本文選題：白色念珠菌 + MP65、Sap2��； 參考：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:白色念珠菌(Candida albicans,CA)是最常見的條件致病菌之一,近二三十年來已成為醫(yī)院內(nèi)感染(如手術(shù)創(chuàng)傷,器官移植和腫瘤治療等免疫抑制劑、廣譜抗生素應(yīng)用)和免疫力低下患者(原發(fā)和繼發(fā)免疫缺陷病,如AIDS)最重要的感染病原之一,系統(tǒng)性感染死亡率為40%~50%。盡管目前有新的抗真菌藥物在應(yīng)用,但是由于它們抗真菌譜窄,出現(xiàn)新的耐藥性,并沒有降低白色念珠菌感染的發(fā)生率和死亡率。因此研究安全有效的免疫制劑會為真菌感染的預(yù)防和治療提供新的途徑,其中真菌疫苗的研究引人矚目。 甘露聚糖-甘露糖蛋白交聯(lián)物是白色念珠菌細(xì)胞壁的重要組成成分,也是重要的粘附素分子,甘露糖蛋白65(65-kD mannoprotein, MP65)是從其酸性提取物中純化出來的主要甘露糖蛋白成分,分子量約65kD,具有很好的免疫原性,其中蛋白核心部分能誘導(dǎo)T細(xì)胞應(yīng)答,對多種念珠菌感染有保護(hù)作用。分泌性酸性蛋白酶2(secretory acid proteinase,Sap2)是白色念珠菌分泌產(chǎn)生的一種最重要的胞外蛋白酶,與白色念珠菌營養(yǎng)作用、粘附、侵襲和破壞組織屏障密切相關(guān),是白色念珠菌致病的重要毒力因子。Sap2是大多數(shù)白色念珠菌廣泛存在的一種抗原,以可溶性抗原形式存在于血清中,念珠菌病人的血清中存在高滴度的抗Sap2的抗體。研究表明以Sap2的白色念珠菌提取物免疫小鼠,可以減弱該菌對粘膜的感染。 因此本研究選取編碼MP65、Sap2蛋白的基因,構(gòu)建其真核表達(dá)質(zhì)粒及原核表達(dá)質(zhì)粒,以真核質(zhì)粒免疫小鼠,分析MP65、Sap2的免疫原性以及其DNA疫苗在抵抗白色念珠菌系統(tǒng)性感染中的免疫保護(hù)作用,為進(jìn)一步研究該蛋白功能及研制其真菌疫苗提供實(shí)驗(yàn)基礎(chǔ)。 方法:1.原核、真核表達(dá)質(zhì)粒的構(gòu)建:熱酚法提取白色念珠菌總RNA,經(jīng)RT-PCR方法擴(kuò)增獲得mp65、sap2目的基因,克隆至pMD18-T載體上進(jìn)行測序,測序結(jié)果正確后,經(jīng)BamH I、EcoR I和EcoR I、XhoI酶切再次鑒定,最后將其分別克隆至原核表達(dá)質(zhì)粒pGEX4T-2、pET32a和真核表達(dá)質(zhì)粒pcDNA3.1中。 2.目的蛋白的表達(dá)、純化與鑒定:將重組原核表達(dá)質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)異丙基-β-D-硫代半乳糖苷酶(IPTG)誘導(dǎo)、超聲裂解菌體、SDS-PAGE電泳觀察結(jié)果,獲得重組表達(dá)的包涵體蛋白。低濃度尿素梯度透析與金屬鎳螯合層析柱純化回收重組蛋白后,對重組蛋白進(jìn)行定量測定。Western blot、ELISA方法初步鑒定其抗原特異性及反應(yīng)性。 3.真核表達(dá)質(zhì)粒的大量提取:堿裂解法分別大量提取真核表達(dá)質(zhì)粒pcDNA3.1-mp65和pcDNA3.1-sap2,純化后定量備用。 4.動物免疫:取40只4周齡BALB/c小鼠隨機(jī)分成4組,每組10只,雌雄各半,分別命名為pcDNA3.1-mp65質(zhì)粒組、pcDNA3.1-sap2質(zhì)粒組、pcDNA3.1-mp65+pcDNA3.1-sap2聯(lián)合質(zhì)粒組及PBS對照組,股四頭肌接種真核表達(dá)質(zhì)粒,每只100μg/次,每次間隔2周,加強(qiáng)免疫3次,每次免疫前Qg眥取血收集血清。 5.免疫效果檢測:間接ELISA法檢測各免疫組血清特異性抗-MP65、抗-Sap2 IgG;流式細(xì)胞術(shù)檢測小鼠脾臟內(nèi)CD4+、CD8+淋巴細(xì)胞變化情況。 6.攻毒實(shí)驗(yàn):末次免疫后20天尾靜脈注射給予小鼠致死量白色念珠菌1×106個/只,15天內(nèi)觀察存活率。結(jié)果:1.經(jīng)RT-PCR方法克隆出全長為1140bp的mp65和1197bp的sap2基因,其核苷酸序列與GenBank中公布的序列基本同源。 2 .成功構(gòu)建了原核表達(dá)質(zhì)粒pGEX4T-2-mp65、pET32a-sap2及真核表達(dá)質(zhì)粒pcDNA3.1-mp65、pcDNA3.1- sap2。構(gòu)建的原核表達(dá)質(zhì)粒pGEX4T-2-mp65和pET32a-sap2轉(zhuǎn)化BL-21,經(jīng)SDS-PAGE和Western blot證實(shí),分別誘導(dǎo)表達(dá)出66KD左右的GST融合蛋白和His融合蛋白。表達(dá)的蛋白主要以包涵體形式存在。 3.真核表達(dá)質(zhì)粒pcDNA3.1-mp65和pcDNA3.1-sap2免疫動物后,各免疫組小鼠血清IgG水平隨免疫時間延長,抗體滴度逐漸升高。末次免疫后,抗-MP65 IgG最高效價可達(dá)到1:1600,抗-Sap2 IgG最高效價可達(dá)到1:3200。流式細(xì)胞術(shù)檢測結(jié)果經(jīng)方差分析顯示,兩組質(zhì)粒免疫后均能誘導(dǎo)小鼠脾臟內(nèi)CD4+T細(xì)胞百分含量增加,與PBS對照組比較有顯著性差異(P0.05)。