JP2001238676A - Chicken type monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a chicken type monoclonal antibody suitable for mass production by a genetic recombination method and to obtain a new plasmid vector therefore a new primer therefor and further a new chicken type monoclonal antibody. SOLUTION: This new method for producing a chicken type monoclonal antibody is characterized by using an expression vector comprising a gene encoding an scFv of the chicken type monoclonal antibody transduced thereinto in an expression vector comprising a gene encoding a chicken Cλ chain (L chain constant region) transduced thereinto in the method for producing the chicken type monoclonal antibody by the genetic recombination method. The chicken type monoclonal antibody is produced by the method. Furthermore, a new expression vector pCPDS is used when the chicken type monoclonal antibody is produced by the genetic recombination method and a new primer CLF1 is used therefor. The method for amplifying the gene using the primer is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a recombinant chicken monoclonal antibody by a gene recombination method, an expression vector used therefor, a recombinant chicken monoclonal antibody produced by the method, and a recombinant chicken monoclonal. The present invention relates to a primer for amplifying a gene encoding a variable region of an antibody, and a method for amplifying a gene using the primer. The present invention also relates to a novel oligonucleotide used as a primer for amplifying a gene encoding a variable region of a recombinant chicken monoclonal antibody.

[0002]

2. Description of the Related Art A monoclonal antibody is a single antibody that recognizes only a specific part of an antigen. This technology is a core technology in the biotechnology field along with gene recombination. Is rapidly spreading and developing. Monoclonal antibodies are widely used such as mouse and rat types, but chicken type has already been proposed to some extent by the inventors. The great advantage of the chicken type is that antibodies that are difficult to produce in mammals such as mice can be produced.

[0003] Chickens are phylogenetically lower than mammals, but since they are animals with extremely fine immunity like mammals, many useful chicken antibodies have been produced so far. . On the other hand, it has been experienced that when an antibody capable of recognizing a biological component highly conserved among mammals including humans cannot be produced using a mammal, it can be produced as a chicken antibody. for that reason,
As one means of mass-controlling chicken antibodies, a method has been developed in which laying hens are immunized with a specific antigen, and then antibodies that have migrated to eggs are generated and used as yolk antibodies (polyclonal antibodies). No monoclonal antibody can be produced by the method.

[0004] Although mouse monoclonal antibodies have been used in a very wide range of basic and applied fields, there have been few successful cases of monoclonal antibodies other than mouse and rat types that have been applied. As an example of application to monoclonal antibodies other than mouse and rat types, chicken monoclonal antibodies developed by the present inventors by the cell fusion method have already been reported. As described above, a great advantage of chickens is that antibodies that are difficult to produce using mice or rats are easily obtained as chicken antibodies, and that they can also be produced as monoclonal antibodies. Examples of successful chicken monoclonal antibodies using the cell fusion technique by the present inventors include chicken monoclonal antibodies recognizing N-glycolylneuraminic acid (NeuGc) and prion protein (PrP) highly conserved in mammals. And the like. NeuGc is an antigen that serves as a human cancer marker and is present in almost all mammals except humans, and thus cannot produce antibodies in animals generally used as immunized animals such as mice, rats and rabbits.
PrP is known to be an abnormal form of a mad cow disease or a human CJD pathogen, but PrP has 90% or more amino acid sequence homology between mammals. On the other hand, since the homology between mammals and chickens is only on the order of 30%, there have been few successful cases of producing mouse monoclonal antibodies that recognize mammalian PrP so far. The present inventors have already confirmed that NeuGc and mammalian P
We succeeded in creating chicken mAbs that can recognize rP, and could use them for human cancer diagnosis and mad cow disease / human CJD research.

However, the greatest drawback of the chicken monoclonal antibody previously developed by the present inventors was that the hybridomas (fused cells) producing the antibody had low antibody-producing ability. As described above, chicken-type monoclonal antibodies can produce monoclonal antibodies that are difficult to produce in other mammals, but the antibody-producing cells (hybridomas) often have lower antibody-producing abilities than mice and rats. It was a shortcoming and a major obstacle to its practical use.

On the other hand, in recent years, a technique for expressing a recombinant antibody (recombinant antibody) on the surface of a phage by utilizing genetic engineering technology has been developed. This phage display antibody technology was established in 1991 by Wim
In a system developed by Inter, et al. (Winter,
G., et al., Nature, 349, 293-299 (1991)), an antibody gene was isolated from non-immunized human peripheral blood lymphocytes, and VH and VL genes were artificially shuffled to obtain diversified sc.
Fv (single chain fragment of variable region) antibody was expressed as a phage fusion protein to obtain a specific antibody (Marks, JD, et al., J. Mol. Biol., 222, 581).
-597 (1991)). This technique was highly evaluated as a technique for producing a humanized antibody that can avoid immunity and replaces the cell fusion method. At present, many practical antibodies have been produced using highly immunized mouse splenocytes, and anti-PrP antibodies have been produced in the same manner (Williamson, RA, et al., Proc. Nat.
l. Acad. Sci. USA., 99, 7279-7282 (1996)).

[0007] Examples of using chickens include the following reports from two groups in Japan and overseas. Davies et al. Established an antibody gene derived from a non-immunized sac of Fabricius and three types of specific antibodies from an expression vector fd-tet-DOG1, and this technique can be applied to non-mammalian animals. (Davis, ED, et al., J. Imm
unol. Methods, 186,125-135 (1995)). However, the reactivity of the produced antibody is extremely low, and there is a drawback that only a phage display antibody can be used due to the structure of the expression vector. On the other hand, Yamanaka et al. Reported the construction of a specific phage display antibody showing sufficient reactivity using an antibody V region gene derived from mouse albumin-immunized spleen cells and an independently developed expression vector pPDS. The use of an immune spleen cell-derived antibody gene library was emphasized in the preparation of (A. Yamanaka, HI, et al.,
J. Immunol., 157, 1156-1162 (1996)). The pPDS used here was developed as a mouse antibody expression vector (Yamanaka, HI, et al., J. Biochem., 11).
7, 1218-1227 81995)), a phagemid vector pBlu for cloning commercially available from STRATA GENE.
It is based on scriptII.

FIG. 1 shows the structure of the expression vector pPDS. pPDS utilizes the lactose operon (Lac) promoter to induce the expression of an integrated scFv antibody cDNA. Downstream of the promoter, a ribosome binding site (RBS) and M13 g
It is designed so that the leader sequence (g31) of eneIII is artificially ligated, and the antibody gene integrated at the cloning site consisting of EagI and BssHII can be expressed as a reconstituted phage body through the periplasm of Escherichia coli. Further downstream, a mouse Cκ chain gene is ligated in preparation for the expression of the Fab antibody, which leads to a structural site of cp3, which is a termination codon. This Cκ chain is also a very effective tag for verifying whether the expression of the fusion protein has been performed successfully. In addition, this pPDS vector has a TAG between the Cκ chain and gene III so as to be able to cope with the expression of soluble scFv or Fab antibody.
(Amber sequence) A stop codon is inserted, and is usually expressed as a phage display antibody by using a supE E. coli strain, but when cultured in a non-suppressor strain, a soluble antibody is secreted into the medium. Become like

[0009] Since such an expression vector is derived from a chicken antibody gene in a soluble antibody,
An antibody against mouse Cκ chain had to be used for the detection, and when a mouse antibody was used in a sandwich ELISA or the like, accurate measurement of a chicken antibody was impossible.
Since the present inventors have received pPDS from Dr. Yamanaka and intend to utilize them for producing phage display antibodies from antibody-producing chicken hybridomas, this problem has been a matter to be solved.

[0010]

SUMMARY OF THE INVENTION The present invention provides a method for producing a chicken monoclonal antibody suitable for mass production by a gene recombination method. In addition, the present invention provides a novel plasmid vector and a novel primer therefor. Further, the present invention provides a novel chicken monoclonal antibody.

[0011]

Means for Solving the Problems The present invention relates to a method for producing a chicken monoclonal antibody by a gene recombination method, wherein an expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced, The present invention relates to a novel method for producing a chicken monoclonal antibody using an expression vector into which a gene encoding a scFv of a chicken monoclonal antibody has been introduced, and a chicken monoclonal antibody produced by the method.
The present invention also relates to a novel expression vector used in the method. Further, the present invention provides a method for amplifying a gene encoding VL or scFv of a chicken monoclonal antibody, wherein one of the primers is a restriction enzyme Bs.
A primer for amplifying a gene encoding a scFv of a chicken monoclonal antibody, which has a nucleotide sequence capable of being cleaved by sHII, and a VL or scFv of a chicken monoclonal antibody using the primer.
And a method for amplifying a gene encoding Also,
The present invention relates to the VL or s of chicken monoclonal antibody.
The present invention relates to a novel oligonucleotide useful as a primer for amplifying a gene encoding cFv.

