JPH08116978A - Preparation of antibody fab library - Google Patents

Abstract

PURPOSE: To obtain the subject vector containing a cistron having a linker containing a site for accepting the gene of H-chain Fd constituting Fab and a cistron having a site for accepting an L-chain gene and useful for the preparation of a library for producing Fab fragment. CONSTITUTION: This phagimide vector to give an expression library of an antibody Fab fragment by transforming E.coli is produced by forming the 1st cistron containing a tac-SD promoter, a pectate-dissolving leader sequence, a linker part having a site for accepting a gene selected from a gene coding an H-chain Fd and a gene coding an L-chain constituting an antibody Fab fragment and a termination site from the 5'-site in the order and the 2nd cistron containing a tac-SD promoter, a pectate-dissolving leader sequence, a linker site having a site for accepting the other gene selected from a gene coding an H-chain Fd and a gene coding an L-chain, a GeneIII gene coding a pilus protein and a termination site from the 5'-site in the order.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for producing an antibody Fab fragment, a recombinant phagemid vector used therefor,
The present invention relates to a method for producing an antibody Fab library, primers for cloning genes encoding H chain Fd and L chain constituting Fab fragments, and a method for obtaining these genes. Specifically, the gene of the Fab fragment of the antibody was cloned from lymphocytes collected from organs or peripheral blood, and recombinant phage (phagemid)
A method for preparing a library and a method for obtaining an antibody Fab protein that binds to a target antigen from the library.

[0002]

BACKGROUND OF THE INVENTION A commonly used method for obtaining monoclonal antibodies is a method of immortalizing antibody-producing cells. The most representative one is the cell fusion method, and the sensitization B
It is a method of establishing a monoclonal antibody-producing cell line by fusing cells with a myeloma cell line.
(Ref 2: Nature 256: 495-497, 1975). Cells that serve as fusion partners for antibody-producing B cells include P3 × 63.653-Ag8
(ATCC CRL1580), SP2 / O (ATCC1581), K6H6-B5 (Ref 3: J
Exp Med 168: 475-489, 1988) and the like, but these are derived from rodents, and those derived from humans and established as partner cells are not known. Many attempts have been made to fuse lymphocytes derived from animals other than rodents with partner cells established in mice and rats. For example, in the case of human-derived cells, the probability of successful fusion is extremely low, and Even if fusion is possible, long-term passage is very difficult (Ref 4: J Immunol Methods 95: 123-126, 19).
86). There are numerous reports on the development of human monoclonal antibodies, but the number of successful cases is extremely small.
(Ref 5: J Immunol 10: 278-285, 1991, 6: Hybridoma
5: 33-41, 1986).

Another method for immortalizing cells is to directly immortalize antibody-producing B cells using B cell oncogenic virus. In the case of human B cells, EBV (Epstein-Barr virus) is used (Ref 7: The Medical
Bulletin [Naito Medical Research Foundation] 2: 30-35, 1984). The problem with this method is that after infection, even if the antibody-producing cell clone shows growth, the antibody-producing ability is lost in most cases with continued passage, and the immunoglobulin subclass (from IgG ) It often happens that it shifts to IgM type. Further, when this method is viewed as a part of a pharmaceutical manufacturing method, the use of an oncogenic virus has a very serious problem, and it is difficult to take safe measures against contamination of these viral DNAs.

Another reason why it is difficult to obtain human antibody-producing cells is that humans cannot sensitize the living body. If you have a history of infection with bacteria or viruses, you usually have antibodies to these foreign antigens in your serum. However, the number of B cells producing the desired antibody in peripheral blood is very small, and it is difficult to immortalize the cells by cell fusion or EBV transformation even though they can be detected. In addition, there is a high possibility that clones with high binding affinity (affinity) will be lost during the B cell cloning process. None of the human monoclonal antibodies developed by this method have been approved as therapeutic agents,
Only a few are in clinical trials.

Under these circumstances, experiments have recently been attempted to immortalize antibodies at the gene level using phage (or phagemid) vectors (Reference 8: JBiol Chem, 267: 16007- 16010, 1992, 9: Science
240: 1041-1043, 1988, 10: Science 246: 1275-1281, 1
989). The research itself of incorporating a specific antibody gene into a phage and expressing it in E. coli is not novel. On the other hand, attempts to make a large number of antibody genes into a library are relatively recent trends. That is, an attempt is made to rescue an antibody gene from a relatively small number of antibody-producing lymphocytes, or to make a library of as many antibody genes as possible from peripheral blood or lymph nodes expressing various antibodies.

[0006] A method for expressing a foreign protein as a fusion protein on the coat protein of a phage particle is reported by Smith et al., University of Missouri, USA (Ref. 11: Science 228: 1315-).
1317, 1985), followed by antibody and growth hormone (Ref 12: Gene 8: 309-314, 199) in line with the improvement of M13 line phage.
0) and other foreign proteins have been tried.
These are generally phage display systems (P
hage Display System) or a combination antibody library (Combinational Antibody Library). When these methods are applied to an antibody, the whole protein is not necessary, and only the gene of the antibody fragment containing the V region which is the binding site for the antigen may be made into a library.

There are roughly two methods for excising the antibody gene cDNA and introducing it into a phage. One is the H chain V region (HV) and the L chain V region (L
V) coding sequences, each of which is shuffled to create any combination of H chain and L chain, which is artificially linked by a peptide linker and incorporated into a phage (phagemid) to obtain a single chain FV ( scF
V)) (Ref 12: Nature 352: 624).
-628, 1991, representative vector: pCANTAB5). In the other, Fab (Note: Fa) consisting of an arbitrary combination of H chain and L chain is obtained by cloning the H chain Fd gene and the L chain gene, respectively, and independently integrating them into a phage vector.
See definition 1: b in Immunology, by JohnWiley & Sons,
New York, 1992) (see 10: Science 246: 1275-1281, 1989, 13: Proc Natl A).
cad Sci USA 88: 7978-7982, 1991, Representative Vectors:
pCOMB3).

[0008] The technique of immortalizing the antibody produced by B cells of peripheral blood, lymph nodes and spleen at the gene level using a phage (phagemid) vector can rescue all antibody genes including a very small population. there is a possibility. In particular, there are great expectations for its application to human monoclonal antibodies. As an example of application to human antibodies using scFV, results from the study of autoantibodies were found by Winter et al., MRC Research Institute in the UK (Ref. 14: EM).
BO J 12: 725-734, 1993). In the application example using Fab,
A human monoclonal antibody to the hepatitis B surface antigen (see 15: Proc Natl Acad Sci USA 89: 3175-31) by a group led by Lerner et al.
79, 1992), anti-thyroglobulin autoantibodies (Ref 16: Auto
immunity 12: 135-141, 1992), anti-HIV antibody (Ref 17:
Proc Natl Acad Sci USA 91: 3809-3813, 1994). On the other hand, the technique of immortalizing antibodies at the gene level can be used in combination with cell immortalization techniques such as EBV transformation. In the case of EBV, since it induces transient cell growth, it is possible to increase the probability of gene rescue from an antibody-producing strain with a low population, and finally to utilize a recombinant gene, which causes a problem of viral DNA contamination. There is no.

[0009]

Although many studies have been reported on the phage vector system, the phages or phagemids that have been published so far are not yet fully functional, and there is much room for improvement.

For example, in the case of scFV, the VDJ binding region of the H chain, which is considered to be the most important domain of the antigen recognition site
Since the vicinity of (CDR3) and the N terminus of the L chain need to be linked by an artificial linker, they do not always have the original antibody conformation. Ie scFV
When the whole antibody is reconstructed using the chimeric antibody technique, the V region gene identified as is difficult to reproduce its binding activity. As a countermeasure to this problem, a linker having a flexible structure such as (Gly ₄ Ser) ₃ is typically used (Ref 18: Proc Natl Acad Sci USA 85: 5879-588.
3, 1988) has been used, but the binding affinity is usually decreased by more than one digit, and scF is used for the purpose of therapeutic drug development.
The use of V libraries remains questionable (Ref 19:
Nucleic Acids Res 19: 4133-4137, 1991).

On the other hand, in the case of Fab, such a problem does not occur because the natural conformation is directly reflected, and it can be said that Fab is suitable for the development of therapeutic drugs. However, for the construction of Fab, two heterogeneous genes, that is, the H chain Fd gene and the L chain gene need to be independently formed into a library, and the protein expressed based on each gene is S-. It is necessary to combine with S. Therefore, in this case, the phage vector needs to efficiently integrate each gene into the two gene introduction sites, and a sufficient number of independent clones to enable the library formation of numerous antibody genes. It is required that the antibody gene can be stably maintained in the vector. The phagemid vectors that have been published so far do not have the ability to sufficiently meet such demands.

What is required in the selection of the phage vector system is two different genes (H chain Fd gene,
L-chain gene) integration efficiency is high, in order to obtain an antibody gene group library, the capacity including independent clones is high, and an Escherichia coli strain having excellent growth ability can be selected as a host. When using, for example, λ phage as a vector, it is very troublesome to clone a gene of interest to construct a library, and there is also a problem in the capacity to contain an independent clone of the library (Ref 11: Science 246 : 1275-
1281, 1989).

For this reason, the use of the phagemid vector is superior as a methodology. The phagemid is packaged in viral virions, can be cloned in single chain and infects E. coli male strains to form colonies on selection plates. Therefore, in addition to being excellent in operability, it is highly adaptable to the case where the capacity requirement for library formation is large, such as an antibody fragment. Lerner of Scripps Laboratories and Chang of Protein Design Labs have published not only phages, but also examples of using M13-based phagemid vectors (Reference 20: Methods 2: 119-124, 19).
91, (pCOMB3), 21: J Immunol 147: 3610-3614, 1991).

What is required in the construction of the phagemid vector system is the expression of the Fd gene and the L chain gene and the method for constructing the Fab protein which is a heterodimer, the Fab protein expression format which facilitates the screening of the target clone, the Fab protein and the It is possible to correspond to the Fd gene and the L chain gene in the phagemid via the ciliary protein which is the origin of the gene. Fab
The most common method for the expression of E. coli is to secrete Fab into the periplasm between the outer and inner membranes of infected E. coli, which is the p3 signal peptide (Signal sequence of Gene III gene: Ref 22: Virology 132: 445- 455, 198
4), pectate lysis leader sequence (Ref 9: Science 240: 1
041-1043, 1988), E. coli thermostable enterotoxin signal sequence (Ref 23: Biotechnology 9: 1373-1377, 199).
1) etc. are used. The Fd protein is pIII (gene III protein) or pVIII (gene
VIII protein) and expressed as a fusion protein
(Ref 22: Virology 132: 445-455, 1984, 24: Science 2
28: 1315-1317, 1985) is often used, but this is Fd.
Since the Fab protein can be expressed in a state in which it is linked to the ciliary protein on the outer shell of the phage particle having the gene encoding the protein, it can directly correspond to the protein and its underlying gene. Fd gene and L
The lacZ promoter is the most commonly used promoter for the expression of chain genes for all genes (Ref 11: Science 228: 1315-1317, 1985). l
It is known that the acZ promoter always weakly induces expression even when IPTG (isopropyl-β-D-thiogalactopyranoside) is not used. Lac repressor as E. coli host
Although the use of (lacI ^Q ) can be used to avoid it, it tends to be an obstacle to the operation when a recombinant of the Fd gene and the L chain gene is produced. These promoters, leader (signal) sequences, and types of fusion proteins greatly affect the function, expression and screening efficiency, and stability of phage vectors. The first object of the present invention is to construct a phagemid vector for Fab library having excellent performance.

