赖型钩端螺旋体Loa22重组质粒的蛋白表达和对巨噬细胞的毒性作用

Expression and Identification of Gene Loa22 from the Virulent Strain of Serovar Lai of L.interrogans and Its Cytotoxicity to Macrophages

目的对赖型钩端螺旋体Loa22基因进行表达和功能研究。方法构建赖型钩端螺旋体Loa22成熟肽重组质粒,经异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行蛋白诱导表达,SDS-PAGE和Western Blotting检测目标蛋白表达情况。经亲和层析获得目标蛋白并作用于小鼠巨噬细胞ANA-1,检测细胞上清液中乳酸脱氢酶(LDH)活力、四氮唑复合物(XTT)吸光度值和细胞凋亡率以评价其细胞毒性。结果成功构建Loa22成熟肽原核表达质粒,通过鉴定并纯化得到Loa22成熟肽,该蛋白使培养ANA-1细胞上清液中LDH活力升高,XTT吸光度值下降,细胞凋亡率增加。结论Loa22对ANA-1具有明显的毒性效性,该基因可能是致病钩体的一个重要毒力相关基因。

Objective To study the function of Loa22 gene from virulent serovar Lai. L. interrogans by expressing its protein. Methods The recombinant plasmid with Loa22 was constructed and its expression was induced by IPTG. The expression product was identified by SDS-PAGE and Western Blotting and purified with GST-affinity chromatography. The purified protein was applied to mice macrophages ANA-1 cells purified by GST-affinity column and exerted to ANA-1 cells. The cytotoxicity of purified protein to ANA-1 was evaluated by detecting LDH activity in culture medium, XTT aborbtion and apoptosis ration. Results The recombinant plasmid with Loa22 mature peptide was constructed successfully and the protein was purified subsequently. Significant higher level of LDH activity and apoptosis ratio and lower XTT absorption were observed by comparison of expression protein treated cells and those untreated. Conclusion Loa22 had a toxic effect on ANA- 1 and the gene Loa22 might be a virulent-related gene of L. interrogans.