CN100999732A

CN100999732A - Process of producing anti-humen CD20 monoclone antibody by animal mammary gland

Info

Publication number: CN100999732A
Application number: CN 200610172293
Authority: CN
Inventors: 李宁; 汤波; 余姗姗
Original assignee: BEIJING JIPULIN BIOTECHNOLOGY Co Ltd
Current assignee: Beijing Jifulin Biotechnology Co., Ltd.
Priority date: 2006-12-30
Filing date: 2006-12-30
Publication date: 2007-07-18

Abstract

The present invention discloses process of producing anti-human CD20 monoclonal antibody in animal mammary gland bioreactor. The process of producing anti-human CD20 monoclonal antibody in animal mammary gland bioreactor is superior to available process of producing in mammal cell line, and has higher expression amount, chimeric antibody with higher capacity of combining with human B lymphoma cell surface antigen CD20 and obviously lowered production cost.

Description

A kind of method of utilizing animal's mammary gland to produce anti-humen CD 20 monoclone antibody

Technical field

The present invention relates to the genetically engineered field, relate to a kind of method of utilizing animal's mammary gland to produce anti-humen CD 20 monoclone antibody particularly.

Background technology

When treatment lymphocytic hyperplasia disease, main employing was based on the chemotherapy of cell killing in the past.Along with immunology and molecular biological progress, people begin to adopt some immunoregulation druges such as Interferon, rabbit, cytohormone to wait and treat the lymphocytic hyperplasia disease, but effect is undesirable, and very strong side effect is arranged.Recent years, the treatment that monoclonal antibody is used for malignant tumour is the target that medical circle is made great efforts always.

CD20 antigen originates from the cytocerastic pre B cell phase of B, is present in the bone-marrow-derived lymphocyte surface of normal and canceration, and is not present in other cells, so CD20 itself just provides treatment B lymphoma or leukemic target.Anti-CD-20 monoclonal antibody in case with the CD20 specific combination, its Fc part then combines with effector cell's (as having Cytotoxic T cell, natural killer cell), cause cytolysis send out should, thereby cause necrocytosis, promptly produce so-called antibody-dependent cellular cytotoxicity (ADCC) effect.

1994, the Britain scientist at first selected for use the anti-humen CD 20 monoclone antibody in mouse source to treat the B lymphoma, has obtained first-stage success, but this mouse source antibody causes people's immunological rejection easily.Afterwards, people had been merged the constant region of human immunoglobulin gene and the variable region of mouse-anti people CD20 gene, produced the chimeric anti-CD 20 monoclonal antibody of people mouse.Compare with mouse source antibody, this chimeric monoclonal antibody has the human antibody constant region, can reduce immunogenicity, and can combine with people's C1Q and cause complement dependent cytotoxicity (CDCC) effect, can cause two kinds of effects of CDCC and ADCC simultaneously, and this antibody also has the long transformation period, can prolong the effect time.1997, the medicine of or follicular non-Hodgkin formula B lymphoma (LG/F NHL) rudimentary as treatment through drugs approved by FDA based on the cancer therapy drug Rituximab of people mouse inosculating antibody humen CD 20 monoclone antibody becomes the milestone of mab treatment malignant tumour.Rituximab has now become the lymphadenomatous standard drug of treatment non-Hodgkin formula B, Rituximab global marketing volume was nearly 3,000,000,000 dollars in 2005, become the global marketing volume and occupy deputy bio-pharmaceutical, and supply falls short of demand, therefore a lot of research institutions and biotech firm research and develop anti-humen CD 20 monoclone antibody, in the hope of sharing its enormous profits.At present, the research and development of anti-humen CD 20 monoclone antibody concentrate on the humanization modified and more effective production method.Humanization modified mainly is that mouse source variable region amino acid sequence is transformed into part or all of people's source sequence, as US2003/0219433A1 and CN200510082842.7, thereby obtains the better monoclonal antibody drug of result of treatment.

At present, medicinal antibody preparation mainly is to express by the mammal cell line that transforms, and the 9 one-tenth above products in world market all are to adopt clone to cultivate to produce.Mammal cell line can produce has complete active antibody, yet it is too high to adopt this method to produce required cost in enormous quantities, and cell expressing antibody production low (0.1-1 grams per liter), the purifying process complexity, thereby the present head and shoulders above throughput of demand that has caused therapeutic antibodies, therefore the defective of traditional monoclonal antibody production technique becomes increasingly conspicuous.

The present invention is characteristics of utilizing transgenic animal technical costs low (being the 1/100-1/10 of mammal cell line culture technique) and galactophore biological reactor output height (1-20 grams per liter), make up the mammary gland expression vector of anti-humen CD 20 monoclone antibody, be transferred in the mammalian genes group and obtain transgenic animal, thereby in its mammary gland, efficiently express foreign proteins such as monoclonal antibody.

Summary of the invention

(1) technical problem that will solve

The purpose of this invention is to provide a kind of method of utilizing animal mammary gland bioreactor to produce anti-humen CD 20 monoclone antibody.

(2) technical scheme

Used carrier among the present invention, clone and reagent source: pBC1 carrier (available from Invitrogen company), pCI-neo (available from Progema company), the pC γ and the pC κ carrier (available from Transcending Biotechnologies company) that contain human immunoglobulin heavy chain and variable region of light chain cDNA sequence, synthetic and the sequencing of sequence and primer is given birth to the worker by Shanghai and is finished, the Taq enzyme, the T4DNA ligase enzyme, restriction endonuclease is all available from Dalian TaKaRa company, anti-human IgG two resists available from sigma company, human IgG ELISA detection kit is available from Bethyl company, the somatic cell clone agents useful for same is all available from Sigma company, and the human B lymphocyte oncocyte is that the Daudi cell is available from Beijing consonance medical university Institute of Basic Medical Sciences's cell centre.

Used enzyme is cut, is connected among the present invention, the step of normal experiment operations such as recovery, conversion, pcr amplification, extraction genome, electroporation transfection method, liposome transfection method, microinjection, semen transformation, Western blotting, Southern blotting sees " molecular cloning (third edition) " (2000, Science Press) or reagent corresponding box specification sheets for details; The transgenic mice preparation process is referring to " mice embryonic operation experiments guide " (work such as A. Na Ji, 2004, Science Press).

