本文關(guān)鍵詞：A族鏈球菌表面蛋白Fba單克隆抗體對應(yīng)表位的鑒定及研究　出處：《河北醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： A族鏈球菌(GAS) Fba蛋白 單克隆抗體 抗原表位 噬菌體肽庫 FH

【摘要】： 目的:A族鏈球菌(Group A Streptococcus,GAS)是一種常見致病菌,可引起嚴(yán)重的侵襲性感染及感染后變態(tài)反應(yīng)性疾病。青霉素一直是抗鏈球菌感染的首選藥物,但隨著抗菌藥的大量應(yīng)用、乃至濫用,耐藥菌株逐漸增多,加之鏈球菌本身的免疫逃逸,鏈球菌感染在臨床上仍然呈現(xiàn)反復(fù)性及難以治愈的問題。誘導(dǎo)產(chǎn)生高效價(jià)抗體仍然是預(yù)防、控制鏈球菌感染的有效措施,故疫苗的研制成為預(yù)防此菌感染的又一切入點(diǎn)。但由于GAS血清型眾多及其表面某些免疫原性強(qiáng)的蛋白可誘導(dǎo)嚴(yán)重的自身免疫反應(yīng)等弊端,目前還沒有理想的疫苗問世[1]。 Fba蛋白(Fibronectin-binding protein a)是2001年由Terao Y.等人新發(fā)現(xiàn)的一種表達(dá)于GAS表面的蛋白[2],廣泛分布于多種型別的鏈球菌表面,并且在不同型別的鏈球菌中具有很高的同源性。Fba蛋白屬于Fn(Fibronectin,Fn,纖連蛋白)結(jié)合蛋白,利用Fn作為橋梁,與上皮細(xì)胞、內(nèi)皮細(xì)胞上的相關(guān)受體結(jié)合,從而介導(dǎo)細(xì)菌進(jìn)入宿主細(xì)胞[3]。Fba蛋白還可與補(bǔ)體調(diào)節(jié)因子FH、FHL-1結(jié)合,阻止補(bǔ)體C3在菌體表面沉積,借此逃避補(bǔ)體溶菌、抵抗巨噬細(xì)胞的調(diào)理吞噬[4,5]。以往對Fba蛋白的研究表明其有良好的免疫原性并可誘發(fā)保護(hù)性免疫應(yīng)答[6,7]。鑒于此,Fba蛋白有望成為GAS疫苗的候選蛋白。 本室致力于Fba蛋白的特性及其在鏈球菌逃避免疫攻擊的作用研究。本室前期已經(jīng)制備了2株抗Fba蛋白的單克隆抗體(Monoclonal antibody,McAb),其中McAb2能特異性的結(jié)合野生型GAS,并證明McAb2只與Fba分段蛋白中Fba68-161段結(jié)合,初步預(yù)測表位是以104~108位氨基酸為核心的肽段。本課題以McAb2結(jié)合Fba蛋白的特異性區(qū)段為基礎(chǔ),利用生物信息學(xué)方法預(yù)測McAb2所對應(yīng)的表位,借助基因工程手段獲取了含有相應(yīng)表位的融合蛋白及預(yù)期表位缺失蛋白,并利用噬菌體肽庫篩選McAb2結(jié)合的關(guān)鍵位點(diǎn),進(jìn)而對該結(jié)合位點(diǎn)是否與FH和Fba蛋白的結(jié)合位點(diǎn)相重疊進(jìn)行了研究。為進(jìn)一步探索Fba蛋白在GAS致病機(jī)制中的作用,鑒定McAb2的功能及制備表位肽疫苗奠定了基礎(chǔ)。 方法: 1針對McAb2結(jié)合Fba蛋白的特異區(qū)段,利用生物信息學(xué)方法預(yù)測其所對應(yīng)的表位,確定涵蓋此表位的三個(gè)重疊表位片段,采用搭橋PCR擴(kuò)增該基因片段并克隆表達(dá),Western blot檢測融合蛋白與McAb2的結(jié)合。 2構(gòu)建相應(yīng)表位缺失的基因片段并克隆表達(dá),Western blot及ELISA檢測突變蛋白與McAb2是否結(jié)合。 3合成表位重疊肽片段,dot-ELISA檢測各段與McAb2的結(jié)合。 4利用噬菌體7肽庫篩選McAb2對應(yīng)的優(yōu)勢氨基酸。 5通過競爭ELISA檢測McAb2是否能競爭FH與Fba蛋白的結(jié)合。 6利用血漿吸附實(shí)驗(yàn)[5]檢測McAb2能否阻止人血漿中的FH與Fba蛋白結(jié)合。 結(jié)果: 1成功構(gòu)建了pGEX-4T-2與預(yù)測的三條基因片段84~101aa、95~114aa、101~118aa的原核表達(dá)質(zhì)粒,并分別得以表達(dá)。Western blot結(jié)果顯示,Fba95-114和Fba101-118與McAb2有明顯的雜交帶。 2成功構(gòu)建表位缺失的基因片段68~161aa△96~118aa與pGEX-4T-2的重組原核表達(dá)質(zhì)粒,并得以成功表達(dá)。Western blot顯示McAb2不與Fba68-161△96-118結(jié)合。間接ELISA結(jié)果與上述結(jié)果一致。 3合成三段重疊肽分別是Fba蛋白的第100~112、104~116和108~120位氨基酸多肽,dot-ELISA結(jié)果顯示第100~112位的氨基酸多肽與McAb2的結(jié)合能力最強(qiáng)。 