本文選題：結(jié)核分枝桿菌 + 特異性抗原��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：結(jié)核病(TB)仍然是威脅全球人類健康一個重大的挑戰(zhàn)。在2012年估計有新增結(jié)核病例860萬,死于結(jié)核病有130萬人,其中有30萬人為HIV陽性。據(jù)估計,全球范圍內(nèi)的結(jié)核病發(fā)病率和與人類免疫缺陷病毒(HIV)共感染的病例數(shù)在未來數(shù)十年內(nèi)將大幅增加。在世界低收入國家的負(fù)擔(dān)較重,特別是在東南亞和撒哈拉以南的非洲。 在這些高負(fù)擔(dān)的地區(qū),結(jié)核病診斷的基石仍然是痰涂片鏡檢。在HIV陰性的患者中,常規(guī)臨床檢測敏感性的變化在35%和80%之間,但在艾滋病毒感染的患者中,敏感性低至20%。此外,在后一組中,痰稀缺性是常見的,這就需要增加操作過程,如誘導(dǎo)痰或支氣管鏡進(jìn)行樣品采集。傳統(tǒng)的痰涂片檢查在兒童和肺外結(jié)核的患者中的用處也有限。分枝桿菌培養(yǎng)需要幾個星期才能提供結(jié)果,價格昂貴且需要專門的實驗室設(shè)施,在大多數(shù)高負(fù)擔(dān)地區(qū)仍無法使用。當(dāng)前,較新的T細(xì)胞定量檢測[γ-干擾素的釋放試驗(IGRAs)]在高負(fù)擔(dān)的地區(qū)沒有實用性,同時不能區(qū)分潛伏的活動性結(jié)核病。核酸擴(kuò)增試驗(NAATs)有限的特異性,使之在結(jié)核高負(fù)擔(dān)地區(qū)無法廣泛使用。結(jié)核病診斷仍然需要價格低廉,快速,易于推廣的檢測技術(shù)和產(chǎn)品。 迄今為止,大多數(shù)已開發(fā)的方法是基于特定循環(huán)抗體的檢測。在很長一段時間內(nèi),肺結(jié)核的血清學(xué)診斷一直處于研究階段,但現(xiàn)在仍然沒有一個具有廣泛臨床應(yīng)用價值的檢測方法�？乖瓩z測幾乎可以肯定證實活動性結(jié)核的存在。相比之下,抗體檢測并不能確定活動性結(jié)核,甚至在清除感染半年后也能檢測到抗體反應(yīng)。我們應(yīng)該集中更多的力量在直接檢測體液中抗原的基礎(chǔ)上來開發(fā)檢測方法。這樣的檢測方法對患者的診斷及治療過程中的隨訪可能是有用的。因為在疾病活躍的階段,結(jié)核病具有高度的傳染性,對這種疾病快速,特異的檢測,不僅可以遏制結(jié)核病的蔓延,同時將幫助醫(yī)生開展恰當(dāng)、及時的治療,又可以減少多重耐藥(MDR)以及普遍耐藥(XDR)結(jié)核分枝桿菌菌株進(jìn)化的機(jī)會。 1983年,在腦脊液中第一次檢測到結(jié)核抗原,自此以后陸續(xù)有學(xué)者在痰液和腦脊液中通過ELISA、乳膠凝集法檢測到結(jié)核分枝桿菌抗原。近期文獻(xiàn)報導(dǎo)則以ELISA和膠體金等檢測血清、CSF和尿中的PPD衍生物及其特定的菌體成份,如38kD抗原LAM等,其敏感性和特異性分別在20%-94.0%和78.6%-100%。但這些抗原都比較單一,而且大多數(shù)商業(yè)抗原檢測試劑盒不能滿足臨床的需要,因此這些結(jié)核分枝桿菌抗原的檢測方法未能廣泛應(yīng)用于診斷活動性結(jié)核病。 本研究構(gòu)建了原核重組質(zhì)粒pBVIL1-38KD、pBVIL1-ESAT6、 pBVIL1-CFP10,經(jīng)溫度誘導(dǎo)在大腸桿菌中高效表達(dá),用離子交換層析方法純化蛋白；融合蛋白IL1-38KD+ESAT6+CFP10、GST-38KD+ESAT6+CFP10經(jīng)溫度、IPTG誘導(dǎo)表達(dá)后,用離子交換層析、親和層析方法純化蛋白。將純化得到的蛋白免疫動物,獲得了高效價的單克隆及多克隆抗體。選用Protein G親和層析法純化抗體,同時以純化得到的抗體分別作為包被抗體和檢測抗體建立雙抗體夾心檢測抗原ELISA法,在此基礎(chǔ)上建立了化學(xué)發(fā)光檢測結(jié)核分枝桿菌抗原的定量檢測技術(shù),并進(jìn)行初步臨床評價。 本研究共分五部分進(jìn)行： 1、以結(jié)核分枝桿菌H37Rv基因組DNA為模板,經(jīng)過PCR擴(kuò)增出結(jié)核分枝桿菌38KD, ESAT6, CFP10基因。通過分子克隆方法,成功構(gòu)建結(jié)核分枝桿菌38KD、ESAT6、CFP10基因的原核表達(dá)質(zhì)粒pBVIL1-38KD, pBVIL1-ESAT6, pBVIL1-CFP10。通過42℃水浴誘導(dǎo)質(zhì)粒pBVIL1-38KD, pBVIL1-ESAT6, pBVIL.1-CFP10在原核表達(dá)系統(tǒng)中有效表達(dá)出約53kDa的IL1-38KD、21.3kDa的IL1-ESAT6、21.1kDa的IL1-CFP10重組蛋白。通過離子交換層析法對IL1-38KD, IL1-ESAT6, IL1-CFP10重組蛋白進(jìn)行純化,經(jīng)間接EILSA法對純化的蛋白進(jìn)行了活性的初步鑒定,結(jié)果顯示克隆表達(dá)的蛋白具有很好的靈敏度和特異性。 2、本研究是基于實驗室成功地構(gòu)建了pBVIL1-38KD+ESAT6+CFP10、 PGEX-38KD+ESAT6+CFP10質(zhì)粒的基礎(chǔ)上。誘導(dǎo)表達(dá)后采用離子交換層析及親和層析對融合蛋白進(jìn)行純化,獲得了高濃度、高純度的融合蛋白。對蛋白的活性進(jìn)行了鑒定,與各分片段蛋白相比,提高了檢測敏感性,并具有很好的特異性。 3、將純化的蛋白免疫大耳白兔,得到了兔抗結(jié)核分枝桿菌特異性抗原(38KD、 ESAT6、CFP10和融合抗原38KD+ESAT6+CFP10)的多克隆抗體,效價分別為1：32000,1：16000,1：32000,1：128000。將純化的蛋白免疫BALB/c小鼠,得到了鼠抗結(jié)核分枝桿菌特異性抗原的單克隆抗體,抗38kD的陽性克隆共4株：BBA171、BFE624、BBG411、BEA831,抗體滴度效價分別為1：256000、1：128000、1：256000、1：128000；抗ESAT6的陽性克隆1株：BFF1024,抗體滴度效價為1：512000；抗CFP10的陽性克隆1株：BBG1028,抗體滴度效價為1：256000。 