猪Toll样受体基因的变异及其与支原体肺炎感染的关系

Toll样受体(TLRs)作为重要的模式识别受体,在动物的天然免疫中发挥重要作用,是机体抵抗感染的第一道屏障;TLRs基因变异会改变机体对病原的抗性或易感性,本试验旨在探讨TLR2、TLR4基因突变与支原体肺炎感染的关系。试验以国外引进猪品种(长白、大白、皮特兰)、中国地方猪品种(梅山、二花脸、姜曲海、金华)以及江苏省培育猪品种(苏钟猪)为材料,通过PCR-SSCP技术检测实验猪群TLR2、TLR4基因SNPs分布,结合动物攻毒实验及文献报道,对其中部分SNP不同基因型猪的肺部巨噬细胞做LPS感染实验,借助RT-PCR跟踪不同时间点猪TLR2、TLR4基因以及促炎性因子TNF-α、IL-1β的表达变化,揭示猪Toll样受体基因的变异与支原体肺炎感染的关系。结果表明,本实验猪群TLR4基因存在4个SNP位点:T611A、G960A、G962A、C1027A,其中G960A为同义突变,其余为有义突变;位点C1027A在中、外猪品种间呈现明显的碱基分布差异。LPS诱导下的猪肺部巨噬细胞TLR2、TLR4基因及促炎性因子TNF-α、IL-1β均表现不同程度的表达上调;其中,位点C1027A的CC型猪TLR2、TLR4基因表达水平及增速均显著高于AC型猪(P<0.01),TNF-α、IL-1β的表达水平则显著低于AC型猪(P<0.01),提示C等位基因极可能是猪抗支原体肺炎感染的优势基因。

Genetic Variation of Toll-like Receptors and Mycoplasma pneumoniae Infection in Pigs

As significant pattern-recognition receptors,Toll-like receptors(TLRs)play an important role in the innate immunity and serve as the first barrier in the host defense against microbial pathogens.Variation of TLR genes would affect the resistance or susceptibility to diseases in human beings and animals.The purpose of this article is to analyze the correlation between the genetic variation of Toll-like receptors and Mycoplasma pneumoniae Infection in pigs.In this study,PCR-SSCP was applied to detect the distribution of single nucleotide polymorphisms(SNPs)in Western Commercial pig breeds(Landrace,Large White,Pietrain),Chinese indigenous pig breeds(Meishan,Erhualian,Jiangquhai,Jinhua)and Jiangsu cultivated breed(Suzhong pig).And then porcine alveolar macrophages(PAMs)of different genotype were stimulated by LPS and the expression levels of TLR2,TLR4 and pro-inflammatory cytokines TNF-α,IL-1β at different time points were detected by real-time PCR to reveal the relationship between mutations in porcine TLRs and Mycoplasma pneumonia infection.Results show that:(1)Overall three non-synonymous SNPs(T611A,G962A,C1027A)and a synonymous SNPs(G960A)were found in TLR4 coding sequence,and the distribution of C1027A was biased in Western pig and Chinese indigenous pig populations.(2)The expression of TLR2,TLR4 and TNF-α,IL-1β were enhanced at different levels by LPS stimulation.TLR2,TLR4 gene expression and growth rate of CC-genotype in 1 027 bp of TLR4 were significantly higher than AC-genotype pigs(P<0.01),whereas TNF-α,IL-1β expression was significantly lower than that of AC groups(P<0.01).Thus,alleles C in 1 027 bp(C 1 027 A)of TLR4 may probably be the resistance gene to M.pneumonia infections in pigs.