含凝血因子FXa识别位点的融合表达体系的构建

The System of Fusion Expression Including the Site of Cleavage by FXa

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BL2 1,用异丙基硫代 β D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 2 0 %。重组蛋白以可溶形式存在。融合蛋白经FXa切割后 。

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.