摘要
提取鸭肝炎病毒RNA作为模板,进行RT-PCR扩增,得到723 bp的VP3基因,将纯化的VP3基因片段与pMD18-T载体进行连接,构建DHVVP3基因克隆重组质粒。然后定向插入到pET-32a(+)表达载体,筛选获得原核表达载体pET-32a-VP3。用IPTG诱导表达,经SDS-PAGE电泳和Western blot分析表明,VP3蛋白得到正确表达,其分子量为47.1 ku,表达的蛋白能与鸭肝炎阳性血清发生特异性反应。
The VP3 gene of duck hepatitis virus（DHV） was amplified by reverse transcription - polymerase chain reaction（RT - PCR） yielding a product of 723 bp cDNA fragment. Using T - A cloning technique, the PCR product was cloned into pMD18 - T and pET - 32a（ ＋ ） vectors to obtain recombinant plasmid pET - 32a- VP3. Analyzed by SDS- PAGE and Western blot, VP3 protein was expressed in Escherichia coli BL21 （ DE3 ） at a high level after being induced with Isopropylthio - β - D - galactoside （IPTG）. The fusion protein had a molecular mass of approximately 47.1 ku and could react with positive serum against DHV.
出处
《山东农业科学》
2008年第8期14-16,20,共4页
Shandong Agricultural Sciences
基金
山东省自然基金项目(Z2006D06)
关键词
鸭肝炎病毒
VP3基因
克隆
表达
Duck hepatitis virus
VP3 gene
Cloning
Expression
