四种结核潜伏感染蛋白制备及免疫学特性研究

发布时间：2018-04-28 07:25

本文选题：结核分枝杆菌 + 潜伏感染　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：目的：获得结核分枝杆菌潜伏感染相关重组蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，并评价其免疫学特性及其应用价值。方法：应用基因工程技术克隆、表达并纯化结核分枝杆菌潜伏感染相关蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，并对蛋白表达条件进行优化。进一步用纯化的重组蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c刺激376例中国人PBMC，其中包括未感染结核健康对照组103例、结核潜伏感染组51例、活动性结核病组116例、BCG接种组49例（Rv1813c无此组）、非结核呼吸疾病组57例，用ELISPOT技术检测分泌IFN-γ的效应T细胞斑点数（SFC），比较其在不同人群中的差异，评价它们在抗结核潜伏感染细胞免疫应答中的作用；用ELISA方法检测未感染结核的健康对照组72例、结核潜伏感染组36例及活动性结核病组62例共计170例人血清中4种蛋白特异性抗体IgG的水平，并比较组间差异，评价4种潜伏感染相关蛋白在抗结核体液免疫方面的作用；以潜伏感染组效应T细胞SFC为阳性组，以活动性结核病组SFC为阴性组，绘制各个潜伏抗原鉴别结核活动性感染与潜伏感染的ROC曲线，在特异度80%及90%两个点取SFC的cut-off值，以此评价并且比较潜伏感染蛋白对结核活动性感染与潜伏感染的鉴别诊断效能。结果：1、本研究制备并获得纯度较高的重组蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，其纯度均达到95%以上。2、重组蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c刺激中国潜伏感染人群后分泌IFN-γ的效应T细胞数显著高于活动性结核病组（p0.05），也显著高于未感染正常对照组（p0.05）；Rv2029c、Rv2659c、Rv1813c在非结核呼吸疾病组显著低于结核潜伏感染组（p0.05），Rv2628在非结核呼吸疾病组低于潜伏感染组，但差异不明显（p0.05），，与未感染健康对照组无显著差别（p0.05）；BCG接种3个月后PPD阳转者与健康对照组潜伏蛋白特异的SFC值进行比较，两者之间差异不明显（p0.05），BCG接种前后潜伏感染蛋白刺激的SFC值无显著差异（p0.05）。3、Rv1813c蛋白特异的IgG值在活动性结核病组显著高于未感染的健康对照组和潜伏感染组（p0.05），而其余3个蛋白特异的IgG水平在这三组间差异不显著（p0.05）。4、ROC曲线分析结果：Rv2029c蛋白的曲线下面积最高达到89.1%，高于Rv2659c（69.9%）、Rv2628（68.8%）及Rv1813c（39%）。Rv2029c、Rv2659c及Rv2628蛋白诱导潜伏感染组产生细胞免疫应答的阳性率最高。进一步评价各潜伏蛋白对鉴别结核活动感染与潜伏感染效能，Rv2029c灵敏度最高，在潜伏感染人群中的阳性率达到80%以上，但在未感染结核的健康组和活动性结核病组的假阳性率较高；Rv2659c虽然灵敏度不及Rv2029c，但其在未感染结核的健康组和活动性结核病组的假阳性率低；Rv2628的灵敏度与Rv2659c相当，但其假阳性率高于Rv2659c，低于Rv2029c。结论：1、本研究成功地构建了四种结核潜伏感染相关蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c高表达的基因工程菌株，获得的重组蛋白纯度高。2、Rv2029c、Rv2659c、Rv2628及Rv1813c可被结核潜伏感染人群识别，诱导其效应T细胞产生的免疫应答强于活动性结核病患者。3、重组Rv2659c蛋白通过ELISPOT技术诊断结核潜伏感染、鉴别活动性结核感染的效能最高，有望成为针对中国人群结核潜伏感染的新型诊断标志物。4、Rv2029c、Rv2659c和Rv2628不诱导机体产生体液免疫应答，Rv1813c诱导活动性结核病患者产生高水平的特异性抗体，可能在结核病致病机制中发挥作用。总之，我们的研究结果为其潜伏感染相关抗原的研究奠定了基础，也为潜伏感染诊断标志物及潜伏感染疫苗的研究提供了丰富的实验依据。
[Abstract]:Objective: to obtain recombinant protein Rv2029c, Rv2659c, Rv2628 and Rv1813c associated with latent infection of Mycobacterium tuberculosis, and to evaluate its immunological characteristics and its application value.
Methods: gene engineering technique was used to clone, express and purify the latent infection related proteins Rv2029c, Rv2659c, Rv2628 and Rv1813c of Mycobacterium tuberculosis, and optimize the protein expression conditions. The purified recombinant protein Rv2029c, Rv2659c, Rv2628 and Rv1813c were used to stimulate 376 cases of Chinese PBMC, including non tuberculosis healthy controls. There were 103 cases, 51 cases of tuberculosis latent infection group, 116 cases of active tuberculosis group, 49 cases of BCG inoculation group (Rv1813c without this group) and 57 cases of non tuberculosis respiratory disease group. The number of T cell spots (SFC) secreted by IFN- gamma was detected by ELISPOT technique, and the difference in different population was compared and the role of them in the cellular immune response to the latent infection of tuberculosis was evaluated. 72 healthy controls, 72 cases of uninfected tuberculosis, 36 cases of latent tuberculosis and 62 patients with active tuberculosis were used to detect the level of 4 protein specific antibody IgG in 170 human serum, and the difference between the groups was compared, and the effect of 4 latent infection related proteins in the anti tuberculosis humoral immunity was evaluated. The T cell SFC was used as the positive group, and the active tuberculosis group SFC was negative group. The ROC curve of the latent antigen to identify the active and latent infection of tuberculosis was plotted, and the cut-off value of SFC was taken at the specificity of 80% and 90% two points. In order to evaluate and compare the differential diagnostic efficacy of latent infection protein to the active and latent infection of tuberculosis.
