西尼罗病毒多表位基因的融合表达及其免疫保护作用研究

Fusion expression of multiepitope of West Nile virus and its immunoprotection

目的研究西尼罗病毒（WNV）多表位基因的融合表达及其免疫保护作用。方法利用生物信息软件Biosun分析西尼罗全基因序列，确定表位基因片段并选择合适片段连接构成多表位基因。采用重叠PCR方法扩增该基因，构建多表位基因的原核重组表达载体pET-43a-M，进行融合表达和纯化。将表达蛋白加弗氏佐剂免疫小鼠并进行攻毒试验，观察其保护作用。结果分子克隆和构建了原核重组表达质粒pET-43a-M，表达和纯化了融合蛋白Nus-M，该蛋白免疫后的小鼠能够部分抵御西尼罗病毒的攻击。结论构建的西尼罗病毒多表位基因能够在原核细胞内表达，表达产物有较弱的免疫保护反应，为该病毒多表位抗原的串联及多表位疫苗研究提供了试验资料，但需要进一步改进。

Objective To investigate the fusion expression of the multiepitope of West Nile virus （WNV） and its immtmoprotection. Methods The whole gene of WNV was analyzed with bioinformation software Biosun and the potential epitope genes were confirmed. The most possible genes were chosen and the multiepitope was constructed through linkers. The multiepitope was amplified by PCR. The PCR products was cloned into pET-43a（＋） vector, and the vector was transformed into E. coli BL21 cells and the multiepitope fusion protein was expressed. The multiepitope fusion protein was analyzed by Western blot with serum against WNV. To observe the immunoprotection of the recombinant proteins against WNV challenge, the Nus-M were purified and hypodermically injected into mice with Fretmd＇s adjuvant. Different groups of vaccinated mice were intracerebrally with WNV at a dose of 100 LD50. Results The recombinant protein can be expressed in eukaryotic ceils and induce immtmopro-tection in mice. Conclusion The recombinant orotein can induce a feeblish immune response against WNV.