CN103173457A - Sequences of variable regions of anti-CD20 monoclonal antibody and method for preparing same - Google Patents

Abstract

The invention relates to sequences of variable regions of an anti-CD20 monoclonal antibody. The sequences comprise a nucleotide sequence 51802-VH and a nucleotide sequence 51802-VL. A method for preparing the sequences comprises the following steps of: immunizing a mouse by taking a CD20 protein as an antigen, detecting to obtain the mouse of which the corresponding antibody expression is positive, separating the spleen cell of the mouse, fusing the spleen cell with the myeloma cell, culturing the spleen cell and the myeloma cell in an HAT culture medium, detecting to obtain the positive antibody expression cloning, extracting the total RNA (ribonucleic acid) of the positive hybridoma cell, carrying out inverse transcription by taking the mRNA (messenger ribonucleic acid) in the total RNA as a template to obtain the cDNA (complementary deoxyribonucleic acid) of the gene of the corresponding antibody, obtaining the heavy-chain variable region and the light-chain variable region of the corresponding antibody by using a specific primer through PCR (polymerase chain reaction), and cloning and testing the sequences.

Description

Anti-CD-20 monoclonal antibody variable region sequences and preparation method thereof

Technical field

The present invention relates to the biological medicine technology field, more specifically, relate to anti-CD-20 monoclonal antibody variable region sequences and preparation method thereof.

Background technology

The CD20 molecule is the distinctive sign in human B lymphocyte surface, on the bone-marrow-derived lymphocyte surface, with the oligomer form, exists.The structure of CD20 is similar to some ionic channels, and each CD20 monomer is 4 cross-films, and the N end of molecule and C end are at cytoplasmic homonymy, can form polymer, CD20 can form calcium channel on the bone-marrow-derived lymphocyte film, and CD20 antibody, under the effect of BCR, can form calcium ionic current.After the B cell activation, BCR-CD20 complex dissociation, phosphorprotein, caldesmon can temporarily be enrolled into CD20, thereby participate in the conduction of intracellular signal.

In human body, all there is CD20 antigen in nearly all normal or malignant B.But there is not CD20 antigen in other hemocytes as T lymphocyte, NK cell and granulocyte etc.Therefore, CD20 antigen is a desirable target antigen of mab treatment.Now synthesize a kind of specific antibody for CD20 antigen, be called anti-CD20, be used for the treatment of the B cell non-Hodgkin's that the state of an illness is slow, aggressive is low, the efficient 30%-50% that reaches.

Human body has immune response to mouse source antibody, and this is also the major obstacle that mouse source antibody is applied to clinical treatment.By the genetic engineering antibody technology can be on gene level engineered antibody, reduce the immunogenicity of mouse source 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 the 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

As mentioned above, anti-CD20 is applied to the clinical treatment of bone-marrow-derived lymphocyte knurl, evident in efficacy, toxic side effect is little, nowadays the analysis and research that resist CD20 are more and more, based on this background, the objective of the invention is to build the nucleotide sequence of variable region of heavy chain of anti-CD-20 monoclonal antibody and the nucleotide sequence of variable region of light chain, the same method that the above-mentioned nucleotide sequence of preparation is provided.

For achieving the above object, the present invention carrys out immune mouse with CD20 albumen as antigen, then detects corresponding antibodies and expresses the mouse of being positive; get the spleen of expressing the mouse of being positive; separating Morr. cell, then merge splenocyte and myeloma cell, obtain hybridoma; After cultivating in the HAT substratum, detection obtains the antibody expression positive colony, extract the total RNA of positive hybridoma cell, the mRNA of take in total RNA is template, reverse transcription obtains the cDNA of corresponding antibodies gene, obtain corresponding antibodies variable region of heavy chain and variable region of light chain by pcr amplification again, realize thus the present invention.

The invention provides following every:

The anti-CD-20 monoclonal antibody variable region sequences, comprise two kinds of nucleotide sequences, and described nucleotide sequence is respectively 51802-VH and 51802-VL.

