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,每次免疫前內眦取血收集血清。
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(P<0.05)。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(P<0.05)。
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。
Objective: Candida albicans is the most common opportunistic fungal pathogen which has become one of the most important pathogen of nosocomial infections (application of immunosuppressive agents and broadspectrug antibiotic from surgical trauga, organ transplanting, cancer-treatment) and immunocompromised patients (primary and secondary immunodeficiencies, for example AIDS) during the last few decades, mortality of caused systemic infection is 40%~50%. Although new antifungal drugs were applicated in clinic, the incidence and mortality of infection still can’t be reduced because of the narrow spectrug of anti-fungal medicine and the occurrence of new drug resistance to Candida albicans. Therefore the study of safe and effective immune agents will provide a new promising approach to fungal infection prevention and treatment, in which fungi vaccine research was the focus.
Mannan-mannoprotein conjugates are not only an important component of Candida albicans cell wall but also an important adhesin molecule. MP65 is the main mannoprotein be purified from the acidic extraction. The protein core part can indu ce T cell response and have a wide range of protective effects on many kinds of candida infection. And Sap2 is the most important extracellular protease secreted by Candida albicans under certain conditions, which play a crucial role in the nutrition of Candida albicans and is closely related to adhesion, invasion and destruction of host tissue barrier, so it is an important virulence factor of Candida albicans. As a widespread antigen component existed in the majority of Candida albicans, its existence form of serum is soluble. It is found that the Candida patient's serum retain high titer of Sap2 antibodies. Studies had verified that Sap2 extraction of Candida albicans can weaken invasion to mucosal tissue in mice immunized with them.
For the reason above, in this research we selected the gene encoding MP65 and Sap2 protein, constructed the recombinant eukaryotic and prokaryotic expression plasmid of them. BALB/c mice were immunized with eukaryotic plasmids, it is aim to analyse immunogenicity of MP65, Sap2 as well as the immunization protective effect of resistance to Candida albicans systemic infection which provided an experimental foundation in further research and production of fungi vaccine.
Method: 1. Construction of eukaryotic and prokaryotic expression plasmid: Candida albicans total RNA was extracted by hot phenol method. Then the mp65 and sap2 gene were obtained by RT-PCR and cloned into pMD18-T vector for sequence identification. After receiving the correct sequence results and re-identification by BamHI, EcoR I and EcoR I, XhoI digestion, the target gene were cloned into prokaryotic expression plasmid pGEX4T-2, pET32a and eukaryotic expression plasmids pcDNA3.1 respectively.
2. Expression, purification and identification of purpose protein: Recombinant prokaryotic expression plasmids were transfered into E. coli BL21 and induced by IPTG. Then BL-21 cell transformed were crumbled by ultrasonics and the results were analized by SDS-PAGE electrophoresis. The recombinant protein were expressed mainly as the form of inclusion body and were purified by gradient dialysis with low concentrations of urea and nickel chelate column effectively. Utilize Western blot method for preliminary identification of its antigen specificity and reactivity.
3. Large quantity acquisition of Eukaryotic expression plasmid: Eukaryotic expression plasmid pcDNA3.1-mp65 and pcDNA3.1-sap2 were extracted by alkaline lysis method separately, and were served after purification and quantitation.
4. Animal immunization: Forty 4 week-old BALB/c mice were divided into four groups randomly, among each group the male and female was equally. Four groups were respectively named with pcDNA3.1-mp65 plasmid group, pcDNA3.1-sap2 plasmid group, pcDNA3.1-mp65 + pcDNA3.1-sap2 plasmid group and PBS control group. Quadricepse of leg were injected with eukaryotic expression plasmid, 100μg per mouse, total 4 times at interval two weeks. Serum were harvested from the inn -er canthal vein blood before every time of immunization.
4. Detection of the immune effect: anti-MP65 and anti-Sap2 IgG in the serum were detected by indirect ELISA. CD4+, CD8+ lymphocyte phenotypes in the spleen were analyzed by Flow cytometry.
5. Candida albicans challenge experiment: 20 days after the last boost, BALB/c mice were injected via tail vein with 1×106 lethal dose of Candida albicans, the survival were observed for 15 days and evaluate the protective effect of the DNA vaccines.
Results: 1. Mp65 and sap2 gene were obtained successfully by RT-PCR. The sequence analysis shows that the mp65 (1140bp) and sap2 (1197bp) gene were homologous basically with the ones in GenBank.
2. Prokaryotic expression plasmid pGEX4T-2-mp65 and pET32a-sap2, eukaryotic expression plasmid pcDNA3.1-mp65, pcDNA3.1-sap2 had been constructed successfully. Prokaryotic expression plasmid pGEX4T-2-mp65 and pET32a-sap2 were transformed to BL-21. GST and His fusion protein with approximate molecular mass of 66KD were expressed and identified by SDS-PAGE and Western blot. The main expression form of the protein was inclusion body.
3. The titers of IgG in serum increased gradually after animals were immunized with pcDNA3.1-mp65 and pcDNA3.1-sap2 following time extending. After the last immunization, the highest titer of anti-MP65 IgG was 1:1600, while the highest titer of anti-Sap2 IgG was 1:3200. Flow cytometry results showed that percentage of CD4+T cell increased in both mp65 and Sap2 plasmid group after immunization of mice, which exists significant difference compared with the PBS control group by analysis of variance (P<0.05). And no statistically significant difference was found in the percentage of CD8+ T cell between immune groups.
4. Fifteen days after the Candida albicans impact, the survival rate of mice immunized with pcDNA3.1-mp65 plasmid or pcDNA3.1-sap2 plasmid was 20% respectively, survival rate of mice immunized with pcDNA3.1-mp65 +pcDNA3.1-sap2 plasmid was 40 %, and PBS group survival rate was 0% ,which exists significant difference by Chi-square test (P <0.05).
Conclusion:
1. Prokaryotic expression plasmid pGEX4T-2-mp65, pET32a-sap2 and eukaryotic expression plasmid pcDNA3.1 -mp65, pcDNA3.1-sap2 of mp65 and sap2 gene of Candida albicans were constructed successfully.
2. Prokaryotic expression plasmids of mp65 and sap2 gene were induced and expressed fusionin protein in E. coli. BL-21 successfully.
3. Recombinant eukaryotic expression plasmid of mp65 and sap2 gene can stimulate animals producing humoral and cellular immune response.
4. Both mp65 and sap2 recombinant eukaryotic expression plasmid have a certain protective effect against systemic Candida albicans infection.
引文
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