博卡病毒VP2基因在昆虫细胞sf9中表达及临床标本筛查  

Expression of recombinant VP2 gene in insect sf9 cells and screening of clinical specimens

作　　者：滕灵方[1] 林峰[1] 郑美云[1] 郑昌华[1] 吴锋[1] 曾爱平[1] 黄恩佩[1] 莫一涵[1] 郑敏巧[1] 李旭阳[1] 侯建毅[1] 

机构地区：[1]温州医学院附属温岭医院,浙江温州317500

出　　处：《中华实验和临床病毒学杂志》2009年第6期427-429,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（No.30771913）;国家高技术研究发展“八六三”计划基金（No.2007AA02Z400）.

摘　　要：目的表达人博卡病毒（HBoV）vP2蛋白，用于临床标本HBoV的筛查。方法将VP2基因重组于杆状病毒基因组，重组的杆状病毒感染昆虫细胞sf9，用HA抗体进行Western Blot鉴定重组融合vP1蛋白表达。通过电镜观察重组蛋白颗粒。以vR2蛋白作为抗原对176例临床样本进行免疫印迹筛查。结果在昆虫细胞中vB蛋白的表达量约占细胞总蛋白的60％以上，VP2蛋白相对分子质量为60×10^3。电镜观察博卡病毒vP，蛋白形成病毒样颗粒（VLP），其大小在20nm左右；采用免疫印迹技术从临床标本中筛查出4例患者HBoV阳性，阳性率达到2.28％（4／176）。结论本研究成功的在昆虫细胞中表达了博卡病毒VR蛋白；表达vP2蛋白具有一定抗原性，为开展人博卡病血清学研究方法建立提供基础。Objective To clone and express VP2 gene from HBoV, and the expressed VP2 protein was as the antigen in order to detect serum from children in Wenling area with lower respiratory tract infections. Methods The VP2 gene was recombined with the genome of Baculovirus, which infected the insect cell. The fusion protein with HA tag was applied to confirm the specificity of expressed protein. Furthermore, the recombinant protein was observed using electron microscopy. The 176 serum from children in Wenling area with lower respiratory tract infections was screened using Western blot. Results The expressed VP2 protein was more than 60% in total proteins from insect cell, and MWt about 60×10^3. The virus-like particle （VLP） was observed using electron microscopy, and size about 20nm. The 176 serum from children in wenling area with lower respiratory tract infections was screened using Western blot. The HBoV positive rate was 2.28% （4/176）. Conclusion The VP2 protein from human bocavirus was expressed in insect cell successfully. Through HA tag the VP2 protein was specific, and then the assay using SDS-PAGE with Western blot could detect and screen the antibody in serum from children with lower respiratory tract infections rapidly and accurately.

关 键 词：细小病毒科 基因表达调控 免疫印迹法 

分 类 号：R3[医药卫生—基础医学]