Results: 1, the purity of recombinant protein Rv2029c, Rv2659c, Rv2628 and Rv1813c were obtained and the purity of the recombinant protein reached more than 95%.2. The recombinant protein Rv2029c, Rv2659c, Rv2628 and Rv1813c stimulated the secretory IFN- gamma in the latent infection population of China. The number of T cells was significantly higher than that of the active tuberculosis group (P0.05), and was significantly higher than that of the non infection. The normal control group (P0.05), Rv2029c, Rv2659c, Rv1813c in the non tuberculosis respiratory disease group was significantly lower than the latent infection group (P0.05), Rv2628 in the non tuberculosis respiratory disease group was lower than the latent infection group, but the difference was not significant (P0.05), no significant difference with the uninfected healthy control group (P0.05); BCG inoculation 3 months after the PPD positive control group and the healthy control group. The specific SFC value of latent protein was not significant (P0.05). The SFC value of latent infection protein stimulation before and after BCG inoculation was not significantly different (P0.05).3, and the specific IgG value of Rv1813c protein in active tuberculosis group was significantly higher than that of uninfected healthy control group and latent infection group (P0.05), while the remaining 3 protein specific IgG water was in the active tuberculosis group. The difference between the three groups was not significant (P0.05).4, ROC curve analysis results: the maximum area under the curve of Rv2029c protein reached 89.1%, higher than Rv2659c (69.9%), Rv2628 (68.8%) and Rv1813c (39%).Rv2029c, Rv2659c and Rv2628 protein induced latent infection group had the highest positive rate of cell immune response. Further evaluation of the latent protein was identified. Tuberculosis active infection and latent infection efficiency, Rv2029c sensitivity is the highest, the positive rate in the latent infection population is above 80%, but the false positive rate of the healthy group and the active tuberculosis group not infected with tuberculosis is higher; Rv2659c is less sensitive than Rv2029c, but it is in the health group and the active tuberculosis group that is not infected with tuberculosis. The positive rate is low, the sensitivity of Rv2628 is similar to that of Rv2659c, but the false positive rate is higher than Rv2659c, lower than Rv2029c.
Conclusion: 1, four genetic engineering strains with high expression of Rv2029c, Rv2659c, Rv2628 and Rv1813c were successfully constructed. The obtained recombinant protein was highly.2, Rv2029c, Rv2659c, Rv2628 and Rv1813c could be identified by the latent infection of tuberculosis, and the immune response produced by its effect T cells was stronger than that of activity. .3, the recombinant Rv2659c protein is used to diagnose the latent infection of tuberculosis through ELISPOT technology and the highest efficacy in identifying active tuberculosis infection. It is expected to be a new diagnostic marker for latent infection of tuberculosis in Chinese population,.4. Rv2029c, Rv2659c and Rv2628 do not induce body fluid immune response. Rv1813c induces active TB patients. A high level of specific antibodies may play a role in the pathogenesis of tuberculosis. In a word, our results provide a basis for the study of its latent infection related antigens, and also provide a rich experimental basis for the study of latent infection markers and latent infection vaccines.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52

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