丁酸梭菌对不同遗传背景仔猪TLR2信号通路差异调控的分子机制

Molecular mechanism of Clostridium butyricum differentially regulating TLR2 signaling pathway in piglets with different genetic backgrounds

为了研究丁酸梭菌对不同遗传背景断奶仔猪TLR2信号通路差异调控的分子机制,试验采用PCR方法从二元杂交黑猪和三元杂交白猪回肠中扩增出TLR2全基因,并对其进行克隆和鉴定,利用BioEdit软件分析两种仔猪TLR2基因与GenBank中人、黑猩猩、水牛、家牛、绵羊、马、藏羚羊和宽吻海豚8个物种的同源性,使用MEGA 4.0软件进行聚类分析,邻接法构建系统进化树,使用ProtParam、ProtScale和TMHMM在线工具分别预测分析蛋白质结构、疏水性和跨膜区,并应用DNAStar软件比较两种仔猪TLR2蛋白序列差异。结果表明:PCR扩增获得二元杂交黑猪和三元杂交白猪的TLR2全基因,大小均为2358 bp,且目的基因正确连接到了pMD19-T载体中。二元杂交黑猪和三元杂交白猪TLR2基因序列与人、黑猩猩、水牛、家牛、绵羊、马、藏羚羊和宽吻海豚8个物种的同源性分别为78%~82%和79%~82%,亲缘关系较远,而两种仔猪在同一个分支内。二元杂交黑猪和三元杂交白猪TLR2基因的开放阅读框编码蛋白的氨基酸数目均为785个,等电点分别为7.53和7.78,不稳定指数分别为43.76和42.86,平均亲水系数分别为-0.152和-0.146,二元杂交黑猪和三元杂交白猪TLR2蛋白的疏水性分值相同,但富含疏水性氨基酸的区域不同。TLR2蛋白的第1~588位氨基酸位于细胞膜外,具有识别微生物的功能,且两种仔猪的TLR2蛋白氨基酸在第13,16,17,19~21,143,164,248,249,296位不同。说明TLR2蛋白胞外识别区域氨基酸的差异可能会影响丁酸梭菌刺激产生的信号强度,进而影响丁酸梭菌对不同遗传背景仔猪TLR2信号通路的差异调控。

In order to investigate molecular mechanism of Clostridium butyricum differentially regulating TLR2 signaling pathway in piglets with different genetic backgrounds, the whole TLR2 gene was amplified from ileum of binary hybridization black piglets and ternary hybridization white piglets by PCR, and the TLR2 genes were cloned and identified. BioEdit software was used to analyze the homology of TLR2 genes between the two kinds of piglets and eight species in GenBank including human, chimpanzee, buffalo, domestic cattle, sheep, horse, Tibetan antelope and bottlenose dolphin. MEGA 4.0 was used for cluster analysis, and adjacency method was used for construction phylogenetic tree. ProtParam, ProtScale and TMHMM online tools were used to predict and analyze protein structure, hydrophobicity and transmembrane region respectively, and DNAStar software was used to compare the differences of TLR2 protein in the two kinds of piglets. The results showed that the entire TLR2 genes of binary hybridization black piglets and ternary hybridization white piglets was amplified by PCR, and the size of TLR2 gene was 2 358 bp, and the target gene was correctly connected to pMD19-T vector. The homologies of binary hybridization black piglets and ternary hybridization white piglets to humans, chimpanzees, buffaloes, cattle, sheep, horses, Tibetan antelopes and bottlenose dolphins were 78%-82% and 79%-82%, respectively. They are distantly related and both species are in the same branch. The number of amino acids encoded by TLR2 open reading frame was 785, and the isoelectric points of TLR2 in black and white piglets were 7.53 and 7.78, respectively. The instability indexes were 43.76 and 42.86, respectively, and the average hydrophilic coefficients were-0.152 and-0.146, respectively. The hydrophobicity scores of TLR2 protein in binary hybridization black piglets and ternary hybridization white piglets were the same, but the regions rich in hydrophobic amino acids were different. The amino acids from 1 th to 588 th of TLR2 protein were located outside the cell membrane, which were used to recognize the microorganisms, and the TLR2 protein amino acids in the two kinds of piglets were different in the 13 th, 16 th, 17 th, 19 th to 21 th, 143 th, 164 th, 248 th, 249 th and 296 th positions. It indicated that the difference of amino acids in the extracellular recognition region of TLR2 protein might affect the generation of signal intensity by Clostridium butyricum stimulation, and thus affected the differential regulation of TLR2 signaling pathway by Clostridium butyricum in piglets with different genetic backgrounds.