猪生长激素基因分泌型表达载体的构建及表达  

Construction of a Secretory Expression System for the Overexpression of Porcine Growth Hormone cDNA Gene in E. coli

作　　者：朱宝利[1] 黄涛[1] 贾锋[1] 周顺伍[1] 齐顺章[1] 

机构地区：[1]北京农业大学动物生物化学教研室,北京100094

出　　处：《农业生物技术学报》1993年第1期50-56,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金

摘　　要：为了使猪生长激素cDNA基因在大肠杆菌中的表达产物分泌到细胞周质中,本文采用了Kunkel报道的寡聚核苷酸指导的定点突变法,将猪生长激素cDNA基因与大肠杆菌外膜蛋白基因(ompA)的信号序列组装成嵌合基因,并在PL-PR双启动子的控制下进行诱导表达。结果表明表达产物占细菌总蛋白的20％左右;经western blot法证实有少量游离的猪生长激素被分泌到细胞周质中。透射电镜观察结果证明表达产物的大部分是以包涵体的形式存在,N-末端氨基酸分析结果表明表达产物的信号肽已被切除,但尚未证实表达产物形成的包涵体是在细胞的周质中,还是在胞液中。In order to construct a secretory expression vector for porcine growth hormone(pGH) production in E. coli, the pGH cDNA gene was firstly inserted downstream of the ompA signal sequence in piN Ⅲ ompA1. The undesired nucleotide sequence between the 3'-terminal codon(GCC) of the ompA signal sequence and the 5'-terminal codon(TTC) of the mature pGH gene was deleted using oligonucleotide mediated site-directed mutagenesis. The resulted ompA-pGH fused gene was cloned into the expression vector pJW2 by placing it under the control of P_L-P_R tandem promoter. Following transformation of E. coli strain JA221, high-level expression of pGH gene was acheived by inducing it at 42℃. The SDS-PAGE of the total bacterial protein showed that the expression product had a molecular weight similar to that of mature porcine growth hormone and the western blot analysis confirmed that it was truely pocine growth hormone. The expression efficiency was approximately 20% as estimated by gel scanning. The further analysis showed that the amount of expression products is considerably low in the periplasmic space, and the majority was found in form of inclusion bodies. However, the N-terminal aminoacid is an alanine as determined, indicating that the signal peptide had been cleaved off during the biosynthesis of the pGH.

关 键 词：猪 生长激素 基因表达 表达载体 

分 类 号：S828.2[农业科学—畜牧学]