东亚钳蝎氯离子通道毒素BmK CTa在大肠杆菌中的异源表达及其对宿主菌增殖的影响

目的在大肠杆菌中异源表达东亚钳蝎氯离子通道毒素BmK CTa,并观察其对宿主菌增殖的影响。方法构建BmK CT基因原核表达质粒pExSecI-rBmK CTa,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot进行鉴定。光密度法检测含不同质粒的大肠杆菌BL21(DE3)及空菌在37℃,LB液体培养基中的生长速率。结果重组原核表达质粒pExSecI-rBmK CTa经PCR、双酶切和测序证明构建正确。目的蛋白的表达量占全菌总蛋白的19.94%,为可溶性表达,且具有良好的反应原性。rBmK CTa的异源表达显著抑制了大肠杆菌在对数生长期的增殖。结论rBmK CTa在大肠杆菌BL21(DE3)中获得了可溶性表达,且对大肠杆菌的生长具有显著的抑制作用。提示BmK CT可能特异性地作用于宿主菌的氯离子通道,对原核生物的氯离子通道有抑制作用。

Heterologous Expression of Chloride Ion Channel Toxin BmK CTa from Buthus martensii Karsch in E. coli and Its Inhibitory Effect on Proliferation of Host Bacterial Strain

Objective To express BmK CTa, a chloride ion channel toxin from Buthus martensii Karsch in E. coli and study its inhibitory effect on the proliferation of host bacterial strain. Methods Recombinant plasmid pExSecI-BmK CTa was constructed and transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. The growth rates of E. coli BL21(DE3)carrying various plasmids in liquid LB medium at 37℃ were monitored by densitometry. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pExSecI-BmK CTa was constructed correctly. The expressed product in a soluble form contained 19. 94% of total somatic protein and showed good reactogenicity. The heterologous expression of rBmK CTa inhibited the proliferation of E. coli BL21(DE3) in logarithmic growth phase significantly. Conclusion rBmK CTa was expressed in a soluble form in E. coli BL21(DE3) and showed significantly inhibitory effect on the growth of host bacterial strain, indicating that BmK CT might specifically inhibit the chloride ion channel of prokaryotic organisms.