JP3908257B2 - Oligonucleotides for chicken monoclonal antibodies - Google Patents

Description

  The present invention relates to a primer for amplifying a gene encoding a variable region of a recombinant chicken monoclonal antibody, and a gene amplification method using the primer. The present invention also relates to a novel oligonucleotide used as a primer for amplifying a gene encoding the variable region of a recombinant chicken monoclonal antibody.

A monoclonal antibody is a single antibody that recognizes only a specific part of an antigen. This technology is a key technology in the biotechnology field along with genetic recombination, and diagnostic and therapeutic drugs using this technology are rapidly spreading. Evolving.
Monoclonal antibodies are widely used, such as mouse and rat types, but there are some examples that the inventors have already proposed for chicken types. A great advantage of this chicken type is that it is possible to produce antibodies that are difficult to produce in mammals such as mice.

  Although chickens are phylogenetically lower than mammals, they are animals with extremely elaborate immunity similar to mammals, and so many useful chicken antibodies have been prepared so far. On the other hand, when an antibody capable of recognizing a biological component highly preserved between mammals including humans cannot be produced using a mammal, it has also been experienced that it can be produced as a chicken antibody. For this reason, as one means for preparing a large amount of chicken antibody, a method has been developed in which a laying hen is immunized with a specific antigen, and then an antibody transferred to the egg is produced and used as an egg yolk antibody (polyclonal antibody). Monoclonal antibodies cannot be produced by this method.

Mouse monoclonal antibodies are used in a very wide range of basic and application fields, but there are few successful examples of monoclonal antibodies other than mouse and rat antibodies. As an example of application with monoclonal antibodies other than mouse and rat, chicken monoclonal antibodies by the cell fusion method developed by the present inventors have already been reported. As described above, the great advantage of chickens is that antibodies that are difficult to produce using mice or rats can be easily obtained as chicken antibodies, and can also be produced as monoclonal antibodies.
Examples of successful chicken monoclonal antibodies using cell fusion technology by the present inventors include chicken monoclonal antibodies that recognize N-glycolylneuraminic acid (NeuGc) and prion protein (PrP) highly conserved in mammals. Chicken monoclonal antibody that recognizes). NeuGc is an antigen that serves as a human cancer marker, and is present in almost all mammals except humans. Therefore, it cannot produce antibodies in animals that are generally widely used as immunized animals such as mice, rats, and rabbits. In addition, PrP is known for its abnormal type to be a pathogen of mad cow disease or human CJD, but PrP has an amino acid sequence homology of 90% or more among mammals. On the other hand, since the homology between mammals and chickens is only about 30%, there have been few successful examples of mouse monoclonal antibody production that recognizes mammalian PrP produced so far. The present inventors have already succeeded in producing chicken-type mAbs capable of recognizing NeuGc and mammalian PrP, and were able to utilize them for human cancer diagnosis and mad cow disease / human CJD research.

However, the biggest drawback of the chicken-type monoclonal antibody previously developed by the present inventors is that the antibody-producing ability of the hybridoma (fusion cell) that produces the antibody is low.
As described above, a chicken monoclonal antibody can produce a monoclonal antibody that is difficult to produce in other mammals, but its antibody-producing cells (hybridomas) have a lower ability to produce antibodies than mice and rats. It was a drawback and a major obstacle to commercialization.

  On the other hand, in recent years, a technique for expressing a recombinant antibody (recombinant antibody) on the phage surface by utilizing genetic engineering technology has been developed. This phage display antibody technology is a system developed by Winter et al. Of MRC Laboratory in 1991 (see Non-Patent Document 1), in which an antibody gene is isolated from non-immune human peripheral blood lymphocytes and artificially VH, VL A scFv (single chain fragment of variable region) antibody diversified by gene shuffling was expressed as a phage fusion protein to obtain a specific antibody (see Non-Patent Document 2). This technique has been highly evaluated as a humanized antibody production technique that can avoid immunity and is replaced with a cell fusion method. At present, many practical antibodies have been prepared using highly immunized mouse spleen cells, and anti-PrP antibodies have also been prepared (see Non-Patent Document 3).

  Examples of using chickens include reports from the following two groups in Japan and overseas. Davies et al. Established three specific antibodies from an antibody gene derived from a non-immune Fabricius sac and an expression vector fd-tet-DOG 1, and this technique can be similarly applied to non-mammals and immune It was proved that it can be created avoiding (see Non-Patent Document 4). However, the reactivity of the produced antibody is extremely low, and there is a drawback that only a phage display antibody can be obtained from the structure of the expression vector. On the other hand, Yamanaka et al. Reported the construction of a specific phage display antibody exhibiting sufficient reactivity using an antibody V region gene derived from mouse albumin immunized spleen cells and an expression vector pPDS originally developed. Emphasis was placed on the use of an immune spleen cell-derived antibody gene library (see Non-Patent Document 5). The pPDS used here was developed as a mouse antibody expression vector (see Non-Patent Document 6) and is based on the cloning phagemid vector pBluescript II commercially available from STRATA GENE.

FIG. 1 shows the structure of the expression vector pPDS. pPDS is designed to induce the expression of scFv antibody cDNA incorporated using its lactose operon (Lac) promoter. Downstream of the promoter, a ribosome binding site (RBS) and an M13 geneIII leader sequence (g3l) are artificially linked, and the antibody gene incorporated at the cloning site consisting of EagI and BssHII is reconstituted via the periplasm of E. coli. It is designed to be expressed as a phage body. Further downstream, a mouse Cκ chain gene is linked in preparation for Fab antibody expression, leading to the structural site of cp3 and serving as a stop codon. This Cκ chain is a very effective tag for verifying whether the expression of the fusion protein is successful. In addition, this pPDS vector has a TAG (amber sequence) stop codon inserted between the Cκ chain and geneIII so that it can cope with the expression of soluble scFv or Fab antibody. Is expressed as a phage display antibody, but when cultured in a non-suppressor strain, a soluble antibody is secreted into the medium.
Since it is such an expression vector, even if it is derived from a chicken antibody gene in a soluble antibody, an antibody against a mouse Cκ chain must be used for detection. In the case of using a mouse antibody in a sandwich ELISA or the like, An accurate measurement of the antibody was not possible. This problem was also a matter to be solved because the present inventors received the pPDS grant from Dr. Yamanaka and tried to use it for the production of phage display antibodies from antibody-producing chicken hybridomas.

Winter, G., et al., Nature, 349, 293-299 (1991) Marks, J. D., et al., J. Mol. Biol., 222, 581-597 (1991) Williamson, R. A., et al., Proc. Natl. Acad. Sci. USA., 99, 7279-7282 (1996) Davis, E. D., et al., J. Immunol. Methods, 186, 125-135 (1995) Yamanaka, H. I., et al., J. Immunol., 157, 1156-1162 (1996) Yamanaka, H.I., et al., J. Biochem., 117, 1218-1227 81995)

  The present invention provides a method for producing a chicken monoclonal antibody suitable for mass production by gene recombination. The present invention also provides a novel plasmid vector and a novel primer therefor. Furthermore, the present invention provides a novel chicken monoclonal antibody.

