本文選題：結(jié)核分枝桿菌 + 潛伏感染�。� 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：目的：獲得結(jié)核分枝桿菌潛伏感染相關(guān)重組蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，并評價其免疫學(xué)特性及其應(yīng)用價值。 方法：應(yīng)用基因工程技術(shù)克隆、表達并純化結(jié)核分枝桿菌潛伏感染相關(guān)蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，并對蛋白表達條件進行優(yōu)化。進一步用純化的重組蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c刺激376例中國人PBMC，其中包括未感染結(jié)核健康對照組103例、結(jié)核潛伏感染組51例、活動性結(jié)核病組116例、BCG接種組49例（Rv1813c無此組）、非結(jié)核呼吸疾病組57例，用ELISPOT技術(shù)檢測分泌IFN-γ的效應(yīng)T細胞斑點數(shù)（SFC），比較其在不同人群中的差異，評價它們在抗結(jié)核潛伏感染細胞免疫應(yīng)答中的作用；用ELISA方法檢測未感染結(jié)核的健康對照組72例、結(jié)核潛伏感染組36例及活動性結(jié)核病組62例共計170例人血清中4種蛋白特異性抗體IgG的水平，并比較組間差異，評價4種潛伏感染相關(guān)蛋白在抗結(jié)核體液免疫方面的作用；以潛伏感染組效應(yīng)T細胞SFC為陽性組，以活動性結(jié)核病組SFC為陰性組，繪制各個潛伏抗原鑒別結(jié)核活動性感染與潛伏感染的ROC曲線，在特異度80%及90%兩個點取SFC的cut-off值，以此評價并且比較潛伏感染蛋白對結(jié)核活動性感染與潛伏感染的鑒別診斷效能。 結(jié)果：1、本研究制備并獲得純度較高的重組蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c，其純度均達到95%以上。2、重組蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c刺激中國潛伏感染人群后分泌IFN-γ的效應(yīng)T細胞數(shù)顯著高于活動性結(jié)核病組（p0.05），也顯著高于未感染正常對照組（p0.05）；Rv2029c、Rv2659c、Rv1813c在非結(jié)核呼吸疾病組顯著低于結(jié)核潛伏感染組（p0.05），Rv2628在非結(jié)核呼吸疾病組低于潛伏感染組，但差異不明顯（p0.05），，與未感染健康對照組無顯著差別（p0.05）；BCG接種3個月后PPD陽轉(zhuǎn)者與健康對照組潛伏蛋白特異的SFC值進行比較，兩者之間差異不明顯（p0.05），BCG接種前后潛伏感染蛋白刺激的SFC值無顯著差異（p0.05）。3、Rv1813c蛋白特異的IgG值在活動性結(jié)核病組顯著高于未感染的健康對照組和潛伏感染組（p0.05），而其余3個蛋白特異的IgG水平在這三組間差異不顯著（p0.05）。4、ROC曲線分析結(jié)果：Rv2029c蛋白的曲線下面積最高達到89.1%，高于Rv2659c（69.9%）、Rv2628（68.8%）及Rv1813c（39%）。Rv2029c、Rv2659c及Rv2628蛋白誘導(dǎo)潛伏感染組產(chǎn)生細胞免疫應(yīng)答的陽性率最高。進一步評價各潛伏蛋白對鑒別結(jié)核活動感染與潛伏感染效能，Rv2029c靈敏度最高，在潛伏感染人群中的陽性率達到80%以上，但在未感染結(jié)核的健康組和活動性結(jié)核病組的假陽性率較高；Rv2659c雖然靈敏度不及Rv2029c，但其在未感染結(jié)核的健康組和活動性結(jié)核病組的假陽性率低；Rv2628的靈敏度與Rv2659c相當，但其假陽性率高于Rv2659c，低于Rv2029c。 結(jié)論：1、本研究成功地構(gòu)建了四種結(jié)核潛伏感染相關(guān)蛋白Rv2029c、Rv2659c、Rv2628及Rv1813c高表達的基因工程菌株，獲得的重組蛋白純度高。2、Rv2029c、Rv2659c、Rv2628及Rv1813c可被結(jié)核潛伏感染人群識別，誘導(dǎo)其效應(yīng)T細胞產(chǎn)生的免疫應(yīng)答強于活動性結(jié)核病患者。3、重組Rv2659c蛋白通過ELISPOT技術(shù)診斷結(jié)核潛伏感染、鑒別活動性結(jié)核感染的效能最高，有望成為針對中國人群結(jié)核潛伏感染的新型診斷標志物。4、Rv2029c、Rv2659c和Rv2628不誘導(dǎo)機體產(chǎn)生體液免疫應(yīng)答，Rv1813c誘導(dǎo)活動性結(jié)核病患者產(chǎn)生高水平的特異性抗體，可能在結(jié)核病致病機制中發(fā)揮作用�？傊�，我們的研究結(jié)果為其潛伏感染相關(guān)抗原的研究奠定了基礎(chǔ)，也為潛伏感染診斷標志物及潛伏感染疫苗的研究提供了豐富的實驗依據(jù)。
[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.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R52

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