本文選題：HP-PRRSV + 補體系統(tǒng)��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬高致病性藍耳病是由豬繁殖與呼吸綜合征(Porcine Reproductive Respiratory Syndrome,PRRS)病毒的變異株引起的一種急性高致死性傳染病,又稱豬“高熱病”。豬繁殖與呼吸綜合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)持續(xù)性感染會引起免疫抑制,引發(fā)細菌或病毒的繼發(fā)性感染,從而增加治療難度。因此若能從遺傳水平提高豬對PRRSV的抵抗力,進而實施抗病育種是解決PRRS危害的可行措施。補體系統(tǒng)是先天性免疫的重要組成部分,是機體抵抗病原體的第一道防線,在抗病毒中具有重要作用。然而研究豬感染PRRSV后補體系統(tǒng)的應(yīng)答反應(yīng)及與機體互作鮮有報道,僅見肖書奇(Xiao et al.,2010)和周平(Zhou et al.,2011)分別發(fā)表的兩篇文章涉及到轉(zhuǎn)錄水平上部分補體基因的表達信息。前者芯片結(jié)果顯示約克夏與長白雜交豬感染經(jīng)典北美PRRSV后4天和7天采肺組織樣,補體激活因子是表達上調(diào)的,補體抑制因子是表達下調(diào)的,補體系統(tǒng)處于持續(xù)激活狀態(tài);而后者結(jié)果表明通城豬HP-PRRSV感染后5天,肺泡巨噬細胞中補體抑制因子CLU顯著上調(diào),補體激活的關(guān)鍵因子CFP與C3的表達被下調(diào),補體系統(tǒng)受到抑制。因此,為更深入研究感染PRRSV后豬補體系統(tǒng)的應(yīng)答反應(yīng)與功能,本研究以課題組前期已開展的通城豬與大白豬人工感染試驗材料和感染前后轉(zhuǎn)錄組分析數(shù)據(jù)為基礎(chǔ),篩選到了高致病性豬藍耳病毒(Highly Pathogenic PRRS,HP-PRRSV)感染機體后補體系統(tǒng)差異表達基因,并對補體系統(tǒng)開展以下研究:(1)補體通路中的基因分析:以通城豬與大白豬HP-PRRSV感染組各3頭及對照組各3頭的肺泡巨噬細胞RNA為材料,利用Illumina Hi Seq TM2000進行深度測序,并用轉(zhuǎn)錄組測序(RNA Sequencing,RNA-Seq)數(shù)據(jù)分析通用軟件Tophat 2.0對測序產(chǎn)出reads進行定位。然后統(tǒng)計轉(zhuǎn)錄本和基因的差異表達情況,用閾值Fold Change大于等于2或小于等于2,同時P值小于等于0.01,篩選HP-PRRSV感染前后補體系統(tǒng)差異表達基因。測序結(jié)果顯示:通城豬中補體基因C3AR、C4A、C5與絲氨酸相關(guān)蛋白酶1(MBL-associated serine protease,MASP-1)基因被上調(diào),會有利于補體凝集素途徑的激活;而補體CFP基因的下調(diào)與CFH的上調(diào),會對補體旁路途徑產(chǎn)生抑制;補體抑制因子CD55、CD59、CLU與C4BPB的上調(diào),會抑制補體轉(zhuǎn)化酶和攻膜復(fù)合物的形成,從而對整個補體系統(tǒng)產(chǎn)生抑制作用。大白豬中,補體凝集素途徑MASP-1基因的上調(diào)、經(jīng)典途徑C1R上調(diào)以及C4A與C3AR的上調(diào),有利于補體系統(tǒng)經(jīng)典途徑和凝集素途徑的激活;補體旁路途徑抑制子CFH的上調(diào)及穩(wěn)定轉(zhuǎn)化酶的CFP的下調(diào),會對補體旁路途徑激活產(chǎn)生抑制作用;補體抑制因子C1R、CD55、CD59、CLU、C4BPB被上調(diào),會抑制轉(zhuǎn)化酶及攻膜復(fù)合物的形成,對整個補體系統(tǒng)產(chǎn)生抑制。