结核分枝杆菌抗原定量检测技术的建立及其初步评价

发布时间：2018-06-20 20:32

本文选题：结核分枝杆菌 + 特异性抗原　；参考：《中国人民解放军军事医学科学院》2014年硕士论文

【摘要】：结核病(TB)仍然是威胁全球人类健康一个重大的挑战。在2012年估计有新增结核病例860万,死于结核病有130万人,其中有30万人为HIV阳性。据估计,全球范围内的结核病发病率和与人类免疫缺陷病毒(HIV)共感染的病例数在未来数十年内将大幅增加。在世界低收入国家的负担较重,特别是在东南亚和撒哈拉以南的非洲。在这些高负担的地区,结核病诊断的基石仍然是痰涂片镜检。在HIV阴性的患者中,常规临床检测敏感性的变化在35%和80%之间,但在艾滋病毒感染的患者中,敏感性低至20%。此外,在后一组中,痰稀缺性是常见的,这就需要增加操作过程,如诱导痰或支气管镜进行样品采集。传统的痰涂片检查在儿童和肺外结核的患者中的用处也有限。分枝杆菌培养需要几个星期才能提供结果,价格昂贵且需要专门的实验室设施,在大多数高负担地区仍无法使用。当前,较新的T细胞定量检测[γ-干扰素的释放试验(IGRAs)]在高负担的地区没有实用性,同时不能区分潜伏的活动性结核病。核酸扩增试验(NAATs)有限的特异性,使之在结核高负担地区无法广泛使用。结核病诊断仍然需要价格低廉,快速,易于推广的检测技术和产品。迄今为止,大多数已开发的方法是基于特定循环抗体的检测。在很长一段时间内,肺结核的血清学诊断一直处于研究阶段,但现在仍然没有一个具有广泛临床应用价值的检测方法。抗原检测几乎可以肯定证实活动性结核的存在。相比之下,抗体检测并不能确定活动性结核,甚至在清除感染半年后也能检测到抗体反应。我们应该集中更多的力量在直接检测体液中抗原的基础上来开发检测方法。这样的检测方法对患者的诊断及治疗过程中的随访可能是有用的。因为在疾病活跃的阶段,结核病具有高度的传染性,对这种疾病快速,特异的检测,不仅可以遏制结核病的蔓延,同时将帮助医生开展恰当、及时的治疗,又可以减少多重耐药(MDR)以及普遍耐药(XDR)结核分枝杆菌菌株进化的机会。 1983年,在脑脊液中第一次检测到结核抗原,自此以后陆续有学者在痰液和脑脊液中通过ELISA、乳胶凝集法检测到结核分枝杆菌抗原。近期文献报导则以ELISA和胶体金等检测血清、CSF和尿中的PPD衍生物及其特定的菌体成份,如38kD抗原LAM等,其敏感性和特异性分别在20%-94.0%和78.6%-100%。但这些抗原都比较单一,而且大多数商业抗原检测试剂盒不能满足临床的需要,因此这些结核分枝杆菌抗原的检测方法未能广泛应用于诊断活动性结核病。本研究构建了原核重组质粒pBVIL1-38KD、pBVIL1-ESAT6、 pBVIL1-CFP10,经温度诱导在大肠杆菌中高效表达,用离子交换层析方法纯化蛋白；融合蛋白IL1-38KD+ESAT6+CFP10、GST-38KD+ESAT6+CFP10经温度、IPTG诱导表达后,用离子交换层析、亲和层析方法纯化蛋白。将纯化得到的蛋白免疫动物,获得了高效价的单克隆及多克隆抗体。选用Protein G亲和层析法纯化抗体,同时以纯化得到的抗体分别作为包被抗体和检测抗体建立双抗体夹心检测抗原ELISA法,在此基础上建立了化学发光检测结核分枝杆菌抗原的定量检测技术,并进行初步临床评价。本研究共分五部分进行： 1、以结核分枝杆菌H37Rv基因组DNA为模板,经过PCR扩增出结核分枝杆菌38KD, ESAT6, CFP10基因。通过分子克隆方法,成功构建结核分枝杆菌38KD、ESAT6、CFP10基因的原核表达质粒pBVIL1-38KD, pBVIL1-ESAT6, pBVIL1-CFP10。