CN103173457B

CN103173457B - Sequences of variable regions of anti-CD20 monoclonal antibody and method for preparing same

Info

Publication number: CN103173457B
Application number: CN201310066338.2A
Authority: CN
Inventors: 吴纯; 李静文; 邹建炫; 汪伟; 杨春花; 周延庆; 李顺玲; 孙其玲; 洪扬; 陈媛
Original assignee: Abgent Biotechnology Suzhou Co Ltd
Current assignee: Wuxi Apptec Suzhou Co Ltd
Priority date: 2013-03-04
Filing date: 2013-03-04
Publication date: 2015-01-28
Anticipated expiration: 2033-03-04
Also published as: CN103173457A

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 biomedicine technical field, more specifically, relate to anti-CD-20 monoclonal antibody variable region sequences and preparation method thereof.

Background technology

CD20 molecule is the distinctive mark in human B lymphocyte surface, exists with oligomeric forms on bone-marrow-derived lymphocyte surface.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, polymer can be formed, CD20 can form calcium channel on bone-marrow-derived lymphocyte film, and CD20 antibody, under the effect of BCR, can form calcium ionic current.After B cell activation, BCR-CD20 complex dissociation, phosphorprotein, caldesmon temporarily can be enrolled into CD20, thus participate in the conduction of intracellular signal.

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

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 genetic engineering antibody technology can on gene level engineered antibody, reduce the immunogenicity of mouse source antibody, improve antibodies specific, stability and avidity.Carry out humanization modified to mouse source antibody, antibody variable region and human antibody constant domain can be retained, improve affinity of antibody; Or engineered antibody structure, build the scFv that only retains antibody variable region or the Fab containing antibody variable region and portion constant district, antibody percent absorption in vivo and transformation period can be improved.

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 resisting CD20 get more and more, based on this background, the object 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 samely provide the method preparing above-mentioned nucleotide sequence.

For achieving the above object, the present invention CD20 albumen carrys out immune mouse as antigen, then detects that corresponding antibodies expresses the mouse of being positive; get the spleen of expressing mouse of being positive; separating Morr. cell, then merges splenocyte and myeloma cell, obtains hybridoma; After cultivating in HAT substratum, detection obtains antibody expression positive colony, extract positive hybridoma cell total serum IgE, with the mRNA in total serum IgE for 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 the present invention thus.

The invention provides following every:

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

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

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

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

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

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

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

(1) preparation of hybridoma: the BALB/c mouse first using CD20 proteantigen immunization. Female health, the mouse that after picking out immunity, antibody expression is positive, get its spleen cell, then by the Mouse spleen cells of separation and myeloma cell fusion, obtain hybridoma;

(2) screening of monoclonal cell: above-mentioned hybridoma is cultivated in HAT substratum, carry out ELISA to the monoclonal cell of ELISA tests positive to sieve again, then the ELISA positive monoclonal cell filtered out is carried out WB to sieve again, again WB positive cell is carried out subclone, through 2 subclones, filtering out can the monoclonal cell of stably excreting antibody, will determine strain, frozen after the monoclonal cell enlarged culturing of the positive;

(3) acquisition of hybridoma anti-CD-20 monoclonal antibody: the subclass hypotype of the monoclonal cell first prepared by authentication step (2), then the monoclonal cell of the positive is expelled in Mice Body and carries out ascites production, then by the ascites of generation by obtaining anti-CD-20 monoclonal antibody after chromatography purification;

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

The invention provides anti-CD-20 monoclonal antibody variable region sequences, and 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.; In for the preparation of the treatment of bone-marrow-derived lymphocyte knurl and diagnostic medicine, there is important value.

Accompanying drawing explanation

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

Wherein 1-5 band represents 8ug/mL respectively, 4ug/mL, 3ug/mL, 1ug/mL, 0.5ug/mL; "-" represents negative serum; "+" represents 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 VH gene, and swimming lane 2 is VL gene, and swimming lane 3 is DL2000DNA Marker.

Fig. 3 .407CT20.1.2 clonal antibody variable region VH and VL object fragment are building up to enzyme on pMD18-T carrier and cut qualification 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 embodiments are only for illustration of the present invention; instead of limitation of the present invention; simple modifications to preparation method of the present invention under topic before design of the present invention, all belongs to the scope of protection of present invention to the utilization of variable region nucleotide sequence of the present invention.

The preparation of embodiment 1. anti-CD-20 monoclonal antibody variable region sequences and qualification

1, hybridoma preparation

1.1 animal immune

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

1.2 collect bone-marrow-derived lymphocyte

After supplementary immunization 3 days, mouse was plucked eyeball and gets blood, and positive control made by centrifugal rear acquisition serum; Get mouse spleen under sterile state, and be placed on by spleen 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; Collect the cell suspension after filtering in centrifuge tube, abandon supernatant liquor after centrifugal, then cannot be used up full substratum suspension cell, gets 1 × 10 ⁸individual cell, at room temperature places stand-by.

