中国美利奴羊TLR2基因多态性及其与布鲁氏菌病的相关性分析

试验旨在研究Toll样受体2（Toll-like receptor，TLR2）基因的多态性及其与中国美利奴羊布鲁氏菌病易感性的相关性。利用生物信息学方法对NCBI上公布的绵羊TLR2基因序列进行比对，选出多态位点丰富的片段进行扩增，运用PCR-SSCP的方法对206个中国美利奴布鲁氏菌病阴性样本和80个中国美利奴羊布鲁氏菌病阳性样本进行TLR2基因的多态性检测，然后对不同等位基因的PCR产物进行测序，确定该基因的多态性位点，经卡方检验分析每个SNP位点的等位基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性，利用生物信息学软件分析RNA二级结构及蛋白质的二级结构。结果表明，在279 bp的序列中共检测到3个SNPs，分别为：C1731T、G1737C和G1749T，均未引起对应氨基酸的改变，属于无义突变。这些位点在病例组和对照组之间的等位基因频率及基因型频率均不存在显著差异（P>0.05）。各突变位点均能引起RNA二级结构和最小自由能的改变，而蛋白质的二级结构均未改变。由此得出，中国美利奴羊TLR2基因的3个SNPs位点（C1731T、G1737C和G1749T）与中国美利奴羊布鲁氏菌病易感性无相关性。

Polymorphism of TLR2 Gene in Chinese Merino Sheep and Its Correlation Analysis with Brucellosis

This experiment was aimed to study the correlation between Toll-like receptor 2 (TLR2) gene polymorphisms and susceptibility to brucellosis of China Merino sheep.Bioinformatics methods were used for comparing the TLR2 gene sequences of sheep which were published on NCBI,and selected abundant fragments of polymorphic loci for amplification.The method of PCR-SSCP was used to detect the polymorphism of TLR2 gene from 206 brucellosis negative samples and 80 brucellosis positive samples of Chinese Merino sheep,the PCR products of different alleles were sequenced to determine the polymorphic loci of TLR2 gene.The correlation between allele frequency,genotype frequency and polymorphism of each SNP and susceptibility to brucellosis were analyzed by Chi square test.Bioinformatics analysis software was used to analyze the secondary structures of RNA and protein.The results showed that 3 SNPs (C1731T,G1737C and G1749T) were detected in the sequence of 279 bp,which didn't lead to the change of corresponding amino acids,and belonged to nonsense mutation.There was no significant difference in allele frequency and genotype frequency among the cases and controls (P>0.05).The secondary structures and the minimum free energy of RNA were changed at each mutation site,but the secondary structures of protein was not changed.In conclution,there was no correlation between 3 SNPs (C1731T,G1737C and G1749T) of TLR2 gene and brucellosis susceptibility of Chinese Merino sheep.