抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证

目的:构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体并制备和验证抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型。方法:利用PCR法扩增出抗人p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L,然后分别将嵌合抗体重链基因H和嵌合抗体轻链基因L连接到乳腺特异性表达质粒pBC1,从而构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L。分别将抗p185~(erbB2)人鼠嵌合抗体ChAb26乳腺特异表达载体pBC1-H和pBC1-L线性化,然后使用原核显微共注射法获得8只转基因FVB小鼠,通过鼠尾直接PCR鉴定其转基因阳性。通过RT-PCR、荧光定量PCR鉴定转基因小鼠乳腺组织中抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达。使用小鼠乳汁采集器收集其乳汁并通过Western blot和夹心ELISA等实验鉴定抗p185~(erbB2)人鼠嵌合抗体ChAb26是否获得表达。结果:经测序验证,抗p185~(erbB2)人鼠嵌合抗体ChAb26的嵌合重链基因H和嵌合轻链基因L分别与乳腺特异表达质粒pBC1正确正向连接。鼠尾直接PCR结果显示所获8只转基因FVB小鼠均为转基因双阳性小鼠,且抗p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L在它们的后代中稳定遗传,它们的后代中转基因小鼠双阳性率约为30%; RT-PCR和荧光定量PCR的结果显示,转基因双阳性小鼠及其双阳性后代的乳腺组织中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达; Western blot和ELISA等实验结果显示,转基因双阳性小鼠乳汁中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的蛋白质表达,而且抗p185~(erbB2)人鼠嵌合抗体ChAb26与羊抗人κ链抗体和羊抗人Ig G Fc-HRP抗体均能特异性结合。结论:成功构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L和制备了抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型,为今后抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因牛乳腺生物反应器的研究奠定了理论和技术基础。

The Preparation and Validation of p185 erb B2 Human-mouse Chimeric Antibody ChAb26 Transgenic Mice Mammary Gl and Bioreactor

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.