汉滩病毒M、S基因部分片段嵌合基因真核载体的构建与基因免疫

Construction and genetic immunization of eukaryotic expression vector containing chimeric competent frag-ments of Hantaan virus M and S segments

目的在本室前期工作的基础上,进一步进行汉滩病毒M基因G2片段与S基因0.7Kb片段嵌合基因基因免疫的研究。方法构建汉滩病毒76-118株M基因G2片段与S基因5′端700bp片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7。用该质粒免疫Balb/c小鼠,并用ELISA法及淋巴细胞增殖实验,检测基因免疫后的免疫应答效果。结果限制性内切酶鉴定结果表明真核表达载体的构建正确。用pcDNA3.1-G2S0.7直接免疫小鼠,可诱导产生抗汉滩病毒核蛋白NP及糖蛋白GP特异性的抗体,抗体效价分别为1200及180。淋巴细胞增殖实验表明,嵌合基因免疫小鼠脾细胞对NP及GP的增殖指数,均明显高于对照组。结论汉滩病毒M基因G2片段及S基因0.7kb片段的嵌合基因,既可刺激机体产生特异性的抗汉滩病毒体液免疫应答,也可刺激机体产生特异性的细胞免疫应答,本研究为进一步进行汉滩病毒基因疫苗的研究奠定了实验基础。

Aim To investigate the expression and im muno -genicity of re-combinant eukaryotic expression ve ctor containing chimeric gene of the G2fragment of Hantaan virus M segment a nd 0.7Kb fragment of S segment.Methods Recombinant eukaryotic expression vector pcDNA3.1-G2S0.7was constructed by cloning chimeric gene containing G2fragment of M segment and 0.7Kb fragment of S segme nt into pcDNA3.1(+).Then Balb /c mice were immunized by the pcD NA3.1-G2S0.7.ELISA was used to detect the sera antibody tite rs of immunized mice.Meanwhile,the stimulation index(SI )of spleencytes to nucleoprotein(NP)and glycopro-tein(GP)were detected by MTT method.Results Restriction enzyme analysis showed that eukaryotic exp ression vector pcDNA3.1-G2S0.7was successfully constructed.ELISA re sults revealed that the pcDNA3.1-G2S0.7could induce immunized mice t o produce specific antibody against NP and GP.MTT results showed that the SI of immunized mouse spleen-cytes to NP and GP were significantly higher than that of control mice.Conclusion It is suggested that the chimeric gen e of Hantaan virus con-taining G2fragment of M segment and 0.7Kb fragment of S segment can directly stimulate Balb /c mice to pr oduce specific humoral immunity and cellular immunity in against Hantaan virus.