人LRG1基因的原核表达及生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of Human LRG1 Gene

为了探讨LRG1基因结构与功能,对该基因进行克隆并构建到原核表达载体上,并对其进行表达及生物信息学分析。用Trizol法提取人肝癌HepG2细胞总RNA后,PCR扩增得到LRG1片段,经鉴定后将目的基因与原核表达载体pET28a连接,经诱导表达获得His-LRG1蛋白。LRG1基因cDNA片段大小为1 044 bp,编码347个氨基酸;成功构建pET28a原核表达载体,经多次不同条件诱导后,得到大小约40 kD目的蛋白;利用软件对LRG1蛋白的一级、二级结构进行了预测,分析总结得LRG1基因编码的蛋白是一个不稳定且具有亲水性的蛋白,可与多种信号开关相互作用。LRG1属于高度保守的富亮氨酸重复家族成员,其原核表达载体不易诱导产生大量目的蛋白,克隆表达该基因有利于验证其结构与功能关系。

In order to explore the structure and function of LRG1 gene,this gene was cloned and constructed to prokaryotic expression vector,and the expression and bioinformatics analysis were performed.The total RNA was extracted from HepG2 cells by Trizol method,and LRG1 fragment was obtained by PCR amplification.After identification,the target gene was connected to the prokaryotic expression vector pET28a,and His-LRG1 protein was obtained through induction expression.The cDNA fragment of LRG1 gene was 1 044 bp,which encoding 347 amino acids.The prokaryotic expression vector pET28a was successfully constructed,and the target protein was obtained with the size of about 40 kD after several inductions under different conditions.The primary and secondary structure of LRG1 protein were predicted by the software,and the results showed that LRG1 gene encoding protein was an unstable but hydrophilic protein,which could interact with a variety of signal switches.LRG1 belonged to highly conservative family members with leucine-rich repeat,and its prokaryotic expression vector was not easy to induce large amount of target proteins.Cloning and expression of the gene might be beneficial to verify the relationship between structure and function.