抗p185erbB2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  

The Preparation and Validation of p185erbB2 Human-mouse Chimeric Antibody ChAb26 Transgenic Mice Mammary Gl and Bioreactor

作　　者：梁振鑫 刘芳[2] 张玮[1] 刘庆友[2] 李力[1] LIANG Zhen-xin;LIU Fang;ZHANG wei;LIU Qing-you;LI Li(Affiliated Tumor Hospital of Guangxi Medical University,Regional High-Defense Tumor Early Prevention Research Department Key Laboratory of Ministry of Education ,Nanning 530021,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西医科大学附属肿瘤医院区域性高发肿瘤早期防治研究教育部重点室验室,南宁530021 [2]广西大学动物科学技术学院,南宁530004

出　　处：《中国生物工程杂志》2019年第8期40-51,共12页China Biotechnology

基　　金：广西科技攻关项目(1598013-2)资助项目

摘　　要：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IgG 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 Sal I and Not I. After,they become the pBC1-H-linear plasmid and pBC1-L-linear plasmid. Secondly,the pBC1-H-linear plasmid and p BC1-L-linear plasmid to be coinjectioned 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 50 kDa heavy chain specific band and a 25 kDa 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

关 键 词：erbB2原癌基因 嵌合抗体 转基因小鼠 乳腺生物反应器 

分 类 号：Q819[生物学—生物工程]