炭疽新疫苗研究

发布时间：2018-06-17 02:13

本文选题：炭疽杆菌 + 保护性抗原　；参考：《武汉大学》2010年博士论文

【摘要】：炭疽是一种古老的疾病,它的致病菌,炭疽芽孢杆菌也是细菌学中发现最早的病原菌之一。炭疽杆菌是革兰氏阳性杆状菌,生命周期中能形成孢子。炭疽杆菌的主要毒力因子是一个三组分的毒素,包括三种蛋白：保护性抗原PA,致死因子LF,水肿因子EF。三个蛋白由毒力质粒pXO1编码,单独存在时没有毒性。三个蛋白两两结合后可形成两种炭疽毒素,即致死毒素(PA+LF)和水肿毒素(PA+EF)。几乎所有的真核细胞表面都有PA的受体,因此,一旦进入体内,PA就可结合在细胞表面受体,然后在类furin蛋白酶的作用下,在PA保守序列164RKKR167处水解成一个氨基端片段PA20(分子量20kDa)和一个羧基端片段PA63(分子量63kDa)。PA20游离出去,PA63可自我组装成一个环状的七聚体,插入细胞膜形成一个孔道。然后,EF和／或LF与PA63七聚体结合,被转运到胞内,发挥细胞毒效应。三种蛋白(EF, LF和PA)中,只有PA诱导的抗体具有保护性,因此PA也是炭疽疫苗的一个主要免疫原,PA的制备早期从炭疽杆菌培养物中获取,安全性难以保证。现在的主流是从大肠杆菌中制备重组PA。在我们的研究中,使用一种新的表达系统,大肠杆菌株Rosetta 2(DE3),高效表达重组PA蛋白,克服了PA基因含有66个大肠杆菌稀少密码子(占PA基因共773个密码子的9.0%)而带来的低表达问题,无需重新优化密码子合成PA基因。rPA在Rosetta 2(DE3)表达后,形成的包涵体经过Triton X-100和2M尿素洗涤,5M尿素溶解,透析,以及60%硫酸铵沉淀,最终在AKTA Purifier 10,使用Phenyl Sepharose High Performance介质,一步疏水层析纯化到无标签的重组PA。SDS-PAGE分析纯度大于99%。得率为每升培养物约13 mg。该rPA具有和天然PA相同的生物活性,体外可与rLF结合,杀伤巨噬细胞。在以rPA作为组分的炭疽疫苗中,用不同处理方式的芽孢或者菌体与之配伍,肌内免疫BALB/c小鼠,初步研究了不同组分疫苗的药理学机制。实验结果表明：(1)未匀浆芽孢,即甲醛灭活芽孢,免疫后的IgG效价,脾淋巴细胞增殖的刺激指数和ELISPOT阳转率均要优于高压芽孢和Co60芽孢；(2)rPA+铝佐剂免疫小鼠后,诱导B细胞分泌rPA抗体到血液中发挥功能,但不能促进T细胞增殖和分泌IFN-γ。这说明rPA°单组分主要发挥的是体液免疫而不是细胞免疫,通过血清中的高滴度rPA抗体中和毒素起到保护作用；(3)不同处理方式的芽孢或者菌体与rPA联合免疫后,芽孢或者菌体的体液免疫和细胞免疫都很强烈,都可促进rPA的细胞免疫功能,产生rPA抗原特异性T淋巴细胞,但rPA抗体水平还不太稳定；(4)小鼠腹腔攻毒炭疽弱毒株A16R的试验中,含有rPA组分的疫苗都能产生保护力。单纯的芽孢组分也能对小鼠产生保护作用,无需rPA的参与,单纯的菌体保护力很弱,对两倍最少致死量的腹腔攻毒剂量没有保护力。这表明,芽孢中的某些抗原成分参与了炭疽免疫保护,提高了疫苗的保护效率。而菌体,虽然与芽孢有交叉抗原,但这种交叉抗原和菌体特异性的抗原都不能提供很好的保护力。因此,rPA配伍芽孢作为目前较为理想的疫苗组分,为我们提供了一种炭疽新疫苗研究的可行思路。
[Abstract]:Anthrax is an ancient disease. Its pathogenic bacteria, Bacillus anthracis, are also one of the earliest pathogenic bacteria found in bacteriology. Anthrax is a Gram-positive bacillary bacilli, which can form spores in the life cycle. The main virulence factor of Bacillus anthracis is a three component toxin, including three kinds of protein: protective antigen PA, lethal factor LF The three protein of the edema factor EF. is encoded by the virulence plasmid pXO1 and has no toxicity when it exists alone. The combination of three proteins 22 can form two kinds of anthrax toxin, that is, lethal toxin (PA+LF) and edema toxin (PA+EF). Almost all the eukaryotic cells have PA receptors on the surface of the eukaryotic cells. Therefore, once into the body, PA can be combined on the cell surface receptor. Then, under the action of furin like protease, the amino terminal fragment PA20 (molecular weight 20kDa) and a carboxyl terminal fragment PA63 (molecular weight 63kDa).PA20 are dissociated from the PA conservative sequence 164RKKR167, and PA63 can be self assembled into a circular seven polymer and inserted into the cell membrane to form a channel. Then, EF and / or LF and PA63 seven polymers It is transported to the cell and plays the cytotoxic effect.
