CN105646704B - Anti- p185erbB2 human mouse chimeric antibody ChAb26, mammary gland specific expression vector, transgenosis FVB mouse and preparation method thereof - Google Patents

Anti- p185erbB2 human mouse chimeric antibody ChAb26, mammary gland specific expression vector, transgenosis FVB mouse and preparation method thereof Download PDF

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CN105646704B
CN105646704B CN201510996843.6A CN201510996843A CN105646704B CN 105646704 B CN105646704 B CN 105646704B CN 201510996843 A CN201510996843 A CN 201510996843A CN 105646704 B CN105646704 B CN 105646704B
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chain gene
pbc1
chab26
mouse
chimeric antibody
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CN105646704A (en
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李力
梁振鑫
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Guangxi Medical University
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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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • 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
    • 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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

Abstract

The invention discloses a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26 is mainly made of heavy chain gene H and light chain gene L, its heavy chain gene H and light chain gene L are connected respectively on mammary specific expression plasmid pBC1, so that building obtains p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.After pBC1-H and pBC1-L linearisation, imports FVB mouse fertilized egg and produce transgenosis FVB mouse, using it as galactophore biological reactor, anti-p185 is obtained by mammary specific expressionerbB2Human mouse chimeric antibody ChAb26.The antibody reduces the immunogenicity of source of mouse monoclonal antibody, produces medicinal recombinant protein by gained transgenosis FVB mouse and has many advantages, such as high, at low cost, the active height of yield, easy scale, can be shortened new drug market periods and market potential is huge.

Description

Anti- p185erbB2 human mouse chimeric antibody ChAb26, mammary gland specific expression vector, turn Gene FVB mouse and preparation method thereof
Technical field
The technology of the present invention belongs to bioengineering field more particularly to a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26, cream Gland specific expression vector, transgenosis FVB mouse and preparation method thereof.
Background technique
Shih etc. (1981) scientist has found a kind of new proto-oncogene from rat embryo neuroblast for the first time, and Name neu gene.It is then compared by nucleic acid sequence analysis and chromosome spectrum analysis finds neu proto-oncogene and proto-oncogene There are high homologies by erb B.Therefore, neu proto-oncogene is considered as a kind of related gene of erb B proto-oncogene, and by It is named as erb B2/HER2.The proto-oncogene erb B2 of the finders such as Coussens is located at 2 areas 1 of No. 17 chromosome long arms of people Band (17q21), coded product are receptor tyrosine kinase (RTK, the receptor tyrosine that molecular weight is 185kDa ), thus the p185 albumen that is otherwise known as kinase.A kind of transmembrane glycoprotein that it is made of 1255 amino acid residues.RTK is One superfamily (superfamily), member include erb B proto-oncogene coded product EGF-R ELISA (EGFR, Epidermal growth factor receptor) and erb B2/HER2 receptor, erb B3/HER3 receptor and erb B4/HER4 receptor etc..Erb B receptor is widely distributed in the cell membrane of all epithelial cells in addition to vascular tissue On；Erb B2 receptor is common in the various coelomic epitheliums of normal person, galandular epithelium and embryo, and erb B2 receptor is deposited in them In faint expression；Erb B3 receptor is except in addition to human hematopoietic system is not expressed, at human body majority position, presence is expressed；erb B4 receptor is except in addition to glomerulus and peripheral nerve are not expressed, presence is expressed in its hetero-organization is equal.Film outskirt, film inner region The primary structure of erb B2 receptor is constituted with transmembrane region (TM, transmembrane).Film outskirt have specificity, it with match Body combines, and receives signal, contains a large amount of cysteine residues repetitive sequences；Film inner region determines the biology effect intracellular of growth factor It answers.Transmembrane region major function is anchored receptor and the allosteric effect for leading to receptor, generally constitutes one by 22-26 amino acid residue A α helicoidal structure, very hydrophobic；The functional areas of erb B2 receptor are located at C-terminal, and main includes the binding site and junket of ATP Histidine kinase functional areas.The mutation of erb B family member is relatively common in human tumor, especially erb B2, their master Wanting function is stimulating cellular growth.
The activation of erbB2 receptor is related to tumour generation, and in addition to the specific stage of development such as embryonic development, erb B2 is just Often the expression quantity in tissue is very low；And in contrast, in the Several Kinds of Malignancy cell such as breast cancer, oophoroma and lung cancer Amplification and the p185 of erb B2 gene are had been found thaterbB2The overexpression of albumen.Amplification and p185 due to erb B2 geneerbB2 The overexpression of albumen so as to cause erb B2 receptor activation, and enhances the signal transduction of its mediation, eventually leads to normal cell Vicious transformation occurs and forms malignant tumour.
Trastuzumab(Be otherwise known as Trastuzumab, the entitled Herceptin of Chinese) it is to act on erb The Humanized monoclonal specific antibody (humanized mAb) of B2 albumen is current most popular anti-erb B2 high table The high potency drugs of the tumour reached.Herceptin energy specific bond is in the area erb B2 receptor extracellular subdomain IV, to block The formation of erb B2 homodimer and heterodimer, and mediate the endocytosis of erb B2 receptor and degrade in lysosome, it hinders The function of disconnected erb B2 receptor, to interfere the phosphorylated-activated of the one end erb B2COOH tail and inhibit MAPK and PI3K letter Number approach, inhibits the growth of cell.Studies have shown that Herceptin be to the erb B2 receptor of people it is specific, act only on The cell line of erb B2 gene overexpression.Its main mechanism is the IgGI Fc end (Crystalline by it Fragment, crystallizable fragment) efficiently mediate antibody rely on cytotoxicity (antibody-dependent cellular Cytotoxicity, ADCC) kill the tumour cell that erbB2 is overexpressed.Herceptin exclusive use can inhibit cancer The growth of cell, but it and the combination of other cellulotoxic chemotherapeutics more show therapeutic effect outstanding, and the mechanism of action is Herceptin can check the receptor-mediated signal transduction of erb B2 so as to cause the anti-apoptotic proteins table such as Bcl-2 and survivin Up to downward, so that malignant cell improves the sensibility of the cellulotoxic chemotherapeutics of Doxorubicin, taxol etc..But Trastuzumab is that the mammalian cell (Chinese hamster ovary cell CHO) being incubated in aseptic culture medium by suspension produces, and is Humanized monoclonal antibodies derived from a kind of recombinant DNA.Its production cost is high to cause tumor patient high medical expense, Anti- p185 chimeric antibody, which is prepared, by mammary gland bioreactor of transgenic animals very likely effectively solves this disadvantage.
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 are special Property expression vector, transgenosis FVB mouse and preparation method thereof, gained human mouse chimeric antibody reduces exempting from for source of mouse monoclonal antibody Epidemic disease source property, producing medicinal recombinant protein by gained transgenosis FVB mouse has high, at low cost, active high, the easy scale of yield Change, can be shortened the advantages that new drug market periods and market potential are huge.
In order to solve the above technical problems, the invention adopts the following technical scheme: anti-p185erbB2Human mouse chimeric antibody ChAb26 is mainly made of heavy chain gene H and light chain gene L, and heavy chain gene H and light chain gene L are respectively provided with sequence table The base sequence of SEQ.ID.No.3 and SEQ.ID.No.6.
Above-mentioned anti-p185erbB2The mammary gland specific expression vector of human mouse chimeric antibody ChAb26 is pBC1-H and pBC1-L, PBC1 is mammary specific expression plasmid.
