CN1150331C - A group of efficient expression carriers for humanized antibody of mammal cell - Google Patents

Abstract

The present invention relates to a group of high-efficiency expression carriers of humanized antibodies of mammalia cells for production. The present invention comprise twelve kinds of carriers which are formed by the oriented combination of different elements. The present invention is mainly characterized in that an expression element of a eukaryotic cell is formed by the oriented combination of a strong promoter EF-1 alpha, light and heavy chain constant-region genes of antibodies originating from genome DNA or messenger RNA, internal ribosome recognition site points and genes of dihydrofolate reductase in different forms. The present invention can be used for cloning a variable region for antibodies or a Fab gene, and the permanent high-efficiency expression of antibody genes of different sources can be obtained after the carriers enter China hamster ovary (CHO) cells. The present invention has the use that the high-efficiency expression carriers can be used for continuously carrying out scale production of IgG antibodies of whole human beings or people-mouse chimeric antibody preparation which are used for clinical treatment.

Description

One group of carriers for humanized antibody of mammal cell efficient expression vector

Background technology

Since B lymphocyte hybridoma cell-fusion techniques in 1975 came out, monoclonal antibody was used for fundamental research as a class, laboratory diagnosis, and the novel product of clinical treatment and prevention, its applying value and DEVELOPMENT PROSPECT have obtained general affirming.The researchdevelopment of molecular biology and molecular immunology has caused the generation and the development of genetic engineering antibody.The phage antibody gene pool technology rise of rising the beginning of the nineties at the end of the eighties and the development in whole genetic engineering antibody technical study field have had breakthrough and have more and more demonstrated its significance and practice prospect studies on Monoclonal Antibody.Human genetically engineered antibody is as class novel specific treatment bio-pharmaceutical or passive immunization vaccine, makes it more and more be subjected to investing the boundary and pays close attention to and show its immeasurable commercial value and social benefit.The development research of world today's human genetically engineered antibody has obtained remarkable progress and has stepped into substantive applied research and development phase by the fundamental research stage.At present in the biological products that drugs approved by FDA is gone on the market or awaited the reply, various forms of monoclonal antibodies and genetic engineering antibody account for certain proportion, in 67 kinds of biological products with the approval listing, treatment and prevention have 9 kinds with monoclonal antibody and genetic engineering antibody at present.The medium antibody that awaiting the reply has 35 kinds.Genetic engineering antibody is indubitable as the prospect of the biological medicine of a class.

The phage antibody gene pool technology side of rising the beginning of the nineties at the end of the eighties has become the focus of world today's human genetically engineered antibody research.From the antibody expression form of antibody library screening mainly based on Fab section antibody and scFv.Its ultimate principle is that the repertoire antibody light chain gene group of pcr amplification and a complete set of heavy chain gene group are inserted in the phage vector, forms phage antibody library after helper phage infects.Obtain the specific antibody gene by using specific antigens antagonist storehouse to carry out the long-pending screening of richness.Nineteen ninety Scripps institute obtains the human immunoglobulin gene storehouse first and obtains people's anti-tetanus toxin monoclone antibody from the donor peripheral blood B cell of tetanus toxin immunity.So far this technology is extensively with making up mouse and human immunoglobulin gene storehouse and being mature on the whole.Thereby set up antibody library by the phage vector expression system and obtain the existing many successful reports of specific gene engineered antibody at home and abroad, these from the antibody of phage antibody library nothing more than two kinds of forms, i.e. Fab antibody or scFv single-chain antibody form.

It is not to be used for clinical diagnosis that people source or humanized antibody are developed topmost purpose, but is used for clinical antitumor and anti-virus infection as a kind of novel therapeutic biotechnology pharmaceutical preparation.Above-mentioned Fab and scFv antibody are usually at expression in escherichia coli, its natural antibody characteristic and stability in vivo are all poor than whole antibody IgG form, and lack the Fc section constant region function that natural antibody has, by it in anti-virus infection, as a kind of treatment antibody, the IgG whole antibody is far superior to Fab and scFv incomplete antibody.Though phage antibody library technique is increasingly mature, the full IgG engineered antibody system in its downstream is fully matured not as yet then, especially in eukaryotic cell rapidly, stablize, efficiently express aspect the genetically engineered whole antibody.Comparatively successful whole antibody expression system is the expressing cho cell system, though the development of the carrier system of this expression system is numerous, and the available use of commercial-free carrier so far.

