结核分枝杆菌免疫优势抗原蛋白PPE68的制备及血清学诊断的研究
发布时间：2019-06-29 07:03
【摘要】： 结核病是由结核分枝杆菌引起的一种能在人类、家畜、家禽、野生动物(如鹿、羚羊、獾和各种野鸟)之间传播的疾病。20世纪以来，由于人口剧增、耐药性的产生、人类免疫缺陷病毒感染流行、免疫抑制剂的使用等原因，结核病日益严重，在全球范围内又死灰复燃，成为传染病的首位杀手。目前，结核诊断的金标准仍是临床检查结合细菌培养和涂片直接镜检再辅以X线检查。这些方法均依赖于病人的临床症状，因而不可能发现亚临床感染的早期病人。因此，结核病的快速、准确诊断，是全球控制结核病的重要措施。 寻找到结核分支杆菌的特异性抗原，对于结核病的诊断具有重要意义。RD1区是卡介苗减毒传代过程中最先缺失的一段区域，它与结核分枝杆菌毒力相关，因为该区基因所编码的蛋白能被宿主免疫系统高度识别，目前大家对结核分枝杆菌诊断试剂的研究主要集中在RD1区，RD1区一共编码9个蛋白，PPE68是由位于该区的Rv3873所编码的蛋白。研究表明PPE68能够被淋巴细胞所识别，激发出强烈的细胞免疫，刺激小鼠脾淋巴细胞增殖，诱导大量IIFN-γ产生，，可以认为PPE68是除ESAT-6和CFP-10以外的第3种重要的免疫优势抗原。 本研究利用PCR技术从结核杆菌H37Rv国际标准株中扩增Rv3873基因，并将其定向克隆pGEX-4T-1中，构建重组表达质粒pGRv3873并转化大肠杆菌JM109，用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后Western blot鉴定，鉴定成功后采用谷胱苷肽—S—转移酶融合蛋白纯化试剂盒对rPPE68进行纯化，SDS-PAGE鉴定纯化的rPPE68，然后将rPPE68免疫新西兰大白兔，制备rPPE68多克隆抗体，建立起以PPD、rPPE68为包被抗原检测结核病人血清中特异性抗体的间接ELISA检测方法，为了能进一步区分病人是否为现症感染，本实验建立了兔抗rPPE68多克隆抗体作为包被抗体检测结核病人血清中特异性抗原的双抗体夹心ELISA检测方法，为结核病的早确诊、早治疗提供了一种新的技术思路。在另一方面，本研究将Rv3873基因定向克隆至真核表达载体pcDNA3.1(+)中，构建成Rv3873基因真核表达载体，为进一步研究新型结核杆菌DNA疫苗奠定了基础．在另一方面，本研究将Rv3873基因定向克隆至真核表达载体pcDNA3.1(+)中，构建成Rv3873基因真核表达载体，为进一步研究新型结核杆菌DNA疫苗奠定了基础。 本研究共分五部分进行： 1．通过设计结核分枝杆菌Rv3873特异性引物，用PCR方法从结核分枝杆菌H37Rv国际标准株中扩增出目的基因Rv3873，克隆入高效原核表达载体pGEX-4T-1中，成功构建原核重组质粒pGRv3873。 2．原核重组质粒pGRv3873，经IPTG诱导，成功表达出约63kDa的rPPE68融合蛋白，并对蛋白进行了包涵体溶解，使其变为可溶性蛋白。 3．纯化rPPE68融合蛋白，用它免疫新西兰大白兔，成功制备到特异性、高效价的兔抗rPPE68多克隆抗体。 4．以结核菌素纯化蛋白衍生物(PPD)为包被抗原，通过对血清稀释度、封闭液、底物作用时间及阴阳判定方法等方面的摸索，建立了优化后的PPD-ELISA程序，参考此方法，建立了以结核分枝杆菌特异性蛋白rPPE68为包被抗原检测结核病人血清中特异性抗体的间接ELISA检测方法．分别检测到PPD、rPPE68的灵敏度为57％、30％，特异性为73.6％、92.7％，分析后认为，rPPE68与PPD比较，在结核病的诊断上显示了更高的特异性，但是灵敏度却欠佳。本部分研究还建立了以包被多克隆抗体检测结核特异性抗原的双抗体夹心ELISA方法，经检测发现，抗rPPE68的灵敏度较低，只有13％，特异性却很高，达到93.6％，认为以结核特异性抗体来检测病人血清中的结核特异性抗原的ELISA检测方法是一种诊断是否为结核现症感染的可行的新技术方法。 5．将pGRv3873中目的基因Rv3873亚克隆入pcDNA3.1(+)真核表达载体，成功构建真核重组质粒pcRv3873，为进一步研究该基因的免疫原性和免疫保护性奠定了基础。 综上所述，本研究选择结核分枝杆菌RD1区Rv3873编码蛋白PPE68为研究目的蛋白，以此制备得到多克隆抗体，建立起ELISA方法对结核患者血清、健康人群血清以及其他非结核肺病患者血清进行检测，通过与PPD检测上述人群的对比，为结核杆菌血清学的诊断提供了重要的实验数据。结果说明rPPE68与抗rPPE68单独进行诊断研究意义不大，但是可以把它作为结核血清学组合候选抗原之一。
[Abstract]:Tuberculosis is a disease that can be spread between humans, livestock, poultry, wild animals (such as deer, antelope, and various wild birds) caused by Mycobacterium tuberculosis. Since the 20th century, the generation of drug resistance, the prevalence of human immunodeficiency virus infection, For reasons such as the use of immunosuppressants, tuberculosis is becoming more and more serious, with a resurgence in the global range, becoming the first killer of infectious diseases. At present, the gold standard for diagnosis of tuberculosis is still a clinical examination combined with bacterial culture and smear direct microscopic examination and supplemented with X-ray examination. These methods rely on the clinical symptoms of the patient, and it is therefore not possible to find an early patient with a sub-clinical infection. Therefore, the rapid and accurate diagnosis of tuberculosis is an important measure of global tuberculosis control. The specific antigen of the Mycobacterium tuberculosis is found, and the diagnosis tool for the tuberculosis It is of great significance that the RD1 region is the first missing region in the attenuated passage of the BCG vaccine, which is related to the virulence of the Mycobacterium tuberculosis, because the protein encoded by the gene can be highly recognized by the host immune system, and the main focus of the research on the