The construction method of above-mentioned mammary gland specific expression vector, by anti-p185erbB2The heavy chain of human mouse chimeric antibody ChAb26 Gene H and light chain gene L is connected respectively on mammary specific expression plasmid pBC1, so that building obtains p185erbB2People mouse is embedding Close antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
The construction method of above-mentioned mammary gland specific expression vector is carried out by following operation:
<1>to contain anti-p185erbB2The pUC57/pBCN-H- of human mouse chimeric antibody ChAb26 heavy chain H and light chain gene L F2A-L-ployA-EGFP-Neo plasmid is template, under the effect of LA Taq archaeal dna polymerase, respectively with H-F/H-R and L-F/ L-R is that primer amplification goes out anti-p185erbB2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L；The program of PCR reaction For 94 DEG C of 5min；94℃30sec；50℃30sec；69℃2min30sec；35cycles；69 DEG C of 7min, 4 DEG C of ∞；
<2>the heavy chain gene H and light chain gene L amplified step<1>using Ago-Gel QIAquick Gel Extraction Kit is recycled Purifying；
<3>the heavy chain gene H and light chain gene L by step<2>recycling are connected respectively on pEASY-T1 cloning vector, are obtained Obtain recon pEASY-T1-H and pEASY-T1-L；
<4>digestion is carried out to the recon pEASY-T1-H and pEASY-T1-L that step<3>obtains using Xho I and used Digestion products heavy chain gene H and light chain gene L is carried out recovery purifying by ethanol precipitation；
<5>by the heavy chain gene H and light chain gene L of step<4>recycling respectively and in advance through Xho I digestion and CIP alkalinity The mammary specific expression plasmid pBC1 of phosphatase dephosphorylation is connected under T7 connection enzyme effect, and building obtains anti-p185erb B2 Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
Primer H-F/H-R and L-F/L-R be respectively provided with sequence table SEQ .ID.No.1/SEQ.ID.No.2, The base sequence of SEQ.ID.No.4/SEQ.ID.No.5.
Transgenosis FVB mouse, specifically expresses anti-p185erbB2Human mouse chimeric antibody ChAb26.
The preparation method of above-mentioned transgenosis FVB mouse, by p185erbB2Human mouse chimeric antibody ChAb26 mammary specific expression After carrier pBC1-H and pBC1-L linearisation, imports FVB mouse fertilized egg and obtain.
The preparation method of above-mentioned transgenosis FVB mouse is imported using the micro- co-injection of maritonucleus.
The preparation method of above-mentioned transgenosis FVB mouse is carried out by following operation:
<1>use Not I and Sal I by anti-p185erb B2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector PBC1-H and pBC1-L are linearized, and anti-p185 is obtainederb B2Human mouse chimeric antibody ChAb26 transgenic mouse milk is special Property expression linear carrier pBC1-H-linear and pBC1-L-linear；
<2>mammary specific expression linear carrier pBC1-H-linear and pBC1-L-linear is micro- total by protokaryon The mode of injection carries out fertilized eggs co-injection；After injection, by zygote transplation to receptor FVB system Mouse Uterus, gestation F0 generation double positive transgenic FVB mouse are obtained afterwards.
Anti- p185 is obtained using above-mentioned transgenosis FVB mouseerbB2The method of human mouse chimeric antibody ChAb26, by following operation It carries out: after maternal rat delivery 10 days, milk acquisition being carried out to transgenosis female rat using mouse milker；Then, by collected cream Juice is transferred to respectively in sterilizing Ep pipe, and 4 DEG C, 4000 × g is centrifuged 20min；After centrifugation, middle layer clear liquid is gently sucked out, Up to anti-p185erbB2Human mouse chimeric antibody ChAb26 solution.
Current source of mouse monoclonal antibody there are aiming at the problem that, be based on galactophore biological reactor and chimeric antibody principle, hair Bright people devises a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26 and its mammary gland specific expression vector, and establish accordingly The construction method of mammary gland specific expression vector and the preparation method of transgenosis FVB mouse.Anti- p185erbB2Human mouse chimeric antibody ChAb26 is mainly made of heavy chain gene H and light chain gene L, and heavy chain gene H and light chain gene L are respectively provided with sequence table The base sequence of SEQ.ID.No.3 and SEQ.ID.No.6.It is special that its heavy chain gene H and light chain gene L are connected respectively to mammary gland Property expression plasmid pBC1 on, so that building obtains 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 linearisation, imports FVB mouse fertilized egg and produce Transgenosis FVB mouse obtains anti-p185 by mammary specific expression using it as galactophore biological reactorerbB2People mouse is embedding Close antibody ChAb26.Since pBC1 carrier has the structure of mammary specific expression, for constructing galactophore of transgenic animal biology It can great expression recombinant protein or antibody when reactor.According to data, raw by the transgenic mouse milk of pBC1 vector construction The recombinant protein content of object reactor expression can reach 35g/L (Youngetal, 1997), transgenic goat mammary gland bioreactor The recombinant protein content of device expression can reach 20g/L (Zeomek, 1998).
The present invention can greatly reduce source of mouse list for the tumour cell and human mouse chimeric antibody of erbB2 receptor overexpression The immunogenicity of clonal antibody, so as to effectively avoid antiantibody reaction from occurring；With the cell culture for preparing medicinal recombinant protein Or the traditional technologies such as microorganism fermentation tank are compared, gained transgenosis FVB mouse produces medicinal recombinant protein and has through the invention High, at low cost, active high, the easy scale of yield can be shortened the advantages that new drug market periods and market potential are huge.Meanwhile it answering The biological therapy for the malignant tumour that can be overexpressed with the present invention for erbB2 receptors such as oophoromas has carried out positive exploration.
Detailed description of the invention
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 electrophoresis of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L Scheme, M is DNA marker III in figure；1,2 is heavy chain gene H；3,4 be 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 solution PCR qualification result of recombinant clone plasmid pEASY-T1-H in embodiment 1, and M is DNA in figure marker 1k；1,2,8,10,11,14 is positive colony recon；3~7,9,12~13,15~18 be negative clone.
Fig. 6 is the bacterium solution PCR qualification result of recombinant clone plasmid pEASY-T1-L in embodiment 1, and M is DNA in figure marker 1k；1,6,7 is positive colony recon；3~5,8~9 be negative clone.
Fig. 7 is recombinant clone plasmid pEASY-T1-L/pEASY-T1-H and its XhoI digestion products electrophoretogram, and M is in figure DNA marker III；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 sequencing and its nucleotide homology compare analysis As a result.
Fig. 9 is anti-p185erbB2Human mouse chimeric antibody ChAb26 light chain gene L sequencing and its nucleotide homology compare analysis As a result.
Figure 10 is the electrophoretogram of XhoI digestion pBC1 skeleton, and M is λ DNA marker in figure；1~6 is pBC1 plasmid (line Property)；7 be pBC1 plasmid (ring-type).
Figure 11 is XhoI digestion recombinant clone plasmid pEASY-T1-L electrophoretogram, and M is 1k DNA marker in figure；P1, P2 is pEASY-T1 cloned plasmids；L1, L2 are Chimeric Antibody Light Chain Gene L.
Figure 12 is XhoI digestion recombinant clone plasmid pEASY-T1-H electrophoretogram, and M is 1kDNA marker in figure；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 solution PCR qualification result, and M is DNA markerIII in figure；2, 9 be positive colony；1,3~8,10~15 be negative clone.
Figure 16 is mammary gland specific expression vector pBC1-H bacterium solution PCR qualification result, and M is DNA markerIII in figure；2, 3,7,9,11 be positive colony；Isosorbide-5-Nitrae~6,8,10 be negative clone.