The present inventors aimed at the production of chicken anti-prion protein (PrP) panel monoclonal antibody which could be a tool for analyzing the pathological condition of prion disease and elucidating the mechanism of prion conversion, and firstly aimed at cell fusion. Prion protein (Pr
P) PrP-specific chicken monoclonal antibody H which has been shown to be effective in recognizing residues 25-29 (RPKPG)
Using UC2-13 as a test material, recombination by phage display antibody technology using a pPDS expression vector and establishment of large-scale preparation were performed.

The present inventors first prepared a chicken monoclonal antibody recognizing mammalian prion protein (PrP) residues 23-231 as follows. Recombinant human prion protein (PrP) 23-231
(About 100 μg) was immunized intraperitoneally to a 4-week-old inbred chicken White Leghorn H-B15 chicken, and its spleen was removed to prepare immunized splenocytes. This was fused with the TK-deficient, ouabain-resistant chicken B cell line MuH1 by the PEG method to obtain a fused cell (hybridoma).
An antibody gene (VH and VL regions) prepared from the spleen lymphocytes of the antibody-producing hybridoma or immunized chicken obtained in this manner is converted into a single chain scFv (single chain fragment of V) via a flexible linker.
region). As a plasmid vector for expressing this scFv, Yamanaka (currently Rohto Pharmaceutical)
Has a pPDS (see FIG. 1), but when pPDS is used, the C region (Cκ) of the mouse immunoglobulin L chain is expressed on the N-terminal side of the scFv, and the V region is
The region becomes a mouse recombinant antibody.

Since the specificity (reactivity) of the antibody is determined by the V region, there is no problem with the reactivity. However, detection without background staining such as HUC2-13 is obtained when the pPDS vector is used. I couldn't. The expression vector pPDS used for the production of the recombinant antibody was designed and constructed by Yamanaka et al. As a mouse antibody expression vector. The scFv antibody to be expressed is composed of an antibody V region having a very high mutation frequency, and is stable. Mouse Cκ chains have been introduced as detection tags because of their difficulty in detection. Therefore, in the recombinant antibody produced using the pPDS vector, an anti-mouse κ chain antibody had to be used for detection while the antigen recognition site was derived from a chicken antibody, and as a result, background staining had occurred. . In the recombinant antibody, as long as the mouse Cκ chain is used as a detection tag, (1)
Sandwich ELI commonly used for high-sensitivity detection and quantitative experiments
In the case of performing SA, if a mouse antibody is used as a capture antibody, the antibody is detected as a background. (2) In the case of immunohistochemical staining using a mouse as a model animal, the immunity of the mouse is The problem that non-specific detection of globulin occurs.

Therefore, the present inventors have solved the above-mentioned problems, and aimed to utilize chicken-type antibodies for prion disease analysis and further extensive research so that they can be detected with anti-chicken Ig antibodies. An attempt was made to construct a novel expression vector in which the mouse Cκ chain in pPDS was replaced with a chicken Cλ chain (L chain constant region). For the purpose of further simplifying the purification, a vector for expressing a pure chicken recombinant antibody into which a tag for purification (FLAG) was introduced was constructed. FIG. 2 shows the outline of the construction of a chicken recombinant antibody expression vector (pCPDS).

First, the chicken Cλ chain gene was cloned. Chicken Cλ chain was amplified by PCR based on cDNA synthesized from normal chicken spleen cells.
A band of about 340 bp, which is considered to be a chain gene, was obtained (FIG. 3).
A). FIG. 3 is a photograph instead of a drawing showing these results. “M1” in FIG. 3 is pUC118 Hinf
I indicates digestion, and “M2” indicates digestion with φx174 HaeIII. This band was ligated after treatment with a restriction enzyme, followed by transformation. The next day, six clones were randomly prepared for plasmids by the alkaline SDS method, and the presence or absence of the insert was confirmed. As a result, all the inserts were confirmed. When the nucleotide sequence of one of the six clones (clone # 1) was determined, it completely matched the previously reported Cλ chain encoding 114 amino acids (see FIG. 4). Therefore, this clone was used as a template for preparing a construct for vector reconstruction.

Based on the cloned chicken Cλ chain gene whose base sequence was determined, a chicken Cλ chain gene to be used in the reconstructed construct was prepared by PCR. As a result, many by-products were observed, but about 360 b
At the position of p, a band presumed to have the FLAG sequence added to the chicken Cλ chain was detected (see FIG. 3B). This band was purified and used as a template for overlap PCR with the geneIII gene.

On the other hand, amplification of the geneIII gene from pPDS was performed using the PCR method. As a result, about 60
A band presumed to be the geneIII gene was detected at the position of 0 bp (see FIG. 3C). This band is purified and F
This was used as a template for overlap PCR with the chicken Cλ gene to which the LAG sequence was added.

The overlap PCR between the chicken Cλ chain and the gene III gene is performed by FLAG added to the chicken Cλ chain gene 3 ′ and 5 ′ to the gene III gene.
Made through the sequence. As a result, a band at which the chicken Cλ chain and the geneIII gene seemed to be linked at a position of about 950 bp was detected (see D in FIG. 3). This band was purified, treated with restriction enzymes, ligated to pPDS treated with EagI and EcoRI, and transformed into E. coli.

After transformation into E. coli, 15 clones were insert-checked by amplification of the chicken Cλ chain gene.
As a result, amplification of chicken Cλ chain was confirmed at a position of about 360 bp in 12 clones (see FIG. 5). FIG. 5 is a photograph replacing the drawing showing the result. “M” in FIG.
UC118 HinfI shows digestion. These clones were pCPDS1, 2, 3, 4, 5, 6, 7,
They were named 8, 9, 10, 11, and 12, respectively. Phage bodies were prepared from these 12 clones without containing the scFv gene, and the expressed proteins were examined. Confirmation of expression of chicken Cλ chain and phage by sandwich ELISA using anti-mouse IgG (H + L) and anti-chicken IgG (H + L) as capture antibodies, and a peroxidase-labeled anti-M13 antibody as a detection antibody. Did. As a positive control, p
A phage body expressed from PDS was used. as a result,
When anti-chicken IgG (H + L) was used as a capture antibody in the remaining 10 clones excluding pCPDS8 and 10,
The ELISA value (OD492) was an average of 0.6 reactivity (FIG. 6). FIG. 6 shows the results, where the solid black indicates the case where anti-mouse IgG (H + L) was used as the capture antibody, and the white outline indicates the case where anti-chicken IgG (H + L) was used as the capture antibody. These clones had an average value of as low as 0.17 when anti-mouse IgG (H + L) was used as a capture antibody, and were clearly lower than the reactivity with pPDS. It was found to be a reconstructed vector that accurately expressed the construct for use.

The structure of the obtained expression vector pCPDS is shown in FIG. Using the reconstructed vector pCPDS7 in which the expression of the correct chicken Cλ chain and the reconstituted phage was confirmed in the above-described experiment, the phage display antibody and the soluble HUC2-13 gene encoding the chicken antibody shown in FIGS. Type antibodies were prepared. The prepared recombinant antibody was tested for its reactivity with the H25 peptide together with HUC2-13. As a result, when the primary antibody was used without dilution, ELISA values (OD492) exceeded 1.5 for all antibodies. If diluted, HU
With the soluble antibody prepared from C2-13 and cell disruption, almost no decrease in ELISA titer was observed up to 25-fold dilution. On the other hand, the phage display antibody and the soluble antibody from the culture supernatant tended to decrease, and a positive ELISA titer was not obtained at a dilution of 125 times. The soluble antibody prepared from the cell disruption was diluted to HUC2 from 125-fold dilution.
The ELISA titer was −13 (see FIG. 14).

FIG. 14 is a graph showing the result.
In FIG. 14, a black square indicates a chicken-type phage display antibody, a gray diamond indicates a chicken-type soluble antibody (culture supernatant), and a gray circle indicates a chicken-type soluble antibody (disrupted cells). , And the gray triangle indicates HUC2-13 (native antibody).

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

In addition, pPDS was used to construct the reconstructed vector.
Based on the vector, it was possible to exchange constructs using appropriate restriction enzyme sites. pPDS does not have an appropriate restriction enzyme site from the scFv antibody cloning site to the EcoRI immediately after the geneIII stop codon, and thus EagI-EcoI
The construct had to be replaced between RIs or between BssHII and EcoRI. Chicken Cλ chain and S
Careful attention was paid to the connection site of the cFv antibody gene to approximate the natural VL-Cλ amino acid sequence, and a restriction enzyme having high cleavage efficiency and not cleaving the vector or ScFv antibody gene and a site where the restriction enzyme was installed were selected. In order to examine this cutting position, the binding between the Vλ region and the Cλ region was summarized. This is shown in Table 1.