Although many studies have been reported on library formation of antibody genes using the phage vector system, most of them are targeted at mouse antibodies and relatively few at human antibodies. When preparing a library of antibody fragments, as many (preferably all) H chain Fd genes and Ls as possible are derived from the lymphocytes from which they are derived.
The chain gene needs to be rescued and cloned correctly. When cloning the H chain Fd gene and the L chain gene, PCR (Polymer
ase Chain Reaction) Primer design is extremely important. The types of restriction enzyme cleavage sequences used at the 5'and 3'ends of the primer must basically satisfy the following three requirements. That is, in the base sequence other than the H chain Fd gene and L chain gene introduction site in the phagemid vector, which does not exist in the base sequence of the human monoclonal antibody Fab fragment or exists only with a probability that can be almost ignored. Does not exist,
The ligation efficiency between the used DNA fragment having a restriction enzyme cleavage sequence and the phagemid is high. Data analysis was carried out using EMBL and GenBank, which are DNA databases.
24, 1991, reference 24: J Immunol 151: 6954-6961, 1993), such as XhoI, SpeI, SacI, XbaI, and ApaLI, which are relatively low in human antibody Fab fragments, but cannot be ignored. It shows the frequency of occurrence (maximum about 5% for each restriction enzyme cleavage sequence), which is a major obstacle to library construction. A second object of the invention is the development of accurate and efficient PCR primers for cloning human monoclonal antibodies.

[0016]

DISCLOSURE OF THE INVENTION The present inventors have developed a phagemid vector that enables a more accurate and efficient library construction of antibody Fab genes in comparison with the previously reported phage (phagemid) vector system. It was constructed. The basic structure of this phagemid vector is a recombinant phage vector capable of tandemly introducing two different genes constituting Fab. As a result of database analysis, a restriction enzyme cleavage sequence at the gene insertion site was selected that has the lowest frequency of appearance in the antibody gene and has the highest ligation efficiency. Also, in order to enable accurate and efficient cloning of the Fab gene, P that effectively corresponds to the diversity of H chain Fd gene and L chain gene
A CR primer group was designed and synthesized. We also prepared two types of vector, a first expression vector in which the ciliated protein is fused with Fd protein and a second expression vector in which the ciliated protein is fused with the L chain. This is because it provides an auxiliary means for enhancing the phagemid library as a measure against the problem that the capacity is difficult to increase or the gene is easily deleted.

In summary, the present invention is a phagemid vector for expressing an antibody Fab fragment, which has a phagemid vector sequence as a backbone and first and second cistrons, one cistron being the Fab. Is a phagemid vector having a linker site having a site for introducing a gene encoding H chain Fd, which constitutes the above, and a cistron having a linker site having a site for introducing a gene encoding L chain, respectively.

The present invention also provides a phagemid vector for expressing a heterodimer composed of different peptide monomers, which comprises (a) a tac-SD promoter, a pectate lysis leader sequence, and a first peptide monomer in order from the 5 ′ side. A first cistron having a linker site having a site for introducing the first gene to be encoded and a termination site, and (b) encoding a tac-SD promoter, a pectate lysis leader sequence, and a second peptide monomer from the 5'side. Is a phagemid vector having a linker site having a site for introducing the gene, a GeneIII gene encoding a cilia protein, and a second cistron having a termination site.

Examples of the heterodimer include antibody Fab fragments in which the first peptide monomer is H chain Fd and the second peptide monomer is L chain.
The pectate lysis leader sequence is derived from MAFF301614 which is a strain of the bacterial strain Erwinia carotovora subspecies atroceptica and encodes the amino acid sequence shown in SEQ ID NO: 1.
A sequence, or a DNA sequence derived from the strain MAFF301629 or MAFF301630 of the same bacterial strain and encoding the amino acid sequence shown in SEQ ID NO: 2 can be mentioned.

Further, as the site for introducing the first gene and the site for introducing the second gene, SfiI, NotI,
Examples include restriction enzyme recognition sites selected from NheI, FseI, or AscI.

The present invention also provides the production of an antibody Fab fragment, which comprises transforming Escherichia coli with the phagemid vector and infecting this transformant with a helper phage to rescue the phagemid.

The present invention further provides the H of the antibody by the PCR method.
A primer for amplifying a chain Fd gene, which comprises 5 '
The sense primer corresponding to the end is composed of any one or a combination of two or more of the oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 3 to 8, and the antisense primer corresponding to the 3'end is SEQ ID NOS: 9 to 9.
The primer consisting of any one or a combination of two or more of the oligonucleotides having the nucleotide sequence shown in 13, and the sense primer corresponding to the 5'end,
An antisense primer consisting of any one or a combination of two or more of the oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 14 to 23, and the antisense primer corresponding to the 3 ′ end is an oligo having the nucleotide sequence shown in SEQ ID NOs: 24 or 25. Primers consisting of any one or a combination of two nucleotides are provided.

Further, according to the present invention, tac-SD is arranged in order from the 5'side.
Promoter, pectate lysis leader sequence, antibody Fa
b segment, a first cistron having a linker site having a site for introducing one of a gene encoding H chain Fd or a gene encoding L chain, and a first cistron, and a tac-SD promoter in order from the 5 ′ side A peptate lysis leader sequence, a linker site having a site for introducing the other of the H chain Fd-encoding gene and the L chain-encoding gene, a GeneIII gene encoding ciliated protein, and a second cistron having a termination site. Prepare a phagemid vector having
A gene encoding an H chain Fd is randomly inserted into one of the linker sites of the first and second cistrons,
A gene encoding an L chain is randomly inserted into the linker site of the other cistron, and Escherichia coli is transformed with the phagemid vector pool into which the H chain Fd gene and the L chain gene are respectively inserted. This is a method for constructing an expression library of Fab fragments.

Furthermore, the present invention is a method for obtaining a gene encoding an H chain Fd and a gene encoding an L chain which constitute an antibody Fab fragment having binding affinity for an antigen, the method comprising the following steps: provide.

From the 5'side, tac-SD promoter,
A pectate lysis leader sequence, a linker site having a site for introducing a gene encoding H chain Fd, and a first cistron having a termination site, and tac- in order from the 5 ′ side.
A first phagemid vector having an SD promoter, a pectate lysis leader sequence, a linker site having a site for introducing a gene encoding an L chain, a GeneIII gene encoding a cilia protein, and a second cistron having a termination site; From the 5'side, a tac-SD promoter, a pectate lysis leader sequence, a linker site having a site for introducing a gene encoding an L chain, and a first cistron having a termination site, and in order from the 5'side
tac-SD promoter, pectate lysis leader sequence, linker site having a site for introducing a gene encoding H chain Fd, GeneII encoding cilia protein
A second phagemid vector having an I gene and a second cistron having a termination site is prepared; an antibody Fab fragment of the antibody Fab fragment is prepared by using one of the first and the second phagemid vectors. An expression library is prepared, and from this library, a phagemid vector producing an antibody Fab fragment having a binding affinity for an antigen is selected; a gene encoding an H chain Fd or an L chain is selected from the selected phagemid vector. Cut out one of the encoded genes;
The excised gene is inserted into one linker site of the other phagemid vector of the first and second phagemid vectors, and when the excised gene is the H chain Fd gene, the L chain gene is present in the other linker site. At random, and when the excised gene is an L chain gene, the H chain d gene is randomly inserted at the other linker site; Escherichia coli is transformed with the obtained phagemid vector pool, and A phagemid vector is selected that produces an antibody Fab fragment that has a high binding affinity for the antigen.

The present invention will be described in detail below.

<1> Construction of Phagemid Vector of the Present Invention The phagemid vector of the present invention contains in its tandem a cistron that controls the expression of two different genes. The first cistron is ta containing the SD sequence from the 5'side.
c-SD promoter, pectate lysis leader sequence
(pelB), the first gene introduction site, and the termination site. The second cistron contains a tac-SD promoter containing an SD sequence from the 5'side, a pectate lysis leader sequence (pelB), a second gene introduction site, GeneIII encoding a ciliated protein, and a termination site.

The pelB gene is atroseptica (Erwinia carotovora subs. At), which is a subspecies of the bacterium Erwinia carotovora.
roseptica) strains MAFF301614, MAFF301629 and MAFF301630 (all of which are National Institute of Resources, Agriculture, Forestry and Fisheries). The pelB derived from MAFF301614 is one whose sequence has been determined or whose sequence has been deduced (Erwinia chrysanthemum (Erwinia chrys
anthemi) EC19, Erwinia ca
rotovora) EC) differs in the amino acid encoded by the 10th codon from the start codon, and the other 2
The two strains had the same encoded amino acid sequence (Ref 25: J Bacteriol 169: 4379-4383,
1987).

Erwinia carotovora is a pectate-lytic pathogenic bacterium, and it is known that a cosmid fused with a fragment of this gene is strongly expressed in Escherichia coli, and that the bacterium-derived substance is induced in the periplasm. (Ref 31:
Rev Phytopathol 18: 361-387, 1980, 32: Gene 35: 63-
70, 1985). Based on these studies, the sequence of pelB has been studied for two types of strains to obtain a leader sequence for inducing the periplasm of foreign proteins expressed in E. coli (Erwinia chrysanthem.
i EC19, Erwinia carotovora EC: Ref 25: J Bacteriol
169: 4379-4383,1987). The inventors have discovered that at, a subspecies of the bacterium Erwinia carotovora, which has a strong pectate lytic activity,
roseptica (Erwinia carotovora subs.atroseptica)
The pelB leader sequence was cloned from strains MAFF301614, MAFF301629 and MAFF301630 (origin: National Institute of Resources, Ministry of Agriculture, Forestry and Fisheries).

In the present invention, MAFF is used for the leader of H chain.
The sequence derived from 301614 (pelB-14) was used, and the sequence derived from MAFF301630 (pelB-30) was used for the leader of the L chain. These sequences further modified the DNA sequences to create restriction enzyme sites for the introduction of the first and second genes, and for rational joining of the introduced genes with the pelB sequence.

In the second cistron, the second gene is expressed on the 3 ′ side of the fd phage-derived G so that it is expressed in a form fused to the minor coat protein of the outer shell of the phage particle.
The DNA encoding eneIII was ligated. As the backbone of the phagemid vector, pUC-based vector
(Reference 26: Meth Enzymol 153: 3-11.1987). This is because a DNA fragment larger than that of M13-based phage can be cloned, and IG (Intergenic Region) is inserted, so that single-stranded DNA is preferentially wrapped in the phage particle as a genome by infection with M13 helper phage. This is because it has the characteristics described below. In addition, pUC
The backbone derived from the system vector contains the ampicillin resistance gene (amp ^r ), col E1 ori, and IG region.