Preferably, the hybridoma cell strain that described nucleotide sequence 51802-VH and 51802-VL are 407CT20.1.2 by preserving number is prepared from.

Preferably, 117 amino acid of described 51802-VH coding, molecular weight is 13273D, wherein the zone of CDR1, CDR2 and CDR3 is respectively 26aa-33aa, 51aa-60aa and 99aa-106aa.

Preferably, 113 amino acid of described 51802-VL coding, molecular weight is 12297D, wherein the zone of CDR1, CDR2 and CDR3 is respectively 27aa-37aa, 55aa-57aa and 94aa-102aa.

Preferably, also comprise the nucleotide sequence that there is the same acid sequence product with above-mentioned two kinds of nucleotide sequences.

Preferably, also comprise through one or several Substitutions, disappearance or still there is the nucleotide sequence that there is identical activity with the aminoacid sequence of described nucleotide sequence generation after adding.

Prepare the method for anti-CD-20 monoclonal antibody variable region sequences, comprise the following steps:

(1) preparation of hybridoma: the BALB/c mouse of at first using the female Sexual health of CD20 proteantigen immunity, pick out the mouse that the rear antibody expression of immunity is positive, get its spleen cell, then the mouse spleen cell and the myeloma cell that separate are merged, obtain hybridoma;

(2) screening of monoclonal cell: above-mentioned hybridoma is cultivated in the HAT substratum, the monoclonal cell of ELISA tests positive is carried out to ELISA to be sieved again, then the ELISA positive monoclonal cell filtered out being carried out to WB sieves again, again the 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) acquisition of hybridoma anti-CD-20 monoclonal antibody: the subclass hypotype of the prepared monoclonal cell of authentication step (2) at first, then positive monoclonal cell is expelled in Mice Body and carries out ascites production, then the ascites of generation is obtained to anti-CD-20 monoclonal antibody after by chromatography purification;

(4) clone of anti-CD-20 monoclonal antibody heavy chain and chain variable region gene: total RNA of monoclonal cell used in extraction step (3), the mRNA of take in total RNA is template, reverse transcription obtains cDNA, and finally the clone obtains variable region of heavy chain and the variable region of light chain of anti-CD-20 monoclonal antibody.

The invention provides the anti-CD-20 monoclonal antibody variable region sequences, and the cloning and sequencing method, it can be used for for the preparation of recombinant antibodies, ScFv antibody, humanized antibody, chimeric antibody, bi-specific antibody, single domain antibody etc.; There is important value in the treatment for the preparation of the bone-marrow-derived lymphocyte knurl and diagnostic medicine.

The accompanying drawing explanation

The western blot figure of Fig. 1 .407CT20.1.2 clonal antibody

Wherein the 1-5 band means respectively 8ug/mL, 4ug/mL, 3ug/mL, 1ug/mL, 0.5ug/mL; "-" means negative serum; "+" means positive serum.

The RT-PCR product agarose gel electrophoresis map analysis result of Fig. 2 .407CT20.1.2 clonal antibody variable region VH and VL

Wherein swimming lane 1 is the VH gene, and swimming lane 2 is the VL gene, and swimming lane 3 is DL2000DNA Marker.

Fig. 3 .407CT20.1.2 clonal antibody variable region VH and VL purpose fragment are building up to enzyme on the pMD18-T carrier and cut evaluation agarose gel electrophoresis figure

Wherein swimming lane 1 is pMD18-T/VH, and swimming lane 2 is pMD18-T/VL, and swimming lane 3 is DL2000DNAMarker.

Embodiment

Below in conjunction with drawings and Examples, the present invention is described further; be understood that; these embodiment are only for illustrating the present invention; rather than limitation of the present invention; before design of the present invention under topic to preparation method's of the present invention simple modifications, the utilization of variable region nucleotide sequence of the present invention is all belonged to the scope of protection of present invention.