[0025]

[Table 1]

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

The chicken Cλ chain was obtained from chicken spleen cells by g
eneIII prepared the gene from pPDS. On the other hand, F
The LAG-amber sequence is introduced into a part of the primer,
Although it was decided to ligate at the time of λ chain amplification, it was judged that it was dangerous to use the primer having as many as 54 bases at the time of Cλ chain amplification from a complicated group of spleen cell-derived cDNAs.
-Decided to perform amber ligated PCR. EagI-B
ssHII scFv antibody gene cloning site-chicken Cλ chain-FLAG-amber sequence (TAG) -g
All reconstruction constructs up to eneIII are KOD
It was prepared by a PCR method using a DNA polymerase. E
Ligation was performed on pPDS treated with agI and EcoRI, and finally a vector expressing chicken Cλ chain and reconstituted phage body of 10 clones was obtained (see FIG. 6).

FIG. 15 shows an outline for producing a pure chicken recombinant antibody of the present invention based on the above experiments. This will be described in order. (1) Preparation of antibody V region genes (VH and VL) RNA was extracted from antibody-producing hybridomas, and RT-P
Based on cDNA synthesized by CR (reverse transcription PCR),
Amplification is performed using a synthetic primer set (a primer set for VH amplification and a primer set for VL amplification) shown in Table 2 below. (2) Construction of scFv After purifying the VH and VL amplification products obtained in the experiment of (1), VH and VL were converted to a linker (scFv linker) (-
Ligation using (Gly-Gly-Gly-Ser) _3- ) (assembly reaction). (3) Re-amplification of scFv and treatment with restriction enzymes Primers shown in Table 2 below for re-amplification of scFv
The set (CHB and CLF1) was used to re-amplify the scFv constructed in (2), and after purifying the scFv, insert it into the plasmid vector pCPDS using the restriction enzymes (EagI and BssHII). Prepare fragments (inserts).

(4) pCPD of insert (scFv)
Insertion into S (Ligation) pCPDS and the insert are adjusted to 1: 1 to 10 (molar ratio), and ligation is performed using Ligation Kit ver. 1 (TAKARA). (5) Transformation of Escherichia coli Escherichia coli (XL1-blue, 100 μl) is transformed using pCPDS (10 μl) into which the insert has been inserted. (6) Selection of transformed E. coli expressing the insert A plurality of E. coli colonies are arbitrarily selected from the grown E. coli colonies, scFv is amplified using a primer set, and the transformed E. coli expressing the insert is selected. . (7) Preparation of Phage-Expressing Antibody A helper phage is infected to the selected E. coli to prepare a phage-expressing antibody. (8) Vanishing antibody for selection of specific phage antibody After reacting the phage-expressed antibody with a plate on which immobilized antigen (prion protein) has been immobilized, only the reacted phage-expressed antibody is recovered and infected to E. coli for specific Target phage antibodies. (9) Preparation of solubilized antibody Specific phage antibody was converted to non-pressor E. coli (SOLA
Or HB2151). Twenty-four hours after infection, the E. coli culture supernatant and the cultured cells are collected, and the supernatant and the cell-disrupted extract are used as solubilized antibodies. (10) Generation of solubilized antibody The solubilized antibody is affinity-purified using an anti-FLAG antibody-bound agarose gel.

Thus, the expression vector p of the present invention
By using CPDS, a pure chicken recombinant antibody can be produced. The production of the scFv for producing the chicken antibody of the present invention will be described in more detail. In mammals and birds, the mechanism of obtaining diversity of antibody genes is different. In mammalian antibody genes, one of many V genes is reconstructed.
In the avian antibody gene, only one antibody gene functions, and a pseudo-V gene is randomly inserted into this functional antibody gene. FIG. 16 schematically shows antibody H chains in mammals (upper part in FIG. 16) and birds (lower part in FIG. 16). In a mammalian antibody gene, one of a number of V genes is reconstructed, so that the nucleotide sequence on the 5 ′ side also varies, and primers must be prepared depending on the type of antibody. There is only one functioning antibody gene, and the pseudo-V gene corresponding to the type of antibody is randomly inserted into this functional antibody gene. Also basically does not fluctuate.

Therefore, when an antibody gene is to be amplified from an antibody-producing cell by PCR, it is not known which of a large number of mammalian V genes is used. A large number of primers must be synthesized and prepared on the V gene side). On the other hand, the bird V gene has one functional V gene as a base.
Because the base sequence is known in advance, one pair of primers, one on the 5 'side and one on the 3' side, can be used for all antibody-producing cells. It is a big feature. The primers for birds disclosed in the present invention are shown in Table 2 below.

[0032]

[Table 2]

By using the primer set shown in Table 2, basically any antibody can be used.
It becomes possible to amplify the antibody genes of birds, preferably chickens. Among them, the primer represented as CLF1 has a novel nucleotide sequence, and a site (gcg) that can be cleaved by the restriction enzyme BssHII in the middle thereof.
cgc). This nucleotide sequence is shown as SEQ ID NO: 1 in the sequence listing. FIG.
Schematically shows the usage status of this primer CLF1, and the left side of the upper part of FIG. 17 is the VL region,
Immediately to the right is the Cλ region. As mentioned above,
In the expression vector pCPDS, since the VL region and the Cλ region are linked by the restriction enzyme BssHII, the state of the 3 ′ end of the VL region and the 5 ′ end of the Cλ region are shown. The portion surrounded by a square frame of the base sequence shown by the circle 2 in FIG. 17 is a position that can be cleaved by the restriction enzyme BssHII. As described above, in birds, the nucleotide sequence at the end of the VL region varies depending on the type of antibody. Therefore, by using this primer CLF1, the VL region and VH
-ScFv consisting of linker-VL can also be amplified.

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

[0035]

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

Example 1 Design of Vector for Expression of Chicken Recombinant Antibody
Performed based on pPDS vector. The mouse Cκ chain gene already inserted in pPDS is replaced with the chicken Cλ chain gene, and the sequence of the VL and Cλ linkage is germline
And a restriction enzyme site for cloning was selected so that the gene used was not cleaved (see Table 1). In addition, EagI was used so that the vector could be assayed for the expression of chicken Cλ chain even without the insert (Mock).
The subsequent spacer sequence was newly redesigned. Downstream of the chicken Cλ chain is a FLAG sequence (DY
KDDDDDK) and TAG (amb) so that it can be expressed as a soluble antibody between the FLAG sequence and geneIII.
er) Stop codon was inserted. G from EagI site
The construct prepared up to eneIII was ligated to pPDS treated with EagI and EcoRI to obtain a chicken recombinant antibody expression vector (see FIG. 2).

Example 2 Isolation of Chicken Cλ Chain Gene (1) Design and Synthesis of Chicken Cλ Chain Gene Cloning Primer The chicken Cλ chain gene cloning primer was obtained from OLIGO 4.05 Primer Analys based on the previously reported Cλ chain base sequence.
Designed using is Software (National Bioscinece)
Outsourced to Hokkaido System Science for synthesis. (2) Amplification and cloning of chicken Cλ chain gene Amplification of chicken Cλ chain gene was performed by RT-PCR from normal chicken spleen cells according to the method described later. PCR
The reaction solution was extracted with phenol, extracted with phenol / chloroform, concentrated by ethanol precipitation, and redissolved in 20 μl of DW. 16 U / μl BamHI and 20 U /
1 μl of each HindIII was added, and 37
The mixture was treated with a restriction enzyme at 2 ° C. for 2 hours, and concentrated by ethanol precipitation. The sample after concentration was redissolved in 10 μl of DW,
After electrophoresis using 1.5% agarose gel, UltraCle
Purified in an and redissolved in 10 μl of DW. After the concentration measurement, the mixture was mixed with pBluescript II SK (-) so that the molar ratio was vector: insert = 1: 3, and TA
Ligation was performed at 16 ° C. for 3 hours using KARA Ligation Kit ver. After completion of the ligation, 5 μl of the reaction solution was transformed into competent cells, and 40 μl was plated on a 2 × YT plate containing ampicillin. next day,
The obtained colony was isolated, cultured in a small amount in a 2 × YT medium containing ampicillin, and a plasmid was prepared by an alkali-SDS method. The plasmid was treated with BamHI and HindIII to confirm the presence or absence of an insert. The nucleotide sequence was determined according to a standard method (see FIG. 4).

