家鸡Leptin成熟肽cDNA的克隆、重组蛋白表达及纯化  

Chicken Leptin Mature Peptide Fusion Protein:Gene Construction,Expression and Purification

作　　者：施振旦[1] 邵西兵[1] 方梅霞[1] 于迎春[1] 刘颖[1] 陈楠[1] 

机构地区：[1]华南农业大学动物科学学院,广东广州510642

出　　处：《中国兽医学报》2004年第3期258-260,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目 ( 3 970 0 10 2 ) ;广东省自然科学基金资助项目 (粤科基〔1997〕2 3 )

摘　　要：从 18周龄鸡卵巢组织中抽提总 RNA,使用六聚体随机引物反转录后 ,用鸡 L eptin(瘦素 )特异性引物扩增出鸡L eptin编码区第 5 2～ 4 6 0 bp的长度为 4 0 9bp的 c DNA片段。根据鸡 L eptin的 3′端第 4 6 0～ 4 92 bp序列设计了 3条部分相互重叠并且顺序串联延伸的下游反义引物 ,并在最后 1条引物 3′端连接上 Eco R 切点 ;在以上用于反转录扩增的 5′端引物的 5′端连接一 Bam H 切点。用该 5′端引物分别与 3个 3′端引物配对 ,利用反转录扩增出的 4 0 9bp的L eptin c DNA片段作为第 1模板进行扩增 ,扩增产物再作为模板与下一引物对再次扩增。经 3次扩增得到编码鸡 L ep-tin成熟肽的全长 4 5 1bp的 c DNA序列。将该 4 5 1bp的 L eptin c DNA序列经 Bam H 和 Eco R 双酶切后 ,克隆入表达质粒 p RSET A的 Bam H 和 Eco R 两酶切位点之间 ,构建成表达质粒 p L ep- SCAU。转化有重组表达质粒 p L ep-SCAU的大肠杆菌 BL 2 1(DE3)在 L B培养基中培养后 ,经 IPTG诱导表达出相对分子质量为 2 0 10 0的鸡 L eptin融合蛋白和少量 4 0 2 0 0的 L eptin融合蛋白。L eptin融合蛋白的表达在 IPTG浓度为 0 .0 5 mmol/ L 时达到最高 ,占总菌体蛋白的 32 .6 %。用 Ni- NTA凝胶从 7L 发酵培养菌裂解液中纯化出 180 mA chicken cDNA fragment of 409 bp was amplified by RT-PCR with total RNA extracted from 18-wk old chicken ovaries,random hexomal primers to reverse transcribe the first chain cDNAs,and chicken leptin specific primers,which covered from 52 to 460 nucleotide of coding region of chicken leptin.Three sequentially overlapping and extending 3′ primers were also designed according to the sequences from 460 to 492 bp in chicken leptin cDNA.The last 3′ primer was extended with a 3′ EcoRⅠ restriction site and the 5′ primer in RT-PCR was also added a BamHⅠ site.The 3′ primers were paired sequentially in relaying manner with the 5′ primer to amplified chicken leptin cDNA fragments,using the 409 bp RT-PCR product as the first template and the resultant PCR product as template for the next reaction.Thus a 451 bp full length chicken leptin mature peptide cDNA fragment was produced following 3 PCR reactions,which was inserted into the BamHⅠ and EcoRⅠ cloning sites of expression plasmid pRSET A,to generate the chicken leptin fusion protein expressing plasmid pLep-SCAU.Following incubation and induction by IPTG,E.coli BL21(DE3) transformed with pLep-SCAU produced 2 chicken leptin fusion proteins corresponding to the theoretical sizes of (20 100) and (40 200).The expression level was highest under induction by 0.05 mmol/L IPTG,at 32.6% of the total bacterial protein.Approximately 180 mg of chicken leptin fusion protein was purified by Ni-NTA sepharose from bacterial lysate from 7 litres of fermentation culture.

关 键 词：鸡 Leptin成熟肽 CDNA 克隆 重组蛋白 表达 纯化 基因 瘦素 

分 类 号：S831.2[农业科学—畜牧学] Q78[农业科学—畜牧兽医]