綜上所述,從轉(zhuǎn)錄組數(shù)據(jù)看出,通城豬與大白豬的補體系統(tǒng)都激活同時又受到抑制,但是由于通城豬中檢測到補體系統(tǒng)三條途徑交匯點C5基因的上調(diào),而大白豬中沒有檢測到,因此推斷通城豬補體系統(tǒng)激活程度比大白豬高一些。(2)補體系統(tǒng)差異表達基因的定量檢測分析:以通城豬與大白豬HP-PRRSV感染組各3頭及對照組各3頭的肺泡巨噬細胞編碼脫氧核糖核酸(Coding deoxyribonucleic acid,c DNA)為模板,通過熒光實時定量驗證了通城豬與大白豬中補體差異表達基因關(guān)鍵因子在感染HP-PRRSV后的表達情況,定量結(jié)果顯示:在通城豬與大白豬中補體基因C3AR、C4A、CFH、CD55、CD59與CLU呈上調(diào)表達,而補體基因CFP下調(diào)表達,與轉(zhuǎn)錄組數(shù)據(jù)基本一致即補體系統(tǒng)激活同時又受到抑制。(3)HP-PRRSV感染通城豬與大白豬7天后免疫組化檢測補體C3d在肺組織中的分布:結(jié)果顯示HP-PRRSV感染的通城豬與大白豬中都檢測到了補體C3d的大量沉積,而陰性對照組未檢測到陽性信號,并根據(jù)有關(guān)文獻報道補體C3d是補體系統(tǒng)激活標志,得出結(jié)論通城豬與大白豬補體系統(tǒng)被激活。(4)補體系統(tǒng)中心因子C3在HP-PRRSV感染后血清中的變化:以HP-PRRSV感染的通城豬與大白豬各3頭為材料,以HP-PRRSV感染0天的血清為對照,用酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)檢測血清中補體C3的水平變化,結(jié)果顯示:通城豬中感染后5天與7天的C3水平顯著高于感染前;在大白豬中補體C3水平一直處于穩(wěn)定,且感染HP-PRRSV后的C3水平與感染前差異不顯著。通城豬與大白豬相比,通城豬在感染后第5天補體C3水平顯著高于大白豬第5天血清中補體C3水平,感染后第7天補體C3水平極顯著高于大白豬第7天水平,這說明HP-PRRSV感染后通城豬補體系統(tǒng)激活程度比大白豬高,同時相關(guān)文獻報道血清中補體C3水平升高利于機體清除病毒,因此也推斷出通城豬與大白豬相比可能更有效的清除藍耳病毒。本研究從轉(zhuǎn)錄水平與蛋白水平初步探索了對HP-PRRSV感染通城豬與大白豬后補體系統(tǒng)的應(yīng)答反應(yīng):從轉(zhuǎn)錄數(shù)據(jù)來看HP-PRRSV感染通城豬與大白豬后,補體系統(tǒng)激活的同時又受到補體抑制因子的抑制。蛋白水平上免疫組化檢測到補體系統(tǒng)激活標志蛋白C3d在通城豬與大白豬肺組織中大量沉積,表明補體系統(tǒng)都被激活;而ELISA測定血清中補體C3水平在品種間存在差異:感染高致病性藍耳病毒后通城豬第5天與第7天血清中補體C3水平都顯著高于大白豬第5天和第7天水平。蛋白水平上結(jié)合免疫組化結(jié)果表明HP-PRRSV感染后,通城豬與大白豬補體系統(tǒng)都被激活,但通城豬補體系統(tǒng)激活程度比大白豬高,且可能更有效的清除藍耳病毒,希望這些結(jié)果為今后研究藍耳病毒與補體互作以及PRRSV抗病育種提供一定的參考。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28

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本文編號：2026738