CN104059151A - Anti-CD138 monoclonal antibody variable region sequence and preparation method thereof - Google Patents

Abstract

The invention provides sixteen nucleotide sequences of an anti-CD138 monoclonal antibody variable region sequence and a preparation method of the anti-CD138 monoclonal antibody variable region sequence. In addition, a sequencing process is carried out. The anti-CD138 monoclonal antibody variable region sequence can be used for preparing recombinant antibodies, ScFv antibodies, humanized antibodies, chimeric antibodies, dual-specific antibodies and single-domain antibodies; can be used for preparing antibody drugs; has a great potential value in aspects of tumor diagnosis, illness state progress, metastatic potential, prognostic evaluation and the like; is an important improvement of development of oncogene in the technical field of biological medicine; and enables a more comprehensive knowledge of values in prediction, diagnosis, treatment and prognosis of cancer to be obtained in the industrial field.

Description

Anti-CD138 variable region of mab sequence and preparation method thereof

Technical field

The present invention relates to biological medicine, relate in particular to a kind of anti-CD138 variable region of mab sequence and preparation method thereof.

Background technology

CD138 is a kind of proteoglycan, by interacting to regulate cell behavior with heparin binding growth factor, the dissolvable matrix multiple effector that becomes to grade.There are 4 members in Syndecans family, is respectively Syndecan-1 ,-2 ,-3 ,-4, wherein extensive to the research of Syndecan-I (CD138).

Sydecan-1 (CD138) molecule is cross-film bonding proteoglycan family member, endochylema section, cross-film section and extracellular fragment, consists of.CD138 is the macromolecular complex that a class is very complicated, molecular weight is 85～92kD approximately, by core protein and a class glycoconjugate that the glycosaminoglycan chains covalent attachment of branch does not form, also claim proteoglycan, proteoglycan is present in all Mammalss, is the important composition composition of intercellular substance, cytoplasmic membrane.

CD138 molecule belongs to I type transmembrane protein, adheres to the film outskirt at place, a hydrophobic transmembrane and a short C end cytoplasmic domain be comprised of a N end signal peptide, one containing glycosaminoglycan.Cross-film district and the cytoplasmic domain of CD138 are high conservatives, and cytoplasmic domain peptide chain is shorter, 13 amino acid, consist of, and district is closely connected with cross-film.The function of cytoplasmic domain part is also not clear at present.

Transmembrane protein glycan CD138 can express the tumor cell surface in various sources, comprises myelomatosis, ovarian cancer, mammary cancer, liver cancer, Hokdkin disease and the lymphoma relevant with some HIV.After normal cell cancerates, the expression of surface of cell membrane CD138 molecule often changes, and has certain dependency with grade malignancy, the prognosis of tumour.CD138 is a kind of adhesion molecule, and it reduces or disappearance at tumour cell Membrane surface expression, and the behaviors such as growth, differentiation of cell are got muddled, and has caused oncocyte amount reproduction, shows extremely strong invasion and attack activity and transfer characteristics.

The generation of CD138 and tumour, development have certain dependency, and have certain relation with somatotype, the differentiation of tumour by stages, can judge tumour somatotype according to CD138 expression degree in cancerous tissue, and take corresponding treatment means.Because CD138 can promote cell-cell, adhesion between cell-matrix, growth of tumour cell, invasion and attack and the characteristic shifting, therefore can be used for immunity or the gene therapy of tumour, its aspect such as evaluation in diagnosing tumor, disease progression, metastatic potential and prognosis has very large potential value.Therefore,, along with molecular biological development, the deepening continuously of oncogene research, believes prediction, diagnosis, the treatment of this gene pairs cancer, the value of prognosis can be familiar with more comprehensively.

Mouse source monoclonal antibody can cause human antimouse antibody reaction in human body.The development of genetic engineering antibody technology make people can be on gene level engineered antibody, reduce the immunogenicity of antibody, improve antibodies specific, stability and avidity.Mouse source antibody is carried out humanization modified, can retain antibody variable region and human antibody constant region and merge, improve affinity of antibody; Or engineered antibody structure, build the ScFv that only retains antibody variable region or the Fab that contains antibody variable region and part constant region, can improve antibody percent absorption and the transformation period in vivo.

Summary of the invention

The invention solves deficiency of the prior art, anti-CD138 variable region of mab nucleotide sequence is provided, the method for preparing above-mentioned nucleotide sequence is provided simultaneously.

Invention thinking of the present invention: use CD138 albumen as antigen, after immune mouse, detection obtains corresponding antibodies and expresses the mouse of being positive, get positive mouse spleen, after separating Morr. cell, merge splenocyte and myeloma cell, after cultivating in defined medium, detection obtains antibody expression positive colony, extract the total RNA of positive hybridoma cell, the mRNA of take in total RNA is template, and reverse transcription obtains the cDNA of corresponding antibodies gene, then obtains corresponding antibodies variable region of heavy chain and variable region of light chain with specific primer PCR.

Technical scheme of the present invention is: anti-CD138 variable region of mab sequence, it is characterized in that, comprise 16 kinds of nucleotide sequences, described nucleotide sequence is respectively 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH, 59166 (2.2)-VL.

