猪传染性胃肠炎病毒蛋白激活TLRs对PK-15细胞FcRn表达影响的研究
发布时间：2018-09-04 10:53
【摘要】：免疫系统是机体在进化过程中形成的一种可以通过多种屏障来识别“自己”和“非己”的系统。黏膜免疫是抵抗外源入侵的第一道屏障。当“非己”成分入侵机体后可以被机体的模式识别受体(PRRs)所识别,并引起信号转导,从而促进某些核转录因子的激活并入核进而调节相关基因的表达。其中第一个被确认的模式识别受体就是TLRs家族。本研究一方面通过构建TGEV结构蛋白和非结构蛋白真核表达载体研究其对细胞FcRn表达的影响,并挖掘病毒相关蛋白的功能域;另一方面研究TGEV、UV-TGEV以及TGEV蛋白引起细胞FcRn表达上调的相关TLRs信号通路。取得的主要研究成果如下:1.TGEV对细胞FcRn表达的影响TGEV感染PK-15细胞或IPEC-J2细胞后,通过荧光定量PCR、双荧光素酶报告系统以及Western Blot实验对细胞FcRn的mRNA和蛋白表达进行检测。结果表明TGEV能引起细胞FcRn mRNA和蛋白表达显著上调。2.TGEV蛋白对细胞FcRn表达的影响本研究通过紫外线灭活TGEV,并用不同剂量的UV-TGEV刺激PK-15细胞后通过双荧光素酶报告系统和Western Blot检测发现UV-TGEV能够显著上调FcRn mRNA和蛋白表达水平。说明TGEV的结构蛋白参与了细胞FcRn表达的调控。与TGEV活病毒刺激相比,UV-TGEV上调细胞FcRn程度较低,这说明除病毒核酸以外,TGEV的非结构蛋白也可能参与了对FcRn的表达调控。3.TGEV蛋白真核表达载体的构建本研究通过提取TGEV WH-1株的RNA,反转录获得cDNA,并以此为模板,应用TGEV各蛋白基因的特异性引物分别进行扩增,并将扩增产物回收后经酶切连接到pCAGGS-HA真核表达载体,通过PCR扩增、酶切鉴定及测序验证准确后,再分别将各蛋白重组表达质粒转染PK-15细胞。通过荧光定量PCR和Western Blot检测FcRn的表达水平。结果表明结构蛋白M、N和E以及非结构蛋白nsp1、nsp5、nsp7、nsp8、nsp9、nsp15均可以上调细胞FcRn的表达。4.TGEV M和N蛋白截短表达载体的构建为了探究M蛋白和N蛋白调控FcRn表达的结构功能域,本研究通过构建不同的截短蛋白表达质粒并转染PK-15细胞,通过Western Blot和荧光定量PCR实验对细胞FcRn表达水平进行检测。最终确定N蛋白有效功能域位于156-198位氨基酸,M蛋白有效功能域位于219-262位氨基酸。5.探究TGEV上调FcRn表达的相关TLRs信号通路本研究对TGEV、UV-TGEV以及TGEV蛋白上调细胞FcRn的表达是否通过激活TLRs相关通路进行调控展开了研究。首先将MyD88和TRIF负调控质粒转染PK-15细胞,再应用TGEV和UV-TGEV刺激细胞,然后通过检测细胞FcRn表达水平的变化,证实了TGEV和UV-TGEV均能通过TLRs通路上调细胞FcRn蛋白的表达;通过荧光定量PCR检测证实了TGEV感染细胞能够激活TLR1、TLR2、TLR3、TLR4和TLR6的表达,UV-TGEV能够激活TLR1和TLR2。进一步将能上调FcRn表达的各种TGEV蛋白表达质粒分别转染细胞后发现nsp1、nsp5、nsp7、nsp8、nsp9、nsp15等六种非结构蛋白后均能显著激活TLR1、TLR2、TLR4和TLR6,结构蛋白E能显著激活TLR2、TLR4和TLR6,截短蛋白N1能显著激活TLR1、TLR4和TLR6,截短蛋白M2能激活TLR2和TLR4。
[Abstract]:Mucosal immunity is the first barrier against foreign invasion. When "non-self" components invade the body, they can be recognized by the body's pattern recognition receptors (PRRs) and cause signal transduction, thereby promoting the body's ability to recognize itself and its non-self. Some nuclear transcription factors are activated and incorporated into the nucleus to regulate the expression of related genes. The first recognized pattern recognition receptor is the TLRs family. On the one hand, the up-regulation of FcRn expression induced by TGEV, UV-TGEV and TGEV proteins was studied. The main results were as follows: 1. The effect of TGEV on FcRn expression in PK-15 cells or IPEC-J2 cells infected with TGEV was studied by fluorescence quantitative PCR, double luciferase reporting system and Western Blot assay. The results showed that TGEV could induce a significant up-regulation of FcRn mRNA and protein expression. 2. The effect of TGEV protein on FcRn expression in PK-15 cells was studied by UV inactivation of TGEV and stimulation of PK-15 cells with different doses of UV-TGEV. The expression level of FcRn mRNA and protein was up-regulated, indicating that the structural protein of TGEV was involved in the regulation of FcRn expression. Compared with the stimulation of TGEV, the level of FcRn up-regulated by UV-TGEV was lower, indicating that the non-structural protein of TGEV might also be involved in the regulation of FcRn expression besides viral nucleic acid. In this study, the RNA of TGEV WH-1 strain was extracted, and the cDNA was obtained by reverse transcription. As a template, the specific primers