人乙酰肝素酶大亚基蛋白在大肠杆菌中融合表达.docVIP

人乙酰肝素酶大亚基蛋白在大肠杆菌中融合表达 　 作者：李燕杰，李吉学，凌焱，汤国营，林艳丽，朱厚础 【关键词】 人乙酰肝 　　Cloning, expression and characterization of human heparanse Cterminal subunit protein in E. coli 　　【Abstract】AIM: To construct the expression vector for heparanase (HPA) Cterminal subunit and obtain enough protein for further study. METHODS: Full encoding gene of heparanase protein was isolated from the human platenta cDNA library and its expressing vector pGEX2TK50 ku HPA was constructed by cloning its Cterminal subunit gene into the pGEX2TK vector. After transformed by the expression vector, E.coli BL21 (PlyS, DE3) was induced to express the target protein. RESULTS: E.coli BL21 (PlyS, DE3, pGEX2TK50 ku HPA) began to express the target protein fused by GST protein and Cterminal subunit protein after 1 h induction and kept producing it until 3 h after induction when the target protein reached a maximum. No degradation of produced protein was observed in the hetero E.coli cells. The target protein was expressed mostly in inclusive bodies. When growing at lower temperature, some fused protein was detected soluble in cell lysis. CONCLUSION: The Cterminal subunit protein of heparanase fused with GST protein has been successfully expressed in E.coli BL21 (PlyS, DE3). The protein may be a good antigen in preparing the antibody for heparanase and in further study of its function. 　　【Keywords】 heparanase; Cterminal subunit; pGEX2TK; gene expression 　　【摘要】 目的： 乙酰肝素酶在肿瘤转移、血管生成、组织发生等过程中起重要作用，其羧基端大亚基蛋白是该酶的重要组成部分，但由于此蛋白在体内含量少，易降解，很难直接从组织内得到一定数量的蛋白. 本研究利用工程菌表达乙酰肝素酶的大亚基蛋白以期得到相当数量的蛋白，以便进一步用于该酶的抗体制备和功能研究. 方法： 从人胎盘cDNA文库中分离乙酰肝素酶蛋白全长编码基因，通过PCR和酶切等方法将乙酰肝素酶大亚基的基因克隆入原核表达载体pGEX2TK中，测序鉴定序列完全正确后，转化大肠杆菌BL21（PlyS,DE3）进行表达. 结果： 工程菌BL21（PlyS, DE3, pGEX2TK50 ku HPA）成功表达出乙酰肝素酶大亚基融合蛋白. 诱导1 h后工程菌即开始表达大亚基融合蛋白，在诱导3 h后表达蛋白量达到最高，随着时间的延长，目的蛋白没有被降解. 诱导剂IPTG的浓度对其表达量及其表达形式无显著影响. 此蛋白的表达形式主要为包涵体形式. 低温诱导可见少量可溶蛋白存在. 结论： 在大肠杆菌中成功表达了大亚基的融合蛋白. 此蛋白可以用于进一步乙酰肝素酶的抗体制备、免疫鉴定、功能活性等研究. 　　【关键词】 乙酰肝素酶；羧基端大亚基；pGEX2TK；基因表达 　　0引言 　　乙酰肝素酶（heparanase, HPA）是迄今发现的惟一能特