In a preferred embodiment of the present invention, further comprise, prepared by the hybridoma that described nucleotide sequence 59166 (1.1)-VH and 59166 (1.1)-VL are 587CT11.3.6.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (1.2)-VH and 59166 (1.2)-VL are 587CT11.3.6.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (1.1)-VH and 58208 (1.1)-VL are 480CT5.4.3.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (1.2)-VH and 58208 (1.2)-VL are 480CT5.4.3.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (2.1)-VH and 58208 (2.1)-VL are 480CT13.4.3.2.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (2.2)-VH and 58208 (2.2)-VL are 480CT13.4.3.2.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (2.1)-VH and 59166 (2.1)-VL are 587CT7.3.6.5.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (2.2)-VH and 59166 (2.2)-VL are 587CT7.3.6.5.2 by preserving number.

In a preferred embodiment of the present invention, further comprise, also comprise the nucleotide sequence with described 16 kinds of nucleotide sequences with same acid sequence product.

In a preferred embodiment of the present invention, further comprise, also comprise through one or several Substitution, disappearance or after adding and still there is the nucleotide sequence that the aminoacid sequence producing with described nucleotide sequence has identical activity.

In a preferred embodiment of the present invention, further comprise, described nucleotide sequence 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH and 59166 (2.2)-VL can be used for Dispersal risk.

In a preferred embodiment of the present invention, further comprise, above-mentioned antibody comprise following one or more: recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bi-specific antibody and single domain antibody.

In a preferred embodiment of the present invention, further comprise that a kind of antibody drug comprises above-mentioned antibody and pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, further comprise, an expression vector, comprises above-mentioned nucleotide sequence: 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH, 59166 (2.2)-VL.

In a preferred embodiment of the present invention, further comprise that a kind of expressive host is loaded with expression vector claimed in claim 8.

A method of preparing above-mentioned anti-CD138 variable region of mab sequence, comprises the following steps:

(1) preparation of hybridoma: by the BALB/c mouse of myelomatosis tumour cell or the immune female Sexual health of CD138 proteantigen difference, pick out the mouse that the rear antibody expression of immunity is positive, get its spleen cell, then separated mouse spleen cell and myeloma cell are merged, form hybridoma;

(2) screening of monoclonal cell: the hybridoma in step (1) is cultivated in HAT substratum, the monoclonal cell of ELISA tests positive is carried out to ELISA to be sieved again, then filtering out ELISA positive monoclonal cell carries out WB and sieves again, again WB positive cell is carried out to subclone, through 2 subclones, filter out can stably excreting antibody monoclonal cell, will after positive monoclonal cell enlarged culturing, determine strain, frozen;

(3) preparation of anti-CD138 monoclonal antibody: the subclass hypotype of the monoclonal cell of preparation in first authentication step (2), again positive monoclonal cell is expelled to and in Mice Body, carries out ascites production, then the ascites of generation is obtained to anti-CD138 monoclonal antibody after by chromatography purification;

(4) clone of anti-CD138 variable region of mab sequence gene: total RNA of monoclonal cell used in extraction step (3), obtain mRNA, take described mRNA as template again, reverse transcription obtains cDNA, and finally clone obtains anti-CD138 monoclonal antibody variable region of heavy chain and variable region of light chain;

(5) the variable region fragment in step (4) is building up in carrier to the evaluation of checking order.

The defect that solves prior art of the present invention, has following beneficial effect: the invention provides 16 kinds of nucleotide sequences of anti-CD138 variable region of mab sequence, and the preparation method of anti-CD138 variable region of mab sequence, and check order; It can be used for preparing recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bi-specific antibody and single domain antibody, can be applicable on Dispersal risk medicine, at the aspects such as evaluation of diagnosing tumor, disease progression, metastatic potential and prognosis, there is very large potential value; Be an important advance of oncogene development in biomedicine field, make to the value of the prediction of cancer, diagnosis, treatment, prognosis, can be familiar with more comprehensively in industry field.

Accompanying drawing explanation

Fig. 1 is that anti-CD138 monoclonal antibody (480CT5.4.3) immunoblotting (WB) detects figure.

Fig. 2 is that anti-CD138 monoclonal antibody (480CT13.4.3.2) immunoblotting (WB) detects figure.

Fig. 3 is that anti-CD138 monoclonal antibody (587CT7.3.6.5) immunoblotting (WB) detects figure.

Fig. 4 is that anti-CD138 monoclonal antibody (587CT11.3.6) immunoblotting (WB) detects figure.

Wherein, the 1-4 in Fig. 1-4 respectively is antibody concentration 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml.

Fig. 5 is that anti-CD138 monoclonal antibody (clone 480CT5.4.3) IF detects figure.

Fig. 6 is that anti-CD138 monoclonal antibody (clone 480CT13.4.3.2) IF/FC detects figure.

Fig. 7 is that anti-CD138 monoclonal antibody (clone 587CT7.3.6.5) IF/FC detects figure

Fig. 8 is the RT-PCR product agarose gel electrophoresis map analysis result figure of 480CT5.4.3 clonal antibody variable region VH and VL.

Wherein swimming lane 1 is DL2000DNAMarker; Swimming lane 2 is VH gene; Swimming lane 3 is VL gene; Swimming lane 4 is RT-PCR amplification VH gene negative control; Swimming lane 5 is RT-PCR amplification VL gene negative control.

Fig. 9 is the RT-PCR product agarose gel electrophoresis map analysis result figure of 480CT13.4.3.2 clonal antibody variable region VH and VL.

Wherein swimming lane 1 is DL2000DNAMarker; Swimming lane 2 is VH gene; Swimming lane 3 is VL gene; Swimming lane 4 is RT-PCR amplification VH gene negative control; Swimming lane 5 is RT-PCR amplification VL gene negative control.

