牛轮状病毒VP4基因与LTB基因的融合表达及免疫原性研究

PCR扩增轮状病毒VP4基因和大肠杆菌LTB基因编码区,将其克隆至pET32a载体上,构建pET32a-VP4-LTB质粒,转化大肠杆菌BL21(DE3),PCR、酶切鉴定后进一步测序,测序结果表明融合基因片段由2637 bp组成,为编码879个氨基酸残基的多肽。经IPTG诱导和SDS-PAGE检测发现表达产物分子量约为118.9 KD,以包涵体的形式存在。融合蛋白经镍离子螯合次氨基三乙酸(Ni-NTA)亲和层析纯化后免疫小鼠,结果所产生的抗体效价高于单独VP4蛋白的免疫结果但差异不显著(P0.5),但与对照组相比差异显著。表明所表达的融合蛋白具有较好的免疫原性,为研制牛轮状病毒亚单位疫苗奠定了基础。

Construction ,Expression and Immunogenicity of Fusion Gene VP4 Gene of Bovine Rotavirus and LTB Gene

The coding region of rotavirus VP4 gene and LTB gene was obtained by PCR amplification. The plasmid pET32a -VP4-LTB containing VP4-LTB fusion gene was constructed by cloning VP4 and LTB gene into pET32a, which was further confirmed by restriction enzyme digestion and nucleotide sequence. SDS-PAGE analysis demonstrated that the recombinant plasmid could express the VP4-LTB fusion protein in form of inclusion body , and its molecular weight was around 118.9 KD. The fusion protein of VP7-LTB and VP7 were purified by Ni-NTA. Vaccine were prepared and immune to mouse.The result showed that the antibody tilter of fusion protein VP4-LTB was higher than that of VP4, but the difference was not significant(P>0.5), compared with control group there was significant difference (P<0.1).The results demonstred that VP4-LTB fusion protein had a better immunogenicity and could act as a candidate vaccine against bovine rotavirus infection.