Summary of the invention

The present invention relates to one group of carriers for humanized antibody of mammal cell efficient expression vector preparation and application, its principal character is the strong eukaryotic cell expression promotor EF-1a of collection, the antibody constant region gene, eukaryotic cell expression amplification gene DHFR gene and ribosome internal binding site IRES gene are one, make and to be cloned into this carrier system by the cloning site that is provided with in the carrier at arbitrary antigenic specific antibody variable region gene or Fab gene and from the arbitrary antigenic specific antibody variable region gene or the Fab gene of the different genera in other sources from phage selection, the transfection cells of mamma animals, behind Chinese suslik ovary (CHO) cell commonly used, under the condition of screening pressing, set up to express the production clone of whole antibody, might the becoming the commercialization antibody preparation of consequent whole antibody and be used to prevent clinically or treat corresponding disease.

One group of carriers for humanized antibody of mammal cell efficient expression vector preparation of the present invention statement and uses thereof, main contents comprise following some:

1, one group of carriers for humanized antibody of mammal cell efficient expression vector is shown in Figure of description 1 and accompanying drawing 2.Fig. 1 is a cells of mamma animals human antibody efficient expression vector skeleton carrier synoptic diagram, comprises Figure 1A and Figure 1B.Fig. 2 is a cells of mamma animals human antibody efficient expression vector expression cassette synoptic diagram, and " heavy chain A1, B1 " shows skeleton carrier A and the total expression vector box 1 of B among the figure, by that analogy.Wherein symbol EF-1a is a promotor, CH1, and CH2 and CH3 are respectively CH1, and 2 and 3.BGHpolyA is an ox somatomedin poly-A tail, and DHFR is a dihydrofolate reductase gene, and IRES is the ribosome internal binding site.This group comprises 12 kinds of carriers that combined by the different elements orientation, and its principal character is made up of following crucial DNA element:

1) the promotor EF-1 α of promotor gene expressive function is by force arranged.

2) from IgG1 gene heavy chain total length constant region gene or Fc gene and humanized IgG gene Kappa constant region of light chain gene or the Lambda constant region of light chain gene of human lymphocyte mRNA.

3) from the IgG gene heavy chain total length constant region gene that comprises intron and the humanized IgG gene Kappa constant region of light chain gene of human lymphocyte genomic dna.

4) be used for respectively the solemnly restriction enzyme site of chain variable region gene, Kappa chain variable region gene or Lambda chain variable region gene.

5) be positioned at the dihydrofolate reductase gene that is used for goal gene screening and amplification in goal gene downstream.

6) the ribosome internal binding site (IRES) between goal gene downstream and amplification gene dihydrofolate reductase gene.

2, one group of carriers for humanized antibody of mammal cell efficient expression vector, except that the plain resistant gene of the blue or green enzyme of total ammonia benzyl and e. coli dna duplicate the primary element, comprise 12 kinds of carrier systems that combine by the different elements orientation, its feature is summarised among the table 1, mainly is made up of following primary expression box:

1) be used for the expression cassette that heavy chain gene is expressed:

The composition of expression cassette 1 is successively by promotor EF-1 α, the antibody gene leader, the cloning site BssHII and the BstEII of heavy chain variable region gene (VH), the IgG gene heavy chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ox somatomedin poly-A tail (BGHPoly A), initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

Expression cassette 2 or 3 composition are successively by promotor EF-1 α, the antibody gene leader, the cloning site XhoI of heavy chain variable region gene (VH) and NheI or heavy chain Fd gene can swell site XhoI and SpeI, IgG1 gene heavy chain total length constant region gene or Fc base from human lymphocyte mRNA, BGH Poly A, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

The composition of expression cassette 4 is successively by promotor EF-1 α, the antibody gene leader, heavy chain can be distinguished the cloning site BssHII and the BstEII of gene (VH), the IgG gene heavy chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

Expression cassette 5 or 6 composition are successively by promotor EF-1 α, the antibody gene leader, the cloning site XhoI of heavy chain variable region gene (VH) and NheI or heavy chain Fd gene can swell site XhoI and SpeI, IgG1 gene heavy chain total length constant region gene or Fc base from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 polyA).