The method of utilizing animal's mammary gland to produce anti-humen CD 20 monoclone antibody of the present invention is characterized in that it comprises the steps:

(1) according to (Sequences of Proteins of Immunological Interest (ed5) .NIH Publication No.9 such as Kabat EA, 1-3242,1991) the monoclonal antibody weight chain variabl area sequence of the mouse-anti people CD20 of Gong Buing and its leader peptide sequence, the weight chain variabl area sequence of synthetic mouse-anti humen CD 20 monoclone antibody, be inserted in the pC γ carrier that contains human normal immunoglobulin gamma heavy chain constant region, make up the heavy chain mammary gland expression vector pBC1-HuG1F that obtains anti-CD20 human mouse chimeric antibody;

(2) according to (Sequences of Proteins of Immunological Interest (ed5) .NIH Publication No.9 such as Kabat EA, 1-3242,1991) the monoclonal antibody light chain variable region sequence of the mouse-anti people CD20 of Gong Buing and its leader peptide sequences, the light chain variable region sequence of synthetic mouse-anti humen CD 20 monoclone antibody, be inserted in the pC κ carrier that contains human normal immunoglobulin κ constant region of light chain, make up the light chain mammary gland expression vector pBC1-HuK1F that obtains anti-CD20 human mouse chimeric antibody;

(3) with NotI and SalI difference double digestion heavy chain mammary gland expression vector pBC1-HuG1F and light chain mammary gland expression vector pBC1-HuK1F, recovery contains the gene fragment of chimeric antibody heavy chain and contains the gene fragment of chimeric antibody light chain, the gene fragment that reclaims is dissolved in TE solution respectively, the mixed in molar ratio such as solution that will contain above-mentioned two kinds of gene fragments again, it is 1-2.5 μ g/ml that mixing solutions is diluted to final concentration, then described mixing solutions is expelled in the zygote protokaryon of mouse, again with zygote transplation in the female mouse uterine tube of the false pregnancy of estrus synchronization, produce filial generation, identify, obtain transgenic mice, utilize its mammary gland to produce anti-humen CD 20 monoclone antibody;

(4) with SalI respectively enzyme cut light chain mammary gland expression vector pBC1-HuK1F, heavy chain mammary gland expression vector pBC1-HuG1F and double alternative carrier pCE-EGFP-IRES-NEO (seeing Chinese invention patent CN03160031X), reclaim the gene fragment that contains the chimeric antibody light chain respectively, contain the gene fragment of chimeric antibody heavy chain and contain green fluorescent protein and the gene fragment of two selection markers of neomycin resistance, the gene fragment that reclaims is dissolved in TE solution respectively, that will reclaim contains the segmental solution of chimeric antibody light chain gene and contains mixed in molar ratio such as the segmental solution of chimeric antibody heavy chain gene again, the solution that will contain above-mentioned two kinds of gene fragments then and the solution that contains green fluorescent protein and neomycin resistance double-tagging gene fragment of recovery is 3-10 in molar ratio: 1 mixes, again the mixing solutions that makes is transferred in the fetal fibroblast of heavy livestock, makes the transgenosis donorcells; Again described donorcells nuclear is moved in the ovocyte that has removed nuclear, merge, cultivate, make the transgene clone blastaea, then described clone's blastaea is moved into the intrauterine of the acceptor heavy livestock of estrus synchronization, produce filial generation, identify, obtain the large transgenic domestic animal, utilize its mammary gland to produce anti-humen CD 20 monoclone antibody.

Method of the present invention, wherein the recovery of the gene fragment described in step (3) and (4) is to adopt first electroelution to reclaim, and the fragment that has reclaimed is made the method for secondary recovery of test kit again.Because when external source fragment during greater than 10kb, conventional agarose gel electrophoresis recovery method is easy to generate dna degradation, the integrity when influencing exogenous origin gene integrator to the animal gene group.

Method of the present invention, wherein the marker gene described in the step (4) is except that green fluorescent protein and neomycin resistance, can also adopt other marker gene that is used for the mammalian cell screening, as thymidine kinase gene, dihydrofolate reductase gene, paraxin hexanoyl transferase gene, xanthine-guanine transferring enzyme (XGPRT) gene etc.

The concrete operations of the secondary recovery of gene fragment of the present invention are as follows:

Electroelution reclaims: behind the agarose gel electrophoresis, downcut the gel that needs to reclaim DNA band position, pack in the dialysis tubing of having handled well, add an amount of electrophoretic buffer, amount of buffer is not advisable for just not living blob of viscose, and dialysis tubing is placed electrophoresis chamber, makes the direction of dialysis tubing and blob of viscose vertical with electrophoretic direction, the side that gel is close to is near negative pole, (voltage 1.5v/cm 3hr), makes the DNA sample all run out of blob of viscose to electrophoresis, be affixed on a side of dialysis tubing, counterelectrophoresis 30sec, the electrophoretic buffer in the sucking-off dialysis tubing places the centrifuge tube of 1.5ml, gets supernatant, add the 3M NaAc of 1/10 times of volume and the dehydrated alcohol of 2 times of volumes, place 0.5～1hr with deposit D NA for-20 ℃, then 12,000rpm, 4 ℃ of centrifugal 20min, deposit D NA, with 70% washing with alcohol precipitation, vacuum is drained back recovery fragment and is dissolved in TE solution (10mmol/LTris.cl (pH7.4), 0.1mmol/L EDTA, with the preparation of Milli-Q water) ,-20 ℃ of preservations are standby.

Test kit secondary recovery: after the DNA that gets the above-mentioned recovery of 2 μ g carries out agarose gel electrophoresis, downcut the gel that needs to reclaim DNA band position, the gel of the above dna fragmentations of the recyclable 10kb of company such as employing Qiagen reclaims test kit (as Qiagen KIT, BIO101 etc.) and reclaims.

Take the secondary recovery method to significantly improve and reclaim DNA purity and integrity, obtain the dna fragmentation of purity height and nothing degraded.

Method of the present invention, wherein the method that is adopted in the fetal fibroblast of heavy livestock that mixing solutions is transferred to described in the step (4) is electroporation transfection method or liposome transfection method.

Method of the present invention, wherein the gene fragment that contains the chimeric antibody light chain that will reclaim described in the step (4) can also adopt microinjection or semen transformation with the method that the gene fragment that contains the chimeric antibody heavy chain imports heavy livestock.

Method of the present invention, wherein said heavy livestock are ox, sheep or pig.Preferably, described heavy livestock is an ox.

