CAJ | 학술논문

目的通过对猪带绦虫谷胱甘肽转移酶GST的表达,对其免疫性进行初步研究.方法利用生物信息学从猪带绦虫成虫cDNA质粒文库中筛选出谷胱甘肽转移酶(GST)基因,将其克隆到原核表达载体pET28a(+),经过双酶切、PCR鉴定后,异丙基-β-D半乳糖苷(IPTG)诱导表达,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,重组后的蛋白用His-镍蛋白纯化柱纯化,纯化的重组蛋白用蛋白印迹进行免疫学分析,Western blotting鉴定该蛋白免疫反应性.结果成功构建了重组体,并得到高纯度蛋白,该蛋白可被感染猪带绦虫的病人及猪、感染牛带绦虫病人及感染亚带绦虫病人血清所识别.结论猪带绦虫谷胱甘肽转移酶可在原核系统中获得具有免疫反应性的高效表达.
목적 통과대저대조충곡광감태전이매GST적표체,대기면역성진행초보연구.방법 이용생물신식학종저대조충성충cDNA질립문고중사선출곡광감태전이매(GST)기인,장기극륭도원핵표체재체pET28a(+),경과쌍매절、PCR감정후,이병기-β-D반유당감(IPTG)유도표체,병통과십이완기광산납-취병희선알응효전영(SDS-PAGE)감정,중조후적단백용His-얼단백순화주순화,순화적중조단백용단백인적진행면역학분석,Western blotting감정해단백면역반응성.결과 성공구건료중조체,병득도고순도단백,해단백가피감염저대조충적병인급저、감염우대조충병인급감염아대조충병인혈청소식별.결론 저대조충곡광감태전이매가재원핵계통중획득구유면역반응성적고효표체.
In the present study, the prokaryotic expression of glutathione transferase (GST) gene from Taenia solium and its immunological properties were investigated by means of biological informatics methods. The GST gene from T.solium was screened from the cDNA plasmid library of the adult worms. This gene was cloned into prokaryotic expression plasmid pET28a(+) and then expressed in E.coli BL21(DE3) after double enzyme digestion, PCR identification and IPTG induction. The expressed product was identified by SDS-PAGE and the recombinant protein was purified through purification column of His-Ni~(2+) protein. Meanwhile, the immunoreactivity of the purified protein was analyzed by Western blot assay. In these ways, the recombinants were successfully constructed and the highly purified proteins were obtained. It was demonstrated that these proteins could be recognized by sera of patients infected with T.asitica and T.rhynchus saginatus. From these observations, it is evident that highly efficient expression of GST of Taenia solium with definite immunoreactivity can be demonstrated in the prokaryotic expression system.