Example 3 Preparation of Chicken Cλ Chain Gene for Ligation Reaction (1) Design and Synthesis of Chicken Cλ Chain Gene Amplification Primer The chicken Cλ chain gene amplification primer used as the construct of the reconstructed vector was a scFv antibody gene cloning. Incorporate EagI and BssHII so as to become a site, and add a sense primer with attention to frameshift so that expression can be carried out even with an empty vector, and add a FLAG-amber-geneIII-5 ′ terminal sequence to ligate with geneIII isolated from pPDS. An antisense primer was designed and synthesized. (2) Amplification of chicken Cλ chain gene After determining the nucleotide sequence, a clone that was completely matched by referring to a data bank was prepared in large amounts by the cesium chloride method. Using the prepared sample as a template, a chicken Cλ chain gene for overlap PCR was prepared. The PCR reaction solution was extracted with phenol and extracted with phenol / chloroform, concentrated by ethanol precipitation, and redissolved in 10 μl of DW. After electrophoresis of the reaction solution on a 1.5% agarose gel, Ultra
Purified with Clean, redissolved in 10 μl of DW, VCS
-A sample for overlap PCR with M13-derived gene III was used.

Example 4 Preparation of geneIII derived from VCS-M13 for ligation reaction (1) Design and synthesis of geneIII amplification primers For geneIII amplification from pPDS, a chicken Cλ chain and a FLAG-amber sequence can be linked. Ec immediately after the sense primer and the stop codon of geneIII.
An antisense primer into which oRI was introduced was designed and synthesized. (2) Amplification of geneIII GeneIII derived from VCS-M13 was prepared by PCR from the pPDS vector. The PCR reaction solution was extracted with phenol and extracted with phenol / chloroform, concentrated by ethanol precipitation, and redissolved in 10 μl of DW. After electrophoresis of this reaction solution on a 1.5% agarose gel, UltraC
Purified by lean, redissolved in 10 μl of DW, and used as a sample for overlap PCR with chicken Cλ chain.

Example 5 Chicken Cλ chain and gene III
Ligation reaction between chicken Cλ chain and gene III was carried out at a molar ratio of 1:
The assembly reaction and the reamplification reaction were performed based on the template mixed in Step 1. The PCR reaction solution was extracted with phenol and extracted with phenol / chloroform, concentrated by ethanol precipitation, and redissolved in 10 μl of DW. After electrophoresis of this reaction solution on a 1.5% agarose gel, UltraCl
Purified with ean and redissolved in 10 μl of DW.

Example 6 Removal of mouse Cκ chain and gene III from pPDS vector and chicken Cλ chain-gen
Insertion of e3 In order to insert chicken Cλ chain-geneIII into the pPDS vector, restriction enzyme treatment was performed with 10 U / μl of EagI and 10 U / μl of EcoRI, and further, BAP treatment was performed to prevent intramolecular ligation. The reaction solution after the BAP treatment was subjected to electrophoresis using a 1.0% agarose gel, purified by UltraClean, redissolved in 10 µl of DW, and used as a sample for reconstruction. Also, chicken Cλ
Strand-geneIII was similarly treated with a restriction enzyme, purified and
μl of the reconstitution sample was prepared. After measuring the concentration of the prepared reconstitution sample, the mixture was mixed so that the molar ratio of the vector and the insert was 1: 3, and the TAKARA Ligation Kit was used.
After ligation at 16 ° C for 3 hours at r.1, the reaction mixture 5
μl was transformed into 50 μl of competent cells. The transformed E. coli was plated at 40 μl on a 2 × YT plate containing ampicillin, and cultured at 37 ° C. overnight. The inserted gene was confirmed by amplification of the chicken Cλ chain by PCR using the colony as a template.

Example 7 Confirmation of Expression of Reconstituted Phage Body Expression of reconstituted phage body was confirmed by sandwich ELISA.
A method was used. Anti-chicken IgG (H + L) antibody (Kirk
egaard and Perry Laboratories) and anti-mouse IgG
(H + L) antibody (Kirkegaard and Perry Laboratorie
s) was immobilized at 0.5 μg / well overnight at 4 ° C., and after blocking, 50 μl of the reconstituted phage body without scFv antibody expression was
1 / well was added and reacted at 37 ° C. for 1 hour. After the reaction, a 3,500-fold diluted HRPO-labeled anti-M13 antibody was reacted at 37 ° C. for 1 hour to develop color using o-phenylenediamine as a substrate. After the reaction for 10 minutes, the absorbance at 492 nm was measured with a microplate reader MPR-A4i.

Example 8 Preparation of HUC2-13 (1) Test cell line A thymidine kinase-deficient, ouabain-resistant chicken B cell line MuH1 was used as a parent strain and a mammalian porin protein PrP 25-29 prepared by cell fusion. Anti-PrP chicken monoclonal antibody HUC2-13 recognizing residues
Production hybridomas were used. The cells were 10% F
IMDM containing BS (Fetal bovine serum) (SIGMA) (I
scove's modified Dullbecco's medium) (GIBCO BRL)
The culture was maintained at 38.5 ° C. in a 5% CO ₂ incubator. The culture supernatant was used as anti-PrP chicken monoclonal antibody HUC2-13. (2) Amplification of antibody V gene by RT-PCR (2-1) RNA extraction and cDNA synthesis RNA of the test cells was obtained using ISOGEN-LS (Nippon Gen.
Using e), isolation was performed according to the attached protocol. The isolated RNA is diethylpyrocarbonate-distilled water (DEPC-DW)
Redissolved in 20 μl. 1st strand cDN
Synthesis of A was performed using the Oligo-dT primer method. 500 μg / ml for 1-5 μg of total RNA solution
Oligo dT15 primer (Boehringer Mammhei
m) was added thereto and adjusted to 12 μl with distilled water (DW). This mixture was incubated at 70 ° C. for 10 minutes, cooled on ice, and 4.0 μl of 5 × reverse transcription buffer (GIBCO BRL) was added in 0.1 M dithiothreitol.
itol, DTT) (GIBCO BRL) 2.0 μl, 5 mM
2.0 μl of dNTPs (GIBCO BRL) was added and incubated at 42 ° C. for 2 minutes. Thereafter, 200 U / μl reverse transcriptase (Superscript II RT) (GIBCO
BRL) and add 50 μl at 42 ° C., followed by 7 μl.
Incubate at 0 ° C. for 15 minutes. Synthesized cDNA
Was stored at -20 ° C. (2-2) Amplification and purification of VH, VL antibody gene and linker gene VH, VL by PCR (polymerase chain reaction) method
For amplification of the VL antibody gene, the cD synthesized in (2-1) was used.
For amplification of a linker gene, a pair of sense primer and reverse primer, KOD DNA polymerase (TOYOBO), Clo
ned Pfu DNA polymerase (Perkin Elmer)
Was used. As a positive control, an anti-mouse albumin monoclonal antibody 3C8 (chicken recombinant antibody) prepared by Yamanaka et al. Was tested. The PCR reaction solution of the VH and VL antibody genes was concentrated by phenol extraction, phenol / chloroform extraction, and ethanol precipitation with addition of 2 mg of 10 mg / ml glycogen.
Electrophoresis was performed using a 1.5% agarose gel (Wako Pure Chemical Industries, Ltd.). The resulting band is Ultra Clean (MO
(BIO laboratories, Inc.) according to the attached protocol, and eluted with 20 μl of DW. The PCR reaction solution of the linker gene was used in the same manner as in the purification of the VH and VL antibody genes. However, a 3.0% agarose gel was used for electrophoresis.