diagnostic reagent of the mycobacterium tuberculosis is In the RD1 region, a total of 9 proteins were encoded in the RD1 region, and the PPE68 was formed by Rv3873 located in the region The study shows that the PPE68 can be recognized by the lymphocytes, which can stimulate the proliferation of the spleen and lymphocytes of the mouse, induce a large number of IFN-1 production, and it can be considered that the PPE68 is the third important exemption other than the ESAT-6 and the CFP-10. In this study, the Rv3873 gene was amplified from the International Standard of Mycobacterium tuberculosis H37Rv by PCR, and the recombinant expression plasmid pGRv3873 was cloned into pGEX-4T-1 and transformed into E. coli JM109, and the expression was induced with isopropyl-1-D-thiogalactosylate (IPTG). The purified rPPE68 was purified by SDS-PAGE, and then the rPPE68 was used to immunize New Zealand white rabbits to prepare rPPE68doc. an indirect ELISA detection method for detecting the specific antibody in the serum of the tuberculosis human serum by using the PPD and the rPPE68 as an antigen, and in order to further The method of double-antibody sandwich ELISA for detecting the specific antigen in the sera of the patients with tuberculosis was established by using the anti-rPPE68 polyclonal antibody of the rabbit as a coating antibody, and the early diagnosis and the early treatment of the tuberculosis were established. In that aspect of the present study, the Rv3873 gene is directionally cloned into the eukaryotic expression vector pcDNA3.1 (+) to construct the eukaryotic expression vector of the Rv3873 gene, so as to further study the novel tuberculosis rod. On the other hand, the Rv3873 gene was directionally cloned into the eukaryotic expression vector pcDNA3.1 (+) to construct the eukaryotic expression vector of the Rv3873 gene. The bacterial DNA vaccine lays the foundation This study is divided into five parts:1. The target gene Rv3873 is amplified from the international standard strain of Mycobacterium tuberculosis H37Rv by the PCR method by designing the specific primer of Mycobacterium tuberculosis Rv3873, and then cloned into the high-efficiency prokaryotic expression vector pGEX-4. In T-1, a prokaryotic recombinant plasmid pGRv3872.2 was successfully constructed.2. The recombinant plasmid pGRv3873 was constructed and the rPPE of about 63 kDa was successfully expressed by IPTG. 68. The fusion protein was fused and the inclusion body of the protein was dissolved to make it a soluble protein.3. The rPPE68 fusion protein was purified and used A rabbit anti-rPPE68 polyclonal antibody with specificity and high efficiency was successfully prepared by immunizing New Zealand white rabbits. The optimized PPD-ELISA procedure was established by using the method of time and the determination of yin and yang. The sensitivity of rPPE68 was 57%,30%, 73.6% and 92.7% respectively, and the specificity was 73.6% and 92.7% respectively. In comparison with PPD, rPPE68 showed higher specificity in the diagnosis of tuberculosis, but the sensitivity was poor. The part of this study also established a double-antibody sandwich ELISA method for detecting the specific antigen of tuberculosis by using the coated polyclonal antibody. It is found that the sensitivity of anti-rPPE68 is low, only 13%, the specificity is very high, reaching 93.6%, it is considered that the