The construction of the phagemid vector of the present invention will be described below.

(A) Outline of structure of phagemid vector for constructing Fab library (1) First expression vector (pRPLS / Fab-I) Example of structure of phagemid vector for constructing Fab library (pRPLS / Fab-I) : See Example 1) is shown in FIG. This vector contains two cistrons, the first and the second. The first cistron contains tac-SD promoter, pelB-30, a linker site having a first gene insertion site, and a termination site from the 5'side, and the second cistron contains tac-SD promoter, pelB-14 from the 5'side. , A linker site having a second gene insertion site, a Gene III gene, and a termination site.

In this example, the restriction enzyme site of the first gene insertion site has NheI and AscI from the 5 ′ side, and the second gene insertion site has SfiI and NotI from the 5 ′ side. pRPL
E. coli DH5 carrying S / Fab-I (named pRPLS / Fab-1)
Has been deposited with the deposit number of FERM P-14578 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science since October 11, 1994.

(2) Second Expression Vector (pRPLS / Fab-II) Another example of the structure of the phagemid vector for constructing the Fab library (pRPLS / Fab-II: see Example 1) is shown in FIG. The first cistron contains tac-SD promoter, pelB-14, a linker site having a first gene insertion site, and a termination site from the 5'side, and the second cistron contains tac-SD promoter, pelB-30 from the 5'side. , A linker site having a second gene insertion site, a Gene III gene, and a termination site. It has SfiI and NotI from the 5'side as the restriction enzyme site of the first gene insertion site, and NheI and AscI from the 5'side as the second gene insertion site. pRPLS / Fab-II
E. coli DH5 (designated pRPLS / Fab-2), which retains
Deposited under the accession number FERM P-14579 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science since October 11, 4th.

(B) Promoter, leader sequence, Ge
Preparation of neIII gene A method for synthesizing or cloning the GeneIII gene encoding tac-SD promoter, pelB leader sequence and ciliated protein, which is necessary as a preparation for constructing a phagemid vector, will be described.

(1) Cloning of the pelB gene The pelB sequence is Erwinia carotovora, for example Erwinia.
From the MAFF301614 strain, MAFF301629 strain, and MAFF301630 strain, which are the strains of carotovora subsp.
It is obtained by amplification by the CR method. The oligonucleotide used for the primer is SEQ ID NO: 29.
And those having the sequences shown in 30. MAFF301614 strain-derived pelB amino acid sequence obtained by using this primer (hereinafter referred to as "pelB-14") and D
The NA sequence is shown in SEQ ID NO: 1 and the pelB derived from the MAFF301629 strain and the pelB derived from the MAFRF301630 strain (hereinafter referred to as "pelB-3
The sequence "0") is shown in SEQ ID NO: 2.

(2) Cloning of tac-SD promoter The tac-SD promoter is 3'of the tac promoter.
A promoter having an SD sequence (ribosome binding site) in the downstream, which is commercially available (eg, tac Promoter GenBl
ock: Pharmacia) can be used. This promoter is used as a promoter independent of each of the first cistron and the second cistron contained in the expression vector of the present invention. Therefore, the Fd gene and the L chain gene cloned into the expression vector of the present invention are
In both cases, strong expression is induced by IPTG.

PRPLS / Fab-I and pRP obtained in Examples described later
In LS / Fab-II, tac in the first cistron
As restriction enzyme cleavage sequences at both ends of the -SD promoter, AflIII was used on the 5'side and BamHI on the 3'side. In addition, the restriction enzyme cleavage sequences at both ends of the tac-SD promoter in the second cistron include HindII on the 5'side.
BamHI was used for the I and 3'sides.

(3) Cloning of GeneIII Gene The GeneIII gene was cloned into fd phage (eg ATCC3700).
From 0), it is obtained by amplification by the PCR method. The primers used include oligonucleotides having the sequences shown in SEQ ID NOs: 26 and 27. GeneIII
For pRPLS / Fab-I, fusion with Fd protein, p
In RPLS / Fab-II, it was used for fusion with L chain protein.

<2> Setting of restriction enzyme cleavage sequences to be retained at the 5'and 3'ends of H chain Fd gene and L chain gene Analyzes of human antibody Fab and L chain DNA sequences using EMBL and GenBank databases. However, not only C area and framework area but also CDR (C
omplementarity Determining Region) also has a restriction enzyme cleavage sequence that is likely to appear, and it is very important to select the restriction enzyme cleavage sequences to be attached to both ends of the H chain Fd gene and L chain gene that form Fab in constructing the library. It turned out to be. The sites having these restriction enzyme cleavage sequences on the phagemid vector are Fd and L
Since it becomes the site for introducing the chain gene, it was necessary to select a site with high ligation efficiency. As a result, SfiI,
NotI, AscI, NheI and FseI were selected. The use of NheI is inappropriate for cloning mouse antibody genes, but any of the four combinations is possible for antibody genes of other animals. The SfiI site is created by modifying the DNA sequence near the 3'end of the pelB-14 sequence used as the leader sequence for the H chain without changing the encoded amino acid sequence (or inducing the L chain). It is possible to modify the same with the pelB-30 array. Although Tth111I was not found in at least the analyzed database, it was excluded from the selection of restriction enzyme sites because its ligation efficiency was extremely low.

<3> Primer for cloning human H-chain Fd gene and L-chain gene Fa based on lymphocytes obtained from peripheral blood or spleen
When trying to create a b gene library, the number of independent clones generated is enormous. Furthermore, the H chain gene and the L chain gene are separately separated from each other for convenience of cloning.
In order to obtain A and prepare each cDNA, the number of independent clones consisting of the combination is in principle their multiplier. For example, when 10 ⁷ types of natural antibody genes are present, the number of recombinant genes is 10 ⁷ × 10 ^7, which is 10 ¹⁴ .

Extensive research has been conducted on the gene sequence of human antibodies, and in addition to the EMBL and GenBank databases, it has also been edited as a data book by Kabat et al. Of NIH, USA (Ref. 27: Sequences of protei).
ns of immunological interest, 1991). Human antibody Fa
There are several papers on primers as PCR templates for cloning b gene (Ref 16: Autoimmunity 12: 135-141, 1992, 19: Nucleic.
Acids Res 19: 4133-4137, 1991), it is hard to say that it has enough homology to compare and collate in the database. The 5'sequence of the Fd gene differs in consensus sequence depending on the VH family, and there are further mutations to be considered in the mRNA sequence resulting from rearrangement of the gene. Similarly, regarding the L chain 5 ′ sequence, there are differences in the consensus sequence between the Vκ family and the Vλ family, and there are further mutations to be considered in the mRNA sequence resulting from the rearrangement of genes.

The previously reported primers for cloning human antibodies are (Ref 16: Autoimmunity 12: 135-141, 1992, 19: Nucle
ic Acids Res 19: 4133-4137, 1991, 28: Biochem Bioph
y Res Com 160: 1250-1256, 1989), it does not have sufficient correspondence to the diversity of 5'-side DNA sequences, and it is completely insufficient for preparing a library. In addition, judging from the size of primer degeneracy (degeneracy: the number of combinations of sequences contained in a primer), there are some that are actually considered unsuitable for use in PCR. In the present invention, an efficient primer having sufficient compatibility with the diversity of these sequences and minimizing degeneracy was developed. In the present invention, Fd is set so as to cleave at the amino-terminal side of the S—S bond where H chains bind to each other to form a homodimer, and designed to obtain an original antibody conformation by binding to an intact L chain. did.

(A) Human Antibody Fab Gene Cloning Primer A primer as a PCR template for cloning the Fab gene is designed so that the Fd gene constituting the H chain and the L chain gene can be completely cloned. Preferably.

(1) Primer for Fd gene cloning The 3'side sequence of the Fd gene is unique depending on the isotype of the antibody and, in the case of IgG, depending on its subclass. For example, regarding the Fd gene antisense primer, IgG1 is SEQ ID NO: 9, and IgG2 is SEQ ID NO: 1
0, IgG3 is SEQ ID NO: 11, IgG4 is SEQ ID NO: 12, I
gM can be amplified by the PCR method using a primer having the nucleotide sequence of SEQ ID NO: 13. The degeneracy of each sequence is 1.

The 5'side sequence of the Fd gene has a consensus sequence characteristic of the VH family and a certain degree of diversity. For this reason, the design of the sense primer was generally made to correspond to the VH family, but when comparing DNA sequences within 8 from the 5'end of the Fd gene, designing for each VH family limits the total number of primers. In some cases, it is not always efficient. For example, in the case of the VH1 family, the majority (about 80%) can be PCR-amplified with the sense primer of SEQ ID NO: 3, while the rest can be efficiently supplemented by using the sense primer of SEQ ID NO: 4. The sense primer of SEQ ID NO: 5 basically targets the VH2 family and VH6 family, but also includes most of the VH4 family. Therefore, the primers shown in SEQ ID NOS: 3 to 8 are designed in consideration of accurately rescue all antibody genes, labor of library construction, and capacity thereof.

Table 1 shows each sense primer and VH.
Indicates the family correspondence.

[0049]

[Table 1]

(2) Primer for L chain gene cloning As an antisense primer for cloning the L chain gene by the PCR method, SEQ ID NO:
An oligonucleotide having the sequence shown in SEQ ID NO: 25 for the 24 and λ chains can be mentioned. Unique sequence for κ chain (1 for degeneracy), 3 for λ chain
It is a sequence in which bases at certain positions can be replaced (degeneracy is 8). The 5'sequence of the L chain gene has different sequence characteristics depending on the type (κ, λ). For the κ chain, design sense primers for each Vκ family, and for the λ chain,
Although it is basically based on the λ family, it was more efficient to give cross-ability between the families depending on the sequence characteristics as a result of comparing amino acid sequences within 8 amino acids from the 3'end.
Examples of the kappa chain gene sense primer include the nucleotide sequences set forth in SEQ ID NOS: 14 to 17, and examples of the λ chain gene sense primer include the nucleotide sequences of SEQ ID NOS: 18 to 23.

Table 2 shows each sense primer and L.
The correspondence between the chain type and the Vκ family and Vλ family is shown.

[0052]

[Table 2]

(B) Cloning of antibody Fab gene from lymphocytes The method for cloning the antibody Fab gene from lymphocytes will be specifically described below.

(1) Preparation of mRNA and synthesis of first-strand cDNA Lymph cells were isolated from the peripheral blood of a healthy individual, and the cells were collected on the 8th day after activation of proliferation by EBV virus infection to prepare mRNA. Oligo- (dT) for Fd gene,
First strand cDNA in the presence of reverse transcriptase (reverse transcriptase) using random primers
Synthesize A. Similarly, Oligo- (d
T), First-strand cDNA is synthesized in the presence of reverse transcriptase (reverse transcriptase) using random primers.

(2) PCR amplification of cDNA 2-1. PCR amplification of cDNA of Fd gene Pooled VH sense primer (SEQ ID NO: 3 to
7) and a pool of antisense primers corresponding to IgG subclasses (SEQ ID NOs: 9 to 12)
PCR of gG Fd gene cDNA was performed.