4、以單克隆、多克隆抗體為包被和標(biāo)記抗體,創(chuàng)建雙抗體夾心結(jié)核分枝桿菌抗原ELISA檢測法。對試劑各反應(yīng)條件進(jìn)行了優(yōu)化,得到了結(jié)核分枝桿菌抗原檢測方法的最佳反應(yīng)條件：包被抗體與酶標(biāo)抗體分別為兔抗38KD+ESAT6+CFP10多抗與HRP-兔抗38KD+ESAT6+CFP10多抗；包被抗體濃度為0.25ug/ml;樣品與稀釋液按50ul+50ul稀釋；酶標(biāo)抗體濃度為1：600。同時建立了抗原ELISA法的標(biāo)準(zhǔn)曲線(y=0.0218x+0.1387,R2=0.9933),確定了最低檢測限(1.11ng/ml)。在結(jié)核分枝桿菌抗原ELISA方法的基礎(chǔ)上,建立了結(jié)核分枝桿菌抗原化學(xué)發(fā)光定量的檢測方法,標(biāo)準(zhǔn)曲線(y=15583x+89992,R2=0.9962),確定了最低檢測限(0.46ng/ml)。 5、對建立的結(jié)核抗原化學(xué)發(fā)光定量檢測技術(shù)進(jìn)行初步臨床評價,對接受抗結(jié)核治療的患者在不同時間段血清中抗原濃度的檢測,結(jié)果顯示患者血清中的結(jié)核分枝桿菌特異性抗原會隨著治療時間而逐漸降低。經(jīng)過對結(jié)核患者痰結(jié)核分枝桿菌培養(yǎng)上清中結(jié)核特異性抗原的檢測結(jié)果,抗原化學(xué)發(fā)光技術(shù)與結(jié)核分枝桿菌培養(yǎng)陽性的符合率較高為92%,同時在結(jié)核分枝桿菌培養(yǎng)陰性的上清中,抗原的檢出率能達(dá)到27.11%。定量分析后結(jié)果顯示,在培養(yǎng)的這段時間內(nèi)結(jié)核分枝桿菌特異性抗原38KD、ESAT6、CFP10的分泌量主要集中在0.75ng/ml～40ng/ml之間。結(jié)核分枝桿菌特異性抗原化學(xué)發(fā)光檢測技術(shù)與商業(yè)化抗體檢測試劑盒共同檢測應(yīng)用,抗原與抗體檢測有一定的互補(bǔ)性。 綜上所述,本研究建立的結(jié)核分枝桿菌抗原化學(xué)發(fā)光定量檢測方法,可用于結(jié)核病的早期輔助診斷�？乖瓩z測能區(qū)分結(jié)核患者的既往感染與感染,同時可用于結(jié)核病患者治療的療效評價,具有一定的臨床應(yīng)用價值。
[Abstract]:Tuberculosis (TB) remains a major challenge to global human health. In 2012, 8 million 600 thousand new cases of tuberculosis were estimated. There were 1 million 300 thousand people who died of tuberculosis, of which 300 thousand were HIV positive. It is estimated that the global incidence of tuberculosis and the number of cases co infected with human immunodeficiency virus (HIV) will be larger in the next few decades. The increase is heavier in the low income countries of the world, especially in Southeast Asia and sub Saharan Africa.
In these high burdens, the cornerstone of tuberculosis diagnosis remains the sputum smear examination. In HIV negative patients, the changes in routine clinical sensitivity are between 35% and 80%, but in the patients with HIV infection, the sensitivity is low to 20%., and in the latter group, the phlegm deficiency is common, which requires an increase in the operation process, such as lure. Samples of sputum or bronchoscopy are collected. Traditional sputum smears are also limited in children and patients with extrapulmonary tuberculosis. Mycobacterium culture takes weeks to provide results, expensive and specialized laboratory facilities, which are still unusable in most high burdens. Current, new T cell quantitative detection [ Interferon - gamma release test (IGRAs)] is not practical in high burden areas and can not distinguish latent active tuberculosis. The limited specificity of nucleic acid amplification test (NAATs) makes it not widely used in the area of tuberculosis high burden. The diagnosis of tuberculosis still needs low price, rapid and easy to popularize detection techniques and products.
