[目的]克隆获得牛轮状病毒VP4全基因,并进行真核表达.[方法]采用RT-PCR技术,从牛轮状病毒总RNA中扩增出VP4基因,将其重组到含有Flag标签的pcDNA3.1(+)载体中,经PCR、酶切和测序鉴定正确后,通过Lipofect2000TM转染293T细胞,细胞增殖后经RT-PCR和Western Blot检测VP4基因的表达.[结果]获得了VP4基因的阳性重组真核表达载体pcDNA3.1(+)-VP4;转染293T细胞后经RT-PCR和Western Blot检测表明,该基因的重组表达载体构建正确,可在293T细胞内瞬时表达.[结论]VP4基因的重组真核表达载体构建成功并可在真核表达细胞内表达,该研究为VP4基因的DNA疫苗的研发及应用奠定了基础.%[ Qbjective ] To achieve the successful cloning and eukaryotic expression of the full-length VP4 gene of bovine rotavirus. [ Method ] VP4 gene was amplified from the total RNA in bovine rotavirus by RT-PCR technique, then cloned into pcDNA3. 1 ( + ) vector with Flag TAQ. The reeombinant vector was confirmed by PCR, restricting enzyme digestion and DNA sequence. The recorabinant plasmid was trans-fecled into 293T cells through Lipofect2000? The expressions of VP4 gene were confirmed by RT-PCR technique and Western Blot. [ Result] The reeombinant plasmid pcr)NA3. l( + )-VP4 was constructed, the detection by RT-PCR and Western Blot indicated thai the reeombinant plasmid pcDNA3.1( + )-VP4 has been constructed successfully and could express in 293T cells. [Conclusion] The reeombinant vector containing VP4 gene was constructed successfully and could express in eucaryotic cells. The study lays foundation for the development and application of DNA vaccine of VP4 gene.
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