【摘要】 目的构建结核杆菌Rv0757基因原核表达重组质粒pET28a-Rv0757,并研究表达的目标蛋白对小鼠骨髓巨噬细胞株ANA-1细胞的作用。方法将扩增出的Rv0757基因重组到原核表达质粒pET28a(+),并通过SDS-PAGE和Western blot鉴定诱导表达的目的蛋白。纯化目标蛋白后将目标蛋白PhoP作用于小鼠ANA-1细胞,检测活细胞数、乳酸脱氢酶(lactate dehydrogenase,LDH)、一氧化氮(NO)和细胞凋亡指标。结果成功构建出pET28a-Rv0757原核表达质粒,诱导表达的目标蛋白经SDS-PAGE和Western blot鉴定分析,其相对分子质量约为32×103。目的蛋白作用于小鼠ANA-1细胞后对活细胞数和细胞上清LDH活力无明显影响,但可以明显抑制细胞释放NO和细胞凋亡。结论本研究成功构建了原核表达载体pET28a-Rv0757,并成功表达出目标蛋白,该蛋白对小鼠ANA-1细胞没有毒性损伤,但可以抑制细胞释放NO和细胞的凋亡。更多还原

【Abstract】 Objective To construct recombinant plasmid pET28a-Rv0757 with Mycobacterium tuberculosis(Mtb)Rv0757gene,and to determine the effect of the protein of Rv0757 gene on ANA-1cells.Methods We recombined the amplified Rv0757 gene into theprokaryotic plasmid pET28a(+).The expressed product was identified by SDS-PAGE and Western blot.Murine macrophages were treated with purified protein PhoP.Survived cells,lactic dehydrogenase(LDH),nitric oxide(NO),and cell apoptosis were detected after the treatment.Results We successfully constructed the recombinant plasmid pET28a-Rv0757.The target protein was confirmed by SDSPAGE and Western blot.The protein had no significant effect on cell numbers and LDH activities in the culture supernatant,but it inhibited the release of NO and apoptosis of ANA-1cells.Conclusion pET28a-Rv0757 plasmid with prokaryotic expression was successfully constructed.The targetprotein has no toxicity on macrophages,but it can inhibit NO release and cell apoptosis of ANA-1.更多还原