JP2013233096A - Gene ligation method and method for preparing single chain variable fragment using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid ligation method ligating two kinds of DNA or DNA library precisely and in high efficiency, and a screening method of a gene encoding a functional protein utilizing the ligation method, and a mass production method of the functional protein.SOLUTION: To a 5' side of a first DNA reverse primer and a 5' side of a second DNA forward primer, respectively, a linker sequence is provided and 5' terminal is phosphorylated. After amplifying each of the first and the second DNAs with PCR, the phosphorylated terminal chain is λ exonuclease-digested until the linker region becomes a single chain, annealed and substitution synthesized with BstDNA polymerase, and a double-stranded fusion DNA is produced. By selecting two kinds of DNA libraries as the first and the second DNAs, a double-stranded fusion DNA library is prepared, thereby a fusion protein library can be prepared by combining with a phage display method, so that the functional protein can be screened using target functionality as an index. Further by repeating the screening, the target functional protein can be selectively obtained. For example, a single chain antibody having high linkage activity to a target substance can be produced.

Description

  The present invention relates to a gene ligation method and a method for producing a single chain antibody using the same.

1. Highly functional single chain antibody 1-1. Usefulness of single-chain antibody The monoclonal antibody invented in 1970 was a mouse antibody and did not produce clinical effects as expected, but in the 1990s human immunoglobulin genes were expressed in CHO cells and the like. Since the development of this technology, expectations for antibody drugs have increased again and research and development have been activated. While transgenic mice that directly produce human antibodies have also been developed, technological development for screening optimal antibodies from an enormous amount of antigen library by phage display has also progressed, and antibody production technology has progressed dramatically. In particular, the achievement in the antibody drug field is remarkable, and the development of new drugs is progressing one after another. The importance of antibodies, including their use as diagnostics and research tools, has been increasing recently.

  In particular, a single chain variable fragment (scFv) in which only heavy chain (VH) and light chain (VL) antigen recognition sites are linked has a molecular weight of approximately 25 kDa, which is much larger than IgG (molecular weight is 150 kDa). It is small, highly stable, has good infiltration into tissues, and is extremely useful. It can be expressed in E. coli and can be mass-produced. If expressed in a mammalian cell, it can be used to elucidate the physiological function of the antigen in the cell by inhibiting the antigen function in the cell. Moreover, it is possible to increase binding activity and specificity by introducing a mutation into the gene. Many applications are expected, such as fusion with other proteins to function, use as multimers, and expression in cells.

1-2. Problems in the production of single-chain antibodies In order to produce functional antibodies more effectively and efficiently, there is a lack of technology to select functional optimal antibodies using evolutionary engineering techniques such as phage display. I wo n’t. In the case of the phage display method, the phage displaying the optimal antibody finally selected is expressed in a large amount as a recombinant antibody in E. coli as it is, or the gene is isolated and linked to an appropriate vector and then E. coli or CHO cells. And so on. Antibodies displayed on phage include Fab and single chain antibodies.
Single-chain antibodies are very useful molecules because of their size, and VL and VH antibody genes are isolated from hybridomas that produce antibodies that bind to antigens, and VL and VH antibody genes are linked and incorporated into phagemids. VL and VH antibody genes are isolated from spleen and lymphoid tissues of immunized animals, VL and VH antibody genes are ligated, incorporated into phagemids, expressed on phage, and combined by panning A technique of isolating a phage displaying a single chain antibody having activity and obtaining a single chain antibody having binding activity is used. In the latter case, using a cDNA obtained from the spleen of an experimental animal such as a mouse, a “single-chain antibody library” with a huge sequence variation that retains the antibody diversity of immune cells in vivo is created. To do. By repeating the panning step with the target substance in the single chain antibody library, it is possible to concentrate single chain antibodies having high binding properties with the target substance, which is extremely effective for the production of functional single chain antibodies. In creating a “single chain antibody library”, it is important to link VH and VL so that there is no mutation, deletion, or shift of the gene. : CDR region), it is necessary to produce a single-chain antibody so that production is not hindered and the diversity is maintained.

In order to produce a single chain antibody or a single chain antibody library, it is necessary to isolate the VH derived from the antibody heavy chain gene and the VL derived from the light chain gene, and then link them via an appropriate linker. There are mainly two methods as described below (see FIG. 1).
(1) Two-step cloning method: A method in which VH and VL are amplified by the PCR method, VL is first inserted into a phagemid vector and increased in E. coli, and then VH is inserted into a phagemid and cloned in E. coli (non-patent literature) 1).
(2) VL-VH assembly method: A method in which VH and VL are each amplified by the PCR method and then ligated, and the ligated product is incorporated into a phagemid (Non-patent Document 2).
However, the method (1) takes many steps, takes a long time, and when the starting gene is not a single antibody gene but is applied to the above antibody library, There is a disadvantage that the size of the obtained antibody library is reduced due to the uneven amplification efficiency of each gene.
The method (2) has few steps, but since it is ligated by PCR, frame shift or deletion is likely to occur, and it is difficult to obtain a functional single chain antibody. In addition, when the start gene is not a single antibody gene but applied to a VH library and a VL library, the antibody recognition sites in the gene have very large variations. It often happens that the PCR reaction is inhibited and only specific genes are amplified, resulting in a significant reduction in the size of the library.
As a method for linking VL and VH genes in (2), usually, a linker region is added to the C-terminal side of VL and the N-terminal of VH, and the overlapping of the linker parts is used to perform VH by overlapping PCR reaction. , VL are widely used (see FIG. 2, note: the order of VH and VL may be reversed). This method is simple, but it is amplified by PCR reaction after each top strand and bottom strand are completely single-stranded, so that non-specific adhesion is likely to occur, and gene deletion, insertion, replacement, There is a drawback that shift is likely to occur. Further, when applying to VH and VL libraries, there is a fatal defect that only clones having a specific sequence among the diversity sequences are selectively amplified.
As described above, the conventional method for producing a functional single-chain antibody has the above-mentioned problems, and it is not easy to produce a high-quality single-chain antibody that recognizes an antigen with high affinity.