2) be used for the expression cassette that light chain gene is expressed:

The composition of expression cassette 1 is successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the XhoI of light chain Ke Jiao district gene (VL), the IgG gene light chain total length constant region gene and the transcription termination signal thereof that comprise intron from the human lymphocyte genomic dna, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

Expression cassette 2 or 3 composition are successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Kappa or Lambda chain variable region gene (Vk or V λ), IgG1 gene Kappa or Lambda light chain total length constant region gene BGH Poly A from human lymphocyte mRNA, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

The composition of expression cassette 4 is successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the XhoI of chain variable region gene (VL), the IgG gene Kappa chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

Expression cassette 5 or 6 composition are successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Kappa or Lambda chain variable region gene (Vk or V λ), IgG1 gene Kappa or Lambda light chain total length constant region gene from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40poly A).

Below table 1 be the summary of above-mentioned one group of carriers for humanized antibody of mammal cell efficient expression vector.

3, the purposes of one group of carriers for humanized antibody of mammal cell efficient expression vector, it is characterized in that: no matter from phage antibody library hybridoma cell strain amplification screening or secretory antibody all can be at the VH in any antigenic antibody human source or mouse source and VL gene (Vk or V λ) by cloning site optionally by being provided with in the carrier, be cloned in the carrier according to any establishment described in claim 1 and 2, thereby in cells of mamma animals such as Chinese hamster ovary celI, obtain the expression of total man's IgG antibody or chimeric antibody.

4, the purposes of one group of carriers for humanized antibody of mammal cell efficient expression vector, it is characterized in that: can set up the permanent express cell system that efficiently expresses the IgG whole antibody and be used to produce full IgG antibody with this carrier system, according to its antigen-binding specificity and the market requirement, can be used as a kind of novel antibody preparation and carry out scale production, have business development and be worth.

One group of carriers for humanized antibody of mammal cell efficient expression vector of table 1

Heavy chain expression carrier main element and putting in order

VH-dhfr1 EF-1 α-guide secretes peptide-VH can swell site (BssHII+BstEII) CH1-intron

-CH2-intron-CH3-BGHpolyA-SV40ori-DHFR-SV40polyA

VH-dhfr2 EF-1 α-guide secrete peptide-VH can swell site (Xho+NheI)-

CH1-CH2-CH3-BGHpolyA-SV40ori-DHFR-SV40polyA

VH-dhfr3 EF-1 α-guide secrete peptide r-VH can swell site (Xho+SpeI)-

Fc-BGHpolyA-SV40ori-DHFR-SV40polyA

VH-IRES-dhfr1 EF-1 α-guide secrete peptide r-VH can swell site (BssHII+BstEII)-

CH1-intron-CH2-intron-CH3-intron-IRES-DHFR-SV40?polyA

VH-IRES-dhfr2 EF-1 α-guide secrete peptide-VH can swell site (XhoI+NheI)-

CH1-CH2-CH3-intron-IRES-DHFR-SV40?polyA

VH-IRES-dhfr3 EF-1 α-guide secrete peptide-VH can swell site (XhoI+SpeI)-

Fc-intron-IRES-DHFR-SV40?polyA

Light chain expression vector main element and putting in order

VK-dhfr1 EF-1 α-guide secretes peptide-VK or VL can swell the site

(EcoRV+XhoI)-CK-intron-CK?polyA-SV40ori-DHFR-SV40?polyA

VK-dhfr2 EF-1 α-guide secretes peptide-VK can swell site (EcoRV+HindIII)-CK-CK

polyA-SV40ori-DHFR-SV40?polyA

VL-dhfr3 EF-1 α-guide secretes peptide-VL can swell site (EcoRV+HindIII)-CL-CK

polyA-SV40ori-DHFR-SV40?polyA

VK-IRES-dhfr1 EF-1 α-guide secretes peptide-VK orVL can swell site (EcoRV+XhoI)-CK-intron