Figure 10 is the RT-PCR product agarose gel electrophoresis map analysis result figure of 587CT7.3.6.5 clonal antibody variable region VH and VL.

Wherein swimming lane 1 is DL2000DNAMarker; Swimming lane 2 is VH gene; Swimming lane 3 is VL gene; Swimming lane 4 is RT-PCR amplification VH gene negative control; Swimming lane 5 is RT-PCR amplification VL gene negative control.

Figure 11 is the RT-PCR product agarose gel electrophoresis map analysis result figure of 587CT11.3.6 clonal antibody variable region VH and VL.

Wherein swimming lane 1 is DL2000DNAMarker; Swimming lane 2 is VH gene; Swimming lane 3 is VL gene; Swimming lane 4 is RT-PCR amplification VH gene negative control; Swimming lane 5 is RT-PCR amplification VL gene negative control.

Figure 12 is 480CT5.4.3 clonal antibody variable region VH and VL object fragment, is building up to enzyme on pMD18-T carrier and cuts evaluation agarose gel electrophoresis figure.

Wherein swimming lane 1 is DL5000DNAMarker; 2 clones that swimming lane 2,3 is pMD18-T/VH; 2 clones that swimming lane 4,5 is pMD18-T/VL.

Figure 13 is 480CT13.4.3.2 clonal antibody variable region VH and VL object fragment, is building up to enzyme on pMD18-T carrier and cuts evaluation agarose gel electrophoresis figure.

Wherein swimming lane 1 is DL5000DNAMarker; 2 clones that swimming lane 2,3 is pMD18-T/VH; 2 clones that swimming lane 4,5 is pMD18-T/VL.

Figure 14 is 587CT7.3.6.5 clonal antibody variable region VH and VL object fragment, is building up to enzyme on pMD18-T carrier and cuts evaluation agarose gel electrophoresis figure.

Wherein swimming lane 1 is DL5000DNAMarker; 2 clones that swimming lane 2,3 is pMD18-T/VH; 2 clones that swimming lane 4,5 is pMD18-T/VL.

Figure 15 is 587CT11.3.6 clonal antibody variable region VH and VL object fragment, is building up to enzyme on pMD18-T carrier and cuts evaluation agarose gel electrophoresis figure.

Wherein swimming lane 1 is DL5000DNAMarker; 2 clones that swimming lane 2,3 is pMD18-T/VH; 2 clones that swimming lane 4,5 is pMD18-T/VL.

Embodiment

In order to make those skilled in the art person understand better the present invention, and above-mentioned advantage of the present invention can be become apparent more, below in conjunction with Figure of description and specific embodiment, the present invention is further detailed explanation.

Embodiment

Step 1: the preparation of hybridoma

(1) animal immune

With myelomatosis tumour cell or the CD138 proteantigen BALB/c mouse of the female Sexual health in 3 of immunity 6-8 age in week respectively, both legs muscle, both shoulders are subcutaneous, abdominal cavity multi-point injection 50-100ug antigen/only, 3 immunity blood sampling in latter 7 days is surveyed Serum Antibody and is tired, blood sampling once weekly, ELISA while selecting serum 1:4000 dilution detects OD value and is greater than 1.0, and serum WB detects positive BALB/c mouse for merging simultaneously, merges first 3 days, do not add the antigen abdominal cavity booster immunization injection of adjuvant, 60ug/ only.

(2) hybridoma preparation

(2.1) collect bone-marrow-derived lymphocyte

After supplementary immunization 3 days, mouse was plucked eyeball bloodletting, and centrifugal rear serum gives over to positive control.By aseptic technique, take out spleen, and spleen is placed in the incomplete substratum of the pre-temperature of 10ml, peel off reticular tissue around, put in 100 order stainless (steel) wires, with the inner core of syringe, grind, grinding limit, limit drips incomplete substratum and rinses.Cell suspension after collection is filtered is in centrifuge tube, centrifugal, abandons supernatant, and the full substratum suspension cell that toos many or too much for use, gets 1 * 10 ⁸individual cell, puts room temperature stand-by.

(2.2) preparation of murine myeloma cell

Merge first 10 days, myeloma cell is taken out from liquid nitrogen, put into rapidly 37 degree water-bath 10min and dissolve completely to refrigerating fulid.Centrifugal, abandon supernatant, put in IMDM perfect medium, 37 ℃, 5%CO ₂cultivate.According to cell growth condition, change liquid, cell cultures expands.2-3d before merging, cell should be in logarithmic phase.Before merging, logarithmic phase murine myeloma cell is collected in centrifuge tube, counting, gets 2 * 10 ⁷-3 * 10 ⁷individual cell, the centrifugal supernatant of abandoning, the full substratum suspension cell that toos many or too much for use, puts room temperature stand-by.

