MP65和Sap2重组表达质粒对小鼠系统性白色念珠菌感染的免疫保护作用研究

发布时间：2018-04-18 19:55

本文选题：白色念珠菌 + MP65、Sap2　；参考：《河北医科大学》2009年硕士论文

【摘要】： 目的:白色念珠菌(Candida albicans,CA)是最常见的条件致病菌之一,近二三十年来已成为医院内感染(如手术创伤,器官移植和肿瘤治疗等免疫抑制剂、广谱抗生素应用)和免疫力低下患者(原发和继发免疫缺陷病,如AIDS)最重要的感染病原之一,系统性感染死亡率为40%~50%。尽管目前有新的抗真菌药物在应用,但是由于它们抗真菌谱窄,出现新的耐药性,并没有降低白色念珠菌感染的发生率和死亡率。因此研究安全有效的免疫制剂会为真菌感染的预防和治疗提供新的途径,其中真菌疫苗的研究引人瞩目。甘露聚糖-甘露糖蛋白交联物是白色念珠菌细胞壁的重要组成成分,也是重要的粘附素分子,甘露糖蛋白65(65-kD mannoprotein, MP65)是从其酸性提取物中纯化出来的主要甘露糖蛋白成分,分子量约65kD,具有很好的免疫原性,其中蛋白核心部分能诱导T细胞应答,对多种念珠菌感染有保护作用。分泌性酸性蛋白酶2(secretory acid proteinase,Sap2)是白色念珠菌分泌产生的一种最重要的胞外蛋白酶,与白色念珠菌营养作用、粘附、侵袭和破坏组织屏障密切相关,是白色念珠菌致病的重要毒力因子。Sap2是大多数白色念珠菌广泛存在的一种抗原,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的抗体。研究表明以Sap2的白色念珠菌提取物免疫小鼠,可以减弱该菌对粘膜的感染。因此本研究选取编码MP65、Sap2蛋白的基因,构建其真核表达质粒及原核表达质粒,以真核质粒免疫小鼠,分析MP65、Sap2的免疫原性以及其DNA疫苗在抵抗白色念珠菌系统性感染中的免疫保护作用,为进一步研究该蛋白功能及研制其真菌疫苗提供实验基础。方法:1.原核、真核表达质粒的构建:热酚法提取白色念珠菌总RNA,经RT-PCR方法扩增获得mp65、sap2目的基因,克隆至pMD18-T载体上进行测序,测序结果正确后,经BamH I、EcoR I和EcoR I、XhoI酶切再次鉴定,最后将其分别克隆至原核表达质粒pGEX4T-2、pET32a和真核表达质粒pcDNA3.1中。 2.目的蛋白的表达、纯化与鉴定:将重组原核表达质粒转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷酶(IPTG)诱导、超声裂解菌体、SDS-PAGE电泳观察结果,获得重组表达的包涵体蛋白。低浓度尿素梯度透析与金属镍螯合层析柱纯化回收重组蛋白后,对重组蛋白进行定量测定。Western blot、ELISA方法初步鉴定其抗原特异性及反应性。 3.真核表达质粒的大量提取:碱裂解法分别大量提取真核表达质粒pcDNA3.1-mp65和pcDNA3.1-sap2,纯化后定量备用。 4.动物免疫:取40只4周龄BALB/c小鼠随机分成4组,每组10只,雌雄各半,分别命名为pcDNA3.1-mp65质粒组、pcDNA3.1-sap2质粒组、pcDNA3.1-mp65+pcDNA3.1-sap2联合质粒组及PBS对照组,股四头肌接种真核表达质粒,每只100μg/次,每次间隔2周,加强免疫3次,每次免疫前Qg眦取血收集血清。 5.免疫效果检测:间接ELISA法检测各免疫组血清特异性抗-MP65、抗-Sap2 IgG;流式细胞术检测小鼠脾脏内CD4+、CD8+淋巴细胞变化情况。 6.攻毒实验:末次免疫后20天尾静脉注射给予小鼠致死量白色念珠菌1×106个/只,15天内观察存活率。结果:1.经RT-PCR方法克隆出全长为1140bp的mp65和1197bp的sap2基因,其核苷酸序列与GenBank中公布的序列基本同源。 2 .成功构建了原核表达质粒pGEX4T-2-mp65、pET32a-sap2及真核表达质粒pcDNA3.1-mp65、pcDNA3.1- sap2。构建的原核表达质粒pGEX4T-2-mp65和pET32a-sap2转化BL-21,经SDS-PAGE和Western blot证实,分别诱导表达出66KD左右的GST融合蛋白和His融合蛋白。表达的蛋白主要以包涵体形式存在。 3.真核表达质粒pcDNA3.1-mp65和pcDNA3.1-sap2免疫动物后,各免疫组小鼠血清IgG水平随免疫时间延长,抗体滴度逐渐升高。末次免疫后,抗-MP65 IgG最高效价可达到1:1600,抗-Sap2 IgG最高效价可达到1:3200。流式细胞术检测结果经方差分析显示,两组质粒免疫后均能诱导小鼠脾脏内CD4+T细胞百分含量增加,与PBS对照组比较有显著性差异(P0.05)。而CD8+ T细胞百分含量无明显差异。两种质粒单独免疫组与联合免疫组比较也无显著性差异。 4.白色念珠菌攻毒15天后,pcDNA3.1-mp65质粒组及pcDNA3.1-sap2质粒组各有20%的存活率,pcDNA3.1-mp65 + pcDNA3.1-sap2联合质粒组存活率达到40%,PBS对照组存活率为0%,经χ2检验有显著差异(P0.05)。结论:1.成功构建了白色念珠菌基因mp65和sap2的原核表达质粒pGEX4T-2-mp65、pET32a-sap2和真核表达质粒pcDNA3.1-mp65、pcDNA3.1-sap2。 2.MP65和Sap2的原核表达质粒在大肠杆菌BL-21中成功表达融合蛋白。 3.MP65与Sap2重组真核表达质粒能诱导动物机体产生体液免疫和细胞免疫。 4.MP65与Sap2两种真核表达质粒对白色念珠菌系统性感染均有一定的免疫保护作用。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392;R519

【共引文献】