结核分枝杆菌特异性抗原Rv2991的克

发布时间：2018-05-24 05:32

本文选题：结核分枝杆菌 + Rv2991　；参考：《郑州大学》2017年硕士论文

【摘要】：背景结核病是一种古老的疾病,是目前传染病的头号杀手,严重威胁人类的健康。结核病的诊断和治疗仍是世界性难题,控制及预防结核病的关键是早期快速诊断结核分枝杆菌感染。目前临床上的诊断方法,并不能早期快速诊断出结核病。临床上常用影像学检查和细菌学检查,敏感度较低,且耗时长,结核菌素皮肤试验假阳性率极高。目前建立在细胞免疫基础上的γ干扰素释放实验(IGRA)近年来得到了迅速发展,但是其并不能区分活动性和潜伏性结核病。暂时还没有发现一种结核分枝杆菌抗原对所有结核病人体内的抗体都能检测出来,因此寻找新型的抗原标志物成为国内外研究的热点。目的本研究旨在克隆表达重组蛋白Rv2991,探索其是否具有结核杆菌特异性抗原的性质,并且评价单一抗原Rv2991和复合ESAT-6/CFP-10/Rv3615c在体液免疫和细胞免疫诊断价值,尤其对于菌阴肺结核诊断的应用价值。方法通过TubercuList检索出Rv2991基因序列,以此为模板,用Primer5.0软件设计引物,然后通过分子克隆方法用pET28a与Rv2991获得重组质粒pET28a-Rv2991,转化入BL21(DE3)PlysS感受态细胞进行表达与纯化,得到重组蛋白Rv2991,然后采用Triton X-114相分离法去除内毒素,显色基质鲎试剂法测量内毒素含量,BCA法测量蛋白浓度。ELISA法评价Rv2991在体液免疫诊断中的敏感性和特异性。分别用Rv2991、复合抗原ESAT-6/CFP-10/Rv3615c作为抗原刺激单个核淋巴细胞(PBMCs),ELISPOT检测斑点形成细胞数,评价其在细胞免疫诊断中的价值。结果1.以重组抗原Rv2991、ESAT-6、CFP-10、复合抗原ESAT-6/CFP-10/Rv3615c为刺激抗原,检测肺结核患者和健康体检者血清IgG水平,这四种抗原在结核组的IgG水平均高于健康对照组,差异具有统计学意义(P0.05)。Rv2991引起肺结核患者血清中IgG抗体的敏感性(24.0%)与CFP-10(26.0%)相当,略低于ESAT-6(32%);Rv2991 IgG抗体的特异性(91.4%)高于ESAT-6(82.9%)和CFP-10(85.7%);复合抗原ESAT-6/CFP-10/Rv3615c引起肺结核患者血清中IgG抗体的敏感性均高于单一抗原。2.Rv2991、ESAT-6、CFP-10、ESAT-6联合CFP-10的敏感性分别为63.2%、73.7%、68.4%、81.6%;特异性分别为93.3%、90.0%、90.0%、86.7%。复合抗原ESAT-6/CFP-10/Rv3615c和ESAT-6、CFP-10检测肺结核病患者、非结核性肺部疾病患者及健康人,对于其中一个抗原,结核菌抗原阳性率在正常对照组、非结核性肺部疾病组2组间比较,差异无统计学意义,而结核病组均高于前2组。复合抗原ESAT-6/CFP-10/Rv3615c的敏感性(87.7%)高于ESAT-6/CFP-10(82.7%)、ESAT-6(76.5%)及CFP-10(74.1%),两两比较差异均无统计学意义(P0.05);总特异性分别为81.8%(54/66)、84.8%(56/66)、84.8%(56/66)、87.9%(58/66),两两比较差异无统计学意义(P0.05)。复合抗原ESAT-6/CFP-10/Rv3615c、EAST-6肽段库和CFP-10肽段库在菌阳肺结核组的阳性率依次为94.0%(47/50)、90.0%(45/50)、78.0%(39/50),在菌阴肺结核组的阳性率依次为65%(13/20)、45.0%(9/20)、60.0%(12/20)。结论(1)本研究成功克隆表达重组蛋白Rv2991,去除原核表达所产生的内毒素,并测量去除后内毒素含量符合免疫试验的应用要求。(2)Rv2991有望成为结核病诊断的候选抗原,复合抗原ESAT-6/CFP-10/Rv3615c有较高的敏感性和特异性,尤其对于菌阴肺结核的诊断具有重要的诊断价值。
[Abstract]:Background tuberculosis is an ancient disease and is the leading killer of infectious diseases. It is a serious threat to human health. Diagnosis and treatment of tuberculosis is still a worldwide problem. The key to the control and prevention of tuberculosis is the early and rapid diagnosis of Mycobacterium tuberculosis infection. At present, the clinical diagnosis method can not quickly diagnose tuberculosis. Disease. Clinical examination and bacteriological examination, low sensitivity, long time and high false positive rate of tuberculin skin test. IGRA, based on cellular immunity, has developed rapidly in recent years, but it does not distinguish between active and latent tuberculosis. Now a Mycobacterium tuberculosis antigen can be detected in all tuberculosis patients, so finding new antigen markers has become a hot spot at home and abroad. The purpose of this study is to clone and express the recombinant protein Rv2991, to explore whether it has the properties of the specific antigen of tuberculosis, and to evaluate the single antigen Rv2991 and the specific antigen. The value of compound ESAT-6/CFP-10/Rv3615c in the diagnosis of humoral and cellular immunity, especially for the diagnosis of bacterial negative pulmonary tuberculosis. Methods the Rv2991 gene sequence was retrieved by TubercuList, and the primers were designed by Primer5.0 software, and then the recombinant plasmid pET28a-Rv2991 was obtained by molecular cloning and pET28a and Rv2991. The expression and