CAJ | 학술논문

采用PCR技术扩增TAT-Hlbs融合基因,并通过基因操作构建了融合基因的原核重组载体pET-28a-TAT-Hlbs.将阳性重组质粒转化至受体菌Rosseta(DE3)感受态细胞中,以IPTG诱导表达,表达产物经聚丙烯酰胺凝胶电泳检测和Western-blot检测.结果表明:重组菌能够表达目的蛋白,软件分析结果表明表达蛋白约占菌体蛋白的40%,上清表达量约为20%.上清蛋白经纯化后,Western-blot结果显示,His-Tag单克隆抗体可以很好地与所表达的蛋白带特异性结合.所获得的融合蛋白以高效胞质可溶形式表达.
채용PCR기술확증TAT-Hlbs융합기인,병통과기인조작구건료융합기인적원핵중조재체pET-28a-TAT-Hlbs.장양성중조질립전화지수체균Rosseta(DE3)감수태세포중,이IPTG유도표체,표체산물경취병희선알응효전영검측화Western-blot검측.결과표명:중조균능구표체목적단백,연건분석결과표명표체단백약점균체단백적40%,상청표체량약위20%.상청단백경순화후,Western-blot결과현시,His-Tag단극륭항체가이흔호지여소표체적단백대특이성결합.소획득적융합단백이고효포질가용형식표체.
TAT-H1bs fusion gene was obtained through PCR amplification, and recombinant expression plasmid pET-28a-TAT-H1bs was constructed by gene operation technology. The correct recombinant expression plasmid was transformed into the host strain Rosseta induced by IPTG. The specific protein expressed was detected by SDS-PAGE and Western blot.The result showed that the targeted protein was detected in Rosseta(DE3), the fusion protein was expressed at high level amounting to 40% of the total bacterial protein analyzed by computer software. The amount in supernatant is about 20%. After the supernatant protein was purified, Western blot showed the monoclonal antibody anti-His-Tag could react to the targeted protein expressed specifically. The fusion protein was expressed highly in cytoplasmic fraction in Rosseta(DE3).