The heavy chain mammary gland expression vector pBC1-HuG1F of anti-CD20 human mouse chimeric antibody of the present invention, its building process and plasmid map are as shown in Figure 1.

The light chain mammary gland expression vector pBC1-HuK1F of anti-CD20 human mouse chimeric antibody of the present invention, its building process and plasmid map are as shown in Figure 1.

(3) beneficial effect

Adopt method provided by the present invention, the expression amount that utilizes the animal's mammary gland reactor to produce anti-humen CD 20 monoclone antibody is significantly higher than the expression amount that adopts mammal cell line, wherein, the expression amount of the anti-humen CD 20 monoclone antibody of transgenic mice can reach 17.72 ± 0.40mg/ml, the expression amount of the anti-humen CD 20 monoclone antibody of transgenic cattle can reach 14.28 ± 0.90mg/ml, and the ability of the chimeric antibody specific combination people B lymphoma cell surface antigen CD20 of gained also is higher than the antibody that adopts the mammal cell line expression to obtain, production cost can produce huge economic benefit and social effect than adopting mammal cell line to produce remarkable reduction.

Description of drawings

Fig. 1 is the heavy chain mammary gland expression vector pBC1-HuG1F and the anti-CD20 people of anti-CD20 human mouse chimeric antibody

The building process of the light chain mammary gland expression vector pBC1-HuK1F of mouse chimeric antibody;

Fig. 2 cuts pC γ-HuG with the XhoI enzyme, the electrophorogram of pC κ-HuK and pBC1, wherein, M represents 1kb gradient dna molecular amount standard,

swimming lane

1,2,3 is respectively the enzyme of pC κ-HuK positive plasmid, pC γ-HuG positive plasmid, mammary gland expression vector pBC1XhoI and cuts the result, 1.4kb band is heavy chain cDNA, the 750bp band is a light chain cdna;

Fig. 3 is that PCR identifies that positive HuG1F-pBC1 cloned plasmids heavy chain cDNA inserts and insert the electrophorogram of direction of fragments, wherein, M represents 1kb gradient dna molecular amount standard, P1-P6 is the positive PCR result of HuG1F-pBC1,01-06 is direction PCR result, 01 and 02 is positive colony in the right direction, P6 and 06 negative contrast;

Fig. 4 is that PCR identifies that positive HuK1F-pBC1 cloned plasmids light chain cdna inserts and insert the electrophorogram of direction of fragments, wherein, M represents 1kb gradient dna molecular amount standard, P1-P8 is the positive PCR result of HuK1F-pBC1,01-08 is direction PCR result, wherein 03,05,06 and 07 is positive colony in the right direction, P8 and 08 negative contrast;

Fig. 5 is that HuG1F-pBC1 and HuK1F-pBC1 carrier S alI/NotI enzyme cut back to close structure iron, and wherein, globin separaant, beta-casein promotor and beta-casein 3 ' control region all are the sequence on the commercialization mammary gland expression vector pBC1;

Fig. 6 is the electrophorogram that PCR detects the double-stranded positive transgenic mice of integrating of F1, wherein, M represents 1kb gradient dna molecular amount standard, and swimming lane 1-6 is respectively the 19th, 26,35,43,50, No. 36 mouse PCR results, swimming lane 7 is the PCR result of HuG1F-pBC1 and HuK1F-pBC1 equal proportion mixed solution, and the 1.5kb band is the heavy chain of antibody amplified fragments, and the 850bp band is the light chain of antibody amplified fragments;

Fig. 7 is the integration Southern hybridization figure that detects heavy chain of antibody and light chain gene in the transgenic mice genome, and wherein, M represents 1kb gradient dna molecular amount standard, and N is the wild-type mice contrast; P1, P5 are respectively the positive plasmid contrast results of hybridization that is equivalent to 1,5 copy number, 19,26,35,36,43,50 is the positive mouse 19 that F0 integrates for two strands, 26,35,36,43,50 genomic hybridization results, the 1.4kb band is the fragment that the heavy chain of antibody carrier integrated on the genome is come out by probe hybridization, the 750bp band is the fragment that the light chain of antibody carrier is come out by probe hybridization;

Fig. 8 is the electrophorogram that PCR detects the double-stranded transgenic positive cell of integrating, wherein, M represents 1kb gradient dna molecular amount standard, swimming lane 1-9 is respectively different cell clone point PCR results, swimming lane 10 is HuG1F-pBC1 and HuK1F-pBC1 equal proportion mixed solution PCR result, swimming lane 11 negative contrast 1.5kb bands are heavy chain of antibody amplified fragments, and the 850bp band is the light chain of antibody amplified fragments; Fig. 9 is that PCR detects the double-stranded transgenic cattle electrophorogram of integrating, wherein, M represents 1kb gradient dna molecular amount standard, TC1 and TC2 are two clened cows PCR result, swimming lane P is HuG1F-pBC1 and HuK1F-pBC1 equal proportion mixed solution PCR result, the negative contrast of swimming lane N, 1.5kb band are the heavy chain of antibody amplified fragments, and the 850bp band is the light chain of antibody amplified fragments;

Figure 10 is the integration Southern hybridization figure that detects heavy chain of antibody and light chain gene in the transgenic cattle genome, wherein, M represents 1kb gradient dna molecular amount standard, P1-4 is respectively and is equivalent to 1,2, the positive plasmid contrast results of hybridization of 5 and 10 copy numbers, the negative ox contrast of N, TC1 and TC2 are the transgene clone ox, and the 1.4kb band is the fragment that the heavy chain of antibody carrier integrated on the genome is come out by probe hybridization, and the 750bp band is the fragment that the light chain of antibody carrier is come out by probe hybridization;

Figure 11 is the electrophorogram that PCR detects the double-stranded transgenic sheep of integrating, wherein, M represents 1kb gradient dna molecular amount standard, TG1 and TG2 are two transgenic sheep PCR detected result, swimming lane P is HuG1F-pBC1 and HuK1F-pBC1 equal proportion mixed solution PCR result, the negative contrast of swimming lane N1,1.5kb band are the heavy chain of antibody amplified fragments, and the 850bp band is the light chain of antibody amplified fragments;

Figure 12 is the electrophorogram that PCR detects the double-stranded transgenic pig of integrating, wherein, M represents 1kb gradient dna molecular amount standard, TP1 and TP2 are two transgenic pig PCR result, swimming lane HG and HK are respectively HuG1F-pBC1 and HuK1F-pBC1 plasmid PCR result, the negative contrast of swimming lane N, 1.5kb band are the heavy chain of antibody amplified fragments, and the 850bp band is the light chain of antibody amplified fragments;