Preparation and the evaluation of embodiment 1. anti-CD-20 monoclonal antibody variable region sequences

1, hybridoma preparation

1.1 animal immune

BALB/c mouse with the female Sexual health in 3 of simultaneously immunity of CD20 proteantigen 6-8 age in week, after 3 immunity, within 7 days, take a blood sample and survey tiring of Serum Antibody, while selecting the 1:4000 dilution, ELISA detects the serum that the OD value is greater than 1.0, the BALB/c mouse of serum WB tests positive is for merging simultaneously, merge before 3 days, with the antigen abdominal cavity booster immunization injection that does not add adjuvant, injected dose is 50ug/.

1.2 collection bone-marrow-derived lymphocyte

After supplementary immunization 3 days, mouse was plucked eyeball and gets blood, and centrifugal rear acquisition serum is made positive control; Get mouse spleen under sterile state, and spleen is placed in the incomplete substratum of the pre-temperature of 10mL, remove reticular tissue, be placed in 100 order stainless (steel) wires, grind with the inner core of syringe, grinding limit, limit drips incomplete substratum and rinses; Cell suspension after collect filtering, in centrifuge tube, is abandoned supernatant liquor after centrifugal, and the full substratum suspension cell that then toos many or too much for use, get 1 * 10 ⁸individual cell, at room temperature place stand-by.

1.3 the preparation of murine myeloma cell

Merge first 10 days, the myeloma cell is taken out from liquid nitrogen, put into rapidly 37 ℃ of water-baths, in 1min, make frozen storing liquid dissolve fully; Centrifugal, abandon supernatant liquor, put in the IMDM perfect medium, 37 ℃, 5%CO ₂the cell enlarged culturing; 2-3 days before merging, cell should be in logarithmic phase.Before merging, the logarithmic phase murine myeloma cell is collected in centrifuge tube, counting, get 2 * 10 ⁷-3 * 10 ⁷individual cell, the centrifugal supernatant liquor of abandoning, the full substratum suspension cell that toos many or too much for use, put room temperature stand-by.

1.4 cytogamy

50%PEG is put to 37 ℃, 5%CO before merging ₂adjust temperature in cell culture incubator, 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, add the incomplete substratum of 30mL, centrifugal 10min under the condition of rotating speed 1200rpm, abandoning supernatant, gently at the bottom of the attack pipe, make cell mass loose, rotate equably centrifuge tube on one side, draw the PEG fusogen of the pre-temperature of 1mL on one side with the 1mL valinche, in during the about 2cm in end place slowly adds cell along tube wall from pipe of centrifuge tube, the limit edged rotates centrifuge tube, be controlled at 60s from joining the time added, jog centrifuge tube 30s, standing 60s, then add immediately the incomplete nutrient solution of 20mL, make the PEG dilution and lose the short effect of melting, concrete addition is within first minute, to add 1mL, within second minute, add 4mL, within subsequently three minutes, remaining liq is added, standing 10min in 37 ℃ of water-baths, 1200rpm, 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.

1.5 the screening of monoclonal cell

Cell is cultivated in the HAT substratum, and the myeloma cell of not merging and the lymphocyte do not merged are dead gradually; The hybridoma merged can be survived and breed in the HAT substratum.At 37 ℃, 5%CO ₂cultivate 7-10 days in cell culture incubator; 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 sealed, and hatches 1h for 37 ℃; After washing 5 times, add the sheep anti-mouse igg of HRP mark, hatch 1h for 37 ℃; Add the TMB nitrite ion after washing, then add 2M sulfuric acid termination reaction, carry out reading on microplate reader; ELISA is detected to positive clone to be chosen into 24 porocyte culture plates cultivations, carrying out ELISA after 3 days sieves again, the ELISA positive colony filtered out is carried out to WB to be sieved again, the 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.

2, the acquisition of hybridoma anti-CD-20 monoclonal antibody and biological assay

2.1 the acquisition of hybridoma anti-CD-20 monoclonal antibody

The cell conditioned medium liquid of determining the monoclonal cell of strain is identified to the subclass hypotype of monoclonal antibody with U.S. company BD Mouse Monoclonal Antibody Isotyping Kit, then positive monoclonal cell is expelled in Mice Body and carries out ascites production, the ascites of production obtains antibody after by chromatography purification.

