本文選題：炭疽桿菌 + 保護(hù)性抗原��； 參考：《武漢大學(xué)》2010年博士論文

【摘要】：炭疽是一種古老的疾病,它的致病菌,炭疽芽孢桿菌也是細(xì)菌學(xué)中發(fā)現(xiàn)最早的病原菌之一。炭疽桿菌是革蘭氏陽性桿狀菌,生命周期中能形成孢子。炭疽桿菌的主要毒力因子是一個(gè)三組分的毒素,包括三種蛋白：保護(hù)性抗原PA,致死因子LF,水腫因子EF。三個(gè)蛋白由毒力質(zhì)粒pXO1編碼,單獨(dú)存在時(shí)沒有毒性。三個(gè)蛋白兩兩結(jié)合后可形成兩種炭疽毒素,即致死毒素(PA+LF)和水腫毒素(PA+EF)。幾乎所有的真核細(xì)胞表面都有PA的受體,因此,一旦進(jìn)入體內(nèi),PA就可結(jié)合在細(xì)胞表面受體,然后在類furin蛋白酶的作用下,在PA保守序列164RKKR167處水解成一個(gè)氨基端片段PA20(分子量20kDa)和一個(gè)羧基端片段PA63(分子量63kDa)。PA20游離出去,PA63可自我組裝成一個(gè)環(huán)狀的七聚體,插入細(xì)胞膜形成一個(gè)孔道。然后,EF和／或LF與PA63七聚體結(jié)合,被轉(zhuǎn)運(yùn)到胞內(nèi),發(fā)揮細(xì)胞毒效應(yīng)。 三種蛋白(EF, LF和PA)中,只有PA誘導(dǎo)的抗體具有保護(hù)性,因此PA也是炭疽疫苗的一個(gè)主要免疫原,PA的制備早期從炭疽桿菌培養(yǎng)物中獲取,安全性難以保證�，F(xiàn)在的主流是從大腸桿菌中制備重組PA。在我們的研究中,使用一種新的表達(dá)系統(tǒng),大腸桿菌株Rosetta 2(DE3),高效表達(dá)重組PA蛋白,克服了PA基因含有66個(gè)大腸桿菌稀少密碼子(占PA基因共773個(gè)密碼子的9.0%)而帶來的低表達(dá)問題,無需重新優(yōu)化密碼子合成PA基因。rPA在Rosetta 2(DE3)表達(dá)后,形成的包涵體經(jīng)過Triton X-100和2M尿素洗滌,5M尿素溶解,透析,以及60%硫酸銨沉淀,最終在AKTA Purifier 10,使用Phenyl Sepharose High Performance介質(zhì),一步疏水層析純化到無標(biāo)簽的重組PA。SDS-PAGE分析純度大于99%。得率為每升培養(yǎng)物約13 mg。該rPA具有和天然PA相同的生物活性,體外可與rLF結(jié)合,殺傷巨噬細(xì)胞。 在以rPA作為組分的炭疽疫苗中,用不同處理方式的芽孢或者菌體與之配伍,肌內(nèi)免疫BALB/c小鼠,初步研究了不同組分疫苗的藥理學(xué)機(jī)制。實(shí)驗(yàn)結(jié)果表明：(1)未勻漿芽孢,即甲醛滅活芽孢,免疫后的IgG效價(jià),脾淋巴細(xì)胞增殖的刺激指數(shù)和ELISPOT陽轉(zhuǎn)率均要優(yōu)于高壓芽孢和Co60芽孢；(2)rPA+鋁佐劑免疫小鼠后,誘導(dǎo)B細(xì)胞分泌rPA抗體到血液中發(fā)揮功能,但不能促進(jìn)T細(xì)胞增殖和分泌IFN-γ。這說明rPA°單組分主要發(fā)揮的是體液免疫而不是細(xì)胞免疫,通過血清中的高滴度rPA抗體中和毒素起到保護(hù)作用；(3)不同處理方式的芽孢或者菌體與rPA聯(lián)合免疫后,芽孢或者菌體的體液免疫和細(xì)胞免疫都很強(qiáng)烈,都可促進(jìn)rPA的細(xì)胞免疫功能,產(chǎn)生rPA抗原特異性T淋巴細(xì)胞,但rPA抗體水平還不太穩(wěn)定；(4)小鼠腹腔攻毒炭疽弱毒株A16R的試驗(yàn)中,含有rPA組分的疫苗都能產(chǎn)生保護(hù)力。單純的芽孢組分也能對小鼠產(chǎn)生保護(hù)作用,無需rPA的參與,單純的菌體保護(hù)力很弱,對兩倍最少致死量的腹腔攻毒劑量沒有保護(hù)力。這表明,芽孢中的某些抗原成分參與了炭疽免疫保護(hù),提高了疫苗的保護(hù)效率。而菌體,雖然與芽孢有交叉抗原,但這種交叉抗原和菌體特異性的抗原都不能提供很好的保護(hù)力。因此,rPA配伍芽孢作為目前較為理想的疫苗組分,為我們提供了一種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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;PIKA Provides an Adjuvant Effect to Induce Strong Mucosal and Systemic Humoral Immunity Against SARS-CoV[J];Virologica Sinica;2011年02期

相關(guān)博士學(xué)位論文 前2條

1 卞廣林;基于漢遜酵母細(xì)胞的新型乙肝表面抗原疫苗的研究[D];復(fù)旦大學(xué);2009年

2 曹莎;炭疽致死毒素?zé)o毒突變體的篩選及其免疫原性和抗毒素機(jī)理研究[D];華中農(nóng)業(yè)大學(xué);2009年

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