含肠道病毒71型VP1基因片段RNA病毒样颗粒的构建  

CONSTRUCTION OF VIRUS-LIKE PARTICLES CONTAINING THE VP1 GENE FRAGMENT OF EV71

作　　者：张文帅[1] 崔仑标[1] 葛以跃[1] 戚宇华[1] 单军[1] 李显[1] 史智扬[1] 

机构地区：[1]卫生部肠道病原微生物重点实验室江苏省疾病预防控制中心微生物科,南京210009

出　　处：《现代预防医学》2010年第3期515-518,共4页Modern Preventive Medicine

基　　金：江苏省国际科技合作项目(BZ2008068)

摘　　要：[目的]构建含肠道病毒71型(EV71)VP1基因片段的抗核糖核酸酶的病毒样颗粒。[方法]利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-MS2。将EV71VP1基因片段连接到中间载体噬菌体基因的下游,构建原核表达载体pET32a-MS2-EV71。将重组质粒pET32a-MS2-EV71转化宿主菌,经IPTG诱导表达获得病毒样颗粒,对病毒样颗粒进行透射电镜观察、RT-PCR检测和稳定性试验。[结果]重组质粒pET32a-MS2-EV71经PCR、双酶切鉴定和测序分析后证实构建成功;透射电镜观察到直径大约23nm的圆形病毒样颗粒;RT-PCR和稳定性试验结果表明,诱导产生的病毒样颗粒含EV71VP1基因片段,并且稳定性良好。[结论]成功构建了含EV71VP1基因片段的病毒样颗粒,稳定性良好,可作为病毒检测时标准品和质控品使用。[Objective] To construct RNase-resistant virus-like particles containing the VP1 gene fragment of enterovirus type 71（EV71）.[Methods] The coatprotein and maturase gene of E.coli bacteriophage MS2 was amplified by PCR,then the gene was cloned into pET32a to construct the intermediate vector pET32a-MS2.Prokaryotic expression vector pET32a-MS2EV71 was constructed by cloning the VP1 gene fragment of EV71 into the downstream of bacteriophage gene of pET32a-MS2.To obtain virus-like particles,the recombinant plasmid pET32a-MS2-EV71 was transformed into E.coli BL21 strain and induced by IPTG.Then the virus-like particles were observed by TEM,tested by RT-PCR and stable experiment.[Results] The recombinant plasmid pET32a-MS2-EV71 was successfully constructed.The expression products observed by transmission electron microscope（TEM） were circular virus-like particles,and the diameter of these particles was about 23nm.The results of RT-PCR and stability test showed that the virus-like particles contained the VP1 gene fragment of EV71 and had good stability.[Conclusion] A new virus-like particles containing the VP1 gene fragment of EV71 is successfully constructed and has good stability,it could be used as the standard and quality control in the area of virus detection.

关 键 词：MS2噬菌体 EV71 病毒样颗粒 

分 类 号：R373.2[医药卫生—病原生物学]