagttacgc aga 43 <210> 358 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 358 gccatggcgc gccaatagct agccgaacct gaagtcaccc aga 43 <210> 359 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 359 gccatggcgc gccaatagct agccgaagct gacatctacc aga 43 <210> 360 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 360 gccatggcgc gccaatagct agccatatct ggagtctccc aca 43 <210> 361 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 361 gccatggcgc gccaatagct agccgatgct ggaatcaccc aga 43 <210> 362 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 362 gccatggcgc gccaatagct agccaatgct ggtgtcactc aga 43 <210> 363 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 363 gccatggcgc gccaatagct agccagtgct gtcgtctctc aa 42 <210> 364 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 364 gccatggcgc gccaatagct agccgacgct ggagtcacac aa 42 <210> 365 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 365 gccatggcgc gccaatagct agccgatggt ggaatcactc agt 43 <210> 366 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 366 gccatggcgc gccaatagct agccactgct gggatcaccc ag 42 <210> 367 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 367 gccatggcgc gccaatagct agccgaagct ggagttgccc agt 43 <210> 368 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 368 gccatggcgc gccaatagct agccgaagct ggagtggttc agt 43 <210> 369 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 369 gccatggcgc gccaatagct agccgaagct ggagttactc agt 43 <210> 370 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 370 gccatggcgc gccaatagct agccgatgct gtagttacac aa 42 <210> 371 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 371 gccatggcgc gccaatagct agccgatgct ggagttatcc agt 43 <210> 372 <211> 42 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 372 gccatggcgc gccaatagct agccggtgct ggagtctccc ag 42 <210> 373 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 373 gccatggcgc gccaatagct agccgatgct gatgttaccc aga 43 <210> 374 <211> 43 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 374 gccatggcgc gccaatagct agcctctcag actattcatc aat 43 <210> 375 <211> 21 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 375 caggcacacc agtgtggcct t 21 <210> 376 <211> 36 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 376 caccttagag ctcttagagt ctctcagctg gtacac 36 <210> 377 <211> 33 <212> DNA <213> Artificial <220> <223> Primer sequence <400> 377 gacattatgc atgatctctg cttctgatgg ctc 33

Claims (58)

a) the nucleotide sequence of interest is amplified by a complex molecular amplification method using a template derived from an individual of an isolated single cell or a homologous cell, b) a method of joining a plurality of discontinuous nucleotide sequences of interest, including binding the nucleotide sequences of interest amplified in step a). The method of claim 1, wherein the nucleotide sequence of interest comprises a variable region encoding sequence and the binding results in a cognate pair of the variable region encoding sequence. 3. The method of claim 1, wherein the nucleotide sequence of interest comprises an immunoglobulin variable region encoding sequence and the binding results in a cognate pair of light chain variable region encoding sequences joined with a heavy chain variable region encoding sequence. Way. 3. The alpha chain variable region encoding sequence or delta chain variable region encoding sequence of claim 1, wherein the nucleotide sequence of interest comprises a T cell receptor variable region encoding sequence, and wherein the binding is associated with a beta chain variable region encoding sequence. Generating a cognate pair consisting of a gamma chain variable region encoding sequence associated with a) amplifying the nucleotide sequence of interest by a complex molecular amplification method using templates derived from individuals of genetically different cells, b) a method of joining any of a plurality of discontinuous nucleotide sequences of interest, including binding the nucleotide sequences of interest amplified in step a). The method of claim 5, wherein the individual of the cell is lysed. 7. The method of claim 5 or 6, wherein the nucleotide sequence of interest comprises a variable region encoding sequence and the binding results in a combinatorial library of pairs of variable region encoding sequences. 8. The method of claim 7, wherein the nucleotide sequence of interest comprises an immunoglobulin variable region encoding sequence and wherein the binding produces a combinatorial library of light chain variable region and heavy chain variable region encoding sequence pairs. 8. The method of claim 7, wherein the nucleotide sequence of interest comprises a T cell receptor variable region encoding sequence and the binding produces a combinatorial library of α chain variable region and β chain variable region encoding sequence pairs. 10. The method according to any one of claims 1 to 9, wherein the complex molecular amplification method is complex RT-PCR amplification. The method of claim 10, wherein the complex RT-PCR amplification is a two step process comprising a separate reverse transcription (RT) step prior to the complex PCR amplification. The method of claim 10, wherein the complex RT-PCR amplification is performed in a single step comprising initially adding all the components necessary to perform both reverse transcription (RT) and complex PCR amplification to a single vessel. The method of claim 1, wherein the binding of the nucleotide sequence of interest is performed in the same vessel as the complex molecule amplification. The method of claim 10, wherein the binding of the nucleotide sequence of interest is performed with complex PCR amplification using a complex overlap-extension primer mix. The method of claim 1, wherein the binding of the nucleotide sequence of interest is carried out by ligation. 