The preparation of 1.3 murine myeloma cells

Merge first 10 days, myeloma cell is taken out from liquid nitrogen, put into rapidly 37 DEG C of water-baths, in 1min, make frozen storing liquid dissolve completely; Centrifugal, abandon supernatant liquor, put in IMDM perfect medium, 37 DEG C, 5%CO ₂cell expansion is cultivated; 2-3 days before merging, cell should be in logarithmic phase.Collect in centrifuge tube by logarithmic phase murine myeloma cell before merging, counting, gets 2 × 10 ⁷-3 × 10 ⁷individual cell, centrifugally abandons supernatant liquor, and cannot be used up full substratum suspension cell, and it is stand-by to put room temperature.

1.4 cytogamy

50%PEG is put 37 DEG C, 5%CO before merging ₂temperature is adjusted, by 2 × 10 in cell culture incubator 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 attack pipe, make cell mass loose, rotate centrifuge tube equably on one side, while draw the PEG fusogen of the pre-temperature of 1mL with 1mL valinche, slowly add in cell along tube wall at the 2cm place that is about at the bottom of pipe of centrifuge tube, limit edged rotates centrifuge tube, from joining the time controling that adds at 60s, jog centrifuge tube 30s, leave standstill 60s, then the incomplete nutrient solution of 20mL is added immediately, PEG is diluted and loses and short melt effect, concrete addition adds 1mL in first minute, within second minute, add 4mL, within subsequently three minutes, remaining liq is added, 10min is left standstill in 37 DEG C of water-baths, 1200rpm, centrifugal 10min, supernatant discarded, add the perfect medium containing HAT, make cell suspension, be taped against on 96 porocyte culture plates, put 37 DEG C, 5%CO ₂in cell culture incubator.

The screening of 1.5 monoclonal cells

Cell is cultivated in HAT substratum, the myeloma cell of not merging and the lymphocyte do not merged dead gradually; The hybridoma merged can be survived and breed in HAT substratum.At 37 DEG C, 5%CO ₂7-10 days is cultivated in cell culture incubator; Use antigen coated enzyme plate, 37 DEG C of bags, by 2h, are then closed with 2%BSA; Drawing the cell conditioned medium growing clone in 96 orifice plates is added in the enzyme plate closed, and hatches 1h for 37 DEG C; After washing 5 times, add the sheep anti-mouse igg of HRP mark, hatch 1h for 37 DEG C; Add TMB nitrite ion after washing, then add 2M sulfuric acid termination reaction, microplate reader carries out reading; ELISA being detected positive clone chooses into 24 porocyte culture plates cultivations, carry out ELISA after 3 days to sieve again, the ELISA positive colony filtered out is carried out WB sieve again, WB positive cell carries out subclone, through 2 subclones, filtering out can the monoclonal cell of stably excreting antibody, will determine strain, frozen after the mono-clonal enlarged culturing of the positive.

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

The acquisition of 2.1 hybridoma anti-CD-20 monoclonal antibodies

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

2.2Western blot detects the specificity of antibody

Get processed cell pyrolysis liquid, gel carries out vertical SDS-PAGE, 120v90min, after electrophoresis terminates, take off gel, be placed on pvdf membrane, albumen semidrying is transfected on pvdf membrane, 10v, 120min; Pvdf membrane after transfer printing room temperature in 5% skim-milk is closed 2h; Be dissolved in by antibody in 3mL confining liquid, 4 DEG C are spent the night; Wash 3 times with washings, each 5min, dilute the sheep anti-mouse igg of HRP mark with confining liquid, incubated at room temperature 2h; Wash 3 times with washings again, chemical illuminating reagent be added on pvdf membrane, be exposed to darkroom on X film, by development and fixing, result is reflected on film; Scan film, analytical results, its result is with reference to Fig. 1.

2.3 monoclonal antibody affinity costants measure

With coating buffer by antigen diluent, add enzyme plate respectively, 100 microlitres/hole, 37 DEG C of 2h, PBS wash 5 times, pat dry, and add 2%BSA confining liquid 200 microlitres/hole, and 4 DEG C are spent the night, and wash 5 times, pat dry; Join bag after antibody is carried out gradient dilution from 4ug/mL by good enzyme plate, 100 microlitres/hole, 37 DEG C of 1h, wash 5 times, pat dry; Add the goat anti-mouse igg antibody of HRP mark, working fluid 100 microlitres/hole, 37 DEG C of 1h, wash 5 times, pat dry; Add TMB nitrite ion, 37 DEG C of reaction 5-10min, add stop buffer 50 microlitres/hole, measure OD value immediately, go out monoclonal antibody affinity costant according to formulae discovery in microplate reader with 450nm wavelength.