of TGEV protein genes were used to amplify the amplified products respectively. The recombinant products were then digested and linked to pCAGGS-HA eukaryotic expression vector by enzyme digestion, PCR amplification, enzyme digestion and sequencing to verify their accuracy. The expression of FcRn in PK-15 cells was detected by fluorescence quantitative PCR and Western Blot. The results showed that structural proteins M, N and E, as well as non-structural proteins nsp1, nsp5, nsp7, nsp8, Nsp9 and nsp15 could up-regulate the expression of FcRn in PK-15 cells. In this study, different truncated protein expression plasmids were constructed and transfected into PK-15 cells. The expression level of FcRn was detected by Western Blot and fluorescence quantitative PCR. The effective domain of N protein was located at 156-198 amino acids and the effective domain of M protein was located at 219-262 amino acids. 5. To explore the up-regulation of FcRn by TGEV. In this study, we investigated whether the up-regulation of FcRn expression by TGEV, UV-TGEV and TGEV proteins was mediated by activation of TLRs-related pathways. It was confirmed that both TGEV and UV-TGEV could up-regulate the expression of FcRn protein through the TLRs pathway, and that TGEV-infected cells could activate the expression of TLR1, TLR2, TLR3, TLR4 and TLR6, and that UV-TGEV could activate TLR1 and TLR2, and further up-regulate the expression of various TGEV protein plasmids in FcRn-infected cells. TLR1, TLR2, TLR4 and TLR6 were significantly activated by p1, nsp5, nsp7, nsp8, Nsp9 and nsp15. Structural protein E could significantly activate TLR2, TLR4 and TLR6, truncated protein N1 could significantly activate TLR1, TLR4 and TLR6, truncated protein M2 could activate TLR2 and TLR4.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：2221867
[Abstract]:Mucosal immunity is the first barrier against foreign invasion. When "non-self" components invade the body, they can be recognized by the body's pattern recognition receptors (PRRs) and cause signal transduction, thereby promoting the body's ability to recognize itself and its non-self. Some nuclear transcription factors are activated and incorporated into the nucleus to regulate the expression of related genes. The first recognized pattern recognition receptor is the TLRs family. On the one hand, the up-regulation of FcRn expression induced by TGEV, UV-TGEV and TGEV proteins was studied. The main results were as follows: 1. The effect of TGEV on FcRn expression in PK-15 cells or IPEC-J2 cells infected with TGEV was studied by fluorescence quantitative PCR, double luciferase reporting system and Western Blot assay. The results showed that TGEV could induce a significant up-regulation of FcRn mRNA and protein expression. 