【摘要】：白念珠菌是一種常見的條件致病菌,也是念珠菌感染中居于首位的病原菌。近年來,隨著大量免疫抑制劑、化療藥物和廣譜抗生素的廣泛應(yīng)用,侵襲性念珠菌感染(invasive candidiasis, IC)的發(fā)病率和死亡率不斷上升。現(xiàn)階段仍然缺乏能早期、敏感而特異性檢測侵襲性白念珠菌感染的方法。國內(nèi)外研究已經(jīng)把注意力集中于檢測患者血液中的白念珠菌特異性抗原成分及其代謝產(chǎn)物上。白念珠菌烯醇化酶即是目前研究較多的一類免疫優(yōu)勢抗原,檢測病人血清中烯醇化酶抗原及其相應(yīng)抗體可以區(qū)分侵襲性感染和單純的定植,為IC的診斷提供有力的依據(jù)。 目的： 應(yīng)用分子克隆和原核表達(dá)技術(shù),獲得白念珠菌烯醇化酶的全長重組蛋白及N端肽段ENO1-319P；制備相應(yīng)的多克隆抗體；為建立快速診斷侵襲性念珠菌感染新的血清學(xué)檢測方法提供試劑原料。 方法： 以白念珠菌C1標(biāo)準(zhǔn)株基因組DNA作為模板,用PCR法擴(kuò)增烯醇化酶的全長DNA序列,以pET28a(+)為載體,構(gòu)建重組表達(dá)質(zhì)粒,轉(zhuǎn)化大腸埃希菌BL21(DE3),IPTG誘導(dǎo)重組融合蛋白表達(dá)。用抗his6標(biāo)簽的單克隆抗體和白念珠菌抗體陽性的病人血清進(jìn)行Western blot鑒定,TALON金屬親和柱層析純化重組蛋白。以純化的融合蛋白作為抗原分別免疫家兔和山羊制備多克隆抗血清,并檢測抗體特異性。另通過念珠菌與人體細(xì)胞內(nèi)烯醇化酶氨基酸序列的同源性比對尋找同源性低的區(qū)域,并通過原核表達(dá)技術(shù)獲得序列特異性更高的肽段EN01-319P,,并建立檢測病人血清中ENO1-319P抗體的ELISA方法以評估其在侵襲性念珠菌病早期診斷中的價(jià)值。 結(jié)果： 先獲得了含白念珠菌烯醇化酶全長基因的重組表達(dá)載體和相應(yīng)工程菌株,測序結(jié)果證明克隆的烯醇化酶基因序列完全正確,經(jīng)IPTG誘導(dǎo)后能高效表達(dá)重組融合蛋白。使用重組蛋白制備了針對白念珠菌烯醇化酶的兔抗血清及羊抗血清。后來又成功克隆了白念珠菌烯醇化酶N端1-319位氨基酸的肽段,獲得的重組肽段經(jīng)病人血清Western blot鑒定表明有良好的抗原性,以之建立的ELISA抗體檢測方法敏感性為86.4%,特異性95.0%,IC患者陽性率為84.8%(56/66),健康人陽性率為3.0%(6/200)。 結(jié)論： 成功克隆了白念珠菌烯醇化酶及其N端肽段ENO1-319P并在大腸埃希菌中獲得高效表達(dá),并以純化的重組蛋白制備抗ENO1多克隆抗體。為IC的早期診斷建立檢測白念珠菌烯醇化酶抗原和相應(yīng)抗體的血清學(xué)方法打下了基礎(chǔ)。
[Abstract]:Candida albicans is a common conditional pathogen and the first pathogen in Candida infection. In recent years, with the widespread use of a large number of immunosuppressive agents, chemotherapeutic drugs and broad-spectrum antibiotics, the incidence and mortality of invasive Candida invasively infected with (invasive candidiasis, IC) continue to increase. There is still a lack of early, sensitive and specific detection of invasive Candida albicans infection. Studies at home and abroad have focused on the detection of Candida albicans specific antigen components and their metabolites in the blood of patients. Candida albicans enolase is a kind of immune dominant antigen. Detection of enolase antigen and its antibody in serum of patients can distinguish invasive infection from simple colonization, and provide a powerful basis for the diagnosis of IC. Objective: to obtain the full-length recombinant protein of Candida albicans enolase and N-terminal peptide ENO1-319P; to prepare polyclonal antibodies by molecular cloning and prokaryotic expression. To establish a new serological detection method for rapid diagnosis of invasive Candida infection. Methods: the genomic DNA of Candida albicans C1 standard strain was used as template, the full length DNA sequence of enolase was amplified by PCR, and the recombinant expression plasmid was constructed by using pET28a () as vector. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3). IPTG induced the expression of recombinant fusion protein. Anti-his6 labeled monoclonal antibody and Candida albicans antibody positive patient serum were identified by Western blot, and the recombinant protein was purified by TALON metal affinity column chromatography. The purified fusion protein was used as antigen to immunize rabbits and goats respectively to prepare polyclonal antiserum and to detect the specificity of antibody. In addition, the homology of amino acid sequence between Candida spp and human cell enolase was compared to find the region with low homology, and the peptide EN01-319P,, with higher sequence specificity was obtained by prokaryotic expression technique. In order to evaluate the value of ELISA in the early diagnosis of invasive candidiasis, a ELISA method was established to detect the ENO1-319P antibody in the sera of patients with invasive candidiasis. Results: the recombinant expression vector containing the full-length enolase gene of Candida albicans and the corresponding engineering strain were obtained. The sequencing results showed that the cloned enolase gene sequence was completely correct and the recombinant fusion protein was highly expressed after induction by IPTG. Rabbit antiserum and sheep antiserum against Candida albicans enolase were prepared with recombinant protein. Subsequently, the N-terminal amino acid fragment of Candida albicans enolase was cloned successfully. The results of Western blot showed that the recombinant peptide had good antigenicity. The sensitivity of the established ELISA antibody detection method was 86.4%. The specificity was 95.0%, the positive rate of IC patients was 84.8% (56 / 66) and the positive rate of healthy people was 3.0% (6 / 200). Conclusion: Candida albicans enolase and its N-terminal peptide ENO1-319P were cloned and highly expressed in Escherichia coli. The purified recombinant protein was used to prepare anti-ENO1 polyclonal antibody. It lays a foundation for the establishment of a serological method for the detection of Candida albicans enolase antigen and their antibodies in the early diagnosis of IC.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R379

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