猪A组轮状病毒Vp7基因与双标记表达载体pW425et的重组及在大肠杆菌中的表达

Expression of Recombinant Vp7 Gene of Porcine Rotavirus A with Double Labeling Expression Vector pW425et in Escherichia coli

以猪A组轮状病毒mRNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了981 bp的Vp7基因,通过T-A克隆技术,将PCR产物克隆至pGEM-T载体中,构建出克隆质粒pGEM-T-Vp7。以SacⅠ和KpnⅠ双酶切pGEM-T-Vp7及双标记表达载体pW425et,并将纯化的Vp7基因亚克隆至双标记表达载体pW425et中,构建出可以在乳酸菌与大肠杆菌之间穿梭表达的原核表达重组质粒pW425et-Vp7。将pW425et-Vp7转化至thyA基因缺陷型的大肠杆菌感受态Escherichia coliX13中,经生长功能弥补筛选阳性克隆和SDS-PAGE分析,可见约40 kD的融合蛋白。Western blot分析表明该蛋白具有与猪轮状病毒多克隆抗体的反应原性。

Vp7 gene of Porcine Rotavirus A was amplified by the reverse transcription-polymerase chain reaction （RT - PCR）, the product of which was an approximately 981bp cDNA segment. The PCR product was cloned into pGEM-T vector, by T-A cloning technique. Cloning plasmid pGEM-T-Vp7 and the double labeling expression vector pW425et were digested by Sac Ⅰ and Kpn Ⅰ double enzymes respec- tively. The purified Vp7 gene was subcloned into the vector pW425et. Thus, the recombinant pW425et- Vp7 was constructed, which then was transformed into the competence thyA gene-mutant E. coli X13. Treated lysates of bacterium were loaded directly onto SDS - PAGE, on which approximately 40 kD fusion protein was observed. The protein was further analyzed by Western blot, which indicated that the protein was reactive with the antibody of Rotavirus A. The results lay fotmdation for further studies on the expres- sion in the lactobacillus.