中国生物工程杂志

Objective: To construct the vectors of human-mouse chimeric antibody ChAb26 with anti-p185, which can be expression in transgenic mice mammary gland and to express the human-mouse chimeric antibody ChAb26 with anti- p185 by mammary gland bioreactor of transgenic mice. Methods: Firstly, to obtain heavy chain gene H and light chain gene L of human-mouse chimeric ChAb26 by PCR. Secondly, the heavy chain gene H and the light chain gene L of human-mouse chimeric antibody ChAb26 connection to pBC1 after their serial numbers are true by DNA sequencing technology proved. Finally, using DNA sequencing technology for detection the vectors of human-mouse chimeric antibody ChAb26 with anti-p185, pBC1-H and pBC1-L and firstly, the pBC1-H plasmid and pBC1-L plasmid to be linearized by SalI and NotI.After,they become the pBC1-H-linear plasmid and pBC1-L-linear plasmid. Secondly,the pBC1-H-linear plasmid and pBC1-L-linear plasmid to be co-injectioned into the fertilized eggs of FVB mice by microinjection. Thirdly,using mouse tail direct PCR kit for detection the positive clones of the founder mice and their descendants.Furthermore,using RT-PCR and real-time PCR technique for detection the express mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland.The last,using ELISA and Western blot technique for detection the express of mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland. Results: Sequence analysis showed that the serial numbers of pBC1-H plasmid and pBC1-L plasmid are true.Sequence analysis showed that all of 8 founder mice were the positive clones by mouse tail direct PCR identification.Besides,the positive clones rate of their descendants average value is 30%,from F1 to F4 generation.Western blot result showed that a 50kDa heavy chain specific band and a 25kDa light chain specific band were observed in milk of transgenic mice,this meaning milk of transgenic mice containthe human-mouse chimeric antibody ChAb26 with anti- p185. ELISA result indicated that the milk of transgenic mice can combine with anti-human IgG (Fc specific)-HRP antibody produced in goat and anti-human Kappa light chain antibody produced in goat,this meaning milk of transgenic mice containthe human-mouse chimeric antibody ChAb26 with anti- p185.RT-PCR and real-time PCR technique result indicated that the express mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland. Conclusion: The anti-p185 ^{erb B2} human mouse chimeric antibody ChAb26 transgenic animal mammary gland specific expression vectors pBC1-H and pBC1-L were successfully constructed and the anti-p185 ^{erb B2} human mouse chimeric antibody ChAb26 transgenic mouse mammary gland bioreactor model was prepared for future anti-p185 ^{erb B2}. The human and mouse chimeric antibody ChAb26 transgenic bovine mammary gland bioreactor laid the theoretical and technical basis.