CN105646704A - Anti-p185<erbB2> human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse - Google Patents
Anti-p185<erbB2> human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse Download PDFInfo
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- CN105646704A CN105646704A CN201510996843.6A CN201510996843A CN105646704A CN 105646704 A CN105646704 A CN 105646704A CN 201510996843 A CN201510996843 A CN 201510996843A CN 105646704 A CN105646704 A CN 105646704A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
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- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
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- Veterinary Medicine (AREA)
- Environmental Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses an anti-p185<erbB2> human-mouse chimeric antibody ChAb26. The anti-p185<erbB2> human-mouse chimeric antibody ChAb26 is mainly composed of a heavy chain gene H and a light chain gene L, and the heavy chain gene H and the light chain gene L are respectively connected to a mammary gland specific expression plasmid pBC1 in order to obtain p185<erbB2> human-mouse chimeric antibody ChAb26 mammary gland specific expression vectors pBC1-H and pBC1-L. The pBC1-H and pBC1-L are linearized, the linearized pBC1-H and pBC1-L are introduced to FVB mouse zygotes to produce a transgenic FVB mouse, and the anti-p185<erbB2> human-mouse chimeric antibody ChAb26 can be obtained through mammary gland specific expression with the transgenic FVB mouse as a mammary gland bioreactor. The antibody reduces the immunogenicity of the v monoclonal antibody, and a medicinal recombinant protein produced through the transgenic FVB mouse has the advantages of high output, low cost, high activity, easy large-scale formation, shortening of the manufacturing period of new medicines, and huge market potential.
Description
Technical field
The technology of the present invention belongs to bioengineering field, particularly relates to a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26, mammary gland specific expression vector, transgenosis FVB mouse and its preparation method.
Background technology
Shih etc. (1981) scientist finds a kind of new proto-oncogene first from rat embryo neuroblast, and names neu gene. Find that neu proto-oncogene and proto-oncogene erbB exist high homology by nucleic acid sequence analysis comparison and karyomit(e) spectrum analysis subsequently. Therefore, neu proto-oncogene is considered as a kind of genes involved of erbB proto-oncogene, and is named as erbB2/HER2. 2 districts 1 that the proto-oncogene erbB2 of the finders such as Coussens is positioned at people's No. 17 chromosome long arm are with (17q21), receptor tyrosine kinase (the RTK of its coded product to be molecular weight be 185kDa, receptortyrosinekinase), because of and be called as p185 albumen. A kind of transmembrane glycoprotein that it is made up of 1255 amino-acid residues. RTK is a superfamily (superfamily), its member comprises erbB proto-oncogene encodes product EGF-R ELISA (EGFR, and erbB2/HER2 acceptor, erbB3/HER3 acceptor and erbB4/HER4 acceptor etc. epidermalgrowthfactorreceptor). ErbB acceptor is widely distributed on the cytolemma of all epithelial cells except vascular tissue; ErbB2 acceptor is common in the various coelomic epithelium of normal people, glandular epithelium and embryo, and erbB2 acceptor all exists faint expression in them; ErbB3 acceptor is not except expressing in human hematopoietic system, and it all exists expression at the most position of human body; ErbB4 acceptor except not expressing at renal glomerulus and peripheral nerve, its its hetero-organization all in all there is expression. Mo Wai district, Mo Nei district and cross-film district (TM, transmembrane) constitute the main structure of erbB2 acceptor. Mo Wai district has specificity, and it combines with part, received signal, containing a large amount of cysteine residues tumor-necrosis factor glycoproteins; Biological effect in the born of the same parents of Mo Nei district decision somatomedin. ?he In ? that lets down be greedy for money or food ? �� desert strike ? �� fluorenes pull out steathily merchant lead fluorenes a surname's scheme rainwater in puddles cut �� �� ? Hua Wen ? 2-26 amino-acid residue to pieces and form a �� helicoidal structure, highly hydrophobic; The functional zone of erbB2 acceptor are positioned at C-terminal, mainly comprise binding site and the Tyrosylprotein kinase functional zone of ATP. The sudden change of erbB family member is relatively common in human tumor, and especially erbB2, their main function is stimulate cell growth.
The activation of erbB2 acceptor occurs relevant to tumour, and except specific etap such as fetal developments, erbB2 expression amount in the normal tissue is very low; And in contrast, the multiple malignant cells such as mammary cancer, ovarian cancer and lung cancer have all found amplification and the p185 of erbB2 geneerbB2The process LAN of albumen. Due to amplification and the p185 of erbB2 geneerbB2The process LAN of albumen, thus cause erbB2 receptor activation, and strengthen the intracellular signaling of its mediation, finally cause normal cell generation vicious transformation and form malignant tumour.
Trastuzumab(It is called as again Trastuzumab, Chinese Herceptin by name) it is the Humanized monoclonal specific antibody (humanizedmAb) acting on erbB2 albumen, it is the efficient medicine of the tumour of current most popular anti-erbB 2 high expression level. Herceptin energy specific combination is in erbB2 acceptor Bao Wai subdomainIV district, thus block the formation of erbB2 homodimer and heterodimer, and mediate and gulp down in erbB2 acceptor and degrade in lysosome, block the function of erbB2 acceptor, thus hinder the phosphorylation activation of erbB2COOH one end tail and suppress MAPK and PI3K signal pathway, the growth of T suppression cell. Research shows, the erbB2 acceptor of people is specificity by Herceptin, only acts on the clone of erbB2 gene overexpression. Its main mechanism is the IgGIFc end (CrystallineFragment by it, crystallization fragment) mediate antibody relies on efficiently cytotoxicity (antibody-dependentcellularcytotoxicity, ADCC) kills and wounds the tumour cell of erbB2 process LAN. Herceptin is used alone the growth that namely can suppress cancer cell, but it and other cellulotoxic chemotherapeutics coupling more show outstanding result for the treatment of, its mechanism of action is that Herceptin can check the receptor-mediated signal transduction of erbB2 thus cause the inhibitor of apoptosis protein down-regulated expressions such as Bcl-2 and survivin so that the susceptibility of the cellulotoxic chemotherapeutics of Dx, taxol etc. is improved by malignant cell. But, Trastuzumab produces by the mammalian cell (Chinese hamster ovary cell CHO) of suspension culture in aseptic culture medium, is the derivative Humanized monoclonal antibodies of a kind of recombinant DNA. Its production cost is extremely high thus causes tumour patient medical expense expensive, prepares anti-p185 chimeric antibody by mammary gland bioreactor of transgenic animals and very likely effectively solves this shortcoming.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26, mammary gland specific expression vector, transgenosis FVB mouse and its preparation method; gained human mouse chimeric antibody reduces the immunogenicity of mouse resource monoclonal antibody, produces that medicinal recombinant protein has output height, cost is low, activity is high by gained transgenosis FVB mouse, easily mass-producing, can shorten new drug market periods and the advantage such as market potential is huge.
For solving the problems of the technologies described above, the present invention by the following technical solutions: anti-p185erbB2Human mouse chimeric antibody ChAb26, forms primarily of heavy chain gene H and light chain gene L, and heavy chain gene H and light chain gene L has the base sequence of sequence table SEQ .ID.No.3 and SEQ.ID.No.6 respectively.
Above-mentioned anti-p185erbB2The mammary gland specific expression vector of human mouse chimeric antibody ChAb26, for pBC1-H and pBC1-L, pBC1 are mammary specific expression plasmid.