So far, most of the developed methods have been based on specific circulating antibodies. In a long period of time, the serological diagnosis of tuberculosis has been at the research stage, but there is still not a wide clinical application of detection methods. Antigen detection can almost confirm the existence of active tuberculosis. Antibody tests do not determine active tuberculosis and even detect antibody responses after six months of infection. We should concentrate more forces on the development of detection methods based on direct detection of antigen in body fluids. This method may be useful for the diagnosis and treatment of patients. At the active stage of the disease, tuberculosis is highly contagious. Rapid and specific detection of this disease can not only prevent the spread of tuberculosis, but also help doctors to carry out appropriate and timely treatment, and also reduce the opportunity for the evolution of multidrug resistance (MDR) and general resistance (XDR) Mycobacterium tuberculosis (MDR) strains.
In 1983, tuberculosis antigen was first detected in cerebrospinal fluid. Since then, some scholars have detected Mycobacterium tuberculosis antigen by ELISA and latex agglutination in sputum and cerebrospinal fluid. In the recent literature, ELISA and colloidal gold were used to detect serum, CSF and PPD derivatives in urine and specific body components, such as 38kD antigen LAM, and so on. The sensitivity and specificity of these antigens are 20%-94.0% and 78.6%-100%., respectively, but these antigens are relatively single, and most of the commercial antigen detection kits can not meet the clinical needs. Therefore, the detection methods of these Mycobacterium tuberculosis antigens have not been widely used in the diagnosis of active tuberculosis.
In this study, the Prokaryotic Recombinant Plasmid pBVIL1-38KD, pBVIL1-ESAT6, pBVIL1-CFP10 were constructed, and the protein was purified by temperature induction in Escherichia coli. The protein was purified by ion exchange chromatography, the fusion protein IL1-38KD+ESAT6+CFP10, GST-38KD+ESAT6+CFP10 was induced by temperature, IPTG, and purified by ion exchange chromatography and affinity chromatography. The purified protein was immunized with a high efficient monoclonal and polyclonal antibody. The Protein G affinity chromatography was used to purify the antibody, and the purified antibody was used as the inclusion antibody and the detection antibody to establish the double antibody sandwich detection antigen ELISA method respectively. On this basis, the chemiluminescence detection of tuberculosis branch rod was established. Quantitative detection of bacterial antigens and preliminary clinical evaluation.
