HP-PRRSV感染猪后补体系统应答研究

发布时间：2018-06-16 12:42

本文选题：HP-PRRSV + 补体系统　；参考：《华中农业大学》2015年硕士论文

【摘要】：猪高致病性蓝耳病是由猪繁殖与呼吸综合征(Porcine Reproductive Respiratory Syndrome,PRRS)病毒的变异株引起的一种急性高致死性传染病,又称猪“高热病”。猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)持续性感染会引起免疫抑制,引发细菌或病毒的继发性感染,从而增加治疗难度。因此若能从遗传水平提高猪对PRRSV的抵抗力,进而实施抗病育种是解决PRRS危害的可行措施。补体系统是先天性免疫的重要组成部分,是机体抵抗病原体的第一道防线,在抗病毒中具有重要作用。然而研究猪感染PRRSV后补体系统的应答反应及与机体互作鲜有报道,仅见肖书奇(Xiao et al.,2010)和周平(Zhou et al.,2011)分别发表的两篇文章涉及到转录水平上部分补体基因的表达信息。前者芯片结果显示约克夏与长白杂交猪感染经典北美PRRSV后4天和7天采肺组织样,补体激活因子是表达上调的,补体抑制因子是表达下调的,补体系统处于持续激活状态;而后者结果表明通城猪HP-PRRSV感染后5天,肺泡巨噬细胞中补体抑制因子CLU显著上调,补体激活的关键因子CFP与C3的表达被下调,补体系统受到抑制。因此,为更深入研究感染PRRSV后猪补体系统的应答反应与功能,本研究以课题组前期已开展的通城猪与大白猪人工感染试验材料和感染前后转录组分析数据为基础,筛选到了高致病性猪蓝耳病毒(Highly Pathogenic PRRS,HP-PRRSV)感染机体后补体系统差异表达基因,并对补体系统开展以下研究:(1)补体通路中的基因分析:以通城猪与大白猪HP-PRRSV感染组各3头及对照组各3头的肺泡巨噬细胞RNA为材料,利用Illumina Hi Seq TM2000进行深度测序,并用转录组测序(RNA Sequencing,RNA-Seq)数据分析通用软件Tophat 2.0对测序产出reads进行定位。然后统计转录本和基因的差异表达情况,用阈值Fold Change大于等于2或小于等于2,同时P值小于等于0.01,筛选HP-PRRSV感染前后补体系统差异表达基因。测序结果显示:通城猪中补体基因C3AR、C4A、C5与丝氨酸相关蛋白酶1(MBL-associated serine protease,MASP-1)基因被上调,会有利于补体凝集素途径的激活;而补体CFP基因的下调与CFH的上调,会对补体旁路途径产生抑制;补体抑制因子CD55、CD59、CLU与C4BPB的上调,会抑制补体转化酶和攻膜复合物的形成,从而对整个补体系统产生抑制作用。大白猪中,补体凝集素途径MASP-1基因的上调、经典途径C1R上调以及C4A与C3AR的上调,有利于补体系统经典途径和凝集素途径的激活;补体旁路途径抑制子CFH的上调及稳定转化酶的CFP的下调,会对补体旁路途径激活产生抑制作用;补体抑制因子C1R、CD55、CD59、CLU、C4BPB被上调,会抑制转化酶及攻膜复合物的形成,对整个补体系统产生抑制。综上所述,从转录组数据看出,通城猪与大白猪的补体系统都激活同时又受到抑制,但是由于通城猪中检测到补体系统三条途径交汇点C5基因的上调,而大白猪中没有检测到,因此推断通城猪补体系统激活程度比大白猪高一些。(2)补体系统差异表达基因的定量检测分析:以通城猪与大白猪HP-PRRSV感染组各3头及对照组各3头的肺泡巨噬细胞编码脱氧核糖核酸(Coding deoxyribonucleic acid,c DNA)为模板,通过荧光实时定量验证了通城猪与大白猪中补体差异表达基因关键因子在感染HP-PRRSV后的表达情况,定量结果显示:在通城猪与大白猪中补体基因C3AR、C4A、CFH、CD55、CD59与CLU呈上调表达,而补体基因CFP下调表达,与转录组数据基本一致即补体系统激活同时又受到抑制。(3)HP-PRRSV感染通城猪与大白猪7天后免疫组化检测补体C3d在肺组织中的分布:结果显示HP-PRRSV感染的通城猪与大白猪中都检测到了补体C3d的大量沉积,而阴性对照组未检测到阳性信号,并根据有关文献报道补体C3d是补体系统激活标志,得出结论通城猪与大白猪补体系统被激活。(4)补体系统中心因子C3在HP-PRRSV感染后血清中的变化:以HP-PRRSV感染的通城猪与大白猪各3头为材料,以HP-PRRSV感染0天的血清为对照,用酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测血清中补体C3的水平变化,结果显示:通城猪中感染后5天与7天的C3水平显著高于感染前;在大白猪中补体C3水平一直处于稳定,且感染HP-PRRSV后的C3水平与感染前差异不显著。通城猪与大白猪相比,通城猪在感染后第5天补体C3水平显著高于大白猪第5天血清中补体C3水平,感染后第7天补体C3水平极显著高于大白猪第7天水平,这说明HP-PRRSV感染后通城猪补体系统激活程度比大白猪高,同时相关文献报道血清中补体C3水平升高利于机体清除病毒,因此也推断出通城猪与大白猪相比可能更有效的清除蓝耳病毒。本研究从转录水平与蛋白水平初步探索了对HP-PRRSV感染通城猪与大白猪后补体系统的应答反应:从转录数据来看HP-PRRSV感染通城猪与大白猪后,补体系统激活的同时又受到补体抑制因子的抑制。蛋白水平上免疫组化检测到补体系统激活标志蛋白C3d在通城猪与大白猪肺组织中大量沉积,表明补体系统都被激活;而ELISA测定血清中补体C3水平在品种间存在差异:感染高致病性蓝耳病毒后通城猪第5天与第7天血清中补体C3水平都显著高于大白猪第5天和第7天水平。蛋白水平上结合免疫组化结果表明HP-PRRSV感染后,通城猪与大白猪补体系统都被激活,但通城猪补体系统激活程度比大白猪高,且可能更有效的清除蓝耳病毒,希望这些结果为今后研究蓝耳病毒与补体互作以及PRRSV抗病育种提供一定的参考。
[Abstract]:High pathogenic blue ear disease of pigs is an acute high death infectious disease caused by the mutant strain of Porcine Reproductive Respiratory Syndrome (PRRS) virus, also known as "high fever" in pigs. The persistent infection of porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) It can cause immunosuppression, causing secondary infection of bacteria or viruses, thus increasing the difficulty of treatment. Therefore, it is a feasible measure to improve the resistance of pigs to PRRSV from the genetic level and then to carry out the disease resistance breeding. The complement system is an important part of the innate immunity and the first line of defense against the pathogen. However, the response of the complement system after PRRSV infection and the interaction with the body are rarely reported. Only two articles published by Xiao Shuqi (Xiao et al., 2010) and Zhou Ping (Zhou et al., 2011) related to the expression information of the transcriptional upside complement gene. The former chip results show Yue Kexia The lung tissue samples were collected 4 days and 7 days after the infection with the classic