-IRES-DHFR-SV40?polyA

VK-IRES-dhfr2 EF-1 α-guide secretes peptide-VK can swell the site

(EcoRV+HindIII)-CK-IRES-DHFR-SV40?polyA

VL-IRES-dhfr3 EF-1 α-guide secretes peptide-VL can swell the site

(EcoRV+HindIII)-CL-IRES-DHFR-SV40?polyA

…………………………………………………………………………………………………………………………………………

As a kind of treatment antibody, the IgG whole antibody is far superior to Fab and scFv incomplete antibody.Though phage antibody library technique is increasingly mature, the full IgG engineered antibody system in its downstream is fully matured not as yet then, especially in China.What the present invention stated is one group of carriers for humanized antibody of mammal cell efficient expression vector carrier and uses thereof, this carrier system comprises that altogether above-mentioned 12 kinds make up different IgG expression vectors, they have same or analogous skeleton carrier, different separately IgG expresses assembly, can be used to the clone of antibody variable region or Fab gene and expresses and produce the IgG whole antibody fast.This vector gene can obtain to produce total man IgG antibody stably manufactured clone along with plasmid DNA duplicating and stable duplicating in bacterium behind the transfection cells of mamma animals, obtain a kind of clinical treatment antibody preparation of feasibility.

Embodiment

Following preferential embodiment elaborates to the present invention, load does not mean that restriction content of the present invention in these embodiments, be explanation the present invention, used intermediate carrier EF-VH of the intermediate carrier that relates in the vector construction and EF-VL be by Bautz professor friend present, and pSV2-dhfr and pIRESneo be respectively available from American I nvitrogen, Premerg and Clontech company, pTA-IgGH, pTA-IgGK, pAc-K-CH3, pAc-K-Fc and pAc-L-CH3 create for the both sides laboratory makes up.Main strain X LI-Blu is available from Strategene company.Antibody gene gene in the used expression cassette in claims is created from the inventor.Embodiment 1-3 is one group of carriers for humanized antibody of mammal cell efficient expression vector preparation method and carrier feature description thereof; Embodiment 4 is one group of carriers for humanized antibody of mammal cell efficient expression vector applicability feature.

Example 1: primer used in vector construction and the application is by oneself design, and is synthetic in MWG (Germany) commercialization company, below is all primer sequences numberings:

1.BssHII-F

5′TATCCGCGCGCACTCCCCAGGTGCAACTGCTCGAGTCTGGG-3′

2.XbaI-R

5′-TGCTCTAGATTATTTTCCCGGACTTAAGGAGAG-3′

3.BstEII-HR

5′-GGAGACGGTGACCAGGGTTCCCTC-3′

4.XhoI-KR

5′-CTTGATCTCGAGCTTGGTCCCCTGGTC-3′

5.HindIII?R

5′-CGCAAGCTTATTGAATCAAAGGATATATGAC-3′

6.XohI-LR

5′-TAGGACCTCGAGCTTGGTCCCCTCCGCC-3′

7.SpeI-LR

5′-GGACTAGTCCTATGAACATTCTGTAGG-3′

8.SpeI-KR

5′-GGACTAGTCCTAACACTCTCCCCTGTTGA-3′

9.EcoRV-VL2

5′-CCGGATATCGCCCTCACGCAGCCTGCCTCCGTG-3′

10.EcoRV-VL5

5′-CCGGATATCGTG-CTCACGCCGCCCTCAGTG-3′

11.EcoRV-VK1a

5′-CCGGATATCGTGCTCACCCAGTCTCCA-3′

12.EcoRV-VK3a

5′-CCGGATATCGTGATCACGCAGCCTGCCTCCGTG-3′

Example 2: the acquisition of antibody expression box element in the carrier system: 1, skeleton carrier main element EF-1a, DHFR gene and IRES gene thereof be respectively from carrier EF-VH, EF-VL, pSV2-DHFR and IRES.2, carrier VH-dhfr1, VH-IRES-shfr1, VK-dhfr1, the light weight chain constant area gene of the antibody among the VH-IRES-dhfr1 is from contriver Bautz professor laboratory research and development carrier pTA-IgGH, pTA-K.3, carrier VH-dhfr2, VH-dhfr3, VH-IRES-dhfr2, VH-IRES-dhfr3, VK-dhfr2, VK-dhfr3, VH-IRES-dhfr2, the antibody gene among the VH-IRES-dhfr3 is researched and developed carrier pAc-K-CH3, pAc-K-Fc and pAc-L-CH3 jointly from contriver's beam rice virtue and Bautz professor laboratory.

The structure of example 3. expression vectors: as accompanying drawing 1 and accompanying drawing 2, be the skeleton carrier of serial carrier among the present invention and the synoptic diagram of different expression cassettes, it mainly is made up of following committed step:

1) with BssHII and two enzymes of XbaI and carrier EF-VH, electrophoresis reclaims the carrier band, and this carrier contains EF-1a promotor and BGHpolyA tail and new enzyme plain gene (NEO), about 5468 base pairs (bp),

2) be template with carrier pTA-IgGH, increase by PCR method with primer BssHII-F and XbaI-R and contain the weight chain constant area gene of intron gene that electrophoresis reclaims segment, about 1796bp contains VH gene clone site BssHII and BstEII.

3) be template with carrier pAc-K-CH3, increasing by PCR method with primer BssHII-F and XbaI-R does not contain the weight chain constant area gene of intron gene, and electrophoresis reclaims segment, and about 930bp contains VH gene clone site XhoI and NheI.

4) be template with carrier pAc-K-CH3, increasing by PCR method with primer BssHII-F and XbaI-R does not contain the CH Fc gene of intron gene, and electrophoresis reclaims segment, and about 630bp contains Fd gene clone site XhoI and SpeI.

5) with 1) with 2) in segment be connected, obtain intermediate carrier EF-IgGH1.With 1) with 3) in segment be connected, obtain intermediate carrier EF-IgGH2.With 1) with 4) in segment be connected, obtain intermediate carrier EF-IgGH3.

6) with SacI and two enzymes of XbaI and carrier EF-VK, electrophoresis reclaims the carrier band, and this carrier contains EF-1a promotor and CKpolyA tail and new enzyme plain gene (NEO), about 5100 base pairs (bp),

7) being template with carrier pTA-IgGK, increasing by PCR method with primer SacI-F and SpeI-KR and contain the constant region of light chain gene of intron gene, does electrophoresis reclaim segment, approximately? bp contains VL gene clone site EcoRV and XhoI.

8) be template with carrier pAc-K-CH3, increasing by PCR method with primer SacI-F and SpeI-KR does not contain the constant region of light chain gene of intron gene, and electrophoresis reclaims segment, and about 350bp contains VL gene clone site EcoRV and HindIII.

9) be template with carrier pAc-L-CH3, increasing by PCR method with primer SacI-F and SpeI-LR does not contain the light chain Lambda chain constant region gene of intron gene, and electrophoresis reclaims segment, and about 350bp contains VL gene clone site EcoRV and HindIII.

10) with 6) with 7) in segment be connected, obtain intermediate carrier EF-IgGK1.With 6) with 8) in segment be connected, obtain intermediate carrier EF-IgGK2.With 6) with 9) in segment be connected, obtain intermediate carrier EF-IgGL3.

11) with PvuII and two enzymes of BamHI and carrier pSV-dhfr, it is the segment of 1852bp that electrophoresis reclaims length, and this segment contains the initial replicon of SV40 (SV40 ori) among Fig. 1, dihydrofolate reductase gene (DHFR) and SV40polyA tail.This segment is flat with the big segment enzyme benefit of Klenow, and dephosphorylation.

12) with srf1 and two enzymes of Bst11071 and above-mentioned 6 the intermediate carrier EF-IgGH1 of carrier, EF-IgGH2, EF-IgGH3, EF-IgGK1, EF-IgGK2, EF-IgGL3, the plain resistance of new enzyme in the excision original vector is selected gene, electrophoresis reclaims carrier segment separately, and mend with the big segment enzyme of Klenow and put down, and dephosphorylation.

