鹿茸富脯氨酸多肽-APRP基因融合表达载体的构建及表达  

Construction of Fusion Expression Vector and Inducible Expression of the Antler Proline-rich Polypeptide-APRP

作　　者：郭寰宇[1] 董文博[2] 曲晓波[1] 史文婷[1] 郭焱[1] 

机构地区：[1]长春中医药大学,吉林长春130117 [2]吉林大学第一医院二部,吉林长春132013

出　　处：《现代生物医学进展》2016年第23期4419-4422,共4页Progress in Modern Biomedicine

基　　金："十二五"国家科技支撑项目(2011BAI03B02-2)

摘　　要：目的:构建鹿茸富脯氨酸多肽(APRP)基因融合表达载体,并诱导表达融合蛋白GST-APRP。方法:以鹿茸顶端组织总c DNA为模板,PCR方法扩增目的片段,连接p MDTM18-T载体,转化E.coli DH5α进行TA克隆。酶切与测序鉴定合格后,将目的片段与p GEX-6P-1质粒连接,完成p GEX-6P-1-APRP融合表达载体的构建。利用原核表达系统进行初步诱导表达。SDS-PAGE蛋白电泳检测蛋白表达情况。结果:PCR成功扩增出目的基因片段,目的基因片段大小为216 bp。提取表达载体质粒经酶切及测序分析证实p GEX-6P-1-APRR融合表达载体构建成功。利用终浓度为0.5 m MIPTG诱导,融合蛋白成功表达,分子量为35 k Da。结论:目前利用基因工程技术获得特定的鹿茸多肽的报道很少。本研究结果表明,利用原核表达系统可在体外对鹿茸富脯氨酸多肽基因(APRP)进行融合表达,并且融合蛋白GST-APRP以可溶形式表达,有利于进一步研究APRP。这也为研究其他鹿茸多肽单体成分提供重要参考。Objective： To construct the fusion expression vector of antler proline-rich polypeptide（APRP）, and induce the expression of fusion protein GST-APRP. Methods： The APRP segment was amplified by PCR technology with total c DNA template of velvet antler top organization. The product of amplification was linked with p MDTM 18-T vector, and the plasmid was transformed into E.coli DH5 α to accomplish TA clone. The fusion expression vector p GEX-6P-1-APRP was constructed by linking the target segment with p GEX-6P-1 vector after the correct identification of enzyme digestion and sequencing. Prokaryotic expression system was used to induce the expression of protein preliminarily, and expression was detect by SDS-PAGE protein electrophoresis. Results： We amplified the APRP gene fragment by PCR, the length of it was 216 bp, and constructed the fusion expression vector p GEX-6P-1-APRR successfully.We obtained the fusion protein whose molecular weight was 35 k Da by using 0.5 m M IPTG of final concentration. Conclusions： The reports’ quantity of obtaining specific velvet antler polypeptides by genetic engineering technology is rare currently. This study results show that we can induce the fusion expression of antler proline-rich polypeptide（APRP） in vitro, and GST-APRP fusion protein expressed with soluble form, which is helpful for further research of APRP. These efforts are also providing a valuable reference for the researching of other monomer composition of velvet antler polypeptides.

关 键 词：APRP GST-APRP 融合表达载体 诱导表达 

分 类 号：R285.5[医药卫生—中药学] S825[医药卫生—中医学]