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 anti-CD20 antibodies secreting high-affinity, high specific adopting aforesaid method to obtain, 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 serum IgE, take a morsel and 1% non denatured agarose gel electrophoresis detection quantitative with nanodrop, use SuperScript.III First-Strand Synthesis System for RT-PCR kit (article No.: Invitrogen-18080-051) reverse transcription cDNA subsequently, with light chain or the variable region of heavy chain of specific primers amplify anti-CD20 antibodies gene.By the PCR reaction product containing corresponding variable region of heavy chain or variable region of light chain fragment through 1% agarose gel electrophoresis, cut glue and be separated object fragment.After reclaiming test kit (JaRa-GK2042) purifying object product by glue, the purity of 1% agarose gel electrophoresis qualification object fragment; Afterwards, the corresponding variable region of heavy chain obtain recovery and variable region of light chain are cloned into sequencing vector pMD18-T, rear order-checking, carry out homology and structural analysis to sequencing result.

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, hold primer for CD20 antibody heavy chain variable region of increasing in signaling zone and constant region synthesis 5 ' and 3 ', primer sequence is as follows:

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

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

According to the sequence of IgG variable region, hold primer for CD20 antibody chain variable region of increasing in signaling zone and constant region synthesis 5 ' and 3 ', primer sequence is as follows:

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

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

3.1.2 hybridoma Total RNAs extraction

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

Collect logarithmic phase hybridoma 407CT20.1.2, each 2 × 10 ⁶individual, the centrifugal 5min of 800rpm, removes supernatant, and the cell precipitated can be stored in-80 DEG C, or is directly used in RNA extraction;

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

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

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

In above RNeasy spin column, respectively add 700 μ L RW1 solution, the centrifugal 15s of 8000x g, goes filtrate;

In above RNeasy spin column, respectively add 500 μ L RPE solution, the centrifugal 15s of 8000x g, goes filtrate;

In above RNeasy spin column, respectively add 500 μ L RPE solution, the centrifugal 2min of 8000x g, goes filtrate;

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

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

3.1.3 reverse transcription PCR

With hybridoma 407CT20.1.2 total serum IgE for template, with RT-PCR first chain synthesis SuperScript.III test kit (article No. 18080-051) of Invitrgen, reverse transcription cDNA, concrete steps are as follows:

(1) primer and template denaturation

By shown in table 1, be made into 10 μ L systems, 65 DEG C, 5min, places 1min on ice subsequently, is beneficial to primer and is combined with 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

By shown in table 2, be made into 10 μ L systems, add the system of primer and RNA template denaturation, 50 DEG C, 50min; 85 DEG C, 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

1 μ L RNaseH is added, 37 DEG C, 20min in above every individual system.

3.1.4 antibody variable region specific primer PCR

Shown in table 3 and table 4, the cDNA obtained with reverse transcription 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

After the variable region of heavy chain obtained by PCR and variable region of light chain nucleotide fragments product glue reclaim, be building up to respectively on sequencing vector, send order-checking.Concrete steps are as follows:

(1) glue reclaims PCR primer

After PCR primer runs 1% agarose gel electrophoresis, 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 400 μ L in conjunction with liquid B, is placed to gel piece and melts completely in 60 DEG C of water-baths;

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

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

Repeat previous step;

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

Put by GenClean post to 1.5mLEP pipe, add 30-50 μ L elutriant, place 2min, 12,000rpm for 37 DEG C, the centrifugal 1min of room temperature is with wash-out object fragment.

(2) enzyme is cut

Enzyme cuts 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 object fragment	17
10×FD?buffer	2
		Enzyme 1	0.5
Enzyme 2	0.5

Enzyme tangent condition: 37 DEG C, 30min; 85 DEG C, 5min;

Run glue and reclaim long segment in plasmid enzyme restriction system, the antibody object 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 object fragment	7
		10×ligase?buffer	1
ligase	0.5

Condition of contact: 16 DEG C, 4h.