Figure 13 is the Western blotting figure that detects CD 20 antagonizing Chimeric antibody expression in the transgenic mice breast sample, wherein, M is the molecular weight of albumen standard, H is the pure product results of hybridization of human IgG albumen, the negative mouse milk hybridization of N contrast, 26 and 36 is that F0 is for positive mouse 26 and No. 36 milk sample results of hybridization, 21, the 23rd, F1 is for positive mouse 21, No. 23 milk sample results of hybridization, the 50kDa fragment is the heavy chain protein fragment of hybridizing the sex change antibody that comes out, and the 25kDa fragment is the light chain protein fragment of hybridizing the sex change antibody that comes out;

Figure 14 is the Western blotting figure that detects CD 20 antagonizing Chimeric antibody expression in the transgene cow milk sample, wherein, M is the molecular weight of albumen standard, H is the pure product results of hybridization of human IgG albumen, N1 is double-tagging carrier transgenic cattle milk hybridization contrast, N2 is common clone's cow's milk sample results of hybridization, N3 is common cow's milk sample results of hybridization, TC1 and TC2 are transgenic cattle milk sample results of hybridization, the 50kDa fragment is the heavy chain protein fragment of hybridizing the sex change antibody that comes out, and the 25kDa fragment is the light chain protein fragment of hybridizing the sex change antibody that comes out;

Figure 15 is the Western blotting figure that detects CD 20 antagonizing Chimeric antibody expression in the transgenic sheep breast sample, wherein, M is the molecular weight of albumen standard, H is the pure product results of hybridization of human IgG albumen, N is non-transgenic sheep milk hybridization contrast, TG1 is a transgenic sheep milk sample results of hybridization, and the 50kDa fragment is the heavy chain protein fragment of hybridizing the sex change antibody that comes out, and the 25kDa fragment is the light chain protein fragment of hybridizing the sex change antibody that comes out;

Figure 16 is the Western blotting figure that detects CD 20 antagonizing Chimeric antibody expression in the transgenic pig breast sample, wherein, M is the molecular weight of albumen standard, H is the pure product results of hybridization of human IgG albumen, N is non-transgenic pig milk hybridization contrast, TP1 and TP2 are transgenic pig milk sample results of hybridization, and the 50kDa fragment is the heavy chain protein fragment of hybridizing the sex change antibody that comes out, and the 25kDa fragment is the light chain protein fragment of hybridizing the sex change antibody that comes out.

Figure 17 is transgenic mice preparation flow figure.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

The structure of embodiment 1 mammary gland expression vector of the present invention

1.1 the structure of the heavy chain mammary gland expression vector of anti-CD20 human mouse chimeric antibody

According to (Sequences of Proteins of Immunological Interest (ed5) .NIH Publication No.9 1-3242 such as Kabat EA, 1991) the monoclonal antibody weight chain variabl area sequence of the mouse-anti people CD20 of Gong Buing and its leader peptide sequence, the weight chain variabl area sequence of synthetic mouse-anti humen CD 20 monoclone antibody (giving birth to the worker by Shanghai finishes).

Because the 5 ' end of the human normal immunoglobulin gamma heavy chain constant region in the pC γ carrier contains restriction enzyme sites such as XhoI, NheI and ApaI, 3 ' end contains XhoI, and mammary gland expression vector pBC1 foreign gene insertion site is XhoI.In order the synthetic weight chain variabl area sequence to be inserted in the pC γ carrier that contains human normal immunoglobulin gamma heavy chain constant region to be built into the sequence of heavy chain of a complete anti-CD20 human mouse chimeric antibody, need to introduce NheI and two restriction enzyme sites of ApaI at weight chain variabl area sequence 5 ' and 3 ' end respectively, thus design a pair of primer (VHF:5 '-TCT AGC TAG CCG CCA CCA TGG GTT GGA GCC-3 '; VHR:5 '-ATC ATGGGC CCT TGG TGG AGG CTG CAG AGA-3 '), the synthetic weight chain variabl area sequence is carried out pcr amplification, to be connected to pGEM-Teasy (Promega company) carrier after the recovery of PCR product, be numbered pGEM-VH, and check order, utilize NheI and ApaI double digestion pGEM-VH after the proof sequence is correct, reclaim the 420bp fragment, be connected to same with reclaiming product through the pC γ of NheI and ApaI double digestion carrier, cut and check order and identify that positive colony contains the sequence of heavy chain of anti-CD20 human mouse chimeric antibody through enzyme, this carrier called after pC γ-HuG, the XhoI enzyme is cut qualification result and is seen accompanying drawing 2.

With XhoI respectively enzyme cut pC γ-HuG and pBC1, enzyme is cut product reclaim back connection conversion, utilize PCR to identify and insert fragments sequence and direction of insertion, promptly at primer of the XhoI of pBC1 restriction enzyme site 5 ' end design, with the primer VHR clone to be measured that increases, this primer is pBCF:5 '-CTA ATC CCA GAATCT AAG CGA-3 ', positive colony is identified with primer VHF and VHR, the PCR qualification result is seen accompanying drawing 3, the evaluation of checking order then, obtain the heavy chain mammary gland expression vector of anti-CD20 human mouse chimeric antibody, carrier called after pBC1-HuG1F.The building process of the heavy chain carrier of anti-CD20 human mouse chimeric antibody as shown in Figure 1.

1.2 the structure of the light chain mammary gland expression vector of anti-CD20 human mouse chimeric antibody

According to (Sequences of Proteins of Immunological Interest (ed5) .NIH Publication No.9 1-3242 such as Kabat EA, 1991) the monoclonal antibody light chain variable region sequence of the mouse-anti people CD20 of Gong Buing and its leader peptide sequences, the light chain variable region sequence of synthetic anti-CD20 human mouse chimeric antibody (giving birth to the worker by Shanghai finishes).

