白念珠菌烯醇化酶及其N端肽段的原核表达和应用研究

发布时间：2019-04-19 12:01

【摘要】：白念珠菌是一种常见的条件致病菌,也是念珠菌感染中居于首位的病原菌。近年来,随着大量免疫抑制剂、化疗药物和广谱抗生素的广泛应用,侵袭性念珠菌感染(invasive candidiasis, IC)的发病率和死亡率不断上升。现阶段仍然缺乏能早期、敏感而特异性检测侵袭性白念珠菌感染的方法。国内外研究已经把注意力集中于检测患者血液中的白念珠菌特异性抗原成分及其代谢产物上。白念珠菌烯醇化酶即是目前研究较多的一类免疫优势抗原,检测病人血清中烯醇化酶抗原及其相应抗体可以区分侵袭性感染和单纯的定植,为IC的诊断提供有力的依据。目的：应用分子克隆和原核表达技术,获得白念珠菌烯醇化酶的全长重组蛋白及N端肽段ENO1-319P；制备相应的多克隆抗体；为建立快速诊断侵袭性念珠菌感染新的血清学检测方法提供试剂原料。方法：以白念珠菌C1标准株基因组DNA作为模板,用PCR法扩增烯醇化酶的全长DNA序列,以pET28a(+)为载体,构建重组表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导重组融合蛋白表达。用抗his6标签的单克隆抗体和白念珠菌抗体阳性的病人血清进行Western blot鉴定,TALON金属亲和柱层析纯化重组蛋白。以纯化的融合蛋白作为抗原分别免疫家兔和山羊制备多克隆抗血清,并检测抗体特异性。另通过念珠菌与人体细胞内烯醇化酶氨基酸序列的同源性比对寻找同源性低的区域,并通过原核表达技术获得序列特异性更高的肽段EN01-319P,,并建立检测病人血清中ENO1-319P抗体的ELISA方法以评估其在侵袭性念珠菌病早期诊断中的价值。结果：先获得了含白念珠菌烯醇化酶全长基因的重组表达载体和相应工程菌株,测序结果证明克隆的烯醇化酶基因序列完全正确,经IPTG诱导后能高效表达重组融合蛋白。使用重组蛋白制备了针对白念珠菌烯醇化酶的兔抗血清及羊抗血清。后来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,获得的重组肽段经病人血清Western blot鉴定表明有良好的抗原性,以之建立的ELISA抗体检测方法敏感性为86.4%,特异性95.0%,IC患者阳性率为84.8%(56/66),健康人阳性率为3.0%(6/200)。结论：成功克隆了白念珠菌烯醇化酶及其N端肽段ENO1-319P并在大肠埃希菌中获得高效表达,并以纯化的重组蛋白制备抗ENO1多克隆抗体。为IC的早期诊断建立检测白念珠菌烯醇化酶抗原和相应抗体的血清学方法打下了基础。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R379

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