(4) transform

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

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

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

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

(5) bacterium colony PCR identifies

Choose mono-clonal 4-6 in every block reformer plate individual, after point to corresponding resistant LB flat board, be bacterium colony PCR and identify, PCR reaction product is run 1% sepharose and is detected with or without object long 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
		Clone	1

Table 9

(6) upgrading grain

Choose bacterium colony PCR to be accredited as positive mono-clonal and to shake bacterium, after bacterium protected by 20% glycerine, 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, suck supernatant as far as possible;

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

Add 250 μ L BufferB1, reverse 10 times gently to mix, leave standstill 5min and clarify to solution thickness;

Add 350 μ L Buffer N1, reverse immediately repeatedly, fully mix to solution, now occur white flock precipitate;

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

Supernatant liquor after careful absorption is centrifugal is to (avoiding picking up precipitation) with in the centrifugal column of collection tube, and under room temperature, 13,000rpm centrifugal 1min, outwell the waste liquid in collection tube, are placed back in by centrifugal column in collection tube;

In DNA post, add 500 μ L Buffer KB, centrifugal 1min under room temperature, outwells the waste liquid in collection tube, is placed back in by centrifugal column in collection tube;

In centrifugal column, add 500 μ L DNA washings (adding dehydrated alcohol), under room temperature, the centrifugal 1min of 13,000rpm, outwells waste liquid in collection tube, is placed back in by centrifugal column in collection tube, repeats this step once;

Put back to by centrifugal column in supercentrifuge, uncap under 13,000rpm room temperature centrifugal 5-10min, thoroughly to remove residual ethanol.

Be transferred to by centrifugal column in a new 1.5mL centrifuge tube, the middle to DNA post adds the elutriant of 50 μ L60 DEG C preheatings, and room temperature places the centrifugal 1min of 2min, 13000rpm, wash-out plasmid DNA.

(7) enzyme cuts qualification

Identified by the corresponding digestion with restriction enzyme of the plasmid DNA of extraction, 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

Enzyme tangent condition: 37 DEG C, 30min; 85 DEG C, whether 5min, running 1% agarose gel electrophoresis qualification has object band.

(8) plasmid PCR qualification

Enzyme cut the positive plasmid of qualification again PCR qualification whether have object band, 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 cuts qualification positive plasmid	1

Table 12

PCR primer runs 1% agarose gel electrophoresis, if there is positive band, then send order-checking.

4.2 order-checking comparison and structural analyses

51802-VH chain analyzes display by FR-IGMT and CDR-IGMT:

CDR1：GGATTCACCTTCATTACCTACGCC

CDR2：ATAAGAAGTAAAAATAATAATTTTGCAACA

CDR3：GTGAGGGGGTCCTCCTTTGACTTC

Belong to IgH-V10VH10 family

51802-VL chain analyzes display by FR-IGMT and CDR-IGMT:

CDR1：CAGAGTCTCCTAGATGGCAAAGGCATCACTTAT

CDR2：CAGATGTCC

CDR3：GCTCAAAATCTAGAACTTCCGTATACG

Belong to IGKV24/25 subgroup

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

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

51802-VH Nucleotide is:

GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTAGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCATTACCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAGAAATTTGGAATGGGTTGCTCGCATAAGAAGTAAAAATAATAATTTTGCAACATATTATGCCGATTCAGTGAGAGACAGGTTCACCATCTTCAGAGATGATTCACAAAACCTACTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGGGGGTCCTCCTTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

The aminoacid sequence of 51802-VH coding is:

EVQLVESGGGLVQPRGSLKLSCAASGFTFITYAMNWVRQAPGRNLEWVARIRSKNNNFA

TYYADSVRDRFTIFRDDSQNLLYLQMNNLKTEDTAMYYCVRGSSFDFWGQGTTLTVSS

51802-VL nucleotides sequence is classified as:

GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAGCGTCAGCTTCCATCTCCTGCAGGTCTAGTCAGAGTCTCCTAGATGGCAAAGGCATCACTTATTTCAATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGTCTCAGGAGTCCCAAACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTACTACTGTGCTCAAAATCTAGAACTTCCGTATACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT

The aminoacid sequence of 51802-VL coding is:

DIVMTQAAFSNPVTLGASASISCRSSQSLLDGKGITYFNWYLQKPGQSPQLLIYQMSNLV

SGVPNRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTKLEIKR

Claims

1. the encoding sequence of anti-CD-20 monoclonal antibody variable region sequences, is characterized in that, comprise two kinds of nucleotide sequences, described nucleotide sequence is respectively 51802-VH and 51802-VL; Described 51802-VH encodes 117 amino acid, and molecular weight is 13273D, and wherein the region of CDR1, CDR2 and CDR3 is correspondingly respectively 26aa-33aa, 51aa-60aa and 99aa-106aa; Described 51802-VL encodes 113 amino acid, and molecular weight is 12297D, and wherein the region of CDR1, CDR2 and CDR3 is correspondingly respectively 27aa-37aa, 55aa-57aa and 94aa-102aa.

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