16. The method according to any one of claims 1 to 15, wherein further molecular amplification is performed using primer mixes suitable for amplifying the nucleic acid sequence of interest to which it is bound. 15. The method of claim 14, wherein the complex overlap-extension primer mix comprises a primer set, wherein at least one primer set member of each primer set is capable of hybridizing with the overlap-extension tail of the primer set member of the second primer set. A kidney tail. 18. The method of claim 14 or 17, wherein the complex overlap-extension primer mix is a) at least one C _L or J _L primer complementary to the sense strand of an immunoglobulin light chain region encoding sequence, b) one or more V _L 5 ′ primers or V _LL primers complementary to the antisense strand of an immunoglobulin light chain variable region encoding sequence or light chain variable region leader sequence and capable of forming a primer set with the primer (s) in a) above. , c) one or more C _H or J _H primers complementary to the sense strand of an immunoglobulin constant heavy chain domain encoding sequence or heavy chain binding region encoding sequence, and d) one or more V _H 5 ′ primers or V _{HLs that} are complementary to the antisease strand of an immunoglobulin heavy chain variable region encoding sequence or heavy chain variable region leader sequence and that are capable of forming a primer set with the primer (s) in c). And a primer. The primer of claim 18, wherein the primer of step b) is a V _LL primer having at least 90% sequence identity with the gene-specific region of SEQ IDs 93-98, and the primer of step d) is a gene- of SEQ ID 86-92. A V _HL primer having at least 90% identity with a specific region. 20. The method of any one of claims 1 to 19, wherein a single cell, an individual of allogeneic cells, or an individual of genetically different cells is obtained from a lymphocyte containing cell fraction. a) providing a lymphocyte-containing cell fraction from the donor, b) optionally hatching specific lymphocyte individuals from the cell fraction, c) obtaining an individual of isolated single cells, comprising individually distributing the cells from the cell fraction into multiple containers, c) The variable region encoding sequence contained in the individual of an isolated single cell, according to any of claims 1 to 4, or as long as they refer to any one of claims 1 to 4. A method of generating a library of cognate pairs comprising linked variable region encoding sequences, including amplification and binding according to the method of claim 1. 22. The method of claim 21, wherein prior to performing amplification and binding (step d), individual isolated single cells of the single cell individual are expanded to the individual of allogeneic cells. 23. The method of any one of claims 20-22, wherein the lymphocyte-containing cell fraction consists of whole blood, bone marrow, mononuclear cells, or leukocytes. 24. The method of any one of claims 20 to 23, wherein the lymphocyte-containing cell fraction is hatched into cells of the B lymphocyte lineage. 25. The method of any one of claims 20 to 24, wherein the lymphocyte-containing cell fraction or B lymphocyte lineage is hatched into plasma cells. 26. The method of any one of claims 20-25, wherein cells from lymphocyte-containing cell fractions, B lymphocyte lineages or plasma cells are antigen-specificly hatched. 24. The method of any one of claims 20 to 23, wherein the lymphocyte-containing cell fraction is hatched into cells of the T lymphocyte lineage. 24. The method of any one of claims 20 to 23, wherein the antigen-specific T cells are produced by stimulation of lymphocyte-containing cell fractions. 29. The method of any one of claims 1 to 28, further comprising inserting a library of linked nucleotide sequences or cognate pairs into the vector. 30. The method of claim 29, wherein the vector is selected from cloning vectors, shuttle vectors, display vectors or expression vectors. 31. The method of claim 29 or 30, wherein the individual absence of bound nucleotide sequences or cognate pair libraries comprises an immunoglobulin heavy chain variable region encoding sequence associated with a light chain variable region encoding sequence, wherein the sequences are one or more immunoglobulin constant domains. Or in-frame insertion into a vector already containing sequences encoding fragments thereof. 31. The method of claim 29 or 30, wherein the individual absence of the bound nucleotide sequence or cognate pair library comprises a T cell receptor α chain variable region encoding sequence associated with a T cell receptor β chain variable region encoding sequence, wherein the sequences are A method characterized in that it is inserted in-frame into a vector already containing sequences encoding one or more T cell receptor constant domains or fragments thereof. 