重组溶瘤痘苗病毒VG9-HGFK1的构建  

Construction of recombinant oncolytic vaccinia virus VG9-HGFK1

作　　者：程运涛 杨雪 胡泽阳 周惠敏 邓黎莉[4] 胡志刚 CHENG Yun-tao;YANG Xue;HU Ze-yang;ZHOU Hui-min;DENG Li-li;HU Zhi-gang(Wuxi People′s Hospital Affiliated to Nanjing Medical University,Wuxi Children′s Hospital,Jiangsu,Wuxi 214000,China)

机构地区：[1]南京医科大学附属无锡人民医院无锡市儿童医院,江苏无锡214000 [2]南通大学公共卫生学院,江苏南通226000 [3]江苏大学医学院,江苏镇江212000 [4]江苏省原子医学研究所,江苏无锡214000

出　　处：《中国病毒病杂志》2020年第3期184-188,共5页Chinese Journal of Viral Diseases

基　　金：国家自然科学基金青年科学基金项目(81703061);江苏省妇幼健康重点人才项目(FRC201741);无锡市医学重点人才培养项目(ZDRC006);无锡市科技发展资金项目(WX18IIAN027)。

摘　　要：目的构建表达人肝细胞生长因子第一结构域(kringle 1domain of hepatocyle growth factor,HGFK1)基因的重组溶瘤痘苗病毒VG9-HGFK1,并初步检测其对人源宫颈癌细胞(Hela细胞)的杀伤效果。方法将pCB-HGFK1转入大肠埃希菌JM109,挑取单克隆菌落进行测序。以痘苗病毒天坛株广9(VG9)为载体,与pCB-HGFK1质粒共转染HEK 293T/17细胞,在细胞内同源重组,通过筛选表达HGFK1基因的重组溶瘤痘苗病毒VG9-HGFK1,用聚合酶链式反应(PCR)鉴定重组病毒中插入基因HGFK1的表达,用免疫印迹法鉴定VG9-HGFK1感染非洲绿猴肾细胞(BSC-40细胞)内HGFK1基因的表达,并进行药筛、病毒纯化及滴度测定。利用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测VG9、VG9-EGFP及VG9-HGFK1对Hela细胞的杀伤效果。结果收集单个菌落,进行测序鉴定,与目标序列一致。HGFK1基因验证,电泳结果显示,在约800bp处可见条带,HGFK1的大小为818bp,与电泳结果相一致。重组型TK区验证,仅在2291bp处出现条带。免疫印迹结果证实了HGFK1基因的表达。通过滴度测定,VG9-HGFK1滴度为2×107 PFU/ml。MTT结果显示VG9-HGFK1对Hela细胞的杀伤效果高于VG9及VG9-EGFP。结论成功构建了重组溶瘤痘苗病毒VG9-HGFK1,其对Hela细胞有明显的杀伤作用,为后续抗肿瘤实验打下了基础。Objective To construct oncolytic recombinant vaccinia virus VG9-HGFK1expressing HGFK1gene and initially test its killing effect on Hela cells.Methods pCB-HGFK1plasmid was transformed into E.coli JM109,and the monoclonal colonies were selected for sequencing.293Tcells were co-transfected with the vector of vaccinia virus Tiantan strain(VG9)and the plasmid pCB-HGFK1for homologous recombination in cells.Polymerase chain reaction(PCR)identified the expression of the inserted gene HGFK1.Western blot identified the expression of HGFK1gene in VG9-HGFK1infected BSC-40cells.Recombinant virus VG9-HGFK1was used for drug screening,virus purification and titer determination.MTT was used to detect the killing effect of VG9,VG9-EGFP and VG9-HGFK1on human cervical cancer cell line Hela.Results Sequencing of collected single colony was consistent with the target sequence.HGFK1gene in recombinant virus was verified by PCR,as well as the TK region.The result of HGFK1gene in the recombinant virus showed a band about 800bp,while the relative molecular weight of HGFK1was 818bp.The result of TK region showed a single band at 2291bp.Western blot confirmed the expression of HGFK1gene in BSC-40cells.The titer of VG9-HGFK1was 2×10^7 PFU/ml by titer measurement.MTT results showed that the killing effect of VG9-HGFK1on Hela cells was higher than that of VG9and VG9-EGFP.Conclusions The recombinant oncolytic vaccinia virus VG9-HGFK1was successfully constructed.The virus has obvious killing effect on cervical cancer Hela cells.

关 键 词：血管抑制因子 病毒载体 痘苗病毒 天坛株 

分 类 号：R373.9[医药卫生—病原生物学]