The present invention relates to a method for producing a chicken monoclonal antibody by a genetic recombination method, wherein the scFv of a chicken monoclonal antibody is encoded in an expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced. The present invention relates to a novel method for producing a chicken monoclonal antibody characterized by using an expression vector into which a gene has been introduced, and a chicken monoclonal antibody produced by the method.
The present invention also relates to a novel expression vector used in the above method.
Furthermore, the present invention relates to a scFv of a chicken monoclonal antibody characterized in that one of the primers for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody has a base sequence that can be cleaved by the restriction enzyme BssHII. And a method for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody using the primer.
The present invention also relates to a novel oligonucleotide useful as a primer for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody.

More specifically, the present invention relates to the following items (1) to (11).
(1) One of the primers for amplifying the gene encoding VL or scFv of a chicken monoclonal antibody has a base sequence that can be cleaved by the restriction enzyme BssHII, and encodes scFv of a chicken monoclonal antibody Primer for gene amplification.
(2) The primer according to (1), wherein the number of bases is 15 to 30.
(3) The nucleotide sequence is
3'-ttgggactggcaggatccgcggggggtc-5 '
Or the primer as described in said (1) or (2) which is the complementary strand.
(4) The base sequence of the other primer is
5'-ctgatggcggccgtgacgt-3 '
Or the primer in any one of said (1)-(3) which is the complementary strand.
(5) The base sequence of the other primer is
5'-tctgacgtcgcgctgactcagcc-3 '
Or the primer in any one of said (1)-(3) which is the complementary strand.
(6) A method of amplifying a gene encoding a variable region of a chicken monoclonal antibody using the primer according to any one of (1) to (5).
(7) The method according to (6) above, wherein the variable region is a VL region.
(8) The method according to (7), wherein the primer is the primer according to (5).
(9) The method according to (6) above, wherein the variable region is scFv.
(10) The method according to (9), wherein the primer is the primer according to (4).
(11) The nucleotide sequence is
3'-ttgggactggcaggatccgcggggggtc-5 '
Or an oligonucleotide that is a complementary strand thereof.

  The present inventors first obtained by a cell fusion method for the purpose of producing a chicken anti-prion protein (PrP) panel monoclonal antibody that can be used as a tool for pathological analysis of prion disease and elucidation of prion conversion mechanism and its stable use. Phage display using pPDS expression vector using PrP-specific chicken monoclonal antibody HUC2-13, which has been shown to be effective in recognizing 25-29 residues (RPKPG) of mammalian prion protein (PrP) Recombinant antibody technology and mass preparation were established.

The present inventors first prepared a chicken monoclonal antibody that recognizes mammalian prion protein (PrP) 23-231 residues as follows.
Recombinant human prion protein (PrP) 23-231 (about 100 μg) was immunized into the abdominal cavity of a 4-week-old inbred chicken white leghorn species H-B15 chicken, and the spleen was excised to prepare immune splenocytes. This was fused with MuH1, which is a TK-deficient / ouabain-resistant chicken B cell line, by the PEG method to obtain a fused cell (hybridoma).
A scFv (single chain fragment of V region) is prepared by making the antibody gene (VH and VL regions) prepared from the antibody-producing hybridoma or spleen lymphocyte of the chicken thus obtained into a single chain via a flexible linker. did. As a plasmid vector for expressing this scFv, there is already pPDS (see FIG. 1) constructed by Yamanaka (currently Rohto Pharmaceutical). When pPDS is used, the C region of the mouse immunoglobulin L chain ( Cκ) is expressed, and the V region becomes a chicken and the C region becomes a mouse recombinant antibody.

  Since the specificity (reactivity) of the antibody is defined by the V region, there is no problem in reactivity, but detection without background staining such as HUC2-13 was not obtained when the pPDS vector was used. . The expression vector pPDS used for the production of the recombinant antibody was designed and constructed as a mouse antibody expression vector by Yamanaka et al. The expressed scFv antibody is composed of an antibody V region with a very high mutation frequency and is stable. Mouse Cκ chain has been introduced as a tag for detection because it is difficult to detect by detection. Therefore, a recombinant antibody produced using the pPDS vector must use an anti-mouse kappa chain antibody for detection while the antigen recognition site is derived from a chicken antibody, resulting in background staining. . As long as a mouse Cκ chain is used as a detection tag in a recombinant antibody, (1) when sandwich ELISA, which is widely used for high-sensitivity detection and quantitative experiments, is performed, assuming that a mouse antibody is used as a capture antibody, the antibody (2) In the case of immunohistochemical staining using a mouse as a model animal, there arises a problem that the immunoglobulin of the mouse is detected non-specifically.

Accordingly, the present inventors have solved the above-mentioned problems and have been able to detect with anti-chicken Ig antibody in pPDS for the purpose of utilizing a prion disease analysis and further for a broader research. An attempt was made to construct a novel expression vector in which the mouse Cκ chain was replaced with a chicken Cλ chain (L chain constant region). Furthermore, for the purpose of simplifying purification, a pure chicken recombinant antibody expression vector into which a purification tag (FLAG) was introduced was constructed.
FIG. 2 shows an outline of the construction of a chicken recombinant antibody expression vector (pCPDS).
First, the chicken Cλ chain gene was cloned.
When a chicken Cλ chain was amplified by PCR based on cDNA synthesized from normal chicken spleen cells, a band of about 340 bp which was considered to be a Cλ chain gene was obtained (see A in FIG. 3). FIG. 3 is a photograph replacing these drawings showing these results. In FIG. 3, “M1” indicates digestion with pUC118 HinfI, and “M2” indicates digestion with φx174 HaeIII.
This band was ligated after treatment with a restriction enzyme and transformed. On the next day, 6 clones were randomly prepared by alkaline SDS method, and the presence or absence of the insert was confirmed. As a result, all the inserts were confirmed. When the nucleotide sequence of one of the 6 clones (clone # 1) was determined, it was completely in agreement with the previously reported Cλ chain encoding an amino acid of 114 residues (see FIG. 4). Therefore, this clone was used as a template for preparing a vector reconstructing construct.

  Based on the cloned chicken Cλ chain gene whose nucleotide sequence was determined, the chicken Cλ chain gene used for the reconstructed construct was prepared by PCR. As a result, although many by-products were observed, a band that was thought to have added the FLAG sequence to the chicken Cλ chain was detected at a position of about 360 bp (see B in FIG. 3). This band was purified and used as a template for overlapping PCR with the geneIII gene.

  On the other hand, amplification of the geneIII gene from pPDS was performed using the PCR method. As a result, a band considered to be geneIII gene was detected at a position of about 600 bp (see C in FIG. 3). This band was purified and used as a template for overlapping PCR with a chicken Cλ gene to which a FLAG sequence was added.

  The overlapping PCR of the chicken Cλ chain and the gene III gene was performed via the FLAG sequence added to the 3 ′ side of the chicken Cλ chain gene and the 5 ′ side of the gene III gene. As a result, a band where a chicken Cλ chain and a gene III gene were considered to be linked at a position of about 950 bp was detected (see D in FIG. 3). This band was purified, treated with restriction enzymes, ligated to pPDS treated with EagI and EcoRI, and transformed into E. coli.