而CD8+ T細(xì)胞百分含量無明顯差異。兩種質(zhì)粒單獨(dú)免疫組與聯(lián)合免疫組比較也無顯著性差異。 4.白色念珠菌攻毒15天后,pcDNA3.1-mp65質(zhì)粒組及pcDNA3.1-sap2質(zhì)粒組各有20%的存活率,pcDNA3.1-mp65 + pcDNA3.1-sap2聯(lián)合質(zhì)粒組存活率達(dá)到40%,PBS對照組存活率為0%,經(jīng)χ2檢驗(yàn)有顯著差異(P0.05)。 結(jié)論:1.成功構(gòu)建了白色念珠菌基因mp65和sap2的原核表達(dá)質(zhì)粒pGEX4T-2-mp65、pET32a-sap2和真核表達(dá)質(zhì)粒pcDNA3.1-mp65、pcDNA3.1-sap2。 2.MP65和Sap2的原核表達(dá)質(zhì)粒在大腸桿菌BL-21中成功表達(dá)融合蛋白。 3.MP65與Sap2重組真核表達(dá)質(zhì)粒能誘導(dǎo)動物機(jī)體產(chǎn)生體液免疫和細(xì)胞免疫。 4.MP65與Sap2兩種真核表達(dá)質(zhì)粒對白色念珠菌系統(tǒng)性感染均有一定的免疫保護(hù)作用。
[Abstract]:Objective: Candida albicans (Candida albicans CA) is the most common opportunistic pathogen of nearly twenty or thirty years to become nosocomial infections (such as surgical trauma, organ transplantation and tumor treatment and other immunosuppressive agents, broad-spectrum antibiotics) and immunocompromised patients (primary and secondary immunodeficiency diseases, such as infection of AIDS) most important, systemic infection and mortality of 40%~50%. although new antifungal drugs in the application, but because of their narrow antifungal spectrum, the emergence of new drug resistance, and did not decrease the incidence and mortality of Candida albicans infection. So the study of safe and effective immune agents will provide a new way for the prevention and treatment of fungal infections among them, fungi vaccine research focus.
Mannan - mannose protein conjugate is an important component of cell wall of Candida albicans, is also an important adhesion molecule, mannose protein 65 (65-kD mannoprotein MP65) is the main protein component of purified mannose from the acidic extraction, molecular weight of about 65kD, with good immunogenicity, the protein core part can induce T cell response, has a protective effect on Candida albicans infection. A variety of secretory acid proteinase 2 (secretory acid, proteinase, Sap2) is one of the most important extracellular protease secretion of Candida albicans, adhesion and nutrition, Candida albicans, invasion and destruction of the tissue barrier are closely related, is an important virulence factor.Sap2 of Candida albicans is a kind of most pathogenic antigen of Candida albicans exists, in the form of soluble antigen in serum, high serum Candida in patients The anti Sap2 antibody of the titer. The study showed that immunization of mice with Sap2 extract of Candida albicans could weaken the infection of the bacteria to the mucous membrane.