In the three proteins (EF, LF and PA), only PA induced antibodies are protective, so PA is also a major immunogen of the anthrax vaccine. The preparation of PA is early obtained from the anthrax culture, and it is difficult to ensure the safety. Now the mainstream is to prepare recombinant PA. from Escherichia coli in our study and use a new expression system. Enterobacteriaceae Rosetta 2 (DE3), which efficiently expressed recombinant PA protein, overcame the low expression problem of the PA gene containing 66 rare codon of Escherichia coli (9% of the PA codon of the PA gene). It did not need to reoptimize the PA gene.RPA in Rosetta 2 (DE3), and the inclusion bodies formed by Triton X-100 and 2M urea washing. Polyester, 5M urea dissolving, dialysis, and 60% ammonium sulfate precipitation, finally in AKTA Purifier 10, using Phenyl Sepharose High Performance medium, one step hydrophobic chromatography purification to unlabeled recombinant PA.SDS-PAGE analysis purity greater than 99%. yield per liter of 13 mg., the same biological activity as natural PA is the same as that of natural PA. Combined, killing macrophages.
In the anthrax vaccine with rPA as a component, the combination of spore or mycelium with different treatments was used to immunization with BALB/c mice in muscle. The pharmacological mechanism of different component vaccines was preliminarily studied. The results showed that: (1) non homogenate spore, formaldehyde inactivated spore, IgG titer after immunization, stimulation index and ELIS of splenic lymphocyte proliferation. The positive rate of POT was better than that of the high pressure spore and Co60 spore; (2) after the rPA+ aluminum adjuvant immunized mice, the induced B cells secreted the rPA antibody to the blood, but it did not promote the proliferation and secretion of IFN- gamma in the T cells. This shows that the single component of rPA is mainly mediated by the humoral immunity rather than the fine cell immunity, and neutralization of the high titer rPA antibody in the serum. Toxin played a protective role; (3) after the combined immunization of spores or mycelia with rPA in different treatments, the humoral immunity and cell immunity of the spores and the mycelium were very strong, all of which could promote the cellular immune function of rPA and produce the rPA antigen specific T lymphocyte, but the rPA antibody was not very stable; (4) the mouse abdominal attack anthrax weakly virulent strain In the A16R test, the vaccine containing the rPA component can produce protective power. The simple sporulation component can also protect the mice, without the need of rPA, the simple bacterial protection is very weak, and the two times the least lethal dose of the abdominal attack dose not protect. This shows that some antigens in the spores are involved in the anthrax immunization protection, The vaccine has improved the efficiency of protection. Although the bacteria have cross antigen with spore, the cross antigen and the specific antigen of the mycelium can not provide good protection. Therefore, rPA compatibility with the spores is an ideal vaccine component, which provides a feasible way of thinking for the study of the new anthrax vaccine.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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