通过42℃水浴诱导质粒pBVIL1-38KD, pBVIL1-ESAT6, pBVIL.1-CFP10在原核表达系统中有效表达出约53kDa的IL1-38KD、21.3kDa的IL1-ESAT6、21.1kDa的IL1-CFP10重组蛋白。通过离子交换层析法对IL1-38KD, IL1-ESAT6, IL1-CFP10重组蛋白进行纯化,经间接EILSA法对纯化的蛋白进行了活性的初步鉴定,结果显示克隆表达的蛋白具有很好的灵敏度和特异性。 2、本研究是基于实验室成功地构建了pBVIL1-38KD+ESAT6+CFP10、 PGEX-38KD+ESAT6+CFP10质粒的基础上。诱导表达后采用离子交换层析及亲和层析对融合蛋白进行纯化,获得了高浓度、高纯度的融合蛋白。对蛋白的活性进行了鉴定,与各分片段蛋白相比,提高了检测敏感性,并具有很好的特异性。 3、将纯化的蛋白免疫大耳白兔,得到了兔抗结核分枝杆菌特异性抗原(38KD、 ESAT6、CFP10和融合抗原38KD+ESAT6+CFP10)的多克隆抗体,效价分别为1：32000,1：16000,1：32000,1：128000。将纯化的蛋白免疫BALB/c小鼠,得到了鼠抗结核分枝杆菌特异性抗原的单克隆抗体,抗38kD的阳性克隆共4株：BBA171、BFE624、BBG411、BEA831,抗体滴度效价分别为1：256000、1：128000、1：256000、1：128000；抗ESAT6的阳性克隆1株：BFF1024,抗体滴度效价为1：512000；抗CFP10的阳性克隆1株：BBG1028,抗体滴度效价为1：256000。 4、以单克隆、多克隆抗体为包被和标记抗体,创建双抗体夹心结核分枝杆菌抗原ELISA检测法。对试剂各反应条件进行了优化,得到了结核分枝杆菌抗原检测方法的最佳反应条件：包被抗体与酶标抗体分别为兔抗38KD+ESAT6+CFP10多抗与HRP-兔抗38KD+ESAT6+CFP10多抗；包被抗体浓度为0.25ug/ml;样品与稀释液按50ul+50ul稀释；酶标抗体浓度为1：600。同时建立了抗原ELISA法的标准曲线(y=0.0218x+0.1387,R2=0.9933),确定了最低检测限(1.11ng/ml)。在结核分枝杆菌抗原ELISA方法的基础上,建立了结核分枝杆菌抗原化学发光定量的检测方法,标准曲线(y=15583x+89992,R2=0.9962),确定了最低检测限(0.46ng/ml)。 5、对建立的结核抗原化学发光定量检测技术进行初步临床评价,对接受抗结核治疗的患者在不同时间段血清中抗原浓度的检测,结果显示患者血清中的结核分枝杆菌特异性抗原会随着治疗时间而逐渐降低。经过对结核患者痰结核分枝杆菌培养上清中结核特异性抗原的检测结果,抗原化学发光技术与结核分枝杆菌培养阳性的符合率较高为92%,同时在结核分枝杆菌培养阴性的上清中,抗原的检出率能达到27.11%。定量分析后结果显示,在培养的这段时间内结核分枝杆菌特异性抗原38KD、ESAT6、CFP10的分泌量主要集中在0.75ng/ml～40ng/ml之间。结核分枝杆菌特异性抗原化学发光检测技术与商业化抗体检测试剂盒共同检测应用,抗原与抗体检测有一定的互补性。综上所述,本研究建立的结核分枝杆菌抗原化学发光定量检测方法,可用于结核病的早期辅助诊断。抗原检测能区分结核患者的既往感染与感染,同时可用于结核病患者治疗的疗效评价,具有一定的临床应用价值。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378.911

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