Therefore, this study selected encoding MP65, Sap2 gene, construct its eukaryotic expression vector and prokaryotic expression plasmid, the plasmid immunized mice, analysis of MP65, the immunogenicity of Sap2 DNA vaccine and its immune protective effect against Candida albicans systemic infection, to provide experimental basis for further study of the protein the function and development of fungal vaccines.
Methods: 1. prokaryotic, eukaryotic expression plasmid: Candida albicans total RNA was extracted by the hot phenol method, RT-PCR method amplified mp65 Sap2 gene, cloned into pMD18-T vector and sequenced. The sequencing result, by BamH I, EcoR I and EcoR I, XhoI digestion identification again, finally they were cloned into prokaryotic expression plasmid pGEX4T-2 and eukaryotic expression plasmid of pET32a pcDNA3.1.
2. the expression of target protein, purification and identification of the Recombinant Prokaryotic expression plasmid was transformed into Escherichia coli BL21 (DE3), the isopropyl thiogalactoside beta -D- enzyme (IPTG) induced by ultrasonic degradation bacteria, SDS-PAGE electrophoresis to observe the results of inclusion body protein recombinant expression. Low concentration of purified recombinant protein urea gradient dialysis and nickel chelate chromatography, the recombinant protein was determined.Western blot ELISA method for preliminary identification of its antigen specificity and reactivity.
3. a large number of eukaryotic expression plasmids were extracted: a large amount of eukaryotic expression plasmid pcDNA3.1-mp65 and pcDNA3.1-sap2 were extracted by alkaline lysis.
4. animal immunity: 40 4 week old BALB/c mice were randomly divided into 4 groups, 10 rats in each group, half male and half female, were named pcDNA3.1-mp65 plasmid group, pcDNA3.1-sap2 plasmid group, pcDNA3.1-mp65+pcDNA3.1-sap2 plasmid group and PBS control group, unit four biceps with eukaryotic expression plasmid, each 100 g/ times, each time for 2 weeks the 3 time interval, strengthen immunity, before each vaccination Qg canthus blood serum was collected.
5. immune effect test: indirect ELISA method was used to detect the specific anti -MP65 and anti -Sap2 IgG in each immune group, and the changes of CD4+ and CD8+ lymphocytes in spleen were detected by flow cytometry.
The 6. challenge test: 20 days after the last immunization of mice given intravenous injection of the lethal dose of Candida albicans 1 * 106 / only, 15 days to observe the survival rate by RT-PCR method. Results: 1. cloned cDNA of 1140bp mp65 and 1197bp Sap2 gene sequence of the nucleotide sequence published in GenBank with the basic homologous.
2. we successfully constructed the prokaryotic expression plasmid pGEX4T-2-mp65 and eukaryotic expression plasmid pcDNA3.1-mp65 pET32a-sap2, prokaryotic expression plasmid pGEX4T-2-mp65 and pET32a-sap2 into BL-21 pcDNA3.1- sap2. constructed by SDS-PAGE and Western blot, respectively, confirmed that the induced expression of GST 66KD fusion protein and His fusion protein. The expressed protein mainly exists in the form of inclusion body.
3. eukaryotic expression plasmid pcDNA3.1-mp65 and pcDNA3.1-sap2 immune animal, the immunized mice serum IgG levels with immune time, antibody titer increased gradually. After the last immunization, the highest titer of anti -MP65 IgG can reach 1:1600 and the highest titer of anti -Sap2 IgG can achieve 1:3200. flow cytometry results of variance analysis showed that the two groups immunization were in mouse spleen CD4+T cell percentage induced increased, there was significant difference between control group and PBS (P0.05). No significant difference in the percentage of cells in CD8+ T. Two kinds of plasmid alone group was immunized with CO immunization group compared with no significant difference.
4. after 15 days of Candida albicans attack, the survival rate of pcDNA3.1-mp65 plasmid group and pcDNA3.1-sap2 plasmid group was 20%, and the survival rate of pcDNA3.1-mp65 + pcDNA3.1-sap2 combined plasmid group reached 40%, while the survival rate of PBS control group was 0%, which was significantly different by chi square test (P0.05).
Conclusion: 1.. The prokaryotic expression plasmid pGEX4T-2-mp65, pET32a-sap2 and eukaryotic expression plasmid pcDNA3.1-mp65, pcDNA3.1-sap2. of Candida albicans mp65 and Sap2 were successfully constructed.
The prokaryotic expression plasmid of 2.MP65 and Sap2 expressed the fusion protein successfully in Escherichia coli BL-21.
The recombinant eukaryotic expression plasmid of 3.MP65 and Sap2 can induce humoral and cellular immunity in animal body.
Two eukaryotic expression plasmids of 4.MP65 and Sap2 have protective effects on the systemic infection of Candida albicans.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392;R519

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