The construction process of above-mentioned mammary gland specific expression vector, by anti-p185erbB2The heavy chain gene H and light chain gene L of human mouse chimeric antibody ChAb26 are connected respectively on mammary specific expression plasmid pBC1, thus build and obtain p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
The construction process of above-mentioned mammary gland specific expression vector, is undertaken by following operation:
<1>with containing anti-p185erbB2The pUC57/pBCN-H-F2A-L-ployA-EGFP-Neo plasmid of human mouse chimeric antibody ChAb26 heavy chain H and light chain gene L is template, under LATaqDNA polysaccharase effect, and the anti-p185 taking H-F/H-R and L-F/L-R as primer amplification goes out respectivelyerbB2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L; The program of PCR reaction is 94 DEG C of 5min; 94 DEG C of 30sec; 50 DEG C of 30sec; 69 DEG C of 2min30sec; 35cycles; 69 DEG C of 7min, 4 DEG C of ��;
<2>use sepharose reclaims heavy chain gene H and the light chain gene L recovery purifying that step<1>is amplified by test kit;
<3>the heavy chain gene H and the light chain gene L that step<2>are reclaimed are connected respectively on pEASY-T1 cloning vector, obtain recon pEASY-T1-H and pEASY-T1-L;
<4>use XhoI recon pEASY-T1-H and pEASY-T1-L that step<3>obtains is carried out enzyme cut and use ethanol precipitation digestion products heavy chain gene H and light chain gene L is carried out reclaim purifying;
<5>heavy chain gene H step<4>reclaimed and light chain gene L respectively with cut the dephosphorylized mammary specific expression plasmid pBC1 with CIP alkaline phosphatase through XhoI enzyme in advance and be connected under T7 ligase enzyme effect, build and obtain anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
Primer H-F/H-R and L-F/L-R has the base sequence of sequence table SEQ .ID.No.1/SEQ.ID.No.2, SEQ.ID.No.4/SEQ.ID.No.5 respectively.
Transgenosis FVB mouse, expresses anti-p185 specificallyerbB2Human mouse chimeric antibody ChAb26.
The preparation method of above-mentioned transgenosis FVB mouse, by p185erbB2After human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L linearizing, import FVB mouse fertilized egg and obtain.
The preparation method of above-mentioned transgenosis FVB mouse, employing is fertilized, and egg nucleus is micro-injects importing altogether.
The preparation method of above-mentioned transgenosis FVB mouse, is undertaken by following operation:
<1>use NotI and SalI by anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L carries out linearizing, obtains anti-p185erbB2Specific expressed linear carrier pBC1-H-linear and pBC1-L-linear of human mouse chimeric antibody ChAb26 transgenic mouse milk;
<2>by the protokaryon micro-mode of injection altogether, mammary specific expression linear carrier pBC1-H-linear and pBC1-L-linear is carried out zygote to inject altogether; After injection, by zygote transplation to, in acceptor FVB system Mouse Uterus, obtaining F0 generation two positive transgenic FVB mouse after gestation.
Above-mentioned transgenosis FVB mouse is used to obtain anti-p185erbB2The method of human mouse chimeric antibody ChAb26, is undertaken by following operation: maternal rat delivery is after 10 days, it may also be useful to the female mouse of transgenosis is carried out milk collection by mouse milking apparatus; Then, the milk collected is transferred to respectively in sterilizing Ep pipe, 4 DEG C, the centrifugal 20min of 4000 �� g;After centrifugal end, sucking-off middle level clear liquid gently, obtains anti-p185erbB2Human mouse chimeric antibody ChAb26 solution.
For current mouse resource monoclonal antibody Problems existing, based on galactophore biological reactor and chimeric antibody principle, inventor has devised a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26 and mammary gland specific expression vector thereof, and establish the construction process of mammary gland specific expression vector and the preparation method of transgenosis FVB mouse accordingly. Anti-p185erbB2Human mouse chimeric antibody ChAb26, forms primarily of heavy chain gene H and light chain gene L, and heavy chain gene H and light chain gene L has the base sequence of sequence table SEQ .ID.No.3 and SEQ.ID.No.6 respectively. Its heavy chain gene H and light chain gene L is connected respectively on mammary specific expression plasmid pBC1, thus builds and obtain p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L. After mammary gland specific expression vector pBC1-H and pBC1-L linearizing, import FVB mouse fertilized egg and produce transgenosis FVB mouse, using it as galactophore biological reactor, obtained anti-p185 by mammary specific expressionerbB2Human mouse chimeric antibody ChAb26. Owing to pBC1 carrier has the structure of mammary specific expression, can great expression recombinant protein or antibody during for building mammary gland bioreactor of transgenic animals. According to data, the recombinant protein content expressed by the transgenic mouse milk bio-reactor of pBC1 vector construction can reach 35g/L (Youngetal, 1997), the recombinant protein content that transgenic goat galactophore biological reactor is expressed can reach 20g/L (Zeomek, 1998).
The present invention is directed to the tumour cell of erbB2 acceptor process LAN and human mouse chimeric antibody can greatly reduce the immunogenicity of mouse resource monoclonal antibody, it is thus possible to effectively avoid anti-antibody reaction to occur; Compared with the conventional art such as the preparation cell cultures of medicinal recombinant protein or microorganism fermentation tank, produce that medicinal recombinant protein has output height, cost is low, activity is high by gained transgenosis FVB mouse of the present invention, easy mass-producing, new drug market periods can be shortened and the advantage such as market potential is huge. Meanwhile, the biotherapy applying the malignant tumour that the present invention can be the erbB2 acceptor process LAN such as ovarian cancer has carried out positive exploration.
Accompanying drawing explanation
Fig. 1 is pUC57/pBCN-H-F2A-L-ployA-EGFP-Neo plasmid map in embodiment 1.
The anti-p185 of Fig. 2erbB2The pcr amplification product electrophorogram of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L, in figure, M is DNAmarkerIII; 1,2 is heavy chain gene H; 3,4 is light chain gene L.
Fig. 3 is recombinant clone plasmid pEASY-T1-H plasmid map in embodiment 1.
Fig. 4 is recombinant clone plasmid pEASY-T1-L plasmid map in embodiment 1.
Fig. 5 is the bacterium liquid PCR qualification result of recombinant clone plasmid pEASY-T1-H in embodiment 1, and in figure, M is DNAmarker1k; 1,2,8,10,11,14 is positive colony recon; 3��7,9,12��13,15��18 is negative clone.
Fig. 6 is the bacterium liquid PCR qualification result of recombinant clone plasmid pEASY-T1-L in embodiment 1, and in figure, M is DNAmarker1k; 1,6,7 is positive colony recon; 3��5,8��9 is negative clone.
Fig. 7 is recombinant clone plasmid pEASY-T1-L/pEASY-T1-H and XhoI digestion products electrophorogram thereof, and in figure, M is DNAmarkerIII; G1 is pEASY-T1-L plasmid; G2 is pEASY-T1-H plasmid;P1, P2 are pEASY-T1 clones; H is heavy chain gene H; L is light chain gene L.
Fig. 8 is anti-p185erbB2Human mouse chimeric antibody ChAb26 heavy chain gene H checks order and nucleotide homology compare of analysis result.
Fig. 9 is anti-p185erbB2Human mouse chimeric antibody ChAb26 light chain gene L checks order and nucleotide homology compare of analysis result.
Figure 10 is the electrophorogram that XhoI enzyme cuts pBC1 skeleton, and in figure, M is �� DNAmarker; 1��6 is pBC1 plasmid (linearly); 7 is pBC1 plasmid (ring-type).
Figure 11 is that XhoI enzyme cuts recombinant clone plasmid pEASY-T1-L electrophorogram, and in figure, M is 1kDNAmarker; P1, P2 are pEASY-T1 cloned plasmids; L1, L2 are Chimeric Antibody Light Chain Gene L.
Figure 12 is that XhoI enzyme cuts recombinant clone plasmid pEASY-T1-H electrophorogram, and in figure, M is 1kDNAmarker; P1��P4 is pEASY-T1 cloned plasmids; H1��H4 is chimeric antibody heavy gene H.
Figure 13 is mammary gland specific expression vector pBC1-L plasmid map in embodiment 1.
Figure 14 is mammary gland specific expression vector pBC1-H plasmid map in embodiment 1.