33. The method of any one of claims 21 to 32, further comprising generating a sub-library by selecting a subset of cognate pairs of linked variable region sequences that encode a binding protein with the desired target specificity. Generating a library of target-specific cognate pairs of variable region encoding sequences. 34. The method of any one of claims 31 to 33, further comprising moving a library of target-specific cognate pairs of cognate pairs or variable region encoding sequences into a mammalian expression vector. The method of claim 34, wherein the mammalian expression vector comprises human immunoglobulin classes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, kappa light chain or lambda light chain, or human T cell receptor alpha, beta, delta and And / or encode one or more constant region domains selected from gamma chains. The method according to any one of claims 29 to 35, a) introducing into a host cell a vector encoding a segment of the bound nucleotide sequence, b) culturing the host cell under conditions suitable for expression, and c) obtaining the expressed protein product from the vector inserted into the host cell. 37. The method of claim 36, wherein the protein product is a monoclonal antibody comprising a cognate pair of light chain variable regions associated with a heavy chain variable region. The method of claim 36, wherein the polypeptide product is a monoclonal T cell receptor comprising a cognate pair of alpha chain variable regions bound to a beta chain variable region. A library of cognate pairs consisting of linked variable region encoding sequences. 40. The library of claim 39, wherein the cognate pairs of variable region encoding sequences are obtained by the method according to claim 21 or any dependent claim of claim 21. 41. The library of claim 39 or 40, wherein the individual absence of cognate pairs comprises an immunoglobulin light chain variable region encoding sequence associated with an immunoglobulin heavy chain variable region encoding sequence. The library of claim 41, wherein the individual absence encodes a full length antibody selected from human immunoglobulin classes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. 41. The TcR gamma chain variable region encoding of claim 39 or 40, wherein the individual absence of cognate pairs is combined with a TcR alpha chain variable region encoding sequence or a TcR delta chain variable region encoding sequence associated with a TcR beta chain variable region encoding sequence. A library comprising sequences. The library of claim 40, wherein the individual members encode full length TcR. A sub-library of cognate pairs of variable region encoding sequences encoding proteins exhibiting the desired binding specificities derived for a particular target. A sub-library encoding a protein exhibiting the desired binding specificity derived for a particular target selected from the library according to any one of claims 39 to 44. A sub-library of cognate pairs of bound immunoglobulin heavy chain variable region and light chain variable region encoding sequences, wherein the immunoglobulin expressable from the library can react or bind with tetanus toxin. 48. An individual of a host cell comprising a library or sub-library according to any one of claims 39 to 47. 49. The individual of claim 48, wherein the cell is a mammalian cell. 50. A recombinant polyclonal protein expressed from a host cell according to claim 48 or 49. Recombinant polyclonal immunoglobulin or fragment thereof capable of reacting or binding to tetanus toxin. The recombinant polyclonal immunoglobulin of claim 51, wherein the individual antibody members are cognate pairs. 52. The method of claim 51 or 52, wherein the SEQ ID pairs 229: 276, 262: 309, 240: 287, 245: 292, 267: 314, 246: 293, 258: 305, 242: 289, 231: 278, 263 : 310, 232: 279, 237: 284, 255: 302, 260: 307, 251: 298, 233: 280, 228: 275, 244: 291, 250: 297, 252: 299, 264: 311, 272: 319 Two or more individual comprising an immunoglobulin heavy chain variable region encoding sequence coupled with an immunoglobulin light chain variable region encoding sequence having at least 90% identity to one individual SEQ ID pair selected from the group consisting of 235: 282 or 238: 285 Recombinant polyclonal immunoglobulin characterized by including a cognate pair. A recombinant immunoglobulin or fragment thereof, wherein the complementarity determining regions (CDR1, CDR2 and CDR3) can react or bind with tetanus toxin derived from one or more of the SEQ ID pairs of claim 33. A pharmaceutical composition comprising the recombinant polyclonal immunoglobulin according to claim 50 as an active ingredient together with one or more pharmaceutically acceptable excipients. A pharmaceutical composition comprising, as an active ingredient, a recombinant polyclonal antibody capable of reacting or binding to tetanus toxin, optionally with a pharmaceutically acceptable excipient. 57. A pharmaceutical composition according to claim 55 or 56 which is used as a medicament. A method of preventing or treating a patient at risk of developing tetanus, comprising administering to a patient in need thereof a composition comprising a recombinant polyclonal antibody capable of reacting or binding to a tetanus toxin.

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