2.2Western blot detects the specificity of antibody

Get processed cell pyrolysis liquid, carry out vertical SDS-PAGE on gel, 120v90min, electrophoresis takes off gel after finishing, and 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 the 3mL confining liquid, and 4 ℃ are spent the night; With washings washing 3 times, each 5min, dilute the sheep anti-mouse igg of HRP mark with confining liquid, under room temperature, hatch 2h; With washings washing 3 times, chemical illuminating reagent is added on pvdf membrane again, is exposed on the X film to darkroom, by developing and photographic fixing, result is reflected on film; Scan film, analytical results, its result is with reference to Fig. 1.

2.3 the monoclonal antibody affinity costant is measured

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

3, the clone of anti-CD-20 monoclonal antibody heavy chain and chain variable region gene

Cell strain used is the hybridoma cell strain of the anti-CD20 antibodies that can secrete high-affinity, high specific of employing aforesaid method acquisition, and corresponding deposit number is 407CT20.1.2, the antibody molecule hypotype IgG1 of its secretion.The 407CT20.1.2 hybridoma of taking the logarithm vegetative period, test kit RNeasy MiniKit(article No. with QIAGEN company: QIAGEN-74106) extract total RNA, take a morsel and quantitatively reach 1% non-sex change agarose gel electrophoresis detection with nanodrop, use subsequently SuperScript.III First-Strand Synthesis System for RT-PCR test kit (article No.: Invitrogen-18080-051) reverse transcription cDNA, with light chain or the variable region of heavy chain of special primer amplification anti-CD20 antibodies gene.To contain the PCR reaction product of corresponding variable region of heavy chain or variable region of light chain fragment through 1% agarose gel electrophoresis, and cut glue and separate the purpose fragment.After reclaiming test kit (JaRa-GK2042) purifying purpose product by glue, 1% agarose gel electrophoresis is identified the purity of purpose 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 rear order-checking, carry out homology and structural analysis to sequencing result.

The relating operation concrete steps are as follows:

3.1RT-PCR amplification CD20 light chain of antibody and variable region of heavy chain

3.1.1 design of primers

According to the sequence of IgG variable region, be used for increasing the CD20 antibody heavy chain variable region at signaling zone and synthetic 5 ' and 3 ' the end primer of constant region, primer sequence is as follows:

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

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

According to the sequence of IgG variable region, be used for increasing the CD20 antibody chain variable region at signaling zone and synthetic 5 ' and 3 ' the end primer of constant region, primer sequence is as follows:

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

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

3.1.2 the total RNA of hybridoma extracts

Extract total RNA with above-mentioned hybridoma 407CT20.1.2, concrete steps are as follows:

Collect logarithmic phase hybridoma 407CT20.1.2, each are 2 * 10 years old ⁶individual, the centrifugal 5min of 800rpm, remove supernatant, and the cell precipitated can be stored in-80 ℃, or be directly used in the RNA extraction;

Every part of cell sample (cell count approximately 2 * 10 ⁶individual) in add 350 μ L RTL solution, vortex 30s;

Add 70% ethanol in above every system, blow and beat gently evenly with the 1mL liquid-transfering gun;

Above system mixed solution is proceeded to RNeasy spin column, and the centrifugal 15s of 8000x g, go filtrate;

Respectively add 700 μ L RW1 solution in above RNeasy spin column, the centrifugal 15s of 8000x g, go filtrate;

Respectively add 500 μ L RPE solution in above RNeasy spin column, the centrifugal 15s of 8000x g, go filtrate;

Respectively add 500 μ L RPE solution in above RNeasy spin column, the centrifugal 2min of 8000x g, go filtrate;

RNeasy spin column is changed in the 2mL collection tube without the RNA enzyme, at full speed centrifugal 1min;

RNeasy spin column is changed in the 1.5mLEP pipe without the RNA enzyme, add the H of 30-50 μ L without the RNA enzyme ₂o, the centrifugal 1min of 8000x g, eluted rna.

