Marc-145细胞内GP5表达对猪繁殖与呼吸综合征病毒感染的影响

发布时间：2018-04-23 05:30

本文选题：猪繁殖与呼吸综合征病毒 + GP5　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的猪的繁殖障碍和呼吸系统的传染病,是目前严重危害养猪业的重要病毒性传染病之一。PRRSV为有囊膜,单股正链RNA病毒,分类属于尼多病毒目,动脉炎病毒科成员。PRRSV基因是由一条15kb的单股正链RNA构成,包括9个开放阅读框(ORF),分别编码2个非结构蛋白(Nsp1和Nsp2),和7个结构蛋白(GP2a、E、GP3、GP4、GP5、M和N)。GP5作为主要结构蛋白含有病毒重要的抗原中和表位,外源性免疫GP5蛋白或利用GP5 DNA疫苗内源性的表达GP5蛋白可以有效抑制PRRSV对猪体的感染。然而,在实际PRRSV感染猪只过程中,GP5表达诱导中和抗体的产生却发生了延迟或是被抑制。这表明,在病毒感染过程中GP5的功能受到了其它PRRSV的蛋白或宿主蛋白的修饰或干扰。为了排除其它PRRSV蛋白的干扰作用,明确GP5蛋白表达在病毒感染过程中的作用,本课题研究的内容和结果如下:1.通过RT-PCR克隆PRRSV ORF5基因片段,利用overlap PCR扩增获得带有Flag标签的ORF5基因片段。构建重组慢病毒表达载体pTRIP-GP5-Flag,将重组质粒和包装载体共转染293T细胞获得重组慢病毒。利用重组慢病毒转导Marc-145细胞,通过嘌呤霉素和单克隆筛选,获得到稳定表达GP5Flag的细胞系。IFA和Western blot检测结果证明GP5Flag在Marc-145细胞内稳定表达。2.利用CCK-8 Kit和Annexin V-FITC Apoptosis Detection Kit分别对筛选获得细胞系进行细胞增殖和细胞凋亡检测,分析GP5蛋白表达对Marc-145细胞活性的影响。检测结果显示,与对照组细胞相比,GP5蛋白在Marc-145细胞内表达不影响Marc-145细胞的增殖,同时也未诱导细胞凋亡。3.为了分析GP5表达对PRRSV感染的影响,我们选择三种不同的PRRSV毒株SD16,VR-2332和JXA1分别感染表达GP5的Marc-145细胞,结果显示GP5的表达显著抑制三种病毒在Marc-145细胞上的增殖,但并不影响PRRSV对Marc-145的吸附。4.I型干扰素基因水平检测结果显示,GP5蛋白的表达显著提高了细胞IFN-β的产生和显著增强了细胞内IFN-β的启动子活性。为了进一步证明GP5蛋白表达诱导IFN-β的产生从而抑制PRRSV的感染,设计合成针对IFN-β的siRNA,通过敲低细胞内IFN-β的表达进而检测PRRSV的感染变化,实验结果显示,当GP5表达细胞IFN-βmRNA水平被敲低后,PRRSV对Marc-145细胞的感染可以得到显著性恢复。综上所述,为了探究GP5的表达对PRRSV感染的影响,本研究构建了GP5稳定表达的Marc-145细胞系,细胞活性测定和病毒感染实验表明GP5蛋白的表达没有影响细胞的活性但却显著抑制了病毒的感染,并且这种对病毒的抑制作用是由GP5蛋白诱导IFN-β产生导致的。该研究通过对PRRSV GP5蛋白表达对宿主的调控分析,明确了GP5蛋白的预表达不利于PRRSV的后期感染,为更加深入了解PRRSV的感染与致病机制提供新的思路。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is a reproductive disorder and respiratory disease caused by porcine reproductive and respiratory syndrome virus. PRRSv is one of the most important viral infectious diseases which seriously endangers the pig industry at present. PRRSv is a envelope, single-stranded RNA virus, which belongs to the order Nidovirus. The member of arteritis virus family. PRRSv gene is composed of a single strand positive strand RNA of 15kb. Nine open reading frames (ORF) encoding two nonstructural proteins, Nsp1 and Nsp2, respectively, and seven structural proteins, GP2aEGP3, GP4, GP5Mand N).GP5, were used as major structural proteins to contain important antigen-neutralizing epitopes of the virus. Exogenous immunizing GP5 protein or endogenous expression of GP5 protein using GP5 DNA vaccine can effectively inhibit the infection of PRRSV to pig body. However, the production of neutralizing antibodies induced by GP5 expression was delayed or inhibited during actual PRRSV infection in pigs. This suggests that the function of GP5 is modified or interfered with by other PRRSV proteins or host proteins during viral infection. In order to eliminate the interference of other PRRSV proteins and clarify the role of GP5 protein expression in the process of virus infection, the contents and results of this study are as follows: 1. The PRRSV ORF5 gene fragment was cloned by RT-PCR, and the ORF5 gene fragment with Flag tag was obtained by overlap PCR amplification. The recombinant lentivirus expression vector pTRIP-GP5-Flag. was constructed and co-transfected into 293T cells to obtain the recombinant lentivirus. The recombinant lentivirus was used to transduction Marc-145 cells. By purine mycin and monoclonal screening, the stable expression of GP5Flag in Marc-145 cell lines. IFA and Western blot were obtained. The results showed that GP5Flag expressed stably in Marc-145 cells. CCK-8 Kit and Annexin V-FITC Apoptosis Detection Kit were used to detect the proliferation and apoptosis of the selected cell lines, and the effect of GP5 protein expression on the activity of Marc-145 cells was analyzed. The results showed that the expression of GP5 protein in Marc-145 cells did not affect the proliferation of Marc-145 cells, nor did it induce apoptosis. In order to analyze the effect of GP5 expression on PRRSV infection, we selected three different PRRSV strains SD16VR-2332 and JXA1 to infect Marc-145 cells expressing GP5, respectively. The results showed that the expression of GP5 significantly inhibited the proliferation of the three viruses on Marc-145 cells. The results showed that the expression of GP5 protein significantly increased the production of IFN- 尾 and the promoter activity of IFN- 尾. In order to further prove that the expression of GP5 protein induces the production of IFN- 尾 and thus inhibits the infection of PRRSV, we designed and synthesized siRNAs against IFN- 尾 and detected the changes of PRRSV infection by knocking down the expression of IFN- 尾 in cells. When the IFN- 尾 mRNA level of GP5 expression cells was knocked down, the infection of Marc-145 cells could recover significantly. In conclusion, in order to investigate the effect of GP5 expression on PRRSV infection, we constructed a Marc-145 cell line with stable expression of GP5. Cell activity test and virus infection test showed that the expression of GP5 protein did not affect the cell activity, but significantly inhibited the virus infection, and the inhibitory effect on the virus was caused by the production of IFN- 尾 induced by GP5 protein. Through the analysis of the regulation of PRRSV GP5 protein expression on the host, it is clear that the pre-expression of GP5 protein is not conducive to the late infection of PRRSV, which provides a new idea for further understanding the infection and pathogenesis of PRRSV.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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