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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 construct prokaryotic expression vector of full-length chikungunya virus envelope protein E2 (CHIKV-E2,1-404 aa) and its trans-membrane hydrophobic region (351-378 aa) deletion mutant E2 (1-350 aa) The effect of transmembrane hydrophobic region on E2 protein expression in E. coli was analyzed. Methods ExPasy prediction software was used to predict the transmembrane hydrophobic region of E2 protein. The corresponding gene sequence was obtained from the CHIKV-E2 amino acid sequence in GenBank database. The full-length CHIKV-E2 gene (404aa) was synthesized in vitro by nucleic acid primers designed for whole-genome synthesis based on OE-PCR. The full-length E2 protein and prokaryotic expression plasmid of its deletion mutant were constructed. The correct two recombinant plasmids were transferred to E. coli BL21 (DE3), induced by IPTG, and the expression of the recombinant plasmid was detected by SDS-PAGE. Results The full-length cDNA encoding CHIKV-E2 (1 ~ 404 aa) with a size of 1 212 bp was successfully synthesized by OE-PCR and the full-length E2 protein and its recombinant plasmid pET21b- 404) and pET21b-E2 (1 ~ 350). After induced by IPTG, SDS-PAGE showed that the expression level of pET21b-E2 (1 ~ 350) fusion protein was significantly higher than that of pET21b-E2 . Conclusion The transmembrane hydrophobic region of E2 protein (351-378 aa) has an important influence on the prokaryotic expression of this protein. The mutant lacking this hydrophobic region has a significantly higher expression level of E2 protein in Escherichia coli than the full-length E2 protein.