神经营养肽NP14基因的合成及其在大肠杆菌中的高表达  

Synthesis of multiple copies of neurotrophic peptide and their high level expressions in E.coli

作　　者：张之勇[1] 苑辉卿[1] 姜安丽[1] 蔡捷[1] 任凯[1] 胡晓燕[1] 孔峰[1] 张建业[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学教研室,山东济南250012

出　　处：《中国病理生理杂志》2007年第12期2304-2308,共5页Chinese Journal of Pathophysiology

基　　金：教育部科学技术研究重点资助项目(No106101)

摘　　要：目的:根据鞘脂激活蛋白原神经营养序列设计并构建短肽(neurotrophic peptide,NP)多拷贝串连表达载体,利用基因工程的方法制备短肽NP。方法:根据大肠杆菌的偏爱密码子设计并合成NP的碱基片段,通过PCR方法合成串联双拷贝2NP片段,经平端连接克隆到载体pUC18中。利用EcoT14I酶切pUC18-2NP后可产生非镜相对称黏性末端,与表达载体pETEcoT一次连接反应,得到一系列含有不同片段拷贝数的表达载体pETE-coT-multiNP,经PCR-array方法进行阳性克隆筛选、测序鉴定后转化入大肠杆菌BL21(DE3)进行原核表达。结果:经IPTG诱导后,在BL21(DE3)菌中高效表达了2、4、8拷贝融合蛋白,并在每个单拷贝之间加入了溴化氢的切割位点,使融合蛋白切割后能够得到单拷贝的短肽。结论:短肽NP在大肠杆菌中获得高效表达,为进一步研究其生物活性奠定了基础。AIM ： To construct and express containing multiple tandem copies of a peptide （ neurotrophic peptide, NP）, which was designed according to the NP sequence of prosaposin. METHODS： DNA sequence of peptide NP was synthesized by the preferred codons of E. coli. Two copies of NP fragments were produced by PCR and inserted into pUC18 vector. The fragments from pUC18 -2NP by EcoT 14I were ligated into tandem multi - NP fragments through self- ligation, and subcloned into pET- EcoT vector, which has a unique EcoT14I cloning site allowing unidirectional insertion of a desired sequence. Multi - NP clones were screened by PCR - array. RESULTS ： The constructs with different repeats of NP were obtained and expressed as fused - proteins at high level in E. coli BI21 （DE3）. In order to get monomer peptide, each copy of peptide was interspersed by a unique site where the fused - protein could be cleaved by cyanogens bromide. CONCLUSION： Peptide NP could be highly expressed in E. coli. This work builds a solid foundation for further study on its bioactivity.

关 键 词：神经营养肽 融合蛋白质类 原核表达 

分 类 号：R363[医药卫生—病理学]