CAJ | 학술논문

目的构建牛分枝杆菌 Mb2277基因的原核表达载体,获得高纯度的融合表达蛋白,并对其表达产物的免疫原性进行初步研究.方法根据GenBank提供的M. Bovis Mb2277基因序列设计引物.以牛分枝杆菌(93006)DNA为模板,通过PCR方法扩增出Mb2277基因片段,以BamHⅠ和EcoR Ⅰ双酶切PCR产物和pET28a(+),后将纯化的Mb2277基因克隆至pET28a(+)中,构建出原核表达质粒,再转化到E.coli BL21(DE3)表达菌株中, IPTG诱导后,收集菌体进行SDS-PAGE,进一步利用Western Blot对表达产物的反应原性进行研究.结果获得了牛结核分枝杆菌Mb2277原核表达质粒,IPTG诱导表达后,SDS-PAGE分析可见约25ku蛋白表达条带,Western Blot结果显示,表达产物与兔抗分枝杆菌抗体具有反应性.结论该蛋白具有较强的反应原性.
목적 구건우분지간균 Mb2277기인적원핵표체재체,획득고순도적융합표체단백,병대기표체산물적면역원성진행초보연구.방법 근거GenBank제공적M. Bovis Mb2277기인서렬설계인물.이우분지간균(93006)DNA위모판,통과PCR방법확증출Mb2277기인편단,이BamHⅠ화EcoR Ⅰ쌍매절PCR산물화pET28a(+),후장순화적Mb2277기인극륭지pET28a(+)중,구건출원핵표체질립,재전화도E.coli BL21(DE3)표체균주중, IPTG유도후,수집균체진행SDS-PAGE,진일보이용Western Blot대표체산물적반응원성진행연구.결과 획득료우결핵분지간균Mb2277원핵표체질립,IPTG유도표체후,SDS-PAGE분석가견약25ku단백표체조대,Western Blot결과현시,표체산물여토항분지간균항체구유반응성.결론 해단백구유교강적반응원성.
To obtain fusion protein of Mycobacterium bovis with high purity, the recombinant prokaryotic expression vector for Mb2277 gene was constructed and the immunogenicity of its products was initially investigated in the present study.A pair of primer was designed according the gene sequence Mb2277 from the genomic DNA of M.Bovis in GenBank. and was amplified by PCR using DNA of M.Bovis 93006 strain as template. The PCR product and pET-28a(+) was then digested by BamHⅠ and EcoR Ⅰdouble enzyme. To constructed a prokaryotic expression plasmid, the purified Mb2277 was cloned to pET28a(+). Then the recombinant plasmid was transformed into competent cell of E.coli BL21(DE3).The bacteria were induced by IPTG and its lysates were analyzed by SDS-PAGE and Western-blotting. In this way, the prokaryotic expression plasmid for M. bovis Mb 2277 protin was obtained, and a expression band with molecular of 25 ku could be found in SDS-PAGE analysis. As demonstrated by Western blotting this expression product showed excellent reactivity with rabbit immune sera against M. bovis.