Figure 15 is mammary gland specific expression vector pBC1-L bacterium liquid PCR qualification result, and in figure, M is DNAmarkerIII; 2,9 is positive colony; 1,3��8,10��15 is negative clone.
Figure 16 is mammary gland specific expression vector pBC1-H bacterium liquid PCR qualification result, and in figure, M is DNAmarkerIII; 2,3,7,9,11 is positive colony; 1,4��6,8,10 is negative clone.
Figure 17 is mammary gland specific expression vector pBC1-L electrophorogram, and in figure, M is DNA �� marker; 1,2 is pBC1-L plasmid.
Figure 18 is mammary gland specific expression vector pBC1-H electrophorogram, and in figure, M is DNA �� marker; 1,2 is pBC1-H plasmid.
Figure 19 is the anti-p185 of mammary gland specific expression vector pBC1-LerbB2Middle chimeric antibody ChAb26 light chain gene L nucleotide sequence homology compare of analysis figure, in figure, underlined in red is XhoI multiple clone site.
Figure 20 is the anti-p185 of mammary gland specific expression vector pBC1-HerbB2Middle chimeric antibody ChAb26 heavy chain gene H nucleotide sequence homology compare of analysis figure, in figure, underlined in red is XhoI multiple clone site.
Figure 21 is mammary specific expression linearized vector pBC1-L-linear plasmid map.
Figure 22 is mammary specific expression linearized vector pBC1-H-linear plasmid map.
Figure 23 is mammary gland specific expression vector pBC1-H plasmid linearization electrophorogram, 1 (A) in figure, and 2 (A) are pBC1-H-linear fragments; 1 (B), 2 (B) are pBR322+Ampicillin fragments; 3 is pBC1-H plasmid; M is �� Marker.
Figure 24 is mammary gland specific expression vector pBC1-L plasmid linearization electrophorogram, and in figure, 1 (A) is pBC1-L-linear fragment; 1 (B) is pBR322+Ampicillin fragment; 2 is pBC1-L plasmid; M is �� Marker.
Figure 25 is that in embodiment 2, F0 is for transgenic mice mouse tail Direct PCR electrophorogram, and in figure, N is wild-type FVB mouse; 1��8 is mouse number; W is ddH2O; M is DNAMarkerIII; NC is FVB mouse internal reference; TgL is Chimeric Antibody Light Chain Gene L portion DNA fragmentation; TgH is chimeric antibody heavy gene H part DNA fragmentation.
Figure 26 is that in embodiment 2, F1 (F0-1) is for transgenic mice mouse tail Direct PCR electrophorogram, and in figure, N is wild-type FVB mouse; 1��10 is mouse number; W is ddH2O; M1 is DNAMarkerIII; M2 is DNAMarkerI; NC is FVB mouse internal reference; TgL is Chimeric Antibody Light Chain Gene L portion DNA fragmentation;TgH is chimeric antibody heavy gene H part DNA fragmentation.
Figure 27 is F2 generation (F0-1) transgenic mice mouse tail Direct PCR electrophorogram in embodiment 2, and in figure, N is wild-type FVB mouse; 1��9 is mouse number; W is ddH2O; M1 is DNAMarkerIII; M1 is DNAMarker100; NC is FVB mouse internal reference; TgL is Chimeric Antibody Light Chain Gene L portion DNA fragmentation; TgH is chimeric antibody heavy gene H part DNA fragmentation.
Figure 28 is F3 generation (F0-1) transgenic mice mouse tail Direct PCR electrophorogram in embodiment 2, and in figure, N is wild-type FVB mouse; 1��11 is mouse number; W is ddH2O; M is DNAMarkerIII; NCFVB mouse internal reference; TgL is Chimeric Antibody Light Chain Gene L portion DNA fragmentation; TgH is chimeric antibody heavy gene H part DNA fragmentation.
Figure 29 is F4 generation (F0-1) transgenic mice mouse tail Direct PCR electrophorogram in embodiment 2, and in figure, N is wild-type FVB mouse; 1��9 is mouse number; W is ddH2O; M is DNAMarkerI; NC is FVB mouse internal reference; TgL Chimeric Antibody Light Chain Gene L portion DNA fragmentation; TgH is chimeric antibody heavy gene H part DNA fragmentation.
Figure 30 is TR-PCR product agarose gel electrophoresis figure in embodiment 2, in figure: N is FVB wild-type mice mammary tissue cDNA; F0 is the two positive mice mammary tissue cDNA of F0 transgenosis; F1 is the two positive mice mammary tissue cDNA of F1 generation transgenosis; F2 is that F2 is for the two positive mice mammary tissue cDNA of transgenosis; M is DNAMarkerI; 1 is the internal reference product of FVB wild-type mice/transgenic mouse milk tissue cDNA; The RT-PCR product of 2 chimeric antibody heavy gene H Partial cDNA fragments; 3 is the RT-PCR product of Chimeric Antibody Light Chain Gene L portion cDNA fragment.
Figure 31 is the solubility curve of fluorescent quantitative PCR experiment internal reference primer in embodiment 2.
Figure 32 is anti-p185 in embodiment 2erbB2The solubility curve of middle chimeric antibody ChAb26 light chain gene L.
Figure 33 is anti-p185 in embodiment 2erbB2The solubility curve of middle chimeric antibody ChAb26 heavy chain gene H.
Figure 34 is the mRNA relative expression quantity figure of chimeric antibody in embodiment 2.
Figure 35 is the expression figure that in embodiment 2, immunohistochemical methods WesternBlot identifies chimeric antibody ChAb26, and in figure, 50KDa is 50KDa heavy chain; 25KDa is 25KDa light chain; MIgG is mouse IgG; HIgG is human IgG; Tg is transgenic mouse milk; NC is wild-type FVB mouse milk.
Figure 36 is the concentration results figure of the expression of sandwich ELISA qualification chimeric antibody ChAb26 in embodiment 3, and IgG is human IgG; P is PBS solution; N is FVB wild-type mice milk; Tg is transgenic mouse milk; 1-3 is multiple hole numbering.
Figure 37 is the expression figure (Nikon200 ��) of the expression immunohistochemical methods qualification chimeric antibody ChAb26 that WesternBlot detects chimeric antibody ChAb26 in embodiment 2, and in figure, A is FVB wild-type mice mammary tissue; B is that F0 is for the two positive mice mammary tissue of transgenosis; C is the two positive mice mammary tissue of F1 generation transgenosis; D is that F2 is for the two positive mice mammary tissue of transgenosis.
Figure 38 in the expression concentration of chimeric antibody (measure) result figure, the figure of sandwich ELISA qualification chimeric antibody ChAb26 in embodiment 21 is 200ng/ml human IgG; 2 is 100ng/ml human IgG; 3 is 50ng/ml human IgG; 4 is 25ng/ml human IgG; 5 is 12.5ng/ml human IgG; 6 is 6.25ng/ml human IgG; 7 is 3.125ng/ml human IgG; 8 is 0ng/ml human IgG;9 is FVB wild-type mice milk; 10 is transgenic mouse milk; A-c is multiple hole numbering.
Figure 39 is ELISA quantitative criterion curve and the regression equation figure of chimeric antibody in embodiment 3 transfer Gene Double positive mice milk, F=29.331 (P < 0.05) in figure; R2=0.83; T=5.416 (P < 0.05).
Figure 40 is the antigen-specific that in embodiment 3, Salmonella measures chimeric antibody, and in figure, A figure is the antigen-specific figure that Salmonella measures chimeric antibody, and 1 is the anti-human erbB2 monoclonal antibody of mouse; 2 is human IgG; 3 is mouse IgG; 4 is PBS; 5 is the two positive mice milk of transgenosis; 6 is FVB wild-type mice milk; A-c is multiple hole numbering; B figure is the antigen-specific figure that Salmonella measures chimeric antibody, and 1 is the anti-human erbB2 monoclonal antibody of mouse; 2 is human IgG; 3 is mouse IgG; 4 is PBS; 5 is the two positive mice milk of transgenosis; 6 is FVB wild-type mice milk; A-c is multiple hole numbering.