2. Gene ligation method (DNA molecule ligation method)
As a method for synthesizing a double-stranded gene by ligating short DNA fragments, various ligation methods are known. Synthesis of short single-stranded DNA covering all gene sequences for both top strand and bottom strand, annealing reaction and ligation (Non-patent Document 3), and part of top and bottom strands alternately overlap There is a method of creating a double-stranded gene by synthesizing and annealing the single-stranded part after polymerase annealing (Non-patent Document 4), but both are low in efficiency and practical because of many gene errors is not. In addition, even if the single-stranded region of the nucleic acid is not completely complementary, it often hybridizes with other single-stranded regions, and it is not easy to proceed with the above reaction with high specificity.
1-2. The overlap PCR described in is a gene ligation method that is often used. In principle, if there are overlapping sequences between fragments to be ligated, dozens of fragments can be ligated at once in a single PCR reaction. However, deletion, insertion, substitution, and shift of genes occur, which is realistic. is not. In addition, when a diversity region exists in the sequence in the DNA fragment to be joined as in a gene library, if a sequence similar to the binding region appears in the diversity region part, the original PCR reaction is inhibited. There is a defect that only clones having a specific sequence are selectively amplified.

  Gibson et al. Developed a method for efficiently ligating double-stranded fragments amplified by PCR (Non-patent Document 5, Patent Document 1). Adjacent fragments have a common sequence of about 40 bases, and the 5 'end of the fragment is digested with T5 exonuclease to make the common portion into a single strand. Then, the part is annealed. In this method, a polymerase is allowed to act to synthesize a complementary strand of the single-stranded portion remaining by annealing, and the two fragments are connected with Taq ligase (ligation enzyme) to link the genes. In normal 5'-3 'exonuclease, since the digestion reaction is carried out quickly, it is difficult to stop the reaction in the middle, and the double strand is completely digested into a single strand and then transferred to the next annealing step ( However, in the case of T5 exonuclease, the digestion efficiency decreases every time several bases are digested. By utilizing this property of T5 exonuclease, it is theoretically possible to shift to the annealing step in the middle of the digestion efficiency drop. However, in practice, it is difficult to accurately control the digestion reaction of T5 exonuclease. Insufficient digestion makes it difficult for the annealing process to specifically associate the two fragments, while too much digestion results in longer single-stranded portions and non-specific binding due to their exposure, which is unexpected A purified product will be produced. Therefore, it is necessary to set the common part of both DNA fragments as long as about 40 bases, and the primer becomes long. If too long a primer is used, there is also a disadvantage that the first stage of PCR amplification is not performed efficiently. In addition, in the ligation step using T5 exonuclease, the 5 ′ end, which is not the ligation part of the two fragments, is also digested to form a single-stranded region and become a sticky end, so unexpected binding between fragments occurs. In addition, when incorporated into a vector, there is a drawback that the subsequent operation is greatly hindered, such as the restriction enzyme treatment cannot be performed directly on both ends of the fragment.

  From the above, it is desired to develop a gene ligation method that can also be applied to the production of a single-chain antibody, which suppresses unnecessary background reaction and ligates genes efficiently without mistakes. When applied to a single-chain antibody library, a method capable of creating a high-quality single-chain antibody that recognizes an antigen with high affinity has been desired.

US release 2010/0035768 Patent No. 4700805

2010scFv by Two-step Cloning Dafine Muller Antibody EngineeringVol.2 Ed. Roland Kontermann and Stefan Dubel Springer 2011 Construction of scFv Fragments from Hybridoma or Spleen Jonas V. Schaefer, Annemarie Honegger and Andreas Pluckthum, Antibody Engineering Vol.1Ed. Roland Kontermann and Stefan Dubel Springer Y. Jigami, M. Murakami, H. Harada, H. Tanaka Gene, 43 (1986), pp. 273-279 Expression of synthetic human-lysozymegene in Saccharomycescerevisiae: use of syntheticchicken-lysozyme signal sequence for secretion and processing Y. Tsuchiya, K. Morioka, J. Shirai, K. Yoshida and S. Inumura Nucleic Acids Symposium Series No. 50 275-276 2006 Comparison of artificial synthesis methods of gene Daniel G Gibson, Lei Young, Ray-Yuan Chuang, J Craig Venter, Clyde AHutchison III & Hamilton O Smith, NATURE METHODS VOL.6 NO.5 MAY2009 343 Enzymatic assembly of DNA molecules up toseveral hundred kilobases

  An object of the present invention is to provide a gene ligation method for accurately and highly efficiently linking two kinds of DNAs or DNA libraries, and screening for a gene encoding a functional protein using the ligation method. It is to provide a method and a method for mass production of functional proteins. In particular, an object is to provide a method for creating a high-quality single-chain antibody that recognizes an antigen with high affinity.

