猪SLA-DRB1基因与PRRSV抗性的关联研究

发布时间：2018-07-03 20:57

本文选题：猪繁殖与呼吸综合征病毒 + SLA-DRB1基因　；参考：《南京农业大学》2016年硕士论文

【摘要】：由猪繁殖与呼吸综合症病毒(PRRSV)引起的猪繁殖与呼吸综合征(PRRS)是一种高度传染性疾病,对世界各国的养猪业造成了巨大的经济损失,主要表现为仔猪的呼吸道疾病和妊娠母猪的繁殖障碍。PRRSV具有很强的变异性并且在猪体内持续性感染,现有的疫苗无法完全控制该疾病的流行,因此遗传抗性育种是控制该病流行的新的研究方向。已有研究表明不同猪品种之间存在PRRSV抗性的差异,姜曲海猪抗性显著高于定远猪,但机制不明。本文对姜曲海猪、定远猪肺泡巨噬细胞(PAM)接种PRRSV,12 h后检测基因表达,发现抗原递呈过程中起重要作用的SLA-DRB1在姜曲海猪中高表达,而定远猪中表达量甚低。在此基础上,采用RNAi技术研究了SLA-DRB1基因敲减后PAMs中PRRSV载量的差异;以及SLA-DRB1上游转录因子CIITA和RFX5在PRRSV攻毒前后mRNA表达水平的差异;并以大白姜曲海杂交攻毒群体及其横交一代攻毒群体为研究对象,分析了SLA-DRB 基因多态性与猪PRRSV易感性的相关性,旨在探索猪对PRRSV抗性差异的潜在机制。主要试验内容包括以下四部分:1.姜曲海猪和定远猪PAMs中感染PRRSV前后SLA-DRB1基因mRNA的转录水平本研究采用肺泡灌流的方法获得姜曲海猪和定远猪的肺泡巨噬细胞(PAMs),纯化后体外感染PRRSV NJGC珠,在感染PRRSV后6 h和12 h收集细胞,采用荧光定量PCR技术检测PRRSV-N基因、SLA-DRB1基因mRNA转录水平。结果表明,攻毒后,定远猪PAM中PRRSV-N基因mRNA的表达水平显著性高于姜曲海猪(P0.05),而SLA-DRB1基因mRNA表达水平均显著低于姜曲海猪(P0.05),同时定远猪PAM中SLA-DRB1基因mRNA表达水平在攻毒前亦显著低于姜曲海猪(P0.05)。2.SLA-DRB1基因敲减的PAM中PRRSV载量为了进一步了解SLA-DRB1基因与PRRSV之间的关系,本研究采用RNAi技术抑制了SLA-RB1基因在PAM中的表达,PRRSV感染SLA-DRB1基因表达抑制后的PAM,12 h后收集细胞,荧光定量PCR技术检测SLA-DRB1基因和PRRSV-N基因mRNA的表达水平。结果显示SLA-DRB1基因的表达受到抑制后,接种PRRSV后12 h的PAM中PRRSV-N基因的表达量显著性上升,即PRRSV的载量显著增加。3.SLA-DRB1基因上游转录因子表达水平为进一步探究SLA-DRB1在不同抗性猪差异表达的机制,对该基因上游起调控作用的转录因子CIITA和RFX5进行了进一步的研究。采用荧光定量PCR技术检测CIITA基因和RFX5基因在接种PRRSR后6h和12h的姜曲海猪和定远猪PAM中的表达量。结果发现,接种PRRSV后6 h和12 h CIITA基因和RFX5基因在姜曲海猪中的表达水平均高于定远猪,且在攻毒后表达水平均高于攻毒前表达水平,其表达趋势与SLA-DRB1相同。4.猪SLA-DRBR基因单核苷酸多态性与PRRSV易感性的关联分析为探索猪SLA-DRB1基因单核苷酸多态性与PRRSV易感性的关系,对猪SLA-DRB1基因启动子区和外显子3进行扩增和测序,筛选多态性位点,分析其与PRRSV易感性的相关性。测序共发现了 8个单碱基突变位点,分别是启动子序列上-12bp处的C/A突变和-27 bp、-29 bp、-56 bp处的C/T突变以及在外显子3上17 bp处的G/A突变、95 bp处的C/T突变、137 bp、232 bp处的G/C突变。针对外显子3 g.232 G/C突变建立PCR-RFLP方法,在所检测的7个群体共315个个体中该突变位点只检测到GG型和GC型两种基因型,在大白猪中只发现了 GG型,大白姜曲海猪群体及横交一代杂交群体中以GC型(0.57/0.62)居多,GG型(0.43/0.38)次之,其他群体均以GG型居多,GC型次之。对该位点大白姜曲海群体和横交一代杂交群体不同基因型与体重及PRRSV载量进行关联分析,发现该位点不同基因型猪血液中PRRSV载量差异不显著(P0.05),大白姜曲海群体中不同基因型在体重变化差异也不显著(P0.05)。针对启动子区的g.-56 C/T突变建立CRS-PCR-RFLP方法,共检测了 7个群体共315个个体,在横交一代群体及长白猪和定远猪中均检测到3种基因型,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,在大白姜曲海群体中只检测到CC型(0.53)和TC型(0.47)两种基因型,其他群体只检测到CC型。分析该位点与攻毒后横交一代群体猪体重变化的相关性发现,CC型个体的平均体重极显著性高于TT型和TC型(P0.01),分析该位点与猪PRRSV易感性的相关性发现横交一代群体中CC基因型的PRRSV病毒载量在接毒后各时间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时达到显著性差异(P0.05)。针对启动子区的另外3个突变位点对横交一代群体采用直接测序的方法进行分型,均检测到了三种基因型,-29 bp处以TC型居多,TT型次之,CC型最少;-27 bp处以TC型居多,CC型次之,TT型最少;-12 bp处以CC型最多,AC型次之,AA型最少。对-29 bp处的突变与PRRSV相对表达量及体重进行相关性分析发现,在接种病毒后7、11、14、21dPRRSV相对表达量CC型显著(P0.05)低于TT型和TC型而体重相关性分析得到CC型个体在不同时间点的平均体重最高,于接毒后14d、21d、28d、42d显著(P0.05)高于TT型和TC型。对-27bp处的突变与PRRSV相对表达量及体重进行相关性分析发现,在接种病毒后4d、7 d、11 d、14 d、21 d和42 dPRRSV相对表达量TT型显著(P0.05)低于CC型和TC型;而体重相关性分析发现TT型个体在不同时间点的平均体重最高,于接毒后21d、28d、42d显著(P0.05)高于CC型和TC型,于接毒后11d、14 d、35 d显著(P0.05)高于TC型。对-12 bp处的突变与PRRSV相对表达量及体重进行相关性分析发现,不同基因型之间PRRSV相对表达量及体重均无显著性差异(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).
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S828

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