Figure 41 is the humanized result figure that in embodiment 3, westernblot analyzes chimeric antibody ChAb26, and in figure, hIgG is human IgG; F0 is that the two positive F0 of transgenosis is for mouse milk; F1 is the two positive F1 generation mouse milk of transgenosis; F2 is that the two positive F2 of transgenosis is for mouse milk; F3 is that the two positive F3 of transgenosis is for mouse milk; NC:FVB wild-type mice milk; MIgG is mouse IgG.
Figure 42 is SKOV3 cell growth curve in embodiment 3.
Figure 43 be SKOV3 apoptosis in embodiment 3 morphological feature (24h, HE dye, Nikon �� 400) figure, figure in A be cell blank control group; B is FVB wild-type mice milk treatment group; C is 0.48mg.L-1ChAb26 treatment group; D is 0.50mg.L-1Herceptin treatment group.
Figure 44 is ultrastructure feature (24h, the H-7650) figure of SKOV3 apoptosis in embodiment 3, and in figure, A is cell blank control group; B is FVB wild-type mice milk treatment group; C is 0.48mg.L-1ChAb26 treatment group; D is 0.50mg.L-1Herceptin treatment group.
Figure 45 is that to detect A in apoptosis (24h) figure, the figure of SKOV3 be cell blank control group to flow cytometry (FCM); B is FVB wild-type mice milk treatment group; C is 0.48mg.L-1ChAb26 treatment group; D is 0.50mg.L-1Herceptin treatment group.
Figure 46 is that to detect A in cell cycle (24h) figure, the figure of SKOV3 be cell blank control group to flow cytometry (FCM); B is FVB wild-type mice milk treatment group; C is 0.48mg.L-1ChAb26 treatment group; D is 0.50mg.L-1Herceptin treatment group.
Embodiment
The anti-p185 of embodiment 1erbB2The structure of human mouse chimeric antibody ChAb26 transgenic mouse milk specific expression vector
1, the acquisition of goal gene
With containing anti-p185erbB2The pUC57/pBCN-H-F2A-L-ployA-EGFP-Neo plasmid of human mouse chimeric antibody ChAb26 heavy chain H and light chain gene L is template (Fig. 1), under LATaqDNA polysaccharase effect, the anti-p185 taking H-F/H-R (SEQ.ID.NO.1/SEQ.ID.NO.2) and L-F/L-R (SEQ.ID.NO.4/SEQ.ID.NO.5) as primer amplification goes out respectivelyerbB2Human mouse chimeric antibody ChAb26 heavy chain gene H (SEQ.ID.NO.3) and light chain gene L (SEQ.ID.NO.6). Through 1% agarose gel electrophoresis qualification, heavy chain gene H is consistent with theoretical value with light chain gene L specific band, and size is respectively 2113bp and 799bp (Fig. 2).
The amplimer of the table 1 goal gene sequence of restriction enzyme XhoI (the underscore part be)
The reaction system of table 2PCR
The program of PCR reaction is: 94 DEG C of 5min;94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C of 2min30sec, 35cycles; 69 DEG C of 7min; 4 DEG C of ��.
PCR primer is detected with 1% agarose gel electrophoresis, then uses DNA sepharose glue to reclaim test kit (TIANgelMidiPurificationKit) and PCR primer is carried out purifying and recovery.
2, the screening of recon
The PCR primer reclaimed is connected with pEASY-T1 cloning vector, thus obtain and connect product pEASY-T1-H (SEQ.ID.NO.7) and pEASY-T1-L (SEQ.ID.NO.8), then will connect product and transform DH5 �� competent cell, get 100 �� L bacterium liquid coated plates, overnight incubation.
Above-mentioned Amp(+)After LB culture dish overnight incubation, it is seen that the bacterium colony being dispersed in, therefrom select 10 single bacterium colonies, it is inoculated in different 10ml Glass tubings, Amp in each pipe(+)The volume of LB nutrient solution is 2.5mL, finally places in constant-temperature table and cultivates 16-20 hour (37 DEG C, 200rpm).
With the general primer M13 of pEASY-T1, taking recombinant plasmid bacterium liquid as template, utilize bacterium liquid PCR method qualification recon. Amplified production is known through 1% agarose gel electrophoresis qualification, in Fig. 5,1,2,8,10,11 and No. 14 bacterium liquid are pEASY-T1-H recombinant clone plasmid bacterial solution, and the specific band of its bacterium liquid pcr amplification is consistent with theoretical value, and size is about 2300bp (Fig. 5). In Fig. 6,1,6 and No. 7 bacterium liquid is pEASY-T1-L recombinant clone plasmid bacterial solution, and the specific band of its bacterium liquid pcr amplification is consistent with theoretical value, and size is about 1000bp (Fig. 6). Finally pEASY-T1-H/L recombinant clone plasmid bacterial solution is carried out little carrying, and use XhoI restriction enzyme that pEASY-T1-H/L recombinant clone plasmid is carried out enzyme and cut, known through 1% agarose gel electrophoresis qualification, pEASY-T1-H/L recombinant clone plasmid band is consistent with theoretical value, being respectively 5930bp and 4615bp, enzyme cuts the pEASY-T1 cloned plasmids band of generation and anti-p185erbB2The band of human mouse chimeric antibody heavy chain gene H or light chain gene L is also consistent with theoretical value, is respectively 3829bp, 2113bp and 799bp (Fig. 7).
The general primer M13 of table 3pEASY-T1
The reaction system of table 4PCR
The program of PCR reaction is: 94 DEG C of 5min; ; 94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C of 2min30sec, 35cycles; 69 DEG C of 7min; 4 DEG C of ��.
By the bacterium liquid correct through above-mentioned preliminary identification, using the general primer M13 of pEASY-T1 plasmid as sequencing primer, Sanger chain termination method is adopted to carry out check order (Hua Da genome company). Finally by the anti-p185 of sequencing result and desired designerbB2The sequence of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L compares. Known anti-p185 by analysiserbB2The anti-p185 of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L sequencing result sequence and desired designerbB2Human mouse chimeric antibody heavy chain gene H consistent with light chain gene L sequence (Fig. 8 and Fig. 9).
Adopt TIANprepMiniPlasmidKit test kit that recombinant plasmid is carried out extracting.
3, the preparation of carrier
Use XhoI digestion with restriction enzyme mammary specific expression plasmid pBC1, and with the mammary specific expression plasmid pBC1 cut without enzyme in contrast, known through 1% agarose gel electrophoresis qualification, mammary specific expression plasmid pBC1 is cut into linear pBC1 plasmid by thorough enzyme, band consistent with theoretical value (Figure 10). Then with alkaline phosphatase CIP, the pBC1 mammary gland specific expression vector after XhoI endonuclease digestion is carried out dephosphorylation reaction.
Use the pEASY-T1-H/pEASY-T1-L plasmid of XhoI endonuclease digestion through checking order correct, and by ethanol precipitation by the purifying of product (H/L chain DNA fragment) and recovery. Known through 1% agarose gel electrophoresis qualification, recombinant clone plasmid pEASY-T1-L or pEASY-T1-H is cut into pEASY-T1 plasmid and the DNA fragmentation of ChAb26 Chimeric Antibody Light Chain Gene L or heavy chain gene H by thorough enzyme, band consistent with theoretical value (Figure 11 and Figure 12).