Depending on the accuracy of the library of interest, antisense primers are IgG1 (SEQ ID NO: 9), IgG1
Those corresponding to 2 (SEQ ID NO: 10), IgG3 (SEQ ID NO: 11) and IgG4 (SEQ ID NO: 12) can be selected and used.
For IgM, SEQ ID NO: 13 is used. For sense primers, divide them into 3 groups (Group 1: SEQ ID NO: 3 and 4, Group 2: SEQ ID NO: 5, Group 3: SEQ ID NO: 6 and 7) and use them in combination with antisense primer. As a result, it is possible to keep degeneracy low.

2-2. PCR amplification of L chain gene cDNA First, the kappa chain was carried out, and then the lambda chain was targeted. V
The κ sense primer and the Vλ sense primer were pooled and used. However, depending on the accuracy of the target library, the sense primers for Vκ are divided into three groups (Group A: SEQ ID NO: 14, Group B: SEQ ID NO: 1).
5, Group C: SEQ ID NOs: 16 and 17), using each κ chain-specific antisense primer (SEQ ID NO: 24) for P
Perform CR. The sense primer for Vλ was also divided into three groups (Group D: SEQ ID NO: 18, 19, Group
E: SEQ ID NO: 20, Group F: SEQ ID NOs: 21 and 22 and 23), and PCR is performed using a λ chain-specific antisense primer (SEQ ID NO: 25).

(3) Incorporation of Fd gene and L chain gene into phagemid vector The PCR product of the Fd gene was purified and digested with, for example, SfiI and NotI, and then the first expression vector (eg pRPLS / Fab-
Ligate to the second gene introduction site in I). Similarly,
The PCR product of the L chain gene is purified, for example, NheI, AscI
After digestion with, the product is ligated to the first gene introduction site of the phagemid vector (pRPLS / Fab-I) into which the Fd gene has been previously incorporated.

(4) Rescue of Fab-expressing phagemid Using the phagemid vector in which the Fd gene and the L chain gene were integrated, E. coli competent cells XL1-
Transform Blue (Takara Shuzo) etc. After growing E. coli, the phagemid is rescued by a helper phage, such as VCS-M13 helper phage (Funakoshi). Fa is present on the coat protein of this phagemid.
b expresses a fusion protein with the gene III cilia protein. The Fab-expressing phagemid can select the desired antibody by panning against the antigen.

(5) Screening of Fab with the second expression vector PCR of Fd gene was carried out by the same procedure as the first expression vector.
After digesting the product with SfiI and NotI, the product was ligated to the first gene introduction site of the second expression vector (eg pRPLS / Fab-II), and then the PCR product of the L chain gene was NheI, As
After digestion with cI, the phagemid vector (pRPLS / Fab-II) having the Fd gene already incorporated therein is randomly ligated to the second gene introduction site to form a library.

The difference from the first expression vector is that the L chain protein is expressed as a fusion protein with the ciliary protein, which allows effective use as a two-step screening method. That is, the H chain Fd gene of Fab having the highest binding affinity to the antigen of interest obtained by panning the first expression vector has been identified,
The Fab gene library was reconstructed by introducing all of the genes into the second expression vector and randomly ligating the L chain gene, and the Fab composed of the combination with the L chain having the highest binding affinity to the antigen of interest. Is a method of screening again. Due to the characteristics of the phage (phagemid) vector, it may not be sufficient to form a library in a single cloning, and therefore it is used as a means for enhancing the library.

<4> Use of Recombinant Phage Library Immortalize the antibodies produced by lymphocytes of the living body and peripheral blood at the gene level and make a library using recombinant phages (phagemids). This recombinant phage library system has the potential to effectively rescue antibody genes from a population of lymphocytes or heterozygous antibody-producing cells that only yield a very small population. There are great expectations for obtaining a gene for a human monoclonal antibody that has a high potential for application in the pharmaceutical field, but it can also be used for antibodies derived from other species. Further, it can be effectively used for making a library of immunoglobulin superfamily genes having a β-sheet structure similar to that of antibodies, for example, TcR gene. It can also be applied to single-chain peptides by using only one cistron.

The phagemid vector into which the Fab gene has been incorporated can infect Escherichia coli and can be increased in large quantities, and can be used as a diagnostic or therapeutic drug. In particular, it is highly useful in the development of human monoclonal antibodies that are difficult to immortalize at the cellular level.

[0064]

EXAMPLES Examples of the present invention will be described below.

[0065]

Example 1 Construction of first expression vector pRPLS / Fab-I and second expression vector pRPLS / Fab-II

(1) Construction of plasmid for antibody gene cloning Phagemid vectors pRPLS / Fab-I and pRPLS / Fab-II
PUC / CV-1 and pUC / CV-2 were prepared as plasmid vectors for antibody gene cloning used in the step of constructing. PUC119 (Ref: Gene 33: 103-119, 1985)
The multi-cloning site (MCS) was modified. pUC / CV-
MCS of 1 is 5'-HindIII-SphI-PstI-SalI-XbaI-BamHI-Sf
iI-NotI-SacI-EcoRI-3 ', pUC / CV-2 MCS is 5'-Hin
dIII-SphI-PstI-SalI-XbaI-BamHI-NheI-AscI-KpnI-SacI
-EcoRI-3 '.

(2) Cloning of pelB leader sequence MAFF301614 strain, MAFF301629 strain, MAFF30 which are strains of the bacterial strain Erwinia carotovora subsp. Atroseptica
The pelB leader sequence was cloned from each of the 1630 strains. As PCR primers, the sense primer defined in SEQ ID NO: 29 and the antisense primer defined in SEQ ID NO: 30 were synthesized.

One colony of MAFF301614 strain was scooped with a platinum loop, and solution-1 (50 mM glucose, 25 mM Tris-HCL, 10 mM E
Suspended in 50 μl of DTA pH8.0) and then solution-2 (0.2 N NaO
H, 1% SDS) 100 μl was added. To 0.1 μl of this solution, add 100 pmole of sense primer (SEQ ID NO: 29) and antisense primer (SEQ ID NO: 30), and add Taq polymerase 2.5
U / 0.5 μl and 2.0 μl MMLV buffer (250 mM Tris-HCL (25 ° C
PH 8.3), 375 mM KCL, 15 mM MgCl2), 100 mM DTT 1.0 μ
l, 10 mM dNTP 0.5 μl (10 mM dATP, dCTP, dGTP, dTTP),
0.25 μl of BSA acetate (4 mg / ml) was added, and sterile water was added so that the final volume was 100 μl.

One drop of mineral oil (Sigma Chemical Co.) was added to the above mixed solution, and PCR was carried out under the following conditions. 94 ° C 5
After heating for 1 minute, 94 ℃ 2 minutes, 60 ℃ 1 minute, 72 ℃ 2 minutes 3
After repeating the step 30 times, the reaction was terminated by treating at 72 ° C. for 10 minutes. Remove the aqueous phase from the reaction solution, add 2 μl of 10 × H solution to 10 μl of it, and add the restriction enzyme BamHI to 12 μl.
U / 1 μl and NotI (20 U / 1 μl) were added, and sterilized water (7 μl) was added, followed by reaction at 37 ° C for 1 hour. The reaction solution was subjected to electrophoresis for 30 minutes using 0.8% agarose gel. Wavelength 254n on gel
A band of about 0.1 kb detected by irradiating with m ultraviolet light was cut out. Put this agarose fragment into a 1.5 ml tube,
Centrifugation was performed at 15,000 rpm for 10 minutes using a centrifuge, and the obtained aqueous solution was separated with a pipette to obtain a DNA solution.

PUC / CV-1 was added to 0.2 μl of this DNA solution.
The plasmid cleaved at both BamHI and NotI sites is 1ng /
0.1 μl was added, 1.8 μl of solution A of DNA ligation kit Ver.1 (Takara Shuzo) and 0.3 μl of solution B were added, and the mixture was reacted at 16 ° C. for 30 minutes. The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. This solution was placed on ice for 2 minutes, then 0.9 ml of SOC medium (Toyobo) was added, and the mixture was shake-cultured at 37 ° C. for 1 hour on a shaker. This solution was centrifuged at 5,000 rpm for 1 minute using a centrifuge and the supernatant was discarded. The competent cells that had settled in the solution remaining in the tube were suspended and plated on two ampicillin plates at a ratio of 1:10. These plates were incubated overnight at 37 ° C. In the generated colony, a new restriction enzyme site between BamHI and NotI
A transformant containing a plasmid having a DNA sequence containing the 0.1 kb pelB gene inserted therein was obtained. When this inserted sequence was sequenced, pelB-14 having the nucleotide sequence shown in SEQ ID NO: 1 was obtained.

When the pelB leader sequence of the MAFF301629 strain and the MAFF301630 strain were cloned and sequenced by the same method, the two had the same DNA sequence and had the sequence shown in SEQ ID NO: 2 (pelB-3.
0).

(3) Modification of pelB leader sequence pelB-14 is a sequence containing a BamHI site 5'upstream and SfiI, NotI near the 3'end for the purpose of use as a leader sequence of the Fd gene. I modified. This modification was performed without changing the encoded amino acid sequence by PCR using a sense primer having the sequence shown in SEQ ID NO: 31 and an antisense primer having the sequence shown in SEQ ID NO: 32 (the nucleotide sequence is Change). The obtained PCR products are BamHI and NotI.
Digest with pUC / CV-1 and then pCV1 / PB-14
I got

PelB-30 was modified to a sequence having a BamHI site at the 5'upstream and NheI and AscI from the 3'end to the downstream for the purpose of use as a leader sequence of the L chain gene. This modification consists of the sense primer having the sequence shown in SEQ ID NO: 31 and the SEQ ID NO:
It was performed by PCR using an antisense primer having the sequence shown in 33. In addition, between the NheI site and the AscI site of the sense primer (SEQ ID NO: 33), 10 bases including a stop codon (TAATGATAGA: in SEQ ID NO: 33, 11 to 2
(It corresponds to the 0th array). P obtained
CR product was digested with BamHI and AscI, and then pUC / CV
It was ligated with -2 to obtain pCV2 / PB-30.

(4) Incorporation of tac-SD Sequence The tac-SD promoter is the
A sequence containing an SD sequence (ribosome binding site) was ligated to the 3'side, which was derived from tac Promoter GenBlock (Pharmacia). Plasmid pD540 (Pharmacia) 100ng / 1μl
Add 10 μH solution (1 μl) to the restriction enzyme BamHI12U / 1 μl and H
IndIII 20U / 1 μl was added, and the mixture was reacted at 37 ° C. for 1 hour. The reaction mixture was subjected to electrophoresis for 30 minutes using 0.8% agarose gel. Approximately detected by irradiating the gel with UV light of 254 nm wavelength
A 0.1 kb band was cut out. 1.5 pieces of this agarose fragment
Place in a ml tube and centrifuge at 15,000 rpm for 10 minutes using a centrifuge, separate the resulting aqueous solution with a pipette, and
NA solution was used.