(2-3) Amplification and Purification of ScFv Antibody Gene In the assembly reaction and the re-amplification reaction for amplifying the scFv antibody gene synthesis, purified VH, VL, and a linker were mixed at a molar ratio of 1: 1: 1. I went. The PCR reaction solution of the scFv antibody gene was extracted with phenol, phenol / chloroform, and 10 mg / m2.
After concentration by ethanol precipitation to which 2 μl of l glycogen was added, electrophoresis was performed using a 1.5% agarose gel. The obtained band was purified by Ultra Clean according to the attached protocol, and eluted with 20 μl of DW. (3) Preparation of expression vector (pPDS) Expression vector for phage display (pPDS) provided by Dr. Hachiro Yamanaka, Rohto Pharmaceutical Co., Ltd. (FIG. 1)
Was transformed into Escherichia coli XL1-Blue by the rubidium chloride method, plated on a 2 × YT plate containing 50 μg / ml ampicillin, and cultured at 37 ° C. overnight. The obtained colonies were selectively collected, and 50 μg /
A large amount of shaking culture was performed in 2 × YT medium containing ml ampicillin. Vector purification from the cultured Escherichia coli was performed by cesium chloride density gradient equilibrium centrifugation. (4) Restriction enzyme treatment and BAP treatment 2 μg of purified scFv gene fragment and vector pPDS
10 μg is 4.0 U of Ea for 1.0 μg of DNA.
gI (New England BioLabs, Inc.) was added at 37 ° C.
For overnight. After the EagI treatment, BssHII (New England BioLabs, Inc.) was added to the reaction mixture at the same ratio, and the mixture was treated at 50 ° C. for 4 hours. After BssHII treatment, 68
The enzyme was inactivated at 20 ° C. for 20 minutes.
After making up to 0 μl, extract with phenol, extract with phenol / chloroform, 10 mg / ml glycogen
After ethanol precipitation with addition of 2 μl, the mixture was redissolved in 30 μl of DW. The vector is alkaline phosphatase (Bacterial) from E. coli to prevent intramolecular ligation.
Dephosphorylation treatment was performed using alkaline phosphatase (BAP) (TOYOBO). In 30 μl of vector, 10 μl of 5 × BAP buffer, alkaline phosphatase (0.4 U
/ Μl) 3 μl and DW 7 μl were added, followed by treatment at 37 ° C. for 3 hours. The reaction solution after the treatment is 300 μl in DW
Phenol extraction, phenol / chloroform extraction, 10 mg / ml glycogen 2μ
Then, ethanol precipitation was performed and redissolved in 30 μl of DW.

(5) Ligation to a vector The purified scFv gene fragment and the vector pPDS were prepared at a molar ratio of 3: 1.
Ligation reaction was performed using ver.1 (TAKARA). In addition, the method followed the attached protocol. (6) Transformation (Rubidium chloride method) Transformation was performed using Escherichia coli XL1 stored at -85 ° C.
Thaw 100 μl of the -Blue competent cell solution in ice, add 10 μl of the ligation solution thereto, and add
Let stand for 0 minutes. Next, it was heat-treated at 42 ° C. for 2 minutes, quickly transferred to ice and allowed to stand for 2 minutes. 1.0m SOC medium
and shake culture at 37 ° C for 1 hour.
The cells were plated on SOBAG plates containing g / ml ampicillin and cultured overnight at 37 ° C. (7) Confirmation of Inserted DNA Fragment Confirmation of the scFv gene, which is the inserted DNA, was carried out in the M13-R of the Lac Z upstream sequence, which is a sequence derived from the pPDS vector and was designed to completely amplify the insertion site, and in the mouse Cκ chain. Using a pair of primers of SLP-1 having a sequence, PCR was performed using colonies as a template. (8) Preparation of phage display antibody After transformation, colonies in which the inserted DNA fragment was confirmed were
Shaking culture was slowly performed overnight at 30 ° C. in 2 ml of an SOC medium containing 50 μg / ml ampicillin. The next day, in 0.8 ml of the culture solution, 0.2 ml of SuperBroth medium containing 100 μg / ml ampicillin, and 2M
37 μl of glucose was added. After shaking culture in a 50 ml tube at 37 ° C. for 3 hours, 5 × 109 pfu helper phage (VCS-M13) was added, and the mixture was cultured for 30 minutes with gentle shaking to infect the phage. After further culturing with shaking for another 30 minutes, 800 × g 1 at room temperature.
After centrifugation for 0 minutes, the supernatant was discarded. The precipitate was resuspended in 1 ml of Super Broth medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin, and then added with 9 ml of the same medium and cultured overnight with vigorous shaking. After cooling the next day, the cells were removed by centrifugation at 800 × g for 10 minutes, and the supernatant was filtered with a 0.45 μm pore filter (Kanto Chemical), and the filtrate was used as a phage display antibody suspension. The antibody was stored at 4 ° C or -20 ° C. Ten colonies were randomly isolated from the obtained colonies, and the presence or absence of the insert (scFv gene fragment) was confirmed by PCR. As a result, inserts were confirmed in 4 out of 10 clones (clone #: 1, 3, 5, and 6). Phage display antibodies were prepared based on the inserts, and these four types of phage display antibodies were used as HUC2p1, HUC2p3, HUC2p5 and H
It was named UC2p6. (9) Preparation of soluble antibody The soluble antibody is an amber sequence non-suppressor strain.
It was prepared by infecting Escherichia coli XL1-Blue SOLR strain (SOLR strain) with a phage display antibody. The SOLR strain is an SO containing 25 μg / ml kanamycin.
The cells were cultured overnight in 2 ml of C medium, and 5 μl of a phage display antibody was added to 200 μl of the culture, followed by infection at 37 ° C. for 1 hour. This culture was diluted with 2 × YT medium, and
The cells were plated on 2 × YT plates containing 0 μg / ml ampicillin, and cultured at 37 ° C. overnight. A scFv antibody gene fragment confirmed by PCR using the obtained colonies as a template was cultured in 2 ml of Super Broth medium containing 50 μg / ml ampicillin. Super Broth medium 4.5 m was added to 500 μl of the culture solution.
l, and ampicillin and kanamycin were added thereto at final concentrations of 100 μg / ml and 50 μg / ml, respectively, followed by shaking culture at 37 ° C. for 2 hours. Thereafter, IPTG was added to a final concentration of 1 mM, and the cells were cultured with shaking for 4 hours. The culture solution was ice-cooled and centrifuged at room temperature for 10 minutes at 800 × g, and the supernatant was collected. The supernatant was further 0.45
The solution is sterilized by filtration with a μm pore filter, and the filtrate is dissolved antibody solution (culture supernatant).
S After stirring with 5 ml, Handy Sonic mod
The solution was subjected to ultrasonic treatment with el UR-20P (Tomy Seiko), and the filtrate was prepared as a soluble antibody solution (microbial cell-disrupted extract). The antibody was stored at 4 ° C or -20 ° C.

Example 9 Expression of HUC2-13 ScFv Antibody Using a Reconstructed Vector The HUC2p3 to ScFv constructed in Example 8 (8)
The antibody gene was amplified using primers designed for the reconstructed vector. The reaction mixture was extracted with phenol, extracted with phenol / chloroform, and concentrated by ethanol precipitation.
Redissolved in 20 μl. Reconstructed vector after large-scale preparation using the cesium chloride method and HUC2p3-derived S
The cFv antibody gene contained 10 U / μl EagI and 10 U
/ Μl of BssHII. Further, the vector was subjected to BAP treatment to prevent intramolecular ligation. The reaction solution after the treatment was subjected to electrophoresis using a 1.0% agarose gel, and purified by UltraClean.
It was redissolved in 10 μl to obtain a sample for reconstitution. After the concentration of the prepared reconstructed sample was measured, it was mixed so that the molar ratio of vector: insert was 1: 3, and TAKARA Lig
Ligation at 16 ° C for 3 hours with ation Kit ver.1,
5 μl of the reaction solution was transformed into 50 μl of competent cells. The transformed Escherichia coli was ampicillin-containing 2 × Y
40 μl was plated on a T plate and cultured at 37 ° C. overnight. The insert was checked by PCR using primers for amplifying the scFv antibody gene and colonies as a template. A phage display antibody was prepared from the colony in which the insert was confirmed, according to the method described in Example 8.

Example 10 Preparation of Soluble Antibody A soluble antibody was prepared according to the method described in Example 8.

Example 11 ELISA The reactivity of the prepared chicken phage display antibody and the soluble antibody was determined using the H25 peptide as an antigen.
Analyzed by LISA. For the primary antibody recombinant antibody and native antibody, 5-fold serial dilutions up to 15,625-fold were used. HR diluted 2,000-fold in secondary antibody
PO-labeled anti-chicken IgG (H + L) (Kirkegaard and
(Perry Laboratories). That is, H25 peptide at a concentration of 5 μg / ml was placed in a 96-well plate at 50 μl / well (0.25 μg / w
ell) and solidified overnight at 4 ° C. to prepare an antigen plate. The antigen plate was prepared by diluting BlockAce (Snow Brand Milk Products) to 25% with PBS at 400 μl /
Add well and block at 37 ° C for 1 hour.
Washed three times with Tween 20 (0.05%). The primary antibody reaction was performed by adding 50 μl / well of the recombinant antibody.
In addition, it was left standing at 37 ° C. for 2 hours. PBS-Twe
After washing five times with En 20 (0.05%), a peroxidase-labeled anti-M13 antibody (Amersham Pharmac.) diluted 3,000-fold with 10% BlockAce was used as a secondary antibody reaction.
ia biotech) and allowed to stand at 37 ° C. for 1 hour. PBS
-After washing 8 times with Tween 20 (0.05%), o-
Color development is performed for 20 minutes using phenylenediamine (when using a peroxidase-labeled secondary antibody) (SIGMA) or p-nitrophenylphosphate (when using an alkaline phosphatase-labeled secondary antibody) (Kirkegaard and Perry Laboratories), and 492 or 405 nm. Microplate reader MPR-A4i (TOS)
OH).

