牛轮状病毒结构蛋白VP4基因的真核表达

Eukaryotic Expression of Bovine Rotavirus Structural Protein VP4 Gene

[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。

[Objective] 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 recombinant vector was confirmed by PCR,restricting enzyme digestion and DNA sequence.The recombinant plasmid was transfected into 293T cells through Lipofect2000TM.The expressions of VP4 gene were confirmed by RT-PCR technique and Western Blot.[Result] The recombinant plasmid pcDNA3.1（＋）-VP4 was constructed,the detection by RT-PCR and Western Blot indicated that the recombinant plasmid pcDNA3.1（＋）-VP4 has been constructed successfully and could express in 293T cells.[Conclusion] The recombinant 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.