本文選題：結(jié)核分支桿菌　切入點(diǎn)：嵌合蛋白　出處：《蘇州大學(xué)》2008年碩士論文

【摘要】： 目的構(gòu)建結(jié)核桿菌嵌合蛋白Ag856A2的原核表達(dá)載體,進(jìn)行原核表達(dá)、純化和抗原性分析,制備該蛋白的單克隆抗體,并鑒定單抗的重要性質(zhì)。方法以DNA疫苗HG856A為模板,經(jīng)PCR擴(kuò)增獲得ag856a2基因,克隆至pET28a質(zhì)粒得到原核表達(dá)質(zhì)粒pET856A2;用該重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),以IPTG誘導(dǎo)目的基因表達(dá),通過SDS-PAGE鑒定其表達(dá)形式;用Ni2+柱親和層析純化嵌合蛋白Ag856A2;分別用Ag85A及ESAT-6的小鼠抗血清對(duì)純化的蛋白Ag856A2進(jìn)行Western Blotting分析其抗原性。用DNA疫苗HG856A和純化的嵌合蛋白Ag856A2免疫BALB/C小鼠,取脾細(xì)胞和骨髓瘤細(xì)胞SP2/0融合,通過HAT培養(yǎng)基選擇性培養(yǎng),間接ELISA法篩選陽性克隆,用有限稀釋法將陽性克隆單克隆化,再用間接ELISA法篩選、鑒定,最終建立穩(wěn)定分泌單克隆抗體的雜交瘤細(xì)胞株;分別用間接ELISA法鑒定單克隆抗體的亞類、效價(jià)及所識(shí)別的抗原,用Western Blotting分析識(shí)別蛋白ESAT-6的單克隆抗體的特異性。結(jié)果成功構(gòu)建了重組質(zhì)粒pET856A2,嵌合蛋白Ag856A2以包涵體形式表達(dá),約占菌體總蛋白的35%;經(jīng)Ni2+柱親和純化的樣品SDS-PAGE分析純度在90%以上,Ag85A及ESAT-6抗血清均可與其發(fā)生特異性反應(yīng)。融合后經(jīng)篩選、克隆化得到6株抗Ag856A2的雜交瘤細(xì)胞,經(jīng)鑒定有五株可同時(shí)與蛋白Ag856A2、Ag85A反應(yīng),為IgG1亞類;一株可同時(shí)與蛋白Ag856A2、ESAT-6反應(yīng),為IgG2b亞類,其培養(yǎng)上清與蛋白ESAT-6反應(yīng)效價(jià)為6400,與蛋白Ag856A2反應(yīng)效價(jià)為12800;該株單克隆抗體經(jīng)Western Blotting鑒定可與蛋白Ag856A2、ESAT-6發(fā)生特異性反應(yīng)。結(jié)論以包涵體形式表達(dá)的嵌合蛋白Ag856A2具有Ag85A及ESAT-6的抗原性;以其DNA疫苗和純化蛋白免疫小鼠后,初步建立起一株分泌抗蛋白ESAT-6單克隆抗體的雜交瘤細(xì)胞株。本研究為進(jìn)一步研究該嵌合蛋白在結(jié)核病亞單位疫苗中的應(yīng)用打下了基礎(chǔ),為抗蛋白ESAT-6單克隆抗體在結(jié)核病早期診斷上的應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Objective to construct the prokaryotic expression vector of the chimeric protein Ag856A2 of Mycobacterium tuberculosis for prokaryotic expression, purification and antigenicity analysis, to prepare the monoclonal antibody against the protein and to identify the important properties of the monoclonal antibody.Methods DNA vaccine HG856A was used as template, ag856a2 gene was amplified by PCR and cloned into pET28a plasmid to obtain prokaryotic expression plasmid pET856A2. The recombinant plasmid was transformed into E. coli BL21DE-3, and the target gene was induced by IPTG, and its expression form was identified by SDS-PAGE.The chimeric protein Ag856A2 was purified by Ni2 affinity chromatography and its antigenicity was analyzed by Western Blotting with the mouse antiserum of Ag85A and ESAT-6.BALB/C mice were immunized with DNA vaccine HG856A and purified chimeric protein Ag856A2. The spleen cells and myeloma cells SP2/0 were fused. The positive clones were screened by indirect ELISA method in HAT medium, and the positive clones were monoclonal by limited dilution method.Then indirect ELISA method was used to screen and identify the hybridoma cell lines secreting monoclonal antibodies stably, and the subclasses, titers and recognized antigens of monoclonal antibodies were identified by indirect ELISA method, respectively.The specificity of monoclonal antibody to recognize protein ESAT-6 was analyzed by Western Blotting.Results the recombinant plasmid pET856A2 was successfully constructed, and the chimeric protein Ag856A2 was expressed in the form of inclusion body, accounting for about 35% of the total bacterial protein, and the purity of the sample SDS-PAGE purified by Ni2 column affinity was more than 90%, and the specific reaction of Ag85A and ESAT-6 antiserum could be observed.After fusion, six hybridoma cells resistant to Ag856A2 were obtained. Five of them could react with the protein Ag856A2, Ag85A, and one could react with Ag856A2, ESAT-6, which were IgG2b subclasses at the same time, and five of them could react with Ag856A2A2Ag85A at the same time, and one of them could react with Ag856A2ESAT-6 at the same time.The supernatant reacted with protein ESAT-6 and protein Ag856A2 with the titer of 6400 and 12800.The monoclonal antibody was identified by Western Blotting to react specifically with protein Ag856A2ESAT-6.Conclusion the chimeric protein Ag856A2 expressed in the form of inclusion body has the antigenicity of Ag85A and ESAT-6. After immunizing mice with its DNA vaccine and purified protein, a hybridoma cell line secreting monoclonal antibody to protein ESAT-6 was established.This study laid a foundation for further study on the application of the chimeric protein in tuberculosis subunit vaccine and laid a foundation for the application of anti-protein ESAT-6 monoclonal antibody in the early diagnosis of tuberculosis.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【參考文獻(xiàn)】

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1 何國鈞,肖和平;肺結(jié)核病診斷進(jìn)展[J];中華結(jié)核和呼吸雜志;2001年08期

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本文編號(hào)：1692764