摘要：
为制备具有免疫原性的猪瘟病毒E2蛋白,在大肠杆菌中表达E2蛋白,并对其进行纯化及免疫原性分析.将E2基因插入原核表达载体pET-28a,构建重组质粒pET-28a-E2,将其转化至大肠杆菌BL21(DE3)感受态细胞进行诱导表达,SDS-PAGE电泳和Western blot分析结果显示,成功表达了可溶性E2蛋白.对IPTG浓度及诱导温度和时间进行优化,结果显示,IPTG终浓度为0.1 mmol/L时,18 °C条件下诱导表达16 h,E2蛋白的可溶性表达量最高.通过分子筛一步纯化E2蛋白,其终质量浓度为0.5 g/L.Western blot分析和间接ELISA结果显示,纯化后的E2蛋白在变性及非变性条件下都能够被猪瘟阳性血清特异性识别,表明纯化后的E2蛋白活性良好.将纯化后的E2蛋白添加弗氏佐剂乳化后免疫BALB/C小鼠,采集血清进行病毒中和试验,结果显示,免疫后56 d 产生较高水平的中和抗体,表明E2蛋白免疫原性良好.综上,成功利用大肠杆菌BL21(DE3)菌株表达了可溶性E2蛋白,且纯化后的E2蛋白免疫原性良好.%The objective of this study was to obtain the immunogenic classical swine fever virus(CSFV)E2 protein using E. coli expression system,and then get the purified E2 protein,followed by estimation of its immunogenicity. The E2 gene was inserted into prokaryotic expression vector pET-28a to construct recombinant plasmid pET-28a-E2.The plasmid was then transformed into E. coli BL21(DE3) competent cells for expression. SDS-PAGE and Western blot results showed that E2 protein was successfully expressed in the cells and proved to be soluble.Through the optimization of IPTG concentration,induction temperature and time,it was found that the highest soluble expression of E2 protein was achieved at 18°Cafter induction with 0. 1 mmol/L IPTG for 16 hours. After one-step purification of size exclusion chromatography,the E2 protein was purified and the final concentration was 0.5 g/L.Western blot and indirect ELISA results showed that the purified E2 protein with or without denaturation could be specifically recognized by the CSFV positive serum,indicating that the purified E2 protein was of good activity.The purified E2 protein was used to immunize BALB/C mice after emulsified with Freund's adjuvant.Blood samples were collected at different time points for virus neutralization experiment. The results showed that the neutralizing antibody was detected after 56 days of immunization,indicating that the E2 protein had good immunogenicity. In conclusion,soluble envelope glycoprotein E2 could be expressed in E.coli BL21(DE3) competent cells,which represented good immunogenicity after purification.