Figure 17 is mammary gland specific expression vector pBC1-L electrophoretogram, and M is DNA λ marker in figure；1,2 is pBC1-L Plasmid.
Figure 18 is mammary gland specific expression vector pBC1-H electrophoretogram, and M is DNA λ marker in figure；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 nucleic acid Sequence homology compares analysis chart, and underlined in red is XhoI multiple cloning sites in figure.
Figure 20 is the anti-p185 of mammary gland specific expression vector pBC1-HerbB2Middle chimeric antibody ChAb26 heavy chain gene H nucleic acid Sequence homology compares analysis chart, and underlined in red is XhoI multiple cloning sites in figure.
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 electrophoretogram, and 1 (A) in figure, 2 (A) are pBC1- H-linear segment；1 (B), 2 (B) are pBR322+Ampicillin segments；3 be pBC1-H plasmid；M is λ Marker.
Figure 24 is mammary gland specific expression vector pBC1-L plasmid linearization electrophoretogram, and 1 (A) is pBC1-L- in figure Linear segment；1 (B) is pBR322+Ampicillin segment；2 be pBC1-L plasmid；M is λ Marker.
Figure 25 is that F0 is for transgenic mice rat-tail Direct PCR electrophoretogram in embodiment 2, and N is wild type FVB mouse in figure； 1~8 is mouse number；W is ddH2O；M is DNA Marker III；NC is FVB mouse internal reference；TgL is chimeric antibody light base Because of L portion DNA fragmentation；TgH is the part chimeric antibody heavy gene H DNA fragmentation.
Figure 26 is that F1 (F0-1) is for transgenic mice rat-tail Direct PCR electrophoretogram in embodiment 2, and N is wild type in figure FVB mouse；1~10 is mouse number；W is ddH2O；M1 is DNA Marker III；M2 is DNA Marker I；NC is FVB Mouse internal reference；TgL is Chimeric Antibody Light Chain Gene L portion DNA fragmentation；TgH is the part chimeric antibody heavy gene H DNA piece Section.
Figure 27 is F2 generation (F0-1) transgenic mice rat-tail Direct PCR electrophoretogram in embodiment 2, and N is wild type in figure FVB mouse；1~9 is mouse number；W is ddH2O；M1 is DNA Marker III；M1 is DNA Marker 100；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 Segment.
Figure 28 is F3 generation (F0-1) transgenic mice rat-tail Direct PCR electrophoretogram in embodiment 2, and N is wild type in figure FVB mouse；1~11 is mouse number；W is ddH2O；M is DNA Marker III；NC FVB mouse internal reference；TgL is chimeric Antibody light chain gene L portion DNA fragmentation；TgH is the part chimeric antibody heavy gene H DNA fragmentation.
Figure 29 is F4 generation (F0-1) transgenic mice rat-tail Direct PCR electrophoretogram in embodiment 2, and N is wild type in figure FVB mouse；1~9 is mouse number；W is ddH2O；M is DNA Marker I；NC is FVB mouse internal reference；TgL chimeric antibody is light Chain gene L portion DNA fragmentation；TgH is the part chimeric antibody heavy gene H DNA fragmentation.
Figure 30 is TR-PCR product agarose gel electrophoresis figure in embodiment 2, and in figure: N is FVB wild-type mice mammary gland group Knit cDNA；F0 is the double positive mice breast tissue cDNA of F0 transgenosis；F1 is the double positive mice breast tissues of F1 generation transgenosis cDNA；F2 is F2 for the double positive mice breast tissue cDNA of transgenosis；M is DNA Marker I；1 be FVB wild-type mice/turn The internal reference product of DNA murine breast tissue cDNA；The RT-PCR product of 2 chimeric antibody heavy gene H partial cDNA fragments；3 It is the RT-PCR product of Chimeric Antibody Light Chain Gene L portion cDNA segment.
Figure 31 is the solubility curve of fluorescent quantitative PCR experiment internal control 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 mRNA relative expression's spirogram of chimeric antibody in embodiment 2.
Figure 35 is the expression figure that immunohistochemistry Western Blot identifies chimeric antibody ChAb26 in embodiment 2, 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 identification chimeric antibody ChAb26 in embodiment 3, and IgG is people IgG；P is PBS solution；N is FVB wild-type mice milk；Tg is transgenic mouse milk；1-3 is multiple holes number.
Figure 37 is the expression immunohistochemistry identification inosculating antibody that Western Blot detects chimeric antibody ChAb26 in embodiment 2 The expression figure (Nikon 200 ×) of body ChAb26, A is FVB wild-type mice breast tissue in figure；B is F0 for the double sun of transgenosis Property mammary gland of mouse tissue；C is the double positive mice breast tissues of F1 generation transgenosis；D is F2 for the double positive mice mammary gland groups of transgenosis It knits.
Figure 38 is the expression (concentration of measurement chimeric antibody) of sandwich ELISA identification chimeric antibody ChAb26 in embodiment 2 Result figure, 1 is 200ng/ml human IgG in figure；2 be 100ng/ml human IgG；3 be 50ng/ml human IgG；4 be 25ng/ml people IgG；5 be 12.5ng/ml human IgG；6 be 6.25ng/ml human IgG；7 be 3.125ng/ml human IgG；8 be 0ng/ml human IgG；9 It is FVB wild-type mice milk；10 be transgenic mouse milk；A-c is multiple holes number.
Figure 39 be in the double positive mice milk of embodiment 3 transgenic the ELISA quantitation curves of chimeric antibody with return Return graph of equation, F=29.331 (P < 0.05) in figure；R2=0.83；T=5.416 (P < 0.05).
Figure 40 is the antigentic specificity of Salmonella measurement chimeric antibody in embodiment 3, and A figure is Salmonella in figure The antigentic specificity figure of chimeric antibody is measured, 1 is the anti-human erbB2 monoclonal antibody of mouse；2 be human IgG；3 be mouse IgG；4 be PBS；5 be to turn Gene Double positive mice milk；6 be FVB wild-type mice milk；A-c is multiple holes number；B figure is that Salmonella measurement is chimeric The antigentic specificity figure of antibody, 1 is the anti-human erbB2 monoclonal antibody of mouse；2 be human IgG；3 be mouse IgG；4 be PBS；5 be the double sun of transgenosis Property mouse milk；6 be FVB wild-type mice milk；A-c is multiple holes number.
Figure 41 is humanized's result figure that western blot analyzes chimeric antibody ChAb26 in embodiment 3, hIgG in figure It is human IgG；F0 is the double positive F0 of transgenosis for mouse milk；F1 is the double positive F1 generation mouse milks of transgenosis；F2 is transgenosis Double positive F2 are for mouse milk；F3 is the double positive F3 of transgenosis 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 is morphological feature (for 24 hours, HE is dyed, Nikon × 400) figure of SKOV3 Apoptosis in embodiment 3, figure Middle A is cell blank control group；B is FVB wild-type mice milk processing group；C is 0.48mg.L-1ChAb26 processing group；D is 0.50mg.L-1Herceptin processing group.
Figure 44 is Ultrastructure Features (for 24 hours, H-7650) figure of SKOV3 Apoptosis in embodiment 3, and A is cell in figure Blank control group；B is FVB wild-type mice milk processing group；C is 0.48mg.L-1ChAb26 processing group；D is 0.50 mg.L- 1Herceptin processing group.
Figure 45 is Apoptosis (for 24 hours) figure of flow cytometry (FCM) detection SKOV3, and A is cell blank control in figure Group；B is FVB wild-type mice milk processing group；C is 0.48mg.L-1ChAb26 processing group；D is 0.50mg.L- 1Herceptin processing group.
