化脓性链球菌表面蛋白Fba的免疫原性研究
发布时间:2018-11-21 13:09
【摘要】: 目的:A族链球菌(GAS)是一类分布广泛的致病菌,能引起多种临床疾病,包括从喉或皮肤的浅表感染到高度侵袭及具有潜在致死的感染,甚至感染后严重的超敏反应肾小球肾炎、风湿性心脏病等。这些疾病都是由链球菌对呼吸道及皮肤的上皮细胞的黏附引起的。GAS可以通过其菌体表面蛋白与人血浆纤连蛋白(Fibronectin)结合,并以此为分子桥梁再与上皮细胞上的相关受体结合,介导细菌进入细胞,从而逃避宿主细胞对它的吞噬和杀伤[1]。 Fba蛋白是一种新发现的表达于GAS表面的蛋白,它至少存在于18个血清型中,并具有结合FHL-1、FH和纤连蛋白的能力。研究表明,Fba蛋白有助于GAS的抗吞噬,与FHL-1结合后能促进GAS进入人体上皮细胞,是一种重要的毒力因子[2,3]。鉴于此,本课题选取了Fba蛋白编码基因为研究对象,构建了Fba原核表达质粒和真核表达质粒,之后诱导原核质粒表达,通过Western blot和ELISA检测重组蛋白。并将真核表达质粒和纯化的重组Fba蛋白免疫小鼠,对它们所诱导的体液免疫和细胞免疫进行评估,以进一步对其免疫原性及Fba在抗GAS感染免疫中的作用进行分析,为进一步研制以其为基础的诊断试剂和疫苗提供依据。 方法:1 PCR方法扩增Fba基因,克隆至pMD18-T载体上进行测序,测序正确后,经BamHI、EcoR I和BamHI、XhoI双酶切后,将其分别克隆至原核表达质粒pGEX4T-2和真核表达质粒pcDNA3.1。 2原核表达质粒通过IPTG诱导,获得了高效表达的蛋白包涵体,采用从SDS凝胶上电洗脱回收的方法进行纯化回收,Western-blot和ELISA鉴定表达的目的蛋白。 3以该蛋白作为抗原包被酶联板检测GAS全菌免疫鼠血清,未免疫鼠血清,抗链“O”阳性病人血清及健康志愿者血清中可能存在的Fba抗体,同时以GAS的M蛋白包板检测以上各类血清中的M抗体作为阳性对照。 4雌性BALB/c小鼠随机分成6组,分别为Fba蛋白免疫组、M蛋白免疫组、pcDNA3.1/fba+Fba蛋白免疫组、pcDNA3.1/fba免疫组、pcDNA3.1空质粒对照组及PBS对照组。ELISA检测各免疫组血清IgG的动态变化水平;脾细胞增殖试验及流式细胞术检测小鼠体内CD4+、CD8+淋巴细胞的变化。给予链球菌攻击评价Fba蛋白及质粒对免疫小鼠的保护功效。 结果:1 PCR方法获得了Fba基因,测序正确后,成功构建了原核表达质粒pGEX4T-2/fba和真核表达质粒pcDNA3.1/fba。 2在大肠杆菌中成功表达了Fba重组蛋白,表达蛋白主要以包涵体形式存在。ELISA和Western-blot证实表达的Fba重组蛋白可与已知兔抗Fba阳性血清产生特异性反应。 3 Fba蛋白可与GAS全菌免疫鼠血清、抗链“O”阳性病人血清发生特异性结合,尽管Fba蛋白做为诊断抗原的检测结果比M蛋白的检测结果的OD值小,但二者的检测结果一致,均为阳性。 4质粒或蛋白免疫后小鼠IgG产生水平以Fba蛋白免疫组增高最为明显,依次为Fba质粒蛋白混合免疫组及Fba质粒组。特异性抗原诱导后体外脾细胞增殖试验显示:pcDNA3.1/fba免疫组增值水平明显高于其它组,流式细胞检测结果与此一致,并显示CD4+、CD8+T细胞水平较对照组有显著性增高。 5链球菌攻击后,PBS组小鼠在4天内全部死亡,而Fba蛋白组、pcDNA3.1/fba+Fba蛋白组及M蛋白组在14天的观察期内均有60%的存活率,pcDNA3.1/fba组为40%。结论:1成功构建了原核表达质粒pGEX4T-2/fba和真核表达质粒pcDNA3.1/fba。 2在大肠杆菌中高效表达了Fba重组蛋白。 3 GAS感染可以诱导人体及动物体产生Fba抗体,即Fba蛋白与M蛋白一样,也具有良好的免疫原性和抗原特异性。因M蛋白及M抗体是人体感染GAS后诱发超敏反应的主要原因,加之其血清型的多样性,使M蛋白作为GAS的候选疫苗受到限制,故可以考虑Fba蛋白作为GAS的候选疫苗及诊断抗原。 4 Fba蛋白能有效地诱导机体产生抗体,Fba真核质粒能有效诱导抗体及Th细胞增殖,且Fba蛋白或基因获得了较强的抗GAS感染的能力,以上结果均提示Fba蛋白很有希望成为GAS的候选疫苗。
[Abstract]:Objective: A family of Streptococcus A (GAS) is a kind of widely distributed pathogenic bacteria, which can cause a variety of clinical diseases, including superficial infection from the throat or the skin to highly invasive and potentially fatal infections, even after infection, such as, for example, glomerulonephritis, rheumatic heart disease, and the like. These diseases are caused by the adhesion of the Streptococcus to the respiratory tract and the epithelial cells of the skin. The GAS can bind to the human plasma fibronectin (Fibronectin) by its bacterial surface protein and bind the molecular bridge with the related receptors on the epithelial cell, and mediate the bacteria to enter the cell, thus avoiding the phagocytosis and killing of the host cell[1]. The Fba protein is a newly discovered protein expressed on the surface of the GAS, which is at least present in 18 serotypes and has binding FHL-1, FH and The research shows that the Fba protein can contribute to the anti-phagocytosis of GAS, and can promote the entry of GAS into human epithelial cells after the combination with FHL-1, which is an important virulence. In view of this, the Fba protein coding gene was selected as the research