基孔肯雅病毒包膜蛋白E2的全基因合成及原核表达

Gene synthesis,cloning,prokaryotic expression and analysis of chikungunya virus E2 glycoprotein

目的通过对全长基孔肯雅病毒包膜蛋白E2(CHIKV-E2,1～404 aa)及其跨膜疏水区(351～378 aa)缺失突变体E2(1～350 aa)进行原核表达,分析跨膜疏水区对E2蛋白在大肠杆菌中表达的影响。方法利用ExPasy预测软件对E2蛋白跨膜疏水区进行预测分析,根据GenBank数据库中CHIKV-E2氨基酸序列获得其对应的基因序列。结合重叠延伸PCR(OE-PCR)原理设计用于全基因合成的核酸引物对CHIKV-E2全长基因(404 aa)进行体外合成,构建全长E2蛋白及其缺失突变体原核表达质粒,将序列正确的两种重组质粒分别转至E.coli BL21(DE3),经IPTG诱导表达,SDS-PAGE检测重组质粒的表达情况。结果 OE-PCR法成功合成了大小为1 212 bp的编码CHIKV-E2(1～404 aa)蛋白的全长基因,构建了全长E2蛋白及其缺失突变体重组表达质粒pET21b-E2(1～404)和pET21b-E2(1～350),经IPTG诱导表达后,SDS-PAGE结果显示缺失突变体pET21b-E2(1～350)融合蛋白表达量较pET21b-E2(1～404)有明显提高。结论 E2蛋白跨膜疏水区(351～378 aa)对该蛋白的原核表达具有重要影响,缺失该疏水区的突变体在大肠杆菌中表达量比全长E2蛋白表达量明显提高。

Objective To analyze the impact of hydrophobic transmembrane domain of chikungunya virus E2 glycoproteins on E2 expression in E. coli by comparing E2 expression with the hydrophobic transmembrane domain-deleted mutant. Methods On-line software ExPasy was used to predict transmembrane domain of E2 protein and optimized gene sequence encoding E2 protein was obtained according to amino acids of the protein from GenBank ; Primers were designed to synthesize E2 gene according to the principle of OE-PCR and prokaryotie expression plasmids of full-length E2 protein and its mutant were constructed. After being transfromed into E. coli BL21 （ DE3 ）, two plasmids in E. coli were induced to express by IPTG and protein expression was identified by SDS-PAGE. Results In the study, the gene encoding CHIKV-E2 （1 - 404 aa） protein was synthesized by OE-PCR and two prokaryotic expression plasmids, and pET21 b-E2 （ 1 - 404 aa） and pET21 b-E2 （ 1 - 350 aa） were constructed successfully. The plasmids were induced by IPTG and SDS-PAGE, which showed that the production of the deletion mutant E2 （ 1 - 350 aa） was much higher than that of pET21 b-E2 （ 1 -404）. Conclusion The hydrophobic transmembrane domain （351 - 378 aa） of E2 protein plays an important role in E2 production in E. coli and production of the mutant with the domain deleted is significantly higher than that of full-length E2 protein.