EV71病毒P1和3CD基因共表达重组杆状病毒双顺反子穿梭载体的构建及鉴定

Construction and identification of recombinant baculovirus transfer co-expression vector of P1 and 3CD genes from EV71

目的通过克隆EV71病毒结构前体蛋白P1和非结构蛋白3CD,构建共表达P1前体蛋白和非结构蛋白3CD的重组杆状病毒表达载体。方法设计合成EMCV病毒IRES序列的特异性引物,克隆至含有CMV启动子的杆状病毒穿梭载体pFastBacDual-CMV上,命名为pFBCMV-IRES。从手足口病重症患者分离EV71病毒株,经RT-PCR技术分别扩增出EV71病毒的P1前体蛋白区与3CD区的片段,定向克隆至杆状病毒穿梭载体pF-BCMV-IRES的IRES序列的上游和下游,随后转化到大肠杆菌DH5α中,经PCR和双酶切鉴定转化菌落。结果通过对重组杆状病毒穿梭载体pFBCMV-IRES-P1-3CD进行酶切和DNA序列分析,证明共表达EV71病毒P1和3CD基因的重组杆状病毒双顺反子穿梭载体构建成功。结论共表达EV71病毒P1和3CD基因重组杆状病毒双顺反子穿梭载体的成功构建,将为下一步制备EV71病毒的类病毒颗粒疫苗打下基础。

Objective To amplify P1 and 3CD genes from patient infected with EV71 and to construct the recom- binant baculovirus transfer co- expression vector of P1 and 3CD. Methods The internal ribosomal entry site （IRES） from encephalomyocarditis virus （EMCV） was amplified and inserted into pFastBacDual vector containing CMV promoter, named pFBCMV - IRES. The cDNA encoding P1 and 3CD were obtained by RT - PCR from EV71 isolated from patient and recombined into pFBCMV -IRES vector. The reconstructed plasmid pFBCMV -IRES -P1 -3CD was confirmed by restriction enzymolysis and PCR. Results The co - expression plasmid pFBCMV - IRES - P1 - 3CD was successfully constructed, which were further confirmed with PCR and double endonucleases. Conclusion The successful construction of recombinant baculovirns transfer vector pFBCMV - IRES - P1 - 3CD establishes experimental basis for construction of virus like particle vaccine of EV71.