After transformation into E. coli, 15 clones were insert checked by amplification of the chicken Cλ chain gene. As a result, amplification of chicken Cλ chain was confirmed at a position of about 360 bp in 12 clones (see FIG. 5). FIG. 5 is a photograph replacing this drawing showing this result. “M” in FIG. 5 indicates digestion with pUC118 HinfI.
These clones were named pCPDS1,2,3,4,5,6,7,8,9,10,11,12, respectively. For these 12 clones, phage bodies were prepared without scFv gene, and the expressed proteins were examined.
Confirmation of expression of chicken Cλ chain and phage body by sandwich ELISA using anti-mouse IgG (H + L) and anti-chicken IgG (H + L) as capture antibodies and peroxidase-labeled anti-M13 antibody as a detection antibody. Did. In addition, the phage body expressed from pPDS was used as a positive control. As a result, when the remaining 10 clones excluding pCPDS8 and 10 were anti-chicken IgG (H + L) as a capture antibody, the reactivity was an ELISA value (OD492) average 0.6 (FIG. 6). FIG. 6 shows the results, where black represents the case where anti-mouse IgG (H + L) was used as the capture antibody, and white represents the case where anti-chicken IgG (H + L) was used as the capture antibody.
These clones had an average value of 0.17 when anti-mouse IgG (H + L) was used as the capture antibody, and the reconstructed vector was clearly lower than the reactivity with pPDS. It was found to be a reconstructed vector that accurately expresses the construct for use.

The structure of the obtained expression vector pCPDS is shown in FIG.
Using the reconstructed vector pCPDS7 in which the expression of the correct chicken Cλ chain and reconstituted phage was confirmed in the above-described experiment, the phage display antibody of the gene HUC2-13 encoding the chicken antibody shown in FIGS. Type antibodies were prepared. The prepared recombinant antibody was tested for reactivity with the H25 peptide in combination with HUC2-13. As a result, when the primary antibody was used without dilution, the ELISA value (OD492) exceeded 1.5 for all antibodies. When diluted, soluble titers prepared from HUC2-13 and cell disruption showed almost no decrease in ELISA titer up to 25-fold dilution. On the other hand, the phage display antibody and the soluble type antibody from the culture supernatant tended to decrease, and a positive ELISA titer could not be obtained at 125-fold dilution. The soluble antibody prepared from the destruction of the cells exceeded the ELISA titer of HUC2-13 from 125-fold dilution (see FIG. 14).

  FIG. 14 is a graph showing the results. In FIG. 14, black square marks indicate chicken-type phage display antibodies, gray rhombus marks indicate chicken-type soluble antibodies (culture supernatant), and gray round marks indicate chicken-type soluble antibodies (cell destruction). Gray triangles indicate HUC2-13 (native antibody).

From the above experiment, it was found that a vector for producing a pure chicken recombinant antibody could be reconstructed. A pure chicken recombinant antibody obtained from this vector has little noise due to a mouse antibody, and can increase the S / N ratio of detection.
In constructing the vector of the present invention, attention should be paid to the following three points: (1) a functional ScFv antibody can be expressed, (2) a vector capable of producing a Fab antibody in the future, and (3) easy purification. did. Considering these three points, a chicken Cλ chain was used instead of the mouse Cκ chain, and an artificial sequence called a FLAG sequence was selected as a purification tag.

  In addition, the reconstructed vector was prepared based on the pPDS vector, and it was possible to replace the construct using an appropriate restriction enzyme site. Since pPDS does not have an appropriate restriction enzyme site from the scFv antibody cloning site to EcoRI immediately after the geneIII stop codon, the construct must be replaced between EagI-EcoRI or BssHII-EcoRI. Choose a restriction enzyme that does not cleave the vector or the ScFv antibody gene and its installation site, while taking care to approximate the natural VL-Cλ amino acid sequence at the ligation site between the chicken Cλ chain and the ScFv antibody gene. did. In order to examine this cutting position, the bond between the Vλ region and the Cλ region was summarized. This is shown as Table 1.

* In Table 1 is a substitutable amino acid, and italic indicates an amino acid having a problem in substitution. The base sequence corresponding to the amino acid sequence shown in 1) is a primer sequence for pPDS, and the base sequence corresponding to the amino acid sequence shown in 2) is a primer sequence for the chicken antibody expression vector of the present invention. Show.
As a result, it became BssHII again. However, when the construct was replaced between BssHII and EcoRI, it was revealed that the validity of the vector could not be confirmed because a frame shift occurred during the expression of the empty vector and the protein was not expressed accurately. It was. Therefore, it was decided to introduce an EagI-EcoRI construct in which a new EagI-BssHII spacer was added.

The chicken Cλ chain was prepared from chicken splenocytes, and gene III was prepared from pPDS. On the other hand, the FLAG-amber sequence was introduced into a part of the primer and ligated at the time of Cλ chain amplification. However, it is dangerous to use a primer having 54 bases from the complicated spleen cell-derived cDNA group at the time of Cλ chain amplification. Therefore, FLAG-amber ligated PCR was performed using the once cloned Cλ chain as a template.
All of the constructs for reconstructing from EagI-BssHII scFv antibody gene cloning site-chicken Cλ chain-FLAG-amber sequence (TAG) -geneIII were prepared by PCR using KOD DNA polymerase. Ligation was performed on pPDS treated with EagI and EcoRI, and finally a vector expressing 10 clones of chicken Cλ chain and reconstituted phage was obtained (see FIG. 6).

FIG. 15 shows an outline for producing a pure chicken recombinant antibody of the present invention based on the above experiment. This will be described in order.
(1) Preparation of antibody V region genes (VH and VL) Based on cDNA extracted from antibody-producing hybridoma and synthesized by RT-PCR (reverse transcription PCR), synthetic primer sets (VH shown in Table 2 below) Amplification primer set for amplification, primer set for VL amplification).
(2) Construction of scFv After purifying the amplification products of VH and VL obtained in the experiment of (1), VH and VL were purified by using a linker (scFv linker) (-(Gly-Gly-Gly-Ser) _3- ). To be linked (assembly reaction).
(3) Re-amplification of scFv and restriction enzyme treatment Using the primer set (CHB and CLF1) shown in Table 2 below for re-amplification of scFv, re-amplification of scFv constructed in (2) was performed. After purification, an scFv inserted gene fragment (insert) is prepared using restriction enzymes (EagI and BssHII) for insertion into the plasmid vector pCPDS.

(4) Insertion (scFv) into pCPDS (ligation)
pCPDS and insert are adjusted to 1: 1 to 10 (molar ratio), and ligation is performed using Ligation Kit ver. 1 (TAKARA).
(5) Transformation of Escherichia coli Escherichia coli (XL1-blue, 100 μl) is transformed using pCPDS (10 μl) with the inserted insert.
(6) Selection of transformed Escherichia coli expressing insert A plurality of E. coli colonies are arbitrarily picked from the grown Escherichia coli colonies, scFv is amplified using a primer set, and transformed E. coli expressing the insert is selected. .
(7) Preparation of phage expression antibody A selected phage is infected with a helper phage to prepare a phage expression antibody.
(8) Panning for selection of specific phage antibodies After reacting phage-expressed antibodies on a plate on which an antigen (prion protein) is immobilized, only the reacted phage-expressed antibodies are collected and infected with Escherichia coli. Specific phage antibodies are selected.
(9) Preparation of solubilized antibody Non-pressor E. coli (SOLA or HB2151) is infected with a specific phage antibody. 24 hours after the infection, the E. coli culture supernatant and the cultured cells are collected, and the supernatant and the cell disruption extract are used as a solubilized antibody.
(10) Production of solubilized antibody The solubilized antibody is affinity purified using an anti-FLAG antibody-conjugated agarose gel.

