重组抗癫痫肽二串体的表达、纯化与鉴定  

Expression,Purification and Identification of Two Repeat of Recombinant Anti-Epilepsy Peptide

作　　者：王宗仁[1] 高碧峰[1] 袁有才[2] 李晶华[1] 张德安[1] 马静[1] 

机构地区：[1]第四军医大学西京医院中医科,陕西西安710032 [2]陕西中医学院,陕西咸阳712046

出　　处：《生物技术通讯》2007年第6期901-904,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30671764)

摘　　要：目的:利用基因工程方法表达抗癫痫肽(AEP)串联体蛋白,并进行纯化、鉴定。方法:PCR扩增AEP及AEP-His基因片段,并分别克隆至测序载体中;测序正确后,利用基因重组技术获得二串体基因,构建毕赤酵母表达载体pPIC9K-AEP2-His,甲醇诱导AEP2-His在毕赤酵母中表达,用Ni-chelating Sepharose FF柱纯化表达的融合蛋白,并用抗His单克隆抗体进行Western blot鉴定。结果:构建了AEP二串体酵母表达载体,获得了纯化的AEP2-His蛋白,其相对分子质量约20 000。结论:用基因串联思路表达小分子多肽是一种可行的方法;AEP2-His蛋白的成功表达为其功能研究奠定了基础。Objective： To expression recombinant tandem repeat of anti-epilepsy peptide（AEP） and to purify and identify the fusion protein. Methods： The core fragment of AEP and AEP-His were amplified by PCR, then were cloned into the vector pMD18T respectively and named pMD18T-AEP and pMD18T-AEP-His that were identified by sequencing. The tandem repeat of AEP2-His was constructed by gene engineering method. The expression plasmid of pPIC9K-AEP2-His was constructed by clonal thechnology. The fusion protein of AEP2-His was expressed induced by methanol in Pichia cells, it was purified by Ni-chelating Sephaharose cloum, and identified by Western blot. Results＇- The expression plasmid of pPIC9K-AEP2-His was constructed and the purified AEP2-His fusion protein was obtained with molecular weight of about 20 kD. Conclusion： To expression short peptide, the method of expressing tandem repeat gene is practical. The successful expression of AEP2-His has established a foundation for its biological activity research.

关 键 词：抗癫痫肽 串联体 融合蛋白 

分 类 号：Q78[生物学—分子生物学] R742.1[医药卫生—神经病学与精神病学]