(2.3) cytogamy

Before merging, 50%PEG is put to 37 ℃, 5%CO ₂in cell culture incubator, adjust temperature.By 2 * 10 ⁷-3 * 10 ⁷individual myeloma cell's suspension and 1 * 10 ⁸individual spleen bone-marrow-derived lymphocyte suspension moves in a 50ml centrifuge tube, adds the incomplete substratum of 30ml, 1500rpm, centrifugal 10min, supernatant discarded.At the bottom of attack pipe, make cell mass loose gently.Rotate equably centrifuge tube on one side, another hand is drawn the PEG fusogen of the pre-temperature of 1ml with 1ml suction pipe, in centrifuge tube bottom, about 2cm place slowly adds in cell along tube wall, limit edged rotates centrifuge tube, the PEG application of sample time is controlled at 60s, jog centrifuge tube 30s, standing 60s, then add immediately the incomplete nutrient solution of 20ml, make PEG dilution and lose the short effect of melting, concrete addition is that a min adds 1ml, the 2nd min adds 4ml, within 3min subsequently, remaining liq is added, put 37 ℃ of standing 10min of water-bath, 1500 revs/min, centrifugal 10min, supernatant discarded, add the perfect medium that contains HAT, make cell suspension, be taped against on 96 porocyte culture plates, put 37 ℃, 5%CO ₂in cell culture incubator.

Step 2: screening positive monoclonal hybridoma

Above-mentioned cell is cultivated in HAT substratum, and the myeloma cell of not merging and the lymphocyte not merging are dead gradually.Only have the hybridoma of fusion in HAT substratum, to survive and to breed.37 ℃, in 5%CO2 cell culture incubator, cultivate 7-10 days.Use antigen coated enzyme plate, 37 ℃ of coated 2h, then seal with 2%BSA.The cell conditioned medium of drawing growth clone in 96 orifice plates is added in the enzyme plate having sealed, and hatches 1h for 37 ℃.Wash after 5 times, add the sheep anti-mouse igg of HRP mark, hatch 1h for 37 ℃.After washing, add TMB nitrite ion, then add 2M sulfuric acid termination reaction.In microplate reader, carry out reading.ELISA is detected to positive clone and choose into 24 porocyte culture plates cultivations, carry out ELISA and sieve again after 3 days, the ELISA positive colony filtering out is carried out to WB and sieve again, WB positive cell carries out subclone.Through 2 subclones, filter out can stably excreting antibody monoclonal cell, will after positive mono-clonal enlarged culturing, determine strain, frozen.

Step 3: the preparation of anti-CD138 monoclonal antibody

(1) biological assay of monoclonal antibody

The cell conditioned medium of determining the monoclonal cell of strain is identified to the subclass hypotype of monoclonal antibody with U.S. company BD MouseMonoclonal Antibody Isotyping Kit, then positive monoclonal cell is expelled to and in Mice Body, carries out ascites production, the ascites of producing obtains antibody after by chromatography purification, adopt Western Blot to identify the specificity of antibody, ELISA identifies the avidity of monoclonal antibody, and concrete operations are as follows:

(1.1) Western Blot detects the specificity of antibody

Get processed cell pyrolysis liquid, carry out vertical SDS-PAGE on gel, 120v90min, after electrophoresis finishes, takes off gel, is placed on pvdf membrane, albumen is transfected on pvdf membrane to 10v, 120min by semidrying.By the room temperature sealing 2h in 5% skim-milk of the pvdf membrane after transfer printing.Antibody is dissolved in 3ml confining liquid, and 4 ℃ are spent the night.With washings washing 3 times, each 5min, dilutes the sheep anti-mouse igg of HRP mark with confining liquid, under room temperature, hatch 2h.With washings washing 3 times, chemical illuminating reagent and pvdf membrane are hatched altogether, do X exposure to darkroom, by developing and photographic fixing, result are reflected on film.Scan film, analytical results, its result is with reference to shown in Fig. 1-7.

(1.2) monoclonal antibody affinity costant is measured

With coating buffer, by antigen diluent, add respectively enzyme plate, 100 microlitres/hole, 37 ℃ of 2h, PBS washing 5 times, pats dry, and adds 2%BSA confining liquid 200 microlitres/hole, and 4 spend night, wash 5 times, pat dry.Antibody from starting to carry out gradient dilution, 4ug/ml is joined in the enzyme plate being coated with, 100 microlitres/hole, and 37 ℃ of 1h, wash 5 times, pat dry, and add the goat anti-mouse igg antibody of HRP mark, working fluid 100 microlitres/hole, 37 ℃ of 1h, wash 5 times, pat dry.Add TMB nitrite ion, 37 ℃ of reaction 5-10min, add stop buffer 50 microlitres/hole, measure OD value immediately in microplate reader with 450nm wavelength, according to formula, calculate monoclonal antibody affinity costant.

Step 4: the clone of anti-CD138 monoclonal antibody heavy chain and chain variable region gene

Cell strain used is the anti-CD138 that can secrete high-affinity, high specific that adopts aforesaid method and obtain or the hybridoma cell strain of CD138 antibody, corresponding deposit number with and the antibody molecule hypotype of secretion as shown in table 1 below:

Table 1

Deposit number	Antibody molecule hypotype
		587CT11.3.6.1	IgG1
587CT11.3.6.2	IgG1
		480CT5.4.3.1	IgG1
480CT5.4.3.2	IgG1
		480CT13.4.3.2.1	IgM
480CT13.4.3.2.2	IgM
		587CT7.3.6.5.1	IgM
587CT7.3.6.5.2	IgM

8 hybridoma 2 * 10^6 that take the logarithm vegetative period, test kit RNeasy Mini Kit(article No. with QIAGEN company: QIAGEN-74106) extract total RNA, and 1% non-sex change agarose gel electrophoresis detection quantitative with Nanodrop takes a morsel, use subsequently SuperScript.IIIFirst-Strand Synthesis System for RT-PCR test kit (article No.: Invitrogen-18080-051) reverse transcription cDNA, with special primer increase light chain or the variable region of heavy chain of anti-CD138 antibody gene.The PCR reaction product that contains corresponding variable region of heavy chain or variable region of light chain fragment, through 1% agarose gel electrophoresis, is cut to the separated object fragment of glue.By glue, reclaim after test kit (JaRa-GK2042) purifying object product, 1% agarose gel electrophoresis is identified the purity of object fragment.Afterwards, corresponding variable region of heavy chain and variable region of light chain that recovery is obtained are cloned into sequencing vector pMD18-T, and check order, and sequencing result is carried out to homology and structural analysis.