0.5 μl of this DNA solution was added to BamHI and Hind
Plasmid pCV1 / PB-14 cleaved at both sites of III was 1 ng /
0.1 μl was added, and 3.6 μl of solution A and 0.6 μl of solution B of DNA Ligation Kit Ver.1 (Takara Shuzo) were added and reacted at 16 ° C. for 30 minutes. The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. This was placed on ice for 2 minutes, then 0.9 ml of SOC medium (Toyobo) was added, and the mixture was shake-cultured at 37 ° C. for 1 hour on a shaker. This was centrifuged at 5,000 rpm for 1 minute using a centrifuge and the supernatant was discarded. The competent cells precipitated with the solution remaining in the tube were suspended and spread at a ratio of 1:10 on two ampicillin plates. These plates were cultured overnight at 37 ° C., plasmids were isolated from the resulting colonies, and a plasmid newly inserted with a DNA containing the tac-SD promoter of about 0.1 kb between the restriction enzyme sites BamHI and HindIII was inserted into pCV1. / PBH1

Similarly, instead of pCV1 / PB-14, pCV2 / P
Using B-30, the DNA of tac-SD promoter
The resulting plasmid inserted into pCV2 / PB-30 was designated as pCV2 / PBL.
I set it to 1.

(5) Cloning of GeneIII gene As a primer for cloning the GeneIII gene connected to the Fd gene, a sense primer having a NotI site (SEQ ID NO: 26) and an antisense primer having an EcoRI site (SEQ ID NO: 27) Was synthesized. Add 100 pmole of each of these sense primer and unsense primer to 0.01 pg of fd phage (ATCC37000) genome and add 2.5 U / 0.5 μl of Taq polymerase (Beringer-Mannheim)
And 2.0 μl MMLV buffer (250 mM Tris-HCL (pH 8.3 at 25 ℃), 3
75mM KCL, 15mM MgCl2), 100mMDTT 1.0μl, 10mM dNTP
0.5 μl (10 mM dATP, dCTP, dGTP, dTTP), BSA acetate (4 mg / m
l) 0.25 μl was added, and sterilized water was added so that the final volume was 100 μl. One drop of mineral oil (Sigma Chemical Co.) was added to these mixed solutions, and PCR was performed under the following conditions. 95 ° C 3
After heating for 1 minute, 95 ℃ 1 minute, 55 ℃ 2 minutes, 72 ℃ 2 minutes 3
The steps were repeated 30 times, and the reaction was terminated by treating at 72 ° C for 10 minutes.

The aqueous phase was taken out from this PCR reaction solution, 10 μl of which was added with 2 μl of 10 × H solution, and the restriction enzyme NotI was added to 20 μl.
U / 1 μl and EcoRI (20 U / 1 μl) were added, and sterilized water (7 μl) was added, followed by reaction at 37 ° C for 1 hour. The reaction solution was subjected to electrophoresis for 30 minutes using 0.8% agarose gel. Wavelength 254nm on gel
The band of about 1.2 kb detected by irradiating with the ultraviolet ray was cut out. This agarose fragment was placed in a 1.5 ml tube and centrifuged at 15,000 rpm for 10 minutes using a centrifuge, and the obtained aqueous solution was separated with a pipette to give a DNA solution.

NotI, EcoR was added to 0.5 μl of this DNA solution.
Plasmid pUC / CV-1 cleaved at both sites of I was 1ng / 0.1μ
l In addition, A of DNA Ligation Kit Ver.1 (Takara Shuzo)
The solution (3.6 μl) and the solution B (0.6 μl) were added and reacted at 16 ° C. for 30 minutes.
The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. Place on ice for 2 minutes and then add SOC medium (Toyobo)
0.9 ml was added, and the mixture was shake-cultured at 37 ° C. for 1 hour with a shaker. The supernatant was discarded by centrifuging at 5,000 rpm for 1 minute using a centrifuge. The competent cells precipitated by the solution remaining in the tube were suspended and spread at a ratio of 1:10 on two ampicillin plates. Incubate these plates at 37 ° C overnight,
Among the plasmids obtained from the generated colonies, a gene of about 1.2 kb between the restriction enzyme sites NotI and EcoRI was newly added.
PCV1 / Fd-G3 with inserted gene-containing DNA
And

On the other hand, GeneIII connected to L gene
As a primer for cloning genes
A sense primer (SEQ ID NO: 28) having a cI site was synthesized and used in combination with an antisense primer (SEQ ID NO: 27) having an already synthesized EcoRI site, and PCR was performed.
The reaction was performed. Hereinafter, the above Gene for Fd gene connection
The GeneIII gene was amplified in the same manner as the amplification of the III gene, and this was inserted between the AscI site and EcoRI site of pUC / CV-2 to obtain pCV2 / FL-G3.

(6) Construction of First Expression Vector (1) A recombinant phagemid vector into which a gene encoding the L chain as the first gene and a gene encoding the H chain Fd as the second gene can be inserted was constructed.

The Gene III gene was excised from pCV1 / Fd-G3 by digesting NotI and EcoRI. This Gene I
II gene, tac-SD promoter and pelB
-14-containing plasmid pCV1 / PBH1 was inserted between NotI and EcoRI sites to obtain pCV1 / PBH2. pe included in pCV1 / PBH2
The SfiI site near the 3'end of IB-14 and the GeneIII
The NotI site attached 5'upstream serves as the site for introducing the H chain Fd gene. Specifically, the plasmid pCV1 / Fd-G3 500ng / 9
1 μl of 10 × H solution was added to ml, and the restriction enzymes EcoRI 15U / 1 μl and NotI 20U / 1 μl were added and reacted at 37 ° C. for 1 hour. The reaction mixture was subjected to electrophoresis for 30 minutes using 0.8% agarose gel.
Approximately 1.2 kb detected by irradiating the gel with UV light with a wavelength of 254 nm
I cut out the band. This agarose fragment was placed in a 1.5 ml tube and centrifuged at 15,000 rpm for 10 minutes using a centrifuge, and the obtained aqueous solution was separated with a pipette to separate DNA.
It was a solution.

0.5 μl of this DNA solution was added to pCV1 / PB-14
Plasmids cleaved at both BamHI and HindIII sites of
ng / 0.1 μl was added, and 6.6 μl of solution A and 1.1 μl of solution B of DNA Ligation Kit Ver.1 (Takara Shuzo) were added and reacted at 16 ° C. for 30 minutes. The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. Place on ice for 2 minutes and then SOC media
(Toyobo) was added in an amount of 0.9 ml and shake-cultured at 37 ° C. for 1 hour with a shaker. The supernatant was discarded by centrifuging at 5,000 rpm for 1 minute using a centrifuge. The remaining solution was used to suspend the precipitated competent cells and spread them on two ampicillin plates at a ratio of 1:10. The restriction enzyme sites EcoRI and N were newly added to the plasmids obtained from the resulting colonies after overnight culture at 37 ° C.
A pUC / PBH2 was obtained by inserting about 1.2 kb of the DNA containing the GeneIII gene into otI.

Next, a sense primer having an AflIII site was prepared so that tac-SD-pelB-30 contained in the plasmid pCV2 / PBL1 could be amplified by modifying the terminal sequence.
An antisense primer (SEQ ID NO: 35) having (SEQ ID NO: 34) and (Stop) -AscI- (Stop) -HindIII site was synthesized. pC
Add 100 pmole of the above primer to 0.1 pg / 1 μl of V2 / PBL1 and add 2.5 U / 0.5 μl of Taq polymerase (Boehringer Mannheim).
2.0 μl MMLV buffer (250 mM Tris-HCL (pH 8.3 at 25 ℃), 37
5 mM KCl, 15 mM MgCl ₂ ), 100 mM DTT 1.0 μl, 10 mM dNTP
0.5 μl (10 mM dATP, dCTP, dGTP, dTTP), BSA acetate (4 mg / m
l) 0.25 μl was added, and sterilized water was added so that the final volume was 100 μl. One drop of mineral oil (Sigma Chemical Co.) was added and PCR was performed under the following conditions. After heating at 94 ℃ for 3 minutes, 94 ℃ 1
The reaction was terminated by repeating the three steps of 30 minutes, 60 ° C. for 2 minutes, and 72 ° C. for 2 minutes 30 times, and then treating at 72 ° C. for 10 minutes.
Remove the aqueous phase from the reaction solution, and add 10 μH solution to 10 μl of it 2
Add μl, add 20 U / 1 μl of restriction enzyme HindIII and 10 U of AflIII
/ 1 μl was added, and 7 μl of sterilized water was added, followed by reaction at 37 ° C for 1 hour. The reaction solution was subjected to electrophoresis for 30 minutes using 0.8% agarose gel. Approximately 0.2 kb by irradiating ultraviolet rays with a wavelength of 254 nm
I cut out the band. This agarose fragment was placed in a 1.5 ml tube and centrifuged at 15,000 rpm for 10 minutes using a centrifuge, and the obtained aqueous solution was separated with a pipette to separate DNA.
It was a solution. For 0.5 μl of this DNA solution, pCV1 / PBH2
The plasmid cleaved at both HindIII and AflIII sites of
1 ng / 0.1 μl was added, 5.4 μl of solution A and 0.9 μl of solution B of DNA Ligation Kit Ver.1 (Takara Shuzo) were added, and the mixture was reacted at 16 ° C. for 30 minutes. The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. Place on ice for 2 minutes and then SOC media
(Toyobo) was added in an amount of 0.9 ml and shake-cultured at 37 ° C. for 1 hour with a shaker. The supernatant was discarded by centrifuging at 5,000 rpm for 1 minute using a centrifuge. The remaining solution was used to suspend the precipitated competent cells and spread them on two ampicillin plates at a ratio of 1:10. A new restriction enzyme site, HindIII, was found in the plasmids obtained from the resulting colonies after overnight culture at 37 ° C.
0.2kb between tac-SD-pelB-30-L and AflIII
The one in which the DNA sequence containing the chain gene introduction site was inserted was designated as pRPLS / Fab-IP.

(7) Construction of the first expression vector (2) A termination linker was inserted between the SfiI site and NotI site of the plasmid pRPLS / Fab-IP. pRPLS / Fa
Add 1 μl of 10 × H solution to 50 ng / 1 μl of bIP and add SfiI 2 restriction enzyme.
0 U / 1 μl was added and reacted at 50 ° C. for 1 hour. Then 10 x NE
1 μl of B (Toyobo) solution was added, 20 U / 1 μl of restriction enzyme NotI was added, and the mixture was reacted at 37 ° C. for 1 hour. The reaction mixture was subjected to electrophoresis for 30 minutes using 0.8% agarose gel. The band of about 4.4 kb was cut out by irradiating with ultraviolet ray of wavelength 254 nm. Put this agarose fragment into a 1.5 ml tube and centrifuge 15,
Centrifugation was performed at 000 rpm for 10 minutes, and the obtained aqueous solution was separated with a pipette to obtain a DNA solution. To this solution, newly synthesized linker A (SEQ ID NO: 37) and linker B (SEQ ID NO: 38) were mixed in equal amounts (500 ng / 1 μl) O. 1 μl was added, and DNA Ligation Kit Ver. 1 (Takara Shuzo) was added. A liquid
1.2 μl and 0.2 μl of solution B were added and reacted at 16 ° C. for 30 minutes. The reaction solution was added to 0.1 ml of Escherichia coli competent cell strain DH5 (Toyobo), reacted for 30 minutes on ice, and then heat-shocked at 42 ° C for 60 seconds. Place on ice for 2 minutes, then add SOC medium (Toyobo) to 0.
9 ml was added and the mixture was shake-cultured at 37 ° C. for 1 hour with a shaker.
The supernatant was discarded by centrifuging at 5,000 rpm for 1 minute using a centrifuge. Suspend the remaining competent cells
Spread 1:10 on 2 ampicillin plates. 3
Of the plasmids obtained from the resulting colonies after overnight culture at 7 ° C, those in which a DNA containing a termination sequence was newly inserted between the restriction enzyme sites SfiI and NotI were pR
It was PLS / Fab-I.

