CAJ | 학술논문

从玉米嫩叶中提取总RNA,通过RT-PCR的方法扩增出玉米谷氨酰胺转胺酶(TGase)全长基因,回收目的片段并测序,该基冈编码区全长1605bp,编码535个氨基酸残基,分子量为60.9kD,与GenBank(登录号:AJ421525)上已发表的序列同源性为100%.按正确的阅读框架将玉米TGase基因片段定向克隆到表达载体pET-28a上,将重组质粒转化剑大肠杆菌Rosetta(DE3)菌株,1mmol/L IPTG诱导融合蛋白表达,经凝胶分析软件测得蛋白表达量约占总蛋白的15%,以His-Tag抗体作为一抗,采用Western-blot方法检测目的蛋白,结果证明所表达的特异蛋白是带有His-Tag的重组融合蛋白,利用Ni~(2+)-NTA琼脂糖树脂亲和层析柱纯化日的蛋白,SDS-PAGE鉴定为单一条带,测得纯化后的TGase酶活力达到16U/mg.
종옥미눈협중제취총RNA,통과RT-PCR적방법확증출옥미곡안선알전알매(TGase)전장기인,회수목적편단병측서,해기강편마구전장1605bp,편마535개안기산잔기,분자량위60.9kD,여GenBank(등록호:AJ421525)상이발표적서렬동원성위100%.안정학적열독광가장옥미TGase기인편단정향극륭도표체재체pET-28a상,장중조질립전화검대장간균Rosetta(DE3)균주,1mmol/L IPTG유도융합단백표체,경응효분석연건측득단백표체량약점총단백적15%,이His-Tag항체작위일항,채용Western-blot방법검측목적단백,결과증명소표체적특이단백시대유His-Tag적중조융합단백,이용Ni~(2+)-NTA경지당수지친화층석주순화일적단백,SDS-PAGE감정위단일조대,측득순화후적TGase매활력체도16U/mg.
In this study,total RNA was extracted from young leaves of maize.Reverse transcription PCR(RT-PCR)method was used to obtain full-lengthnansglutaminase(TGase)gene.The amplified fragment was sequenced to have 1605bp.Thegene consisted of 535 amino acid residues with a calculated molecular mass of 60.9 kD.It was identical to the published TGase gene (GenBankNO.AJ421525).This gene fragment was cloned into pET-28a expression vector.The pET-28a-TGase was transformed into E coli Rosetta(DE3)and expressed in E coil ceils with the induction of 1 mmol/L IPTG.According to quantitative analysis,the expression amount of this protein was approximately 15% of total protein.Western-blot analysis confhTned that this protein was a His-tag recombinant protein.After the purification by Ni~(2+)-NTA affmity chromatography.the purified protein exhibited high purity and one single band was shown in SDS-PAGE.The TGase activity was up to 16U/mg after purification.