白念珠菌Sap2快速检测方法的建立

发布时间：2018-06-03 10:03

本文选题：白念珠菌 + 分泌型天冬氨酸蛋白酶　；参考：《中国人民解放军军医进修学院》2012年硕士论文

【摘要】：研究背景：白念珠菌是常见的条件致病性真菌，可引起浅表的皮肤、粘膜损害，也可侵入深部器官引起侵袭性感染。近年来，侵袭性念珠菌病发病率及病死率显著增加，其中白念珠菌感染最为常见，但由于侵袭性白念珠菌感染临床表现缺乏特异性，早期诊断困难导致许多患者得不到及时救治而致病程迁延或死亡，因此建立一种快速准确的白念珠菌检测方法对于挽救患者的生命具有重大意义。分泌性天冬氨酸蛋白酶2是白念珠菌分泌产生的一种胞外水解酶，是白念珠菌致病的重要毒力因子，能降解细胞外基质、角蛋白、粘蛋白及免疫球蛋白等多种蛋白，为白念珠菌提供营养，并促进白念珠菌黏附上皮细胞，侵入宿主造成组织损伤及协助其逃逸宿主防御系统的破坏。当患者系统性感染白念珠菌时，Sap2以可溶性抗原形式存在于血清中。本研究通过建立乳胶凝集试验来检测侵袭性白念珠菌感染小鼠血清内的Sap2抗原，为Sap2抗原检测对侵袭性白念珠菌感染的早期诊断提供依据。研究内容和目的：构建Sap2重组原核表达载体并表达、纯化出可溶性的蛋白作为抗原；制备多克隆抗体，验证抗体的特异性；建立乳胶凝集法测定侵袭性白念珠菌感染的小鼠血清中Sap2抗原，探讨其对侵袭性白念珠菌感染的诊断价值，同时为进一步制备白念珠菌快速鉴定试剂盒奠定基础。研究方法： 1、玻璃珠法提取白念珠菌基因组DNA为模板，根据SAP2全序列和和原核表达载体pMAL-c2x（+）序列的酶切位点设计引物，经PCR方法获取SAP2目的基因。双酶切SAP2基因与原核表达载体pMAL-c2x（+），连接酶切产物，转化大肠杆菌TOP10感受态细菌，筛选菌落和测序鉴定。 2、将pMAL-c2x/SAP2重组质粒转化大肠杆菌BL21(DE3)感受态细胞，在16℃经异丙基-β-D-硫代半乳糖苷酶（IPTG）诱导表达出可溶性的融合蛋白，经直链淀粉树脂亲和层析、蛋白酶Factor Xa切割标签获得纯化的Sap2蛋白。 3、用可溶性Sap2免疫9只BALB/c小鼠，使其产生致敏B淋巴细胞。初次免疫后分别在第14天、21天、28天加强免疫，共4次，免疫第4次后10天收集尾静脉小量收集小鼠血清，用间接ELISA法检测抗血清效价。待确定高效价抗血清产生后，低温离心大量收集血液。用ProteinG亲和层析柱纯化抗血清，通过SDS-PAGE电泳检测抗体纯度，并利用Western blot检测抗体特异性。 4、通过化学交联法用抗Sap2多克隆抗体致敏聚苯乙烯乳胶，并进行偶联条件优化，同时建立侵袭性白念珠菌感染小鼠模型，在白念珠菌攻击小鼠后的第12h、24h、36h、48h、60h、72h、84h、96h、108h、120h取血，分离血清后与致敏胶乳反应，进行结果判定，并与血培养结果比较。研究结果： 1.经PCR扩增获得的目的基因分子量与预计相同,并定向插入原核表达载体pMAL-c2x（+）中，双酶切后电泳获得预期的SAP2条带，经测序证实为正确的SAP2序列，阅读框架正确，可以进行目的蛋白的诱导表达。 2．重组原核表达载体pMAL-c2x/SAP2经IPTG诱导14h后表达出可溶性的融合蛋白，并经纯化、切除标签后得到32mg目的蛋白。 3、成功制备了抗Sap2多克隆抗体，抗体效价大于1:51200，经过亲和层析法纯化后，Western blot检测结果表明其特异性高。 4、偶联条件优化后证实当温度为37℃时，在加入150mg抗体与乳胶偶联4h后偶联效率最高，且致敏乳胶无自凝性，可重复性好。成功建立侵袭性白念珠菌感染的小鼠模型，在白念珠菌接种后12h乳胶凝集法即可检出Sap2抗原。乳胶凝集法检测抗原的阳性率为96.7%，血培养阳性率为90%，，两者差异无统计学意义（χ2=0.25，P0.05）。乳胶凝集法诊断侵袭性白念珠菌感染的特异性为91.2%，敏感性为96.1%。研究结论: 1.克隆出SAP2基因，成功构建了pMAL-c2x/SAP2原核表达载体。 2.经IPTG低温诱导可以表达出可溶性的MBP-Sap2融合蛋白，通过亲和层析及标签切除成功获得Sap2目的蛋白。 3、rSap2通过动物免疫可以制备多克隆抗体，纯化后抗体特异性高。 4、乳胶凝集试验在机体感染白念珠菌12h后即可检测到Sap2，且操作简便快速，特异性和敏感性高，可以为侵袭性白念珠菌感染的早期阶段提供有价值的诊断依据。
[Abstract]:Research background:
Candida albicans, a common conditional pathogenic fungus, can cause superficial skin, mucous membrane damage and invasive infection. In recent years, the incidence and mortality of invasive candidiasis have increased significantly, among which Candida albicans are the most common, but the clinical manifestations of invasive Candida albicans are lack of specificity. It is of great significance to establish a fast and accurate method for the detection of Candida albicans to save the life of the patients.
Secretory aspartic proteinase 2 is a kind of extracellular hydrolase produced by Candida albicans, an important virulence factor of Candida albicans, which can degrade extracellular matrix, keratin, mucin and immunoglobulin, and provide nutrition for Candida albicans, and promote Candida albicans to adhere to epithelial cells and invade host and cause tissue damage. When the patient's system is infected with Candida albicans, Sap2 exists in the form of soluble antigen in the serum. This study has established a latex agglutination test to detect Sap2 antigen in the serum of invasive Candida albicans in mice, for the early detection of the early detection of invasive Candida albicans infection by Sap2. The diagnosis provides the basis.
Research content and purpose:
To construct the Recombinant Prokaryotic expression vector of Sap2 and to express the soluble protein as antigen, to prepare polyclonal antibody and to verify the specificity of the antibody; to establish the latex agglutination method for the determination of Sap2 antigen in the serum of invasive Candida albicans infection in mice, and to explore its diagnostic value for invasive Candida albicans infection and to further prepare it for further preparation. The rapid identification kit for Candida albicans lays the foundation.
1, the glass bead method extracted Candida albicans genomic DNA as a template, designed primers according to the SAP2 full sequence and pMAL-c2x (+) sequence, and obtained the SAP2 target gene through PCR method. Double enzyme cut SAP2 gene and prokaryotic expression vector pMAL-c2x (+), ligase cut products, transformation of Escherichia coli TOP10 receptive bacteria, screening bacterial colonies. And sequencing identification.