The present inventors, as a method for producing a functional single chain antibody, ligated after amplifying VH and VL by the PCR method, as shown in the above “VL-VH assembly method” in that the number of steps is small. Therefore, the method of incorporating the ligated product into the phagemid was considered suitable. And, aiming to improve the “overlap extension PCR method” used as a general-purpose gene ligation method at that time, the background reaction of the ligation method and the cause of many mistakes have been pursued . As a result, the main reason for reducing the library size when the ligation method is applied to the production of a single chain antibody library is that amplification by PCR is essential for ligation, and that I thought that it was to form a single-stranded DNA causing non-specific binding. Therefore, we have intensively studied a method for joining two DNA fragments without using the PCR method, in which the number of regions that are exposed as single-stranded DNA is reduced and the time during which single-stranded DNA is generated is as short as possible. After trial and error, they came up with a “gene linking method” as a solution.
Specifically, when a VL gene and a VH gene are amplified by PCR, a linker sequence is added to one of the C-terminal and the other N-terminal primers, and phosphoric acid is added to the 5 ′ end. The phosphorylated 5 ′ ends of each of the two types of DNA fragments obtained were digested with λ exonuclease to make the linker portion single-stranded, VL and VH were ligated, and then Bst DNA polymerase was used to make the 3 ′ direction. While removing the remaining complementary strand, a new complementary strand was synthesized (strand displacement synthesis), and a complete double strand of VL and VH linked by a linker could be produced.
When applied to a single VL and VH gene, high-quality VL-VH-linked fragments can be obtained by purification, and Escherichia coli and animal cells can be transformed and mass-produced. In addition, when applied to VL and VH libraries, a single-chain antibody library inserted into a phagemid and transformed into E. coli can be prepared. Therefore, the target functional can be achieved by repeating the panning process using a target substance. Single chain antibodies can be obtained.

Furthermore, by utilizing the property that the λ exonuclease used in the present invention cannot digest Phosphorothioate DNA, the non-specific ( Reaction to the non-phosphorylated 5 ′ end) was prevented. In addition, by converting the portion close to the 3 ′ end of the phosphorylated primer into S, the length of the DNA digested by λ exonuclease can be controlled, and a highly diverse CDR region can be converted into a single strand. Which could be avoided and drastically reduced the background reaction.
The above knowledge was obtained and the present invention was completed.

That is, the present invention is as follows.
[1] In a gene linking method for linking a first DNA and a second DNA via a linker moiety,
Here, the first DNA and the second DNA are the same or different DNA molecules, each being a single DNA molecule or any DNA molecule in a DNA library,
A gene ligation method comprising the following steps (1) to (5);
(1) A step of preparing a first and second DNA amplification primer set, the 5 ′ side of the first DNA amplification reverse primer and the second DNA amplification forward primer 5 ′ Adding a linker sequence to each of the sides and phosphorylating its 5 ′ terminal base;
(2) Amplifying each of the first and second DNAs by PCR to obtain first and second ′ DNAs amplified in a state where one strand has a phosphorylated linker region,
(3) a step of digesting with λ exonuclease until the linker region provided in the 1 ′ and 2 ′ DNA becomes a single strand;
(4) Annealing both of them,
(5) A step of causing a strand displacement reaction in the 3 ′ direction by causing Bst DNA polymerase to act.
[2] In the step (1), any one of 1 to 6 bases from the 3 ′ side in the linker sequence provided in each of the first DNA amplification reverse primer and the second DNA amplification forward primer The method according to [1] above, wherein the base is converted to S.
[3] In the step (1), the 5 ′ terminal base of the first DNA amplification forward primer and the 5 ′ terminal base of the second DNA amplification reverse primer are converted to S. The method according to [1] or [2] above.
[4] DNA or DNA in a DNA library in which the first DNA encodes the H chain variable region of an antibody, and DNA in the DNA library or in which the second nucleic acid encodes the L chain variable region The method according to any one of [1] to [3] above, wherein vice versa or vice versa.
[5] In the method according to any one of [1] to [4], when the first and second DNAs are DNAs in a DNA library, the final products of the steps (1) to (5) are A fusion DNA library comprising a group of fusion DNA molecules in which arbitrary DNA molecules selected from each of the first and second DNA libraries are linked via a linker.
[6] A fusion protein library in which the fusion DNA library according to [5] is linked to a phagemid and transformed into Escherichia coli and associated with the fusion protein on the phage surface.
[7] A fusion protein having a binding activity with a target substance or a gene encoding the same, comprising a panning step for binding the target substance to the surface of the substrate on which the target substance is immobilized with respect to the fusion protein library according to [6] Screening method.
[8] The fusion protein library according to [6] above has a binding activity with a target substance by repeating a panning step, a growth step, and a purification step for binding the target substance to the immobilized substrate surface. Mass production method of high fusion protein.
[9] The method according to [8] above, wherein the fusion protein is a single chain antibody.

In the gene ligation method of the present invention, unlike the conventional overlap extension PCR ligation method, the PCR method is used only when the first VH and VL genes (VH and VL libraries) are prepared. Since no PCR method is used, VH and VL can be linked without gene mutation, deletion or shift. In addition, even when applied to VH and VL libraries, the exonuclease digestion from the 5 ′ end can be controlled, so that even a highly diverse CDR region is not single-stranded, and the linker portion is bound. Since non-specific binding other than that can be prevented, a single-chain antibody library that maintains the original diversity of CDRs can be produced, so that an excellent functional single-chain antibody can be created easily and reliably. Long chains can be joined together by the linking method, and application in a wide range of practical fields is expected. It is also a great advantage that the reaction can be completed in a short time in a single tube, and contamination and sample loss can be minimized.
Furthermore, by converting the 5 ′ end of the non-phosphorylated primer to S during amplification of VL and VH, the reaction of λ exonuclease to non-specific (non-phosphorylated 5 ′ end) can be made zero. By converting the portion near the 3 ′ end of the phosphorylated primer to S, the length of the DNA digested by λ exonuclease can be reliably controlled. That is, DNA from the phosphorylated 5 ′ end to the S-modified portion is digested, and only that portion becomes a single strand. Thereby, only the complementary part of VL and VH can be single-stranded, and the background reaction can be drastically reduced.
In the conventional method using T5 exonuclease (Non-patent Document 5, Patent Document 1), it is difficult to control the digestion of T5 exonuclease, so it was necessary to add a common sequence of at least about 40 bases to the primer used. However, since the digestion with λ exonuclease in the present invention can be completely controlled by raising the reaction temperature, a sufficiently high reaction is obtained with 20 bases in the common part of the primers. In addition, no digestion with λ exonuclease occurs at the end where the ligation is not performed, and there is no concern that the subsequent gene manipulation will be hindered. Further, in the present invention, since only the Bst DNA polymerase can synthesize a complementary strand while peeling off the strand with the phosphate added to the 5 ′ end, and the two fragments can be ligated. Neither a process nor a PCR amplification process is required. The increase in the number of steps increases the probability of gene deletion / insertion / replacement / shift, and this is a fatal defect in library preparation, but it is also an advantage of the present invention that it can be avoided. . In addition, since Bst DNA polymerase has high heat resistance, gene synthesis can be performed at high temperature (65 ° C.). Therefore, annealing at high temperature, that is, annealing under more severe conditions is possible. Therefore, there is an advantage that non-specific binding can be suppressed. On the other hand, in the method using T5 exonuclease, since the polymerase reaction / ligation reaction after annealing is performed at 50 ° C., a nonspecific reaction is likely to occur.