3.1.3 reverse transcription PCR

The total RNA of hybridoma 407CT20.1.2 of take is template, with the synthetic SuperScript.III test kit (article No. 18080-051) of RT-PCR the first chain of Invitrgen, and reverse transcription cDNA, concrete steps are as follows:

(1) primer and template sex change

Press shown in table 1, be made into 10 μ L systems, 65 ℃, 5min, place 1min subsequently on ice, is beneficial to primer and is combined with the RNA template.

Table 1

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

(2) renaturation

Press shown in table 2, be made into 10 μ L systems, add the system of primer and the sex change of RNA template, 50 ℃, 50min; 85 ℃, 5min.

Table 2

Composition	Volume (μ L)
		10×RT?buffer	2
25mM?MgCl2	4
		0.1M?DTT	2
RNase?OUT	1
		SuperScriptIII?RT	1

(3) RNA digestion

Add 1 μ L RNaseH in above every individual system, 37 ℃, 20min.

3.1.4 antibody variable region specific primer PCR

Press shown in table 3 and table 4, 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 3

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

Table 4

4. the order-checking of anti-CD-20 monoclonal antibody heavy chain and chain variable region gene

4.1PCR product cloning, 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 to respectively on sequencing vector after reclaiming, and send order-checking.Concrete steps are as follows:

(1) glue reclaims the PCR product

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

Every 100mg sepharose adds 400 μ L in conjunction with liquid B, is placed to gel piece in 60 ℃ of water-baths and melts fully;

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, remove waste liquid;

Add 500 μ L washingss, 12,000rpm, the centrifugal 1min of room temperature, remove waste liquid;

Repeat previous step;

The GenClean post is put back to collection tube, 12,000rpm, the centrifugal 1min of room temperature;

The GenClean post is put to the pipe to 1.5mLEP, add 30-50 μ L elutriant, place 2min for 37 ℃, 12,000rpm, the centrifugal 1min of room temperature is with wash-out purpose fragment.

(2) enzyme is cut

Enzyme is cut system as shown in table 5 and table 6:

Table 5

Composition	Volume (μ L)
		Plasmid	X(1ug)
10×FD?buffer	5
		Enzyme 1	1
Enzyme 2	1
		H 2O	43-X

Table 6

Composition	Volume (μ L)
		Antibody purpose fragment	17
10×FD?buffer	2
		Enzyme 1	0.5
Enzyme 2	0.5

The enzyme tangent condition: 37 ℃, 30min; 85 ℃, 5min;

Run glue and reclaim long segment in the plasmid enzyme restriction system, the antibody purpose fragment enzyme system of cutting is directly used in connection.

(3) connect

Linked system is as shown in table 7:

Table 7

Composition	Volume (μ L)
		Carrier glue reclaims large fragment	1.5
Antibody purpose fragment	7
		10×ligase?buffer	1
ligase	0.5

Condition of contact: 16 ℃, 4h.

(4) transform

Add 100 μ L DH5 α competent cells, ice bath 30min to linked system;

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

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

The centrifugal 2min of 4000rpm, remove supernatant, stays 100 μ L supernatant re-suspended cell precipitations, is coated with corresponding resistant LB flat board.After liquid-absorbent in plate, be inverted plate, cultivate 12-16h in 370C and bacterium colony can occur.

(5) bacterium colony PCR identifies

Choose on every reformer plate mono-clonal 4-6, point, after corresponding resistant LB flat board, is bacterium colony PCR and is identified, the PCR reaction product is run 1% sepharose and detected and have or not purpose length fragment, and its result is with reference to Fig. 2.

