西尼罗病毒多表位基因免疫保护研究

目的探讨西尼罗病毒多表位基因诱导体液及细胞免疫的可行性及其免疫攻毒保护作用。方法将西尼罗病毒多抗原表位基因克隆到真核表达载体pcDNA3.1中,构建重组真核表达质粒pcDNA3.1-M。通过脂质体转染法将该多表位基因导入BHK细胞中,采用RT-PCR和IFA检测其在细胞内的瞬时表达。将重组质粒通过肌肉注射免疫BALB/c小鼠,通过间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验等,检测小鼠体内特异性体液免疫和细胞免疫的水平。在此基础上进行攻毒试验,分析重组质粒DNA对西尼罗病毒攻击的免疫保护作用。结果重组质粒pcDNA3.1-M能够在真核细胞内表达,采用IFA方法能在细胞内检测到西尼罗病毒特异性抗原。在抗原的刺激下,免疫后的小鼠脾淋巴细胞增殖显著,可诱导特异性CTL应答反应。攻毒试验显示,重组多表位基因能够对西尼罗病毒的攻击起到一定的保护作用,达到50%,中和抗体效价达到1∶50,应用免疫佐剂或应用重组蛋白加强免疫后,其保护率为62.5%,中和抗体效价达到1∶90。结论西尼罗病毒多表位基因可诱发特异性免疫应答,对西尼罗病毒感染具有保护作用,为其基因疫苗研制提供了一定的实验依据。

An investigation on the immuno-protection efficiency of immunization with West Nile Virus multiple epitope gene in mice

To explore the possibility using the multiepitope gene from West Nile Virus(MNV) as immunogen to induce the humoral and cell-mediated immunity and to investigate its protection efficiency against homologous challenges in mice,a sunthetic multiepitope gene was cloned into the eukaryotic expression vector pcDNA3.1 to construct the recombinant plasmid pDNA3.1-M,and then introducted to BHK cells through liposome transfection.The transient expression of products in cells was detected by RT-PCR and IFA assay.And the recombinant plasmid was purified before it was used as immunogen to immunize BALB/c mice.After intrmudcular immunization,the humoral and cell-mediated immunty of the immunized mice were assayed with indirect ELISA,cytotoxic effect of CTL and lymphocyte proliferation assay.Challenge with humologous virus was performed to observe the immuno-protectin effect of the recombinant plasmid gene against WNV.It was demonstrated that this recombinant plasmid pcDNA3.1-M could express efficiently in BHK cells.In these cells,the MNV-specific antigens could be detected by IFA assay.After immunization,marked proliferation of splenic lymphocytes and definite cytotoxic effect of CTL were observed.As demonstrated by the challenge test,this recombinant multiepiyope gene could confer a definite protection to the challenge with homologous virus.When it was used in combination with adjuvant,the rate of protection appeared to be 62.5% with the neutralizing antibody titer of 1∶50.From these observations,it is evident that the recombinant multiepitoprgene can induce specific humoral and cell-mediated immune responses after immunizying to mice and it also confer definite immunoprotection against the challenge with homologous virus.