object, and the expression plasmid and the true nuclear expression plasmid of Fba were constructed, and the expression of the prokaryote was induced by Western blot and ELIS. A. The recombinant protein is detected, and the true nuclear expression plasmid and the purified recombinant Fba protein are immunized with the mouse, and the humoral immunity and the cell immunity induced by the recombinant protein are evaluated to further enhance the immunogenicity and the Fba in the anti-GAS infection immunity. The function of the diagnosis reagent is to further develop the diagnostic reagent based on it. and cloning the Fba gene to the pMD18-T vector for sequencing, and then cloning the Fba gene into a prokaryotic expression plasmid pGEX4T-2 through the BamHI, EcoR I and BamHI and XhoI double enzymes after the sequencing is correct. and the purified recovery is carried out by the method of electroelution and recovery from the SDS gel, West and the like, the protein of interest expressed by ern-blot and ELISA. white as the antigen-coated enzyme-linked plate is used for detecting the Fba antibody which may be present in the serum of the whole-bacteria immune mouse of the GAS, the serum of the non-immunized mouse, the anti-chain 'O' positive patients and the serum of the healthy volunteer, In the same time, the M-antibody in the above-mentioned serum was used as the positive control. The female BALB/ c mice were randomly divided into 6 groups, namely, the Fba protein immunization group, the M protein immune group, the pcDNA3.1/ fba + Fba protein immunization group, group, pcDNA3. 1/ fba immune group, pcDNA3. 1 empty plasmid control group and PBS control group. The dynamic change of serum IgG in each immune group was detected by ELISA. The level of CD4 + in the mice was detected by the proliferation test of the spleen cells and flow cytometry. Changes of CD8 + lymphocytes. The efficacy of Fba protein and plasmid on the protection of the immune mice was evaluated by streptococcal attack. Results: The Fba gene was obtained by 1 PCR method. The prokaryotic expression plasmid pGEX4T-2/ fba and the true nuclear expression plasmid were successfully constructed after the correct sequence. Fba recombinant protein was successfully expressed in E. coli by pcDNA3.1/ fba. 2, and the expression protein was mainly composed of inclusion body. Form Present. ELISA and Western-bl ot璇,
本文编号:2347041
[Abstract]:Objective: A family of Streptococcus A (GAS) is a kind of widely distributed pathogenic bacteria, which can cause a variety of clinical diseases, including superficial infection from the throat or the skin to highly invasive and potentially fatal infections, even after infection, such as, for example, glomerulonephritis, rheumatic heart disease, and the like. These diseases are caused by the adhesion of the Streptococcus to the respiratory tract and the epithelial cells of the skin. The GAS can bind to the human plasma fibronectin (Fibronectin) by its bacterial surface protein and bind the molecular bridge with the related