摘要
根据GenBank中已发表的水貂阿留申病毒(ADV)VP2基因核苷酸序列分别设计合成两对引物,用PCR方法扩增ADV国内分离株VP2基因中主要抗原表位区的两个片段,分别将其克隆到原核表达载体pMAL-c2的多克隆位点中。经酶切、PCR扩增和测序分析证实其已正确插入到表达载体中,且阅读框是正确的,构建原核表达载体pMAL-VPa和pMAL-VPb。阳性重组质粒转化宿主菌TB1,用IPTG进行诱导表达,对表达产物进行SDS-PAGE检测和免疫印迹分析。结果表明两段蛋白均获得了表达,表达产物的分子质量分别约为63、65kD,与理论推测的分子质量一致;并在终浓度为1mmol/L的IPTG诱导下,4h时其表达量达到高峰;Western blot分析表明表达蛋白能被兔抗MBP抗体所识别,具有一定的抗原性。
Two pairs of primers were designed according to the complete genome sequences of Aleutian mink disease parvovirus （ADV） published in GenBank. Two main antigen domains of VP2 gene of ADV strain were amplified by PCR. The amplified DNA products were respectively cloned into the multiple cloning sites of prokaryotic expression vector pMAL-c2. The insert position, the size and the reading frame were confirmed by restriction digestion, PCR and sequence analysis. It was indicated that the prokaryotic expression vectors pMAL-VPa and pMAL-VPb were constructed. The positive recombinant vectors were transformed into recipient germs TB1 for expression by IPTG inducing. The expressed proteins were measured by SDS-PAGE and Western blot. Result showed that both of the two protein fragments could be expressed successfully. The molecular weights of the expressed proteins were separately 63 and 65 KD which agreed with the theoretically presumed. Induced by IPTG at a final concentration of 1 mmol/L, the expressed protein reached the highest quantity after 4 hours of induction. The Western blot results indicated that the expressed antigen proteins could be recognized by the rabbit anti-MBP antiserum and had certain immunoreactivity.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2007年第6期39-41,共3页
Journal of Northeast Forestry University
基金
黑龙江省"十五"科技攻关项目(GB02B506)
关键词
水貂阿留申病毒
VP2基因
抗原表位
原核表达
Aleutian mink disease parvovirus
VP2 genes
Epitope
Prokaryotic expression
