弓形虫SAG1纳米抗体噬菌体文库的构建与鉴定分析  

CONSTRUCTION AND IDENTIFICATION OF PHAGE DISPLAY LIBRARY OF TOXOPLASMA GONDII SAG1 NANOBODY

作　　者：赵宇豪 薛杨继 丁豪杰 丁建祖 郑斌 卓洵辉 楼涤 陈睿 孔庆明 陆绍红 ZHAO Yu-Hao;XUE Yang-Ji;DING Hao-Jie;DING Jian-Zu;ZHENG Bin;ZHUO Xun-Hui;LOU Di;CHEN Rui;KONG Qing-Ming;LU Shao-Hong(Institute of Parasitic Diseases,Hangzhou Medical College,Hangzhou 310013,China)

机构地区：[1]杭州医学院寄生虫病研究所,杭州310013

出　　处：《寄生虫与医学昆虫学报》2020年第4期205-212,共8页Acta Parasitologica et Medica Entomologica Sinica

基　　金：浙江省科技计划重点研发计划(项目编号:2019C03057);浙江省医药卫生科技计划(项目编号:2019PY025);浙江省卫生高层次创新人才培养工程项目。

摘　　要：弓形虫表面抗原1(Surface antigen 1,SAG1)是虫体入侵宿主细胞的重要蛋白,与弓形虫的毒力密切相关,属于弓形虫病诊断和疫苗研究的重要候选分子。本研究表达了弓形虫RH株重组SAG1(Recombinant SAG1,rSAG1)蛋白并免疫双峰驼,分离、提取外周血淋巴单核细胞总RNA,以巢式PCR扩增骆驼重链抗体可变区(VHH),并将其连接至pHEN4载体,构建了SAG1噬菌体纳米抗体库,库容量为3.24×10^(9) cfu/mL,克隆阳性率90%。二轮淘选后富集因子为3.75×10^(3)。随机挑选40个阳性克隆测序,VHH基因编码的氨基酸序列同源性比对分析发现8个phage-ELISA值比较高的阳性克隆聚为3类。亚克隆至pMECS原核表达载体体外表达、纯化后获得8个大小约17 kDa的Anti-SAG1-Nb。免疫印迹结果显示Anti-SAG1-Nb-5能够与弓形虫天然抗原反应并产生明显的特异性条带。生物膜干涉法进一步获得该纳米抗体与SAG1的亲和常数为1.66 nmol/L。本研究首次构建了弓形虫SAG1纳米抗体库,淘选、制备并鉴定了8个Anti-SAG1-Nb,为弓形虫感染的早期检测试剂研制奠定了基础。Toxoplasma gondii(T.gondii)surface antigen 1(SAG1),closely related to the virulence of T.gondii,is an important protein in host cell invasion,and the important candidate for toxoplasmosis diagnosis and vaccine research.The T.gondii RH strain recombinant SAG1(rSAG1)was used to immunize a camel,the total RNA was then isolated from the peripheral blood lymphocyte pools of the camel,and reverse-transcribed into cDNA.VHHs of heavy chain antibody variable region were amplified by nested PCR,and ligated into a phagemid vector pHEN4 for constructing SAG1 phage nanobody(Nb)library.The estimated capacity of this library was 3.24×10^(9) cfu/mL,the positive clone rate was to be 90%,and the enrichment factor was 3.75×10^(3) after two rounds of affinity panning.Forty positive clones were randomly selected and sequenced,the homology analysis of amino acid sequences encoded by VHH indicated that 8 positive clones with higher phage-ELISA value could be classified into 3 different groups.These clones were subcloned into the pMECS prokaryotic expression vector to express the Nb with His-tag,these Nbs were purified through Ni-chelating column chromatography,then 8 Anti-SAG1-Nb with molecular mass around 17 kDa were obtained after purification.Western blotting showed that the Anti-SAG1-Nb-5 might react with the native antigen of T.gondii to form obvious specific bands.ForteBio Octet System analysis further confirmed that the affinity constant of the nanobody with SAG1 was 1.66 nmol/L.The SAG1 phage nanobody library has been firstly generated in present study,and 8 Anti-SAG1-Nbs were prepared and identified,which laid a foundation for developing the early detection reagent of T.gondii infection.

关 键 词：刚地弓形虫 表面抗原蛋白1 纳米抗体 噬菌体展示文库 

分 类 号：R382.5[医药卫生—医学寄生虫学]