基孔肯雅病毒E2蛋白的原核表达及纯化

Prokaryotic expression and purification of E2 protein of Chikungunya virus

目的原核表达并纯化基孔肯雅病毒(Chikungunya virus,CHIKV)E2主要结构蛋白,为预防CHIKV感染及亚单位疫苗的研究奠定基础。方法根据Gen Bank上公布的CHIKV基因序列合成E2基因,并进行密码子优化,以提高原核表达效率。设计引物并以合成的E2基因为模板,PCR扩增CHIKV E2,连接到原核表达载体p ET-28a上,构建重组原核表达质粒p ET-28a-CHIKV E2,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后进行纯化。结果重组原核表达质粒p ET-28a-CHIKV E2经双酶切鉴定构建正确,测序结果与原序列一致。表达的CHIKV E2融合蛋白相对分子质量约为47 000,可与His标签抗体特异性结合,纯化后纯度达95%以上。结论成功在大肠埃希菌中表达了CHIKV E2蛋白,纯化后蛋白纯度较高,为CHIKV亚单位疫苗的免疫试验奠定了基础。

Objective To express the major structural protein E2 of Chikungunya virus（CHIKV）in prokaryotic cells and lay a foundation of prevention of CHIKV infection and development of subunit vaccine. Methods The DNA sequence of CHIKV E2 gene was synthesized according to the CHIKV gene sequence in Gen Bank,of which the codon was optimized to increase the prokaryotic expression efficacy. The CHIKV E2 gene was amplified by PCR with a pair of designed primers using the synthetic E2 gene as a template and inserted into vector p ET-28 a. The constructed prokaryotic expression vector p ET-28 a-CHIKV E2 was transformed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot then purified. Results Restriction analysis proved that recombinant plasmid p ET-28 a-CHIKV E2 was constructed correctly,of which the sequencing result was consistent with that of original sequence. The expressed fusion protein CHIKV E2,with a relative molecular mass of about 47 000,showed specific binding to anti-His tag antibody,and reached a purity of more than 95% after purification. Conclusion CHIKV E2 was successfully expressed in E. coli,which reached a high purity after purification. It laid a foundation of immunization test on CHIKV subunit vaccine.