缺失VIM基因大白猪肾成纤维细胞的构建及其NF-κB和MAPK信号通路的研究

Construction of VIM gene-deficient porcine renal fibroblasts and the effects of the selected monoclonal cells on NF-κB and MAPK singal pathways

波形蛋白(Vimentin,VIM)可介导多种病毒进入机体,为研究病毒的感染机理,本研究将合成的敲除VIM基因的双sgRNA克隆于pX459-puro载体中,构建pX459-puro-V-1和pX459-puro-V-2敲除载体,经电转至大白猪肾成纤维细胞中,经嘌呤霉素筛选出14株单克隆细胞株。将筛选的单克隆细胞株通过PCR鉴定、测序验证、western blot鉴定,结果显示得到1株缺失VIM基因的单克隆细胞。通过100 ng/mL的脂多糖(LPS)分别刺激该单克隆细胞株和可传代大白猪肾成纤维细胞,通过western blot检测NF-κB和MAPK信号通路中p65、p38、JNK、ERK及磷酸化蛋白p-p65、p-p38、p-JNK、p-ERK的表达水平,结果显示经LPS刺激后,缺失VIM基因的单克隆细胞和可传代大白猪肾成纤维细胞中p65、p38、JNK、ERK、p-ERK的表达水平均无明显变化;缺失VIM基因的单克隆细胞中p-p65、p-p38、p-JNK的表达水平明显低于可传代大白猪肾成纤维细胞(P<0.01)。本研究得到了1株缺失VIM基因的单克隆细胞,利用该细胞株首次研究发现VIM基因缺失能够影响宿主细胞中NF-κB和MAPK信号通路中p-p65、p-p38、p-JNK的表达,从而起到降低炎症反应的作用,该缺失VIM基因的单克隆细胞为病毒感染机理的研究提供了良好的实验素材,对相关疫病的防控有重要意义。

VIM(Vimentin) can mediate viruses entry into the host cells. In order to study the mechanism of virus infection,the pX459-puro-VIM knockout vector was constructed by linking the synthesized double sgRNA with p X459-puro vector. The knockout vector was transfected into the prepared porcine kidney fibroblasts by electroporation. By screening with puromycin, 14monoclonal cell lines were selected. The selected monoclonal cell lines were subjected to gene PCR identification, sequencing verification and western blot test to obtain a monoclonal cell line with complete deletion of VIM gene. The expression levels of p65, p38, JNK, ERK and phosphorylated proteins p-p65, p-p38, p-JNK, and p-ERK in the NF-κB and MAPK pathways were analyzed with western blot for both the monoclonal cell line and primary white kidney fibroblasts, which were stimulated with 100ng/mL of LPS. The results showed that post LPS stimulation, the expression levels of p65, p38, JNK, ERK, and p-ERK in both the monoclonal cell line without VIM gene and primary white kidney fibroblasts had no significant differences. The expression levels of p-p65, p-p38 and p-JNK in homozygous VIM-deficient monoclonal cell lines were significantly lower than those in primary renal fibroblasts(P<0.01). In summary, a monoclonal cell line with homozygous deletion of VIM gene was obtained in this study, and it is demonstrated that the expression of p-p65, p-p38 and p-JNK in NF-κB and MAPK pathways were decreased in this obtained cell line resulting the the suppression of the inflammation. This homozygous VIM-deficient monoclonal cell line provides a good experimental material for the study of viral infection mechanism and is of great significance for disease prevention and control.