發(fā)布時間：2018-07-03 20:57

  本文選題：豬繁殖與呼吸綜合征病毒 + SLA-DRB1基因�。� 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：由豬繁殖與呼吸綜合癥病毒(PRRSV)引起的豬繁殖與呼吸綜合征(PRRS)是一種高度傳染性疾病,對世界各國的養(yǎng)豬業(yè)造成了巨大的經(jīng)濟損失,主要表現(xiàn)為仔豬的呼吸道疾病和妊娠母豬的繁殖障礙。PRRSV具有很強的變異性并且在豬體內(nèi)持續(xù)性感染,現(xiàn)有的疫苗無法完全控制該疾病的流行,因此遺傳抗性育種是控制該病流行的新的研究方向。已有研究表明不同豬品種之間存在PRRSV抗性的差異,姜曲海豬抗性顯著高于定遠(yuǎn)豬,但機制不明。本文對姜曲海豬、定遠(yuǎn)豬肺泡巨噬細(xì)胞(PAM)接種PRRSV,12 h后檢測基因表達(dá),發(fā)現(xiàn)抗原遞呈過程中起重要作用的SLA-DRB1在姜曲海豬中高表達(dá),而定遠(yuǎn)豬中表達(dá)量甚低。在此基礎(chǔ)上,采用RNAi技術(shù)研究了SLA-DRB1基因敲減后PAMs中PRRSV載量的差異;以及SLA-DRB1上游轉(zhuǎn)錄因子CIITA和RFX5在PRRSV攻毒前后mRNA表達(dá)水平的差異;并以大白姜曲海雜交攻毒群體及其橫交一代攻毒群體為研究對象,分析了SLA-DRB 基因多態(tài)性與豬PRRSV易感性的相關(guān)性,旨在探索豬對PRRSV抗性差異的潛在機制。主要試驗內(nèi)容包括以下四部分:1.姜曲海豬和定遠(yuǎn)豬PAMs中感染PRRSV前后SLA-DRB1基因mRNA的轉(zhuǎn)錄水平本研究采用肺泡灌流的方法獲得姜曲海豬和定遠(yuǎn)豬的肺泡巨噬細(xì)胞(PAMs),純化后體外感染PRRSV NJGC珠,在感染PRRSV后6 h和12 h收集細(xì)胞,采用熒光定量PCR技術(shù)檢測PRRSV-N基因、SLA-DRB1基因mRNA轉(zhuǎn)錄水平。結(jié)果表明,攻毒后,定遠(yuǎn)豬PAM中PRRSV-N基因mRNA的表達(dá)水平顯著性高于姜曲海豬(P0.05),而SLA-DRB1基因mRNA表達(dá)水平均顯著低于姜曲海豬(P0.05),同時定遠(yuǎn)豬PAM中SLA-DRB1基因mRNA表達(dá)水平在攻毒前亦顯著低于姜曲海豬(P0.05)。2.SLA-DRB1基因敲減的PAM中PRRSV載量為了進(jìn)一步了解SLA-DRB1基因與PRRSV之間的關(guān)系,本研究采用RNAi技術(shù)抑制了SLA-RB1基因在PAM中的表達(dá),PRRSV感染SLA-DRB1基因表達(dá)抑制后的PAM,12 h后收集細(xì)胞,熒光定量PCR技術(shù)檢測SLA-DRB1基因和PRRSV-N基因mRNA的表達(dá)水平。結(jié)果顯示SLA-DRB1基因的表達(dá)受到抑制后,接種PRRSV后12 h的PAM中PRRSV-N基因的表達(dá)量顯著性上升,即PRRSV的載量顯著增加。3.SLA-DRB1基因上游轉(zhuǎn)錄因子表達(dá)水平為進(jìn)一步探究SLA-DRB1在不同抗性豬差異表達(dá)的機制,對該基因上游起調(diào)控作用的轉(zhuǎn)錄因子CIITA和RFX5進(jìn)行了進(jìn)一步的研究。采用熒光定量PCR技術(shù)檢測CIITA基因和RFX5基因在接種PRRSR后6h和12h的姜曲海豬和定遠(yuǎn)豬PAM中的表達(dá)量。結(jié)果發(fā)現(xiàn),接種PRRSV后6 h和12 h CIITA基因和RFX5基因在姜曲海豬中的表達(dá)水平均高于定遠(yuǎn)豬,且在攻毒后表達(dá)水平均高于攻毒前表達(dá)水平,其表達(dá)趨勢與SLA-DRB1相同。4.豬SLA-DRBR基因單核苷酸多態(tài)性與PRRSV易感性的關(guān)聯(lián)分析為探索豬SLA-DRB1基因單核苷酸多態(tài)性與PRRSV易感性的關(guān)系,對豬SLA-DRB1基因啟動子區(qū)和外顯子3進(jìn)行擴增和測序,篩選多態(tài)性位點,分析其與PRRSV易感性的相關(guān)性。測序共發(fā)現(xiàn)了 8個單堿基突變位點,分別是啟動子序列上-12bp處的C/A突變和-27 bp、-29 bp、-56 bp處的C/T突變以及在外顯子3上17 bp處的G/A突變、95 bp處的C/T突變、137 bp、232 bp處的G/C突變。針對外顯子3 g.232 G/C突變建立PCR-RFLP方法,在所檢測的7個群體共315個個體中該突變位點只檢測到GG型和GC型兩種基因型,在大白豬中只發(fā)現(xiàn)了 GG型,大白姜曲海豬群體及橫交一代雜交群體中以GC型(0.57/0.62)居多,GG型(0.43/0.38)次之,其他群體均以GG型居多,GC型次之。對該位點大白姜曲海群體和橫交一代雜交群體不同基因型與體重及PRRSV載量進(jìn)行關(guān)聯(lián)分析,發(fā)現(xiàn)該位點不同基因型豬血液中PRRSV載量差異不顯著(P0.05),大白姜曲海群體中不同基因型在體重變化差異也不顯著(P0.05)。針對啟動子區(qū)的g.-56 C/T突變建立CRS-PCR-RFLP方法,共檢測了 7個群體共315個個體,在橫交一代群體及長白豬和定遠(yuǎn)豬中均檢測到3種基因型,均以 CC 型(0.43/0.62/0.46)居多,TC 型(0.40/0.30/0.36)次之,TT 型(0.17/0.08/0.18)最少,在大白姜曲海群體中只檢測到CC型(0.53)和TC型(0.47)兩種基因型,其他群體只檢測到CC型。分析該位點與攻毒后橫交一代群體豬體重變化的相關(guān)性發(fā)現(xiàn),CC型個體的平均體重極顯著性高于TT型和TC型(P0.01),分析該位點與豬PRRSV易感性的相關(guān)性發(fā)現(xiàn)橫交一代群體中CC基因型的PRRSV病毒載量在接毒后各時間段均不同程度的低于TT基因型和TC基因型,并于4 d、7d和14 d時達(dá)到顯著性差異(P0.05)。針對啟動子區(qū)的另外3個突變位點對橫交一代群體采用直接測序的方法進(jìn)行分型,均檢測到了三種基因型,-29 bp處以TC型居多,TT型次之,CC型最少;-27 bp處以TC型居多,CC型次之,TT型最少;-12 bp處以CC型最多,AC型次之,AA型最少。對-29 bp處的突變與PRRSV相對表達(dá)量及體重進(jìn)行相關(guān)性分析發(fā)現(xiàn),在接種病毒后7、11、14、21dPRRSV相對表達(dá)量CC型顯著(P0.05)低于TT型和TC型而體重相關(guān)性分析得到CC型個體在不同時間點的平均體重最高,于接毒后14d、21d、28d、42d顯著(P0.05)高于TT型和TC型。對-27bp處的突變與PRRSV相對表達(dá)量及體重進(jìn)行相關(guān)性分析發(fā)現(xiàn),在接種病毒后4d、7 d、11 d、14 d、21 d和42 dPRRSV相對表達(dá)量TT型顯著(P0.05)低于CC型和TC型;而體重相關(guān)性分析發(fā)現(xiàn)TT型個體在不同時間點的平均體重最高,于接毒后21d、28d、42d顯著(P0.05)高于CC型和TC型,于接毒后11d、14 d、35 d顯著(P0.05)高于TC型。對-12 bp處的突變與PRRSV相對表達(dá)量及體重進(jìn)行相關(guān)性分析發(fā)現(xiàn),不同基因型之間PRRSV相對表達(dá)量及體重均無顯著性差異(P0.05)。