结核分枝杆菌Rv3084编码酯酶LipR的生物学特性和体内免疫调节功能

  目的  探究结核分枝杆菌（Mycobacterium tuberculosis，MTB）Rv3084编码的酯酶LipR的生物学特性及其在体内的免疫调节功能。  方法  从MTB H37Rv标准毒力株扩增LipR基因，构建重组表达质粒；测序正确的重组质粒转化入大肠杆菌，诱导LipR蛋白表达并纯化，Western blot鉴定；进行LipR酯酶活性检测，分析影响LipR酶活性的因素；将重组质粒于小鼠胫前肌肉注射0.1 mL（含质粒DNA 100 μg）3次（第1，8，15天），末次注射后7 d，处死小鼠，取肺、脾脏进行细胞因子检测。  结果  成功构建重组表达质粒，并发现LipR蛋白在大肠杆菌中主要以包涵体的形式表达，蛋白相对分子质量约为33×103；在标准水解活性实验条件（40 ℃，pH7.5）下，其最适水解底物为4-硝基苯基乙酸酯（pNPA，C2）；以4-硝基苯基丁酸酯（pNPB，C4）作为底物，测得LipR水解活性的最适pH值为8.5，最适反应温度为40 ℃，说明LipR是一种相对耐碱耐热的酯酶；而在不同的除垢剂或金属离子存在的环境下，LipR水解pNPB的活性受到一定程度地抑制。重组质粒注射小鼠后，发现LipR能抑制γ-干扰素（IFN-γ）和白细胞介素（IL）-2的分泌，但IL-10分泌量增加。  结论  酯酶LipR可能参与帮助MTB协同抵抗宿主内苛刻的环境，并充当免疫调节剂，抑制促炎细胞因子分泌。

Mycobacterium Tuberculosis Rv3084 Encodes Functional Esterase and Suppresses the Pro-inflammatory Cytokines in vivo

  Objective  To explore the biological characteristics of the esterase LipR encoded by Mycobacterium tuberculosis (MTB) Rv3084 and its immunomodulatory function in vivo.  Methods  The LipR gene was amplified from MTB H37Rv strain to construct recombinant expression plasmid. After sequencing, the recombinant plasmid was transformed into E. coli for expression and purification of LipR protein. The expressed protein was confirmed with Western blotting assay. The hydrolyzing activity of LipR was detected and the factors affecting LipR enzyme activity were analyzed. Mice were intramuscularly injected with 0.1 mL (containing plasmid DNA 100 μg) recombinant eukaryotic plasmid three times (day 1, 8, and 15); seven days after the last injection, the mice were executed, and the lung and spleen were taken for cytokine detection.  Results  The recombinant expression plasmid was successfully constructed and it was found that LipR protein was mainly expressed in the form of inclusion bodies in E. coli with the relative molecular mass of about 33×103. LipR was demonstrated as an alkaline eurythermic esterase, due to the preference of hydrolyzing short carbon chain esters with optimal hydrolyzing activity on pNP-acetate (pNPA, C2) and the capability in tolerance of high pH and temperature; in the presence of different detergents or metal ions, the activity of LipR hydrolyzing pNP-butyrate (pNPB, C4) was inhibited to some extent. In the mouse model, it was found that LipR could inhibit the secretion of interferon-γ (IFN- γ) and interleukin-2 (IL-2), but to stimulate the secretion of IL-10.  Conclusion  The esterase LipR may be one of the esterases help M. tuberculosis withstand harsh environment inside the host in collaboration, and simultaneously act as an immune modulator to inhibit the secretion of pro-inflammatory cytokines and consequently impact the killing effect of host immune system against M. tuberculosis.