North American PRRSV. The complement activation factor was up regulated, the complement inhibitory factor was down regulated and the complement system was in a continuous activation state, and the latter showed that the complement inhibitory factor CLU in the alveolar macrophage cells was up to be up up and the complement excitation was significantly increased at 5 days after HP-PRRSV infection in Tongcheng pig. The expression of the key living factors CFP and C3 was downregulated and the complement system was suppressed. Therefore, in order to further study the response and function of the pig complement system after infection of PRRSV, this study was based on the artificial infection test materials of the Tongcheng and the White pigs and the transcriptional analysis data before and after the infection. The differentially expressed genes of the complement system after Highly Pathogenic PRRS (HP-PRRSV) infection were carried out, and the following studies were carried out on the complement system: (1) gene analysis in the complement pathway: the RNA of alveolar macrophages from 3 heads of each 3 and 3 of the control group in Tongcheng pig and the HP-PRRSV infection group of the big white pig and Illumina Hi Seq TM200. 0 in depth sequencing, and using RNA Sequencing (RNA-Seq) data analysis, the general software Tophat 2 locals the sequencing output reads. Then the differential expression of the transcript and gene is counted, and the threshold Fold Change is greater than or equal to 2 or less than 2, and the P value is less than 0.01, and the complement system before and after the HP-PRRSV infection is screened. The results showed that the complement gene C3AR, C4A, C5 and serine related protease 1 (MBL-associated serine protease, MASP-1) genes were up-regulated in Tongcheng pig, which would be beneficial to the activation of the complement agglutinin pathway, while the down regulation of the complement CFP gene and the upregulation of CFH would inhibit the complement by-pass pathway, and the complement inhibitory factor CD. 55, the up-regulation of CD59, CLU and C4BPB inhibits the formation of complement invertase and tapping complex and inhibits the whole complement system. In the white pig, the up regulation of the MASP-1 gene of the complement lectin pathway, the up-regulation of the classical pathway C1R and the up regulation of C4A and C3AR are beneficial to the activation of the classical pathway and the agglutinin pathway of the complement system; complement The up regulation of the inhibitor CFH and the down regulation of the CFP of the stable invertase could inhibit the activation of the complement bypass pathway; the complement inhibitory factor C1R, CD55, CD59, CLU, C4BPB were up-regulated, which inhibited the formation of the invertase and the attack membrane complex, and the production inhibition of the whole complement system. The complement system of the big white pigs was activated and suppressed. But because of the up regulation of the C5 gene at the intersection point of the complement system in the Tongcheng pig, the C5 gene was not detected in the big white pig. Therefore, it was concluded that the activation degree of the complement system was higher than that of the large white pig. (2) the quantitative detection and analysis of the differential expression genes of the complement system: The Coding deoxyribonucleic acid (C DNA) of alveolar macrophages (C DNA) of 3 heads of 3 heads and 3 heads in the control group of the city pig and the large white pig is the template. The expression of the key factor of the differential expression gene of the complement in Tongcheng pig and the white pig is quantitatively verified by fluorescence. The quantitative