摘要
目的探讨提高狂犬病病毒(RV)HEP-Flury株糖蛋白(GP)基因在原核细胞中表达的水平和与抗体的反应水平。方法以RVHEP-Flury株的全长质粒为模板,采用PCR方法扩增获得去除信号肽基因的GP(G1)、GP的膜外区基因(G2)。选取膜外区基因中抗原表位富集区基因,对氨基酸密码子进行修饰并合成其序列(G3)。分别将G1、G2及G3基因亚克隆到表达载体pET32a(+),构建融合表达质粒pET32a-G1、pET32a-G2及pET32a-G3,转化表达宿主菌BL21,并利用IPTG诱导表达。经SDS-PAGE,Western-blotting和薄层扫描分析,比较重组蛋白的表达量。结果 G3基因的表达量比G1、G2基因明显提高。结论通过密码子优化,能显著提高G基因在原核细胞中的表达水平。
The aim of this study was to improve prokaryotic expression level of glycoprotein （GP） gene in rabies virus. With the templet of full-length gene plasmid of rabies virus HEP-Flury strains,the gene without signal peptides （G1） and the extra-membrane gene （G2） were obtained by PCR respectively. The codons of glycoprotein gene＇s antigen epitope were modified and the enrich epitope gene was synthesized in order to get the gene G3 and match with the expression system of prokaryotic cell. The gene G1,G2 and G3 were sub-cloned into prokaryotic expression plasmid pET32a （＋） respectively. After these three recombinant plasmids were transformed to BL21,the bacteria were induced by IPTG. The expression proteins were detected with SDS-PAGE,Western -blotting test and thin layer scanning. Results showed that the expression level of gene G3 was notably higher than that of gene G1 and gene G2. And the expression of glycoprotein gene in E. coli could be successfully improved by codon modification.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第5期403-407,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金(30671565)
教育部长江学者和创新团队发展计划(IRT0723)
关键词
狂犬病病毒:糖蛋白基因
基因修饰
原核表达
rabies virus
glycoprotein gene
gene modification
prokaryotic expression
作者简介
通讯作者：郭霄峰，Email：xfguo@scau．edu．cn
