本文選題：結(jié)核分枝桿菌 + Rv2991　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景結(jié)核病是一種古老的疾病,是目前傳染病的頭號(hào)殺手,嚴(yán)重威脅人類的健康。結(jié)核病的診斷和治療仍是世界性難題,控制及預(yù)防結(jié)核病的關(guān)鍵是早期快速診斷結(jié)核分枝桿菌感染。目前臨床上的診斷方法,并不能早期快速診斷出結(jié)核病。臨床上常用影像學(xué)檢查和細(xì)菌學(xué)檢查,敏感度較低,且耗時(shí)長,結(jié)核菌素皮膚試驗(yàn)假陽性率極高。目前建立在細(xì)胞免疫基礎(chǔ)上的γ干擾素釋放實(shí)驗(yàn)(IGRA)近年來得到了迅速發(fā)展,但是其并不能區(qū)分活動(dòng)性和潛伏性結(jié)核病。暫時(shí)還沒有發(fā)現(xiàn)一種結(jié)核分枝桿菌抗原對(duì)所有結(jié)核病人體內(nèi)的抗體都能檢測(cè)出來,因此尋找新型的抗原標(biāo)志物成為國內(nèi)外研究的熱點(diǎn)。目的本研究旨在克隆表達(dá)重組蛋白R(shí)v2991,探索其是否具有結(jié)核桿菌特異性抗原的性質(zhì),并且評(píng)價(jià)單一抗原Rv2991和復(fù)合ESAT-6/CFP-10/Rv3615c在體液免疫和細(xì)胞免疫診斷價(jià)值,尤其對(duì)于菌陰肺結(jié)核診斷的應(yīng)用價(jià)值。方法通過TubercuList檢索出Rv2991基因序列,以此為模板,用Primer5.0軟件設(shè)計(jì)引物,然后通過分子克隆方法用pET28a與Rv2991獲得重組質(zhì)粒pET28a-Rv2991,轉(zhuǎn)化入BL21(DE3)PlysS感受態(tài)細(xì)胞進(jìn)行表達(dá)與純化,得到重組蛋白R(shí)v2991,然后采用Triton X-114相分離法去除內(nèi)毒素,顯色基質(zhì)鱟試劑法測(cè)量內(nèi)毒素含量,BCA法測(cè)量蛋白濃度。ELISA法評(píng)價(jià)Rv2991在體液免疫診斷中的敏感性和特異性。分別用Rv2991、復(fù)合抗原ESAT-6/CFP-10/Rv3615c作為抗原刺激單個(gè)核淋巴細(xì)胞(PBMCs),ELISPOT檢測(cè)斑點(diǎn)形成細(xì)胞數(shù),評(píng)價(jià)其在細(xì)胞免疫診斷中的價(jià)值。結(jié)果1.以重組抗原Rv2991、ESAT-6、CFP-10、復(fù)合抗原ESAT-6/CFP-10/Rv3615c為刺激抗原,檢測(cè)肺結(jié)核患者和健康體檢者血清IgG水平,這四種抗原在結(jié)核組的IgG水平均高于健康對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。Rv2991引起肺結(jié)核患者血清中IgG抗體的敏感性(24.0%)與CFP-10(26.0%)相當(dāng),略低于ESAT-6(32%);Rv2991 IgG抗體的特異性(91.4%)高于ESAT-6(82.9%)和CFP-10(85.7%);復(fù)合抗原ESAT-6/CFP-10/Rv3615c引起肺結(jié)核患者血清中IgG抗體的敏感性均高于單一抗原。2.Rv2991、ESAT-6、CFP-10、ESAT-6聯(lián)合CFP-10的敏感性分別為63.2%、73.7%、68.4%、81.6%;特異性分別為93.3%、90.0%、90.0%、86.7%。復(fù)合抗原ESAT-6/CFP-10/Rv3615c和ESAT-6、CFP-10檢測(cè)肺結(jié)核病患者、非結(jié)核性肺部疾病患者及健康人,對(duì)于其中一個(gè)抗原,結(jié)核菌抗原陽性率在正常對(duì)照組、非結(jié)核性肺部疾病組2組間比較,差異無統(tǒng)計(jì)學(xué)意義,而結(jié)核病組均高于前2組。復(fù)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%),兩兩比較差異均無統(tǒng)計(jì)學(xué)意義(P0.05);總特異性分別為81.8%(54/66)、84.8%(56/66)、84.8%(56/66)、87.9%(58/66),兩兩比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。復(fù)合抗原ESAT-6/CFP-10/Rv3615c、EAST-6肽段庫和CFP-10肽段庫在菌陽肺結(jié)核組的陽性率依次為94.0%(47/50)、90.0%(45/50)、78.0%(39/50),在菌陰肺結(jié)核組的陽性率依次為65%(13/20)、45.0%(9/20)、60.0%(12/20)。結(jié)論(1)本研究成功克隆表達(dá)重組蛋白R(shí)v2991,去除原核表達(dá)所產(chǎn)生的內(nèi)毒素,并測(cè)量去除后內(nèi)毒素含量符合免疫試驗(yàn)的應(yīng)用要求。(2)Rv2991有望成為結(jié)核病診斷的候選抗原,復(fù)合抗原ESAT-6/CFP-10/Rv3615c有較高的敏感性和特異性,尤其對(duì)于菌陰肺結(jié)核的診斷具有重要的診斷價(jià)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R52

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