为构建东方马脑炎病毒E2基因原核表达载体, 完成E2蛋白表达及其免疫活性研究。利用PCR方法扩增E2编码全基因, 大小为1 260 bp, 将酶切后目的片段连接到原核表达载体pET-30a(+)上, 构建成重组质粒pET30a(+)-EEEV-E2, 采用酶切和测序分析方法鉴定正确的重组质粒转化到大肠杆菌BL21中, 诱导E2蛋白表达, 并用SDS-PAGE电泳和Western-blotting分析和鉴定目的蛋白; 最后, 用纯化的E2蛋白免疫BALB/c小鼠, 小鼠随机分成4组: PBS对照组、弗氏佐剂对照组、E2蛋白免疫组和E2蛋白+弗氏佐剂免疫组, 每组小鼠免疫2次, 两次免疫间隔时间为14天, 免疫剂量均为100 μL/只; 小鼠初次免疫后第10天, 用细胞因子ELISA试剂盒检测血清中IL-6、IL-12与TNF-α的浓度, 加强免疫后第14天, 用EEEV的假病毒检测血清中E2蛋白抗体的中和作用。结果表明完成了E2基因的原核表达载体pET30a(+)-E2构建和成功表达了带有His标签的E2融合蛋白, 蛋白以包涵体形式存在, 大小为53.0 kDa; 免疫小鼠血清中产生了高水平的IL-6、IL-12与TNF-α和具有较强中和作用的E2蛋白抗体。研究结果为今后E2蛋白作为基因工程亚单位疫苗的研究提供了重要参考。

In order to construct the prokaryotic expression vector of E2 gene from Eastern equine encephalitis virus and to express E2 protein, the PCR technique amplified the E2 gene encoding the whole protein by the template of the recombinant pFastBacTM1-C-E vector. The enzyme-digested PCR product was inserted into pET30-a(+) vector, here designated as pET30-a(+)-EEEV-E2. After the pET30a(+)-EEEV-E2 vector was exactly identified by the restriction enzyme digestion and the sequence analysis, it was transformed into competence E. Coli BL21 (DE3). The recombinant bacterium expressed E2 protein by IPTG inducing, the SDS-PAGE and Western-blotting assay were used to test the bacterial lysates. Finally, the mice were immunized twice purified E2 protein, the BALB/c mice were randomly divided into PBS groups, freund’s adjuvant groups, E2 protein groups, E2 protein emulsified with freund’s adjuvant groups. The BALB/c mice were immunized two times with 100 μL dose by intramuscular injection of back, 14 days between the two immunes. On the tenth day after the primary immune, the ELISA kit was used to quantitatively analyz the concentration of IL-6、IL-12 and TNF-α from mice sera, on the fourteenth day after the secondary immunization, neutralization of antibody against E2 protein from mice sera was detected by pseudovirus of EEEV. The results indicated the prokaryotic expression vector pET-30a (+)-EEEV-E2 was successfully constructed, and the E2 protein was expressed in the form of inclusion bodies in E.Coli. The concentration of IL-6、IL-12 and TNF-α of experimental groups were of significant difference compared with the control group in the whole experiment(P﹤0.01), and the antibodies from immunized mice sera were of effective neutralization function, which could provide with favorable foundation for E2 protein as a genetic engineering subunit vaccine.