Figure 46 is cell cycle (for 24 hours) figure of flow cytometry (FCM) detection SKOV3, and A is cell blank control in figure Group；B is FVB wild-type mice milk processing group；C is 0.48mg.L-1ChAb26 processing group；D is 0.50mg.L- 1Herceptin processing group.
Specific embodiment
The anti-p185 of embodiment 1erb B2The building of human mouse chimeric antibody ChAb26 transgenic mouse milk specific expression vector
1, the acquisition of target gene
To contain anti-p185erbB2The pUC57/pBCN-H-F2A- of human mouse chimeric antibody ChAb26 heavy chain H and light chain gene L L-ployA-EGFP-Neo plasmid is template (Fig. 1), under the effect of LA Taq archaeal dna polymerase, respectively with 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) is that primer amplification goes out to resist p185erb B2Human mouse chimeric antibody ChAb26 heavy chain gene H (SEQ.ID.NO.3) and light chain gene L (SEQ.ID.NO.6).Through The identification of 1% agarose gel electrophoresis, heavy chain gene H and light chain gene L specific band are consistent with theoretical value, and size is respectively 2113bp and 799bp (Fig. 2).
The amplimer (sequence that underscore part is restriction enzyme Xho I) of 1 target gene of table
The reaction system of 2 PCR of table
The program of PCR reaction are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C of 2min30sec, 35cycles； 69℃7min；4℃∞.
PCR product is detected with 1% agarose gel electrophoresis, then uses DNA Ago-Gel glue reclaim reagent Box (TIANgel Midi Purification Kit) carries out purification and recovery to PCR product.
2, the screening of recon
The PCR product of recycling is connect with pEASY-T1 cloning vector, to obtain connection product pEASY-T1-H (SEQ.ID.NO.7) and pEASY-T1-L (SEQ.ID.NO.8) connection product then, is converted into DH5 α competent cell, takes 100 μ L bacterium solution coated plate, overnight incubation.
Above-mentioned Amp(+)After LB culture dish overnight incubation, it is seen that the bacterium colony being dispersed in therefrom is selected 10 single colonies, is inoculated into In different 10ml glass tubes, Amp in each pipe(+)The volume of LB culture solution is 2.5mL, finally places and cultivates in constant-temperature table 16-20 hours (37 DEG C, 200rpm).
Recon is identified using bacterium solution PCR method using recombinant plasmid bacterium solution as template with pEASY-T1 universal primer M13.Expand Volume increase object identifies that 1,2,8,10,11 and No. 14 bacterium solution is pEASY-T1-H weight it is found that in Fig. 5 through 1% agarose gel electrophoresis Group cloned plasmids bacterium solution, the specific band of bacterium solution PCR amplification is consistent with theoretical value, and size is about 2300bp (Fig. 5).Fig. 6 In, 1,6 and No. 7 bacterium solution is pEASY-T1-L recombinant clone plasmid bacterial solution, the specific band and theoretical value of bacterium solution PCR amplification Unanimously, size is about 1000bp (Fig. 6).It is finally small to the progress of pEASY-T1-H/L recombinant clone plasmid bacterial solution to mention, and use XhoI restriction enzyme carries out digestion to pEASY-T1-H/L recombinant clone plasmid, can through the identification of 1% agarose gel electrophoresis Know, pEASY-T1-H/L recombinant clone plasmid band is consistent with theoretical value, respectively 5930bp and 4615bp, what digestion generated PEASY-T1 cloned plasmids band and anti-p185erbB2The band of human mouse chimeric antibody heavy chain gene H or light chain gene L also with theory Value is consistent, respectively 3829bp, 2113bp and 799bp (Fig. 7).
3 pEASY-T1 universal primer M13 of table
The reaction system of 4 PCR of table
The program of PCR reaction are as follows: 94 DEG C of 5min；；94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C of 2min30sec, 35cycles； 69℃7min；4℃∞.
It will be used through the correct bacterium solution of above-mentioned preliminary identification, using pEASY-T1 plasmid universal primer M13 as sequencing primer (Huada gene company) is sequenced in Sanger chain termination method.Finally by the anti-p185 of sequencing result and desired designerbB2People The sequence of mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L are compared.Through anti-p185 known to analysiserbB2People The anti-p185 of mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L sequencing result sequence and desired designerbB2People mouse is chimeric Antibody heavy chain gene H (Fig. 8 and Fig. 9) consistent with light chain gene L sequence.
Recombinant plasmid is stripped using TIANprep Mini Plasmid Kit kit.
3, the preparation of carrier
Using Xho I digestion with restriction enzyme mammary specific expression plasmid pBC1, and it is special with the mammary gland without digestion Anisotropic expression plasmid pBC1 is identified through 1% agarose gel electrophoresis it is found that mammary specific expression plasmid pBC1 quilt as control The linear pBC1 plasmid of thorough digestion, band are consistent with theoretical value (Figure 10).Then with alkaline phosphatase CIP to through in Xho I PBC1 mammary gland specific expression vector after enzyme cutting digestion carries out dephosphorylation reaction.
Correct pEASY-T1-H/pEASY-T1-L plasmid is sequenced using Xho I endonuclease digestion, and heavy by ethyl alcohol Shallow lake method is by the purification and recovery of product (H/L chain DNA segment).It identifies through 1% agarose gel electrophoresis it is found that recombinant clone plasmid PEASY-T1-L or pEASY-T1-H by thorough digestion at pEASY-T1 plasmid and ChAb26 Chimeric Antibody Light Chain Gene L or again The DNA fragmentation of chain gene H, band are consistent with theoretical value (Figure 11 and Figure 12).
4, mammary gland specific expression vector is constructed
Through Xho I digestion and phosphoric acid is removed by the DNA fragmentation of heavy chain gene H or light chain gene L and in advance using T7 ligase PBC1 plasmid connection after change, constructs anti-p185erbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H/ pBC1-L.And convert DH5 α competence bacteria and amplification cultivation is carried out to it, finally bacterium solution is carried out using universal primer pBC1-R/F PCR identification.It identifies through 1% agarose gel electrophoresis it is found that successfully constructing 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), as a result such as Figure 15 and Figure 16.Figure 15 In, the amplified band of the bacterium solution PCR of 2 and No. 9 bacterium solutions is consistent with theoretical value, about 1000bp.In Figure 16,2,3,7,9 and No. 11 bacterium The amplified band of the bacterium solution PCR of liquid is consistent with theoretical value, about 2300bp.Finally to pBC1-L/pBC1-H recombinant clone plasmid bacterial Liquid progress is small to be mentioned, and is identified through 1% agarose gel electrophoresis it is found that pBC1-L/pBC1-H recombinant clone plasmid band and theoretical value Unanimously, respectively 22421bp and 23735bp (Figure 17 and Figure 18).It will be through the correct bacterium solution of bacterium solution PCR preliminary identification, with pBC1 Universal primer pBC1 F and pBC1 R send Huada gene company to be sequenced as sequencing primer.It is analyzed through being compared to sequencing result It is found that anti-p185erbB2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L sequencing result sequence and desired design Anti- p185erbB2Human mouse chimeric antibody ChAb26 heavy chain gene H is consistent with light chain gene L sequence, and is positive connection, wherein Xho I multiple cloning sites are with red underscore signalment (Figure 19 and Figure 20).Finally use the big extraction reagent kit of QIAGEN (QIAGENPlasmid Maxi Kits) carries out plasmid to mammary gland specific expression vector pBC1-H/pBC1-L and mentions greatly, convenient for opening Open up the experiment such as subsequent linear property, microinjection.