Example 12 Preparation of Antibody V Region Genes (VH and VL) RNA was extracted from antibody-producing hybridomas and RT-P
Based on cDNA synthesized by CR (reverse transcription PCR),
Using the synthetic primer sets (VH amplification primer set and VL amplification primer set) shown in Table 2,
Amplify H and VL regions. (VH amplification conditions) 10 × KOD buffer 15 μl 25 mM MgCl ₂ 3 μl 2.0 mM dNTPS 4 μl Primer CHB (10 nmol / ml) * 5 μl Primer CHSF (10 nmol / ml) * 5 μl cDNA 1 μl KOD polymlase 0.5 μl. The amplification conditions use primers CLSB and CLF1. The above PCR mixture is prepared, set in a thermal cycler, and VH and VL are amplified under the following PCR cycle conditions. (PCR cycle conditions) ・ 98 ° C. 10 seconds × 1 time ・ (98 ° C. 15 seconds, 60 ° C. 7 seconds, 74 ° C. 20 seconds) × 30
Times ・ 74 ℃ 30sec ・ 4 ℃

Example 13 Construction of scFv After purifying the VH and VL amplification products obtained in Example 12, VH and VL were replaced with a linker (scFv link).
l) (assembly reaction). (Ligation conditions) 10 × KOD buffer 1 2.5 μl 25 mM MgCl ₂ 2.1 μl 2.0 mM dNTPs 10 μl VH (0.125 pmol / μl) 1 μl VL (0.125 pmol / μl) 1 μl scFv linker (0.125 pmol / μl) KDO polymerase 0.3 μl DW 7.1 μl Make a PCR mixture or more, set it in a thermal cycler, and ligate VH and VL under the following PCR cycle conditions.・ 98 ° C for 10 seconds x 1 time ・ (98 ° C for 15 seconds, 65 ° C for 30 seconds) x 7 times ・ 74 ° C for 30 seconds x 1 time ・ 4 ° C

Example 14 Re-amplification of scFv and restriction enzyme treatment Using the primer set (CHB and CLF1) shown in Table 2 for re-amplification of scFv, the scFv constructed in Example 13 was re-amplified. After purifying the scFv, a scFv-inserted gene fragment (insert) is prepared using restriction enzymes (EagI and BssHII) for insertion into the plasmid vector pCPDS. (Reamplification reaction) 10 × KOD buffer 1 2.5 μl 2.0 mM dNTPs 4 μl scFv primer CHB (10 nmol / ml) 3 μl scFv primer CLF1 (10 nmol / ml) 3 μl KOD polymerase 0.3 μl DW 12.2 μl PCR conditions 10 seconds x 1 time (15 seconds at 98 degrees Celsius, 7 seconds at 65 degrees Celsius, 20 seconds at 74 degrees Celsius) x 30 times x 30 seconds at 74 degrees Celsius x 4 degrees C (restriction enzyme treatment)-treatment with scFv restriction enzyme 48 μl EagI (50 units / μl) with DW Treatment at 37 ° C. overnight ・ Treatment of pCPDS vector with restriction enzymes pCMDS vector (5 μg) xμl 10 × NEBuffer3 9 μl DW 88 μl EagI (50u) its / [mu] l) after the vector overnight processing and electrophoresed at 37 ° C. digestion confirmed the completeness, 2 [mu] l of BssII (20units / μl)
And digest at 50 ° C. for 3-4 hours. -Treat at 68 ° C for 20 minutes to inactivate the enzyme. -Purification of scFv-pCPDS is BA to prevent self-ligation
P-treated and purified.

Example 15 p of insert (scFv)
Insertion into CPDS (Ligation) The pCPDS and the insert were adjusted to 1: 1 to 10 (molar ratio), and Ligation Kit ver.1 (TAKAR (TAKAR)
Ligation is performed using A)). Vector X μl Insert Y μl 5 × ligation buffer 1 μl 5 μl with DW A buffer 20 μl B buffer 5 μl Treatment at 16 ° C. for 4 to overnight.

Example 16 Transformation of Escherichia coli Escherichia coli (XL1-blue, 100 μl) is forward transformed according to the method described in Example 8 using pCPDS (10 μl) into which the insert has been inserted. The transformed E. coli expressing the insert is arbitrarily selected from a plurality of grown E. coli colonies, and the scFv is amplified using a primer set to select the transformed E. coli expressing the insert.

Example 17 Preparation of Phage-Expressed Antibody A helper phage is infected to the selected Escherichia coli, and a phage-expressed antibody is prepared according to the method described in Example 8.

Example 18 Vanishing Antigen (Prion Protein) for Selection of Specific Phage Antibody After a phage-expressed antibody was reacted with a plate on which a prion protein had been immobilized, only the reacted phage-expressed antibody was recovered, and E. coli was infected. To select specific phage antibodies. Panning is a high affinity antibody selection method using an antigen-antibody reaction. For example, this is performed as follows.
Maltose-binding protein fusion recombinant human PrP23-231 (MBP-HuPr) at a concentration of 5 μg / ml.
P) was added to a 96-well plate at 50 μl / well (0.
(25 μg / well) each, and immobilized at 4 ° C. overnight to prepare an antigen-coated plate. Antigen coated plate 1
25% BlockAce 400 μl / we in 6 wells
Add 1 l, block at 37 ° C for 1 hour, and add PBS-Tw
Washed three times with een 20 (0.05%). After washing
Phage display antibody suspension 1.6 ml, Blo
A mixture of ckAce (704 μl, 5 M NaCl, 96 μl) was added at 150 μl / well, and the mixture was added at 37 ° C. 2
After reacting for hours, PBS-Tween 20 (0.05%)
And washed 15 times. After washing, add 50 μl of eluate
1 / well, and after reaction at room temperature for 15 minutes, 2M
Neutralized with 3 μl / well of Tris. After neutralization, the reaction solution was recovered and XL1-cultured to an OD600 of 0.5 to 1.0.
2 ml of Blue was infected for 1 hour at 37 ° C. A part of the solution after the infection was plated on a SOBAG plate containing 100 μg / ml ampicillin for titer measurement, and the eluted phage titer was measured. After the insert was checked by PCR, the insert ratio was added to the eluted phage titer to determine the positive eluted phage titer. The remaining infection solution contained Super containing 50 μg / ml ampicillin.
400 μl of Broth medium and 37 μ of 2M glucose
After culturing at 37 ° C. for 2 hours, 5 × 109 pfu helper phage (VCS-M13) was infected for 1 hour. After infection, the supernatant was removed by centrifugation at 800 × g for 10 minutes, and a suspension containing 100 μg / ml ampicillin, 50 μg / ml kanamycin and 25 μg / ml tetracycline was removed.
The cells were cultured overnight in 10 ml of perBroth medium. After cooling the next day, the cells were removed by centrifugation at 800 xg for 10 minutes.
The supernatant was filtered through a 0.45 μm pore filter, and the filtrate was used as the primary panned phage display antibody suspension, which was used as the next panning sample.

Example 19 Preparation of Solubilized Antibody According to the method described in Example 8, a specific phage antibody was isolated from non-pressor E. coli (SOLA or HB215).
Infect 1). Twenty-four hours after infection, the E. coli culture supernatant and the cultured cells are collected, and the supernatant and the cell-disrupted extract are used as solubilized antibodies. The solubilized antibody is affinity-purified using an anti-FLAG antibody-bound agarose gel.

[0057]

Industrial Applicability The present invention provides a pure avian recombinant antibody, preferably a chicken recombinant antibody. Chicken-type monoclonal antibodies can produce monoclonal antibodies that cannot be produced in mammals, and recognize N-glycolylneuraminic acid, an antigen serving as a human cancer marker, prion protein related to prion disease, etc. It can be applied to many ecosystem macromolecules, including the examples described above, and is expected to be applied to various testing and diagnostic agents. Therefore, application to various diagnostic agents and therapeutic agents for cancer, prion disease and the like is expected. However, a major drawback of the chicken type is that a fusion cell method for producing a monoclonal antibody generally cannot provide a necessary amount for practical use. The present invention succeeded for the first time in producing large amounts of substantially pure chicken-type antibodies by genetic recombination. That is, the present invention has made it possible to efficiently produce a large amount of chicken monoclonal antibodies using a novel plasmid vector and primers, and provides an antibody, its production method, a vector, a primer set, and the like. It is. The bird antibody gene has a relatively fixed base sequence at the 3 ′ end and the 5 ′ end, so that the gene can be amplified using the primers provided by the present invention regardless of the type of antibody. The present invention provides a method for easily preparing monoclonal antibodies against various antigens by means of genetic recombination.