4, mammary gland specific expression vector is built
Use T7 ligase enzyme by the DNA fragmentation of heavy chain gene H or light chain gene L with in advance through XhoI enzyme cut with dephosphorylation after pBC1 plasmid be connected, build anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H/pBC1-L. And transform DH5 �� competence bacteria and it is carried out amplification cultivation, finally use general primer pBC1-R/F to carry out bacterium liquid PCR and identify. Through 1% agarose gel electrophoresis qualification it will be seen that successfully build anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-L or pBC1-H (SEQ.ID.NO.9 and SEQ.ID.NO.10), result is such as Figure 15 and Figure 16. In Figure 15,2 is consistent with theoretical value with the amplified band of the bacterium liquid PCR of No. 9 bacterium liquid, about 1000bp. In Figure 16,2,3,7,9 is consistent with theoretical value with the amplified band of the bacterium liquid PCR of No. 11 bacterium liquid, about 2300bp. Finally pBC1-L/pBC1-H recombinant clone plasmid bacterial solution is carried out little carrying, identify it will be seen that pBC1-L/pBC1-H recombinant clone plasmid band is consistent with theoretical value through 1% agarose gel electrophoresis, be respectively 22421bp and 23735bp (Figure 17 and Figure 18). By the bacterium liquid correct through bacterium liquid PCR preliminary identification, Hua Da genome company is sent to check order using general primer pBC1F and pBC1R of pBC1 as sequencing primer. Through to sequencing result compare of analysis it will be seen that anti-p185erbB2The anti-p185 of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L sequencing result sequence and desired designerbB2Human mouse chimeric antibody ChAb26 heavy chain gene H is consistent with light chain gene L sequence, and is just to connection, and wherein XhoI multiple clone site marks (Figure 19 and Figure 20) especially with red underscore. Finally use the big extraction reagent kit of QIAGEN (QIAGENPlasmidMaxiKits) mammary gland specific expression vector pBC1-H/pBC1-L is carried out plasmid to carry greatly, it is convenient to carry out the experiments such as follow-up Linearity, microinjection.
The general primer of table 5pBC1
The reaction system of table 6PCR
The program of PCR reaction is: 94 DEG C of 5min; 94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C, 2min30sec, 35cycles; 69 DEG C of 7min; 4 DEG C of ��.
The anti-p185 of embodiment 2erbB2The preparation of human mouse chimeric antibody ChAb26 transgenosis FVB mouse
1, anti-p185erbB2The linearizing of human mouse chimeric antibody mammary gland specific expression vector pBC1-H/pBC1-L
Adopt the anti-p185 that embodiment 1 is built by SalI and NotI enzymes double zyme cuttingerbB2Human mouse chimeric antibody ChAb26 mammary specific expression plasmid pBC1-H/pBC1-L carries out linearizing, and result is as shown in figure 23 and figure 24. Mammary specific expression plasmid pBC1-H (23735bp) or pBC1-L (22421bp) are under the acting in conjunction of restriction enzyme NotI and SalI, linearized respectively is pBC1-H-linear fragment (17861bp, or pBC1-L-linear fragment (16547bp, SEQ.ID.NO.12) SEQ.ID.NO.11).
2, micro-injection altogether prepares transgenic mice
Under match industry (Suzhou) biotech company assists, linearizing mammary specific expression plasmid pBC1-H-linear and pBC1-L-linear concentration dilution to suitable concn (at least needing containing 5-10ugDNA) are carried out the micro-injection altogether of protokaryon and prepares transgenic mice.Inject 200 FVB system mouse fertilized eggs, after injection, by wherein 100 zygote transplations to 5 that form is good acceptor FVB system's Mouse Uterus (uterine tube). Through the qualification of mouse tail Direct PCR, obtaining F0 after gestation for positive transgenic mouse two in mouse is 8, wherein male and female half and half.
3, mouse tail direct PCR method qualification transgenic mice
After mouse is born 4 weeks, cut its 0.2��0.5cm tail point tissue, good fortune border, Chengdu MouseTailDirectPCRKit is used to carry out positive transgenic mouse qualification, wherein with TgHF/R (SEQ.ID.NO.13/SEQ.ID.NO.14) and TgLF/R (SEQ.ID.NO.15/SEQ.ID.NO.16) two to primer as detection primer, controlF/R (SEQ.ID.NO.17/SEQ.ID.NO.18) is as internal reference primer. Reaction carries out 1% agarose gel electrophoresis analytical results after terminating. Taking TgHF/R and TgLF/R two, primer all there is the mouse DNA of amplification as two positive; Only primer is had amplification for single positive by one, and the mouse DNA that primer amplification band is more weak is all regarded as feminine gender without amplification or two by primer by two. Have detected 8 F0 mouse altogether, find that 8 transgenic mices are the two positive mice of transgenosis entirely through PCR detection, namely contain the mouse (Figure 25) of chimeric antibody ChAb26 heavy chain gene H and light chain gene L. In them 4 female two positive mouse are numbered respectively F0-1 to F0-4,4 only male two positive mouse be numbered F0-5 to F0-8 respectively. Using two for these 8 transgenosiss positive mice as Founder mouse, carry out expanding population and gene pure after their sexual maturity, and respectively to below Founder mouse four generation mouse carry out PCR detection by generation. Use controlF/R, TgHF/R and TgLF/R tri-to primer, each the young mouse produced is detected. The F1 to F4 of F0-1 female mice for the mouse tail Direct PCR qualification result of mouse respectively as shown in Figure 26, Figure 27, Figure 28 and Figure 29. To F0 for 4 mouse (F0-1, F0-2, F0-4 and F0-8 in 8 two positive transgenic mouse, and F0-3, F0-5, F0-6 and F0-7 tetra-F0 are for transgenic mice dead generation without male offspring) all mouse of breeding of F1 to F4 generation analyze known, it is similar that the offspring that F0-2, F0-4 breed with F0-8 is bred the two positive rate mean values in 98 mouse to F0-1, is about 30%. Finally obtain the two positive mice (F0-1, F0-2, F0-4 and F0-8) of 4 transgenosiss and breed 57 female pair of positive transgenic mice progeny altogether, 52 male pair of positive transgenic mice progeny.
The primer of table 7 mouse tail Direct PCR
The reaction system of table 8PCR
The program of PCR reaction is: 94 DEG C of 5min; 94 DEG C of 30sec, 60 DEG C of 35sec, 72 DEG C of 1mi, 35cycles; 72 DEG C of 7min; 4 DEG C of ��.
4, the expansion of transgenic mice is bred
For the two positive FVB mouse of transgenosis, the transgenosis F0 obtained is carried out expansion breed. Mode is as follows: F0 mouse and wild-type FVB mouse hybrid, obtains F1 generation mouse; Choose and get the two positive offspring of transgenosis, carry out mating in brood, obtain F2 for mouse, choose and get wherein two positive individuals, carry out sib mating, obtain F3 for mouse. The F3 of two positive is carried out hybridization in compatriot for mouse, obtains F4 for mouse.
5, the mRNA of the two positive mice of RT-PCR method qualification transgenosis expresses
By the mammary tissue of lactation to transgenosis F0 to F2 generation two positive mice, with the mammary tissue of wild-type mice lactation in contrast, carry out RT-PCR detection.With house-keeping gene m ��-ActinF/R (SEQ.ID.NO.19/SEQ.ID.NO.20), Hup/Hlw (SEQ.ID.NO.21/SEQ.ID.NO.22) and Lup/Llw (SEQ.ID.NO.23/SEQ.ID.NO.24) three, the cDNA that primer pair obtains is carried out RT-PCR and identify F1, F2 pair of positive mice that transgenosis FVB mouse breeds. Taking Hup/Hlw and Lup/Llw two, primer all there is the mouse DNA of amplification as two positive; Only primer is had amplification for single positive by one, and the mouse DNA that primer amplification band is more weak is all regarded as feminine gender without amplification or two by primer by two. Visible through 2% agarose gel electrophoresis, transgenosis F0 to F2 lactation generation two positive mice and lactation wild-type mice mammary tissue four samples in, all there is internal reference cDNA object band to produce at 203bp place, in transgenosis F0 to F2 lactation generation pair positive mice three samples, all there is anti-p185 at 134bp placeerbB2Human mouse chimeric antibody ChAb26 heavy chain gene H part segment cDNA object band produces, and all there is anti-p185 at 73bp placeerbB2The cDNA part fragment object band of human mouse chimeric antibody ChAb26 light chain gene L produces, and lactation does not find corresponding object band (Figure 30) in FVB wild-type mice mammary tissue. Result shows, lactation, the two positive mice mammary tissue of transgenosis existed anti-p185erbB2The expression of the mRNA of human mouse chimeric antibody ChAb26 light chain gene L and heavy chain gene H.
