超表达猪STING蛋白细胞株的建立及其对猪瘟病毒增殖的影响

Establishment of swine stimulator of interferon gene cell line and its influences on CSFV replication

为构建表达猪干扰素刺激因子(STIN G)的真核表达载体(pEGFP-STING),进而用细胞转染和亚克隆筛选的方法建立超表达STING蛋白的猪脐静脉血管内皮细胞(SU VEC)细胞株。通过RT-PCR扩增STING基因,构建表达STING的真核表达载体pEGFP-STING,通过Lipofectam ine 2000介导转染至永生化猪血管内皮细胞(SUVECs)中表达。在转染的基础上添加1 500 g/m L G418进行抗性筛选,获得稳定表达STING蛋白的细胞克隆,进一步用荧光共聚焦显微镜观察STING蛋白在细胞内的定位,建立稳定表达STING蛋白的SUVEC细胞株。在此基础之上,猪瘟病毒(CSFV)感染48 h后,通过Real-time PCR法检测稳定表达猪STING蛋白对猪瘟病毒复制的影响。PCR、酶切和测序结果表明,成功扩增猪STING基因并正确插入载体pEGFP-C1中,基因序列与Gen Bank的完全一致。转染SUVECs细胞后,STING蛋白在宿主细胞内被成功表达,并且主要表达在细胞质中。经G418抗性筛选出表达STING蛋白的细胞株后,经RT-PCR和W estern-blot检测表明,STING蛋白在SUVEC细胞中被稳定转录和表达,定位于SUVEC细胞的内质网上面。Real-time PCR检测表明,STING蛋白的超表达促进了CSFV的复制。本研究通过构建猪STING蛋白的真核表达载体实现了STING蛋白在SUVEC细胞中的超表达,建立了稳定表达STING蛋白的SUVEC细胞株。

To construct a eukaryotic expression vector pEGFP-STING for porcine stimulator of interferon genes（STING）,and establish the cell line for STING protein in SUVECs, the porcine STING gene was successfully amplified by reverse transcription polymerase chain reaction and cloned into the p EGFP-C1 expression vector according the vector information. The plasmids p EGFP-STING and p EGFP-C1 were transfected into SUVECs by Lipofectamine 2000.Then the selective media containing 1 500 g/m LG418 was added to select cell clones stably expressing GFPSTING. Then the location of STING was detected by the confocal laser scanning microscope. By the PCR,enzyme digestion and sequencing analyses,STING gene was successfully cloned into the pEGFP-C1 vector. Fluorescence microscope showedthat STING protein was localized in the cytoplasm and located to the endoplasmic reticulum.Further study showed that the overexpession of STING protein could enhance classical swine fever virus（CSFV）,and replication by Real-time PCR detection. Porcine STING protein was successfully over-expressed and the stably cell lines expressing GFPSTING were established,which can enhance the replication of CSFV. This study will lay the foundation for elucidating the function of porcine STING protein.