4利用隨機(jī)噬菌體7肽庫篩選的結(jié)果顯示McAb2針對的優(yōu)勢氨基酸是位于Fba基因序列的第100~110位氨基酸中的ITPDL。 5競爭ELISA數(shù)據(jù)經(jīng)統(tǒng)計(jì)學(xué)處理,證實(shí)McAb2能部分封閉FH與野生型GAS的結(jié)合。 6血漿吸附實(shí)驗(yàn)證實(shí)野生型GAS能結(jié)合人血漿中的FH,并且McAb2可以部分阻止FH與Fba蛋白的結(jié)合。 結(jié)論: 1成功克隆、表達(dá)了Fba蛋白McAb2所對應(yīng)的表位肽段及預(yù)期表位缺失蛋白,初步確定了McAb2對應(yīng)的表位區(qū)段。 2利用噬菌體展示技術(shù),確定了McAb2所針對的優(yōu)勢氨基酸。 3 McAb2能夠阻止或部分阻止FH與Fba蛋白的結(jié)合,為進(jìn)一步鑒定McAb2的功能,深入研究GAS的致病機(jī)制提供了有力的依據(jù)。
[Abstract]:Objective: Streptococcus A (Group A Streptococcus, GAS) is a common pathogen that can cause severe invasive infection after infection and allergic diseases. Penicillin has been the drug of choice against Streptococcus infection, but with a large number of applications, and abuse of antibiotics, drug resistant strains increased gradually, and the immune Streptococcus itself the escape in clinic is still repeated and difficult to cure. Streptococcal infection induced high antibody titer is prevention, effective measures to control the streptococcal infection, the vaccine to prevent the infection become another entry point. But because the serotypes of GAS are numerous and some surface immunogenic protein induction of severe autoimmune reaction and other defects, there is no ideal vaccine [1].
Fba protein (Fibronectin-binding protein A) is a kind of expression in 2001 by Terao Y. et al found on the surface of GAS protein [2], Streptoccus are widely distributed in a variety of types, and has a very high homology of.Fba protein belongs to Fn in different serotypes (Fibronectin, Fn, fibronectin binding protein). The use of Fn as a bridge, and the epithelial cells with receptors on endothelial cells, which is mediated by the bacteria into the host cell [3].Fba protein also interacts with complement regulatory factor FH, FHL-1 binding, prevent complement C3 deposition in the cell surface, thereby avoiding bacteriolytic resistance of macrophages, phagocytosis of [4,5]. conditioning of previous research on Fba protein showed that the have good immunogenicity and can induce protective immune responses to [6,7]. in view of this, the Fba protein is expected to become the candidate protein GAS vaccine.