The primer of table 9RT-PCR or fluorescent PCR detection
The reaction system of table 10PCR
The program of PCR reaction is: 94 DEG C of 5min; 94 DEG C of 30sec, 61 DEG C of 30sec, 72 DEG C of 30sec, 35cycles; 72 DEG C of 7min; 4 DEG C of ��.
6, the mRNA of quantitative fluorescent PCR qualification transgenosis FVB two sun mouse expresses
By to transgenosis F0 to F2 generation lactation two positive mice mammary tissue, with m ��-ActinF/R (SEQ.ID.NO.19/SEQ.ID.NO.20), Hup/Hlw (SEQ.ID.NO.21/SEQ.ID.NO.22) and Lup/Llw (SEQ.ID.NO.23/SEQ.ID.NO.24) three, primer pair transgenosis F0 to F2 is detected for the mammary tissue of the lactation of transgenic mice, wherein with the mammary tissue of wild-type mice lactation in contrast. Taking Hup/Hlw and Lup/Llw two, primer all there is the mouse DNA of amplification as two positive; Only primer is had amplification for single positive by one, and the mouse DNA that primer amplification band is more weak is all regarded as feminine gender without amplification or two by primer by two. Result shows, transgenosis F0 to F2 lactation generation two positive mice and lactation wild-type mice mammary tissue four samples in, house-keeping gene m ��-ActinF/R primer all can amplify single and sharp-pointed solubility curve (Figure 31). In transgenosis F0 to F2 lactation generation pair positive mice three samples, light chain detection primer Lup/Llw all can amplify single and sharp-pointed solubility curve (Figure 32), and lactation, wild-type mice sample of breast tissue failed to amplify solubility curve. In transgenosis F0 to F2 lactation generation pair positive mice three samples, heavy chain detection primer Hup/Hlw all can amplify single and sharp-pointed solubility curve (Figure 33), and lactation, wild-type mice sample of breast tissue failed to amplify solubility curve. In sum, fluorescent quantitative PCR experiment shows that lactation, the two positive mice mammary tissue of transgenosis existed anti-p185erbB2The expression (Figure 34) of the mRNA of human mouse chimeric antibody ChAb26 light chain gene L and heavy chain gene H.
The anti-p185 of embodiment 3erbB2The acquisition of human mouse chimeric antibody ChAb26 and qualification
1, anti-p185erbB2The acquisition of human mouse chimeric antibody ChAb26
Maternal rat delivery is after 10 days, it may also be useful to the female mouse (lactation) of transgenosis is carried out milk collection by mouse milking apparatus (WAT2006);Then the milk collected in collection tube is transferred to respectively in sterilizing Ep pipe, 4 DEG C, the centrifugal 20min of 4000 �� g; After centrifugal end, milk is divided into three layers, upper strata oyster white be butterfat, lower floor's insoluble substance, middle level is clear liquid. Penetrating Ep pipe with 1ml disposable syringe at middle level lower edge, sucking-off middle level clear liquid gently, is anti-p185erbB2Human mouse chimeric antibody ChAb26 solution.
2, Westernblot detects the expression of chimeric antibody ChAb26 in the two positive mice milk of transgenosis
With human IgG albumen as positive control, detected the expression of chimeric antibody ChAb26 in transgenic mice and wild-type FVB mouse milk by Westernblot. In the two positive mice milk of human IgG protein groups and transgenosis, heavy chain of antibody and light chain specific band occur at relative molecular mass 50KDa and 25KDa position, and mouse IgG negative control group and wild-type FVB mouse milk group, not finding heavy chain of antibody and light chain specific band (Figure 35), the chimeric antibody ChAb26 constructed by explanation can express in transgenic mouse milk.
3, sandwich ELISA method measures the expression of chimeric antibody ChAb26 in the two positive mice milk of transgenosis
With human IgG albumen as positive control, PBS solution is as negative control, the expression of results display of chimeric antibody ChAb26 in transfer Gene Double positive mice milk and EVB wild-type mice milk is measured by sandwich ELISA method, transgenic mouse milk is positive reaction, and EVB wild-type mice milk is negative reaction, namely containing chimeric antibody ChAb26 in transgenic mouse milk, EVB wild-type mice milk is not containing chimeric antibody ChAb26 (Figure 36 and table 11). This shows anti-p185erbB2Human mouse chimeric antibody ChAb26 light chain gene L and heavy chain gene H all can express in transgenic mouse milk tissue, and can correctly be assembled into active antibody, and is secreted into extracellular well. Visible, Success in Experiment constructs anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland-specific expression vector pBC1-H and pBC1-L, and the success anti-p185 of preparationerbB2Human mouse chimeric antibody ChAb26 transgenic mouse milk bio-reactor model.
Table 11 sandwich ELISA analyzes anti-p185erbB2The expression of human mouse chimeric antibody ChAb26
Wherein, 1:F=5.4E2, P < 0.05; 2:PBS group and EVB wild-type mice milk group are without significant difference; 3: human IgG albumen and other three groups exist significant difference; 4: transgenic mouse milk and other three groups exist significant difference.
4, chimeric antibody ChAb26 in Use immunohistochemistrySP SP detection transgenic mouse milk
Use chimeric antibody ChAb26 in the Use immunohistochemistrySP SP two positive mice mammary gland of detection transgenosis, result display (Figure 37), in lactation wild-type EVB mouse mammary tissue, the tenuigenin of gland body or cytolemma are not dyed to yellow or brown, namely show that there is not chimeric antibody ChAb26 lactation in wild-type EVB mouse mammary gland expresses. And the tenuigenin of gland body or cytolemma are all colored yellow or brown during F0, F1 and F2 are for the two positive mice mammary tissue of transgenosis in lactation, during even F0 is for transgenic mouse milk Tissue cavities in lactation, observe the chimeric antibody ChAb26 being dyed to brown of big area. Show that F0, F1 and F2 existed chimeric antibody ChAb26 for gland body in the two positive mice mammary tissue of transgenosis and expressed lactation, namely successfully prepare anti-p185erbB2Human mouse chimeric antibody transgenic mouse milk bio-reactor.
The anti-p185 of embodiment 4erbB2The qualification of human mouse chimeric antibody ChAb26 related immunological characteristic
1, sandwich ELISA method detects the concentration of chimeric antibody ChAb26 in the two positive mice milk of transgenosis
Enzyme plate use coating buffer anti-human for sheep �� chain antibody (10 �� g/ml) is wrapped quilt, then by the concentration of chimeric antibody ChAb26 in the sandwich ELISA method two positive mice milk of detection transgenosis. The OD450 value (table 12 and Figure 38) that the human IgG albumen of use standard records through ELISA experiment has made typical curve, and calculates its correlation regression equation Y=0.04 ��+0.496 (Figure 39). The OD value of two for each transgenosis positive mice milk sample is substituted into regression equation calculation, after conversion, obtains anti-human p185 in milkerbB2The content of human mouse chimeric antibody ChAb26. Through computational analysis it will be seen that anti-human p185 in the two positive mice milk of transgenosiserbB2The content of human mouse chimeric antibody ChAb26 is at 0.231mg.L-1-0.248mg.L-1(table 13). Anti-human p185 in the two positive mice milk of transgenosiserbB2The average content of human mouse chimeric antibody ChAb26 is 0.240mg.L-1��
Table 12ELISA measures the OD450 value of each concentration human IgG albumen
Wherein, P < 0.05.
