| Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus(PRRSV),is a highly contagious disease,the main symptoms are respiratory tract disease of piglets and reproductive barriers of pregnancy sow.It caused great economic losses to the pig industry around the world when this disease appeared.Due to PRRSV has a strong variation and it can persistently infected in pigs,the existing vaccines can’t effectively control the infection.So choose strong resistance varieties through genetic resistance breeding pigs is effective as a means of suppressing the disease.Some research showed that different pig breeds have different resistance to PRRSV,the resistance in Jiangquhai pig is higher than that in Dingyuan pig,but the mechanism is unclear.In this paper,PRRSV was inoculated into pulmonary alveolar macrophage(PAM)of Jiangquhai pig and Dingyuan pig,the genes expression were detected 12 h later,the data showed that theSLA-DRB1gene played an important role in the process of antigen presented has different expression,its mRNA transcriptional level in Jiangquhai pig was higher than that in Dingyuan pig.Due to this,the RNAi was used to knockdown the expression of SLA-DRB1 gene in PAM,and the copies of PRRSV was determined;thenthe expression of some transcription factors of SLA-DRB1 such as CIITA and RFX5weredetermined;at last,the associations between SLA-DRB1 polymorphisms and the resistance to PRRSV was analyzed.The main experiment contents includes the following four parts:1.The SLA-DRB1transcriptional levels in PAMs ininfected PRRSV of Jiangquhai pig and Dingyuan pig.PAMs were separated and purified from Dingyuan pig and Jiangquhai pig with the method of alveolar perfusion.Cultured PAMs were infected with PRRSV NJGC.Realtime PCR was carried out to determine the mRNA transcriptional levels of PRRSV-N and SLA-DRB1 in PAMs post-infection 6 h and 12 h.The results showed that PRRSV-NmRNA transcriptional level in the PAMs of Dingyuan pig is significantly higher than that of Jiangquhai pig(P<0.05),but the mRNA transcriptionalof SLA-DRBI is significantly lower than that of Jiangquhai pig(P<0.05),meanwhile the mRNA expression of SLA-DRB1 in the PAMs of Dingyuan pig is significantly lower than that of Jiangquhai pig non-infection(P<0.05).2.The differenceof PRRSV copy in PAM after SLA-DRB1 gene knockdown In order to study the relationship of the SLA-DRB1 gene and PRRSV,RNAi was used to inhibit the SLA-DRB1 gene expression in PAM,then the PRRSV-N and SLA-DRB1 mRNA transcriptional level in PAM were determined after PRRSV infection 12 h.The mRNA transcriptional level of PRRSV-N gene significantly increased when the SLA-DRBlwas knockdown.3.The expression levels of SLA-DRB1 upstream regulatory factorsTo further explore the mechanism of differential expression of SLA-DRB1 in different resistant pigs,we determined mRNA transcriptional levels of CIITA gene and RFX5 gene by real-time PCR after PRRSV inoculation 6 h and 12 h in PAM of Jiangquhai pigs and Dingyuan pigs.The results showed that CIITA gene and RFX5 gene expression levels in Jiangquhai pigs were higher than those in Dingyuan pigs after inoculation PRRSV 6 h and 12 h,and the expression levels of these two genes were higher after inoculation then before inoculation,which was the same as the expression pattern of HLA-DRB1.4.SLA-DRB1 gene single nucleotide polymorphism analysis associated with susceptibility to PRRSVIn order to explore SLA-DRB1 gene SNPs with susceptibility to PRRSV,SLA-DRB1 gene promoter region and exon 3 was amplified and sequenced to screen polymorphisms,Then the association between SNPs and the susceptibility to PRRSV was analyze.Sequencing found a total of eight single-base mutations,they are the promoter sequence of-12 bp C/A,-27 bp T/C,-29 bp T/C,-56 bp C/T and the exon 3 of 17 bp G/A,95 bp C/T,137 bp G/C and 232 bp G/C.For exon 3 g.232 G/C mutation construct PCR-RFLP method,total of seven groups 315 individuals were detected only found GG and GC two genotypes.In Large White only found GG genotype,the F1 of Jiangquhai x Large White and transverse cross-generation hybrid population with GC type(0.57/0.62)majority,then the GG genotype(0.43/0.38),followed by other groups are GG type mostly,GC type second.The sites of different genotypes analysis of PRRSV load and weight iin the groups of F1 of Jiangquhai x Large White and transverse cross-generation hybrid population,found that the PRRSV loading was not significant(P>0.05)in different genotypes pig blood of two population,and the different genotypes in weight change was not significant difference(P>0.05)transverse cross-generation hybrid population,The CRS-PCR-RFLP method was constructedfor promoter sequence g.-56 T/C mutation,seven population including 315 individuals were divided into three genotypes,CC,TT and TC.In the transverse generation population,Landrace pigs and Dingyuan pigs were detected three genotypes,CC(0.43/0.62/0.46)majority,TC(0.40/0.30/0.36)followed and TT(0.17/0.08/0.18)the least,in F1 of Jiangquhai x Large White only found CC(0.53)and TC(0.47)two genotypes.Analysis of the correlation of this site with pig’s weight found that the weight of CC genotype was significantly higher than of TT and TC genotype(P<0.01).We analyzed the correlation of the mutation in this site and PRRSV susceptibility,found thatthe PRRSV unit copy of CC genotype was lower than the TT and TC genotypes in transverse cross-generation hybrid population,and reaches a significant level(P<0.05)in 4 d,7 d and 14 d.For the other three mutations in the promoter region of SLA-DRB1 by direct sequencing method in the population of cross-generation hybrid of Jiangquhai x Large White,there are all including three genotypes,TT type most,TC type second,CC type least in g.-29 bp;TCtype most,CCtype second,TT type least in g.-27 bp;CCtype most,ACtype second,AA type least in g.-12 bp.Analysis of the correlation of the mutation of g.-29 bp with PRRSV susceptibility and pig’s weight,the result showed that at PRRSV inoculation?d,11 d,14 d,21d,PRRSV loading of CC were significantly lower(P<0.05)than that of TT and TC genotype and weight-related analysis showed that CC genotype was the highest average weight at different points after inoculation,at 21 d,28 d,42 d was significantly(P<0.05)higher than the TT genotype and TC type.Analysis of the correlation of the mutation of g.-27 bp with PRRSV susceptibility and pig’s weight,the result showed that at PRRSV inoculation 4 d,7 d,11 d,14 d,21d and 42 d,PRRSV loading of TT were significantly lower(P<0.05)lower than that of CC and TC genotype and weight-related analysis showed that TT genotype was the highest average weight at different points after inoculation,at 21 d,28 d,42 d was significantly(P<0.05)higher than the CC genotype and TC type,at 11d,14d,35d was significantly(P<0.05)higher than TC type.Analysis of the correlation of the mutation of g.-12 bp with PRRSV susceptibility and pig’s weight,the result showed that among the different genotypes,the PRRSVloading and body weight were not significantly different(P>0.05). |
