北京化工大学学报（自然科学版）
北京化工大學學報（自然科學版）
북경화공대학학보（자연과학판）
JOURNAL OF BEIJING UNIVERSITY OF CHEMICAL TECHNOLOGY(NATURAL SCIENCE EDITION)
2010年
3期
102-105
,共4页
豹蛙酶%大肠杆菌%基因克隆%结构鉴定
豹蛙酶%大腸桿菌%基因剋隆%結構鑒定
표와매%대장간균%기인극륭%결구감정
onconase%Escherichia coli%cloning%characterization
根据豹蛙酶的氨基酸序列,采用大肠杆菌偏爱密码子设计引物,重叠延伸PCR法合成出目的基因,约350bp.将得到的片断克隆于载体pGEM-T中并测序,再连接至表达载体pET-22b(+)中,重组质粒转入大肠杆菌Rosetta中.转化的重组大肠杆菌用终浓度1mmoL/L的IPTG诱导外源基因表达,采用Tricine-SDS-PAGE系统,分析目标蛋白主要以包涵体形式存在,其质量分数可达48.8%.利用液相电喷雾串联质谱方式(LC-ESI-MS/MS)鉴定蛋白,通过LCQ DECA XP Plus系统进行蛋白序列的分析,其覆盖率达到35%,确定了其蛋白质一级结构的正确性.
根據豹蛙酶的氨基痠序列,採用大腸桿菌偏愛密碼子設計引物,重疊延伸PCR法閤成齣目的基因,約350bp.將得到的片斷剋隆于載體pGEM-T中併測序,再連接至錶達載體pET-22b(+)中,重組質粒轉入大腸桿菌Rosetta中.轉化的重組大腸桿菌用終濃度1mmoL/L的IPTG誘導外源基因錶達,採用Tricine-SDS-PAGE繫統,分析目標蛋白主要以包涵體形式存在,其質量分數可達48.8%.利用液相電噴霧串聯質譜方式(LC-ESI-MS/MS)鑒定蛋白,通過LCQ DECA XP Plus繫統進行蛋白序列的分析,其覆蓋率達到35%,確定瞭其蛋白質一級結構的正確性.
근거표와매적안기산서렬,채용대장간균편애밀마자설계인물,중첩연신PCR법합성출목적기인,약350bp.장득도적편단극륭우재체pGEM-T중병측서,재련접지표체재체pET-22b(+)중,중조질립전입대장간균Rosetta중.전화적중조대장간균용종농도1mmoL/L적IPTG유도외원기인표체,채용Tricine-SDS-PAGE계통,분석목표단백주요이포함체형식존재,기질량분수가체48.8%.이용액상전분무천련질보방식(LC-ESI-MS/MS)감정단백,통과LCQ DECA XP Plus계통진행단백서렬적분석,기복개솔체도35%,학정료기단백질일급결구적정학성.
Based on the amino acid sequence of onconase and the preferred codons of the bacterium Escherichia coli, the target gene was synthesized by PCR-driven overlap extension of about 350bp and linked into the pGEM-T vector. After sequencing, the gene was subcloned into the expression vector pET-22b(+) to obtain the recombinant expression vector, which was transformed into Rosetta. Positive colonies were confirmed by PCR and sequencing. In order to check the gene expression in Rosetta, tricine-SDS-PAGE was performed on the target protein. The results showed that the gene was expressed successfully with a content of 48.8%. Moreover, the proposed primary structure of the expressed protein was shown to be correct, with a coverage of 35% , by characterization by LC-ESI-MS/MS with the LCQ DECA XP Plus system.
