结核分枝杆菌持留相关PcaA和Acr蛋白的表达纯化及初步应用

Expression of Persistent Protein PcaA and Acr of Mycobacterium tuberculosis in Escherichia coli and Their Purification Application

目的结核分枝杆菌（M．tb）潜伏感染状态是造成结核病诊断和研究的难点之一，目前的检测方法很难将潜伏感染结核病（LTBI）和活动性结核病（ATB）区分开。有文献报道分枝菌酸环丙烷合成酶（PcaA）和小分子热休克晶体蛋白（Acr）两个基因在M．tb持留状态下高表达，因此可能和菌的潜伏感染相关，有作为新的诊断靶标的可能，因此我们对它们开展了有关的研究和应用，以期为LTBI的诊断提供新的研究方向和方法。方法首先针对目的基因设计引物，从基因组上扩增其全序列，构建克隆和表达载体，纯化分离蛋白。将蛋白作为抗原应用于免疫学的检测，用酶联免疫吸附测定（ELISA）方法检测血清中两种蛋白抗体的表达水平，最终经过统计学处理，分析LTBI和ATB患者之间的血清学的差异，寻找可应用于l晒床诊断分析的方法和策略。结果成功构建PcaA和Acr蛋白的克隆和表达载体，并获得表达和纯化的蛋白。血清的PcaA和Acr抗体水平检测结果显示健康对照（HC）与ATB之间差异有统计学意义，而ATB和LTBI之间差异并无统计学意义，即使将两个蛋白检测结果进行并联分析后也未显示差异有统计学意义。结论PcaA和Acr蛋白可以在大肠杆菌中成功表达并顺利纯化，PcaA蛋白有较高的抗原特异性和免疫反应性，可用于以后对结核病的检测分析中；虽然用血清学诊断时不能完整的区分LTBI和ATB，但可用于鉴别结核分枝杆菌感染与否。

Objective Mycobacterium tuberculosis （M. tb） latent infection was one major reason which made tuberculosis hard to diagnosis and research. It was very hard to discern latent tuberculosis infection （LTBI） from active tuberculosis （ATB） by using current detection methods. PcaA and Acr were highly expressed under M.tb latent infection some paper reported, which maybe connect with latent infection and possible as a new diagnosis target. So we carried on some research and application, to provide new diagnosis tool and method for LTBI. Methods We designed primer and amplifying target gene full sequence from genome, constructing protein expression vector and doing purification. We detected the antibodies against PeaA and Acr antigens in the patients serum by enzyme-linked immunosorbent assay （ELSIA）, and analyzed the difference between LTBI and ATB after statistical treatment, to find methods and strategies which can be used to clinical diagnosis. Results We successfully constructed expression vector and got purification protein. There showed significantly difference at the antibody level of PcaA and Acr protein in the ATB and normal heathy people scrums, but no clearly difference between LTBI and ATB serums, even after parallel analysis. Conclusions The recombinant PeaA and Acr protein can be successfully expressed in E. coli and smoothly been purified. PcaA has a good antigenic specificity and immunorcactivity, which can be used to tuberculosis detection and analysaiton. Although we can＇ t distinguish LT＇BI and AT＇B by using these two proteins, they maybe can be used to identify M.th infection or not.