5 pBC1 universal primer of table
The reaction system of 6 PCR of table
The program of PCR reaction are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 50 DEG C of 30sec, 69 DEG C, 2min30sec, 35cycles； 69℃7min；4℃∞.
The anti-p185 of embodiment 2erbB2The preparation of human mouse chimeric antibody ChAb26 transgenosis FVB mouse
1, anti-p185erbB2The linearisation of human mouse chimeric antibody mammary gland specific expression vector pBC1-H/pBC1-L
The anti-p185 for being constructed embodiment 1 using Sal I and Not I enzymes double zyme cuttingerbB2Human mouse chimeric antibody ChAb26 mammary specific expression plasmid pBC1-H/pBC1-L is linearized, as a result as shown in figure 23 and figure 24.Mammary gland is special Anisotropic expression plasmid pBC1-H (23735bp) or pBC1-L (22421bp) are in the common of restriction enzyme Not I and Sal I Under effect, it is linearized respectively as pBC1-H-linear segment (17861bp, SEQ.ID.NO.11) or pBC1-L-linear Segment (16547bp, SEQ.ID.NO.12).
2, micro- co-injection method prepares transgenic mice
In the case where match industry (Suzhou) biotech company assists, mammary specific expression plasmid pBC1-H- will be linearized Linear and pBC1-L-linear concentration dilution to suitable concentration (at least needing DNA containing 5-10ug) carries out the micro- total note of protokaryon The method of penetrating prepares transgenic mice.Inject 200 FVB system mouse fertilized eggs, after injection, will wherein 100 forms it is good In zygote transplation to 5 receptor FVB system Mouse Uterus (fallopian tubal).It is identified through rat-tail Direct PCR, F0 is obtained after gestation for small Double positive transgenic mouse are 8 in mouse, wherein half male and half female.
3, rat-tail direct PCR method identifies transgenic mice
After mouse is born 4 weeks, its 0.2~0.5cm tail point tissue is cut, Chengdu good fortune border Mouse Tail Direct is used PCR Kit carries out the identification of positive transgenic mouse, wherein with TgH F/R (SEQ.ID.NO.13/SEQ.ID.NO.14) and TgL Two pairs of primers of F/ R (SEQ.ID.NO.15/SEQ.ID.NO.16) are as detection primer, control F/R (SEQ.ID.NO.17 / SEQ.ID.NO.18) it is used as internal control primer.After reaction, it carries out 1% agarose gel electrophoresis and analyzes result.With TgH F/R It is double positive for having the mouse DNA of amplification to primer with TgL F/R two；Only pair of primers has amplification to be single positive, and Two pairs of primers regard as feminine gender without amplification or two pairs of weaker mouse DNAs of primer amplification band.It is small that 8 F0 are had detected altogether Mouse detects 8 transgenic mices of discovery by PCR and is all the double positive mices of transgenosis, that is, contains chimeric antibody ChAb26 heavy chain The mouse (Figure 25) of gene H and light chain gene L.The double positive mouse of 4 females in them are numbered respectively as F0-1 to F0-4,4 It is F0-5 to F0-8 that male double positive mouse are numbered respectively.Using the double positive mices of this 8 transgenosis as Founder mouse, to it Carry out after sexal maturity expanding population and gene pure, and four generation mouse below to Founder mouse proceeds by generations respectively PCR detection.Using control F/R, TgH F/R and TgL F/R tri- to primer, each newborn mouse produced is detected. The F1 to F4 of F0-1 female mice for mouse rat-tail Direct PCR qualification result respectively such as Figure 26, Figure 27, Figure 28 and Figure 29 institute Show.To 4 mouse (F0-1, F0-2, F0-4 and F0-8, and F0-3, F0-5, F0-6 in 8 double positive transgenic mouse of F0 generation With tetra- F0 of F0-7 for transgenic mice death infertility) F1 to F4 generation bred all mouse analysis it is found that F0-2, It is similar that the offspring that F0-4 and F0-8 are bred and F0-1 breed double positive rate average values in 98 mouse, and about 30%.Most The double positive mices (F0-1, F0-2, F0-4 and F0-8) of 4 transgenosis are obtained afterwards has bred the double positive transgenics of 57 females altogether Mice progeny, the double positive transgenic mice progenies of 52 males.
The primer of 7 rat-tail Direct PCR of table
The reaction system of 8 PCR of table
The program of PCR reaction are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 60 DEG C of 35sec, 72 DEG C of 1mi, 35cycles；72 ℃ 7min；4℃∞.
4, the expansion breeding of transgenic mice
The transgenosis F0 of acquisition has been carried out expanding breeding for the double positive FVB mouse of transgenosis.Mode is as follows: F0 mouse with Wild type FVB mouse hybrid obtains F1 generation mouse；The double positive offsprings of picking transgenosis, it is brood it is interior mate, obtain F2 generation Mouse, wherein double positive individuals, progress sib mating obtain F3 for mouse to picking.The F3 of double positives is subjected to compatriot for mouse Interior hybridization obtains F4 for mouse.
5, the mRNA expression of the double positive mices of RT-PCR method identification transgenosis
By the breast tissue of the nursing period to transgenosis F0 to F2 generation double positive mices, with wild-type mice nursing period Breast tissue carries out RT-PCR detection as control.With house-keeping gene m β-Actin F/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) it is bis- to carry out F1, F2 that RT-PCR identification transgenosis FVB mouse is bred by the cDNA that three pairs of primer pairs obtain Positive mice.The mouse DNA for having amplification to primer with Hup/Hlw and Lup/Llw two is double positive；Only pair of primers There is amplification to be single positive, and two pairs of primers regard as yin without amplification or two pairs of weaker mouse DNAs of primer amplification band Property.Through 2% agarose gel electrophoresis as it can be seen that generation nursing period transgenosis F0 to F2 double positive mices and nursing period wild-type mice In four samples of breast tissue, there are the generation of internal reference cDNA purpose band, nursing period transgenosis F0 to F2 Dai Shuanyang at 203bp In property three samples of mouse, there is anti-p185 at 134bperbB2Human mouse chimeric antibody ChAb26 heavy chain gene H Partial Fragment CDNA purpose band generates, and there is anti-p185 at 73bperbB2The portion cDNA of human mouse chimeric antibody ChAb26 light chain gene L Fragment section purpose band generates, and does not find corresponding purpose band (Figure 30) in nursing period FVB wild-type mice breast tissue. The result shows that there are anti-p185 for the double positive mice breast tissues of nursing period transgenosiserbB2Human mouse chimeric antibody ChAb26 light chain base Because of the expression of the mRNA of L and heavy chain gene H.
9 RT-PCR of table or the primer of fluorescent PCR detection
The reaction system of 10 PCR of table
The program of PCR reaction are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 61 DEG C of 30sec, 72 DEG C of 30sec, 35cycles； 72℃ 7min；4℃∞.