In this way, a pure chicken recombinant antibody can be produced by using the expression vector pCPDS of the present invention. The production of scFv for producing the chicken antibody of the present invention will be described in more detail.
Mammals and birds have different antibody gene diversity acquisition mechanisms, and mammalian antibody genes reconstruct one of many V genes. In avian antibody genes, only one antibody gene functions. And pseudo-V genes are randomly inserted into this functional antibody gene.
FIG. 16 schematically shows antibody H chains in mammals (upper part of FIG. 16) and birds (lower part of FIG. 16). In mammalian antibody genes, one of many V genes is reconstructed, so the 5 'base sequence also fluctuates, and a primer must be prepared depending on the type of antibody. In avian antibody genes, There is only one functioning antibody gene, and a pseudo-V gene corresponding to the type of antibody is randomly inserted into this functional antibody gene. Also basically does not fluctuate.

Therefore, when trying to amplify an antibody gene from antibody-producing cells by PCR, it is not known which mammalian V gene is used from among many V genes. A large number of primers must be synthesized and prepared.
On the other hand, since the avian V gene has a single functional V gene as a base, its base sequence is known in advance, so one pair of primers, one at the 5 ′ side and one at the 3 ′ side, The ability to deal with antibody-producing cells is also a major feature when using avian antibodies.
Table 2 below shows the avian primers disclosed in the present invention.

By using the primer set shown in Table 2, it is possible to amplify the antibody genes of birds, preferably chickens, basically with any antibody. Among these, the primer shown as CLF1 has a novel base sequence, and is characterized by having a site (gcgcgc) that can be cleaved by the restriction enzyme BssHII in the middle thereof. This base sequence is shown in SEQ ID NO: 1 in the sequence listing.
FIG. 17 schematically shows how the primer CLF1 is used. The upper left side of FIG. 17 is the VL region, and the right side is the Cλ region. As described above, in the expression vector pCPDS, since the VL region and the Cλ region are bound by the restriction enzyme BssHII, the state of binding between the 3 ′ end of the VL region and the 5 ′ end of the Cλ region is shown. A portion surrounded by a square frame of the base sequence indicated by circle 2 in FIG. 17 is a position that can be cleaved by the restriction enzyme BssHII.
As described above, since the base sequence at the end of the VL region varies depending on the type of antibody in birds, scFv consisting of the VL region and VH-linker-VL can be amplified by using this primer CLF1. Become.

  As described above, the present invention provides a novel primer set for producing a pure chicken recombinant antibody, a novel expression vector pCPDS, and a pure chicken recombinant antibody produced using the same.

The present invention provides a pure avian recombinant antibody, preferably a chicken recombinant antibody.
Chicken monoclonal antibodies can produce monoclonal antibodies that cannot be produced by mammals, and recognize N-glycolylneuraminic acid, an antigen that is a human cancer marker, prion protein related to prion disease, etc. It can be applied to many biological macromolecules including the above example, and is expected to be developed into various tests and diagnostic agents. Therefore, application to various diagnostic agents and therapeutic agents such as cancer and prion diseases is expected. However, a major drawback of the chicken type is that the fusion cell technique for producing monoclonal antibodies generally cannot provide the necessary amount for practical use. The present invention has succeeded in producing a large amount of a substantially pure chicken antibody by genetic recombination for the first time. That is, the present invention enables efficient and large-scale production of chicken monoclonal antibodies using a novel plasmid vector and primers, and provides antibodies, production methods, vectors, primer sets, etc. It is.
In addition, the avian antibody gene has a relatively fixed base sequence at the 3 'end and 5' end, and can be amplified using the primer provided by the present invention regardless of the type of antibody. Thus, the present invention provides a method by which monoclonal antibodies against various antigens can be easily prepared by genetic recombination means.

  EXAMPLES The present invention will be described more specifically with reference to examples below, but the present invention is not limited to these examples.

Design of Vector for Expression of Chicken Recombinant Antibody The design of the vector for expression of chicken recombinant antibody was based on the pPDS vector. Replacing the mouse Cκ chain gene already inserted into pPDS with the chicken Cλ chain gene, in which the VL and Cλ-linked sequences approximate the germline amino acid sequence and the restriction enzyme site for cloning is not cleaved. Selected (see Table 1). In addition, the spacer sequence after EagI was newly redesigned so that the vector assay can be performed by the expression of chicken Cλ chain even in the absence of insert (Mock). A FLAG sequence (DYKDDDDK), which is a purification tag, was inserted downstream of the chicken Cλ chain, and a TAG (amber) stop codon was inserted between the FLAG sequence and geneIII so that it could be expressed as a soluble antibody. The construct prepared from the EagI site to geneIII was ligated to pPDS treated with EagI and EcoRI, to obtain a chicken recombinant antibody expression vector (see FIG. 2).

Isolation of chicken Cλ chain gene (1) Design and synthesis of primer for cloning chicken Cλ chain gene Primer Analysis Software (National Bioscinece) is used as a primer for cloning chicken Cλ chain gene. Designed and synthesized by outsourcing to Hokkaido System Science.
(2) Amplification and cloning of chicken Cλ chain gene The chicken Cλ chain gene was amplified by RT-PCR according to the method described later from normal chicken spleen cells. The PCR reaction solution was subjected to phenol extraction, phenol / chloroform extraction, ethanol precipitation concentration, and redissolved in 20 μl of DW. 1 μl each of 16 U / μl BamHI and 20 U / μl HindIII was added to the solution, treated with a restriction enzyme at 37 ° C. for 2 hours, and concentrated by ethanol precipitation. The concentrated sample was redissolved in 10 μl of DW, electrophoresed using a 1.5% agarose gel, purified by UltraClean, and redissolved in 10 μl of DW. After concentration measurement, the mixture was mixed with pBluescript II SK (−) so that the molar ratio was vector: insert = 1: 3, and ligated with TAKARA Ligation Kit ver. 1 at 16 ° C. for 3 hours. After completion of ligation, 5 μl of the reaction mixture was transformed into competent cells, and 40 μl was plated on ampicillin-containing 2 × YT plates. On the next day, the obtained colonies were isolated and cultured in a small amount in ampicillin-containing 2 × YT medium, and plasmids were prepared by alkali-SDS method, followed by restriction enzyme treatment with BamHI and HindIII to confirm the presence or absence of the insert.
The base sequence was determined according to a standard method (see FIG. 4).

Preparation of chicken Cλ chain gene for ligation reaction (1) Design and synthesis of primers for chicken Cλ chain gene amplification Primer for amplification of chicken Cλ chain gene to construct a reconstructed vector is EagI, A sense primer that incorporates BssHII and is frame-shifted so that it can be expressed even in an empty vector, and an antisense primer that adds a FLAG-amber-geneIII-5 ′ end sequence to allow ligation to geneIII isolated from pPDS Designed and synthesized.
(2) Amplification of chicken Cλ chain gene After determination of the base sequence, clones that were completely matched with the data bank were prepared by the cesium chloride method. Using the prepared sample as a template, a chicken Cλ chain gene for overlap PCR was prepared. The PCR reaction solution was subjected to phenol extraction, phenol / chloroform extraction, ethanol precipitation concentration, and redissolved in 10 μl of DW. This reaction solution was electrophoresed on a 1.5% agarose gel, purified with UltraClean, redissolved in 10 μl of DW, and used as a sample for overlapping PCR with gene-III derived from VCS-M13.