(8) Construction of second expression vector pRPLS / Fab-II A recombinant phagemid vector into which a gene encoding H chain Fd as a first gene and a gene encoding L chain as a second gene can be inserted was constructed.

Tac-SD-pelB-14 for H chain expression
The sequence ends of were modified and amplified. Already synthesized Afl III
PCR was performed using pCV1 / pBH1 as a template, using a sense primer having a site (SEQ ID NO: 34) and an antisense primer having a newly synthesized SfiI-NotI- (Stop) -HindIII site (SEQ ID NO: 36).

The GeneIII gene cut out from pCV2 / FL-G3 was inserted between the AscI site and EcoRI site of pCV2 / PBL1 to obtain pCV2 / PBL2. The above PCR reaction product was inserted between the AflIII site of this pCV2 / PBL2 and HindIII to obtain the second expression vector pRPLS / Fab-II.

[0089]

Example 2 Cloning of antibody Fab gene from human peripheral blood lymphocytes An example of cloning of IgG1 subclass is shown below.

(1) Collection of Lymph Cells and Separation of mRNA 10 ml of peripheral blood was collected from a healthy person with high hCMV (human cytomegalovirus) neutralizing activity, and Ficoll Paque (Pharmacia) was collected.
Were used to separate lymphocytes. The obtained lymphocytes were infected with EBV to immortalize the cells, and the cells were seeded at 10 ³ / well in a 96-well culture plate. RPMI16 for medium
40% supplemented with 10% fetal bovine serum was used to activate lymphokine (IL-
4: 30ng / ml, IL-5: 20ng / ml, IL-6: 30pg / ml) was added. The cells become active after 1 week, and at that time, the lymphocytes with high antibody-producing ability in the culture supernatant were taken out, seeded on another culture plate at 10 ² / well, and allowed to grow again. Cells
Micro-FastTrack mRNA at the time of increasing to 5 × 10 ⁵ / well
From the cells using ISOLATION KIT (Invitrogen)
RNA was extracted. The obtained mRNA sample was frozen and stored at -70 ° C.

(2) Synthesis of first-strand cDNA 300 μl of mRNA sample was placed in a 1.5 ml Eppendorf tube and
It was centrifuged at 5,000 rpm for 10 minutes. After discarding the supernatant, 1 ml of 70% ethanol was added to the precipitate for washing, and the mixture was centrifuged at 15,000 rpm for 1 minute. After discarding the supernatant again, the precipitate was air dried for 30 minutes. To this, 10 μl of RNase-free DEPC water (water treated with diethyl borocarbonate) was added and heated at 37 ° C. for 5 minutes. 35 μl of this
Reverse transcription reaction using RNA PCR kit (Takara Shuzo)
(RT-PCR) was performed. The primers are random 9mer and Oli
I used go- (dT). The reaction temperature and time were 30 ° C. for 10 minutes, 42 ° C. for 30 minutes, 90 ° C. for 5 minutes, and 5 ° C. for 5 minutes.
The reaction solution R was obtained by the reverse transcription reaction.

(3) PCR amplification of double-stranded cDNA The obtained single-stranded cDNA was subjected to PCR amplification using an RNA PCR kit (Takara Shuzo). The immunoglobulin subclass IgG was targeted.

(A) PCR amplification of Fd gene cDNA The sense primers were VHBACK1 (SEQ ID NO: 3) and VHBACK2.
(SEQ ID NO: 4), VHBACK3 (SEQ ID NO: 5), VHBACK4 (SEQ ID NO: 6), VHBACK5 (SEQ ID NO: 7), VHBACK6 (SEQ ID NO: 8) 1
An equal volume of 1 μl was used with 00 pmole/1 μl. Strictly speaking, it is desirable to change the molar ratio in consideration of degeneracy, but tests with equal amounts were also sufficient to prove the effectiveness of library formation. Antisense primer is FdFOR1
(SEQ ID NO: 9), FdFOR2 (SEQ ID NO: 10), FdFOR3 (SEQ ID NO: 1)
1), FdFOR4 (SEQ ID NO: 12) was used at 100 pmole / 1 μl in 1 μl aliquots. The reaction temperature and time were 90 ° C for 5 minutes, 95 ° C for 90 seconds, 50 ° C for 90 seconds, 72 ° C for 150 seconds, and the steps were repeated 30 times, followed by 72 ° C for 10 minutes to complete the reaction. . The liquid volume used for the reaction was 100 μl, and the obtained solution was used as the reaction liquid Gp.

When the amplified product was confirmed by agarose electrophoresis, a band of about 0.6 to 0.7 kb was observed.

(B) PCR amplification of L chain gene cDNA PCR amplification was first carried out for the κ chain and then for the λ chain. The sense primer for κ chain is VκBACK1 (SEQ ID NO: 14), VκBACK2 (SEQ ID NO: 15), VκBACK3 (SEQ ID NO: 1)
6), VκBACK4 (SEQ ID NO: 17) was used at 100 pmole / 1 μl in 1 μl aliquots. Strictly speaking, it is desirable to change the molar ratio in consideration of degeneracy, but tests with equal amounts were also sufficient to prove the effectiveness of library formation. The antisense primer was CpmFOR1 (SEQ ID NO: 24) at 100pmole /
1 μl was used with 1 μl. The reaction temperature and time are 95 ℃ for 5 minutes, then 95 ℃ for 90 seconds, 50 ℃ for 90 seconds and 72 ℃ for 150 seconds.
The second step was repeated 30 times, and the reaction was allowed to proceed at 72 ° C. for 10 minutes to finish. The liquid volume used for the reaction was 100 μl, and the obtained product was used as the reaction liquid Kp.

The sense primers for λ chain are VλBACK1 (SEQ ID NO: 18), VλBACK2 (SEQ ID NO: 19), VλBACK3 (SEQ ID NO:
20), VλBACK4 (SEQ ID NO: 21), VλBACK5 (SEQ ID NO: 22), V
λBACK6 (SEQ ID NO: 23) was used at 100 pmole / 1 μl in 1 μl aliquots. Strictly speaking, it is desirable to change the molar ratio in consideration of degeneracy, but tests with equal amounts were also sufficient to prove the effectiveness of library formation. The antisense primer is 100 pmole / 1 μl of CλFOR1 (SEQ ID NO: 25).
1 μl was used. The reaction temperature and time were 95 ° C for 5 minutes, then 95 ° C for 90 seconds, 50 ° C for 90 seconds, 72 ° C for 150 seconds, and the steps were repeated 30 times, followed by 72 ° C for 10 minutes to complete the reaction. . The liquid volume used for the reaction was 100 μl, and the obtained product was used as the reaction liquid Lp.

Amplification products of both κ and λ chains were confirmed by agarose gel electrophoresis.
A 7 kb band was observed.

(4) pRPLS / of Fd gene and L chain gene
Incorporation into Fab-I (A) Cleavage of PCR product by restriction enzyme The PCR product of Fd gene (reaction solution Gp) was Sfi at 5 '.
A NotI site was added to the I and 3'sites, which were used for cloning after cutting with the respective restriction enzymes. First, 2 μl of 10 × NEB2 solution was added to 10 μl of reaction solution H, and the restriction enzyme Sf was added.
17 U / 1 μl of iI was added, and 7 μl of sterilized water was added, followed by reaction at 50 ° C. for 1 hour. Add 2 μl of 10 × NEB2 solution and add the restriction enzyme.
After adding 20 U / 1 μl of NotI, the mixture was reacted at 37 ° C. for 1 hour. After the reaction, the DNA was electrophoresed for 30 minutes using 0.8% agarose gel. A band of about 0.6 kb was cut out by irradiating with an ultraviolet ray having a wavelength of 254 nm. A DNA solution of this agarose fragment was separated and purified using QIAEX Gel Extraction Kit (QIAGEN), which is a DNA extraction kit. Finally, the DNA sample is 20μ
l of TE solution (10 mM Tris-HCl PH8.0, 1 mM EDTA). This TE solution was used as solution G. Next, regarding the PCR product of the κ chain gene (reaction solution Kp),
The 5'sense primer has an NheI site and the 3'antisense primer has an AscI site. Therefore, these two restriction enzymes were digested and used for cloning. First, add 2 μl of 10 × NEB4 solution to 10 μl of reaction solution Kp, and add restriction enzyme.
17 U / 1 μl of AscI was added, and 7 μl of sterilized water was added, followed by reaction at 37 ° C. for 1 hour. Further, 2 μl of 10 × NEB2 solution was added, and 20 U / I μl of the restriction enzyme NheI was added, followed by reaction at 37 ° C. for 1 hour. After the reaction, the DNA was electrophoresed for 30 minutes using 0.8% agarose gel. A band of about 0.6 kb was cut out by irradiating with an ultraviolet ray having a wavelength of 254 nm. DNA fragments were separated and purified from the agarose block using QIAEX Gel Extraction Kit (QIAGEN), which is a DNA extraction kit. Finally, the DNA sample was recovered in 20 μl of TE solution (10 mM Tris-HCL PH8.0, 1 mM EDTA). This TE solution was designated as solution K.

(B) Ligation to pRPLS / Fab-I 10 μg of pRPLS / Fab-I was taken, 10 μl of 10 × NEB2 solution was added, 170 U / 10 μl of restriction enzyme SfiI was added, and 80 μl of sterilized water was added, followed by 1 at 50 ° C. Reacted for hours. Further 10 x NEB2 solution 10 μl
Was added, and 20 U / 10 μl of the restriction enzyme NotI was added, followed by reaction at 37 ° C. for 1 hour. After the reaction, the DNA was electrophoresed for 30 minutes using 0.8% agarose gel. A band of about 4.4 kb was cut out by irradiating with an ultraviolet ray having a wavelength of 254 nm. This agarose block is a DNA extraction kit, QIAEX Gel Extraction.
The DNA solution was separated and purified using Kit (QIAGEN). Finally, the DNA sample was 20 μl TE solution (10 mM Tris-HCl PH
8.0, 1 mM EDTA). To 1 μl of this, add 5 μl of solution G purified Fd gene, add 36 μl of solution A and 6 μl of solution B of DNA Ligation Kit Ver.1 (Takara Shuzo), and add 16 μl.
The reaction was carried out at 30 ° C for 30 minutes.

