稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用  

Establishment of Mouse CD40L Stably Expressed NIH3T3 Cell Lines and Its Application in the Culture and Activation of B Lymphocytes

作　　者：郎巧利 何琦琳 黄楠 余琳 杨希 LANG Qiaoli;HE Qilin;HUANG Nan;YU Lin;YANG Xi(Chongqing Academy of Animal Sciences,Key Laboratory of Pig Industry Sciences,Chongqing Research Center for the Development and Utilization of Medical Animal Resources,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,农业部养猪科学重点实验室,养猪科学重庆市市级重点实验室,重庆市医用动物资源的开发与利用工程计算研究中心,重庆402460

出　　处：《中国细胞生物学学报》2020年第1期118-124,共7页Chinese Journal of Cell Biology

基　　金：重庆市科研院所绩效激励引导专项(批准号:cqjxjl201709);重庆荣昌农牧高新技术产业研发专项(批准号:19255);国家自然科学基金(批准号:5167070727)资助的课题。

摘　　要：该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。Recombinant plasmid pcDNA3.1-mCD40L was constructed and transfected into NIH3T3 cells to express mouse CD40L.The transfected cells were selected with G418 to obtain stably transfected cell lines.The RNA of stable transfected cells was isolated and the mRNA expression level of Neo gene was detected by RT-PCR.The expression level of CD40L protein in the culture supernatant of transfected cells was detected by ELISA analysis.RT-PCR revealed that Neo gene could be expressed in the transfected cells.ELISA results showed that the concentration of CD40L in the supernatant of the stable transfected cell line NIH3T3-mCD40L was 1.286 ng/mL.Further activity studies showed that the addition of NIH3T3-mCD40L cells,IL-2 and IL-21 could successfully culture and stimulate B cells to produce specific antibodies.NIH3T3-mCD40L stable cell lines was successfully obtained.This study lays a good foundation for generation of monoclonal antibody from B lymphocytes.

关 键 词：鼠CD40L 稳定转染 NIH3T3细胞 饲养细胞 

分 类 号：Q813.11[生物学—生物工程]