Method for producing single chain antibody (scFv) Single chain antibody production: VH and VL ligation by overlap extension PCR Production of single-chain antibodies: using λ exonuclease Control of λ exonuclease activity by primer S-activation VH and VL antibody gene ligation by λ exonuclease method Gene sequence of chicken single-chain antibody prepared by λ exonuclease method VH and VL antibody gene ligation from antibody gene library by λ exonuclease method Diversity of single-chain antibody libraries prepared from antibody gene libraries by λ exonuclease method Binding properties of single-chain antibodies obtained by lambda exonuclease method Gene sequence of rabbit IgG recognition single chain antibody obtained from VH and VL libraries by λ exonuclease method

1. Gene ligation method of the present invention
1-1. Basic gene ligation method The gene targeted by the “gene ligation method” of the present inventors is basically a ligation method between double-stranded DNA molecules (fragments). It is a technique that can be used for binding DNA fragments and also when DNA libraries are included. In the present invention, when referring to a DNA library, a gene library, etc., it is of course the case that the DNA library is composed of a group of DNA molecules having a completely randomized region. A group of DNA genes that hold is also referred to as a DNA library. For example, spleen-derived VH and VL gene groups are also referred to as VH and VL gene libraries. Further, when linking VH and VL gene libraries, it means linking of arbitrary genes constituting each of VH and VL gene libraries.
As an embodiment of the present invention, as a typical example, when VH and VL genes are linked, and when a gene in VH and VL gene libraries is linked to produce a functional single chain antibody (scFv) Will be described in detail. For the production of a functional single-chain TCR (scTCR: International Immunology Volume 11, Issue 5 p.745-751. 1999) that links the Vα and Vβ genes derived from the T cell receptor using the same technique. Can also be used. In addition, it can be applied to the binding of domain genes in receptors having various domain structures.

1-1-1. Preparation of double-stranded DNA fragments Two types of double-stranded DNA fragments (two types of genes) to be bound are prepared. The length of the double-stranded DNA fragment can be any length as long as it can be amplified by the PCR method, but is usually 100 to 3000 bases, preferably 150 to 2000 bases, more preferably 200. -1000 bases in length. If necessary, each is amplified in advance and purified before use. When two regions on the same cDNA or genomic DNA are selected as double-stranded fragments, they can be amplified simultaneously. As an amplification method at that time, an amplification method by PCR is generally preferable.
Here, when a double-stranded DNA fragment having a complete randomized region is to be obtained, a known randomizing method such as an error-introducing PCR method (Proc. Natd. Acad. Sci. USA Vol. 89, pp. 3576). -3580, April 1992), a library of two types of double-stranded DNA fragments having a randomized region is formed, and each fragment group is purified.

1-1-2. Amplification of phosphorylated double-stranded DNA fragment Next, on the 5 ′ side of the reverse primer for amplification of one fragment and the 5 ′ side of the other forward primer, a linker part for providing a common sequence is provided, The 5 ′ end is phosphorylated. A technique for adding phosphoric acid to the 5 ′ end is a well-known technique, and a technique for adding phosphoric acid from ATP in a solvent using T4 Polynucleotide kinase is common.
Each double-stranded DNA fragment is amplified using the above phosphorylated primer, and a linker-fused double-stranded DNA fragment in which the 5 ′ end of the sense strand or antisense strand is phosphorylated is obtained and purified. In the case of a library, purification is performed for each library consisting of each fragment group.

1-1-3. Single-Strand Digestion and Annealing of Phosphorylated Linker Fusion Double-Stranded DNA Fragment A λ exonuclease (EC 3.1.11.3) is allowed to act on a buffer mixed with two types of phosphorylated linker-fused double-stranded DNA fragments (EC 3.1.11.3). (For example, 37 ° C., 30 seconds) The 5 ′ end phosphorylated strand is digested at least about 15 to 20 bases from the 5 ′ end including the linker portion. However, when the double-stranded DNA fragment has a random region, λ exonuclease digestion stops before the random region because the original diversity may be impaired if the random region becomes a single-stranded state. Adjust as follows. In λ exonuclease digestion, λ exonuclease can be deactivated and the reaction can be stopped by raising the temperature, so it can be adjusted sharply, and if the temperature of the system is raised to 70 ° C to 75 ° C, , Λ exonuclease digestion reaction is completed. In order to anneal the linker part in a single-stranded state, the double-stranded DNA fragment is annealed in the linker part by lowering the temperature to 65-50 ° C.

1-1-4. Add Bst DNA polymerase (DNA-directed DNA Polymerase EC 2.7.7.7) to the linking system of linker-fused double-stranded DNA fragments, and in the 3 'direction for each of the double-stranded DNA fragments annealed at the linker. While removing the remaining complementary strand, a new complementary strand is synthesized to produce a complete double strand in which two types of double-stranded DNA fragments are linked via a linker.

