高通量表达系统在EV71单链抗体表达中的应用  

Construction and application of EV71 ScFv high-throughput cassette expression system

作　　者：余秋凡 陶焱炀 李靖欣[2] 王圆媛 张黎[2] 朱凤才[1,3,4] Yu Qiufan;Tao Yanyang;Li Jingxin;Wang Yuanyuan;Zhang Li;Zhu Fengcai(School of Public Health,Southeast University,Nanjing 210009,China;Department of Vaccine Clinical Evaluation,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;National Health Commision Key laboratory of Enteric Pathogenic Microbiology,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;Center for Global Health,Nanjing Medical University,Nanjing 210009,China)

机构地区：[1]东南大学公共卫生学院,南京210009 [2]江苏省疾病预防控制中心疫苗临床评价所,南京210009 [3]江苏省疾病预防控制中心国家卫生健康委员会肠道病原微生物重点实验室,南京210009 [4]南京医科大学全球健康中心,210009

出　　处：《中华实验和临床病毒学杂志》2019年第6期641-645,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：江苏省疾病预防控制中心科教强业项目(JKRC2016020)。

摘　　要：目的 建立从PCR产物直接表达ScFv抗体的方法,将该方法用于噬菌体抗体库淘选后ScFv抗体的高通量表达。方法 PCR扩增CMV启动子、ScFv和BGH Poly A尾基因片段,使用重叠PCR(overlapping PCR)将上述3个片段依次连接成1个线性的抗体表达盒。瞬时转染293T细胞,并用Western blot、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和间接免疫荧光抗体试验(indirect immunofluorescent antibody,IFA)检测抗体表达盒中ScFv的表达和抗体的活性。从噬菌体库淘选出的阳性克隆中挑取96个抗体,用该表达盒进行高通量表达,并对表达效果进行评价。结果 成功扩增出3个基因片段,并将3个片段连接成1个线性的表达盒。线性表达盒可直接在293T细胞中瞬时表达,表达上清经Western blot检测可见约相对分子质量为38×103的条带;ELISA和IFA结果均显示表达出来的ScFv抗体能与其对应的抗原特异性结合。同时该系统可以用于噬菌体抗体库中抗体基因的高通量表达。结论 成功构建了ScFv的线型表达盒,并实现了抗体的快速高通量表达。Objective To construct a method to express ScFv antibody from PCR products,and use it in phage display for high-throughput ScFv expression.Methods Cytomegalovirus(CMV)promotor,ScFv and BGH-Poly A gene fragments were amplified by PCR.Overlapping PCR was used to form a tandemly linear cassette gene.By transiently transfected into 293T cells,ScFv antibodies expression of cassette gene were tested by Western blot,enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescent antibody(IFA).Ninety-six clones of antibody genes in phage library were selected and expressed by cassette expression system.The expression level was evaluated and analyzed.Results Three fragments were obtained and a cassette expression system formed.Cassette expression system worked successfully in 293T cells,as a Mr.38×103 brand could observed in Western blot assay.The expressed antibody could specifically bind to its antigen by result of ELISA and IFA.This cassette expression system could also be used in phage display for high-throughput panning.Conclusions The cassette expression system was constructed successfully and high-throughput antibody expression has been achieved.

关 键 词：ScFv抗体 抗体表达盒 重叠PCR 高通量表达 

分 类 号：Q78[生物学—分子生物学]