CAJ | 학술논문

目的分泌表达风疹病毒E1-374糖蛋白并检测其免疫原性.方法将E1-374蛋白的cDNA插入表达载体pGAPZαA中构建出表达质粒pGAPZαA-E1-374,经BlnⅠ酶线性化后通过电转的方法导入毕赤酵母菌,博来霉素筛选阳性菌落.间接免疫荧光和Western Blot检测E1-374蛋白的表达及免疫反应性.免疫小鼠后应用间接ELISA检测风疹病毒IgG抗体.结果 SDS-PAGE和Western Blot显示E1-374蛋白的相对分子质量为46.89×103.ELISA法检测免疫小鼠的抗血清为阳性.建立的间接ELISA方法的最佳工作条件是抗原包被浓度为5.5μg/ml,血清最佳稀释度为1:100,板内变异系数值为0.36％～12.45％.结论 E1-374蛋白能够引起小鼠体液免疫应答,是风疹亚单位疫苗的重要候选成分,并可应用于风疹病毒IgG抗体的检测.
목적 분비표체풍진병독E1-374당단백병검측기면역원성.방법 장E1-374단백적cDNA삽입표체재체pGAPZαA중구건출표체질립pGAPZαA-E1-374,경BlnⅠ매선성화후통과전전적방법도입필적효모균,박래매소사선양성균락.간접면역형광화Western Blot검측E1-374단백적표체급면역반응성.면역소서후응용간접ELISA검측풍진병독IgG항체.결과 SDS-PAGE화Western Blot현시E1-374단백적상대분자질량위46.89×103.ELISA법검측면역소서적항혈청위양성.건립적간접ELISA방법적최가공작조건시항원포피농도위5.5μg/ml,혈청최가희석도위1:100,판내변이계수치위0.36％～12.45％.결론 E1-374단백능구인기소서체액면역응답,시풍진아단위역묘적중요후선성분,병가응용우풍진병독IgG항체적검측.
Objective To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein.Methods The cDNA of protein E1-374 was cloned into the expression vector pGAPZαA and transformed into Pichiapastoris GS1l5 cells by electrotransfection.The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot.Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein.Results SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 103.Antiserum (1:100) and E1-374 (5.5 μg/ml) was chosen for ELISA optimization.The intra-assay coefficient of variation for the ELISA was 0.36％-12.45％.Conclusion Protein E1-374 was highly expressed in Pichia pastoris cells,and it was a good choice to prepare rubella virus recombinant protein vaccines.