receptors on the epithelial cell, and mediate the bacteria to enter the cell, thus avoiding the phagocytosis and killing of the host cell[1]. The Fba protein is a newly discovered protein expressed on the surface of the GAS, which is at least present in 18 serotypes and has binding FHL-1, FH and The research shows that the Fba protein can contribute to the anti-phagocytosis of GAS, and can promote the entry of GAS into human epithelial cells after the combination with FHL-1, which is an important virulence. In view of this, the Fba protein coding gene was selected as the research object, and the expression plasmid and the true nuclear expression plasmid of Fba were constructed, and the expression of the prokaryote was induced by Western blot and ELIS. A. The recombinant protein is detected, and the true nuclear expression plasmid and the purified recombinant Fba protein are immunized with the mouse, and the humoral immunity and the cell immunity induced by the recombinant protein are evaluated to further enhance the immunogenicity and the Fba in the anti-GAS infection immunity. The function of the diagnosis reagent is to further develop the diagnostic reagent based on it. and cloning the Fba gene to the pMD18-T vector for sequencing, and then cloning the Fba gene into a prokaryotic expression plasmid pGEX4T-2 through the BamHI, EcoR I and BamHI and XhoI double enzymes after the sequencing is correct. and the purified recovery is carried out by the method of electroelution and recovery from the SDS gel, West and the like, the protein of interest expressed by ern-blot and ELISA. white as the antigen-coated enzyme-linked plate is used for detecting the Fba antibody which may be present in the serum of the whole-bacteria immune mouse of the GAS, the serum of the non-immunized mouse, the anti-chain 'O' positive patients and the serum of the healthy volunteer, In the same time, the M-antibody in the above-mentioned serum was used as the positive control. The female BALB/ c mice were randomly divided into 6 groups, namely, the Fba protein immunization group, the M protein immune group, the pcDNA3.1/ fba + Fba protein immunization group, group, pcDNA3. 1/ fba immune group, pcDNA3. 1 empty plasmid control group and PBS control group. The dynamic change of serum IgG in each immune group was detected by ELISA. The level of CD4 + in the mice was detected by the proliferation test of the spleen cells and flow cytometry. Changes of CD8 + lymphocytes. The efficacy of Fba protein and plasmid on the protection of the immune mice was evaluated by streptococcal attack. Results: The Fba gene was obtained by 1 PCR method. The prokaryotic expression plasmid pGEX4T-2/ fba and the true nuclear expression plasmid were successfully constructed after the correct sequence. Fba recombinant protein was successfully expressed in E. coli by pcDNA3.1/ fba. 2, and the expression protein was mainly composed of inclusion body. Form Present. ELISA and Western-bl ot璇,
本文编号:2347041
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