Bacterium colony PCR system is as shown in table 8 and table 9:

Table 8

Composition	Volume (μ L)
		H 2O	15.8
10×pfu?buffer(Mg 2+)	2
		dNTP(10mM)	1
Taq	0.2
		Sense?primer(20uM)	0.5
Anti-sense?primer(20uM)	0.5
		The clone	1

Table 9

(6) upgrading grain

Choose bacterium colony PCR and be accredited as positive mono-clonal and shake bacterium, after 20% glycerine is protected 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, collect thalline, sucks as far as possible supernatant;

Add 250 μ L BufferA1 (having added RNase A), vortex shakes abundant suspension bacterial cell;

Add 250 μ L BufferB1, reverse gently 10 times to mix, standing 5min clarifies to the solution thickness;

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

Centrifuge tube is gone to supercentrifuge, at room temperature 13, if adularescent precipitation in the centrifugal 10min(supernatant of 000rpm, but recentrifuge);

Careful draw supernatant liquor after centrifugal to (avoiding picking up precipitation) in the centrifugal column with collection tube, under room temperature 13, the centrifugal 1min of 000rpm, outwell the waste liquid in collection tube, and centrifugal column is relay in the recovery collector;

Add 500 μ L Buffer KB in the DNA post, centrifugal 1min under room temperature, outwell the waste liquid in collection tube, and centrifugal column is relay and reclaims in collector;

Add 500 μ L DNA washingss (having added dehydrated alcohol) in centrifugal column, under room temperature, the centrifugal 1min of 13,000rpm, outwell waste liquid in collection tube, and centrifugal column is relay and gets back in collection tube, repeats this step once;

Centrifugal column is put back in supercentrifuge, and the centrifugal 5-10min that uncaps under 13,000rpm room temperature, thoroughly to remove residual ethanol.

Centrifugal column is transferred in a new 1.5mL centrifuge tube, adds the elutriant of 50 μ L60 ℃ preheatings 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 the respective limits endonuclease digestion, and system is as table 10:

Table 10

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

The enzyme tangent condition: 37 ℃, 30min; 85 ℃, 5min, run 1% agarose gel electrophoresis and identify whether the purpose band is arranged.

(8) plasmid PCR is identified

Enzyme cut identify positive plasmid again PCR identify whether the purpose band is arranged, reaction system is as shown in table 11 and table 12:

Table 11

Composition	Volume (μ L)
		H 2O	14.8
10×pfu?buffer(Mg 2+)	2
		dNTP(10mM)	1
taq	0.2
		Sense?primer(20uM)	0.5
Ant?i-sense?primer(20uM)	0.5
		Enzyme is cut the evaluation positive plasmid	1

Table 12

The PCR product runs 1% agarose gel electrophoresis, if positive band is arranged, send order-checking.

4.2 order-checking comparison and structural analysis

The 51802-VH chain is analyzed and is shown by FR-IGMT and CDR-IGMT:

CDR1：GGATTCACCTTCATTACCTACGCC

CDR2：ATAAGAAGTAAAAATAATAATTTTGCAACA

CDR3：GTGAGGGGGTCCTCCTTTGACTTC

Belong to IgH-V10VH10 family

The 51802-VL chain is analyzed and is shown by FR-IGMT and CDR-IGMT:

CDR1：CAGAGTCTCCTAGATGGCAAAGGCATCACTTAT

CDR2：CAGATGTCC

CDR3：GCTCAAAATCTAGAACTTCCGTATACG

Belong to the IGKV24/25 subgroup

51802-VH 117 amino acid of encoding, wherein the zone of CDR1, CDR2 and CDR3 is respectively 26aa-33aa, 51aa-60aa and 99aa-106aa, molecular weight is about 13273D.

51802-VL 113 amino acid of encoding, wherein the zone of CDR1, CDR2 and CDR3 is respectively 27aa-37aa, 55aa-57aa and 94aa-102aa, molecular weight is about 12297D.

