IFITM2的克隆表达及其抗病毒作用初探

Cloning and expression of IFITM2 gene and preliminary study on its antiviral activity

目的:构建含有ifitm2基因的重组质粒,在人胚肾HEK293T细胞中表达,并验证IFITM2蛋白的抗病毒作用。方法:参照Genbank ifitm2基因序列,设计上下游引物,通过RT-PCR技术扩增ifitm2基因,并将扩增产物连接至pMD18-T-sim-ple载体上,目的基因测序正确后进一步亚克隆至真核表达载体pVAX1上,转染HEK293T细胞,通过RT-PCR,蛋白免疫印迹(Western blot)和激光共聚集显微镜(LCSM)验证外源基因在细胞内的表达情况。同时HEK293T细胞过表达IFITM2蛋白,利用含有EGFP基因的VSV-G假膜病毒感染细胞,通过Western blot检测IFITM2蛋白对病毒的抑制作用。结果:成功获得ifitm2基因,测序正确,将其连接到pVAX1载体中,经酶切鉴定,证明含有ifitm2基因的重组质粒构建成功,Western blot和LCSM分析表明,目的基因能够在HEK293T细胞中表达,且呈广泛分布。过表达IFITM2蛋白后,荧光蛋白分析表明,该蛋白对病毒感染具有一定的抑制作用。结论:成功构建了含有ifitm2基因的重组真核表达质粒,该质粒可以在真核细胞中得到有效表达,且对VSV-G假膜病毒具有一定抑制作用,该研究为进一步探讨IFITM2蛋白的抗病毒作用及其分子机制提供了实验及理论基础。

Objective： To construct the recombinant plasmid containing ifitm2 gene and detect its expression in HEK293T cells, and to investigate its antiviral activity. Methods ： The primers of ifitm2 gene were designed and synthesized according to its gene sequence published on Genbank. Then ifitm2 gene was obtained by RT-PCR and subcloned to pMD18-T simple vector. After sequen- cing, the PCR product was inserted into the eukaryotic expression vector pVAX1, resulting into pV-IFITM2, which was transfected into HEK293 cells. The expression of IFITM2 was detected by RT-PCR, Western blot and laser scanning confocal microscope （LCSM）. Simultaneously, VSV-G protein-pseudotype virus containing EGFP was used to infect HEK293T cells over-expressed IFITM2 protein. The antiviral activity of IFITM2 was analyzed by Western blot. Results： The ifitm2 gene was obtained and inserted into pVAXI, and the recombinant plasmid containing ifitm2 gene was constructed successfully. The results of Western blot and LSCM showed that IF- ITM2 was expressed in HEK293 cells and distributed ubiquitously. The result of expression of EGFP contained VSV-G pseudotype virus showed that IFITM2 inhibited the infection of VSV-G pseudotype virus in a certain extent. Conclusion ： The recombinant eukaryotic ex- pression plasmid containing ifitm2 gene was successfully constructed and the effective expression of IFITM2 protein in HEK293 cells was accomplished, which could restrict the VSV-G pseudotype virus infection. This study provided the experimental and theoretical ba- sis for the research of the antiviral effect and molecule mechanism of IFITM proteins.