Because the 5 ' end of the human normal immunoglobulin constant region of light chain in the pC κ carrier contains restriction enzyme sites such as XhoI, NheI and BsiWI, 3 ' end contains XhoI, and mammary gland expression vector pBC1 foreign gene insertion site is XhoI.For the light chain variable region sequence being inserted in the pC κ carrier that contains the human normal immunoglobulin constant region of light chain to be built into the sequence of light chain of a complete anti-CD20 human mouse chimeric antibody, need to introduce NheI and two restriction enzyme sites of BsiWI at variable region sequences 5 ' and 3 ' end respectively, thus design a pair of primer (VLF:5 '-TCT AGG TAG CCG CCA CCA TGG ATT TTC AGG-3 '; VLR:5 '-TGA CTC GTA CGT TTG ATT TCC AGC TTG GTC C-3 ') synthetic light chain variable region sequence is carried out pcr amplification, to be connected to pGEM-Teasy (Promega company) after the recovery of PCR product, be numbered pGEM-VL, and check order, utilize NheI and BsiWI double digestion pGEM-VL after the proof sequence is correct, reclaim the 420bp fragment, be connected to same on the pC κ of NheI and BsiWI double digestion carrier with reclaiming product, cut and check order and identify that positive colony contains the sequence of light chain of anti-CD20 human mouse chimeric antibody through enzyme, this carrier called after pC κ-HuK, the XhoI enzyme is cut qualification result and is seen accompanying drawing 2.

With XhoI respectively enzyme cut pC κ-HuK and pBC1, enzyme is cut product reclaim back connection conversion, utilize PCR to identify that (primer is pBCF and VLR, VLF and VLR) and order-checking evaluation positive colony in the right direction, the PCR qualification result is seen accompanying drawing 4, obtain the light chain mammary gland expression vector of anti-CD20 human mouse chimeric antibody, carrier called after pBC1-HuK1F.The light chain vector construction process of anti-CD20 human mouse chimeric antibody as shown in Figure 1.

Embodiment 2 express anti-humen CD 20 monoclone antibodies transgene mouse model foundation and detect 2.1 dual-gene micro-injections altogether and produce transgenic mices

Produce transgenic mice with microinjection, therefore linear ratio annular integration efficiency height is wanted earlier the expression vector enzyme that builds is cut into and linearly just can be carried out microinjection.With NotI and SalI double digestion light chain expression vector pBC1-HuK1F and heavy chain expression carrier pBC1-HuG1F, remove the prokaryotic vector part, reclaim 16.5kb and contain the exogenous genetic fragment of chimeric antibody light chain and the exogenous genetic fragment that 17.2kb contains the chimeric antibody heavy chain, reclaim fragment structure and see accompanying drawing 5.The recovery of exogenous genetic fragment adopts first electroelution to reclaim, and carries out the method for secondary recovery with test kit to reclaiming fragment then.Concrete operations are as follows:

Electroelution reclaims: behind the agarose gel electrophoresis, downcut the gel that needs to reclaim DNA band position, pack in the dialysis tubing of having handled well (Huamei Bio-Engrg Co.), add an amount of electrophoretic buffer, amount of buffer is not advisable for just not living blob of viscose, and dialysis tubing is placed electrophoresis chamber, makes the direction of dialysis tubing and blob of viscose vertical with electrophoretic direction, the side that gel is close to is near negative pole, (voltage 1.5v/cm 3hr), makes the DNA sample all run out of blob of viscose to electrophoresis, be affixed on a side of dialysis tubing, counterelectrophoresis 30sec, the electrophoretic buffer in the sucking-off dialysis tubing places the centrifuge tube of 1.5ml, gets supernatant, add the 3M NaAc of 1/10 times of volume and the dehydrated alcohol of 2 times of volumes, place 0.5～1hr with deposit D NA for-20 ℃, then 12,000rpm, 4 ℃ of centrifugal 20min, deposit D NA, with 70% washing with alcohol precipitation, vacuum is drained back recovery fragment and is dissolved in TE solution (10mmol/L Tris.cl (pH7.4), 0.1mmol/L EDTA, with the preparation of Milli-Q water) ,-20 ℃ of preservations are standby.

Test kit secondary recovery: after the DNA that gets the above-mentioned recovery of 2 μ g carries out agarose gel electrophoresis, downcut the gel that needs to reclaim DNA band position, adopt the gel recovery test kit (Qiagen KIT, Qiagen company produces) of the above dna fragmentation of recyclable 10kb to reclaim.

The gene fragment that reclaims is dissolved in TE solution (10mmol/L Tris.Cl (pH7.4), 0.1mmol/L EDTA, with the preparation of Milli-Q water), with the OD ratio under ultraviolet spectrophotometer mensuration 260nm and the 280nm, and calculating concentration, OD260/OD280 can be used for injecting mouse fertilized egg more than 1.8.The mixed in molar ratio such as solution that will contain two kinds of exogenous genetic fragments, and to be diluted to final concentration be 1.0-2.5 μ g/ml (i.e. the concentration of two kinds of exogenous genetic fragments respectively for 0.5-1.25 μ g/ml), mixing solutions is expelled in the zygote protokaryon, again with zygote transplation in the female mouse uterine tube of the false pregnancy of estrus synchronization, the result is as shown in table 1 in preparation, shows that the method for secondary recovery can significantly improve the preparation efficiency of transgenic mice.

The transgenic mice preparation efficiency of the different DNA recovery methods of table 1 relatively

Treatment process	Electroelution	Secondary recovery	Ordinary method (once reclaiming)
Treatment process	Electroelution	Secondary recovery	Ordinary method (once reclaiming)	The birth number of mice	43	112	52
The transgenic mice number	10	46	6	The birth number of mice	43	112	52
The transgenic mice number	10	46	6	The transgenic mice ratio	24.0％	41.1％	11.5％

2.2 the PCR of transgenic mice detects

Get 2 age in week transgenic mice tail organize sample, extract genome.Genome with extraction is a template, with the primer that is positioned at both sides, XhoI site on the pBC1 (pBC1xF1:5 '-GAT TGA CAA GTAATA CGC TGT TTC CTC-3 ', pBC1xF2:5 '-CAT CAG AAG TTA AAC AGCACA GTT AG-3 '), in the positive mouse that two strands is integrated, can amplify the 850bp fragment that contains light chain gene and the 1.5kb fragment of heavy chain gene simultaneously, with common mouse (non-transgenic mouse) genome is that the PCR of template reacts negative contrast, is that the PCR of template reacts positive contrast with carrier pBC1-HuG1F and pBC1-HuK1F.Amplification condition is: 94 ℃, and 30sec; 58 ℃, 30sec; 72 ℃, 90sec; 35 circulations.Use 1.0% agarose electrophoresis observations then.PCR detects 6 two strandss and integrates mouse altogether in F0 generation 52 mouse, the results are shown in accompanying drawing 6, the 19, and 26,35,43,50, No. 36 mouse are the transgenic positive mouse of integrating pBC1-HuG1F and pBC1-HuK1F gene.

