藏绵羊溶菌酶1基因的克隆、原核表达及生物信息学分析

试验旨在研究藏绵羊溶菌酶(lysozyme,LZM)1基因在瘤胃、瓣胃、皱胃、气管、肝脏和肾脏各组织中的表达情况、进化关系及抑菌活性.采用RT-PCR技术克隆藏绵羊LZM1基因,定量PCR检测LZM1基因在瘤胃、瓣胃、皱胃、气管、肝脏和肾脏中的表达情况,并将该基因的氨基酸序列与普通牛胃LZM等氨基酸序列进行比对,根据邻位连接法构建系统进化树;将该基因插入pET-32a质粒后构建得到pET-32a-LZM1重组质粒,转化大肠杆菌BL21(DE3)感受态细胞并诱导表达,SDS-PAGE法鉴定表达产物;采用比浊法测定LZM1蛋白的活性;采用琼脂糖平板扩散法研究异源表达蛋白的抑菌活性.结果表明,从藏绵羊瘤胃、瓣胃、皱胃、气管、肝脏和肾脏各组织中成功克隆藏绵羊LZM1基因,其在瘤胃、瓣胃、皱胃、气管、肝脏和肾脏中均有表达,在皱胃中表达量最高;其氨基酸序列与普通牛胃LZM(GenBank登录号:NM_001080339.1)序列的同源性最高(96%);重组蛋白LZM1分子质量约为35 ku;其比活力约为400 U/mL;该重组LZM1对金黄色葡萄球具有良好的抑菌活性.

Cloning,Prokaryotic Expression and Bioinformatic Analysis of Stomach Lysozyme 1 Gene from Tibetan Sheep

The experiment was aimed to study the expression of the Tibetan sheep lysozyme (LZM) 1 gene in rumen, omasum, abomasum, trachea, liver and kidney and the evolutionary relationship between LZM1 and other lysozymes, and its antibacterial activity.The LZM1 gene cDNA sequence was cloned by RT-PCR method, the expression of LZM1 gene was detected by qPCR.Amino acids sequence alignment was performed between LZM1 and cattle stomach lysozyme, Tibetan sheep stomach lysozyme 2, human stomach lysozyme, et al.And phylogenetic tree was constructed based on Neighbor-Joining, the cloned gene was inserted into pET-32a vector to construct a prokaryotic expression vector pET-32a-LZM1.Recombinant expression vector was transformed into E.coli BL21 (DE3) followed by IPTG (isopropyl-β-D-thiogalactoside) inducing.Heterologous expressed LZM1 was analyzed by SDS-PAGE.The bacteriolytic activity of recombinant protein was observed by turbidimetry and agarose plate diffusion experiment.The results showed that LZM1 gene was successfully cloned from rumen, omasum, abomasums, trachea, liver and spleen of Tibetan sheep.The LZM1 gene was expressed in rumen, omasum, abomasums, trachea, liver and spleen, and the mRNA level of LZM1 in abomasus was higher than in other tissues.The highest amino acids sequence similarity (96%) was observed between LZM1 and cattle stomach lysozyme (No:NM_001080339.1), the recombinant protein LZM1 was correctly expressed, with the molecular mass of 35 ku.The bacteriolytic activity of recombinant protein was 400 U／mL.The result of the agarose diffusion assay indicated that the recombinant protein possessed a certain antibacterial activity against Staphylococcus aureus.