1-2. Conversion of DNA fragment into S (Phosphorothioate)
The λ exonuclease used in the present invention has the property that it cannot digest S (Phosphorothioate) DNA . S-treatment at one location of two kinds of DNA fragment amplification primers in the process (convert one or more phosphoric acid oxygens of 1 to 6 bases into S. For example, -PO _{4 is changed} to -PO ₃ S Hereinafter, simply “S” may be performed.
Specifically, for the non-phosphorylated primer, the 5 ′ end is converted to S, thereby preventing the reaction of λ exonuclease to non-specific (non-phosphorylated 5 ′ end).
On the other hand, with regard to the phosphorylated primer, the position of the phosphorylated primer is converted to S by converting one or more of the phosphoric acid groups of 1 to 6 bases in the vicinity of the 3 ′ end (2 to 8 bases from the 3 ′ end) Since the digestion of λ exonuclease stops, the length of DNA exposed in a single-stranded state can be accurately controlled.
In the present invention, since Bst DNA polymerase that synthesizes DNA by strand displacement reaction is selected, even if there is S-modified DNA in the double-stranded portion after annealing, the complementary strand synthesis reaction is not hindered. S-DNA does not remain in the finally obtained double-stranded DNA fragment.

1-3. Therefore, the gene ligation method of the present invention can be expressed as follows.
In a gene linking method for linking a first DNA and a second DNA via a linker moiety,
Here, the first DNA and the second DNA are the same or different DNA molecules, each being a single DNA molecule or any DNA molecule in a DNA library,
A gene ligation method comprising the following steps (1) to (5);
(1) A step of preparing a first and second DNA amplification primer set, the 5 ′ side of the first DNA amplification reverse primer and the second DNA amplification forward primer 5 ′ Adding a linker sequence to each of the sides and phosphorylating its 5 ′ terminal base;
Here, any one of 1 to 6 bases from the 3 ′ side in the linker sequence provided in each of the first DNA amplification reverse primer and the second DNA amplification forward primer may be converted to S. preferable. At the same time, it is more preferable that the 5 ′ terminal base of the first DNA amplification forward primer and the 5 ′ terminal base of the second DNA amplification reverse primer are converted to S.
(2) Amplifying each of the first and second DNAs by PCR to obtain first and second ′ DNAs amplified in a state where one strand has a phosphorylated linker region,
(3) a step of digesting with λ exonuclease until the linker region provided in the 1 ′ and 2 ′ DNA becomes a single strand;
(4) Annealing both of them,
(5) A step of causing a strand displacement reaction in the 3 ′ direction by causing Bst DNA polymerase to act.
By using the gene ligation method as a method for ligating DNA in the first and second DNA libraries, the first and second DNA libraries can be used as the final products of steps (1) to (5). A fused DNA library consisting of a group of fused DNA molecules in which arbitrary DNA molecules selected from the above are linked via a linker can be prepared.
The fusion DNA library can be used for screening functional proteins and genes encoding the same in various screening systems.
For example, since the fusion DNA library can be linked to a phagemid and transformed into E. coli to produce a fusion protein library associated with the fusion protein on the phage surface, a screening method using the fusion protein library, and The mass production method of the obtained functional protein is described.

2. Screening method for target functional protein The double-stranded DNA fragment linked by the linker obtained in the above step is appropriately connected to a vector, and the target fusion protein can be mass-produced in transformed cells.
When applied to a library of double-stranded DNA fragments, a double-stranded DNA fragment library linked by a linker is produced. Typically, these are linked to a phagemid, E. coli is transformed, each of them is mass-produced, and then the protein displayed on the phage surface is screened using the target function as an indicator. For example, for the purpose of obtaining a protein having a strong affinity for a specific target substance, a clone having a strong binding activity can be obtained by a screening step in which a phage is bound to the surface of a substrate on which the target substance is immobilized. A ribosome assay method other than the phage display method may be screened by a cell-free display method.

As a typical fusion protein library, the above 1-3. The fusion DNA library obtained in step 1 was linked to phagemid, E. coli was transformed, and the fusion protein library related to the fusion protein on the phage surface was used to bind the functional protein of the present invention, particularly the target substance. The screening method for fusion proteins having can be expressed as follows.
1-3. The fusion DNA library obtained in (1) is linked to phagemid, E. coli is transformed, and the fusion protein library associated with the fusion protein on the phage surface is bound to the substrate surface on which the target substance is immobilized. A method for screening a fusion protein having a binding activity with a target substance or a gene encoding the same.
Moreover, the mass production method of the fusion protein having the binding activity with the target substance can be expressed as follows.
1-3. A panning step of linking the fusion DNA library obtained in step 1 to phagemid, transforming E. coli, and binding the target substance to the immobilized substrate surface to the fusion protein library associated with the fusion protein on the phage surface; A method for mass production of a fusion protein having a high binding activity to a target substance by repeating a growth step and a purification step.

3. Linking VH and VL genes
3-1. "VL-VH assembly method"
As a typical example of the gene ligation method developed by the present inventors, a case where it is applied to a double-stranded fragment of a VH gene and a VL gene (FIG. 3) will be specifically described below. The case where it is applied to the VH gene and the VL gene may be particularly referred to as “VL-VH assembly method”.
(1) First, the VL gene and the VH gene are each amplified by PCR. At this time, the devices (a) and (b) are added to the primers at the C end of VL and the N end of VH.
(A) A linker sequence is added to the primer.
(B) Add phosphoric acid to the 5 ′ end.
Here, since the linker sequence is usually 9 to 60 bases, preferably about 21 to 51 bases, the length of the whole primer is usually 24 to 75 bases, preferably 36 to 66 bases, respectively. The technique of adding phosphoric acid to the 5 ′ end is a well-known technique, and in this example, ATP-derived phosphoric acid was added to the 5 ′ end with the kinase according to the specification of the kit for T4 Polynucleotide kinase.
Using the VL and VH forward and reverse primers (SEQ ID NOs: 1, 3, 5, and 7) shown in [Table 1] below, the VL and VH genes are amplified by PCR reaction from the cDNA from the hybridoma. The following method may be applied to the VL and VH genes. In addition, if VL and VH genes are amplified from a cDNA library derived from the spleen of an experimental animal by PCR reaction and the VL and VH gene fragments are purified, VL and VH libraries retaining the original diversity of antibodies in the CDR portion are prepared. Therefore, the following method may be applied to the VL and VH libraries.
At that time, if an experimental animal is immunized with a target substance in advance, a VL and VH library capable of creating a single-chain antibody having a stronger binding activity to the target substance can be produced.
The obtained VH gene having a linker region to which phosphate was added at the 5 ′ end and the VL gene having a linker antisense region to which phosphate was added to the 5 ′ end on the complementary strand side (the VL and VH genes were opposite) Or each may be a library. The same shall apply hereinafter).

