【摘要】： 結(jié)核病是由結(jié)核分枝桿菌引起的一種能在人類、家畜、家禽、野生動(dòng)物(如鹿、羚羊、獾和各種野鳥)之間傳播的疾病。20世紀(jì)以來，由于人口劇增、耐藥性的產(chǎn)生、人類免疫缺陷病毒感染流行、免疫抑制劑的使用等原因，結(jié)核病日益嚴(yán)重，在全球范圍內(nèi)又死灰復(fù)燃，成為傳染病的首位殺手。目前，結(jié)核診斷的金標(biāo)準(zhǔn)仍是臨床檢查結(jié)合細(xì)菌培養(yǎng)和涂片直接鏡檢再輔以X線檢查。這些方法均依賴于病人的臨床癥狀，因而不可能發(fā)現(xiàn)亞臨床感染的早期病人。因此，結(jié)核病的快速、準(zhǔn)確診斷，是全球控制結(jié)核病的重要措施。 尋找到結(jié)核分支桿菌的特異性抗原，對(duì)于結(jié)核病的診斷具有重要意義。RD1區(qū)是卡介苗減毒傳代過程中最先缺失的一段區(qū)域，它與結(jié)核分枝桿菌毒力相關(guān)，因?yàn)樵搮^(qū)基因所編碼的蛋白能被宿主免疫系統(tǒng)高度識(shí)別，目前大家對(duì)結(jié)核分枝桿菌診斷試劑的研究主要集中在RD1區(qū)，RD1區(qū)一共編碼9個(gè)蛋白，PPE68是由位于該區(qū)的Rv3873所編碼的蛋白。研究表明PPE68能夠被淋巴細(xì)胞所識(shí)別，激發(fā)出強(qiáng)烈的細(xì)胞免疫，刺激小鼠脾淋巴細(xì)胞增殖，誘導(dǎo)大量IIFN-γ產(chǎn)生，，可以認(rèn)為PPE68是除ESAT-6和CFP-10以外的第3種重要的免疫優(yōu)勢抗原。 本研究利用PCR技術(shù)從結(jié)核桿菌H37Rv國際標(biāo)準(zhǔn)株中擴(kuò)增Rv3873基因，并將其定向克隆pGEX-4T-1中，構(gòu)建重組表達(dá)質(zhì)粒pGRv3873并轉(zhuǎn)化大腸桿菌JM109，用異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)表達(dá)后Western blot鑒定，鑒定成功后采用谷胱苷肽—S—轉(zhuǎn)移酶融合蛋白純化試劑盒對(duì)rPPE68進(jìn)行純化，SDS-PAGE鑒定純化的rPPE68，然后將rPPE68免疫新西蘭大白兔，制備rPPE68多克隆抗體，建立起以PPD、rPPE68為包被抗原檢測結(jié)核病人血清中特異性抗體的間接ELISA檢測方法，為了能進(jìn)一步區(qū)分病人是否為現(xiàn)癥感染，本實(shí)驗(yàn)建立了兔抗rPPE68多克隆抗體作為包被抗體檢測結(jié)核病人血清中特異性抗原的雙抗體夾心ELISA檢測方法，為結(jié)核病的早確診、早治療提供了一種新的技術(shù)思路。在另一方面，本研究將Rv3873基因定向克隆至真核表達(dá)載體pcDNA3.1(+)中，構(gòu)建成Rv3873基因真核表達(dá)載體，為進(jìn)一步研究新型結(jié)核桿菌DNA疫苗奠定了基礎(chǔ)．在另一方面，本研究將Rv3873基因定向克隆至真核表達(dá)載體pcDNA3.1(+)中，構(gòu)建成Rv3873基因真核表達(dá)載體，為進(jìn)一步研究新型結(jié)核桿菌DNA疫苗奠定了基礎(chǔ)。 本研究共分五部分進(jìn)行： 1．通過設(shè)計(jì)結(jié)核分枝桿菌Rv3873特異性引物，用PCR方法從結(jié)核分枝桿菌H37Rv國際標(biāo)準(zhǔn)株中擴(kuò)增出目的基因Rv3873，克隆入高效原核表達(dá)載體pGEX-4T-1中，成功構(gòu)建原核重組質(zhì)粒pGRv3873。 2．原核重組質(zhì)粒pGRv3873，經(jīng)IPTG誘導(dǎo)，成功表達(dá)出約63kDa的rPPE68融合蛋白，并對(duì)蛋白進(jìn)行了包涵體溶解，使其變?yōu)榭扇苄缘鞍住?3．純化rPPE68融合蛋白，用它免疫新西蘭大白兔，成功制備到特異性、高效價(jià)的兔抗rPPE68多克隆抗體。 4．以結(jié)核菌素純化蛋白衍生物(PPD)為包被抗原，通過對(duì)血清稀釋度、封閉液、底物作用時(shí)間及陰陽判定方法等方面的摸索，建立了優(yōu)化后的PPD-ELISA程序，參考此方法，建立了以結(jié)核分枝桿菌特異性蛋白rPPE68為包被抗原檢測結(jié)核病人血清中特異性抗體的間接ELISA檢測方法．分別檢測到PPD、rPPE68的靈敏度為57％、30％，特異性為73.6％、92.7％，分析后認(rèn)為，rPPE68與PPD比較，在結(jié)核病的診斷上顯示了更高的特異性，但是靈敏度卻欠佳。本部分研究還建立了以包被多克隆抗體檢測結(jié)核特異性抗原的雙抗體夾心ELISA方法，經(jīng)檢測發(fā)現(xiàn)，抗rPPE68的靈敏度較低，只有13％，特異性卻很高，達(dá)到93.6％，認(rèn)為以結(jié)核特異性抗體來檢測病人血清中的結(jié)核特異性抗原的ELISA檢測方法是一種診斷是否為結(jié)核現(xiàn)癥感染的可行的新技術(shù)方法。 5．將pGRv3873中目的基因Rv3873亞克隆入pcDNA3.1(+)真核表達(dá)載體，成功構(gòu)建真核重組質(zhì)粒pcRv3873，為進(jìn)一步研究該基因的免疫原性和免疫保護(hù)性奠定了基礎(chǔ)。 綜上所述，本研究選擇結(jié)核分枝桿菌RD1區(qū)Rv3873編碼蛋白PPE68為研究目的蛋白，以此制備得到多克隆抗體，建立起ELISA方法對(duì)結(jié)核患者血清、健康人群血清以及其他非結(jié)核肺病患者血清進(jìn)行檢測，通過與PPD檢測上述人群的對(duì)比，為結(jié)核桿菌血清學(xué)的診斷提供了重要的實(shí)驗(yàn)數(shù)據(jù)。結(jié)果說明rPPE68與抗rPPE68單獨(dú)進(jìn)行診斷研究意義不大，但是可以把它作為結(jié)核血清學(xué)組合候選抗原之一。
[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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 劉東旭;鹿結(jié)核菌野毒株與卡介苗差異基因文庫的構(gòu)建[D];吉林農(nóng)業(yè)大學(xué);2012年

本文編號(hào)：2507639