Preparation of gene III derived from VCS-M13 for ligation reaction (1) Design and synthesis of primer for gene III amplification For gene III amplification from pPDS, termination of sense primer and gene III that can be ligated to chicken Cλ chain via FLAG-amber sequence An antisense primer having EcoRI introduced immediately after the codon was designed and synthesized.
(2) Amplification of geneIII VCS-M13-derived geneIII was prepared by PCR from a pPDS vector. The PCR reaction solution was subjected to phenol extraction, phenol / chloroform extraction, ethanol precipitation concentration, and redissolved in 10 μl of DW. This reaction solution was electrophoresed on a 1.5% agarose gel, purified with UltraClean, redissolved in 10 μl of DW, and used as a sample for overlapping PCR with chicken Cλ chain.

Ligation reaction of chicken Cλ chain and gene III Ligation of chicken Cλ chain and gene III was performed by an assembly reaction and a re-amplification reaction based on a template mixed at a molar ratio of 1: 1. The PCR reaction solution was subjected to phenol extraction, phenol / chloroform extraction, ethanol precipitation concentration, and redissolved in 10 μl of DW. This reaction solution was electrophoresed on a 1.5% agarose gel, purified with UltraClean, and redissolved in 10 μl of DW.

Removal of mouse Cκ chain and geneIII from pPDS vector and insertion of chicken Cλ chain-gene3 In order to insert chicken Cλ chain-geneIII into pPDS vector, restriction enzyme treatment was performed with 10 U / μl EagI and 10 U / μl EcoRI. In order to prevent intramolecular ligation, BAP treatment was performed. The reaction solution after the BAP treatment was electrophoresed using a 1.0% agarose gel, purified with UltraClean, redissolved in 10 μl of DW, and used as a sample for reconstruction. Similarly, chicken Cλ chain-gene III was treated with a restriction enzyme and purified to prepare 10 μl of a sample for reconstruction. The prepared reconstituted sample was measured for concentration, mixed at a molar ratio of vector: insert of 1: 3, ligated with TAKARA Ligation Kit ver.1 at 16 ° C for 3 hours, and 5 µl of the reaction mixture was competed. 50 μl was transformed. The transformed E. coli was plated on ampicillin-containing 2 × YT plates at 40 μl and cultured overnight at 37 ° C.
The inserted gene was confirmed by amplification of the chicken Cλ chain by PCR using a colony as a template.

Confirmation of expression of reconstituted phage bodies Expression of reconstituted phage bodies was confirmed by sandwich ELISA. Anti-chicken IgG (H + L) antibody (Kirkegaard and Perry Laboratories) and anti-mouse IgG (H + L) antibody (Kirkegaard and Perry Laboratories) are immobilized at 0.5 μg / well overnight at 4 ° C. After blocking, the inserted gene can be confirmed. Based on the obtained colonies, 50 μl / well of reconstituted phage bodies without scFv antibody expression was added and reacted at 37 ° C. for 1 hour. After the reaction, HRPO-labeled anti-M13 antibody diluted 3,500 times was reacted at 37 ° C. for 1 hour to develop color using o-phenylenediamine as a substrate. After 10 minutes of reaction, the absorbance at 492 nm was measured with a microplate reader MPR-A4i.

Preparation of HUC2-13 (1) Test cell line Anti-thrombin kinase deficient and ouabain-resistant chicken B cell line MuH1 is used as a parent strain to recognize residues 25-29 of mammalian Puorin protein PrP prepared by cell fusion. A PrP chicken monoclonal antibody HUC2-13 producing hybridoma was used. The cells were cultured and maintained in a 38.5 ° C., 5% CO ₂ incubator using IMDM (Iscove's modified Dullbecco's medium) (GIBCO BRL) containing 10% FBS (Fetal bovine serum) (SIGMA). The culture supernatant was used as an anti-PrP chicken monoclonal antibody HUC2-13.
(2) Amplification of antibody V gene by RT-PCR (2-1) Extraction of RNA and synthesis of cDNA The RNA of the test cells was isolated using ISOGEN-LS (Nippon Gene) according to the attached protocol. The isolated RNA was redissolved in 20 μl of diethylpyrocarbonate-distilled water (DEPC-DW).
The synthesis of 1st strand cDNA was performed using the Oligo-dT primer method. 1.0 μl of 500 μg / ml Oligo dT15 primer (Boehringer Mammheim) was added to 1 to 5 μg of total RNA solution, and prepared to 12 μl with distilled water (DW). This mixture was incubated at 70 ° C. for 10 minutes, and after ice cooling, 4.0 μl of 5 × reverse transcription buffer (GIBCO BRL), 2.0 μl of 0.1M dithiothreitol (DTT) (GIBCO BRL), 2.0 μl of 5 mM dNTPs (GIBCO BRL) was added and incubated at 42 ° C. for 2 minutes. Thereafter, 1.0 μl of 200 U / μl reverse transcriptase (Superscript II RT) (GIBCO BRL) was added and incubated at 42 ° C. for 50 minutes and then at 70 ° C. for 15 minutes. The synthesized cDNA was stored at -20 ° C.
(2-2) Amplification and purification of VH and VL antibody genes and linker genes For amplification of VH and VL antibody genes by PCR (polymerase chain reaction) method, the cDNA synthesized in (2-1) is amplified by linker genes. The plasmid pLINK constructed for linker supply was used as a template, and a pair of sense primer and reverse primer, KOD DNA polymerase (TOYOBO), and Cloned Pfu DNA polymerase (Perkin Elmer) were used. As a positive control, anti-mouse albumin monoclonal antibody 3C8 (chicken recombinant antibody) produced by Yamanaka et al. Was used. PCR reaction solutions of VH and VL antibody genes were extracted by phenol extraction, phenol / chloroform extraction, ethanol precipitation with addition of 2 mg of 10 mg / ml glycogen, and then electrophoresis using 1.5% agarose gel (Wako Pure Chemical). did. The obtained band was purified with Ultra Clean (MO BIO laboratories, Inc.) according to the attached protocol, and eluted with 20 μl of DW. The linker gene PCR reaction solution was used in the same manner as the purification of the VH and VL antibody genes. However, 3.0% agarose gel was used for electrophoresis.

