CAJ | 학술논문

目的利用枯草芽孢杆菌系统表达肠道病毒71型(EV71)病毒VP1蛋白.方法使用基因特异性引物扩增VP1开放读码框(ORF),构建包含VP1完整开放读码框的pSac-VP1穿梭载体.根据枯草芽孢杆菌表达系统双交叉同源重组的特点,将EV71的结构抗原基因VP1整合在枯草芽孢杆菌sacA染色体,并经测序鉴定.使用VP1特异性抗体,通过蛋白印记(Western-Blot)鉴定枯草芽孢杆菌中VP1蛋白的表达情况.结果成功克隆EV71的VP1基因,长度1361 bp,并将其克隆入穿梭载体pSac-Kan.测序结果显示VP1基因成功整合如sacA染色体.Western-Blot证实VP1抗原在枯草芽孢杆菌的成功表达,相对分子质量约35×103.结论利用枯草芽孢杆菌表达系统成功制备EV71病毒VP1抗原,为后续EV71诊断试剂、疫苗研发奠定基础.
목적 이용고초아포간균계통표체장도병독71형(EV71)병독VP1단백.방법 사용기인특이성인물확증VP1개방독마광(ORF),구건포함VP1완정개방독마광적pSac-VP1천사재체.근거고초아포간균표체계통쌍교차동원중조적특점,장EV71적결구항원기인VP1정합재고초아포간균sacA염색체,병경측서감정.사용VP1특이성항체,통과단백인기(Western-Blot)감정고초아포간균중VP1단백적표체정황.결과 성공극륭EV71적VP1기인,장도1361 bp,병장기극륭입천사재체pSac-Kan.측서결과현시VP1기인성공정합여sacA염색체.Western-Blot증실VP1항원재고초아포간균적성공표체,상대분자질량약35×103.결론 이용고초아포간균표체계통성공제비EV71병독VP1항원,위후속EV71진단시제、역묘연발전정기출.
Objective To express the recombinant VP1 protein of enterovirus 71 in bacillus subtilis expression system.Methods We first amplified ORF of VP1 gene with specific primer and constructed pSac-VP1 vector.According to the characteristic of homologous recombination in bacillus subtilis,we integrated EV71 VP1 gene into bacillus subtilis sacA chromosome identified with sequencing.VP1 protein expression was identified with Western-Blot assay using specific antibody.Results VP1 gene fragment was amplified successfully with 1361 bp and inserted into pSac-Kan vector.After homologous recombination,the VP1 gene was integrated into bacillus subtilis sacA chromosome.It was proved that the recombinant VP1 proteins could be expressed in bacillus subtilis with molecular weight about 35 × 103.Conclusion We expressed recombinant VP1 of EV71 in bacillus subtilis system,it would benefit the diagnosis and vaccine research of EV71 in the future.