中文摘要：

以猪A组轮状病毒mRNA为模板，应用优化的反转录聚合酶链式反应（RT—PCR）技术，扩增了2331 bp的Vp4全长基因，通过T-A克隆技术，将PCR产物克隆至克隆载体pGEM-T中，构建质粒pGEM-T-Vp4。以SalⅠ和KpnⅠ双酶切pGEM—T—Vp4．和以胸苷酸合成酶基因（thymidylate synthase，thyA）为选择压力的非抗生素抗性的穿梭表达载体pW425t，并将纯化的Vp4基因亚克隆至表达载体pW425t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pW425t-Vp4。将pW425t-Vp4转化至thyA基因缺陷型的大肠杆菌感受态E．coli X13中，通过生长功能弥补筛选阳性克隆，SDS-PAGE分析，可见约87.67Ku融合蛋白。Western blot分析表明，该蛋白具有与轮状病毒多克隆抗体的反应原性，研究结果为pW425t-Vp4在乳酸菌受体菌株中表达提供科学依据。

英文摘要：

The full Vp4 gene of porcine rotavirus A was amplified from MA 104 cell infected with rotavirus by the reverse transcription-polymerase chain reaction （RT-PCR）, the product was a 2 331 bp eDNA fragment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Recombinant plasmid pGEM-T-Vp4 and pW425t, the prokaryotic expression shuttle vector between E.coli and Lactobacillus, was digested with Sal Ⅰ and Kpn Ⅰ enzymes respectively. The purified Vp4 gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-Vp4 was constructed, and then was transformed into the competence thyA gene-mutant E, coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, and approximately 87.67 Ku fusion protein was observed. The protein was further analyzed using Western blot, the result indicated that the protein was reactive with the antibody of rotavirus A. This result lay the foundation for expression of recombinant pW425t-Vp4 in lactobacillus.