2.3 the Southern blotting of transgenic mice detects

F0 generation 5 transgenic mices of integrating two strands with the PCR test positive, strand afterbody tissue also extracts genome, get about 10 μ g genomes, digest with the XhoI restriction endonuclease, the negative contrast of wild-type mice genome of cutting with the XhoI enzyme, the positive contrast of the pBC1-HuG1F that the XhoI enzyme is cut, pBC1-HuK1F injection carrier segments, hybridization probe is that the XhoI enzyme is cut the heavy chain gene fragment of the 1.4kb that reclaims behind pBC1-HuG1F, the pBC1-HuK1F carrier and the light chain gene fragment of 750bp.With various sample low pressure electrophoresis at a slow speed, adopt the alkali transfer method that DNA is transferred to nylon membrane from agarose, the one side that nylon membrane is had a DNA is inwardly, be rolled into tubular and put into hybrid pipe, add prehybridization solution, be not shorter than 1hr, add the isotope-labeled probe of α-P32dCTP through heat denatured in 65 ℃ of prehybridizations, 65 ℃ of hybridization 12～16hr, wrap with preservative film after washing film, put in the magazine, press the X-ray sheet in the darkroom, in-70 ℃ of radioautograph, 12 hours towards the X-ray sheet developing observations.Southern blotting result as shown in Figure 7, the result shows that positive mouse Southern blotting detected result is consistent with the PCR detected result.

Embodiment 3 expresses the preparation and the detection of the transgenic cattle of anti-humen CD 20 monoclone antibody

3.1 the preparation of transgene clone ox

Get 5 monthly age holstein cow fetus ear tissues,, set up bovine fetal fibroblast system through former foster, the cultivation of going down to posterity of being commissioned to train, vitro culture operation such as freezing.

With SalI respectively enzyme cut light chain mammary gland expression vector pBC1-HuK1F, heavy chain mammary gland expression vector pBC1-HuG1F and double alternative carrier pCE-EGFP-IRES-NEO (seeing Chinese invention patent CN03160031X), reclaim the gene fragment that contains the chimeric antibody light chain respectively, contain the gene fragment of chimeric antibody heavy chain and contain green fluorescent protein and the gene fragment of two selection markers of neomycin resistance, the gene fragment that reclaims is dissolved in TE solution respectively, that will reclaim contains the segmental solution of chimeric antibody light chain gene and contains mixed in molar ratio such as the segmental solution of chimeric antibody heavy chain gene again, the solution that will contain above-mentioned two kinds of gene fragments then and the solution that contains the double-tagging gene fragment of recovery is 3-10 in molar ratio: 1 mixes, be transferred to the mixing solutions that makes in the fetal fibroblast of ox with the electroporation infection protocol again, identify through G418 screening and PCR, be defined as the transgenosis donorcells, the PCR detected result is seen accompanying drawing 8.

Collect the ovary of the ox that grows up from the slaughterhouse, the ovarian follicle that the cut-off footpath is 2～8 millimeters, the recovery form is even, ovarian cumulus-the ovocyte of compact structure-complex body, ovarian cumulus-ovocyte-complex body is put into four orifice plates that contain ripe liquid (M199+10%FBS+0.01U/ml bFSH+0.01U/ml bLH+1 μ g/ml estradiol) with 50 pieces/hole, at 38.5 ℃, after maturation is cultivated 18～20h in the 5%CO2 incubator, the pipe that sophisticated ovocyte is put into 0.1% Unidasa vibrates behind 2～3min, blow and beat gently with Glass tubing again, cumulus cell and ovocyte are broken away from fully, select complete form, tenuigenin evenly and the ovocyte of discharging first polar body be the somatic cell nuclear acceptor.

The ovocyte that will have first polar body moves into and contains in the operation liquid of M199+10%FBS+7.5 μ g/ml cytochalasin B, under 200 power microscopes, above polar body, zona pellucida cut an osculum with a glass needle, again with internal diameter be 20 μ m Glass tubing with first polar body with and the ovocyte of below in karyomit(e) absorb in the lump, the solution of putting into M199+20%FBS again give a baby a bath on the third day after its birth all over after, place incubator standby.

The donorcells (being the transgenic cell that above-mentioned commentaries on classics has double-stranded gene of antibody and labeled vector) of serum starvation 2～4d is digested 2～4min with 0.25%trypsin, the selection diameter is that the somatocyte of 10～12 μ m has gone its immigration in the ovocyte zona pellucida of nuclear with 20 μ m diameter Glass tubings, put it into Zimmerman liquid (.2003.Nat Biotechnol 21 (2) such as Brophy B: put into integration slot behind balance 3～5min 157-162.) then, rotating ovum makes donorcells vertical with electric field with the ovocyte contact surface, field intensity in DC pulse is 2.5kV/cm simultaneously, burst length is 10 μ s, pulse number is 2 times, recurrent interval is after merging (fusion instrument is the ECM-2001 of BTX company) under the condition of 1s, rapidly reconstructed embryo to be moved in the M199+10%FBS liquid.Again reconstructed embryo is put into 5 μ mol/L Ionomycin liquid, changed to behind the 4min in the 1.9mmol/L 6-DMAP liquid, move into again behind the 4h in the CR1aa+5%FBS liquid, at 38.5 ℃, 5%CO ₂Cultivated 2 days in the incubator.

Clone's blastaea of the 7d that form is good moves into the intrauterine of the receptor cow of estrus synchronization.What receptor cow was selected all is the multiparity cow, and the 60d after transplanting carries out rectum and detects, to determine pregnancy rate.

3.2 the detection of positive transgene clone ox

Obtain the clened cows of 2 survivals altogether by body-cell neucleus transplanting method, positive transgene clone ox PCR detects and the detection of Southern trace detects with transgenic mice, PCR result as shown in Figure 9, Southern trace result as shown in Figure 10, the result shows that this two transgene clone ox all is integrated with pBC1-HuG1F and pBC1-HuK1F gene, promptly is integrated with the heavy chain gene and the light chain gene of recombinant antibodies.

