人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达  

Cloning and prokaryotic expression of human lysozyme N-terminal fragment/Exendin-4 chimeric peptide

作　　者：王明[1] 刘宇婷[2] 潘霞明[2] 曾可静[2] 朱元昌 周羽竝[1] 刘誉[1] 

机构地区：[1]暨南大学医学院生物化学教研室,广东广州510632 [2]暨南大学医学院临床医学系,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2009年第6期595-600,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省科技计划项目(2006B35502010);暨南大学引进人才启动基金项目(51204004)

摘　　要：目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化。方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列。以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达。表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽。结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%。Western blotting检测显示重组蛋白为单一清晰条带。重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽。结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白。Aim：To clone, express and purify chimeric peptide of human lysozyme N-terminal fragment/exendin-4. Methods： Human lysozyme N-terminal fragment （1-74aa）and exendin-4 gene sequences were linked by using recombinant PCR. The linker sequence between the lysozyme fragment and exendin-4 contained a site recognized by thrombin and another site by dipeptidyl peptidase IV （DPP IV）. The hLYZ（N74）-Ex4 gene was inserted into prokaryotic expression vector pET-32a（ ＋ ）, transformed into E. coli BL21 （DE3） and induced with IPTG. The expressed products were purified by Ni- NTA affinity chromatography and dialysis, identified by western blotting and then digested with enterkinase to release the chimeric peptide. Results： DNA sequence analysis showed that the recombinant plas- mid pET-32a/hLYZ（N74）-Ex,4 was constructed correctly. The target protein was expressed mainly in inclusions and reached a maximum level of about 30% of total somatic proteins after induction with 0. 6mmol/L IPTG at 37 ℃for 4 h. Western blotting analysis suggested the recombinant protein was a clear band with expected molecular weight. The purified recombinant protein was cleaved into two fragments by enterokinase, the Trx-His tag and the chimeric peptide hLYZ （ N74 ） -exendin- 4. Conclusion： The pro- karyotie expression vector for hLYZ（N74）- exendin-4 fusion gene was successfully constructed, and the recombinant protein was highly expressed in E. coll. , which laid a foundation for their subsequent functional study.

关 键 词：Exendin 4 人溶菌酶 嵌合多肽 原核表达 糖尿病 

分 类 号：R587.1[医药卫生—内分泌] Q78[医药卫生—内科学]