摘要
目的:以人类多肽:N-乙酰氨基半乳糖转移酶2(ppGalNAc-T2)为研究对象,利用载体pGEX-5X-3在大肠杆菌(E.Coli)BL21中原核表达其编码序列,并对表达产物进行纯化、复性及酶活检测。方法:首先利用PCR技术从克隆载体pDONR201-T2得到ppGalNAc-T2全长编码序列,将其亚克隆至原核表达载体pGEX-5X-3,形成重组表达质粒pGEX-5X-3/T2。重组质粒转化大肠杆菌DH5α,测序鉴定后转化BL21,异丙基硫代半乳糖(IPTG)诱导后,表达产物为主要以包涵体形式存在的融合蛋白。对其复性后用谷胱甘肽-琼脂糖(G lutath ione-Sepharose)4B亲和层析柱进行纯化。然后根据ppGalNAc-T2的催化功能,设计底物,建立酶促反应体系,利用高效液相色谱(HPLC)进行酶活分析。结果:成功构建了原核表达重组载体pGEX-5X-3/T2,通过蛋白质电泳检测到分子量约为90 kD的融合蛋白的表达,利用亲和层析得到纯化的酶蛋白,并经W estern印迹得以验证。反应后体系上柱洗脱后出现酶促反应的产物洗脱峰。结论:成功构建了重组表达载体pGEX-5X-3/T2,ppGalNAc-T2全长编码序列被成功表达、纯化及复性,检测到具有酶活性。
Objective： To express the full-length encoding sequence of human polypeptide ： N-Acetylga-lactosaminyhransferase2 in Escherichia coli using vector pGEX-5X-3, and analyze its enzyme activity after purification and renaturation. Methods： The cDNA encoding ppGalNAc-T2, which was amplified from plasmid pDONR201-T2 by PCR, was subcloned into prokaryotic expression vector pGEX-5X-3, resulting in the recombinant plasmid pGEX-5X-3/T2 which was subsequently transformed into Escherichia coli BL21. A fusion protein was expressed after the induction by IPTG. After testified by western blot, we purified the expression products by Glutathione-Sepharose affinity chromatography. After renaturation, its enzyme activity was assayed by Reverse Phase High Performance Liquid Chromatography （RP-HPLC）. Results： The plasmid pGEX-5X-3/T2 was reconstructed successfully. The fusion protein was expressed and testified by SDSPAGE and western blot. The purified enzyme was obtained by affinity chromatography and the HPLC showed its activity. Conclusion： The ppGalNAc-T2 which has certain enzyme activity was successfully expressed, purified and renaturized.
出处
《江苏大学学报（医学版）》
CAS
2006年第2期93-96,共4页
Journal of Jiangsu University：Medicine Edition
基金
国防基础科研计划资助项目(K0102061501-03)
江苏省高校自然科学研究基金资助项目(01KJB310001)