13) with 11) in DHFR gene segment and 12) in the different carriers segment be connected, the heavy chain expression carrier VH-dhfr1 that lists in the acquisition table 1 (the heavy chain A1 among Fig. 2), VH-dhfr2 (heavy chain A2) and VH-dhfr3 (heavy chain A3) and light chain expression vector VK-dhfr1 (the light chain A1 among Fig. 2), VK-dhfr2 (light chain A2) and VL-dhfr3 (light chain A3).

14) with PvuII and two enzymes of HindIII and carrier pSV-dhfr, electrophoresis reclaims the carrier segment that length is about 4660bp, and this segment contains among Fig. 1 b, dihydrofolate reductase gene (DHFR) and SV40polyA tail.This segment is flat with the big segment enzyme benefit of Klenow, and dephosphorylation.

15) with EcoR1 and two enzymes of SmalI and carrier pIRESlneo, it is the segment of 952bp that electrophoresis reclaims length, and this segment contains the ribosome internal binding site (IRES) among Fig. 1 b, this segment is mended with the big segment enzyme of Klenow put down, and dephosphorylation.

16) with 14) in carrier segment and 15) in the different carriers segment be connected, obtain intermediate carrier pSV-dhfr-IRES.

17) with BamHi enzyme and carrier pSV-dhfr-IRES, electrophoresis reclaims the insertion segment that length is about 2464bp, and this segment contains among Fig. 1 b, in ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40polyA tail.This segment is flat with the big segment enzyme benefit of Klenow, and dephosphorylation.

18) with two enzymes of XbaI and Bst11071 and carrier regulations 5) described in intermediate carrier EF-IgGH1, EF-IgGH2, EF-IgGH3, BGHpolyA in the excision original vector and the plain resistance of new enzyme are selected gene, electrophoresis reclaims carrier segment separately, and mend with the big segment enzyme of Klenow and put down, and dephosphorylation.

19) with two enzymes of SpeI and Bst11071 and carrier regulations 10) described in intermediate carrier EF-IgGK1, EF-IgGK2, EF-IgGL3, the plain resistance of new enzyme in the excision original vector is selected gene, electrophoresis reclaims carrier segment separately, and mend with the big segment enzyme of Klenow and put down, and dephosphorylation.

20) with 17) in pSV2-dhfr-IRES insert segment and 18) in EF-IgGH1, EF-IgGH2, three of EF-IgGH3 carrier-pellet disconnection respectively connect, obtain carrier VH-IRES-dhfr-1 (the heavy chain B1 among Fig. 2), VH-IRES-dhfr2 (heavy chain B2) and VH-IRES-dhfr3 (heavy chain B3) in the table 1.

21) with 17) in pSV2-dhfr-IRES insert segment and 19) in EF-IgGK1, EF-IgGK2, three of EF-IgGL3 carrier-pellet disconnection respectively connect, obtain the carrier VK-IRES-dhfr-1 in the table 1, (the light chain B1 among Fig. 2), VK-IRES-dhfr2 (light chain B2) and VL-IRES-dhfr3) (light chain B3)

22) finally obtain in sum, the cells of mamma animals efficient expression vector of 12 kinds of different structures described in the table 1 altogether.

Example 4: the efficient transient expression of humanized IgG antibody in eukaryotic cell: in order to test the characteristic of this carrier system, we are with the single-chain antibody scFv gene and the Fab gene that derive from the phage antibody library screening of this laboratory development, be cloned into the different carriers of this carrier system respectively according to the composition of its light chain composition, be built into different IgG expression vectors.With vector plasmid DNA and the LipofectAMINE Plus cotransfection Cos7 cell of Llipofactin Min transfection reagent box (GIBCO_BRLKit), obtain the expression of the complete humanized IgG antibody of reorganization with purifying.Fig. 3 is the expression level demonstration of the complete humanized IgG antibody of reorganization with different carriers in the carrier system of the present invention.