Concrete operation step is as follows:

(1) RT-PCR amplification CD138 light chain of antibody and variable region of heavy chain

Design of primers:

According to the sequences of variable region of 8 clone's hypotypes, in signaling zone and constant region synthetic 5 ' and 3 ', hold the primer CD138 antibody heavy chain variable region that is used for increasing respectively, primer sequence is as follows:

VHF(5 '-ACTAGTCGACATGGVTTGGSTGTGGAMCTTGCYATTCCT-3 '), contain Sal I restriction enzyme site;

VHR(5 '-CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG-3 '), contain Hind III restriction enzyme site.

According to the sequences of variable region of 8 clone's hypotypes, in signaling zone and constant region synthetic 5 ' and 3 ', hold the primer CD138 antibody chain variable region that is used for increasing respectively, primer sequence is as follows:

LHF(5 '-ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3 '), contain Sal I restriction enzyme site;

LHR(5 '-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3 '), contain Hind III restriction enzyme site.

The total RNA of hybridoma extracts:

With the test kit of QIAGEN company (article No.: 74106) extract total RNA, concrete steps are as follows:

1, collect logarithmic phase hybridoma each 2 * 10 ⁶individual, the centrifugal 5min of 800rpm, removes supernatant, and precipitation is that cell can be stored in-80 ℃, or is directly used in RNA extraction.

2, in every part of cell (2 * 10^6) sample, add 350ulRTL solution, vortex 30s.

3, in above system, add 70% ethanol, with 1mL liquid-transfering gun, blow and beat gently evenly.

4, above system mixed solution is proceeded to RNeasy spin column, the centrifugal 15s of 8000x g, goes filtrate.

5, in above RNeasy spin column, respectively add 700uL RW1 solution, the centrifugal 15s of 8000x g, goes filtrate.

6, in above RNeasy spin column, respectively add 500uL RPE solution, the centrifugal 15s of 8000x g, goes filtrate.

7, in above RNeasy spin column, respectively add 500uL RPE solution, the centrifugal 2min of 8000x g, goes filtrate.

8, RNeasy spin column is transferred in the 2ml collection tube without RNA enzyme, at full speed centrifugal 1min.

9, RNeasy spin column is transferred to without RNA enzyme 1.5mlEP pipe in, add 30～50uL without the H of RNA enzyme ₂o, the centrifugal 1min of 8000x g, eluted rna.

Reverse transcription PCR:

With the synthetic SuperScript.III test kit (article No. 18080-051) of RT-PCR the first chain of Invitrgen, the total RNA of the hybridoma of take is template, reverse transcription cDNA, and concrete steps are as follows:

1, primer and template sex change

Press table 2, be made into 10uL system, 65 ℃, 5min, places 1min subsequently on ice, is beneficial to primer and is combined with RNA template.

Table 2

Composition	Volume uL
		RNA	x(<1ug,>100ng)
Primer Oligo (dT) 20	1
		dNTP	1
Without RNA enzyme H 2O	8-x

2, renaturation

Press table 3, be made into 10uL system, add the system of primer and the sex change of RNA template, 50 ℃, 50min; 85 ℃, 5min.

Table 3

Composition	Volume uL
		10×RT buffer	2
25mM MgCl2	4
		0.1M DTT	2
RNase OUT	1
		SuperScript III RT	1

3, RNA digestion

In above every individual system, add 1uLRNaseH, 37 ℃, 20min.

Antibody variable region specific primer PCR:

Press the system in table 4 and table 5, the cDNA that the reverse transcription of take obtains is template, with special primer synthetic antibody heavy chain and variable region of light chain.

Table 4

Composition	Volume uL
		H 2O	37.4
10×pfu buffer(Mg 2+)	5
		dNTP(10mM)	1
pfu	0.4
		taq	0.2
Sense primer(20uM)	1
		Anti-sense primer(20uM)	1
cDNA	4

Table 5

Step 5: PCR product cloning and order-checking

The variable region of heavy chain that PCR is obtained and variable region of light chain nucleotide fragments product glue are building up on sequencing vector respectively after reclaiming, and order-checking, and concrete steps are as follows.

1, glue reclaims PCR product

PCR product runs after 1% agarose gel electrophoresis, and object band is reclaimed in rubber tapping, reclaims test kit (article No. GK-2042) reclaim with JaRa glue.Step is as follows:

Every 100mg sepharose adds 400uL Binding Solution B, is placed to gel piece and melts completely in 60 ℃ of water-baths;

Above-mentioned mixed solution is transferred to cover to be had in the GenClean post of 2mL collection tube, and room temperature is placed 2min, and the centrifugal 1min of 6000rpm, removes waste liquid;

Add 500uL Wash Solution, 12000rpm, the centrifugal 1min of room temperature, removes waste liquid;

Repeat previous step;

GenClean post is put back to collection tube, 12000rpm, the centrifugal 1min of room temperature;

GenClean post is put to the pipe to 1.5mLEP, add 30～50uLElution Buffer, place 2min for 37 ℃, 12000rpm, the centrifugal 1min of room temperature is with wash-out object fragment.

