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-CP。将口蹄疫病毒(FMDV)的内部核糖体结合位点(IRES)保守序列连接到中间载体噬菌体基因的下游,构建原核表达载体pCPES。将重组质粒pCPES转化宿主菌BL21(DE3),1 mmol/L IPTG诱导表达。蔗糖密度梯度离心纯化表达产物。透射电镜观察到直径大约26 nm的圆形病毒样颗粒。检测病毒样颗粒的稳定性并进行RT-PCR鉴定。结果表明该病毒样颗粒含口蹄疫病毒IRES RNA序列,并且稳定性良好,构建的病毒样颗粒可以作为RNA病毒检测时的标准品和质控品使用。The Coat protein and Maturase gene of E.coli bacteriophage MS2 was amplified by PCR,then the gene was cloned into pET32a to construct the intermediate vector pET32aCP.The conservative sequence of FMDV internal ribosome entry site(IRES) was cloned into the downstream of pET32aCP bacteriophage gene to construct the prokaryotic expression vector pCPES.The recombinant plasmid pCPES transformed into E.coli strain BL21(DE3) was induced to express with 1mmol/L IPTG.The expression products were purified by sucrose density gradient centrifugation.The expression products observed by TEM were circular viruslike particles,and the diameter of these particles was about 26nm.The stability of viruslike particles was detected,and the viruslike particles was identified by RTPCR.The results showed that the viruslike particles contain the FMDV IRES RNA and have good stability.The viruslike particles have great prospect as the standard and quality control in the area of RNA virus detection.福建省科技厅资助项目(2003N051