The reaction product was subjected to phenol extraction and ethanol precipitation, and then dissolved by adding 9 μl of sterile distilled water. to this
1 μl of 10 × NEB4 solution was added, and 34 U / 2 μl of the restriction enzyme AscI was added, followed by reaction at 37 ° C. for 1 hour. Further, add 1 μl of 10 × NEB2 solution, add 40 U / 2 μl of restriction enzyme NheI, and then at 37 ° C.
The reaction was carried out for 1 hour. About 15 bp A generated by restriction enzyme digestion
Nucleic acid purification kit MicroS to remove small scI-NheI fragments
Purification was performed according to the protocol using S-300HR Column of pin Columns (Pharmacia) to obtain 13 μl of DNA solution. To this, add 5 μl of the purified κ chain gene solution K,
DNA Ligation Kit Ver.1 (Takara Shuzo) A Solution 108μ
1 and 18 μl of solution B were added and reacted at 16 ° C. for 30 minutes. The reaction product was phenol-extracted and ethanol-precipitated, and then dissolved by adding 10 μl of sterilized distilled water to E. coli competent cell DH5 strain.
After adding to 0.1 ml and reacting on ice for 30 minutes, heat shock was applied at 42 ° C. for 60 seconds. Place on ice for 2 minutes and then SOC media
(Toyobo) was added in an amount of 0.9 ml and shake-cultured at 37 ° C. for 1 hour with a shaker. The supernatant was discarded by centrifuging at 5,000 rpm for 1 minute using a centrifuge. The remaining solution was used to suspend the competent cells, which were precipitated, and the suspension was seeded on two ampicillin plates at a ratio of 1:10. After culturing at 37 ° C. overnight, it was confirmed that the Fd gene and the L chain (κ) were both incorporated in the plasmid obtained from the resulting colony.

(5) pRPLS / of Fd gene and L chain gene
Incorporation into Fab-II (A) Cleavage of PCR product by restriction enzyme PCR product of L chain gene (λ chain: reaction solution Lp) 5 ′
NheI site and 3'AscI site were added, and they were used for cloning after cutting with each restriction enzyme.
First, 2 μl of 10 × NEB2 solution was added to 10 μl of reaction solution L, 17 U / 1 μl of restriction enzyme AscI was added, and 7 μl of sterilized water was added.
Allowed to react for hours. Further, 2 μl of 10 × NEB2 solution was added, and 20 U / 1 μl of the restriction enzyme NheI was added, followed by reaction at 37 ° C. for 1 hour.
After the reaction, the DNA was electrophoresed for 30 minutes using 0.8% agarose gel. A band of about 0.6 kb was cut out by irradiating with an ultraviolet ray having a wavelength of 254 nm. A DNA solution of this agarose fragment was separated and purified using QIAEX Gel Extraction Kit (QIAGEN), which is a DNA extraction kit. Finally the DNA sample
It was recovered in 20 μl of TE solution (10 mM Tris-HCl PH8.0, 1 mM EDTA). This TE solution was designated as solution L

(B) Ligation to pRPLS / Fab-II 10 μg of pRPLS / Fab-II was taken, 10 μl of 10 × NEB2 solution was added, 170 U / 10 μl of restriction enzyme SfiI was added, and 80 μl of sterilized water was added, followed by 1 at 50 ° C. Reacted for hours. Further 10 x NEB2 solution 10 μl
Was added, and 20 U / 10 μl of the restriction enzyme NotI was added, followed by reaction at 37 ° C. for 1 hour. After the reaction, the DNA was electrophoresed for 30 minutes using 0.8% agarose gel. A band of about 4.4 kb was cut out by irradiating with an ultraviolet ray having a wavelength of 254 nm. This agarose block is DNA
Extraction kit, QIAEX Gel Extraction Kit (QIAGEN)
Was used to separate and purify the DNA solution. Finally, the DNA sample was 20 μl of TE solution (10 mM Tris-HCl PH8.0, 1 mM EDTA).
Recovered. To 1 µl of the solution, 5 µl of a solution G of purified Fd gene was added, and DNA Ligation Kit Ver.1 was added.
36 μl of solution A (Takara Shuzo) and 6 μl of solution B were added and reacted at 16 ° C. for 30 minutes.

The reaction product was subjected to phenol extraction and ethanol precipitation, and then dissolved by adding 9 μl of sterile distilled water. to this
After adding 1 μl of 10 × NEB4 solution and adding 34 U / 2 μl of the restriction enzyme AscI, the mixture was reacted at 37 ° C. for 1 hour. In addition to this 10 × NEB2
After adding 1 μl of the solution and adding 40 U / 2 μl of the restriction enzyme NheI, 37 ℃
And reacted for 1 hour. About 15 bp generated by restriction enzyme digestion
Nucleic acid purification kit Mic to remove small AscI-NheI fragments
Purified according to the protocol using S-300HR Column of roSpin Columns (Pharmacia), and 12 μl of DNA
A solution was obtained. To this, 5 μl of the purified λ chain gene solution L was added, and A of DNA ligation kit Ver.1 (Takara Shuzo) was added.
The liquid (102 μl) and the liquid B (17 μl) were added and reacted at 16 ° C. for 30 minutes.

The reaction product was extracted with phenol and precipitated with ethanol, dissolved in 10 μl of sterile distilled water, added to 0.1 ml of Escherichia coli competent cell DH5 strain, reacted on ice for 30 minutes, and then heat-shocked at 42 ° C. for 60 seconds. Was added. After placing on ice for 2 minutes, 0.9 ml of SOC medium (Toyobo) was added, and the mixture was shake-cultured at 37 ° C. for 1 hour on a shaker. Using a centrifuge 5,
The supernatant was discarded by centrifugation at 000 rpm for 1 minute. Suspend the competent cells that had precipitated with the remaining solution and
Sprinkled on a piece of ampicillin plate. After culturing at 37 ° C. overnight, it was confirmed that the Fd gene and the L chain (λ) were both incorporated in the plasmid obtained from the resulting colony.

References are shown below. Reference 1: Immunology, by JohnWiley & Sons, New York,
1992 Reference 2: Nature 256: 495-497, 1975 Reference 3: J Exp Med 168: 475-489, 1988 Reference 4: J Immunol Methods 95: 123-126, 1986 Reference 5: J Immunol 10: 278-285, 1991 Reference 6: Hybridoma 5: 33-41, 1986 Reference 7: The Medical Bulletin [Naito Medical Research Foundation] 2:
30-35, 1984 Reference 8: J Biol Chem, 267: 16007-16010, 1992 Reference 9: Science 240: 1041-1043, 1988 Reference 10: Science 246: 1275-1281, 1989 Reference 11: Science 228: 1315-1317 , See 1985 12: Gene 8: 309-314, 1990 See 13: Proc Natl Acad Sci USA 88: 7978-7982, 1991 See 14: EMBO J 12: 725-734, 1993 See 15: Proc Natl Sci USA 89: 3175 -3179, 1992 See 16: Autoimmunity 12: 135-141, 1992 See 17: Proc Natl Acad Sci USA 91: 3809-3813, 1994 See 18: Proc Natl Acad Sci USA 85: 5879-5883, 1988 See 19: Nucleic Acids Res 19: 4133-4137, 1991 Reference 20: Methods 2: 119-124, 1991 Reference 21: J Immunol 147: 3610-3614, 1991 Reference 22: Virology 132: 445-455, 1984 Reference 23: Biotechnology 9: 1373- 1377, 1991 Reference 24: J Immunol 151: 6954-6961, 1993 Reference 25: J Bacteriol 169: 4379-4383, 1987 Reference 26: Methods in Enzymol 153: 3-11.1987 Reference 27: Sequences of proteins of immunological int
erest, 1991 Ref 28: Biochem Biophy Res Com 160: 1250-1256, 198.
9 Reference 29: Biochemistry 18: 5294-99, 1979 Reference 30: Meth Enzymol 152: 219-227, 1987

[0106]

INDUSTRIAL APPLICABILITY According to the present invention, antibody F having excellent performance
A phagemid vector for the ab library is obtained.
It also provides accurate and efficient PCR primers for cloning human monoclonal antibodies. Using the phagemid vector and the primer of the present invention, an expression library of antibody Fab fragments can be prepared, and further, antibody Fab against a specific antigen can be produced.
The H chain Fd gene and the L chain gene encoding the b fragment can be selected.

[0107]

[Sequence listing] SEQ ID NO: 1 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear sequence Features Features symbol: sig peptide Location: 1..66 Features Determined method: E Origin organism name: Erwinia Carotovora subs.atroseptica Strain name: MAFF301614 Sequence ATG AAA TAC CTA TTG CCT ACG GCA GCC ACT GGA TTG TTA TTA CTC GCT 48 Met Lys Tyr Leu Leu Pro Thr Ala Ala Thr Gly Leu Leu Leu Leu Ala 1 5 10 15 GCC CAA CCA GCG ATG GCC 66 Ala Gln Pro Ala Met Ala 20

SEQ ID NO: 2 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence features Features symbol: sig peptide Location: 1..66 Features determined Method: E Origin Organism name: Erwinia Carotovora subs.atroseptica Strain name: MAFF301629 Sequence ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG CTA TTA CTC GCT 48 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 GCC CAA CCA GCG ATG GCC 66 Ala Gln Pro Ala Met Ala 20

SEQ ID NO: 3 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..15 Feature determination method: S Feature symbol: primer Location: 22..44 Feature determination method: P sequence AAGGCCCAAC CGGCCATGGC CCAGGTGCAG CTGGTGCAGT CTGG 44

SEQ ID NO: 4 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..15 Characteristic determination method: S Characteristic symbol: primer Location: 22..44 Characteristic determination method: P sequence AAGGCCCAAC CGGCCATGGC CCAGRTYCAG CTGGTGCAGT CTGG 44

SEQ ID NO: 5 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..15 Method by which the feature is determined: S Characteristic symbol: primer Location: 22..44 Method by which the feature is determined: P sequence AAGGCCCAAC CGGCCATGGC CCAGSTRCAG CTGCAGSAGT CRGG 44

SEQ ID NO: 6 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..15 Characteristic determination method: S Characteristic symbol: primer Location: 22..44 Characteristic determination method: P sequence AAGGCCCAAC CGGCCATGGC CSARGTGCAG CTGKTGGAGT CTGG 44

SEQ ID NO: 7 Sequence Length: 44 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence Features Characteristic Symbols : Cleavage-site Location: 3..15 Method by which the feature was determined: S Characteristic symbol: primer Location: 22..44 Method by which the feature was determined: P sequence AAGGCCCAAC CGGCCATGGC CCCAGTGTGA GGTGCAGCTG GTGG 44

SEQ ID NO: 8 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..15 Characteristic determination method: S Characteristic symbol: primer Location: 22..44 Characteristic determination method: P sequence AAGGCCCAAC CGGCCATGGC CCAGGTGCAG CTACAGSAGT GGGG 44

SEQ ID NO: 9 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 11..34 Characteristic determination method: P sequence CTGCGGCCGC TGTGTGAGTT TTGTCACAAG ATTT 34