2, pMAL-c2x/SAP2 recombinant plasmid was transformed into Escherichia coli BL21 (DE3) receptive cells, and soluble fusion protein was induced by isopropyl - beta -D- Thioglucosidase (IPTG) at 16 C. Pure Sap2 protein was obtained by amylose resin affinity chromatography and protease Factor Xa cutting label.
3, 9 BALB/c mice were immunized with soluble Sap2 to produce sensitized B lymphocytes. After the first immunization, the immune system was strengthened in Fourteenth days, 21 days and 28 days respectively. After 10 days of immunization, the tail vein was collected to collect the serum of mice, and the antiserum titer was detected by indirect ELISA method. Blood was purified by ProteinG affinity chromatography. The purity of the antibody was detected by SDS-PAGE electrophoresis, and the specificity of the antibody was detected by Western blot.
4, the polystyrene latex was sensitized with anti Sap2 polyclonal antibody by chemical crosslinking method, and the coupling conditions were optimized. At the same time, the mice model of invasive Candida albicans infected with Candida albicans, 12h, 24h, 36h, 48h, 60H, 72h, 84h, 96h, 108h, 120h after the attack of Candida albicans were taken, and the serum was separated from the sensitized latex, and the results were determined, and the results were determined, and the results were determined, and Comparison of blood culture results.
1. the molecular weight of the target gene was the same as predicted by PCR, and was directed into the prokaryotic expression vector pMAL-c2x (+). The desired SAP2 band was obtained after double enzyme digestion, and the correct SAP2 sequence was confirmed by sequencing. The reading frame was correct and the target protein could be induced to induce expression.
2. the Recombinant Prokaryotic expression vector pMAL-c2x/SAP2 expressed soluble fusion protein after IPTG induced 14h, and purified, and the 32mg target protein was obtained after the label was removed.
3, the anti Sap2 polyclonal antibody was successfully prepared. The titer of the antibody was more than 1:51200. After purification by affinity chromatography, the specificity of Western blot test showed that the antibody was highly specific.
4, when the coupling condition was optimized, it was proved that when the temperature was 37, the coupling efficiency was highest after adding 150mg antibody and latex coupling 4h, and the sensitized latex had no self coagulability, and the reproducibility was good. The mouse model of invasive Candida albicans infection was successfully established. After inoculation of Candida albicans, the Sap2 antigen could be detected by 12h latex agglutination method. Latex agglutination assay was used to detect the resistance of Candida albicans. The positive rate was 96.7% and the positive rate of blood culture was 90%. The difference was not statistically significant (x 2=0.25, P0.05). The specificity of latex agglutination method for the diagnosis of invasive Candida albicans infection was 91.2%, and the sensitivity was 96.1%. study conclusion:
1. SAP2 gene was cloned and the prokaryotic expression vector of pMAL-c2x/SAP2 was successfully constructed.
2. soluble MBP-Sap2 fusion protein can be expressed by IPTG induction at low temperature, and Sap2 target protein can be obtained by affinity chromatography and label removal.
3, rSap2 can prepare polyclonal antibody through animal immunity, and its specificity is high after purification.
4, latex agglutination test can detect Sap2 after infection of Candida albicans 12h, and the operation is simple, rapid, specific and sensitive. It can provide valuable diagnostic basis for early stage of invasive Candida albicans infection.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756

【参考文献】