(2-3) Amplification and purification of scFv antibody gene The assembly reaction and reamplification reaction for scFv antibody gene synthesis amplification were performed by mixing purified VH, VL, and linker at a molar ratio of 1: 1: 1, respectively. It was. The PCR reaction solution of the scFv antibody gene was concentrated by ethanol extraction with phenol extraction, phenol / chloroform extraction, 2 mg of 10 mg / ml glycogen, and then electrophoresed using a 1.5% agarose gel. The obtained band was purified with Ultra Clean according to the attached protocol and eluted with 20 μl of DW.
(3) Preparation of expression vector (pPDS) Transformation of phage display vector (pPDS) (Fig. 1), which was distributed by Dr. Hachiro Yamanaka, Rohto Pharmaceutical Co., Ltd., into Escherichia coli XL1-Blue by the rubidium chloride method After plating on 2 × YT plates containing 50 μg / ml ampicillin, the cells were cultured at 37 ° C. overnight. The obtained colonies were selectively collected and cultured with shaking in a large amount in 2 × YT medium containing 50 μg / ml ampicillin. Vector purification from the cultured Escherichia coli was performed by cesium chloride density gradient equilibrium centrifugation.
(4) Restriction enzyme treatment and BAP treatment 2 μg of purified scFv gene fragment and 10 μg of vector pPDS were added with 4.0 U of EagI (New England BioLabs, Inc.) to 1.0 μg of DNA and treated at 37 ° C. overnight. After EagI treatment, BssHII (New England BioLabs, Inc.) was added to the reaction solution at the same ratio and treated at 50 ° C. for 4 hours. After BssHII treatment, enzyme inactivation treatment was performed at 68 ° C. for 20 minutes, and the reaction solution was made up to 300 μl with DW, followed by phenol extraction, phenol / chloroform extraction, and ethanol precipitation with addition of 2 μl of 10 mg / ml glycogen, Redissolved in 30 μl of DW.
The vector was dephosphorylated using Bacterial alkaline phosphatase (BAP) (TOYOBO) to prevent intramolecular ligation. 10 μl of 5 × BAP buffer, 3 μl of alkaline phosphatase (0.4 U / μl) and 7 μl of DW were added to 30 μl of vector, and the treatment was performed at 37 ° C. for 3 hours. The treated reaction solution was diluted to 300 μl with DW, phenol-extracted, phenol / chloroform extracted, ethanol-precipitated with 2 μl of 10 mg / ml glycogen, and redissolved in 30 μl of DW.

(5) Ligation to vector A purified scFv gene fragment and vector pPDS were prepared so that the molar ratio was 3: 1, and ligation reaction was performed using DNA ligation kit ver.1 (TAKARA). The method followed the attached protocol.
(6) Transformation (rubidium chloride method)
For transformation, 100 μl of Escherichia coli XL1-Blue competent cell solution stored at −85 ° C. was thawed in ice, 10 μl of ligation solution was added thereto, and the mixture was allowed to stand in ice for 30 minutes. Next, it heat-processed for 2 minutes at 42 degreeC, moved to ice quickly, and left still for 2 minutes. 1.0 ml of SOC medium was added and cultured with shaking at 37 ° C. for 1 hour. 10 μl was plated on an SOBAG plate containing 100 μg / ml ampicillin and cultured at 37 ° C. overnight.
(7) Confirmation of Inserted DNA Fragment Confirmation of the scFv gene, which is an inserted DNA, is based on a pPDS vector-derived sequence designed to completely amplify the insertion site, Lac Z upstream sequence M13-R, and mouse Cκ chain The PCR was performed using a colony as a template using a single-pair primer of the sequence SLP-1.
(8) Preparation of phage display antibody After transformation, colonies in which the inserted DNA fragment was confirmed were subjected to slow overnight shaking culture at 30 ° C. in 2 ml of SOC medium containing 50 μg / ml ampicillin. On the next day, 0.2 ml of Super Broth medium containing 100 μg / ml ampicillin and 37 μl of 2M glucose were added to 0.8 ml of the culture solution. After shaking culture in a 50 ml tube at 37 ° C. for 3 hours, 5 × 10 9 pfu of helper phage (VCS-M13) was added and incubated for 30 minutes with gentle shaking to infect the phage. After further shaking culture for 30 minutes, centrifugation was performed at room temperature at 800 × g for 10 minutes, and the supernatant was discarded. The precipitate was resuspended in Super Broth medium containing 1 ml of 100 μg / ml ampicillin and 50 μg / ml kanamycin, and 9 ml of the same medium was added and cultured overnight with vigorous shaking. After cooling the next day, the cells were removed by centrifugation at 800 × g for 10 minutes, the supernatant was filtered with a 0.45 μm pore filter (Kanto Chemical), and the filtrate was used as a phage display antibody suspension. The antibody was stored at 4 ° C or -20 ° C.
Ten colonies were randomly isolated from the obtained colonies, and the presence of an insert (scFv gene fragment) was confirmed by PCR. As a result, inserts were confirmed in 4 out of 10 clones (clone #: 1, 3, 5, and 6). Based on this, phage display antibodies were prepared, and these four types of phage display antibodies were designated as HUC2p1, They were named HUC2p3, HUC2p5 and HUC2p6.
(9) Preparation of Soluble Antibody Soluble antibody was prepared by infecting Escherichia coli XL1-Blue SOLR strain (SOLR strain), which is an amber sequence non-suppressor strain, with a phage display antibody. The SOLR strain was cultured overnight in 2 ml of SOC medium containing 25 μg / ml kanamycin, and 5 μl of phage display antibody was added to 200 μl of the culture solution and infected at 37 ° C. for 1 hour. This culture solution was diluted with 2 × YT medium, plated on a 2 × YT plate containing 100 μg / ml ampicillin, and cultured at 37 ° C. overnight. What confirmed the scFv antibody gene fragment by PCR using the obtained colony as a template was cultured in 2 ml of Super Broth medium containing 50 μg / ml ampicillin. To 500 μl of the culture solution, 4.5 ml of Super Broth medium was added, and ampicillin and kanamycin were added to final concentrations of 100 μg / ml and 50 μg / ml, respectively, followed by shaking culture at 37 ° C. for 2 hours. Thereafter, IPTG was added to a final concentration of 1 mM, and the mixture was further cultured with shaking for 4 hours. The culture solution was ice-cooled and then centrifuged at room temperature at 800 × g for 10 minutes, and the supernatant was recovered. The supernatant was further sterilized by filtration through a 0.45 μm pore filter. The filtrate was stirred with a soluble antibody solution (culture supernatant), and the precipitate was stirred with 5 ml of PBS having the same volume of the culture solution. (Tomy Seiko Co., Ltd.) was sonicated and the filtrate was prepared as a soluble antibody solution (bacteria cell disruption extract). The antibody was stored at 4 ° C or -20 ° C.

Expression of HUC2-13 ScFv Antibody Using Reconstruction Vector The ScFv antibody gene was amplified from HUC2p3 constructed in Example 8 (8) using primers designed for the reconstruction vector. The reaction solution was subjected to phenol extraction, phenol / chloroform extraction, ethanol precipitation concentration, and redissolved in 20 μl of DW.
The reconstructed vector after mass preparation using the cesium chloride method and the ScFv antibody gene derived from HUC2p3 were subjected to restriction enzyme treatment with 10 U / μl EagI and 10 U / μl BssHII. Furthermore, the vector was BAP treated to prevent intramolecular ligation. The treated reaction solution was electrophoresed using 1.0% agarose gel, purified with UltraClean, redissolved in 10 μl of DW, and used as a sample for reconstruction. The prepared reconstituted sample was measured for concentration, mixed at a molar ratio of vector: insert of 1: 3, ligated with TAKARA Ligation Kit ver.1 at 16 ° C for 3 hours, and 5 µl of the reaction solution was used as a competent cell. 50 μl was transformed. The transformed E. coli was plated on ampicillin-containing 2 × YT plates at 40 μl and cultured overnight at 37 ° C. Inserts were checked by PCR using colonies as a template using primers for scFv antibody gene amplification.
The colonies where the insert was confirmed prepared phage display antibodies according to the method described in Example 8.

Preparation of soluble antibody Soluble antibody was prepared according to the method described in Example 8.