Claims (9)

1. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 1, light chain expression vector contains light chain expression box 1, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, the composition of heavy chain expression box 1 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site BssHII and the BstEII of heavy chain variable region gene (VH), the IgG gene heavy chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ox somatomedin poly-A tail (BGH Poly A), initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 polyA); The composition of light chain expression box 1 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the XhoI of chain variable region gene (VL), the IgG gene light chain total length constant region gene and the transcription termination signal thereof that comprise intron from the human lymphocyte genomic dna, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

2. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 2, light chain expression vector contains light chain expression box 2, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, the composition of heavy chain expression box 2 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site XhoI and the NheI of heavy chain variable region gene (VH), IgG1 gene heavy chain total length constant region gene from human lymphocyte mRNA, BGH Poly A, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A); The composition of light chain expression box 2 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Kappa chain variable region gene (Vk), IgG1 gene Kappa light chain total length constant region gene from human lymphocyte mRNA, BGH Poly A, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

3. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 3, light chain expression vector contains light chain expression box 3, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, the composition of heavy chain expression box 3 is followed successively by promotor EF-1 α, the antibody gene leader, heavy chain Fd gene clone site XhoI and SpeI, IgG1 gene heavy chain Fc constant region gene from human lymphocyte mRNA, BGH Poly A, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A); Light chain expression box 3 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Lambda chain variable region gene (V λ), IgG1 gene Lambda light chain total length constant region gene from human lymphocyte mRNA, BGH Poly A, initial son of SV40 virus transcription and promotor (SV40 ori), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

4. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 4, light chain expression vector contains light chain expression box 4, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, heavy chain expression box 4 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site BssHII and the BstEII of heavy chain variable region gene (VH), the IgG gene heavy chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A); Light chain expression box 4 is followed successively by promotor EF-1 α, the antibody gene leader, chain variable region gene (VL) cloning site EcoRV and XhoI, the IgG gene Kappa chain total length constant region gene that comprises intron from the human lymphocyte genomic dna, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

5. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 5, light chain expression vector contains light chain expression box 5, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, heavy chain expression box 5 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site XhoI and the NheI of heavy chain variable region gene (VH), IgG1 gene heavy chain total length constant region gene from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A); Light chain expression box 5 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Kappa chain variable region gene (Vk), IgG1 gene Kappa light chain total length constant region gene from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

6. efficient expression system that is used for producing human antibody at cells of mamma animals, form by heavy chain expression carrier and light chain expression vector, the heavy chain expression carrier contains heavy chain expression box 6, light chain expression vector contains light chain expression box 6, when described heavy chain expression carrier and described light chain expression vector transform cells of mamma animals simultaneously, can give expression to people source whole antibody; Wherein, heavy chain expression box 6 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site XhoI and the SpeI of heavy chain Fd gene, IgG1 gene heavy chain Fc constant region gene from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A); Light chain expression box 6 is followed successively by promotor EF-1 α, the antibody gene leader, the cloning site EcoRV and the HindIII of Lambda chain variable region gene (V λ), IgG1 gene Lambd light chain total length constant region gene from human lymphocyte mRNA, ribosome internal binding site (IRES), dihydrofolate reductase gene (DHFR) and SV40 transcription termination signal (SV40 poly A).

7. any described purposes that is used for producing the efficient expression system of human antibody among the claim 1-6 at cells of mamma animals, it is characterized in that: no matter from phage antibody library hybridoma cell strain amplification screening or secretory antibody all can be at any antigenic antibody variable gene by cloning site optionally by being provided with in the carrier, be cloned in the carrier system of any establishment of describing among the claim 1-6, thereby in cells of mamma animals, obtain the expression of total man's IgG antibody or chimeric antibody.

8. the purposes that is used for producing at cells of mamma animals the efficient expression system of human antibody according to claim 7, wherein, cells of mamma animals is a Chinese hamster ovary celI.

9. according to claim 7 or the 8 described purposes that are used for producing the efficient expression system of human antibody, it is characterized in that: can set up the permanent express cell system that efficiently expresses the IgG whole antibody and be used to produce full IgG antibody with this carrier system at cells of mamma animals.