重组猪生长抑素的基因克隆、表达与纯化
Construction, expression and purification of recombinant somatostatin
王志 1黄同列 1薛晓畅 2张英起2
作者信息
- 1. 第四军医大学药学系学员一队,陕西西安,710033
- 2. 第四军医大学药学系生物技术中心,陕西西安,710033
- 折叠
摘要
目的:在大肠杆菌中克隆表达猪重组生长抑素基因,并进行纯化和鉴定.方法:根据GenBank公布的猪生长抑素基因序列,按照大肠杆菌偏爱密码子设计并合成2条DNA单链,退火后获得猪生长抑素基因序列.克隆至原核表达载体pGEX-4T-1质粒中,转化感受态细胞DH5α,经IPTG诱导表达重组融合蛋白并用GST亲和柱对其进行纯化.结果:重组质粒经酶切鉴定和DNA序列测定证明基因完全正确,经IPTG诱导后在大肠杆菌DH5α中得到高水平表达,表达产物经超声和溶菌酶破碎和Glutathione Sepharose 4B亲和层析纯化获得重组蛋白.结论:猪牛长抑素基因得到了高水平表达,纯化后纯度达到95%以上,为表达产物的大量制备、重组疫苗的进一步优化及在畜牧业生产上的应用研究奠定了基础.
Abstract
AIM: To clone and express pig somatostatin gene in Escherichia. Coli (E. coli) and to purify and identify the fusion protein. METHODS: The gene of pig somatostatin was designed and synthesized according to GenBank data and optimized to be consistent with preferred codens of E, coli. Then the synthesized SS gene was cloned into pGEX-4T-1 prokaryotic expression vector and the plasmid was transformed into E. coli strain DH5α. GST-SS fusion protein was expressed with IPTG induction and purified by GST-affinity chromatography. RESULTS: Restriction enzyme digestion and DNA sequencing showed that the sequence of SS gene was correct. The GST-SS fusion protein was expressed at high level with IPTG induction. After the E. coli was lysed by ultrasonic wave and lysozyme, the recombinant protein was purified with Glutathione Sepharose 4B affinity chromatography. CONCLUSION: The fusion protein GST-SS is effectively expressed in E. coli and the purity is over 95% after affinity chromatography, which lays a good foundation for large-scale production and optimization of the recombinant vaccine for wide application in animal husbandry.
关键词
生长抑素/克隆,分子/基因表达/重组融合蛋白质类Key words
somatostatin/cloning/molecular/gene expression/recombinant fusion proteins引用本文复制引用
基金项目
第四军医大学学员课外科研项目(2007年)
出版年
2009
NETL
NSTL
维普
万方数据