含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建

Construction of the Expression Vector of Viruslike Particles Containing FMDV IRES RNA

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 PC R,then the gene was cloned into pET32a to construct the intermediate vector pET32a-CP. The conservative sequence of FMDV internal ribosome entry site （IRES） was cloned into the downstream of pET32a-CP 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 lmmol/L IPTG. The expression products were purified by sucrose density gradient centrifugation. The expression products observed by TEM were circular virus-like particles, and the diameter of these particles was about 26nm. The stability of virus-like particles was detected, and the virus-like particles was identified by RT-PCR. The results showed that the virus-like particles contain the FMDV IRES RNA and have good stability. The virus-like particles have great prospect as the standard and quality control in the area of RNA virus detection.