tuberculosis-specific antibody is used to detect the serum of the patient. The method of ELISA for detecting the specific antigen of tuberculosis is a new and feasible method for diagnosing the infection of tuberculosis.5. The target gene Rv3873 in pGRv3873 is subcloned into the eukaryotic expression of pcDNA3.1 (+). in conclusion, that Rv3873 encode protein PPE68 in the RD1 region of Mycobacterium tuberculosis is selected as the protein for study, in ord to prepare that polyclonal antibody, the serum of the tuberculosis patient, the serum of the healthy people and the serum of the healthy people are established by the ELISA method, The serum of other patients with non-tuberculosis lung diseases is tested, and the comparison of the above population is detected by the PPD, and the important experimental data is provided for the diagnosis of the serology of the tubercle bacillus.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2507639
[Abstract]:Tuberculosis is a disease that can be spread between humans, livestock, poultry, wild animals (such as deer, antelope, and various wild birds) caused by Mycobacterium tuberculosis. Since the 20th century, the generation of drug resistance, the prevalence of human immunodeficiency virus infection, For reasons such as the use of immunosuppressants, tuberculosis is becoming more and more serious, with a resurgence in the global range, becoming the first killer of infectious diseases. At present, the gold standard for diagnosis of tuberculosis is still a clinical examination combined with bacterial culture and smear direct microscopic examination and supplemented with X-ray examination. These methods rely on the clinical symptoms of the patient, and it is therefore not possible to find an early patient with a sub-clinical infection. Therefore, the rapid and accurate diagnosis of tuberculosis is an important measure of global tuberculosis control. The specific antigen of the Mycobacterium tuberculosis is found, and the diagnosis tool for the tuberculosis It is of great significance that the RD1 region is the first missing region in the attenuated passage of the BCG vaccine, which is related to the virulence of the Mycobacterium tuberculosis, because the protein encoded by the gene can be highly recognized by the host immune system, and the main focus of the research on the diagnostic reagent of the mycobacterium tuberculosis is In the RD1 region, a total of 9 proteins were encoded in the RD1 region, and the PPE68 was formed by Rv3873 located in the region The study shows that the PPE68 can be recognized by the lymphocytes, which can stimulate the proliferation of the spleen and lymphocytes of the mouse, induce a large number of IFN-1 production, and it can be considered that the PPE68 is the third important exemption other than the ESAT-6 and the CFP-10. In this study, the Rv3873 gene was amplified from the International Standard of Mycobacterium tuberculosis H37Rv by PCR, and the recombinant expression plasmid pGRv3873 was cloned into pGEX-4T-1 and transformed into E. coli JM109, and the expression was induced with isopropyl-1-D-thiogalactosylate (IPTG). The purified rPPE68 was purified by SDS-PAGE, and then the rPPE68 was used to immunize New Zealand white rabbits to prepare rPPE68doc. an indirect ELISA detection method for detecting the specific antibody in the serum of the tuberculosis human serum