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly contagious disease. It has caused huge economic losses to the pig industry in the world, mainly as respiratory diseases of piglets and reproductive barrier of gestation sows with strong variability and sustained in pigs. The current vaccine can not completely control the epidemic of the disease. Therefore, genetic resistance breeding is a new research direction to control the epidemic. There has been a study on the difference of PRRSV resistance between different pig breeds. The resistance of ginger to the Dingyuan pig is significantly higher than that of the pig, but the mechanism is not clear. This article is on the alveoli of Jiang Qu sea pig and Dingyuan pig. Cell (PAM) was inoculated with PRRSV, after 12 h, and the expression of gene was detected. It was found that SLA-DRB1, which played an important role in the antigen presentation process, was highly expressed in the Jiang Qu Hai pig, and the expression of the Dingyuan pig was very low. On this basis, the RNAi technology was used to study the difference of PRRSV load in PAMs after the SLA-DRB1 gene knockout, and the SLA-DRB1 upstream transcription factor CIITA and RFX5 in PR. The difference in the expression level of mRNA before and after the attack of RSV was studied, and the correlation between the SLA-DRB gene polymorphism and the susceptibility of porcine PRRSV was analyzed. The purpose of this study was to explore the potential mechanism of the differential resistance of pigs to PRRSV. The main contents of the experiment included the following four parts: 1. ginger koji and the sea pig. The transcriptional level of SLA-DRB1 gene mRNA before and after infection of PRRSV in Dingyuan pig PAMs, the alveolar macrophages (PAMs) of ginger koji and Dingyuan pig were obtained by alveolar perfusion method. After purification, PRRSV NJGC beads were purified in vitro, and 6 h and 12 h were collected after PRRSV infection. The results showed that after attack, the expression level of PRRSV-N gene mRNA in Dingyuan pig PAM was significantly higher than that of Jiang Qu Hai pig (P0.05), and the mRNA expression level of SLA-DRB1 gene was significantly lower than that of ginger koji (P0.05), and the mRNA expression level of SLA-DRB1 gene in Dingyuan pig PAM was significantly lower than that of Jiang Qu sea pig (P0.05) before attack. In order to further understand the relationship between the SLA-DRB1 gene and the PRRSV gene in the subtraction PAM, the RNAi technique was used to suppress the expression of the SLA-RB1 gene in PAM, the PAM of the SLA-DRB1 gene expression in PRRSV infection, and the cells after 12 h, and the fluorescence quantitative PCR technique was used to detect the expression level of the SLA-RB1. The results showed that the expression of SLA-DRB1 gene was inhibited and the expression of PRRSV-N gene in PAM increased significantly in 12 h PAM after PRRSV inoculation, that is, the load of PRRSV increased the expression level of the upstream transcription factor of.3.SLA-DRB1 gene to further explore the mechanism of SLA-DRB1 in the differential expression of different resistant pigs and to regulate the upstream of the gene. The transcriptional factor CIITA and RFX5 were further studied. The expression of CIITA gene and RFX5 gene in the PAM of ginger koji and Dingyuan pig in 6h and 12h after PRRSR were detected by fluorescence quantitative PCR. The results showed that the expression level of 6 h and 12 h genes and the genes in ginger koji were higher than those of Dingyuan pigs. The expression level after attack was higher than that before attack, and its expression trend was associated with the association analysis of single nucleotide polymorphisms of.4. SLA-DRBR gene and PRRSV susceptibility of the same SLA-DRB1 pig SLA-DRBR gene, which was to explore the relationship between single nucleotide polymorphisms of the pig SLA-DRB1 gene and the susceptibility of PRRSV, and the amplification and measurement of the promoter region and exon 3 of the porcine SLA-DRB1 gene. Sequence, screening polymorphic loci and analyzing its correlation with PRRSV susceptibility. 