6, the mRNA expression of the bis- positive mouse of quantitative fluorescent PCR identification transgenosis FVB
By to nursing period transgenosis F0 to F2 generation double positive mice breast tissues, with m β-Actin F/R (SEQ.ID.NO.19/SEQ.ID.NO.20), Hup/Hlw (SEQ.ID.NO.21/SEQ.ID.NO.22) and Lup/Llw Mammary gland of (SEQ.ID.NO.23/SEQ.ID.NO.24) the three couples of primer pair transgenosis F0 to F2 for the nursing period of transgenic mice Tissue is detected, wherein using the breast tissue of wild-type mice nursing period as control.It is right with Hup/Hlw and Lup/Llw two It is double positive that primer, which has the mouse DNA of amplification,；Only pair of primers has amplification to be single positive, and two pairs of primers are without expansion Increase or two pairs of weaker mouse DNAs of primer amplification band regard as feminine gender.The results show that in generation nursing period transgenosis F0 to F2, is double In four samples of breast tissue of positive mice and nursing period wild-type mice, house-keeping gene m β-Actin F/R primer can expand Increase solubility curve single and sharp out (Figure 31).In generation nursing period transgenosis F0 to F2 three samples of double positive mices, light chain Detection primer Lup/Llw can be amplified single and sharp solubility curve (Figure 32), nursing period wild-type mice breast tissue Sample fails to amplify solubility curve.In generation nursing period transgenosis F0 to F2 three samples of double positive mices, heavy chain detection primer Hup/Hlw can be amplified single and sharp solubility curve (Figure 33), and nursing period wild-type mice sample of breast tissue fails Amplify solubility curve.In conclusion fluorescent quantitative PCR experiment shows that the double positive mice breast tissues of nursing period transgenosis exist 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 and identification of human mouse chimeric antibody ChAb26
1, anti-p185erbB2The acquisition of human mouse chimeric antibody ChAb26
After maternal rat delivery 10 days, milk is carried out to transgenosis female rat (nursing period) using mouse milker (WAT 2006) and is adopted Collection；Then milk collected in collection tube is transferred to respectively in sterilizing Ep pipe, 4 DEG C, 4000 × g is centrifuged 20min；Centrifugation knot Milk is divided into three layers after beam, and milky upper layer is butterfat, and lower layer's insoluble matter, middle layer is clear liquid.It is disposable with 1ml Lower edge penetrates Ep pipe to syringe in middle level, and middle layer clear liquid, as anti-p185 is gently sucked outerbB2Human mouse chimeric antibody ChAb26 Solution.
2, Western blot detects the expression of chimeric antibody ChAb26 in the double positive mice milk of transgenosis
It uses human IgG albumen as positive control, transgenic mice and wild type FVB mouse is detected by Western blot The expression of chimeric antibody ChAb26 in milk.In human IgG protein groups and the double positive mice milk of transgenosis, in average molecular matter There is heavy chain of antibody and light chain specific band in the amount position 50KDa and 25KDa, and mouse IgG negative control group and wild type FVB are small Mouse milk group does not find heavy chain of antibody and light chain specific band (Figure 35), illustrates that constructed chimeric antibody ChAb26 can It is expressed in transgenic mouse milk.
3, Sandwich ELISA measures the expression of chimeric antibody ChAb26 in the double positive mice milk of transgenosis
Use human IgG albumen as positive control, PBS solution measures transfer base as negative control, by Sandwich ELISA Because the expression of results of chimeric antibody ChAb26 in double positive mice milk and EVB wild-type mice milk is shown, transgenic mice Milk is positive reaction, and EVB wild-type mice milk is negative reaction, i.e., contains chimeric antibody in transgenic mouse milk ChAb26, EVB wild-type mice milk do not contain chimeric antibody ChAb26 (Figure 36 and table 11).This shows anti-p185erbB2People mouse Chimeric antibody ChAb26 light chain gene L and heavy chain gene H can be expressed in transgenic mouse milk tissue, and can be correct Ground is assembled into active antibody, and is secreted into well extracellular.As it can be seen that Success in Experiment constructs anti-p185erbB2People mouse is embedding Antibody ChAb26 mammary gland-specific expression vector pBC1-H and pBC1-L are closed, and has succeeded and has prepared anti-p185erbB2Human mouse chimeric antibody ChAb26 transgenic mouse milk bioreactor model.
The anti-p185 of 11 sandwich ELISA assay of tableerbB2The 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: people IgG albumen with other three groups there are significant differences；4: transgenic mouse milk with other three groups there are significant differences.
4, chimeric antibody ChAb26 in Use immunohistochemistrySP SP detection transgenic mouse milk
Using chimeric antibody ChAb26 in the double positive mice mammary gland of Use immunohistochemistrySP SP detection transgenosis, (scheme as the result is shown 37), the cytoplasm of body of gland or cell membrane are not dyed to yellow or sepia in nursing period wild type EVB mammary gland of mouse tissue, i.e., Show there is no chimeric antibody ChAb26 expression in nursing period wild type EVB mammary gland of mouse.And turn in nursing period F0, F1 and F2 generation The cytoplasm of body of gland or cell membrane are colored yellow or sepia in Gene Double positive mice breast tissue, or even in nursing period F0 is for the chimeric antibody ChAb26 for being dyed to sepia for observing large area in transgenic mouse milk Tissue cavities.Show to feed Newborn phase F0, F1 and F2 there are chimeric antibody ChAb26 expression, i.e., successfully make for body of gland in the double positive mice breast tissues of transgenosis Standby anti-p185erbB2Human mouse chimeric antibody transgenic mouse milk bioreactor.
The anti-p185 of embodiment 4erb B2The identification of human mouse chimeric antibody ChAb26 related immunological characteristic
1, Sandwich ELISA detects the concentration of chimeric antibody ChAb26 in the double positive mice milk of transgenosis
Sheep anti-human kappa chain antibody (10 μ g/ml) is coated with using coating buffer on ELISA Plate, then passes through sandwich ELISA method Detect the concentration of chimeric antibody ChAb26 in the double positive mice milk of transgenosis.Using the human IgG albumen of standard through ELISA reality The OD450 value (table 12 and Figure 38) that test obtains has made standard curve, and calculate its correlation regression equation Y=0.04 χ+ 0.496 (Figure 39).The OD value of the double positive mice milk samples of each transgenosis is substituted into regression equation calculation, obtains cream after conversion Anti-human p185 in juiceerbB2The content of human mouse chimeric antibody ChAb26.Analysis is computed it is found that in the double positive mice milk of transgenosis Anti-human p185erbB2The content of human mouse chimeric antibody ChAb26 is in 0.231mg.L-1-0.248mg.L-1(table 13).The double sun of transgenosis Anti-human p185 in property mouse milkerbB2The average content of human mouse chimeric antibody ChAb26 is 0.240mg.L-1。
12 ELISA of table measures the OD450 value of each concentration human IgG albumen
Wherein, P < 0.05.
13 ELISA of table measures the OD450 value of the double positive mice milk of transgenosis
2, Salmonella method detects the antigentic specificity of chimeric antibody in the double positive mice milk of transgenosis
Using coating buffer by 10 μ g/ml people's erbB2 extracellular region antigen recombinant proteins, the hole 100ul/ is coated in ELISA Plate.So The antigentic specificity of chimeric antibody in the double positive mice milk of transgenosis is detected by Salmonella method afterwards.The results show that turning base It, also can quilt because chimeric antibody ChAb26 in double positive mice milk can be in conjunction with coated people erbB2 extracellular region antigen protein The identification of goat anti-human igg's Fc segment is positive in Salmonella experiment.The anti-human erbB2 monoclonal antibody of mouse is because cannot be with goat-anti Human IgG FC-HRP in conjunction with and it is negative.When using sheep anti-mouse igg Fc-HRP as secondary antibody, transgenosis is double positive small Though chimeric antibody ChAb26 can be in conjunction with coated people erbB2 extracellular region antigen protein in mouse milk, cannot be with sheep anti-mouse igg Fc-HRP in conjunction with and it is negative.And the anti-human erbB2 monoclonal antibody of mouse is positive in conjunction with sheep anti-mouse igg FC-HRP. No matter secondary antibody is goat anti-human igg Fc-HRP or sheep anti-mouse igg Fc- by FVB wild-type mice milk, human IgG, mouse IgG and PBS HRP negative (Figure 39, Figure 40 and table 14).This shows chimeric antibody ChAb26 tool in the double positive mice milk of transgenosis There is the specificity specifically with people's erbB2 extracellular region antigen binding.
