山羊先天免疫TLR4基因克隆、组织表达分析及其凋亡功能初探

Gene cloning,tissue expression profile and cell apoptosis function analysis of TLR4 gene of innate immunity in goat

为了解山羊TLR4的分子结构、表达特性和免疫调节功能,试验以NCBI中山羊TLR4基因预测序列为模板,采用RACE扩增技术获得初生羔羊TLR4基因c DNA序列,并进行生物信息学分析,利用qRT-PCR技术检测羔羊各组织TLR4基因表达量,构建p EGFP-N1-TRL4重组质粒检测在真核细胞中的凋亡功能。结果表明:克隆得到TLR4基因c DNA序列长2 728 bp,CDS全长2 526 bp,编码841个氨基酸。山羊TLR4蛋白是1个跨膜蛋白,胞外包含2个LRRs-RI、1个LRRs_8及1个TPKR-C2,胞内含有TIL受体结构域、77个潜在磷酸化位点、9个糖基化位点,且与绵羊TLR4氨基酸序列同源性最高。TLR4基因在脾脏中的表达量较高(P<0.05),肝脏、胸腺和肾脏依次减少,颌下腺、肺脏及血液较脾脏显著降低(P<0.05)。说明试验成功构建具有TLR4基因完整CDS序列的p EGFPN1-TLR4重组载体,且能够在HEK293T细胞中正常表达,具有加快细胞凋亡的作用。山羊TLR4基因编码蛋白具有免疫学结构和功能特征,在免疫器官中可能参与细胞凋亡过程。

In order to understand molecular structure, expression profile and immune modulating function of TLR4 of goat, the sequence of goat TLR4 gene from NCBI database was used as a template in this experiment, and the cDNA of TLRg gene of newborn lamb was obtained based on RACE method. Bioinformatics analysis was carried out ; expression levels of TLR4 gene from the tissues of newborn lamb were detected by qRT - PCR method;and apoptosis function of TLRg in eukaryotic cells was determined after recombinant plasmid pEGFP - N1 - TLR4 was con- structed. The results showed that the cDNA length of obtained TLR4 gene was 2 728 bp ; and CDS region was 2 526 bp, which encoded 841 ami- no acids. Goat TLR4 protein was a transmembrane protein, including 2 LRRs - RI, 1 LRRs_8, and 1 TPKR - C2 in extracellular domain and al- so containing a TIL - receptor domain in intracellular domain ,77 potential phosphorylation sites and 9 potential glycosylation sites. Goat TLR4 amino acid sequence had the highest homology with that of sheep. The expression level of TLR4 gene in spleen was higher（P 〈 0.05 ）, followed successively by that in liver, thymus and kidney,and it was significantly lower（ P 〈 0.05 ）in submandibular gland,lung and blood than in spleen. The results suggested that recombinant vector pEGFP - N1 - TLR4 with complete CDS of TLR4 gene was successfully constructed, and could be normally expressed in HEK293T cells, which played a role in the promotion of cell apoptosis. It was concluded that goat TLR4 protein had immunological structure and function characteristics, and may be involved in the process of cell apoptosis in mmune organs.