Table 13ELISA measures the OD450 value of the two positive mice milk of transgenosis
2, Salmonella method detects the antigen-specific of chimeric antibody in the two positive mice milk of transgenosis
Using coating buffer by 10 �� g/ml people erbB2 extracellular region antigen recombinant protein, 100ul/ hole, is coated in enzyme plate. Then by the antigen-specific of chimeric antibody in the Salmonella method two positive mice milk of detection transgenosis. Result shows, and in the two positive mice milk of transgenosis, chimeric antibody ChAb26 can be combined with the people erbB2 extracellular region antigen protein of bag quilt, also can by goat anti-human igg's Fc fragment identification, and be positive reaction in Salmonella is tested. The anti-human erbB2 monoclonal antibody of mouse is not because being combined and the reaction that is negative with goat anti-human igg FC-HRP. When use sheep anti-mouse igg Fc-HRP is anti-as two, though chimeric antibody ChAb26 can be combined with the people erbB2 extracellular region antigen protein of bag quilt in the two positive mice milk of transgenosis, but can not be combined and the reaction that is negative with sheep anti-mouse igg Fc-HRP. And the anti-human erbB2 monoclonal antibody of mouse is combined and the reaction that is positive with sheep anti-mouse igg FC-HRP. FVB wild-type mice milk, human IgG, mouse IgG and PBS no matter two anti-be that goat anti-human igg Fc-HRP or sheep anti-mouse igg Fc-HRP are all negative reaction (Figure 39, Figure 40 and table 14). This shows that in the two positive mice milk of transgenosis, chimeric antibody ChAb26 has the specificity being combined specifically with people erbB2 extracellular region antigen.
Table 14 Salmonella analyzes antigen-specific and the humanized of ChAb26
In table, a: two resist: goat anti-human igg-HRPb: two resists: rabbit anti-mouse igg-HRP.
3, sandwich ELISA method detects the humanized of chimeric antibody ChAb26 in transgenic mouse milk
Enzyme plate use coating buffer anti-human for sheep �� chain antibody (10 �� g/ml) is wrapped quilt, then by chimeric antibody ChAb26 experiment in sandwich ELISA method detection transgenic mouse milk, in the two positive mice milk of transgenosis, chimeric antibody ChAb26 can be combined by �� chain antibody anti-human with the sheep being coated in enzyme plate, also can be combined the reaction that is positive with goat anti-human igg Fc-HRP. And FVB wild-type mice milk is negative reaction in sandwich ELISA is tested, prove that in transgenosis pair positive mice milk, chimeric antibody ChAb26 contains �� chain and the heavy chain Fc section of human IgG, namely has humanized.
4, Salmonella method detects the humanized of chimeric antibody ChAb26 in transgenic mouse milk
By antigen coated in enzyme plate for people erbB2 extracellular region, resisting taking goat anti-human igg Fc-HRP as two carries out in Salmonella experiment, after in the two positive mice milk of transgenosis, chimeric antibody ChAb26 can be combined with the people erbB2 extracellular region antigen being coated in enzyme plate, being combined with goat anti-human igg Fc-HRP is positive reacts and can not combine by rabbit anti-mouse igg Fc-HRP.And FVB wild-type mice milk is negative reaction in Salmonella is tested, prove that in transgenosis pair positive mice milk, chimeric antibody ChAb26 has humanized.
5, the humanized of chimeric antibody ChAb26 in WesternBlot experimental identification transgenic mouse milk
In collection F0 generation, for the two positive mice milk of transgenosis, with FVB wild-type mice milk in contrast, carries out the humanized of chimeric antibody ChAb26 in WesternBlot experimental identification transgenic mouse milk to F3. Result display (Figure 41), in the two positive mice milk of transgenosis, the protein band of chimeric antibody ChAb26 at 50KDa place can be identified by goat anti-human igg Fc-HRP, dyes single specific immune band. The heavy chain of its size position just corresponding human IgG antibody, positive control human IgG also dyes single specific immune band at 50KDa place, there is not single specific immune band herein in negative control mouse IgG, shows that in the two positive mice milk of transgenosis, chimeric antibody ChAb26 contains human IgG light chain constant district. In the two positive mice milk of transgenosis, the protein band of chimeric antibody ChAb26 at 25KDa place by the anti-human �� chain antibody of sheep (rabbit anti-sheep-HRP be two resist) identification, can dye single specific immune band. The light chain of its size position just corresponding human IgG antibody, positive control human IgG also dyes single specific immune band at 25KDa place, there is not single specific immune band herein in negative control mouse IgG, shows that in the two positive mice milk of transgenosis, chimeric antibody ChAb26 contains the IgG �� chain constant region of people. In Gene Double positive mice milk, chimeric antibody ChAb26 has humanized in sum.
The anti-p185 of embodiment 5erbB2The qualification of human mouse chimeric antibody ChAb26 correlation function characteristic
1, the inhibiting rate that SKOV3 cell is grown by CCK-8 test kit detection chimeric antibody ChAb26
The inhibiting rate that SKOV3 cell is grown by CCK-8 test kit detection chimeric antibody ChAb26. Result shows, 0.48mg.L-1ChAb26 group and 0.5mg.L-1Herceptin group, all has significant difference compared with cell blank control group. 0.48mg.L-1ChAb26 group and 0.5mg.L-1Herceptin group is close to the inhibiting rate of ovarian cancer SKOV3 cell proliferation, is respectively 53.63% and 58.43% (table 15)
Table 15 chimeric antibody ChAb26 is to the growth inhibition ratio of SKOV3 cell
Wherein, P < 0.05.
2, the drafting of SKOV3 cell growth curve
With 0.5mg.L-1Herceptin group is as positive control, and cell blank group, as negative control, is respectively 0.48,0.24 and 0.12mg.L with containing chimeric antibody ChAb26-1The two positive mice milk of transgenosis, and after FVB wild-type mice milk acts on SKOV3 cell 24h, 48h and 72h respectively, carry out CCK-8 method detection, using OD450 as ordinate zou, the time is X-coordinate, draws growth curve (Figure 42).
3, anti-p185 is observed in HE dyeingerbB2The morphological feature that human mouse chimeric antibody ChAb26 induction SKOV3 withers and dies
Anti-human p185 is observed in HE dyeingerbB2The morphological feature that human mouse chimeric antibody ChAb26 induction SKOV3 withers and dies. Result shows, 0.48mg.L-1ChAb26 and 0.50mg.L-1Herceptin effect 24h, SKOV3 cell number obviously reduces, and shrinkage phenomenon occurs in cell, and cell volume diminishes, and the connection between cell disappears, and departs from the cell of surrounding, and the concentrated dark dye of caryoplasm, cytoplasm strengthens addicted to Yihong dye. Cell blank control group and FVB wild-type mice milk group cell are without considerable change, cytoplasm and nucleus clear (Figure 43).
4, the anti-p185 of transmission electron microscope observingerbB2The morphological feature that human mouse chimeric antibody ChAb26 induction SKOV3 withers and dies
Result shows, and the SKOV3 cell surface of cell blank control group and FVB wild-type milk treatment group has more microvillus and pseudo-Microfilament, and nucleus volume is bigger, form is irregular, nuclear chromatin enriches, and some cell often has 2-3 kernel, and plastosome and endoplasmic reticulum are more. And use 0.48mg.L-1ChAb26 or 0.50mg.L-1After Herceptin effect 24h, then visible cell withers phenomenon of dying, and main performance is cell shrinkage, chromatin agregation, and under concentrating on nuclear membrane. (Figure 44).