14 Salmonella of table analyzes antigentic specificity and the humanized of ChAb26
In table, a: secondary antibody: goat anti-human igg-HRP b: secondary antibody: rabbit anti-mouse igg-HRP.
3, Sandwich ELISA detects the humanized of chimeric antibody ChAb26 in transgenic mouse milk
Sheep anti-human kappa chain antibody (10 μ g/ml) is coated with using coating buffer on ELISA Plate, then passes through sandwich ELISA method It detects in transgenic mouse milk in chimeric antibody ChAb26 experiment, chimeric antibody in the double positive mice milk of transgenosis ChAb26 can also be positive in conjunction with the sheep anti-human kappa chain antibody for being coated in ELISA Plate in conjunction with goat anti-human igg Fc-HRP Reaction.And FVB wild-type mice milk is negative in sandwich ELISA experiment, it was demonstrated that the double positive mice milk of transgenosis Middle chimeric antibody ChAb26 contain human IgG κ chain and Fc sections of heavy chain, that is, have humanized.
4, Salmonella method detects the humanized of chimeric antibody ChAb26 in transgenic mouse milk
It, in ELISA Plate, is carried out using goat anti-human igg Fc-HRP as secondary antibody direct by people's erbB2 extracellular region antigen coat In ELISA experiment, chimeric antibody ChAb26 can be with the people erbB2 born of the same parents that are coated in ELISA Plate in the double positive mice milk of transgenosis After outskirt antigen binding, it is positive and is unable in conjunction with rabbit anti-mouse igg Fc-HRP in conjunction with goat anti-human igg Fc-HRP.And FVB wild-type mice milk is negative in Salmonella experiment, it was demonstrated that is fitted into the double positive mice milk of transgenosis Antibody ChAb26 has humanized.
5, in Western Blot experimental identification transgenic mouse milk chimeric antibody ChAb26 humanized
F0 generation is collected to F3 for the double positive mice milk of transgenosis, using FVB wild-type mice milk as control, progress The humanized of chimeric antibody ChAb26 in Western Blot experimental identification transgenic mouse milk.(Figure 41) as the result is shown turns Protein band of the chimeric antibody ChAb26 at 50KDa can be known by goat anti-human igg Fc-HRP in Gene Double positive mice milk Not, single specific immune band is dyed.Its size position just corresponds to the heavy chain of human IgG antibody, and positive control human IgG is also Single specific immune band is dyed at 50KDa, negative control mouse IgG does not occur single specific immune band here, Show that chimeric antibody ChAb26 contains human IgG heavy chain constant region in the double positive mice milk of transgenosis.The double positive mices of transgenosis Protein band of the chimeric antibody ChAb26 at 25KDa can be known by sheep anti-human kappa chain antibody (rabbit-anti sheep-HRP is secondary antibody) in milk Not, single specific immune band is dyed.Its size position just corresponds to the light chain of human IgG antibody, and positive control human IgG also exists Single specific immune band is dyed at 25KDa, negative control mouse IgG does not occur single specific immune band, table here IgG κ chain constant region of the chimeric antibody ChAb26 containing someone in the double positive mice milk of bright transgenosis.Gene Double sun in summary Property mouse milk in chimeric antibody ChAb26 have humanized.
The anti-p185 of embodiment 5erb B2The identification of human mouse chimeric antibody ChAb26 correlation function characteristic
1, the inhibiting rate that CCK-8 kit detection chimeric antibody ChAb26 grows SKOV3 cell
The inhibiting rate that CCK-8 kit detection chimeric antibody ChAb26 grows SKOV3 cell.The results show that 0.48mg.L-1ChAb26 group and 0.5mg.L-1Herceptin group 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 oophoroma SKOV3 cell Proliferation, respectively For 53.63% and 58.43% (table 15)
Growth inhibition ratio of the 15 chimeric antibody ChAb26 of table to 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 is as negative control, with containing chimeric antibody ChAb26 is respectively 0.48,0.24 and 0.12mg.L-1The double positive mice milk of transgenosis and FVB wild-type mice milk point SKOV3 cell is not acted on for 24 hours, after 48h and 72h, carry out the detection of CCK-8 method, and using OD450 as ordinate, the time is horizontal seat Mark is drawn growth curve (Figure 42).
3, anti-p185 is observed in HE dyeingerbB2The morphological feature of human mouse chimeric antibody ChAb26 induction SKOV3 apoptosis
Anti-human p185 is observed in HE dyeingerbB2The morphological feature of human mouse chimeric antibody ChAb26 induction SKOV3 apoptosis.As a result It has been shown that, 0.48mg.L-1ChAb26 and 0.50mg.L-1Herceptin is acted on for 24 hours, and SKOV3 cell number significantly reduces, and cell goes out Existing shrinkage phenomenon, cell volume become smaller, and the connection between cell disappears, and are detached from the cell of surrounding, the deep dye of caryoplasm concentration, The thermophilic Yihong dye enhancing of cytoplasm.Cell blank control group and FVB wild-type mice milk group cell without significant change, cytoplasm and Nucleus is clear (Figure 43).
4, the anti-p185 of transmission electron microscope observingerbB2The morphological feature of human mouse chimeric antibody ChAb26 induction SKOV3 apoptosis
The results show that cell blank control group and the SKOV3 cell surface of FVB wild type milk processing group have more micro- suede Hair and pseudo- Microfilament, nucleus volume is larger, and form is irregular, and nuclear chromatin is abundant, some cells often have 2-3 kernel, Mitochondria and endoplasmic reticulum are more.And use 0.48mg.L-1ChAb26 or 0.50mg.L-1After Herceptin effect for 24 hours, then, it is seen that thin Born of the same parents' apoptosis phenomenon, mainly showing is cell shrinkage, chromatin agregation, and is concentrated under nuclear membrane.(Figure 44).
5, AnnexinV-PE/7ADD apoptosis kit assay chimeric antibody induces SKOV3 Apoptosis
AnnexinV-PE/7ADD apoptosis kit assay chimeric antibody induces SKOV3 Apoptosis.The result shows that (figure 45, table 16), 0.48mg.L-1It is 27.3% that ChAb26, which induces the middle and advanced stage apoptosis rate average value of SKOV3 Apoptosis,； 0.50mg.L-1It is 35.75% that Herceptin, which induces the middle and advanced stage apoptosis rate average value of SKOV3 Apoptosis,；Cell blank group and The SKOV3 cell middle and advanced stage apoptosis rate average value of FVB wild-type mice milk processing group is respectively 10.23% and 19.79%, warp SPSS.18 carries out there is significant difference (F=898.71, P < 0.01) between each processing group known to variance analysis.
The middle and later periods apoptotic cell accounting (unit: %) of SKOV3 cell after the induction for 24 hours of table 16
Wherein, F=898.71, P < 0.01.
