森林脑炎病毒包膜E蛋白的原核表达、纯化及其免疫原性初步评价

目的原核表达及纯化森林脑炎病毒(tick-bome encephalitisvirus,TBEV)包膜E蛋白,并初步评价其免疫原性。方法采用RT-PCR法扩增TBEV E蛋白基因,克隆至载体pET-28a,构建重组原核表达质粒pET-28a-TBEV E,转化感受态E.coli BL21(DE3),挑取阳性克隆,扩增后经终浓度为0.1 mmol/L的IPTG诱导,表达产物经12%SDS-PAGE及Western blot鉴定。将鉴定正确的重组蛋白采用镍柱亲和法纯化,铝佐剂吸附后,经小鼠左侧腹腔注射,0.5 mL/只,间隔1周加强免疫1次,末次免疫后7、14、21、28、35、42、49 d,经小鼠眼眶采血,分离血清,ELISA法测定小鼠血清抗体水平。结果经双酶切及测序鉴定,重组质粒pET-28a-TBEV E构建正确。表达产物相对分子质量约55000,主要以包涵体形式表达,表达量约22.3%,且可与小鼠抗TBEV血清发生特异反应,纯化后纯度达90%。末次免疫后21 d,小鼠血清抗体效价达最高,为1∶6400。结论经原核表达系统成功表达并纯化了TBEV E蛋白,该蛋白具有较好的免疫原性,本实验为TBEV诊断制剂及基因工程疫苗的研发奠定了基础。

Prokaryotic expression,purification and immunogenicity of tick-borne encephalitis virus envelope protein E

Objective To express tick-borne encephalitis virus(TBEV)envelop protein E in prokaryotic cells and evaluate its immunogenicity.Methods The E gene of TBEV was amplified by RT-PCR and inserted into vector pET-28 a.The constructed recombinant plasmid pET-28 a-TBEV E was transformed to competent E.coli BL21(DE3),and positive clones were amplified and induced with IPTG at a final concentration of 0.1 mmol/L.The expressed product was identified by 12%SDS-PAGE and Western blot,purified by nickel ion affinity column chromatography and adsorbed with aluminum adjuvant,with which mice were injected by intraperitoneal route,0.5 mL for each,and boosted one week later.Serum samples of mice were collected 7,14,21,28,35,42 and 49 d after booster immunization and determined for antibody level by ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28 a-TBEV E was constructed correctly.The expressed product,with a relative molecular mass of about 55000,mainly existed in a form of inclusion body and contained about 22.3%of total somatic protein,which showed specific reaction with mouse antiTBEV sera and reached a purity of 90%after purification.The serum antibody titer of mice 21 d after booster immunization was 1∶6400.Conclusion TBEV E protein was successfully expressed in prokaryotic expression system and purified,which showed good immunogenicity.It laid a foundation of development of diagnostic kit for TBEV and genetic engineering vaccine.