purification of the BL21 (DE3) PlysS receptive cells were expressed and purified, and the recombinant protein Rv2991 was obtained. Then the endotoxin was removed by Triton X-114 phase separation, and the content of endotoxin was measured by the chromogenic matrix limulus reagent method. The sensitivity and specificity of Rv2991 in the humoral immunodiagnosis were evaluated by BCA method and the protein concentration was measured with Rv2991. Antigen ESAT-6/CFP-10/Rv3615c as an antigen to stimulate mononuclear lymphocyte (PBMCs), ELISPOT to detect the number of dot forming cells and evaluate its value in the diagnosis of cellular immunity. Results 1. the recombinant antigen Rv2991, ESAT-6, CFP-10, compound antigen ESAT-6/CFP-10/Rv3615c were used to stimulate the antigenic, and the serum IgG level in patients with pulmonary tuberculosis and health examination was detected. The IgG level of these four antigens in the tuberculosis group was higher than that in the healthy control group, and the difference was statistically significant (P0.05).Rv2991 caused the sensitivity of IgG antibody in the serum of the patients with tuberculosis (24%) was equivalent to CFP-10 (26%), slightly lower than ESAT-6 (32%); the specificity of Rv2991 IgG antibody (91.4%) was higher than ESAT-6 (82.9%) and CFP-10 (85.7%); the compound antigen ESAT-6/C was ESAT-6/C. The sensitivity of IgG antibody in serum of patients with pulmonary tuberculosis was higher than that of single antigen.2.Rv2991. ESAT-6, CFP-10, ESAT-6 combined CFP-10 sensitivity was 63.2%, 73.7%, 68.4%, 81.6%, respectively, and the specificity was 93.3%, 90%, 90%, 86.7%. compound antigen ESAT-6/ CFP-10/Rv3615c and ESAT-6 respectively, CFP-10 detected pulmonary tuberculosis patients, non tuberculosis There was no significant difference in the positive rate of Mycobacterium tuberculosis between the 2 groups in the normal control group and the non tuberculous pulmonary disease group, but the tuberculosis group was higher than the first 2 groups. The sensitivity of the compound antigen ESAT-6/CFP-10/Rv3615c (87.7%) was higher than that of the ESAT-6/CFP-10 (82.7%), ESAT-6 (76.5%) and CFP-. 10 (74.1%), 22 of the differences were not statistically significant (P0.05); the total specificity was 81.8% (54/66), 84.8% (56/66), 84.8% (56/66), 87.9% (58/66), and 22 had no statistically significant difference (P0.05). The positive rate of the compound antigen ESAT-6/CFP-10/Rv3615c, EAST-6 peptide library and CFP-10 peptide library in the bacteria positive pulmonary tuberculosis group was 94% (47/50), 90. 0% (45/50), 78% (39/50), the positive rate in the bacterial negative pulmonary tuberculosis group was 65% (13/20), 45% (9/20), 60% (12/20). Conclusion (1) this study successfully cloned the recombinant protein Rv2991, removed the endotoxin produced by the prokaryotic expression, and measured the content of the endotoxin in accordance with the application requirements of the immune test. (2) Rv2991 is expected to be a diagnosis of tuberculosis. The candidate antigen, compound antigen ESAT-6/CFP-10/Rv3615c, has high sensitivity and specificity, especially for the diagnosis of Mycobacterium tuberculosis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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