禽流感病毒H7N9人源性单链抗体文库的构建及鉴定  

Development and identification of a human single-chain fragment antibody library against avian influenza A H7N9 virus

作　　者：迟莹[1] 张文帅[1] 焦永军[1] CHI Ying;ZHANG Wen-shuai;JIAO Yong-jun(Jiangsu Provincial Center for Disease Control and Prevention,NHC Key Laboratory of Enteric Pathogenic Microbiology,Jiangsu Nanjing 210009,China)

机构地区：[1]江苏省疾病预防控制中心国家卫生健康委员会肠道病微生物重点实验室

出　　处：《江苏预防医学》2019年第6期611-613,共3页Jiangsu Journal of Preventive Medicine

基　　金：国家自然科学基金青年基金(81601732);国家自然科学基金(31570926);江苏省疾病预防控制中心科教强业工程青年人才(JKRC2016018)

摘　　要：目的构建人源性禽流感病毒H7N9单链抗体文库(scFv),并对文库质量进行鉴定。方法分离11例H7N9恢复期患者外周血中的单个核淋巴细胞(PBMC),提取总RNA,逆转录成cDNA;并以此为模板PCR扩增人源性抗体重链可变区(VH)及轻链可变区(Vκ),再通过重叠PCR法将VH和Vκ基因随机拼接成scFv文库;将构建的scFv文库克隆入噬菌体载体pComb3XSS中,电转化感受态细胞XL1-Blue,制备成完整的人源性禽流感病毒H7N9单链抗体文库;通过计算菌落数量及基因测序检测分析scFv文库的库容量及多样性。结果构建的人源性禽流感病毒H7N9单链抗体文库的库容为1.67×107,测序结果显示scFv文库多样性好,scFv基因阳性率为94.0%,准确率为95.7%。结论成功构建人源性禽流感病毒H7N9单链抗体文库,为后续筛选特异性中和抗体提供了基础。Objective To develop and identify a human single-chain fragment antibody(scFv)library against avian influenza A H7N9 virus.Methods The total RNA was isolated from the peripheral blood mononuclear cell of 11 recovered H7N9 patients and then reverse transcripted into cDNA.The heavy chains(VH)and the light chains(Vk)were amplified by PCR using cDNA as templates and were assembled randomly to scFv library by overlap-PCR.The assembled scFv library was cloned into the pComb3XSS vector and then electro-transformed into XLl-Blue cell to development a complete human scFv library against avian influenza A H7N9 virus.The volume and the divers让y of the constructed scFv library was detected by calculating the colonies and sequencing analysis.Results The volume of the constructed scFv library was 1.67X10^7,sequencing results showed that scFv library had good diversity with cloning efficiency of 94.0%and the accuracy of 95.7%.Conclusion A high-capacity and diverse human scFv library against avian influenza A H7N9 virus has been developed,which will provide a basis for the subsequent screening of specific neutralizing antibodies.

关 键 词：禽流感病毒H7N9 人源性单链抗体 抗体文库(scFv) 

分 类 号：R511.7[医药卫生—内科学]