【摘要】： 目的:A族鏈球菌(GAS)是一類分布廣泛的致病菌,能引起多種臨床疾病,包括從喉或皮膚的淺表感染到高度侵襲及具有潛在致死的感染,甚至感染后嚴(yán)重的超敏反應(yīng)腎小球腎炎、風(fēng)濕性心臟病等。這些疾病都是由鏈球菌對呼吸道及皮膚的上皮細(xì)胞的黏附引起的。GAS可以通過其菌體表面蛋白與人血漿纖連蛋白(Fibronectin)結(jié)合,并以此為分子橋梁再與上皮細(xì)胞上的相關(guān)受體結(jié)合,介導(dǎo)細(xì)菌進(jìn)入細(xì)胞,從而逃避宿主細(xì)胞對它的吞噬和殺傷[1]。 Fba蛋白是一種新發(fā)現(xiàn)的表達(dá)于GAS表面的蛋白,它至少存在于18個血清型中,并具有結(jié)合FHL-1、FH和纖連蛋白的能力。研究表明,Fba蛋白有助于GAS的抗吞噬,與FHL-1結(jié)合后能促進(jìn)GAS進(jìn)入人體上皮細(xì)胞,是一種重要的毒力因子[2,3]。鑒于此,本課題選取了Fba蛋白編碼基因?yàn)檠芯繉ο?構(gòu)建了Fba原核表達(dá)質(zhì)粒和真核表達(dá)質(zhì)粒,之后誘導(dǎo)原核質(zhì)粒表達(dá),通過Western blot和ELISA檢測重組蛋白。并將真核表達(dá)質(zhì)粒和純化的重組Fba蛋白免疫小鼠,對它們所誘導(dǎo)的體液免疫和細(xì)胞免疫進(jìn)行評估,以進(jìn)一步對其免疫原性及Fba在抗GAS感染免疫中的作用進(jìn)行分析,為進(jìn)一步研制以其為基礎(chǔ)的診斷試劑和疫苗提供依據(jù)。 方法:1 PCR方法擴(kuò)增Fba基因,克隆至pMD18-T載體上進(jìn)行測序,測序正確后,經(jīng)BamHI、EcoR I和BamHI、XhoI雙酶切后,將其分別克隆至原核表達(dá)質(zhì)粒pGEX4T-2和真核表達(dá)質(zhì)粒pcDNA3.1。 2原核表達(dá)質(zhì)粒通過IPTG誘導(dǎo),獲得了高效表達(dá)的蛋白包涵體,采用從SDS凝膠上電洗脫回收的方法進(jìn)行純化回收,Western-blot和ELISA鑒定表達(dá)的目的蛋白。 3以該蛋白作為抗原包被酶聯(lián)板檢測GAS全菌免疫鼠血清,未免疫鼠血清,抗鏈“O”陽性病人血清及健康志愿者血清中可能存在的Fba抗體,同時以GAS的M蛋白包板檢測以上各類血清中的M抗體作為陽性對照。 4雌性BALB/c小鼠隨機(jī)分成6組,分別為Fba蛋白免疫組、M蛋白免疫組、pcDNA3.1/fba+Fba蛋白免疫組、pcDNA3.1/fba免疫組、pcDNA3.1空質(zhì)粒對照組及PBS對照組。ELISA檢測各免疫組血清IgG的動態(tài)變化水平;脾細(xì)胞增殖試驗(yàn)及流式細(xì)胞術(shù)檢測小鼠體內(nèi)CD4+、CD8+淋巴細(xì)胞的變化。給予鏈球菌攻擊評價Fba蛋白及質(zhì)粒對免疫小鼠的保護(hù)功效。 結(jié)果:1 PCR方法獲得了Fba基因,測序正確后,成功構(gòu)建了原核表達(dá)質(zhì)粒pGEX4T-2/fba和真核表達(dá)質(zhì)粒pcDNA3.1/fba。 2在大腸桿菌中成功表達(dá)了Fba重組蛋白,表達(dá)蛋白主要以包涵體形式存在。ELISA和Western-blot證實(shí)表達(dá)的Fba重組蛋白可與已知兔抗Fba陽性血清產(chǎn)生特異性反應(yīng)。 3 Fba蛋白可與GAS全菌免疫鼠血清、抗鏈“O”陽性病人血清發(fā)生特異性結(jié)合,盡管Fba蛋白做為診斷抗原的檢測結(jié)果比M蛋白的檢測結(jié)果的OD值小,但二者的檢測結(jié)果一致,均為陽性。 4質(zhì)�；虻鞍酌庖吆笮∈驣gG產(chǎn)生水平以Fba蛋白免疫組增高最為明顯,依次為Fba質(zhì)粒蛋白混合免疫組及Fba質(zhì)粒組。特異性抗原誘導(dǎo)后體外脾細(xì)胞增殖試驗(yàn)顯示:pcDNA3.1/fba免疫組增值水平明顯高于其它組,流式細(xì)胞檢測結(jié)果與此一致,并顯示CD4+、CD8+T細(xì)胞水平較對照組有顯著性增高。 5鏈球菌攻擊后,PBS組小鼠在4天內(nèi)全部死亡,而Fba蛋白組、pcDNA3.1/fba+Fba蛋白組及M蛋白組在14天的觀察期內(nèi)均有60%的存活率,pcDNA3.1/fba組為40%。結(jié)論:1成功構(gòu)建了原核表達(dá)質(zhì)粒pGEX4T-2/fba和真核表達(dá)質(zhì)粒pcDNA3.1/fba。 2在大腸桿菌中高效表達(dá)了Fba重組蛋白。 3 GAS感染可以誘導(dǎo)人體及動物體產(chǎn)生Fba抗體,即Fba蛋白與M蛋白一樣,也具有良好的免疫原性和抗原特異性。因M蛋白及M抗體是人體感染GAS后誘發(fā)超敏反應(yīng)的主要原因,加之其血清型的多樣性,使M蛋白作為GAS的候選疫苗受到限制,故可以考慮Fba蛋白作為GAS的候選疫苗及診斷抗原。 4 Fba蛋白能有效地誘導(dǎo)機(jī)體產(chǎn)生抗體,Fba真核質(zhì)粒能有效誘導(dǎo)抗體及Th細(xì)胞增殖,且Fba蛋白或基因獲得了較強(qiáng)的抗GAS感染的能力,以上結(jié)果均提示Fba蛋白很有希望成為GAS的候選疫苗。
[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，

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