猪源Rab基因shRNA文库与稳定细胞株的构建及其对猪伪狂犬病病毒增殖的影响  

Construction of Porcine Derived Rab Gene shRNA Libraries and Stable Cell Lines and the Effects on PRV Proliferation

作　　者：梁东阁 姚晨 柴雅静 蔡梦攀 褚贝贝[1,2,3] 鲁维飞[1,2,3] 王江[1,2,3] 曾磊[1] 刘忠虎[1,2,3] 明胜利 LIANG Dongge;YAO Chen;CHAI Yajing;CAI Mengpan;CHU Beibei;LU Weifei;WANG Jiang;ZENG Lei;LIU Zhonghu;MING Shengli(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Biochemistry and Nutrition of the Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Growth and Development of Henan Province,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,郑州450046 [2]河南农业大学,农业农村部动物生化与营养重点开放实验室,郑州450046 [3]河南农业大学,河南省动物生长发育调控重点实验室,郑州450046

出　　处：《中国畜牧兽医》2024年第3期1250-1258,共9页China Animal Husbandry & Veterinary Medicine

基　　金：河南农业大学高层次人才专项支持基金(30501322)。

摘　　要：【目的】探究Rab家族蛋白在猪伪狂犬病病毒(Porcine pseudorabies virus,PRV)感染宿主细胞中的作用。【方法】以猪肾上皮细胞(PK15)为试验模型,构建Rab基因shRNA文库,并用嘌呤霉素筛选获得稳定的能够抑制猪源Rab基因表达的细胞株。用实时荧光定量PCR检测细胞敲减效率,通过流式细胞术检测敲减Rab基因对PRV增殖的影响;用Western blotting检测PRV-gB蛋白表达量,使用实时荧光定量PCR检测敲减Rab基因后PRV-gB、PRV-TK基因以及相关细胞炎性因子表达量。【结果】试验成功构建包含13种Rab基因敲减细胞系的shRNA文库。流式细胞术检测结果显示敲减Rab基因能显著抑制PRV-GFP增殖(P<0.05);病毒滴度与Western blotting检测结果表明,敲减Rab基因极显著抑制子代病毒的产生以及PRV-gB蛋白的表达(P<0.01)。实时荧光定量PCR检测结果显示,敲减Rab基因会显著或极显著抑制PRV-gB、PRV-TK基因表达(P<0.05;P<0.01);敲减Rab14和Rab27基因后细胞中IFN-β、ISG15、IL-1β、IL-18基因表达量均显著或极显著升高(P<0.05;P<0.01)。【结论】敲减Rab基因能够抑制PRV增殖。研究结果为进一步研究Rab基因在PRV生活周期中发挥的作用奠定基础。【Objective】The purpose of this study was to investigate the role of Rab family proteins in the infection of Porcine pseudorabies virus(PRV)in host cells.【Method】Porcine renal epithelial cells(PK15)were used as the test model to construct Rab gene shRNA library,and purinomycin was used to screen stable cell lines that inhibited porcine Rab gene expression.Real-time quantitative PCR was used to detect cell knockdown efficiency,and flow cytometry was used to detect the effect of knockdown Rab gene on PRV proliferation.Western blotting was used to detect the expression levels of PRV-gB protein,and Real-time quantitative PCR was used to detect the expression levels of PRV-gB,PRV-TK genes,and related inflammatory factors after Rab gene knockout.【Result】A shRNA library with 13 Rab gene knockdown cell lines was successfully constructed.Flow cytometry analysis showed that knockdown of Rab gene significantly inhibited PRV-GFP proliferation(P<0.05).Viral titer and Western blotting test results showed that Rab gene knockdown extremely significantly inhibited the generation of progeny virus and the expression of PRV-gB protein(P<0.01).The results of Real-time quantitative PCR showed that the expression of PRV-gB and PRV-TK genes could be significantly inhibited after knocking down Rab gene(P<0.05 or P<0.01).The expressions of IFN-β,ISG 15,IL-1βand IL-18 genes were significantly or extremely significantly increased after knocking down Rab 14 and Rab 27 genes(P<0.05 or P<0.01).【Conclusion】Knockdown of Rab gene could inhibit PRV proliferation.The results laid a foundation for further research on the role of Rab gene in PRV life cycle.

关 键 词：Rab基因 猪伪狂犬病病毒(PRV) 慢病毒 shRNA文库 

分 类 号：S858.292[农业科学—临床兽医学]