(2) The phosphorylated 5 ′ ends of the sense and antisense strands of the VL and VH gene fragments are digested with λ exonuclease to make the linker portion single-stranded, and λ exonuclease is inactivated by heat denaturation.
(3) Since the linker portion that has been digested with λ exonuclease to become a single strand has a complementary sequence of VL and VH, after λ exonuclease is deactivated by heat denaturation, an appropriate temperature is obtained. And VL and VH are connected.
(4) Next, the Bst DNA polymerase enzyme is allowed to act to replace the complementary strand remaining in the 3 ′ direction for both the top strand and the bottom strand, and a new complementary strand is synthesized to form a complete double strand. To do.
(5) After the reaction is completed (this reaction is completed in a short time of about 30 minutes), the purified product is subjected to agarose electrophoresis and purified, whereby a high-quality VL-VH linked fragment can be obtained.
(6) When the above method is applied to a VL library and a VH library, the obtained VL-VH linked fragment group is inserted into a phagemid and transformed into E. coli to prepare a single chain antibody library. The target functional single-chain antibody is concentrated by repeating the panning process using the immobilized target substance.

[Table 1] Primer 1) VL forward primer: (SEQ ID NO: 1)
5'-GTGGCCCAGCCGGCCCTGACTCAGCCGTCCTCGGTGTC-3 '
2) S-VL forward primer: (SEQ ID NO: 2)
5'-gaTCGTGGCCCAGCCGGCCCTGACTCAGCCGTCCTCGGTGTC-3 '
(Lowercase part ga is converted to S)
3) VL reverse primer: (SEQ ID NO: 3)
5'-GGAAGATCTAGAGGACTGACCTAGGACGGTCAGG-3 '
4) S-VL reverse primer: (SEQ ID NO: 4)
5'-GGAAGATCTAGAGGACTGACCTAGGACGGTCagG-3 '
(Lowercase AG converted to S)
5) VH forward primer: (SEQ ID NO: 5)
5'-GGTCAGTCCTCTAGATCTTCCGCCGTGACGTTGGACGAG-3 '
6) S-modified VH forward primer: (SEQ ID NO: 6)
5'-GGTCAGTCCTCTAGATCTTCCGCCGTGACGTtgGACGAG-3 '
(S converted to lower case TG)
7) VH reverse primer: (SEQ ID NO: 7)
5'-CCTTTTGCGGCCGCACTAGTGGAGGAGACGATGACTTCGGTCC-3 '
8) S-modified VH reverse primer: (SEQ ID NO: 8)
5'-ccTTTTGCGGCCGCACTAGTGGAGGAGACGATGACTTCGGTCC-3 '
(Lowercase part CC is converted to S)

3-2. Method for Producing Single-Chain Antibody Library As described above, VL and VH gene regions are amplified by PCR reaction using the primers of [Table 1] above from the spleen-derived cDNA library, and the VL and VH genes By purifying the fragments, VL and VH libraries that retain the original diversity of antibodies in the CDR portion can be prepared.
At that time, randomized VL and VH libraries can be prepared by using an existing VH gene and VL gene as a template and randomizing at least one of each CDR region by a method using a degenerate oligonucleotide. . In addition, VL and VH libraries with a higher degree of randomization can also be produced by randomizing CDR regions using degenerate oligonucleotides using spleen-derived VH libraries and VL libraries as templates.

3-3. Method using S-modified DNA Furthermore, in the present invention, it is possible to cause a more specific reaction by using the property that λ exonuclease cannot digest S-modified (Phosphorothioate) DNA (FIG. 4). By converting the 5 ′ end of the non-phosphorylated primer to S during VL and VH amplification, the reaction of λ exonuclease to non-specific (non-phosphorylated 5 ′ end) can be made zero. Further, by converting the portion near the 3 ′ end of the phosphorylated primer into S, the length of the DNA digested by λ exonuclease can be controlled. That is, DNA from the phosphorylated 5 ′ end to the S-modified portion is digested, and only that portion becomes a single strand. Thereby, only the complementary part of VL and VH can be single-stranded, and the background reaction can be drastically reduced.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(Example 1) In order to prepare an amplified naive library for VH and VL , the spleen was extracted from a chicken, RNA was purified, and cDNA was synthesized using it as a template. Using this cDNA as a template, VH and VL genes were each amplified by the PCR method using antibody gene-specific primers [Table 1].
After completion of PCR, agarose gel electrophoresis is performed, and the VL and VH parts are excised and purified. The purified VL and VH were incorporated into a pGEM vector (Promega) by TA cloning and transformed into E. coli DH5α. After transformation, they were plated on LB plates (Sigma Aldrich) and single clones were collected the following morning. This was cultured and the plasmid was purified. The gene sequences of VL and VH were analyzed and confirmed to be VL and VH genes.

