猪NLRC5对PK15细胞中SLA Ⅰ类分子表达的调控研究  

Regulating effect of porcine NLRC5 on the expression of SLA Ⅰ in PK15 cells

作　　者：刘英华 马丽娜 王有祺 高彩霞[2] 陈洪岩[1,2] LIU Ying-hua;MA Li-na;WANG You-qi;GAO Cai-xia;CHEN Hong-yan(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/Innovative Team for Laboratory Animals and Comparative Medicine/National Poultry Laboratory Animal Resource Bank,Harbin 150069,China)

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/实验动物与比较医学创新团队/国家禽类实验动物资源库,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第7期746-752,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0501600);国家自然科学基金项目(31872313);黑龙江省自然科学基金项目(QC2018036);国家重点研发项目省级资金项目(GX18B052)。

摘　　要：为探究猪核苷酸结合寡聚化结构域样受体(NLR)家族端酶募集(CARD)结构域-5(NLRC5)在PK15细胞中的表达情况及其对猪白细胞抗原I类分子(SLA I)表达的调控作用,本研究利用不同终浓度(1μg/mL、1.5μg/mL、2μg/mL)Poly I:C刺激PK15细胞后24 h,通过荧光定量PCR(qPCR)和western blot方法分别检测NLRC5 mRNA转录水平和蛋白的表达水平;设计NLRC5的3条siRNA和对照siRNA分别转染PK15细胞48 h后,分别采用qPCR和western blot检测PK15细胞中NLRC5 mRNA转录水平和蛋白表达水平,从而筛选出最优敲低NLRC5表达的siRNA;将筛选出的最优siRNA和对照siRNA分别转染PK15细胞48 h后,采用qPCR分别检测PK15细胞中NLRC5和SLA I类分子mRNA的转录水平,采用western blot检测PK15细胞中NLRC5蛋白表达水平,通过流式细胞术检测PK15细胞表面SLA I类分子的表达。构建重组质粒pCAGGS-HA-NLRC5并鉴定正确后转染PK15细胞,48 h后,采用qPCR分别检测PK15细胞中NLRC5和SLA I类分子mRNA的转录水平,采用western blot检测PK15细胞中NLRC蛋白表达水平,通过流式细胞术检测PK15细胞表面SLA I类分子表达水平。结果显示,与正常PK15细胞相比,经不同浓度Poly I:C刺激后PK15细胞中NLRC5 mRNA转录水平和蛋白水平均显著升高(p<0.01)。siRNA的筛选结果显示,NLRC5-sus-3为最优siRNA;将其转染PK15细胞后的qPCR和流式细胞术检测结果显示,与转染对照siRNA的PK15细胞相比,敲低NLRC5的PK15细胞中SLA I类分子的mRNA转录水平(p<0.01)和细胞表面SLA I类分子的表达水平均显著降低(p<0.01);重组质粒pCAGGS-HA-NLRC5转染PK15细胞后的结果显示,NLRC5 mRNA转录水平和蛋白水平均显著增高(p<0.01),表明NLRC5在PK15细胞中获得了过表达。qPCR和流式细胞术结果显示,过表达NLRC5后的PK15细胞中SLA I类分子mRNA的转录水平(p<0.01)和其在细胞表面的表达量均升高(p<0.05)。综上所述,经Poly I:C刺激可促进PK15细胞中NLRC5的表达,且NLRC5对SLA I类分子的表达�In order to study the expression of porcine nucleotide-binding oligomerization domain-like receptor(NLR) family caspase recruitment domain(CARD) containing 5(NLRC5) and its regulatory effect on the expression of swine leukocyte antigen class I molecules(SLA I) in PK15 cells, the PK15 cells were stimulated with different concentrations of Poly I:C(1 μg/mL,1.5 μg/mL, 2 μg/mL) for 24 hours, and the mRNA transcription and protein expression level of NLRC5 were detected by fluorescence quantitative PCR(qPCR) and western blot in this study, respectively. Three siRNAs of NLRC5 and control siRNA were designed and transfected into PK15 cells for 48 hours. The mRNA transcription and protein expression levels of NLRC5 in PK15 cells were detected by qPCR and western blot respectively to select the optimal siRNA for knockdown of NLRC5 expression. The optional siRNA and control siRNA were transfected into PK15 cells, for 48 hours, the m RNA transcription levels of SLA class I molecules in PK15 cells were detected by qPCR, the protein expression level of NLRC5 was detected by western blot, and the expression of SLA class I molecules on PK15 cell was detected by flow cytometry. After the recombinant plasmid pCAGGS-HA-NLRC5 was identified correctly, it was transfected into PK15 cells for 48 hours. The mRNA transcription levels of NLRC5 and SLA class I molecules in PK15 cells were detected by qPCR, the protein expression level of NLRC5 were detected by western blot, and the expression of SLA class I molecules on PK15 cell surface was detected by flow cytometry. The results showed that compared with normal PK15 cells, the mRNA transcription and protein expression levels of NLRC5 in PK15 cells were significantly increased after stimulation with different concentrations of Poly I:C(p<0.01). The screening results of siRNA showed that NLRC5-sus-3 was the optimal siRNA. After the optimal siRNA was transfected into PK15 cells, the results of qPCR and flow cytometry showed that the mRNA transcription level of SLA I molecules was signif

关 键 词：PK15细胞 猪NLRC5 SLAⅠ类分子 表达 

分 类 号：S565.2[农业科学—作物学]