results show that the expression of the key factor of the differential expression gene of the complement gene in the city of Tongcheng and the white pig is expressed. The complement genes C3AR, C4A, CFH, CD55, CD59 and CLU were up-regulated in Tongcheng pig and white pig, and the expression of complement gene CFP was down regulated, and the complement system activation was also inhibited by the transcriptional data. (3) the distribution of complement C3d in lung tissue was detected by the immunohistochemical staining of HP-PRRSV infected Tongcheng pig and white pig for 7 days: the results showed HP A large amount of complement C3d deposition was detected in both -PRRSV infected City pigs and large white pigs, and the negative control group did not detect positive signals. According to the relevant literature, the complement system was activated by complement C3d. (4) the central factor C3 of the complement system was in the serum after HP-PRRSV infection. Changes: 3 heads of HP-PRRSV infected Tongcheng pig and large white pig were used as the material, and the serum of HP-PRRSV infected for 0 days was compared, and the level of complement C3 in serum was detected by enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA). The results showed that the level of C3 in 5 days and 7 days after infection in Tongcheng pig was significantly higher than that before infection; at the same time, the level of C3 was significantly higher than that before infection. The level of complement C3 in white pigs remained stable, and the level of C3 after infection with HP-PRRSV was not significantly different from that before infection. The complement C3 level of Tongcheng pig was significantly higher than that of the large white pig in the fifth day after infection, and the level of complement C3 was significantly higher than the seventh day level of the white pig seventh days after infection, and the level of complement C3 was significantly higher than that of the white pig. The activation degree of complement system in Tongcheng pig after Ming HP-PRRSV infection was higher than that of large white pigs. At the same time, the related literature reported that the elevation of complement C3 in serum was beneficial to the organism to clear the virus. Therefore, it was concluded that the Tongcheng pig might be more effective than the white pig to clear the blue ear virus. This study initially explored the sense of HP-PRRSV from the level of transcription and protein. Response to the complement system of dyed Tongcheng pig and white pig: according to the transcriptional data, HP-PRRSV infected Tongcheng pig and white pig were activated by complement system and inhibited by complement inhibition factor. Protein level was detected by immunohistochemical staining of complement system activation marker protein C3d in the lung tissue of Tongcheng pig and white pig. It showed that the complement system was activated and the level of complement C3 in serum was different in ELISA. The level of complement C3 in serum in fifth and seventh days after infection of highly pathogenic cyanobacterium virus was significantly higher than that of fifth days and seventh days in the white pig. The complement system of the big white pigs is activated, but the activation degree of the complement system in Tongcheng pig is higher than that of the large white pig, and it may be more effective to clear the blue ear virus. It is hoped that these results will provide some reference for the study of the interaction between the blue ear virus and complement and the resistance breeding of PRRSV in the future.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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