Next, the 5 ′ end of the reverse primer of VL and the 5 ′ end of the forward primer of VH were treated at 37 ° C. for 30 minutes according to the instruction manual using T4 Polynucleotide kinase (manufactured by NEB) at the following blending ratio. And phosphorylated.
(Primer phosphorylation)
Primer 10 μl
T4 Polynucleotide kinase 2μl
10 x buffer 2 μl
6 μl of water

Using primers and phosphorylated primers, VH and VL genes were amplified from the plasmids of the clones whose sequences were confirmed by PCR under the following conditions.
(PCR reaction)
1 μl of plasmid
3 ′ primer (SEQ ID NO: 1, 5) 0.5 μl
5 ′ primer (SEQ ID NO: 3, 7) 0.5 μl
PCR enzyme (KOD FX TOYOBO) 1 μl
2 x buffer 25 μl
dNTP 8 μl
14 μl of water
(Reaction conditions)
・ 98 ℃ for 1 minute × 1 time (98 ℃ for 10 seconds, 68 ℃ for 30 seconds) × 25 times ・ 68 ℃ for 2 minutes ・ 25 ℃
After completion of the PCR, agarose gel electrophoresis was performed, the VL and VH parts were cut out, and the VL and VH genes were purified.

(Example 2) Gene ligation reaction The phosphorylated part of the purified VL and VH gene was digested with λ exonuclease (manufactured by NEB) at 37 ° C for 30 seconds under the following compounding conditions to obtain complementary VL and VH. The array was exposed.
(Λ exonuclease reaction)
VL 5μl
VH 5μl
λ exonuclease 0.5μl
10 x buffer 2 μl
7.5 μl of water

Incubate the tube at 75 ° C for 30 seconds to inactivate λ exonuclease (NEB), add 2 µl of Bst polymerase (NEB) and 3 µl of 10x buffer, and incubate at 65 ° C for 20 minutes. Thus, the VL and VH genes were annealed, and at the same time, ligation and extension reactions were caused by Bst DNA polymerase to produce a double-stranded gene of VL-VH, that is, scFV. The prepared scFV was subjected to agarose gel electrophoresis, and the result of confirming the band is shown in FIG. The bands of the VL and VH genes disappeared, and the band was shifted to around 750 bp that combined both.

Example 3 Integration of Single Clone scFV into Phagemid A scFV double-stranded gene prepared was excised and purified after agarose gel electrophoresis. Subsequently, using restriction enzymes SfiI and NotI (Promega) according to the instruction manual of the company, the restriction enzyme SfiI and NotI were incorporated into the pCAMTAB5E vector treated with the same restriction enzyme, and transformed into Escherichia coli XL-1 Blue. After transformation, they were plated on LB plates and single clones were collected the following morning. This was cultured and the plasmid was purified. The gene sequence of scFV is analyzed, and the sequences of the VL and VH genes and the linker as the linking part are confirmed. The confirmed gene sequence of scFV is shown in FIG. There were no reading frame shifts, gene deletions, or gene insertions.

(Example 4) A spleen is extracted from a chicken immunized with amplified rabbit IgG of VH and VL libraries , RNA is purified, and cDNA is synthesized using it as a template. Using this cDNA as a template, VH and VL genes were each amplified by PCR using antibody gene-specific primers (SEQ ID NOs: 1, 3, 5, and 7). At this time, primers containing S-formation (SEQ ID NOs: 2, 4, 6, 8, Sigma Aldrich) were also used.
Among the above primers, the 5 ′ end of the VL 3′-side primer (SEQ ID NO: 1, 2, 5, 6) and the 5 ′ end of the VH 5′-side primer (SEQ ID NO: 3, 4, 7, 8) are shown below. Phosphorylation is carried out under the following conditions.
(Primer phosphorylation)
cDNA 1μl
3 ′ primer 0.5 μl
5 ′ primer 0.5 μl
PCR enzyme (KODFX) 1 μl
2 x buffer 25 μl
dNTP 8 μl
14 μl of water
(PCR reaction conditions)
・ 98 ℃ for 1 minute × 1 time (98 ℃ for 10 seconds, 68 ℃ for 30 seconds) × 25 times ・ 68 ℃ for 2 minutes ・ 25 ℃
After completion of PCR, agarose gel electrophoresis is performed, and the VL and VH parts are excised and purified.

(Example 5) VH and VL gene ligation reaction in library The phosphorylated part of the purified VL and VH part genes was digested with λ exonuclease at 37 ° C for 30 seconds to expose the linker sequences of VL and VH.
(Λ exonuclease reaction)
VL 20μl
VH 20μl
λ exonuclease 3μl
10 x buffer 5 μl
2 μl of water
After incubating the tube at 75 ° C. for 30 seconds to inactivate λ exonuclease, 2 μl of Bst DNA polymerase and 3 μl of 10 × buffer were added, and incubated at 65 ° C. for 20 minutes. At the same time as annealing the VH gene, a ligation and extension reaction with BstDNA polymerase is caused to produce a double-stranded gene of scFV. The prepared scFV is subjected to agarose gel electrophoresis, and the scFV is cut out and a band is confirmed. The results are shown in (FIG. 7). As in the case of the single clone, the bands of the VL and VH genes disappeared, and the band was shifted to around 750 bp. When the S-primer was used, the most band shift occurred efficiently. That is, scFv was formed more efficiently.

(Example 6) Integration of scFV gene library into phagemid scFV gene was purified, treated with restriction enzymes SfiI and NotI (manufactured by Promega) according to the manufacturer's instructions, and treated with the same restriction enzyme pCAMTAB5E It was incorporated into a vector by ligation, and transformed into E. coli XL-1 Blue. After transformation, they were plated on LB plates and single clones were collected the following morning. This was cultured and the plasmid was purified. The gene sequence of scFV was analyzed, and the sequences of the VL and VH genes and the linker as the linking portion were confirmed.

(Example 7) Genetic diversity of library A 48 clone colony collected was used to amplify scFV by colony PCR, and the reaction solution was treated using BstOI (Promega) according to the instructions of the company. did. Thereafter, the treatment solution was subjected to agarose gel electrophoresis, and the diversity of the library was confirmed from the cleavage pattern of the 48 clones of the scFV gene. The results are shown in (FIG. 8). All 48 clones had different cleavage patterns with restriction enzymes, indicating that a library with sufficient diversity could be constructed.