by using the PPD and the rPPE68 as an antigen, and in order to further The method of double-antibody sandwich ELISA for detecting the specific antigen in the sera of the patients with tuberculosis was established by using the anti-rPPE68 polyclonal antibody of the rabbit as a coating antibody, and the early diagnosis and the early treatment of the tuberculosis were established. In that aspect of the present study, the Rv3873 gene is directionally cloned into the eukaryotic expression vector pcDNA3.1 (+) to construct the eukaryotic expression vector of the Rv3873 gene, so as to further study the novel tuberculosis rod. On the other hand, the Rv3873 gene was directionally cloned into the eukaryotic expression vector pcDNA3.1 (+) to construct the eukaryotic expression vector of the Rv3873 gene. The bacterial DNA vaccine lays the foundation This study is divided into five parts:1. The target gene Rv3873 is amplified from the international standard strain of Mycobacterium tuberculosis H37Rv by the PCR method by designing the specific primer of Mycobacterium tuberculosis Rv3873, and then cloned into the high-efficiency prokaryotic expression vector pGEX-4. In T-1, a prokaryotic recombinant plasmid pGRv3872.2 was successfully constructed.2. The recombinant plasmid pGRv3873 was constructed and the rPPE of about 63 kDa was successfully expressed by IPTG. 68. The fusion protein was fused and the inclusion body of the protein was dissolved to make it a soluble protein.3. The rPPE68 fusion protein was purified and used A rabbit anti-rPPE68 polyclonal antibody with specificity and high efficiency was successfully prepared by immunizing New Zealand white rabbits. The optimized PPD-ELISA procedure was established by using the method of time and the determination of yin and yang. The sensitivity of rPPE68 was 57%,30%, 73.6% and 92.7% respectively, and the specificity was 73.6% and 92.7% respectively. In comparison with PPD, rPPE68 showed higher specificity in the diagnosis of tuberculosis, but the sensitivity was poor. The part of this study also established a double-antibody sandwich ELISA method for detecting the specific antigen of tuberculosis by using the coated polyclonal antibody. It is found that the sensitivity of anti-rPPE68 is low, only 13%, the specificity is very high, reaching 93.6%, it is considered that the tuberculosis-specific antibody is used to detect the serum of the patient. The method of ELISA for detecting the specific antigen of tuberculosis is a new and feasible method for diagnosing the infection of tuberculosis.5. The target gene Rv3873 in pGRv3873 is subcloned into the eukaryotic expression of pcDNA3.1 (+). in conclusion, that Rv3873 encode protein PPE68 in the RD1 region of Mycobacterium tuberculosis is selected as the protein for study, in ord to prepare that polyclonal antibody, the serum of the tuberculosis patient, the serum of the healthy people and the serum of the healthy people are established by the ELISA method, The serum of other patients with non-tuberculosis lung diseases is tested, and the comparison of the above population is detected by the PPD, and the important experimental data is provided for the diagnosis of the serology of the tubercle bacillus.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 刘东旭;鹿结核菌野毒株与卡介苗差异基因文库的构建[D];吉林农业大学;2012年
本文编号：2507639
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