Embodiment 4 expresses the preparation and the detection of the transgenic sheep of anti-humen CD 20 monoclone antibody

The transgenic sheep preparation process promptly takes somatic cell clone technique to obtain transgenic sheep with the preparation process of transgenic cattle, and transgenic sheep PCR detected result is seen accompanying drawing 11, obtains the transgenic sheep that 1 commentaries on classics has pBC1-HuG1F and the dual-gene structure of pBC1-HuK1F altogether.

Embodiment 5 expresses the preparation and the detection of the transgenic pig of anti-humen CD 20 monoclone antibody

The transgenic pig preparation process promptly takes microinjection to obtain transgenic pig with the preparation process of transgenic mice, and transgenic pig PCR detected result is seen accompanying drawing 12, obtains the transgenic pig that 2 commentaries on classics have pBC1-HuG1F and the dual-gene structure of pBC1-HuK1F altogether.

Embodiment 6 reorganization CD 20 antagonizing Chimeric antibody expression analysis in transgenosis breast sample

6.1 the Western blotting of the transgenic mice that is obtained by embodiment 2 breast sample detects

Gather F0 generation 26, No. 36 mouse, in F1 generation,, 22,23, No. 24 mouse were often newborn, and negative mouse milk is as negative control.Milk was gathered in mouse birth back in one week, before gathering female mouse separated more than 3 hours with newborn mouse, and to the pitocin of abdominal injection doses.Full milk and the newborn sample handled through degreasing demargarinate albumen are with 12% SDS-PAGE gel electrophoresis.Because recombined chimeric antibody contains people source constant region, therefore only need to adopt the anti-human IgG two anti-Western Blot that carry out to analyze, can detect the expression of chimeric antibody in transgenic mice milk of recombinant human.The result as shown in Figure 13, people's pure product IgG behind the sex change electrophoresis, unwind into heavy chain molecule and light chain molecule, difference band with negative mouse milk sample is arranged in F0 code name mouse and F1 mouse, size is 50kDa and 25kDa, show in F0 generation 26 and No. 36, expressed recombinant antibodies in F1 generation 21 and No. 23 transgenic mice milk.

6.2 the Western blotting of the transgenic cattle that is obtained by

embodiment

3,4,5, sheep, pig breast sample detects respectively

The lactagogue pin of producing with Cao Tan pharmaceutical factory, Xi'an carries out the medicine lactagogue by 1/4 dosage to transgene clone ox, sheep, the pig at 6-8 monthly age, is contrast with ordinary milk, goat milk, pig milk, collects the milk sample every day 3 times, collects for two weeks altogether.Western blotting operating process is with transgenic mice breast sample, transgenic cattle, sheep and pig Western blotting detected result are seen accompanying drawing 14 respectively, 15 and 16, the result shows all to express in transgenic cattle, sheep and the pig breast sample that is obtained recombinant anti human CD20 chimeric antibody.

6.3 the external source chimeric antibody is expressed quantitative analysis in the transgenosis breast sample

With sandwich method ELISA the expression amount of chimeric antibody in transgenic mice milk analyzed, the reagent of analyzing usefulness comes from the Human IgG ELISA of Bethyl company detection kit.By solid phase, the anti-human IgG Fc section antibody of horseradish peroxidase (HRP) mark develops the color anti-human IgG Fc section antibody as bag.With the known human serum of IgG concentration as quantitative standards.Each each sample of concentration of measuring is done two repetitions, gets two and repeats average as the one-shot measurement value, and each sample is surveyed concentration three times at least, calculating concentration average and average value standard deviation, and the result is as shown in table 2.

The external source chimeric antibody is expressed quantitative analysis such as mouse breast sample analytic process in transgenosis milk cow, sheep and the pig breast sample, the result is as shown in table 3, TC represents transgenic cattle, TG represents transgenic sheep, TP represents transgenic pig, all is significantly higher than the amount (0.1-1 grams per liter) that adopts the mammal cell line expressing antibodies.

Table 2 transgenic mice antibody expression amount cartogram

The female mouse of transgenic positive	Antibody expression amount (mg/ml)
The female mouse of transgenic positive	Antibody expression amount (mg/ml)	F0-26 F0-36 F1-21 F1-23	7.60±0.30 0.29±0.035 0.3 1±0.024 17.72±0.40

The big domestic animal recombinant antibodies of table 3 transgenosis expression amount cartogram

Transgenic cattle	Antibody expression amount (mg/ml)
Transgenic cattle	Antibody expression amount (mg/ml)	TC1 TC2 TG1 TP1 TP2	11.60±0.30 14.28±0.90 3.36±0.12 5.34±0.70 4.90±0.19

The biological activity assay of embodiment 7 recombinant anti human CD20 chimeric antibodies

Getting cultured human B lymphocyte oncocyte is Daudi cell 125ml, and the centrifugal 10min of 2000rpm removes supernatant liquor, and with 2mlPBS re-suspended cell precipitation, the piping and druming mixing is also with autoclaved screen filtration suspension.The 200ul/ pipe is sub-packed in the 1.5ml centrifuge tube; Add the anti-people CD16 of 6.5 μ l antibody in every centrifuge tube, the anti-people CD32 of 2.2 μ l antibody seals;

Centrifugal back adds antibody expression and detects male transgenosis breast sample (No. 23, transgenic mice breast sample, transgene cow milk sample TC2, transgenic sheep breast sample TG1, transgenic pig breast sample TP2) with 200ulPBS re-suspended cell precipitation, hatches 30min for 4 ℃.As negative control, PBS replaces the milk sample as blank with non-transgenic breast sample.After hatching end, add 800ulPBS, the centrifugal 5min of 2000rpm in 4 ℃ of refrigerated centrifuges abandons supernatant after centrifugal, repeats once with 1mlPBS again, remains low temperature (0-4 ℃) operation in the centrifugal process.

Centrifugal back is precipitated with the 180ulPBS re-suspended cell, adds the anti-humen CD 20 antibody (Ebioscience company, extent of dilution 1: 10) of 20 μ lFITC marks, and 4 ℃ of lucifuges are hatched 30min.Blank replaces antibody to carry out negative staining with PBS.It is centrifugal to hatch the end back.