[Sequence List] SEQUENCE LISTING <110> Japan Science And Technology Corporation <120> Chicken Monoclonal Antibody <130> PA902457 <160> 1 <210> 1 <211> 28 <212> DNA <213> Artificial <400> 13 ' -TTGGGACTGG CAGGATCCGC GCGGGTTC-5 '28

[Brief description of the drawings]

FIG. 1 shows the structure of a known expression vector pPDS.

FIG. 2 shows an outline of construction of a chicken recombinant antibody expression vector (pCPDS) of the present invention.

FIG. 3. Approximately 3 genes likely to be the chicken Cλ chain gene.
A band of 40 bp (A in FIG. 3), a band at about 360 bp that seems to have the FLAG sequence added to the chicken Cλ chain (B in FIG. 3), and a gene III at about 600 bp
It is a photograph instead of a drawing which shows that the band considered to be a gene (C of FIG. 3) and the band (D of FIG. 3) which seemed to have linked the chicken C (lambda) chain of about 950 bp and the geneIII gene were detected. . “M1” in FIG. 3 is pU
Digestion with C118 HinfI, "M2" is φx174
HaeIII indicates digestion.

FIG. 4 shows a comparison between the nucleotide sequence of a chicken Cλ chain gene encoding 114 amino acids and the nucleotide sequence of a Cλ chain obtained by the method of the present invention.

FIG. 5 is a photograph instead of a drawing, showing that amplification of chicken Cλ chain was confirmed at a position of about 360 bp in 12 clones obtained by insertion. “M” in FIG. 5 indicates digestion with pUC118 HinfI.

[Fig. 6] Fig. 6 shows that the recombinant chicken antibodies of 12 clones obtained by the method of the present invention were tested against anti-chicken IgG (H
+ L) as a capture antibody and anti-mouse I
ELI using gG (H + L) as a capture antibody
It shows the SA value (OD492). The solid black indicates the case where anti-mouse IgG (H + L) was used as the capture antibody, and the white outline indicates the case where anti-chicken IgG (H + L) was used as the capture antibody.

FIG. 7 shows the structure of the expression vector pCPDS of the present invention.

FIG. 8 shows the nucleotide sequence of the heavy chain of the V region of the chicken antibody in comparison with HUC2-13.
N in FIG. 8 indicates the base sequence of the D segment, and: indicates a deletion.

FIG. 9 shows the nucleotide sequence of the heavy chain of the chicken antibody V region in comparison with HUC2-13.
N in FIG. 9 indicates the base sequence of the D segment, and: indicates a deletion.

FIG. 10 shows the nucleotide sequence of the V region heavy chain and the amino acid sequence of the V region light chain of the chicken antibody,
3 is shown in comparison with FIG. N in FIG. 10 indicates the base sequence of the D segment, and: indicates a deletion.

FIG. 11 shows the nucleotide sequence of the V region light chain of the chicken antibody in comparison with HUC2-13. In FIG. 11,: indicates a deletion.

FIG. 12 shows the nucleotide sequence of the light chain of the V region of the chicken antibody in comparison with HUC2-13. In FIG. 12,: indicates a deletion.

FIG. 13 shows the diversity acquisition mechanism in the HUC2-13 light chain in comparison with HUC2VL. In FIG. Indicates a mutation,: indicates a deletion,
PV indicates a pseudogene.

FIG. 14 shows the results of an ELISA value (OD492) of an attempt to confirm the reactivity of the recombinant antibody prepared by the method of the present invention with the H25 peptide. In FIG. 14, a black square indicates a chicken-type phage display antibody, a gray diamond indicates a chicken-type soluble antibody (culture supernatant), and a gray circle indicates a chicken-type soluble antibody (disrupted cells). And the gray triangle mark indicates HUC2-
13 (native antibody).

FIG. 15 shows an outline for producing a pure chicken recombinant antibody of the present invention.

FIG. 16 schematically shows the structures of antibody H chain genes in mammals (upper part in FIG. 16) and birds (lower part in FIG. 16).

FIG. 17 schematically shows the usage of the primer CLF1 of the present invention. The left side in the upper part of FIG. 17 is the VL region, and the right side is the Cλ region.

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[Procedure amendment]

[Submission Date] March 3, 2000 (200.3.3)

[Procedure amendment 1]

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[0002]

2. Description of the Related Art A monoclonal antibody is a single antibody that recognizes only a specific portion of an antigen. This technology is a core technology in the biotechnology field along with gene recombination, and uses this technology for diagnostics and therapeutics. The drug is rapidly spreading and developing. Monoclonal antibodies are widely used such as mouse and rat types, but chicken type has already been proposed to some extent by the inventors. The great advantage of the chicken type is that antibodies that are difficult to produce in mammals such as mice can be produced.

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[0003] Chickens are phylogenetically lower than mammals, but as mammals have extremely sophisticated immunity, a large number of useful chicken antibodies have been produced so far. . On the other hand, it has been experienced that when an antibody capable of recognizing a biological component highly conserved among mammals including humans cannot be produced using a mammal, it can be produced as a chicken antibody. Therefore, as one means to adjust chicken antibodies in large quantities,
A method has been developed in which a laying hen is immunized with a specific antigen, and then the antibody transferred to the egg is generated and used as an egg yolk antibody (polyclonal antibody). However, a monoclonal antibody cannot be produced by this method.

[Procedure amendment 5]

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On the other hand, in recent years, a technology for expressing a recombinant antibody (recombinant antibody) on the surface of a phage by utilizing genetic engineering technology has been developed. This phage display antibody technology was launched in 1991 by
(Winter,
G., et al., Nature, 349, 293-299 (1991)), isolated antibody genes from non-immunized human peripheral blood lymphocytes,
ScF diversified by shuffling H and VL genes
v (single chain Fragment of variable region) antibody was expressed as a phage fusion protein to obtain a specific antibody (Marks, JD, et al., J. Mol. Biol., 222, 581-).
597 (1991)). This technique was highly evaluated as a technique for producing a humanized antibody that can avoid immunity and replaces the cell fusion method. At present, a number of practical antibodies have been produced using highly immunized mouse splenocytes, and anti-PrP antibodies have been produced in the same manner (Williamson, RA, et al., Proc. Natl.
Acad. Sci. USA., 99, 7279-7282 (1996)).

[Procedure amendment 6]

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The present inventors first prepared a chicken monoclonal antibody recognizing mammalian prion protein (PrP) residues 23-231 as follows. Recombinant human prion protein (PrP) 23-231
(About 100 μg) was immunized intraperitoneally to a 4-week-old inbred chicken White Leghorn H-B15 chicken, and its spleen was removed to prepare immunized splenocytes. This was fused with the TK-deficient, ouabain-resistant chicken B cell line MuH1 by the PEG method to obtain a fused cell (hybridoma).
An antibody gene (VH and VL regions) prepared from the spleen lymphocytes of the antibody-producing hybridoma or immunized chicken obtained in this manner is converted into a single chain scFv (single chain fragment of V) via a flexible linker.
region). As a plasmid vector for expressing this scFv, Yamanaka (currently Rohto Pharmaceutical)
Has a pPDS (see FIG. 1), but when pPDS is used, the C region (Cκ) of the mouse immunoglobulin L chain is expressed on the N-terminal side of the scFv, and the V region is
The region becomes a mouse recombinant antibody.

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From the above experiments, it was found that the vector for the production of a pure chicken recombinant antibody could be reconstructed. Pure chicken recombinant antibodies obtained from this vector have low noise due to mouse antibodies and can increase the S / N ratio for detection. In constructing the vector of the present invention, (1) a functional ScFv
An antibody can be expressed, (2) a vector capable of producing a Fab antibody in the future, (3) simple purification,
The following three points were noted. In consideration of these three points, chicken Cλ chain was used instead of mouse Cκ chain, and FLAG was used as a purification tag.
An artificial sequence called a sequence was selected.

