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目的:初步研究缺失300~444位氨基酸突变体△IPS-1对IFN-β诱生作用及抗HSV-1病毒作用的影响,进而确定该部位对IPS-1发挥正常功能的作用。方法:通过重叠延伸PCR法获得缺失300~444位氨基酸的△IPS-1,构建突变重组质粒pEF-BOS-FLAG-△IPS-1,并经酶切及测序鉴定。采用磷酸钙体外转染HEK293T真核细胞。经Westernblot检测蛋白的表达;ELISA检测细胞上清中IFN-β分泌量;HSV-1病毒感染分别转染了pEF-BOS-FLAG/IPS-1、pEF-BOS-FLAG/△IPS-1质粒的HEK293T细胞,空斑检测各转染细胞组不同时间段细胞上清的病毒滴度。结果:成功构建了缺失300~444位氨基酸的IPS-1基因重组真核表达载体pEF-BOS-FLAG/△IPS-1;与转染pEF-BOS-FLAG/IPS-1质粒组相比转染了pEF-BOS-FLAG/△IPS-1的细胞在不同时间段其上清中IFN-β分泌量有一定量的减少;空斑测定结果表明转染了pEF-BOS-FLAG/△IPS-1质粒组细胞中病毒滴度明显高于pEF-BOS-FLAG/IPS-1质粒组。结论:△IPS-1与IPS-1相比能使HEK293T细胞IFN-β分泌量明显减少,△IPS-1不能完全抑制HSV-1感染细胞产生的细胞病变效应,该突变体抗HSV-1病毒作用有一定程度的减弱。
OBJECTIVE: To investigate the effect of △ IPS-1, a deletion mutant of amino acid at position 300-444, on the induction of IFN-β and the anti-HSV-1 virus effect, and to determine the effect of this site on the normal function of IPS-1. Methods: △ IPS-1 deletion of amino acids 300-444 was obtained by overlap extension PCR. The recombinant plasmid pEF-BOS-FLAG- △ IPS-1 was constructed and identified by restriction enzyme digestion and sequencing. HEK293T eukaryotic cells were transfected with calcium phosphate in vitro. Western blot was used to detect the expression of IFN-β in the supernatant. The HSV-1 virus was transfected into pEF-BOS-FLAG / IPS-1 and pEF-BOS-FLAG / HEK293T cells and plaque were used to detect the virus titer of the cell supernatant in each transfected cell group at different time points. Results: Recombinant eukaryotic expression vector pEF-BOS-FLAG / △ IPS-1 of IPS-1 gene with amino acids 300-444 amino acids deleted was successfully constructed and transfected into pEF-BOS-FLAG / IPS-1 plasmid group The amount of IFN-β secreted by pEF-BOS-FLAG / △ IPS-1 cells in the supernatant decreased in a certain amount. The plaque assay indicated that the plasmid pEF-BOS-FLAG / △ IPS- The virus titer in the group of cells was significantly higher than that of pEF-BOS-FLAG / IPS-1 plasmid group. Conclusion: △ IPS-1 can significantly reduce the secretion of IFN-β in HEK293T cells compared with IPS-1. △ IPS-1 can not completely inhibit the cytopathic effects of HSV-1 infected cells. The mutant anti-HSV-1 The role of a certain degree of weakening.