SEQ ID NO: 10 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 11..34 Feature determination method: P sequence CTGCGGCCGC TTTGCGCTCA ACTGTCTTGT CCAC 34

SEQ ID NO: 11 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 11..34 Characteristic determination method: P sequence CTGCGGCCGC TGTGTGAGTT GTGTCACCAA GTGG 34

SEQ ID NO: 12 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence characteristics Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 11..34 Feature determination method: P sequence CTGCGGCCGC TGGGGGACCA TATTTGGACT CAAC 34

SEQ ID NO: 13 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 11..34 Characteristic determination method: P sequence CTGCGGCCGC CAGCTCAGCA ATCACTGGAA GAGG 34

SEQ ID NO: 14 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 9..31 Characteristic determination method: P array CCGCTAGCGM CATYCAGWTG ACCCAGTCTC C 31

SEQ ID NO: 15 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..31 Feature determination method: P sequence CCGCTAGCGA TRTTGTGATG ACYCAGWCTC C 31

SEQ ID NO: 16 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..31 Feature determination method: P array CCGCTAGCGA AATTGTGWTG ACGCAGTCTC C 31

SEQ ID NO: 17 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..31 Feature determination method: P sequence CCGCTAGCGA CATCGWGHTG ACCCAGTCTC C 31

SEQ ID NO: 18 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..29 Feature determination method: P sequence CCGCTAGCCA GTCTGYSCTG ACTCAGCCW 29

SEQ ID NO: 19 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 9..29 Characteristic determination method: P sequence CCGCTAGCCA GTCTGTGYTG ACGCAGCCG 29

SEQ ID NO: 20 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Feature symbol: primer Location: 9..29 Characteristic determination method: P array CCGCTAGCMA CKTTATAYTG ACTCAACCG 29

SEQ ID NO: 21 Sequence Length: 29 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence Features Characteristic Symbol : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..29 Feature determination method: P sequence CCGCTAGCCA GACTGTGGTG ACYCAGGAG 29

SEQ ID NO: 22 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Feature determination method: S Feature symbol: primer Location: 9..29 Feature determination method: P sequence CCGCTAGCTC CTATGWGCTG ACTCAGCCA 29

SEQ ID NO: 23 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 9..29 Characteristic determination method: P sequence CCGCTAGCTC TTCTGAGCTG ACTCAGGAC 29

SEQ ID NO: 24 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 11..34 Characteristic determination method: P sequence TTGGCGCGCC ACACTCTCCC CTGTTGAAGC TCTT 34

SEQ ID NO: 25 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 Characteristic determination method: S Characteristic symbol: primer Location: 11..34 Characteristic determination method: P sequence TTGGCGCGCC TGAAMATKCT GTAGSGGCCA CTGT 34

SEQ ID NO: 26 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 4..11 Characteristic determination method: S Characteristic symbol: primer Location: 10..33 Characteristic determination method: P sequence CGGGCGGCCG CAGAAACTGT TGAAAGTTGT TTA 33

SEQ ID NO: 27 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..8 Characteristic determination method: S Characteristic symbol: primer Location: 9..32 Characteristic determination method: P array GTGAATTCCT ATTAAGACTC CTTATTACGC AG 32

SEQ ID NO: 28 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence characteristics Characteristic symbols : Cleavage-site Location: 3..1O Feature determination method: S Feature symbol: primer Location: 13..32 Feature determination method: P sequence TTGGCGCGCC AGCAGAAACT GTTGAAAGTT GT 32

SEQ ID NO: 29 Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..8 Characteristic determination method: S Characteristic symbol: primer Location: 9..35 Characteristic determination method: P sequence TTGGATCCTT CTATTTCAAG GAGACAGTCM TMATG 35

SEQ ID NO: 30 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..8 Feature determination method: S Feature symbol: primer Location: 9..29 Feature determination method: P array CCGAATTCGC CACCCGTATT YGCGGCCAT 29

SEQ ID NO: 31 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..8 Feature determination method: S Feature symbol: primer Location: 9..32 Feature determination method: P sequence GGGGATCCAT GAAATACCTA TTGCCTACGG CA 32

SEQ ID NO: 32 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..10 11..23 Characteristic determination method: S Characteristic symbol: primer Location: 9..40 Characteristic determination method: P sequence CTGCGGCCGC GGCCGGTTGG GCCGCGAGTA ATAACAATCC 40

SEQ ID NO: 33 Sequence length: 47 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence characteristics Characteristic symbols : Cleavage-site Location: 3..10 21..26 Characteristic determination method: S Characteristic symbol: primer Location: 25..47 Characteristic determination method: P sequence CTGGCGCGCC TCTATCATTA GCTAGCCATC GCTGGTTGGG CAGCGAG 47

SEQ ID NO: 34 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence features Characteristic symbols : Cleavage-site Location: 3..8 Characteristic determination method: S Characteristic symbol: primer Location: 9..29 Characteristic determination method: P sequence GGACATGTAC TCCCCATCCC CCTGTTGAC 29

SEQ ID NO: 35 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..8 15..22 Feature determination method: S Feature symbol: primer Location: 15..32 Feature determination method: P array CCAAGCTTCA TTACGGCGCG CCTCTATCAT TA 32

SEQ ID NO: 36 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence features Characteristic symbols : Cleavage-site Location: 3..8 15..22 23..35 Feature determining method: S Characteristic symbol: primer Location: 13..37 Feature determining method: P Sequence CCAAGCTTTA TCACGCGGCC GCGGCCGGTT GGGCCGC 37

SEQ ID NO: 37 Sequence Length: 16 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: No Sequence CGGCCTGATA GTAAGC 16

SEQ ID NO: 38 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: Yes Sequence TTGGCCGGAC TATCATTCGC CGG 23

[0145]

[Brief description of drawings]

FIG. 1 shows the structure and map of pRPLS / Fab-I.

FIG. 2 shows the structure and map of pRPLS / Fab-II.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (C12P 21/02 C C12R 1:19)

Claims (11)

[Claims] 1. A phagemid vector for expressing an antibody Fab fragment, which comprises a first cistron having a linker site having a site for introducing a gene encoding an H chain Fd constituting Fab, and an L chain. Phagemid vector having a second cistron having a linker site having a site for introducing the gene. 2. A phagemid vector for expressing a heterodimer composed of different peptide monomers, which comprises (a) a tac-SD promoter, a pectate lysis leader sequence, and a first peptide monomer in order from the 5 ′ side. A first cistron having a linker site having a site for introducing one gene and a termination site, and (b) a gene encoding a tac-SD promoter, a pectate lysis leader sequence, and a second peptide monomer from the 5'side. A phagemid vector having a linker site having a site to be introduced, a GeneIII gene encoding a cilia protein, and a second cistron having a termination site. 3. The phagemid vector according to claim 2, wherein the heterodimer is an antibody Fab fragment, wherein one of the first peptide monomer and the second peptide monomer is H chain Fd and the other is L chain. 4. The pectate lysis leader sequence is a bacterial strain.
A DNA sequence derived from MAFF301614 which is a strain of Erwinia carotovora subsp. Atroseptica and which encodes the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence derived from strain MAFF301629 or MAFF301630 of the same bacterial strain which is shown in SEQ ID NO: 2 DNA encoding
The phagemid vector according to claim 2, which is a sequence. 5. The site for introducing the first gene and the site for introducing the second gene are SfiI, NotI, NheI or
The phagemid vector according to claim 3, which has a restriction enzyme recognition site selected from FseI and AscI. 6. The phagemid vector according to claim 5, which is pRPLS / Fab-I or pRPLS / Fab-II. 7. A method for producing an antibody Fab fragment, which comprises transforming Escherichia coli with the phagemid vector according to claim 3 and infecting this transformant with a helper phage to rescue the phagemid. 8. A primer for amplifying an H chain Fd gene of an antibody by the PCR method, wherein (a) the sense primer corresponding to the 5'end is an oligonucleotide having a nucleotide sequence shown in SEQ ID NOs: 3 to 8. (B) The antisense primer corresponding to the 3 ′ end is any one or two or more of the oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 9 to 13. A primer consisting of a combination of. 9. A primer for amplifying the L gene of an antibody by the PCR method, comprising: (a) a sense primer corresponding to the 5'end is SEQ ID NO:
An antisense primer comprising any one or a combination of two or more of the oligonucleotides having the nucleotide sequences shown in 14 to 23, and (b) the antisense primer corresponding to the 3'end has the nucleotide sequence shown in SEQ ID NO: 24 or 25. A primer consisting of any one or a combination of two oligonucleotides. 10. A linker site having a site for introducing one of a tac-SD promoter, a peptate lysis leader sequence, and a gene encoding an H chain Fd or an L chain encoding an antibody Fab fragment, in order from the 5 ′ side. , And a first cistron having a termination site,
A tac-SD promoter, a pectate lysis leader sequence, a linker site having a site for introducing the other of the H chain Fd-encoding gene and the L chain-encoding gene, and a ciliary protein-encoding Gen in order from the 5 ′ side.
A phagemid vector having an eIII gene and a second cistron having a termination site is prepared, and a gene encoding H chain Fd is randomly inserted into one of the first and second cistron linker sites,
A gene encoding an L chain is randomly inserted into the linker site of the other cistron, and Escherichia coli is transformed with the phagemid vector pool into which the H chain Fd gene and the L chain gene are respectively inserted. Method for constructing Fab fragment expression library. 11. A method for obtaining a gene encoding an H chain Fd and a gene encoding an L chain which constitute an antibody Fab fragment having binding affinity for an antigen, the method including the following steps: 5 ′ side In order from tac-SD
Promoter, pectate lysis leader sequence, H chain Fd
A first cistron having a linker site having a site for introducing a gene coding for and a termination site, and a site for introducing a tac-SD promoter, a pectate lysis leader sequence, and a gene for coding an L chain in order from the 5 ′ side. Gene encoding a linker site and a cilia protein
A first phagemid vector having a III gene and a second cistron having a termination site, and a linker having a site for introducing a tac-SD promoter, a pectate lysis leader sequence, and a gene encoding an L chain in order from the 5 ′ side. Site, and a first cistron having a termination site, a tac-SD promoter, a pectate lysis leader sequence, a linker site having a site for introducing a gene encoding H chain Fd, GeneIII encoding a ciliated protein in order from the 5 ′ side. A second phagemid vector having a gene and a second cistron having a termination site is prepared; expression of an antibody Fab fragment by the method according to claim 10 using one of the first and second phagemid vectors. Create a library and bind to the antigen from this library Select phagemid vectors that produce antibodies Fab fragments with the sum of;
One of the gene encoding H chain Fd and the gene encoding L chain is excised from the selected phagemid vector; the excised gene is inserted into one of the linker sites of the other phagemid vector of the first and second phagemid vectors. If the excised gene is an H chain Fd gene, the L chain gene is randomly inserted into the other linker site, and the excised gene is L
When the gene is a chain gene, the H chain d gene is randomly inserted into the other linker site; Escherichia coli is transformed with the obtained phagemid vector pool, and an antibody Fab fragment having a high binding affinity for the antigen. A phagemid vector that produces E. coli is selected.