汉滩病毒S、M基因部分片段嵌合基因原核载体的构建及表达产物的鉴定

目的　将汉滩病毒核蛋白 (NP)主要抗原位点活性片段与囊膜糖蛋白G2进行融合原核表达及鉴定 ,为该融合蛋白的真核表达及汉滩病毒基因工程疫苗研究打基础。方法　构建了汉滩病毒 76 - 118株M基因G2 片段与S基因 5’端70 0bp片段的嵌合片段原核表达载体pGEX - 4T1-G2 S0 7,诱导表达相应的融合蛋白 ,用Westernblotting和ELISA方法进行鉴定。结果　酶切结果显示成功构建了原核表达载体 pGEX - 4T1-G2 S0 7。IPTG诱导表达 4h后 ,Westernblotting显示诱导出分子量约 10 0kD的含GST的融合蛋白 (GST -G2 N0 7)。ELISA结果表明该融合蛋白与抗汉坦病毒NP特异性McAb有较高的结合活性 (P/N值为 0 71/ 0 0 7) ,与抗汉滩病毒糖蛋白特异性McAb也有较弱的结合活性 (P/N值为 0 2 1/ 0 0 8)。结论　S、M基因拼接方式是成功的 ,能够表达出有活性的汉滩病毒G2 糖蛋白与NP片段的融合蛋白。

Construction and identification of prokaryotic expression vector containing chimeric fragments of Hantaan virus S and M segments

Aim To establish the basis for further expressing the chimeric competent fragments of Hantaan virus M and S segments in prokaryotic system and provide a new method for developing Hantaan virus gene engineering vaccine Methods Prokaryotic expression vector pGEX 4T1 G 2S0 7 was constructed The fusion protein was expressed by inducing,Western blotting and ELISA methods were used to identify the activity of the protein Results Prokaryotic expression vector pGEX 4T1 G2S0 7 was proved to be successfully constructed by restriction enzyme analysis After having been induced with IPTG for 4 hours,Western blotting analysis showed that GST G 2N0 7 fusion protein about 100KD was induced ELISA results showed that the fusion protein had high combination activity with Hantaan virus nucleoprotein specific McAb(P/N value:0 71/0 07),and low combination activity with Hantaan virus glycoprotein specificMcAb(P/N value:0 21/0 08) Conclusion The prokaryotic expression of the fusion protein encoded by chimeric fragments of Hantaan virus S and M segments is successful