人血清Q型对氧磷酶在大肠杆菌中的表达

目的:应用大肠杆菌原核表达系统高效表达人血清Q型对氧磷酶(PON1)。方法:从pBlueScript-PON1重组质粒中通过PCR扩增得到了人PON1基因,将其亚克隆至原核表达载体pBV220中,构建了重组表达质粒pBV220-PON1,转化大肠杆菌,获得表达菌株。结果:rhPON1重组蛋白以不溶形式存在于包涵体中。凝胶扫描分析表明,重组蛋白表达量约占菌体总蛋白的18%。包涵体经分离、变性、复性等步骤处理后,产物未显示酶活性。结论:原核表达的rhPON1以包涵体形式存在,实现了人PON1在大肠杆菌中的高效表达。

Expression of Q-Type Human Paraoxonase in Escherichia. coli

Objective: To express human paraoxonase(PON1) in E.coli expression system. Methods: PON1 gene was amplified by PCR from pBlueScript-PON1 recombinant plasmid, and then was cloned into pBV220 vector to construct pBV220-PON1 expression plasmid. pBV220-PON1 plasmid was transformed into compenant cells of E.coli, and an expression strain was selected. Results: SDS-PAGE analysis revealed that the human PON1 protein was highly expressed and accumulated up to above 18% of the total amount of bacterial proteins in the form of inclusion body after induction. The inclusion bodies which contained recombinant proteins were isolated, denatured and refolded, however the treated protein did not show any enzyme activities. Conclusion: rhPON1 was expressed in the form of inclusion body in E.coli, recombinant protein was highly expressed.