ELISA
The reactivity of the prepared chicken phage display antibody and soluble antibody was analyzed by ELISA using H25 peptide as an antigen. The primary antibody recombinant antibody and native antibody used a 5-fold serial dilution up to 15,625 times. The analysis was performed by ELISA using HRPO-labeled anti-chicken IgG (H + L) (Kirkegaard and Perry Laboratories) diluted 2,000 times in the secondary antibody.
That is, 50 μl / well (0.25 μg / well) of H25 peptide at a concentration of 5 μg / ml was added to each 96-well plate and solidified at 4 ° C. overnight to obtain an antigen plate. The antigen plate was BlockAce (Snow Brand Milk Products) diluted to 25% with PBS, added with 400 μl / well, blocked at 37 ° C. for 1 hour, and washed 3 times with PBS-Tween 20 (0.05%). The primary antibody reaction was performed by adding 50 μl / well of recombinant antibody and allowing to stand at 37 ° C. for 2 hours. After washing 5 times with PBS-Tween 20 (0.05%), a peroxidase-labeled anti-M13 antibody (Amersham Pharmacia biotech) diluted 3,000-fold with 10% BlockAce was added as a secondary antibody reaction, and allowed to stand at 37 ° C. for 1 hour. did. After washing 8 times with PBS-Tween 20 (0.05%), o-phenylenediamine (when using a peroxidase-labeled secondary antibody) (SIGMA) or p-nitrophenyl phosphate (when using an alkaline phosphatase-labeled secondary antibody) ( The color was developed for 20 minutes using Kirkegaard and Perry Laboratories), and the absorbance at 492 or 405 nm was measured with a microplate reader MPR-A4i (TOSOH).

Preparation of antibody V region genes (VH and VL) RNA was extracted from antibody-producing hybridomas, and based on cDNA synthesized by RT-PCR (reverse transcription PCR), synthetic primer sets (VH amplification primer sets, VH and VL regions are amplified using a primer set for VL amplification.
(VH amplification conditions)
10x KOD buffer 1 5 μl
25 mM MgCl ₂ 3 μl
2.0 mM dNTPS 4 μl
Primer CHB (10 nmol / ml) * 5 μl
Primer CHSF (10 nmol / ml) * 5 μl
cDNA 1 μl
KOD polymerase 0.5 μl
DW 26.5 μl
* Primers CLSB and CLF1 are used as VL amplification conditions.
The above PCR mixture is prepared and set in a thermal cycler, and VH and VL are amplified under the following PCR cycle conditions.
(PCR cycle conditions)
・ 98 ℃ 10 seconds x 1 time ・ (98 degrees C 15 seconds, 60 degrees C 7 seconds, 74 degrees C 20 seconds) x 30 times ・ 74 degrees C 30 seconds ・ 4 degrees C

Construction of scFv After purifying the amplification products of VH and VL obtained in Example 12, VH and VL are ligated using a linker (scFv linker) (assembly reaction).
(Consolidation conditions)
10x KOD buffer 1 2.5μl
2.1 mM of 25 mM MgCl ₂
2.0 mM dNTPs 10 μl
VH (0.125 pmol / μl) 1 μl
VL (0.125 pmol / μl) 1 μl
scFv linker (0.125 pmol / μl) 1 μl
KDO polymerase 0.3 μl
DW 7.1 μl
The above PCR mixture is prepared, set in a thermal cycler, and VH and VL are connected under the following PCR cycle conditions.
・ 98 ℃ 10 seconds x 1 time ・ (98 degrees C 15 seconds, 65 degrees C 30 seconds) x 7 times ・ 74 degrees C 30 seconds x 1 times ・ 4 degrees C

scFv re-amplification and restriction enzyme treatment Using the primer set (CHB and CLF1) shown in Table 2 for scFv re-amplification, the scFv constructed in Example 13 was re-amplified, and the scFv was purified. For insertion into the plasmid vector pCPDS, an scFv insertion gene fragment (insert) is prepared using restriction enzymes (EagI and BssHII).
(Re-amplification reaction)
10 x KOD buffer 1 2.5 μl
2.0 mM dNTPs 4 μl
scFv primer CHB (10 nmol / ml) 3 μl
scFv primer CLF1 (10 nmol / ml) 3 μl
KOD polymerase 0.3 μl
DW 12.2 μl
(PCR temperature conditions)
・ 98 ℃ 10 seconds x 1 time ・ (98 degrees C 15 seconds, 65 degrees C 7 seconds, 74 degrees C 20 seconds) x 30 times ・ 74 degrees C 30 seconds x 1 time ・ 4 degrees C
(Restriction enzyme treatment)
-Restriction enzyme treatment of scFv scFv fragment 20 μl
10 × NEBuffer3 5 μl
48μl with DW
EagI (50 units / μl) Treated overnight at 37 ° C. Restriction enzyme treatment of pCPDS vector pCMDS vector (5 μg) x μl
10 × NEBuffer3 9 μl
88μl with DW
EagI (50 units / μl) Treated overnight at 37 ° C. ・ After confirming that the digestion of the vector was completed by electrophoresis, add 2 μl of BssII (20 units / μl) and digest at 50 ° C. for 3 to 4 hours. .
-Treat at 68 ° C for 20 minutes to inactivate the enzyme.
-Purification of scFv-pCPDS is purified by BAP treatment to prevent self-ligation.

Insert (scFv) into pCPDS (ligation)
pCPDS and the insert are adjusted to 1: 1 to 10 (molar ratio), and ligation is performed using Ligation Kit ver. 1 (TAKARA).
Vector Xμl
Insert Yμl
5 x ligation buffer 1 μl
5 μl with DW
A buffer 20 μl
B buffer 5 μl
Treat at 16 ° C. for 4 to overnight.

Transformation of Escherichia coli Escherichia coli (XL1-blue, 100 μl) is transformed according to the method described in Example 8 using pCPDS (10 μl) with the inserted insert.
A plurality of E. coli colonies are arbitrarily selected from the grown E. coli colonies that express the insert, and scFv is amplified using a primer set to select a transformed E. coli that expresses the insert.

Preparation of phage-expressing antibody A selected Escherichia coli is infected with a helper phage, and a phage-expressing antibody is prepared according to the method described in Example 8.

Panning for selection of specific phage antibodies After reacting phage-expressed antibodies on a plate on which an antigen (prion protein) is immobilized, only the reacted phage-expressed antibodies are collected and infected with Escherichia coli. To select.
Panning is a high affinity antibody selection method using an antigen-antibody reaction. For example, this is performed as follows. 5 μg / ml maltose-binding protein fusion recombinant human PrP23-231 (MBP-HuPrP) was added to a 96-well plate at 50 μl / well (0.25 μg / well) at 4 ° C. overnight, and this was immobilized on an antigen-coated plate. did. 25% BlockAce 400 μl / well was added to 16 wells of the antigen-coated plate, blocked at 37 ° C. for 1 hour, and washed 3 times with PBS-Tween 20 (0.05%). After washing, a mixture of phage display antibody suspension 1.6 ml, BlockAce 704 μl, 5 M NaCl 96 μl was added at 150 μl / well, reacted at 37 ° C. for 2 hours, and washed 15 times with PBS-Tween 20 (0.05%). . To the wells after washing, 50 μl / well of eluate was added, and after 15 minutes of reaction at room temperature, neutralized with 3 μl / well of 2M Tris. After neutralization, the reaction solution was recovered and infected with 2 ml of XL1-Blue cultured at OD600 = 0.5 to 1.0 at 37 ° C. for 1 hour. A part of the solution after infection was plated on a SOBAG plate containing 100 μg / ml ampicillin for titration, and the eluted phage titer was measured. Further, after insert check by PCR, the insert rate was added to the eluted phage titer to determine the positive eluted phage titer. To the remaining infection solution, 400 μl of 50 μg / ml ampicillin-containing Super Broth medium and 37 μl of 2M glucose were added, and after incubation at 37 ° C. for 2 hours, 5 × 109 pfu helper phage (VCS-M13) was infected for 1 hour. After the infection, the supernatant was removed by centrifugation at 800 × g for 10 minutes, and cultured overnight in 10 ml of Super Broth medium containing 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 25 μg / ml tetracycline. After cooling the next day, the cells were removed by centrifugation at 800 × g for 10 minutes, the supernatant was filtered through a 0.45 μm pore filter, and the filtrate was used as the primary panned phage display antibody suspension to prepare the next panning sample.

