A族链球菌表面蛋白Fba单克隆抗体对应表位的鉴定及研究

发布时间：2018-01-14 17:37

本文关键词：A族链球菌表面蛋白Fba单克隆抗体对应表位的鉴定及研究　出处：《河北医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:A族链球菌(Group A Streptococcus,GAS)是一种常见致病菌,可引起严重的侵袭性感染及感染后变态反应性疾病。青霉素一直是抗链球菌感染的首选药物,但随着抗菌药的大量应用、乃至滥用,耐药菌株逐渐增多,加之链球菌本身的免疫逃逸,链球菌感染在临床上仍然呈现反复性及难以治愈的问题。诱导产生高效价抗体仍然是预防、控制链球菌感染的有效措施,故疫苗的研制成为预防此菌感染的又一切入点。但由于GAS血清型众多及其表面某些免疫原性强的蛋白可诱导严重的自身免疫反应等弊端,目前还没有理想的疫苗问世[1]。 Fba蛋白(Fibronectin-binding protein a)是2001年由Terao Y.等人新发现的一种表达于GAS表面的蛋白[2],广泛分布于多种型别的链球菌表面,并且在不同型别的链球菌中具有很高的同源性。Fba蛋白属于Fn(Fibronectin,Fn,纤连蛋白)结合蛋白,利用Fn作为桥梁,与上皮细胞、内皮细胞上的相关受体结合,从而介导细菌进入宿主细胞[3]。Fba蛋白还可与补体调节因子FH、FHL-1结合,阻止补体C3在菌体表面沉积,借此逃避补体溶菌、抵抗巨噬细胞的调理吞噬[4,5]。以往对Fba蛋白的研究表明其有良好的免疫原性并可诱发保护性免疫应答[6,7]。鉴于此,Fba蛋白有望成为GAS疫苗的候选蛋白。本室致力于Fba蛋白的特性及其在链球菌逃避免疫攻击的作用研究。本室前期已经制备了2株抗Fba蛋白的单克隆抗体(Monoclonal antibody,McAb),其中McAb2能特异性的结合野生型GAS,并证明McAb2只与Fba分段蛋白中Fba68-161段结合,初步预测表位是以104~108位氨基酸为核心的肽段。本课题以McAb2结合Fba蛋白的特异性区段为基础,利用生物信息学方法预测McAb2所对应的表位,借助基因工程手段获取了含有相应表位的融合蛋白及预期表位缺失蛋白,并利用噬菌体肽库筛选McAb2结合的关键位点,进而对该结合位点是否与FH和Fba蛋白的结合位点相重叠进行了研究。为进一步探索Fba蛋白在GAS致病机制中的作用,鉴定McAb2的功能及制备表位肽疫苗奠定了基础。方法: 1针对McAb2结合Fba蛋白的特异区段,利用生物信息学方法预测其所对应的表位,确定涵盖此表位的三个重叠表位片段,采用搭桥PCR扩增该基因片段并克隆表达,Western blot检测融合蛋白与McAb2的结合。 2构建相应表位缺失的基因片段并克隆表达,Western blot及ELISA检测突变蛋白与McAb2是否结合。 3合成表位重叠肽片段,dot-ELISA检测各段与McAb2的结合。 4利用噬菌体7肽库筛选McAb2对应的优势氨基酸。 5通过竞争ELISA检测McAb2是否能竞争FH与Fba蛋白的结合。 6利用血浆吸附实验[5]检测McAb2能否阻止人血浆中的FH与Fba蛋白结合。结果: 1成功构建了pGEX-4T-2与预测的三条基因片段84~101aa、95~114aa、101~118aa的原核表达质粒,并分别得以表达。Western blot结果显示,Fba95-114和Fba101-118与McAb2有明显的杂交带。 2成功构建表位缺失的基因片段68~161aa△96~118aa与pGEX-4T-2的重组原核表达质粒,并得以成功表达。Western blot显示McAb2不与Fba68-161△96-118结合。间接ELISA结果与上述结果一致。 3合成三段重叠肽分别是Fba蛋白的第100~112、104~116和108~120位氨基酸多肽,dot-ELISA结果显示第100~112位的氨基酸多肽与McAb2的结合能力最强。 4利用随机噬菌体7肽库筛选的结果显示McAb2针对的优势氨基酸是位于Fba基因序列的第100~110位氨基酸中的ITPDL。 5竞争ELISA数据经统计学处理,证实McAb2能部分封闭FH与野生型GAS的结合。 6血浆吸附实验证实野生型GAS能结合人血浆中的FH,并且McAb2可以部分阻止FH与Fba蛋白的结合。结论: 1成功克隆、表达了Fba蛋白McAb2所对应的表位肽段及预期表位缺失蛋白,初步确定了McAb2对应的表位区段。 2利用噬菌体展示技术,确定了McAb2所针对的优势氨基酸。 3 McAb2能够阻止或部分阻止FH与Fba蛋白的结合,为进一步鉴定McAb2的功能,深入研究GAS的致病机制提供了有力的依据。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】