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

作　　者：马金柱[1,2,3] 王化磊[2] 郑学星[2] 吴红霞[2] 薛向红[2] 王铁成[2] 杨松涛[2] 夏咸柱[2,3] 

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《激光生物学报》2013年第2期147-153,共7页Acta Laser Biology Sinica

基　　金：公益性行业(农业)科研专项(201103032);"十一五"国家科技支撑计划重点项目(2010BAD04B03)

摘　　要：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100μL/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 recom-binant 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 en- zyme 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 bacte-rial 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 ~L 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 anti-body against E2 protein from mice sera was detected by pseudovirus of EEEV. The results indicated the prokaryotic ex-pression 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 differ-ence 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 engi-neering subunit vaccine.

关 键 词：东方马脑炎病毒 原核表达 E2蛋白 免疫活性 

分 类 号：Q78[生物学—分子生物学]