2. The effect of TGEV protein on FcRn expression in PK-15 cells was studied by UV inactivation of TGEV and stimulation of PK-15 cells with different doses of UV-TGEV. The expression level of FcRn mRNA and protein was up-regulated, indicating that the structural protein of TGEV was involved in the regulation of FcRn expression. Compared with the stimulation of TGEV, the level of FcRn up-regulated by UV-TGEV was lower, indicating that the non-structural protein of TGEV might also be involved in the regulation of FcRn expression besides viral nucleic acid. In this study, the RNA of TGEV WH-1 strain was extracted, and the cDNA was obtained by reverse transcription. As a template, the specific primers of TGEV protein genes were used to amplify the amplified products respectively. The recombinant products were then digested and linked to pCAGGS-HA eukaryotic expression vector by enzyme digestion, PCR amplification, enzyme digestion and sequencing to verify their accuracy. The expression of FcRn in PK-15 cells was detected by fluorescence quantitative PCR and Western Blot. The results showed that structural proteins M, N and E, as well as non-structural proteins nsp1, nsp5, nsp7, nsp8, Nsp9 and nsp15 could up-regulate the expression of FcRn in PK-15 cells. In this study, different truncated protein expression plasmids were constructed and transfected into PK-15 cells. The expression level of FcRn was detected by Western Blot and fluorescence quantitative PCR. The effective domain of N protein was located at 156-198 amino acids and the effective domain of M protein was located at 219-262 amino acids. 5. To explore the up-regulation of FcRn by TGEV. In this study, we investigated whether the up-regulation of FcRn expression by TGEV, UV-TGEV and TGEV proteins was mediated by activation of TLRs-related pathways. It was confirmed that both TGEV and UV-TGEV could up-regulate the expression of FcRn protein through the TLRs pathway, and that TGEV-infected cells could activate the expression of TLR1, TLR2, TLR3, TLR4 and TLR6, and that UV-TGEV could activate TLR1 and TLR2, and further up-regulate the expression of various TGEV protein plasmids in FcRn-infected cells. TLR1, TLR2, TLR4 and TLR6 were significantly activated by p1, nsp5, nsp7, nsp8, Nsp9 and nsp15. Structural protein E could significantly activate TLR2, TLR4 and TLR6, truncated protein N1 could significantly activate TLR1, TLR4 and TLR6, truncated protein M2 could activate TLR2 and TLR4.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
【参考文献】
相关期刊论文 前9条
1 Yining He;Zhiwen Xie;Jinglong Dai;Yanjie Cao;Jinlian Hou;Yansheng Zheng;Tianchao Wei;Meilan Mo;Ping Wei;;Responses of the Toll-like receptor and melanoma differentiation-associated protein 5 signaling pathways to avian infectious bronchitis virus infection in chicks[J];Virologica Sinica;2016年01期
2 冷勇;宋振辉;卿家超;翟少华;买买提·艾孜子;;猪传染性胃肠炎病毒M、sM基因重组杆状病毒的构建及病毒样颗粒的组装[J];畜牧兽医学报;2013年09期
3 边葶苈;周继勇;廖敏;;冠状病毒非结构蛋白的研究进展[J];中国动物传染病学报;2013年04期
4 陈丽颖;王艳玲;杨国宇;胡广超;;新生儿Fc受体的研究进展[J];河南农业大学学报;2007年04期
5 李裕;刘文峰;陈松昌;王建敏;万文忠;张顺;;浅谈猪传染性胃肠炎[J];养殖技术顾问;2007年02期
6 王炳艳;吕刚;王楚端;;新生动物免疫球蛋白Fc受体的研究概况[J];中国畜牧杂志;2006年05期
7 方宏清,陈惠鹏;冠状病毒S蛋白的结构和功能[J];生物技术通讯;2003年03期
8 吴国平,尹燕博,吴时友;猪传染性胃肠炎病毒(TGEV)研究进展[J];中国兽医杂志;2003年02期
9 王继科;刘长明;马思奇;王明;魏凤祥;于文涛;周金法;;猪传染性胃肠炎和猪流行性腹泻病毒的免疫电镜诊断的研究[J];中国畜禽传染病;1991年02期
相关博士学位论文 前1条
1 钟波;MITA介导的细胞抗病毒反应信号转导及其调节机制[D];武汉大学;2010年
相关硕士学位论文 前4条
1 徐静;猪传染性胃肠炎病毒分离及部分特性鉴定[D];东北农业大学;2015年
2 姜春霞;猪传染性胃肠炎病毒LJ-12株的分离及鉴定[D];东北农业大学;2013年
3 崔惠娟;猪FcRn在黏膜上皮中的表达与分布研究[D];华中农业大学;2011年
4 董加才;猪胃肠炎病毒的分离鉴定及S基因的克隆、序列分析与原核表达[D];河南农业大学;2008年
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