8 single base mutation sites were found, which were C/A mutations at -12bp in the promoter sequence and -27 BP, -29 BP, C/T mutations at -56 BP, and G/A mutations at the 17 BP in exon 3, 137 mutation, 232 of the mutation. Needles to the outside. The PCR-RFLP method was established by the mutation of the 3 g.232 G/C. In the 315 individuals of the 7 populations, only the GG and GC genotype were detected. Only the GG type was found in the big white pig. The large white ginger koji and the cross generation hybrid population were mostly GC type (0.57/ 0.62), GG type (0.43/0.38), and the other groups were G. G type was the most common and GC type. The correlation analysis of the different genotypes with the weight and the PRRSV load of the crossbred population of this loci was not significant (P0.05), and the difference of the body weight of the different genotypes in the large white ginger group was not significant (P0.05). The g.-56 C/T mutation in the promoter region was established to establish a CRS-PCR-RFLP method. A total of 315 individuals were detected in 7 groups. 3 genotypes were detected in the cross generation group, the long white pig and Dingyuan pig, all of which were CC (0.43/0.62/0.46), TC (0.40/0.30/0.36), and TT (0.17/0.08/0.18) at least, and only in the large white ginger population. Two genotypes of type CC (0.53) and type TC (0.47) were detected, and the other populations only detected the CC type. The correlation between the site and the body weight change of the cross generation pigs after the attack was analyzed. The average weight of the CC type was significantly higher than that of the TT and TC (P0.01). The correlation between the site and the susceptibility of swine PRRSV was found to be found in the CC population in the cross generation population. The PRRSV viral load of genotypes was lower than the TT genotype and TC genotype at various time periods after exposure, and reached significant difference at 4 D, 7d and 14 d (P0.05). Three genotypes, -29 BP, were detected by direct sequencing for the other 3 mutation sites in the promoter region. The majority of TC type, TT type and CC type are the least, -27 BP is mostly TC type, CC type and TT type is the least, -12 BP is the CC type, AC type is the least. The average weight of CC type individuals at different time points was highest, and 14d, 21d, 28d, 42d significantly higher than TT type and TC type after receiving poison. The correlation analysis between the mutation of -27bp and the relative expression and weight of PRRSV found that after inoculation, 7 d, 11, 14, 21, and 42 relative expressions were significant. (P0.05) was lower than CC type and TC type, and the weight correlation analysis found that the average weight of TT individuals at different time points was highest, and 21d, 28d, 42d significantly (P0.05) was higher than CC and TC type after receiving poison, and 11d, 14 d, and higher than the type of TC. There was no significant difference in PRRSV relative expression and body weight between different genotypes (P0.05).
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S828

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