论文部分内容阅读
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。
Objective To express and identify the active fragment of the major antigenic site of Hantaan virus nucleoprotein (NP) and glycoprotein G2, and lay the foundation for the eukaryotic expression of the fusion protein and the research of Hantaan virus genetically engineered vaccine. Methods The prokaryotic expression vector pGEX - 4T1-G2 S0 7 of chimeric fragment of Hantaan virus strain 76-118 and the 70 ’bp fragment of S gene was constructed. The fusion protein was induced and expressed by Western blotting and ELISA Identification. Results The results of enzyme digestion showed that prokaryotic expression vector pGEX - 4T1 - G2 S0 7 was successfully constructed. After IPTG induction for 4h, Western blotting showed that a GST-containing fusion protein (GST-G2 N0 7) with a molecular weight of about 100 kD was induced. The results of ELISA showed that the fusion protein had higher binding activity to anti-Hantaan virus NP-specific McAb (P / N value: 0 71/070) and weak binding to anti-Hantaan virus glycoprotein-specific McAb Activity (P / N value 0 2 1/0 0 8). Conclusion The splicing of S and M genes is successful, and the fusion protein of active glycoprotein of Hantaan virus and NP fragment can be expressed.