2, enzyme is cut

Enzyme is cut system (Fermentas Fast Digest) as shown in table 6-7:

Table 6

Composition	Volume uL
		Plasmid	X(1ug)
10×FD buffer	5
		Enzyme 1	1

Enzyme 2	1
		H 2O	43-X

Table 7

Composition	Volume uL
		Antibody object fragment	17
10×FD buffer	2
		Enzyme 1	0.5
Enzyme 2	0.5

Enzyme tangent condition: 37 ℃, 30min; 85 ℃, 5min; Glue reclaims long segment in plasmid enzyme restriction system, and the antibody object fragment enzyme system of cutting is directly used in connection.

3, connect

Linked system is as table 8:

Table 8

Composition	Volume uL
		Carrier glue reclaims large fragment	1.5
Antibody object fragment	7
		10×ligase buffer	1
ligase	0.5

Condition of contact: 16 ℃, 4h.

4, transform

To linked system, add 100uLDH5 α competent cell, ice bath 30min;

42 ℃ of thermal shock 90s, ice bath 3min;

Add 500uLLB substratum, 37 ℃, 120rpm, recovery 50min;

The centrifugal 2min of 4000rpm, removes supernatant, stays 100uL supernatant re-suspended cell precipitation, is coated with corresponding resistant LB dull and stereotyped.After liquid-absorbent in plate, be inverted plate, in 370 ℃ of cultivations, within 12-16 hour, can there is bacterium colony.

5, bacterium colony PCR identifies

Choose in every reformer plate 4～6 of mono-clonals, point, after corresponding resistant LB flat board, is bacterium colony PCR and is identified, PCR reaction product is run 1% sepharose and detected and have or not object length fragment.Its detected result is as shown in Fig. 8-11, and bacterium colony PCR system is as shown in table 9-10:

Table 9

Composition	Volume
		H 2O	15.8uL
10×pfu buffer(Mg 2+)	2uL
		dNTP(10mM)	1uL
Taq	0.2uL
		Sense primer(20uM)	0.5uL
Anti-sense primer(20uM)	0.5uL
		Clone	1

Table 10

6, upgrading grain

Choose bacterium colony PCR and be accredited as positive mono-clonal and shake bacterium, 20% glycerine is protected after bacterium, with Biomiga-Plasmid Miniprep kit(article No., is BIOMEGA-PD1211-02) upgrading grain.Concrete steps are as follows:

Get the fresh bacterium liquid of 4mLLB, the centrifugal 1min of 1000rpm under room temperature, collects thalline, sucks as far as possible supernatant;

Add 250uL Buffer A1(to add RNaseA), vortex shakes abundant suspension bacterium;

Add 250uL Buffer B1, reverse gently 10 times to mix, standing 5min clarifies to solution thickness;

Add 350uL Buffer N1, reversion immediately repeatedly, fully mixes to solution, now occurs white flocks;

Centrifuge tube is gone to supercentrifuge, if adularescent precipitation in the centrifugal 10min(supernatant of 13000rpm at room temperature, can recentrifuge);

Careful draw supernatant liquor after centrifugal to (avoiding picking up precipitation) in the centrifugal column with collection tube, under room temperature, the centrifugal 1min of 13000rpm, outwells the waste liquid in collection tube, and centrifugal column is relay in recovery collector;

In DNA post, add 500uL Buffer KB, centrifugal 1min under room temperature, outwells the waste liquid in collection tube, and centrifugal column is relay and reclaimed in collector;

In centrifugal column, add 500uL DNA Wash Buffer(to add dehydrated alcohol), under room temperature, the centrifugal 1min of 13000rpm, outwells waste liquid in collection tube, and centrifugal column is relay and got back in collection tube.Repeat this step once;

Centrifugal column is put back in supercentrifuge, and the centrifugal 5～10min that uncaps under 13000rpm room temperature, thoroughly to remove residual ethanol;

Centrifugal column is transferred in a new 1.5mL centrifuge tube, adds the Elution Buffer of 50uL60 ℃ of preheating to the middle of DNA post, room temperature is placed 2min, the centrifugal 1min of 13000rpm, wash-out plasmid DNA;

7, enzyme is cut evaluation

The plasmid DNA of extracting is identified with respective limits endonuclease digestion, and system is as shown in table 11:

Table 11

Composition	Volume uL
		Plasmid	X(500ng)
10×FD buffer	2
		Enzyme 1	0.5
Enzyme 2	0.5
		H 2O	17-X

Enzyme tangent condition: 37 ℃, 30min; 85 ℃, 5min, runs 1% agarose gel electrophoresis and identifies whether there is object band.

8, plasmid PCR is identified

Enzyme cut identify positive plasmid again PCR identify whether there is object band, reaction system as Table 12-13:

Table 12

Table 13

PCR product runs 1% agarose gel electrophoresis, and its detected result, as shown in Figure 12-15, if there is positive band, is sent order-checking.

The above; be only the specific embodiment of the present invention, protection scope of the present invention is not limited to this, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claim was defined.