(Example 8) Isolation of scFV having binding activity All E. coli XL-1 Blue transformation colonies of scFV gene library were recovered from the LB plate and reached 37 ° C and OD (595 nm) of 0.6 in 2 x YT medium. And infected with M13KO7 helper phage (NEB). Thereafter, the cells are cultured overnight at 37 ° C., and the scFV phage library is purified from the culture supernatant by polyethylene glycol precipitation. The purified VL-VH phage library was subjected to the first panning using a plate coated with an antigen (rabbit IgG), and the phage bound to the antigen (rabbit IgG) was eluted. The cells were infected with E. coli XL-1 Blue cultured until (595 nm) reached 0.6. Subsequently, M13KO7 helper phage was infected, and the infected XL-1 Blue was wound on an LB plate, and the next morning, all the E. coli XL-1 Blue transformation colonies were recovered, and the second panning was performed again. Panning was performed three times, and scFV having binding activity was isolated from the E. coli XL-1 Blue transformation clone after the last panning.

(Example 9) Confirmation of binding activity of isolated scFV by ELISA Ten E. coli XL-1 Blue transformation colonies were picked from the LB plate after 3 times of panning, and each was 37 ° C. and OD (595 nm) in 2 × YT medium. Was cultured until 0.63 was reached, and M13KO7 helper phage was infected. Thereafter, the cells are cultured overnight at 37 ° C., and the scFV phage is purified from the culture supernatant by polyethylene glycol precipitation. Disperse the dilution series of scFV phage into a 96-well plate (antigen-coated and then blocked with BSA) coated with antigen (rabbit IgG) and BSA (WAKO) as a negative control, reacted at 37 ° C. for 2 hours, and then washed. . Next, HRP-labeled anti-M13KO7 antibody (GE Healthcare) is diluted 4000 times, dispensed, reacted at 37 ° C. for 1 hour, and then washed. A chromogenic substrate DAB (SIGMA) is added to cause color development at room temperature, and then the absorbance at 450 nm is measured with a plate reader. The measurement results are shown in FIG. Binding activity was seen in No. 3, 5, 8, and 9 among 10 clones.

(Example 10) Sequence confirmation of scFV having binding activity No. 3, 5, 8, and 9 clones that confirmed binding activity were cultured, and the plasmid was purified. The gene sequence of scFV is analyzed, and the sequences of the VL and VH genes and the linker as the linking part are confirmed. As with the single clone, no shift in reading frame, gene deletion, gene insertion, etc. were observed. The result of the sequence analysis of No. 5 is shown in FIG.

[Sequence Listing Free Text]
Sequence number 1: VL forward primer
Sequence number 2: VL forward primer (S)
Sequence number 3: VL reverse primer
Sequence number 4: VL reverse primer (S)
Sequence number 5: VH forward primer
SEQ ID NO: 6: VH forward primer (S)
SEQ ID NO: 7: VH reverse primer
SEQ ID NO: 8: VH reverse primer (S)
SEQ ID NO: 9: DNA sequence of chicken scFv constructed with λ-exonulease method
SEQ ID NO: 10: DNA sequence of chicken scFv spefific to rabbit IgG

Claims (9)

In a gene linking method for linking a first DNA and a second DNA via a linker moiety,
Here, the first DNA and the second DNA are the same or different DNA molecules, each being a single DNA molecule or any DNA molecule in a DNA library,
A gene ligation method comprising the following steps (1) to (5);
(1) A step of preparing a first and second DNA amplification primer set, the 5 ′ side of the first DNA amplification reverse primer and the second DNA amplification forward primer 5 ′ Adding a linker sequence to each of the sides and phosphorylating its 5 ′ terminal base;
(2) Amplifying each of the first and second DNAs by PCR to obtain first and second ′ DNAs amplified in a state where one strand has a phosphorylated linker region,
(3) a step of digesting with λ exonuclease until the linker region provided in the 1 ′ and 2 ′ DNA becomes a single strand;
(4) Annealing both of them,
(5) A step of causing a strand displacement reaction in the 3 ′ direction by causing Bst DNA polymerase to act.   In the step (1), any one of 1 to 6 bases from the 3 ′ side in the linker sequence provided in each of the first DNA amplification reverse primer and the second DNA amplification forward primer is converted to S The method according to claim 1, wherein:   In the step (1), the 5 ′ terminal base of the first DNA amplification forward primer and the 5 ′ terminal base of the second DNA amplification reverse primer are converted to S. Item 3. The method according to Item 1 or 2.   Whether the first DNA is DNA encoding an antibody heavy chain variable region or DNA in a DNA library, and the second nucleic acid is DNA encoding an L chain variable region or DNA in a DNA library 4. The method according to any one of claims 1 to 3, characterized in that: or vice versa.   The method according to any one of claims 1 to 4, wherein when the first and second DNAs are DNAs in a DNA library, they are obtained as final products of the steps (1) to (5). A fused DNA library comprising a fused DNA molecule group in which arbitrary DNA molecules selected from each of the first and second DNA libraries are linked via a linker.   A fusion protein library in which the fusion DNA library according to claim 5 is linked to a phagemid and transformed into Escherichia coli to associate with the fusion protein on the phage surface.   A screening method for a fusion protein having a binding activity to a target substance or a gene encoding the same, comprising a panning step of binding the target substance to the surface of the substrate on which the target substance is immobilized with respect to the fusion protein library according to claim 6.   The fusion protein library according to claim 6, wherein a fusion protein having a high binding activity with a target substance is obtained by repeating a panning step, a proliferation step and a purification step for binding the target substance to the immobilized substrate surface. Mass production method.   The method according to claim 8, wherein the fusion protein is a single chain antibody.