5, AnnexinV-PE/7ADD wither die kit assay chimeric antibody induction SKOV3 apoptosis
AnnexinV-PE/7ADD wither die kit assay chimeric antibody induction SKOV3 apoptosis. Result shows (Figure 45, table 16), 0.48mg.L-1ChAb26 induces the middle and advanced stage apoptosis rate mean value of SKOV3 apoptosis to be 27.3%; 0.50mg.L-1Herceptin induces the middle and advanced stage apoptosis rate mean value of SKOV3 apoptosis to be 35.75%; The SKOV3 cell middle and advanced stage apoptosis rate mean value of cell blank group and FVB wild-type mice milk treatment group is respectively 10.23% and 19.79%, carry out all existing between the known each treatment group of variance analysis significant difference (F=898.71, P < 0.01) through SPSS.18.
The middle and later periods apoptotic cell accounting (unit: %) of SKOV3 cell after 24h induced by table 16
Wherein, F=898.71, P < 0.01.
6, triumphant basilar cell's cycle kit assay chimeric antibody induction SKOV3 cell cycle checks
Result shows (table 17, Figure 46), 0.48mg.L-1ChAb26��0.50mg.L-1After Herceptin and FVB wild-type milk effect SKOV3 cell 24h, all there is rising phenomenon, 0.50mg.L in the SKOV3 cell accounting that versus cell blank group is in the G1 phase-1Herceptin treatment group raises 10.48%, 0.48mg.L-1ChAb26 treatment group and FVB wild-type milk treatment group raise 4.31% and 4.37% respectively. Due to heterogeneity of variance, the SKOV3 cell accounting carrying out being in the known each treatment group of rank test the G1 phase not identical (X entirely2=10.421, P < 0.05), then through comparing between two it will be seen that 0.50mg.L-1Herceptin treatment group all with other each group there is difference; 0.48mg.L-1All there is difference with cell blank control group in ChAb26 treatment group and FVB wild-type milk treatment group, but without difference (F=1.057E4, P < 0.01) between them. 0.48mg.L-1ChAb26��0.50mg.L-1After Herceptin and FVB wild-type milk effect SKOV3 cell 24h, all there is reduction phenomenon, 0.50mg.L in the SKOV3 cell accounting that versus cell blank group is in the G2 phase-1Herceptin treatment group reduces by 13.72%, 0.48mg.L-1ChAb26 treatment group and FVB wild-type milk treatment group reduce by 5.19% and 3.73% respectively. There is difference (F=3.157E4, P < 0.01) in the SKOV3 cell accounting being in the G2 phase between the known each treatment group of variance analysis. In sum, 0.48mg.L-1ChAb26��0.50mg.L-1Herceptin and FVB wild-type milk all can make SKOV3 cell block in G1/S and S/G2 check point to some extent, but blockage effect and fairly obvious, wherein 0.48mg.L-1ChAb26 and FVB wild-type milk makes SKOV3 cell block the effect in G1/S check point without difference.
The cell cycle analysis (unit: %) of SKOV3 cell after 24h induced by table 17
Wherein, a:X2=10.421, P < 0.05; F=1.057E4, P < 0.01;B:F=3.157E4, P < 0.01; A: cell blank control group; B:FVB wild-type mice milk treatment group; C:0.48mg.L-1ChAb26 treatment group; D:0.50mg.L-1Herceptin treatment group.
Claims (10)
1. an anti-p185erbB2Human mouse chimeric antibody ChAb26, forms primarily of heavy chain gene H and light chain gene L, it is characterised in that: described heavy chain gene H and light chain gene L has the base sequence of sequence table SEQ .ID.No.3 and SEQ.ID.No.6 respectively.
2. anti-p185 described in claim 1erbB2The mammary gland specific expression vector of human mouse chimeric antibody ChAb26, it is characterised in that be pBCl-H and pBCl-L, described pBCl is mammary specific expression plasmid.
3. the construction process of mammary gland specific expression vector described in claim 2, it is characterised in that: by anti-p185erbB2The heavy chain gene H and light chain gene L of human mouse chimeric antibody ChAb26 are connected respectively on mammary specific expression plasmid pBCl, thus build and obtain p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBCl-H and pBCl-L.
4. the construction process of mammary gland specific expression vector according to claim 3, it is characterised in that undertaken by following operation:
<1>with containing anti-p185erbB2The pUC57/pBCN-H-F2A-L-ployA-EGFP-Neo plasmid of human mouse chimeric antibody ChAb26 heavy chain H and light chain gene L is template, under LATaqDNA polysaccharase effect, and the anti-p185 taking H-F/H-R and L-F/L-R as primer amplification goes out respectivelyerbB2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L; The program of PCR reaction is 94 DEG C of 5min; 94 DEG C of 30see; 50 DEG C of 30see; 69 DEG C of 2min30sec; 35cycles; 69 DEG C of 7min, 4 DEG C of ��;
<2>use sepharose reclaims heavy chain gene H and the light chain gene L recovery purifying that step<1>is amplified by test kit;
<3>the heavy chain gene H and the light chain gene L that step<2>are reclaimed are connected respectively on pEASY-T1 cloning vector, obtain recon pEASY-T1-H and pEASY-T1-L;
<4>use XhoI recon pEASY-T1-H and pEASY-T1-L that step<3>obtains is carried out enzyme cut and use ethanol precipitation digestion products heavy chain gene H and light chain gene L is carried out reclaim purifying;
<5>heavy chain gene H step<4>reclaimed and light chain gene L respectively with cut the dephosphorylized mammary specific expression plasmid pBCl with CIP alkaline phosphatase through XhoI enzyme in advance and be connected under T7 ligase enzyme effect, build and obtain anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBCl-H and pBCl-L.
5. the construction process of mammary gland specific expression vector according to claim 4, it is characterised in that: described primer H-F/H-R and L-F/L-R has the base sequence of sequence table SEQ .ID.No.1/SEQ.ID.No.2, SEQ.ID.No.4/SEQ.ID.No.5 respectively.
6. a transgenosis FVB mouse, it is characterised in that express anti-p185 specificallyerbB2Human mouse chimeric antibody ChAb26.
7. the preparation method of transgenosis FVB mouse described in claim 6, it is characterised in that: by p185erbB2After human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBCl-H and pBCl-L linearizing, import FVB mouse fertilized egg and obtain.
8. the preparation method of transgenosis FVB mouse according to claim 7, it is characterised in that adopt fertilization egg nucleus is micro-and inject importing altogether.
9. the preparation method of transgenosis FVB mouse according to claim 8, it is characterised in that undertaken by following operation:
<1>use NotI and SalI by anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBCl-H and pBCl-L carries out linearizing, obtains anti-p185erbB2Specific expressed linear carrier pBCl-H-linear and pBCl-L-linear of human mouse chimeric antibody ChAb26 transgenic mouse milk;
<2>by the protokaryon micro-mode of injection altogether, mammary specific expression linear carrier pBCl-H-linear and pBCl-L-linear is carried out zygote to inject altogether; After injection, by zygote transplation to, in acceptor FVB system Mouse Uterus, obtaining F0 generation two positive transgenic FVB mouse after gestation.
10. use transgenosis FVB mouse described in claim 6 to obtain anti-p185erbB2The method of human mouse chimeric antibody ChAb26, it is characterised in that undertaken by following operation: maternal rat delivery is after 10 days, it may also be useful to the female mouse of transgenosis is carried out milk collection by mouse milking apparatus; Then, the milk collected is transferred to respectively in sterilizing Ep pipe, 4 DEG C, the centrifugal 20min of 4000 �� g; After centrifugal end, sucking-off middle level clear liquid gently, obtains anti-p185erbB2Human mouse chimeric antibody ChAb26 solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107589261A (en) * | 2017-09-08 | 2018-01-16 | 苏州立豪生物科技有限公司 | A kind of kit for being used to detect breast cancer |
| CN107589261B (en) * | 2017-09-08 | 2019-02-22 | 上海宝藤生物医药科技股份有限公司 | It is a kind of for detecting the kit of breast cancer |
| US11878065B2 (en) * | 2018-08-08 | 2024-01-23 | Xiaofeng Xia | Reporter protein fused antibodies |
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| CN105646704B (en) | 2019-11-15 |
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