This study is divided into five parts:
1, the Mycobacterium tuberculosis H37Rv genome DNA was used as the template and the Mycobacterium tuberculosis 38KD, ESAT6, and CFP10 genes were amplified by PCR. By molecular cloning, the prokaryotic expression plasmid of Mycobacterium tuberculosis 38KD, ESAT6, CFP10 gene was successfully constructed pBVIL1-38KD, pBVIL1-ESAT6, and pBVIL1-CFP10. was induced by 42 centigrade water bath. SAT6, pBVIL.1-CFP10 effectively expressed 53kDa IL1-38KD, 21.3kDa IL1-ESAT6,21.1kDa IL1-CFP10 recombinant protein in the prokaryotic expression system. The recombinant protein of IL1-38KD, IL1-ESAT6, IL1-CFP10 was purified by ion exchange chromatography, and the activity of the purified protein was preliminarily identified by indirect EILSA method. The results showed that the clone was cloned. The expressed protein has good sensitivity and specificity.
2, this study was based on the successful construction of pBVIL1-38KD+ESAT6+CFP10, PGEX-38KD+ESAT6+CFP10 plasmid. After induction, the fusion protein was purified by ion exchange chromatography and affinity chromatography. The fusion protein with high concentration and high purity was obtained. The activity of the protein was identified and compared with the fragment protein. It has improved sensitivity and specificity.
3, the purified protein was immunized with the rabbit, the polyclonal antibody against the specific antigen of Mycobacterium tuberculosis (38KD, ESAT6, CFP10 and fusion antigen 38KD+ESAT6+CFP10) was obtained. The potency was 1:32000,1:16000,1:32000,1:128000. and the purified protein was immunized with the BALB/c mice, and the specific antigen of Mycobacterium tuberculosis was obtained. The monoclonal antibody and anti 38kD positive clones were 4 strains: BBA171, BFE624, BBG411, BEA831, the titer of antibody titer was 1:256000,1:128000,1:256000,1:128000, the positive clone of anti ESAT6 was 1: BFF1024, the titer of antibody titer was 1:512000, 1 strains of anti CFP10 positive clones: BBG1028, antibody titer titer titer of antibody titer 1:256000.
4, using monoclonal and polyclonal antibody as inclusion and labeling antibody, the detection method of double antibody sandwich Mycobacterium tuberculosis antigen ELISA was established. The reaction conditions of the reagents were optimized. The optimum reaction conditions of the antigen detection method of Mycobacterium tuberculosis were obtained: the antibody and the enzyme labeled antibody were the Rabbit anti 38KD+ESAT6+CFP10 polyclonal and HRP- rabbit respectively. Anti 38KD+ESAT6+CFP10 polyclonal antibody; the concentration of the antibody was 0.25ug/ml; the samples and diluents were diluted by 50ul+50ul; the concentration of the enzyme labeled antibody was 1:600. and the standard curve of the antigen ELISA method (y=0.0218x+0.1387, R2=0.9933) was established, and the minimum detection limit (1.11ng/ml) was determined. On the basis of the ELISA method of Mycobacterium tuberculosis, a conclusion was established. The standard method (y=15583x+89992, R2=0.9962) for the quantitative chemiluminescence detection of Mycobacterium tuberculosis was determined, and the minimum detection limit (0.46ng/ml) was determined.
5, a preliminary clinical evaluation of the established quantitative chemiluminescence detection of tuberculosis antigen was carried out. The results showed that the specific antigen of Mycobacterium tuberculosis in the patient's serum gradually decreased with the time of treatment. The detection results of tuberculosis specific antigen in the bacillus culture supernatant showed that the coincidence rate of antigen chemiluminescence technique and Mycobacterium tuberculosis positive was 92%. At the same time, the detection rate of antigen could reach 27.11%. quantitative analysis in the negative supernatant of Mycobacterium tuberculosis, and the Mycobacterium tuberculosis was found during this period of culture. The secretion of specific antigen 38KD, ESAT6 and CFP10 is mainly concentrated between 0.75ng/ml and 40ng/ml. The specific antigen chemiluminescence detection technology of Mycobacterium tuberculosis and commercial antibody detection kit are used together to detect the complementarity of antigen and antibody detection.
In conclusion, the quantitative chemiluminescence detection method of Mycobacterium tuberculosis established in this study can be used in the early diagnosis of tuberculosis. The antigen detection can distinguish the past infection and infection of tuberculosis patients, and can be used in the evaluation of the curative effect of tuberculosis patients, which has a definite clinical value.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R378.911

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