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(4) pCP of insert (scFv)
Insertion into DS (Ligation) The pCPDS and the insert are adjusted to 1: 1 to 10 (molar ratio), and ligation is performed using Ligation Kit ver. 1 (TAKARA). (5) Transformation of Escherichia coli Escherichia coli (XL1-blue, 100 μl) is transformed using pCPDS (10 μl) into which the insert has been inserted. (6) Selection of transformed E. coli expressing the insert A plurality of E. coli colonies are arbitrarily selected from the grown E. coli colonies, scFv is amplified using a primer set, and the transformed E. coli expressing the insert is selected. . (7) Preparation of Phage-Expressing Antibody A helper phage is infected to the selected E. coli to prepare a phage-expressing antibody. (8) Panning for selection of specific phage antibodies After reacting phage-expressed antibodies on a plate on which an antigen (prion protein) has been immobilized, only the reacted phage-expressed antibodies are recovered and infected with Escherichia coli for specificity. Target phage antibodies. (9) Preparation of solubilized antibody Specific phage antibody was converted to non-pressor E. coli (SOLA
Or HB2151). Twenty-four hours after infection, the E. coli culture supernatant and the cultured cells are collected, and the supernatant and the cell-disrupted extract are used as solubilized antibodies. (10) Generation of solubilized antibody The solubilized antibody is affinity-purified using an anti-FLAG antibody-bound agarose gel.

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Example 18 After reacting a phage-expressed antibody with a plate on which a panning antigen (prion protein) for immobilizing a specific phage antibody was immobilized, only the reacted phage-expressed antibody was recovered and infected with Escherichia coli. To select specific phage antibodies. Panning is a high affinity antibody selection method using an antigen-antibody reaction. For example, this is performed as follows. Maltose binding protein fusion recombinant human PrP23-231 (M
BP-HuPrP) was added to a 96-well plate at 50 μl /
well (0.25 μg / well) at 4 ° C.
This was immobilized overnight and used as an antigen-coated plate. 25% BlockAce 4 in 16 wells of antigen-coated plate
After adding 00 μl / well for 1 hour at 37 ° C., the cells were washed three times with PBS-Tween 20 (0.05%). After washing, phage display antibody suspension
1.6 ml, BlockAce 704 μl, 5 M N
aCl mixed with 96 μl was added to 150 μl / well
Was added and reacted at 37 ° C. for 2 hours. PBS-Tween
Washed 15 times with 20 (0.05%). To the wells after washing, add 50 μl / well of eluate for 15 minutes.
After the reaction at room temperature, the mixture was neutralized with 3 μl / well of 2M Tris. After neutralization, the reaction solution was recovered and OD600 = 0.5-1.
Then, 2 ml of XL1-Blue cultured at 0 ° C was infected for 1 hour at 37 ° C. A part of the solution after the infection was plated on a SOBAG plate containing 100 μg / ml ampicillin for titer measurement, and the eluted phage titer was measured. After the insert was checked by PCR, the insert ratio was added to the eluted phage titer to determine the positive eluted phage titer. To the remaining infection solution, 400 μl of SuperBroth medium containing 50 μg / ml ampicillin and 37 μl of 2M glucose were added, and after culturing at 37 ° C. for 2 hours, 5 × 109 pfu helper phage (VCS-M)
13) was infected for 1 hour. After infection, 800 × g 10
After centrifugation for 10 minutes to remove the supernatant, a SuperBroth medium containing 100 μg / ml ampicillin, 50 μg / ml kanamycin and 25 μg / ml tetracycline 10 ml
For overnight. After cooling the next day, the cells were removed by centrifugation at 800 × g for 10 minutes, the supernatant was filtered with a 0.45 μm pore filter, and the filtrate was used as the primary panned phage display antibody suspension to prepare the next panning sample.

[Procedure amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0057

[Correction method] Change

[Correction contents]

[0057]

The present invention provides a pure avian-type recombinant antibody, preferably a chicken-type recombinant antibody. Chicken-type monoclonal antibodies can produce monoclonal antibodies that cannot be produced in mammals, and recognize N-glycolylneuraminic acid, an antigen serving as a human cancer marker, prion protein related to prion disease, etc. It can be applied to many biological macromolecules, including the examples described above, and is expected to be applied to various testing and diagnostic agents. Therefore, application to various diagnostic agents and therapeutic agents for cancer, prion disease and the like is expected. However, a major drawback of the chicken type is that a fusion cell method for producing a monoclonal antibody generally cannot provide a necessary amount for practical use. The present invention succeeded in producing, for the first time, a large amount of substantially pure chicken-type antibody by a genetic recombination method. That is, the present invention has made it possible to efficiently produce a large amount of chicken monoclonal antibodies using a novel plasmid vector and primers, and provides an antibody, its production method, a vector, a primer set, and the like. It is. The bird antibody gene has a relatively fixed base sequence at the 3 ′ end and the 5 ′ end, so that the gene can be amplified using the primers provided by the present invention regardless of the type of antibody. The present invention provides a method for easily preparing monoclonal antibodies against various antigens by means of genetic recombination.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12N 1/21 // C12N 1/21 (C12P 21/08 (C12P 21/08 C12R 1: 19) C12R 1:19) C12N 15/00 ZNAA F-term (Reference) 4B024 AA01 AA11 AA12 BA41 BA53 CA04 CA20 DA06 EA03 EA04 GA03 GA11 HA08 4B063 QA01 QA13 QA18 QQ08 QQ46 QQ96 QR31 QR62 QS25 4B064 AG27 CA02 DA12 4B065 AA26X AA90X AA98X AB04 AC14 BA02 CA25 CA44 CA46 4H045 AA11 BA10 CA40 DA76 DA86 EA21 EA28 FA72 GA26

Claims (27)

[Claims] 1. A method for producing a chicken monoclonal antibody by a gene recombination method, wherein a chicken monoclonal antibody sc is added to an expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced.
A method for producing a chicken monoclonal antibody, comprising using an expression vector into which a gene encoding Fv has been introduced. 2. The method according to claim 1, wherein the expression vector into which the gene encoding the chicken Cλ chain (L chain constant region) has a tag for purification. 3. The chicken monoclonal antibody scF
3. The method according to claim 1, wherein the restriction enzyme at the position where the gene encoding v is introduced is EagI-BssHII. 4. The method according to claim 1, which is expressed by a phage display method. 5. An expression vector in which a tag (amber) capable of being expressed as a soluble antibody is introduced upstream of a gene encoding a phage protein.
The method described in. 6. An expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced.
6. The method of claim 5, wherein the method is DS. 7. An expression vector for producing a chicken monoclonal antibody by a genetic recombination method,
An expression vector into which a gene encoding a chicken Cλ chain (L chain constant region) has been introduced. 8. The expression vector according to claim 7, wherein the expression vector into which the gene encoding the chicken Cλ chain (L chain constant region) has a tag for purification. 9. The scF of chicken monoclonal antibody
9. The expression vector according to claim 7, wherein the restriction enzyme at the position where the gene encoding v is introduced is EagI-BssHII. 10. The expression vector according to claim 7, wherein the expression vector is chicken pPDS. 11. The sc of chicken monoclonal antibody
The gene encoding Fv is a restriction enzyme EagI-BssH
The expression vector according to any one of claims 7 to 10, which is introduced into position II. 12. The expression vector according to claim 11, wherein the chicken monoclonal antibody scFv can be expressed by a phage display method. 13. The expression vector according to claim 12, wherein a tag (amber) capable of being expressed as a soluble antibody is introduced upstream of a gene encoding a phage protein. 14. A chicken recombinant antibody produced by the method according to claim 1. 15. The antibody according to claim 14, which is a monoclonal antibody against human prion protein (PrP). 16. The antibody according to claim 14, which is a solubilized antibody.
The antibody according to 1. 17. VL of chicken monoclonal antibody
Alternatively, one of the primers for amplifying the scFv-encoding gene has a base sequence that can be cleaved by the restriction enzyme BssHII, and the chicken-type monoclonal antibody scFv-encoding gene amplifying primer. 18. The method according to claim 17, wherein the number of bases is 15 to 30.
The primer according to 1. 19. The base sequence is 3'-ttggggactggcaggatccgcgc.
19. The primer according to claim 17 or 18, which is gggttc-5 'or a complementary strand thereof. 20. The base sequence of the other primer is 5′-ctgatggcggccccgtgacgtt-
The primer according to any one of claims 17 to 19, which is 3 'or a complementary strand thereof. 21. The base sequence of the other primer is 5′-tctgacgtcgcgctgactcagc.
The primer according to any one of claims 17 to 19, which is c-3 'or a complementary strand thereof. 22. A method for amplifying a gene encoding the variable region of a chicken monoclonal antibody using the primer according to any one of claims 17 to 21. 23. The variable region is a VL region.
The method described in. 24. The method according to claim 23, wherein the primer is the primer according to claim 21. 25. The variable region is scFv.
The method described in. 26. The method according to claim 25, wherein the primer is the primer according to claim 20. 27. The base sequence is 3′-ttggggactggcaggatccgcgc.
gggttc-5 ′ or an oligonucleotide that is the complement thereof.