本文選題：白念珠菌 + 分泌型天冬氨酸蛋白酶��； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2012年碩士論文

【摘要】：研究背景： 白念珠菌是常見的條件致病性真菌，可引起淺表的皮膚、粘膜損害，也可侵入深部器官引起侵襲性感染。近年來，侵襲性念珠菌病發(fā)病率及病死率顯著增加，其中白念珠菌感染最為常見，但由于侵襲性白念珠菌感染臨床表現(xiàn)缺乏特異性，早期診斷困難導(dǎo)致許多患者得不到及時(shí)救治而致病程遷延或死亡，因此建立一種快速準(zhǔn)確的白念珠菌檢測(cè)方法對(duì)于挽救患者的生命具有重大意義。 分泌性天冬氨酸蛋白酶2是白念珠菌分泌產(chǎn)生的一種胞外水解酶，是白念珠菌致病的重要毒力因子，能降解細(xì)胞外基質(zhì)、角蛋白、粘蛋白及免疫球蛋白等多種蛋白，為白念珠菌提供營(yíng)養(yǎng)，并促進(jìn)白念珠菌黏附上皮細(xì)胞，侵入宿主造成組織損傷及協(xié)助其逃逸宿主防御系統(tǒng)的破壞。當(dāng)患者系統(tǒng)性感染白念珠菌時(shí)，Sap2以可溶性抗原形式存在于血清中。本研究通過建立乳膠凝集試驗(yàn)來檢測(cè)侵襲性白念珠菌感染小鼠血清內(nèi)的Sap2抗原，為Sap2抗原檢測(cè)對(duì)侵襲性白念珠菌感染的早期診斷提供依據(jù)。 研究?jī)?nèi)容和目的： 構(gòu)建Sap2重組原核表達(dá)載體并表達(dá)、純化出可溶性的蛋白作為抗原；制備多克隆抗體，驗(yàn)證抗體的特異性；建立乳膠凝集法測(cè)定侵襲性白念珠菌感染的小鼠血清中Sap2抗原，探討其對(duì)侵襲性白念珠菌感染的診斷價(jià)值，同時(shí)為進(jìn)一步制備白念珠菌快速鑒定試劑盒奠定基礎(chǔ)。研究方法： 1、玻璃珠法提取白念珠菌基因組DNA為模板，根據(jù)SAP2全序列和和原核表達(dá)載體pMAL-c2x（+）序列的酶切位點(diǎn)設(shè)計(jì)引物，經(jīng)PCR方法獲取SAP2目的基因。雙酶切SAP2基因與原核表達(dá)載體pMAL-c2x（+），連接酶切產(chǎn)物，轉(zhuǎn)化大腸桿菌TOP10感受態(tài)細(xì)菌，篩選菌落和測(cè)序鑒定。 2、將pMAL-c2x/SAP2重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)感受態(tài)細(xì)胞，在16℃經(jīng)異丙基-β-D-硫代半乳糖苷酶（IPTG）誘導(dǎo)表達(dá)出可溶性的融合蛋白，經(jīng)直鏈淀粉樹脂親和層析、蛋白酶Factor Xa切割標(biāo)簽獲得純化的Sap2蛋白。 3、用可溶性Sap2免疫9只BALB/c小鼠，使其產(chǎn)生致敏B淋巴細(xì)胞。初次免疫后分別在第14天、21天、28天加強(qiáng)免疫，共4次，免疫第4次后10天收集尾靜脈小量收集小鼠血清，用間接ELISA法檢測(cè)抗血清效價(jià)。待確定高效價(jià)抗血清產(chǎn)生后，低溫離心大量收集血液。用ProteinG親和層析柱純化抗血清，通過SDS-PAGE電泳檢測(cè)抗體純度，并利用Western blot檢測(cè)抗體特異性。 4、通過化學(xué)交聯(lián)法用抗Sap2多克隆抗體致敏聚苯乙烯乳膠，并進(jìn)行偶聯(lián)條件優(yōu)化，同時(shí)建立侵襲性白念珠菌感染小鼠模型，在白念珠菌攻擊小鼠后的第12h、24h、36h、48h、60h、72h、84h、96h、108h、120h取血，分離血清后與致敏膠乳反應(yīng)，進(jìn)行結(jié)果判定，并與血培養(yǎng)結(jié)果比較。研究結(jié)果： 1.經(jīng)PCR擴(kuò)增獲得的目的基因分子量與預(yù)計(jì)相同,并定向插入原核表達(dá)載體pMAL-c2x（+）中，雙酶切后電泳獲得預(yù)期的SAP2條帶，經(jīng)測(cè)序證實(shí)為正確的SAP2序列，閱讀框架正確，可以進(jìn)行目的蛋白的誘導(dǎo)表達(dá)。 2．重組原核表達(dá)載體pMAL-c2x/SAP2經(jīng)IPTG誘導(dǎo)14h后表達(dá)出可溶性的融合蛋白，并經(jīng)純化、切除標(biāo)簽后得到32mg目的蛋白。 3、成功制備了抗Sap2多克隆抗體，抗體效價(jià)大于1:51200，經(jīng)過親和層析法純化后，Western blot檢測(cè)結(jié)果表明其特異性高。 4、偶聯(lián)條件優(yōu)化后證實(shí)當(dāng)溫度為37℃時(shí)，在加入150mg抗體與乳膠偶聯(lián)4h后偶聯(lián)效率最高，且致敏乳膠無自凝性，可重復(fù)性好。成功建立侵襲性白念珠菌感染的小鼠模型，在白念珠菌接種后12h乳膠凝集法即可檢出Sap2抗原。乳膠凝集法檢測(cè)抗原的陽性率為96.7%，血培養(yǎng)陽性率為90%，，兩者差異無統(tǒng)計(jì)學(xué)意義（χ2=0.25，P0.05）。乳膠凝集法診斷侵襲性白念珠菌感染的特異性為91.2%，敏感性為96.1%。研究結(jié)論: 1.克隆出SAP2基因，成功構(gòu)建了pMAL-c2x/SAP2原核表達(dá)載體。 2.經(jīng)IPTG低溫誘導(dǎo)可以表達(dá)出可溶性的MBP-Sap2融合蛋白，通過親和層析及標(biāo)簽切除成功獲得Sap2目的蛋白。 3、rSap2通過動(dòng)物免疫可以制備多克隆抗體，純化后抗體特異性高。 4、乳膠凝集試驗(yàn)在機(jī)體感染白念珠菌12h后即可檢測(cè)到Sap2，且操作簡(jiǎn)便快速，特異性和敏感性高，可以為侵襲性白念珠菌感染的早期階段提供有價(jià)值的診斷依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R756

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