Study on the properties of the room is dedicated to Fba protein and Streptococcus escape immune attack. This system has already prepared his previous 2 strains of monoclonal antibodies against Fba antigen (Monoclonal antibody, McAb McAb2), which can be combined with wild type specific GAS, and prove that McAb2 only and Fba Fba68-161 protein in combined section preliminary prediction, epitope peptide with 104~108 amino acid is at the core of the issue with McAb2 specific binding domain of Fba protein as the foundation, use biological information to predict the corresponding epitopes of McAb2 methodology, for containing the corresponding epitope fusion protein and epitope protein with expected loss of genetic engineering means, key sites and the use of phage peptide library screening McAb2 binding, and the binding site with FH and Fba protein loci overlap were studied. To further explore the Fba protein in GAS pathogenic effect It lays the foundation for identification of the function of McAb2 and the preparation of epitope peptide vaccine.
Method:
1, according to the specific segment of McAb2 binding to Fba protein, bioinformatics method was used to predict its corresponding epitope, and three overlapping epitope fragments covering this epitope were determined. The gene fragment was amplified by bridging PCR and cloned and expressed, and Western blot was used to detect the binding of fusion protein to McAb2.
2 the gene fragment deletion of the corresponding epitopes was constructed and the expression was cloned. Western blot and ELISA were used to detect the combination of the mutant protein with McAb2.
3 synthetic epitopes overlap peptide fragments, and dot-ELISA was used to detect the combination of each segment with McAb2.
4 a phage 7 peptide library was used to screen the dominant amino acids corresponding to McAb2.
5 the competitive ELISA tests whether McAb2 can compete with the combination of FH and Fba protein.
6 the detection of McAb2 by plasma adsorption test [5] could prevent the binding of FH and Fba protein in human plasma.
Result:
1, we successfully constructed the prokaryotic expression plasmid of pGEX-4T-2 and predicted three gene fragments 84~101aa, 95~114aa and 101~118aa, and expressed.Western blot respectively. The results showed that Fba95-114 and Fba101-118 had obvious hybrids with McAb2.
2 the successful construction of recombinant epitope deletion of 68~161aa gene fragment 96~118aa and pGEX-4T-2 prokaryotic expression plasmid, and the successful expression of.Western blot indicated that McAb2 combined with Fba68-161 96-118. The results of indirect ELISA and the results are consistent.
3, the three segment overlap peptide is the 100~112104~116 and 108~120 amino acid polypeptide of Fba protein. Dot-ELISA shows that the amino acid polypeptide at the 100~112 level has the strongest binding capacity with McAb2.
4 the results of screening by random phage 7 peptide library show that the dominant amino acid against McAb2 is ITPDL. in the 100~110 bit amino acid in the sequence of the Fba gene
5 competitive ELISA data were statistically processed to confirm that McAb2 could partially seal the combination of FH and wild type GAS.
6 plasma adsorption experiments confirmed that wild type GAS could bind to FH in human plasma, and McAb2 could partially prevent the combination of FH and Fba protein.
Conclusion:
1 successfully cloned, expressed the epitope peptide segment corresponding to the Fba protein McAb2 and the expected epitope deletion protein, and preliminarily identified the corresponding epitope segments of the McAb2.
2 a phage display technique was used to determine the dominant amino acid targeted by McAb2.
3 McAb2 can prevent or partially prevent the combination of FH and Fba protein, which provides a powerful basis for further identification of McAb2 function and in-depth study of the pathogenesis of GAS.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙紅蕾;刁昱文;馮新;高宇;劉珊珊;顧敬敏;雷連成;韓文瑜;;噬菌體展示肽庫技術(shù)及其在分子病原細(xì)菌學(xué)研究中的應(yīng)用[J];河北科技師范學(xué)院學(xué)報(bào);2011年01期

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本文編號：1424623