51802-VH Nucleotide is:

GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTAGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCATTACCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAGAAATTTGGAATGGGTTGCTCGCATAAGAAGTAAAAATAATAATTTTGCAACATATTATGCCGATTCAGTGAGAGACAGGTTCACCATCTTCAGAGATGATTCACAAAACCTACTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGGGGGTCCTCCTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

The aminoacid sequence of 51802-VH coding is:

EVQLVESGGGLVQPRGSLKLSCAASGFTFITYAMNWVRQAPGRNLEWVARIRSKNNNFA

TYYADSVRDRFTIFRDDSQNLLYLQMNNLKTEDTAMYYCVRGSSFDFWGQGTTLTVSS

The 51802-VL nucleotides sequence is classified as:

GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAGCGTCAGCTTCCATCTCCTGCAGGTCTAGTCAGAGTCTCCTAGATGGCAAAGGCATCACTTATTTCAATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGTCTCAGGAGTCCCAAACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTACTACTGTGCTCAAAATCTAGAACTTCCGTATACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT

The aminoacid sequence of 51802-VL coding is:

DIVMTQAAFSNPVTLGASASISCRSSQSLLDGKGITYFNWYLQKPGQSPQLLIYQMSNLV

SGVPNRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTKLEIKR

 

 

Claims (7)

1. the anti-CD-20 monoclonal antibody variable region sequences, is characterized in that, comprises two kinds of nucleotide sequences, and described nucleotide sequence is respectively 51802-VH and 51802-VL.

2. anti-CD-20 monoclonal antibody variable region sequences according to claim 1, is characterized in that, the hybridoma cell strain that described nucleotide sequence 51802-VH and 51802-VL are 407CT20.1.2 by preserving number is prepared from.

3. anti-CD-20 monoclonal antibody variable region sequences according to claim 1, it is characterized in that, 117 amino acid of described 51802-VH coding, molecular weight is 13273D, wherein the zone of CDR1, CDR2 and CDR3 correspondingly is respectively 26aa-33aa, 51aa-60aa and 99aa-106aa.

4. anti-CD-20 monoclonal antibody variable region sequences according to claim 1, its feature is being, 113 amino acid of described 51802-VL coding, molecular weight is 12297D, wherein the zone of CDR1, CDR2 and CDR3 correspondingly is respectively 27aa-37aa, 55aa-57aa and 94aa-102aa.

5. anti-CD-20 monoclonal antibody variable region sequences according to claim 1, is characterized in that, also comprises: with above-mentioned two kinds of nucleotide sequences, have the nucleotide sequence of same acid sequence product.

6. anti-CD-20 monoclonal antibody variable region sequences according to claim 5, it is characterized in that, also comprise: still there is the nucleotide sequence that there is identical activity with the aminoacid sequence of described nucleotide sequence generation through one or several Substitutions, disappearance or after adding.

7. prepare the method for the described anti-CD-20 monoclonal antibody variable region sequences of claim 1 to 6 any one, it is characterized in that, comprise the following steps:

(1) preparation of hybridoma: the BALB/c mouse of at first using the female Sexual health of CD20 proteantigen immunity, pick out the mouse that the rear antibody expression of immunity is positive, get its spleen cell, then the mouse spleen cell and the myeloma cell that separate are merged, obtain hybridoma;

(2) screening of monoclonal cell: above-mentioned hybridoma is cultivated in the HAT substratum, the monoclonal cell of ELISA tests positive is carried out to ELISA to be sieved again, then the ELISA positive monoclonal cell filtered out being carried out to WB sieves again, again the 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) acquisition of hybridoma anti-CD-20 monoclonal antibody: the subclass hypotype of the prepared monoclonal cell of authentication step (2) at first, then positive monoclonal cell is expelled in Mice Body and carries out ascites production, then the ascites of generation is obtained to anti-CD-20 monoclonal antibody after by chromatography purification;

(4) clone of anti-CD-20 monoclonal antibody heavy chain and chain variable region gene: total RNA of monoclonal cell used in extraction step (3), the mRNA of take in total RNA is template, reverse transcription obtains cDNA, and finally the clone obtains variable region of heavy chain and the variable region of light chain of anti-CD-20 monoclonal antibody.

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