6, triumphant basal cell's period kit assay chimeric antibody induction SKOV3 cell cycle checks
The result shows that (table 17, Figure 46), 0.48mg.L-1ChAb26、0.50mg.L-1Herceptin and FVB wild type cream After juice acts on SKOV3 cell for 24 hours, the SKOV3 cell accounting that versus cell blank control group is in the G1 phase occurs increasing phenomenon, 0.50mg.L-1Herceptin processing group increases 10.48%, 0.48mg.L-1ChAb26 processing group and the processing of FVB wild type milk Group increases 4.31% and 4.37% respectively.Due to heterogeneity of variance, carry out being in the G1 phase known to rank sum test in each processing group Not all the same (the X of SKOV3 cell accounting2=10.421, P < 0.05), then compared two-by-two it is found that 0.50mg.L- 1Herceptin processing group has differences with other each groups；0.48mg.L-1ChAb26 processing group and the processing of FVB wild type milk Group has differences with cell blank control group, but indifference (F=1.057E4, P < 0.01) between them. 0.48mg.L- 1ChAb26、0.50mg.L-1After Herceptin and FVB wild type milk acts on SKOV3 cell for 24 hours, versus cell blank control Group occurs reducing phenomenon, 0.50mg.L in the SKOV3 cell accounting of G2 phase-1Herceptin processing group reduces by 13.72%, 0.48mg.L-1ChAb26 processing group and FVB wild type milk processing group reduce by 5.19% and 3.73% respectively.Analysis of variance can Know that the SKOV3 cell accounting between each processing group in the G2 phase has differences (F=3.157E4, P < 0.01).In conclusion 0.48mg.L-1ChAb26、0.50mg.L-1Herceptin and FVB wild type milk can be such that SKOV3 cell hinders to some extent It is stagnant in G1/S and S/G2 test point, but blockage effect and fairly obvious, wherein 0.48mg.L-1ChAb26 and FVB wild type cream Juice makes SKOV3 cell block in the effect indifference of G1/S test point.
The cell cycle analysis (unit: %) of SKOV3 cell after the induction for 24 hours of 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 processing group；C:0.48mg.L-1ChAb26 processing group；D:0.50mg.L-1Herceptin processing group.

Claims (5)

1. a kind of anti-p185erbB2Human mouse chimeric antibody ChAb26, the amino acid mainly encoded by heavy chain gene H and light chain gene L Composition, it is characterised in that: the heavy chain gene H and light chain gene L be respectively provided with sequence table SEQ .ID.No.3 and The base sequence of SEQ.ID.No.6；The antibody specific presses following operation preparation: by by SEQ.ID.No.3 and The base sequence of SEQ.ID.No.6 is connected respectively on mammary specific expression plasmid pBC1, constructs to obtain p185erbB2People mouse is chimeric It is small to import FVB after pBC1-H and pBC1-L linearisation by antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L Mouse fertilized eggs produce transgenosis FVB mouse, using it as galactophore biological reactor, are obtained by mammary specific expression anti- p185erbB2Human mouse chimeric antibody ChAb26；Wherein, the construction method of mammary gland specific expression vector pBC1-H and pBC1-L are as follows:
<1>to contain anti-p185erbB2The pUC57/pBCN-H- of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L F2A-L-ployA-EGFP-Neo plasmid is template, under the effect of LA Taq archaeal dna polymerase, respectively with H-F/H-R and L-F/L- R is that primer amplification goes out anti-p185erb B2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L；PCR reaction program be 94℃5min；94℃30sec；50℃30sec；69℃2min30sec；35cycles；69 DEG C of 7min, 4 DEG C of ∞；Wherein, described Heavy chain gene H and light chain gene L is respectively provided with the base sequence of sequence table SEQ .ID.No.3 and SEQ.ID.No.6；
<2>the heavy chain gene H and light chain gene L recovery purifying amplified step<1>using Ago-Gel QIAquick Gel Extraction Kit；
<3>the heavy chain gene H and light chain gene L by step<2>recycling are connected respectively on pEASY-T1 cloning vector, are weighed The sub- pEASY-T1-H and pEASY-T1-L of group；
<4>digestion is carried out to the recon pEASY-T1-H and pEASY-T1-L that step<3>obtains using Xho I and uses ethyl alcohol Digestion products heavy chain gene H and light chain gene L is carried out recovery purifying by the precipitation method；
<5>by the heavy chain gene H and light chain gene L of step<4>recycling respectively and in advance through Xho I digestion and CIP alkaline phosphatase The dephosphorylized mammary specific expression plasmid pBC1 of enzyme is connected under T7 connection enzyme effect, and building obtains anti-p185erb B2People mouse Chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
2. being used to prepare anti-p185 described in claim 1erbB2The mammary gland specific expression vector of human mouse chimeric antibody ChAb26 The construction method of pBC1-H and pBC1-L, it is characterised in that carried out by following operation:
<1>to contain anti-p185erbB2The pUC57/pBCN-H- of human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L F2A-L-ployA-EGFP-Neo plasmid is template, under the effect of LA Taq archaeal dna polymerase, respectively with H-F/H-R and L-F/L- R is that primer amplification goes out anti-p185erbB2Human mouse chimeric antibody ChAb26 heavy chain gene H and light chain gene L；PCR reaction program be 94℃5min；94℃30sec；50℃30sec；69℃2min30sec；35cycles；69 DEG C of 7min, 4 DEG C of ∞；Wherein, described Heavy chain gene H and light chain gene L is respectively provided with the base sequence of sequence table SEQ .ID.No.3 and SEQ.ID.No.6；
<2>the heavy chain gene H and light chain gene L recovery purifying amplified step<1>using Ago-Gel QIAquick Gel Extraction Kit；
<3>the heavy chain gene H and light chain gene L by step<2>recycling are connected respectively on pEASY-T1 cloning vector, are weighed The sub- pEASY-T1-H and pEASY-T1-L of group；
<4>digestion is carried out to the recon pEASY-T1-H and pEASY-T1-L that step<3>obtains using Xho I and uses ethyl alcohol Digestion products heavy chain gene H and light chain gene L is carried out recovery purifying by the precipitation method；
<5>by the heavy chain gene H and light chain gene L of step<4>recycling respectively and in advance through Xho I digestion and CIP alkaline phosphatase The dephosphorylized mammary specific expression plasmid pBC1 of enzyme is connected under T7 connection enzyme effect, and building obtains anti-p185erb B2People mouse Chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L.
3. according to the method described in claim 2, it is characterized by: the primer H-F/H-R and L-F/L-R is respectively provided with sequence The base sequence of table SEQ.ID.No.1/SEQ.ID.No.2, SEQ.ID.No.4/SEQ.ID.No.5.
4. being used to prepare anti-p185 described in claim 1erbB2The preparation side of the transgenosis FVB mouse of human mouse chimeric antibody ChAb26 Method, it is characterised in that carried out by following operation:
<1>the anti-p185 that will be constructed according to claim 2 the method using Not I and Sal IerbB2Human mouse chimeric antibody ChAb26 mammary gland specific expression vector pBC1-H and pBC1-L are linearized, and anti-p185 is obtainederb B2Human mouse chimeric antibody The specific expressed linear carrier pBC1-H-linear and pBC1-L-linear of ChAb26 transgenic mouse milk；
<2>mammary specific expression linear carrier pBC1-H-linear and pBC1-L-linear are passed through into the micro- co-injection of protokaryon Mode carry out fertilized eggs co-injection；After injection, by zygote transplation to receptor FVB system Mouse Uterus, obtained after gestation Obtain F0 generation double positive transgenic FVB mouse.
5. obtaining anti-p185 using transgenosis FVB mouse made from method according to claim 4erbB2Human mouse chimeric antibody The method of ChAb26, it is characterised in that carried out by following operation: after maternal rat delivery 10 days, using mouse milker to transgenosis mother Mouse carries out milk acquisition；Then, collected milk is transferred to respectively in sterilizing Ep pipe, 4 DEG C, 4000 × g is centrifuged 20min； After centrifugation, middle layer clear liquid is sucked out gently to get anti-p185erbB2Human mouse chimeric antibody ChAb26 solution.
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