激光生物学报2013，Vol.22Issue(2)：147-153,7.DOI:10.3969/j.issn.1007-7146.2013.02.009
东方马脑炎E2蛋白原核表达及免疫活性初步研究
Prokaryotic Expression and Preliminary Immunogenicity of E2 Protein from Eastern Equine Encephalitis Virus
摘要
Abstract
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 desig nated 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.关键词
东方马脑炎病毒/原核表达/E2蛋白/免疫活性Key words
eastern equine encephalitis virus/prokaryotic expression/E2 protein/immunocompetence分类
生物科学引用本文复制引用
马金柱,王化磊,郑学星,吴红霞,薛向红,王铁成,杨松涛,夏咸柱..东方马脑炎E2蛋白原核表达及免疫活性初步研究[J].激光生物学报,2013,22(2):147-153,7.基金项目
公益性行业(农业)科研专项(201103032) （农业）
“十一五”国家科技支撑计划重点项目(2010BAD04B03) （2010BAD04B03）
