猪肠道组织RIG-I的表达及其在猪传染性胃肠炎病毒致病机制中作用的初步研究

RIG-I mediates interferon-beta response in porcine infectious gastroenteritis virus infection

为探究天然免疫分子维甲酸诱导基因Ⅰ(RIG-I)在猪肠道组织中的表达情况,评价其在猪传染性胃肠炎病毒(TGEV)致病机制中的作用。本研究经原核表达重组RIG-I蛋白(rRIG-1)并采用SDS-PAGE切胶纯化该蛋白,将其免疫大白兔制备兔RIG-I多克隆抗体,采用间接ELISA检测抗体效价。结果显示获得了效价可达1∶64000的兔RIG-I多克隆抗体。利用该抗体经western blot检测SPF猪各肠道组织中RIG-I的表达。结果显示,制备的多克隆抗体可与猪各肠道中的RIG-I反应,且RIG-I在SPF猪空肠中的表达量最高。利用该抗体经western blot检测TGEV感染的ST细胞及猪空肠中RIG-I的表达水平。结果显示,TGEV感染后ST细胞中RIG-I的表达水平极显著高于空白对照细胞(P<0.01);与正常SPF猪空肠相比,TGEV感染的SPF猪空肠组织中RIG-I蛋白的表达水平明显提高。表明TGEV感染后能够刺激细胞内源性及感染猪肠道组织中RIG-I蛋白的表达水平。将本研究构建的重组质粒pCAGGS-RIG-I-flag和RIG-I干扰RNA(siRIG-I)分别转染ST细胞,采用western blot鉴定RIG-I的过表达和敲低效果后,再利用TGEV感染,24 h后采用qPCR检测RIG-I对上述ST细胞中IFN-β转录水平的影响;收集细胞上清,采用TCID50方法测定病毒滴度。qPCR及病毒滴度的测定结果显示,与转染空载体的对照组相比,RIG-I过表达的ST细胞中IFN-β的转录水平极显著升高(P<0.001),且细胞上清中的病毒滴度下降约75%;与转染NCsiRNA的对照组相比,敲低RIG-I的ST细胞中IFN-β的转录水平极显著下降(P<0.001),且细胞上清中的病毒滴度升高约4.8倍。表明,TGEV感染细胞中的RIG-I能够促进细胞中IFN-β的转录,提高宿主细胞的抗病毒免疫反应,从而抑制病毒的复制。本研究结果证实RIG-I在介导TGEV诱导宿主细胞产生IFN的过程中发挥重要作用,该结果为深入探究TGEV的致病机制奠定了实验基础。

This study was designed to investigate the expression of retinoic acid-induced gene I(RIG-I)in pig intestinal tissues and evaluate its role in the pathogenesis of porcine transmissible gastroenteritis virus(TGEV).Firstly,porcine RIG-I was purified and polyclonal antibody was prepared.The titer of the porcine RIG-I polyclonal antibody was 1∶64000.The expression of RIG-I in pig intestinal tissues was detected by western blot.The results showed that the polyclonal antibody could interact with porcine RIG-I-like receptor,and the expression of porcine RIG-I was the highest in jejunum.Western blot was used to detect the expression level of RIG-I in TGEV infected ST cells and in pig jejunum.The results showed that the expression level of RIG-I in ST cells significantly increased upon TGEV infection,compared with control group,indicating that TGEV infection can stimulate the expression of RIG-I in endogenous cells and intestinal tissues of infected pigs.Further,the pCAGGS-RIG-I-flag recombinant plasmid and siRIG-I interfering RNA were transfected into ST cells,respectively,and then infected with TGEV.The overexpression and knockdown of RIG-I were verified by western blot.After 24 hours,the effect of RIG-I on IFN-βtranscription was detected by qPCR,and the supernatant was collected and the virus titer was determined by TCID50.The results showed that the overexpression of RIG-I significantly induced IFN-βproduction(P<0.01)and reduced the virus titer in supernatant by about 75%,while knockdown RIG-I decreased the transcription level of IFN-βand increased the virus titer in the cell supernatant increased by about 4.8 times.Our findings indicated that porcine RIG-I plays an important role in mediating the induction of IFN-βby TGEV infection,improving the antiviral immune response of host cells,thus inhibiting the replication of virus.These results provides a foundation for further study concerning pathogenic mechanism of TGEV.