Sequence table

59166(1.1)-VH nucleotides sequence is classified as:

GAGGTTCAGCTTCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGACTTCGGTTACTCATTCACTAGTTATTACATGCACTGGGAGCAGCAGAGACATGGACCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTAACAACCAGAAATTCGAGGGCAAGGCCACATCGACTGTAGACAAATCTTCCCCAAACAGCCTGCAGTCACATCCACCAGTATTGCCTACATGGAACTCTGCATATTACTGTTCAAGTGGGATGGACTACTGGGGTCAAGGAACCTCAGTCACCCTATTGACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

59166(1.2)-VH nucleotides sequence is classified as:

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTTAGTGAAGATGTCCTGTGAGTCCAGTGGCATCACCTTTACCAGGTCCTGGGTGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGAAATTTTTCCTGGAAATAGTGATACTAGATATAACGAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCTAGTACAGCCTACATGGAGCTCCGGAGCCTGACACATGAAGACTCTGCGGTCTATTACTGTACAAAAAATGCCTACTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

58208(1.1)-VH nucleotides sequence is classified as:

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAGACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGTCTGGATAAACACCTACACTGGAGCCCCAACATTTGCTGATGACTTCAAGGGACGGTTTGCCCTGTCATTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAAATCGTATGGGTGGTATTTTGATGTGTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

58208(1.2)-VH nucleotides sequence is classified as:

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAGACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGTCTGGATAAACACCTACACTGGAGCCCCAACATTTGCTGATGACTTCAAGGGACGGTTTGCCCTGTCATTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAAATCGTATGGGTGGTATTTTGATGTGTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

58208(2.1)-VH nucleotides sequence is classified as:

CCTGAGGTGATGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTAGTTATTACATGCACTGGGTGAAGCAGAGACATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAATGGCAAAACTATCTACAACCAGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTTCAAGTGGGATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

58208(2.2)-VH nucleotides sequence is classified as:

CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAATAGTGGTAATATTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTGGGACGTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

59166(2.1)-VH nucleotides sequence is classified as:

CCTGAGGTGATGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTAGTTATTACATGCACTGGGTGAAGCAGAGACATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAATGGCAAAACTATCTACAACCAGAAATTCGAGGGCAAGGCCACATCGACTGTAGACAAATCTTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTTCAAGTGGGATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

59166(2.2)-VH nucleotides sequence is classified as:

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTTAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGAAATTAATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCTAGTACAGCCTACATGGAGCTCCGGAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTGCAAGAGGGGATGGCCTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

59166(1.1)-VH encoding amino acid sequence is:

EVQLQQSGTVLARPGTSVTHSLVITCTGSSRDMDHWVKQRPGQGLEWIGANNQKFEGKATSTVDKSSPNSLQSHPPVLPTWNSAYYCSSGMDYWGQGTSVTLLTIGAKAPLSQSP

59166(1.2)-VH encoding amino acid sequence is:

EVQLQQSGPELVKPGALVKMSCESSGITFTRSWVHWVKQSHGKSLEWIGEIFPGNSDTRYNEKFKDKATLTVDKSSSTAYMELRSLTHEDSAVYYCTKNAYFAYWGQGTLVTVSA

58208(1.1)-VH encoding amino acid sequence is:

QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS

58208(1.2)-VH encoding amino acid sequence is:

QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS

58208(2.1)-VH encoding amino acid sequence is:

PEVMKPGASVKISCKASGYSFTSYYMHWVKQRHGKSLEWIGYIDPFNGKTIYNQKFKGKATLTVDKSSSTAYMHLSSLTSEDSAVYYCSSGMDYWGQGTSVTVSS

58208(2.2)-VH encoding amino acid sequence is:

QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS

59166(2.1)-VH encoding amino acid sequence is:

PEVMKPGASVKISCKASGYSFTSYYMHWVKQRHGKSLEWIGYIDPFNGKTIYNQKFEGKATSTVDKSSSTAYMHLSSLTSEDSAVYYCSSGMDYWGQGTSVTVSS

59166(2.2)-VH encoding amino acid sequence is:

EVQLQQSGPELVKPGALVKMSCKASGYTFTDYYMHWVKQSHGKSLEWIGEINPYNGDTFYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGDGLAYWGQGTLVTVSA

59166(1.1)-VL nucleotides sequence is classified as:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAAACACCTATTTATATTGGTACTGCCAGAAACCAGGCCAGTCTCCAAAGCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT

59166(1.2)-VL nucleotides sequence is classified as:

GACATTGTGATGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGACAAGCCTCCATCTCTTGCAGATCAGGTCAGAGCATTGTACACAGAGCTGGAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGTGAGGATATGGGAGTTTATTACTGCTTTCAAGGTACACATGTTCCGCTCACGGGGGGACCAAGCTG

GAAC

58208(1.1)-VL nucleotides sequence is classified as:

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAATAGATTTTCTGGGGTCCCAGACAGGTTCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCT

58208(1.2)-VL nucleotides sequence is classified as:

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAATAGATTTTCTGGGGTCCCAGACAGGTTCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC

58208(2.1)-VL nucleotides sequence is classified as:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTTTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCGTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCT

58208(2.2)-VL nucleotides sequence is classified as:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTTTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCGTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC

59166(2.1)-VL nucleotides sequence is classified as:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACACAGTAATGGAAACACCTATTTATATTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATATGGGAGTTTATTACTGCTTTCAAGGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT

59166(2.2)-VL nucleotides sequence is classified as:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACACAGTAATGGAAACACCTATTTATATTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATATGGGAGTTTATTACTGCTTTCAAGGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC

59166(1.1)-VL encoding amino acid sequence is:

DVVMTQTPLSLPVSLGDGPPSHTGPAKVSVHLAINTYLYWYCQKPGQSPKLIYLVSNLESGVPARFSGRQWIRDRFHTQDQQSGGDAATYYCQHIRELTRSEFGAGTKLELKRA

59166(1.2)-VL encoding amino acid sequence is:

DIVMTQSPASLAVSLGQTSLHLLQIRSEHCTQSWSYMHWNQQKPGQPPRLLLIYRVSNRFSGVPDRFSVGLGQTSPSTSILWRRSEDMGVYYCFQGTHVPLTGGPSWN

58208(1.1)-VL encoding amino acid sequence is:

DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPWTFGGGTKLEIKRA

58208(1.2)-VL encoding amino acid sequence is:

DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPWTFGGGTKLEIK

58208(2.1)-VL encoding amino acid sequence is:

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKRPWTFGGGTKLEIKRA

58208(2.2)-VL encoding amino acid sequence is:

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKRPWTFGGGTKLEIK

59166(2.1)-VL encoding amino acid sequence is:

DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLYWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGVYYCFQGTHVPLTFGAGTKLELKRA

59166(2.2)-VL encoding amino acid sequence is:

DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLYWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGVYYCFQGTHVPLTFGAGTKLELK

Claims (10)

1. anti-CD138 variable region of mab sequence, it is characterized in that, comprise 16 kinds of nucleotide sequences, described nucleotide sequence is respectively 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH, 59166 (2.2)-VL.

2. anti-CD138 according to claim 1 variable region of mab sequence, it is characterized in that, prepared by the hybridoma that described nucleotide sequence 59166 (1.1)-VH and 59166 (1.1)-VL are 587CT11.3.6.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (1.2)-VH and 59166 (1.2)-VL are 587CT11.3.6.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (1.1)-VH and 58208 (1.1)-VL are 480CT5.4.3.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (1.2)-VH and 58208 (1.2)-VL are 480CT5.4.3.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (2.1)-VH and 58208 (2.1)-VL are 480CT13.4.3.2.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 58208 (2.2)-VH and 58208 (2.2)-VL are 480CT13.4.3.2.2 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (2.1)-VH and 59166 (2.1)-VL are 587CT7.3.6.5.1 by preserving number, prepared by the hybridoma that described nucleotide sequence 59166 (2.2)-VH and 59166 (2.2)-VL are 587CT7.3.6.5.2 by preserving number.

3. anti-C138 according to claim 2 variable region of mab sequence, is characterized in that, also comprises the nucleotide sequence with described 16 kinds of nucleotide sequences with same acid sequence product.

4. anti-C138 according to claim 3 variable region of mab sequence, it is characterized in that, also comprise through one or several Substitution, disappearance or after adding and still there is the nucleotide sequence that the aminoacid sequence producing with described nucleotide sequence has identical activity.

5. anti-C138 according to claim 4 variable region of mab sequence, it is characterized in that, described nucleotide sequence 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH and 59166 (2.2)-VL can be used for Dispersal risk.

6. anti-CD138 according to claim 5 variable region of mab sequence, is characterized in that, described antibody comprise following one or more: recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bi-specific antibody and single domain antibody.

7. an antibody drug, is characterized in that, comprises antibody claimed in claim 6 and pharmaceutically acceptable carrier.

8. an expression vector, it is characterized in that, comprise the nucleotide sequence described in claim 1: 59166 (1.1)-VH, 59166 (1.1)-VL, 59166 (1.2)-VH, 59166 (1.2)-VL, 58208 (1.1)-VH, 58208 (1.1)-VL, 58208 (1.2)-VH, 58208 (1.2)-VL, 58208 (2.1)-VH, 58208 (2.1)-VL, 58208 (2.2)-VH, 58208 (2.2)-VL, 59166 (2.1)-VH, 59166 (2.1)-VL, 59166 (2.2)-VH, 59166 (2.2)-VL.

9. an expressive host, is characterized in that, is loaded with expression vector claimed in claim 8.

10. a method of preparing anti-CD138 claimed in claim 1 variable region of mab sequence, is characterized in that, comprises the following steps:

(1) preparation of hybridoma: by the BALB/c mouse of myelomatosis tumour cell or the immune female Sexual health of CD138 proteantigen difference, pick out the mouse that the rear antibody expression of immunity is positive, get its spleen cell, then separated mouse spleen cell and myeloma cell are merged, form hybridoma;

(2) screening of monoclonal cell: the hybridoma in step (1) is cultivated in HAT substratum, the monoclonal cell of ELISA tests positive is carried out to ELISA to be sieved again, then filtering out ELISA positive monoclonal cell carries out WB and sieves again, again WB positive cell is carried out to subclone, through 2 subclones, filter out can stably excreting antibody monoclonal cell, will after positive monoclonal cell enlarged culturing, determine strain, frozen;

(3) preparation of anti-CD138 monoclonal antibody: the subclass hypotype of the monoclonal cell of preparation in first authentication step (2), again positive monoclonal cell is expelled to and in Mice Body, carries out ascites production, then the ascites of generation is obtained to anti-CD138 monoclonal antibody after by chromatography purification;

(4) clone of anti-CD138 variable region of mab sequence gene: total RNA of monoclonal cell used in extraction step (3), obtain mRNA, take described mRNA as template again, reverse transcription obtains cDNA, and finally clone obtains anti-CD138 monoclonal antibody variable region of heavy chain and variable region of light chain;

(5) the variable region fragment in step (4) is building up in carrier to the evaluation of checking order.