Preparation of solubilized antibody According to the method described in Example 8, a specific phage antibody is infected with non-pressor Escherichia coli (SOLA or HB2151). 24 hours after the infection, the E. coli culture supernatant and the cultured cells are collected, and the supernatant and the cell disruption extract are used as a solubilized antibody.
The solubilized antibody is affinity purified using an anti-FLAG antibody-conjugated agarose gel.

The present invention provides a primer set and the like for producing a pure avian recombinant antibody, preferably a chicken recombinant antibody. Chicken monoclonal antibodies can produce monoclonal antibodies that cannot be produced by mammals, and recognize N-glycolylneuraminic acid, an antigen that is a human cancer marker, prion protein related to prion disease, etc. It can be applied to many biological macromolecules including the above example, and is expected to be developed into various tests and diagnostic agents. Therefore, application to various diagnostic agents and therapeutic agents such as cancer and prion diseases is expected. However, a major drawback of the chicken type is that the fusion cell technique for producing monoclonal antibodies generally cannot provide the necessary amount for practical use. The present invention succeeded in producing a large amount of substantially pure chicken antibodies by gene recombination for the first time, and the present invention efficiently produces large amounts of chicken monoclonal antibodies using a novel plasmid vector and primers. The present invention provides a primer set and the like for this purpose.
Therefore, the present invention provides a primer set and the like useful for chicken monoclonal antibodies for various diagnostic agents and therapeutic agents for cancer, prion diseases, etc. have.

FIG. 1 shows the structure of a known expression vector pPDS. FIG. 2 shows an outline of the construction of the chicken recombinant antibody expression vector (pCPDS) of the present invention. FIG. 3 shows a band of about 340 bp that seems to be a chicken Cλ chain gene (A in FIG. 3), a band that seems to have a FLAG sequence added to a chicken Cλ chain at a position of about 360 bp (B in FIG. 3), and a band of about 600 bp. Instead of the figure, it shows that a band that seems to be a geneIII gene at the position (C in FIG. 3) and a band that seems to be linked to the chicken Cλ chain at a position of about 950 bp and the geneIII gene (D in FIG. 3) were detected It is a photograph. In FIG. 3, “M1” indicates digestion with pUC118 HinfI, and “M2” indicates digestion with φx174 HaeIII. FIG. 4 shows a comparison between the base sequence of the chicken Cλ chain gene encoding the 114-residue amino acid and the base sequence of the Cλ chain obtained by the method of the present invention. FIG. 5 is a photograph replacing a drawing, showing that the amplification of chicken Cλ chain was confirmed at a position of about 360 bp in 12 clones obtained by insertion. “M” in FIG. 5 indicates digestion with pUC118 HinfI. FIG. 6 shows ELISA values (OD492) of the recombinant antibodies of 12 clones obtained by the method of the present invention when anti-chicken IgG (H + L) is used as a capture antibody and when anti-mouse IgG (H + L) is used as a capture antibody. ). Black indicates the case where anti-mouse IgG (H + L) is used as the capture antibody, and white indicates the case where anti-chicken IgG (H + L) is used as the capture antibody. FIG. 7 shows the structure of the expression vector pCPDS of the present invention. FIG. 8 shows a comparison of the base sequence of the V region heavy chain of chicken antibody together with HUC2-13. N in FIG. 8 indicates the base sequence of the D segment, and: indicates a deletion. FIG. 9 shows a comparison of the base sequence of the V region heavy chain of chicken antibody together with HUC2-13. N in FIG. 9 indicates the nucleotide sequence of the D segment, and: indicates a deletion. FIG. 10 shows a comparison of the V region heavy chain base sequence and the V region light chain amino acid sequence of chicken antibody together with HUC2-13. N in FIG. 10 indicates the base sequence of the D segment, and: indicates a deletion. FIG. 11 shows a comparison of the base sequence of the V region light chain of chicken antibody together with HUC2-13. In FIG. 11: indicates a deletion. FIG. 12 shows a comparison of the base sequence of the V region light chain of the chicken antibody together with HUC2-13. In FIG. 12, indicates a deletion. FIG. 13 shows the diversity acquisition mechanism in the HUC2-13 L chain in comparison with HUC2VL. In FIG. Indicates a mutation,: indicates a deletion, PV indicates a pseudogene. FIG. 14 shows the result of an ELISA value (OD492) for confirming the reactivity of the recombinant antibody prepared by the method of the present invention to the H25 peptide. In FIG. 14, black square marks indicate chicken-type phage display antibodies, gray rhombus marks indicate chicken-type soluble antibodies (culture supernatant), and gray round marks indicate chicken-type soluble antibodies (cell destruction). Gray triangles indicate HUC2-13 (native antibody). FIG. 15 shows an outline for the production of the pure chicken recombinant antibody of the present invention. FIG. 16 schematically shows the structure of an antibody heavy chain gene in mammals (upper part of FIG. 16) and birds (lower part of FIG. 16). FIG. 17 schematically shows how the primer CLF1 of the present invention is used. The upper left side of FIG. 17 is the VL region, and the right side is the Cλ region.

  The base sequence of the primer of this invention is shown.

Claims (5)

A primer set comprising two kinds of primers for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody,
The following base sequence containing a base sequence that can be cleaved by the restriction enzyme BssHII in one of the primers of the primer set:
3'-ttgggactggcaggatccgcggggggtc-5 '
Or a base sequence that is a complementary strand thereof,
The other primer has the following base sequence:
5'-ctgatggcggccgtgacgt-3 '
Or consisting of a base sequence that is the complementary strand thereof,
A primer set for gene amplification that encodes scFv of a chicken monoclonal antibody. A primer set comprising two kinds of primers for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody,
The following base sequence containing a base sequence that can be cleaved by the restriction enzyme BssHII in one of the primers of the primer set:
3'-ttgggactggcaggatccgcggggggtc-5 '
Or a base sequence that is a complementary strand thereof,
The other primer has the following base sequence:
5'-tctgacgtcgcgctgactcagcc-3 '
Or consisting of a base sequence that is the complementary strand thereof,
A primer set for gene amplification that encodes scFv of a chicken monoclonal antibody.   A method for amplifying a gene encoding a variable region of a chicken monoclonal antibody using the primer set according to claim 1.   The method according to claim 3, wherein the variable region is a VL region.   4. The method of claim 3, wherein the variable region is scFv.

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