摘要
[目的]研究猪的轮状病毒VP4蛋白基因在BL21大肠杆菌中的表达。[方法]以猪肺脏组织中分离的总RNA为模板,采用反转录聚合酶链式反应(RT-PCR)技术扩增VP4蛋白的部分基因,将PCR产物克隆到表达载体pGEX-4T-1中,构建原核表达质粒PGEX-4T-1-VP4并对重组质粒进行酶切和测序鉴定。将重组载体PGEX-4T-1-VP4转化至大肠杆菌BL21(DE3)中进行诱导表达和SDS-PAGE分析,并对重组大肠杆菌进行可溶性分析。[结果]对猪肺脏组织中分离的总RNA进行RT-PCR扩增,获得875 bp的VP4部分基因,重组质粒PGEX-4T-1-VP4经BamHⅠ和XhoⅠ双酶切和测序鉴定后,插入的猪轮状病毒VP4的cDNA编码区序列与已公布的猪GenBank核酸序列比对结果为99.5%;将重组质粒pGEX-4T-1-VP4-转化至大肠杆菌BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约58 kDa融合蛋白带。[结论]该研究为进一步研究轮状病毒的疫苗研制奠定了基础。
[ Objective ] The aim was to study the expression of porcine rotavirus VP4 in Escherichia coli BI21. [ Method] With the total RNA isolated from porcine lung tissues as the template, the partial genes of VP4 protein were amplified by using reverse transcription-polymerase chain reaction （RT-PCR）. PCR products were cloned into the expression vector pGEX-dT-1 to construct the prokaryotic expression plasmid PGEX-4T-1-VP4 and the recombinant plasmid was made for enzyme digestion and sequencing. The recombinant vector PGEX-dT-1-VP4 was transformed into E. coli BL21 （DE3） for induction expression and SDS-PAGE analysis. The solubility analysis of recombinant E. coli was carried out. [ Result] The partial genes of VP4 protein about 875 bp were obtained through RT-PCR amplification on the total RNA isolated from porcine lung tissues. After the recombinant plasmid PGEX4T-1-VP4 was made for double enzyme digestion by BamH I and Xho I and sequenced, the cDNA coding sequence inserted in the porcine rotavirus VP4 had 99.5% similarity to the published nucleic acid sequence of porcine in GenBank. The recombinant plasmid pGEX4T-1-VP4 was transformed into E. coli BL21 （DE3） and induced to express by IPTG. The result of SDS-PAGE showed that the soluble fusion protein with molecular weight of 58 kDa was observed on the SDS-PAGE. [ Conclusion] The study laid the foundation for the further study on vaccine development of porcine rotavirus VP4. K
出处
《安徽农业科学》
CAS
北大核心
2009年第22期10431-10432,10448,共3页
Journal of Anhui Agricultural Sciences
基金
上海市浦江人才计划(PJ[20])
关键词
轮状病毒
VP4基因
克隆
原核表达
Porcine rotavirus
VP4 gene
Cloning
Prokaryotic expression
作者简介
肖长峰（1983-），男，山东淄博人，硕士研究生，研究方向：动物营养及病毒微生物。
通讯作者，研究员。
