含凝血因子FXa识别位点的融合表达体系的构建-东南大学学报医学版

含凝血因子FXa识别位点的融合表达体系的构建

单位：1.南京铁道医学院,生物化学教研室,江苏南京,210009； 2.南京大学,生物化学系医药生物技术国家重点实验室,江苏南京,210093

分类号：Q783.1, Q782, Q786

出版年·卷·期（页码）：2000·19·第三期（149-151）

摘要：

目的:研究含凝血因子FXa识别位点的基因与表达载体pGEX-KG连接后在大肠杆菌中的表达.方法:用PCR对人工合成的抗菌肽X基因的5′端进行改造,引入FXa的酶切位点,用限制性内切酶作用后,连接到含Ptac启动子的表达载体pGEX-KG中,转化大肠杆菌DH5α,用原位杂交法筛选到阳性克隆.转化大肠杆菌BL21,用异丙基硫代βD半乳糖(IPTG)诱导.结果和结论:抗菌肽X基因在大肠杆菌中获高效表达,表达量约占细胞总蛋白的20%.重组蛋白以可溶形式存在.融合蛋白经FXa切割后,产物有抗菌活性.

Objective The experiment was designed to study the expression of the gene in E.coli which including a cleavage site of FXa and was ligased into the expression vector pGEX KG.Method The 5′ end of antibacterial peptides gene X was modified by PCR and the cleavage site of factor Xa was used.The chemically synthesized gene was cloned into the expression vector pGEX KG,transferred into E.coli 5α ,and was highly expressed in E.coli BL21 by IPTG induction.Results and Conclusion This fusion gene was overexpressed in E.coli with an expression level of approximate 20% of total cellular proteins and was produced mainly in the soluble form.About 50% of fusion protein was cleaved after factor Xa digestion,only the factor Xa digested GST X exhibited strong antibacterial activities.

参考文献：

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