Centrifugal back is precipitated with the 800ulPBS re-suspended cell, the piping and druming mixing is also with autoclaved screen filtration suspension, with flow cytometer to painted cell counting, analytical results, the positive antibody of using in the experiment is from Ebioscience company, be by traditional cell culture processes, large scale culturing CH7 hybridoma again from cell culture fluid purifies and separates obtain.In the blank group of experiment, reaction system is under the flow cytometer fluorescence excitation, and the ratio of bone-marrow-derived lymphocyte autofluorescence is 0.05%, and in the experiment negative control group, the non-specific combination ratio of cell is 1.72%.The calculating of chimeric antibody and surface antigen CD20 joint efficiency, by with control group in autofluorescence and non-specific binding data calibration get.With the sum deduction autofluorescence cell number of detected cell that can fluorescence excitation, the non-specific binding reaction numerical value in the negative milk of the deduction sample again.

Experimental result is as shown in table 4, utilize fluorophor FITC negative staining experimental results show that, recombinant antibodies has the vigor of specific combination bone-marrow-derived lymphocyte surface antigen CD20 in the transgenosis breast sample, increase along with the increase of antibody concentration with the B lymphoma cell of CD20 antibodies in the newborn sample, when antibody concentration reaches capacity, lymphoma cell in the reaction system has 98.08%, 95.67%, 99.18% and 99.83% respectively by No. 23 transgenic mice breast samples, TC2 transgene cow milk sample, recombinant antibodies combination in TG1 transgenic sheep breast sample and the TP2 transgenic pig breast sample wherein has 96.87% lymphocyte by the combination of CD20 antibody standard substance in the positive controls.In addition, increase along with antibody concentration in the reaction system, the cell of antibody sandwich increases thereupon, and when the antibody high concentration value, approach to saturation with the lymphocyte bonded, as shown in table 4, prove inosculating antibody physical efficiency specific combination B lymphoma cell surface antigen CD20, and binding ability is higher than the antibody binding capacity of positive control.

The cell of recombinant anti human CD20 antibody is in conjunction with experiment in the table 4 transgenosis breast sample

	Surface antigen CD20 is by non-fluorescin combination/autofluorescence cell numerical value (%)	Saturated concentration antibody and surface antigen CD20 joint efficiency in the breast sample (correction value, %)	Saturation concentration antibodies efficiency ratio (antibody/control antibodies in the newborn sample, %)
				TC2 23 TG1 TP2 positive control negative control blanks	97.34％ 99.75％ 99.18％ 98.83％ 98.54％ 1.72％ 0.05％	95.67％ 98.08％ 97.5 1％ 97.16％ 96.87％ - -	98.76％ 101.25％ 100.66％ 100.30％ 100％ - -

Claims

1, a kind of method of utilizing animal's mammary gland to produce anti-humen CD 20 monoclone antibody is characterized in that it comprises the steps:

(1) weight chain variabl area sequence of synthetic mouse-anti humen CD 20 monoclone antibody is inserted in the pC γ carrier that contains human normal immunoglobulin gamma heavy chain constant region, makes up the heavy chain mammary gland expression vector pBC1-HuG1F that obtains anti-CD20 human mouse chimeric antibody;

(2) the light chain variable region sequence of synthetic mouse-anti humen CD 20 monoclone antibody is inserted in the pC κ carrier that contains human normal immunoglobulin κ constant region of light chain, makes up the light chain mammary gland expression vector pBC1-HuK1F that obtains anti-CD20 human mouse chimeric antibody;

(3) with NotI and SalI difference double digestion heavy chain mammary gland expression vector pBC1-HuG1F and light chain mammary gland expression vector pBC1-HuK1F, recovery contains the gene fragment of chimeric antibody heavy chain and contains the gene fragment of chimeric antibody light chain, the gene fragment that reclaims is dissolved in TE solution respectively, the mixed in molar ratio such as solution that will contain above-mentioned two kinds of gene fragments again, it is 1.0-2.5 μ g/ml that mixing solutions is diluted to final concentration, then described mixing solutions is expelled in the zygote protokaryon of mouse, again with zygote transplation in the female mouse uterine tube of the false pregnancy of estrus synchronization, produce filial generation, identify, obtain transgenic mice, utilize its mammary gland to produce anti-humen CD 20 monoclone antibody;

(4) with SalI respectively enzyme cut light chain mammary gland expression vector pBC1-HuK1F, heavy chain mammary gland expression vector pBC1-HuG1F and double alternative carrier pCE-EGFP-IRES-NEO, reclaim the gene fragment that contains the chimeric antibody light chain respectively, contain the gene fragment of chimeric antibody heavy chain and contain green fluorescent protein and the gene fragment of two selection markers of neomycin resistance, the gene fragment that reclaims is dissolved in TE solution respectively, that will reclaim contains the segmental solution of chimeric antibody light chain gene and contains mixed in molar ratio such as the segmental solution of chimeric antibody heavy chain gene again, the solution that will contain above-mentioned two kinds of gene fragments then and the solution that contains the double-tagging gene fragment of recovery is 3-10 in molar ratio: 1 mixes, again the mixing solutions that makes is transferred in the fetal fibroblast of heavy livestock, makes the transgenosis donorcells; Again described donorcells nuclear is moved in the ovocyte that has removed nuclear, merge, cultivate, make the transgene clone blastaea, then described clone's blastaea is moved into the intrauterine of the acceptor heavy livestock of estrus synchronization, produce filial generation, identify, obtain the large transgenic domestic animal, utilize its mammary gland to produce anti-humen CD 20 monoclone antibody.

2, method according to claim 1, the recovery that it is characterized in that the gene fragment described in step (3) and (4) are to adopt first electroelution to reclaim, and the fragment that has reclaimed are made the method for secondary recovery of test kit again.

3, method according to claim 1 is characterized in that the method that is adopted in the fetal fibroblast of heavy livestock that mixing solutions is transferred to described in the step (4) is electroporation transfection method or liposome transfection method.

4, method according to claim 1 is characterized in that gene fragment that contains the chimeric antibody light chain that will reclaim described in the step (4) and the method that the gene fragment that contains the chimeric antibody heavy chain imports heavy livestock can also adopt microinjection or semen transformation.

5,, it is characterized in that described heavy livestock is ox, sheep or pig according to the described method of each claim of claim 1-4.

6, method according to claim 5 is characterized in that described heavy livestock is an ox.

7, a kind of heavy chain carrier pBC1-HuG1F of anti-CD20 human mouse chimeric antibody is characterized in that its plasmid map as shown in Figure 1.

8, a kind of light chain mammary gland expression vector pBC1-HuK1F of anti-CD20 human mouse chimeric antibody is characterized in that its plasmid map as shown in Figure 1.