2型猪链球菌SSU05-0474蛋白的单克隆抗体制备及抗原表位鉴定

发布时间：2018-01-03 21:12

本文关键词：2型猪链球菌SSU05-0474蛋白的单克隆抗体制备及抗原表位鉴定　出处：《沈阳农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：2型猪链球菌(Streptococcus suis serotype 2,简称SS2)是一种重要的人畜共患病原菌,其感染在全球多个国家发生,引起人和动物的脑膜炎、败血症、关节炎、心内膜炎和中毒性休克综合征等疾病,呈群发或散发流行,给养猪业造成巨大的经济损失,对人类健康也构成严重威胁。为了防治SS2感染,研究者一直致力于其致病机制的研究。目前已经发现多种SS2的毒力因子,包括荚膜多糖、溶菌酶释放蛋白、溶血素、胞外蛋白因子和表面LPXTG蛋白等。其中存在于菌体表面的LPXTG蛋白,通过其N端锚定于菌体表面,从而在感染过程中首先并直接接触宿主细胞或蛋白,在致病性中发挥重要作用。因此,研究LPXTG蛋白的功能是了解SS2致病性的一个重要途径。经过对SS2菌株05ZYH33基因组序列分析发现,基因组编号为SSU05-0474的产物是一种LPXTG蛋白,该蛋白与菌毛骨架蛋白具有较高同源性,因此在本文中简称SsMps蛋白(S. suis Major Pili Subunit protein)。据报道菌毛骨架蛋白在与宿主的相互作用中扮演重要角色,我们的其它研究发现SsMps具有结合宿主一种重要免疫因子的能力,因此很可能在SS2致病性中起作用,研究其功能对了解SS2的致病机制具有重要意义。本研究以2型猪链球菌标准菌株05ZYH33的基因组DNA为模板,通过PCR扩增出1443bp的ssMps基因,经限制性内切酶Nde I和Xho I双酶切后,克隆至表达载体pET22b中,构建pET22b/ssMps重组质粒。重组质粒转化到大肠杆菌BL21(DE3)中后,重组菌通过加入终浓度0.8mmol/L的IPTG,22℃条件下诱导表达,蛋白SsMps最终获得了可溶性表达,表达量约占大肠杆菌BL21(DE3)总蛋白10%,通过亲和层析纯化获得较高纯度的SsMps蛋白,经SDS-PAGE电泳分析,呈现出了一个分子量为45 kDa的蛋白条带,与预期蛋白大小相符。然后经Western-Blot分析验证,证明成功表达并纯化了带有His标签的重组蛋白。以该重组蛋白为免疫原,免疫BALB/C雌鼠,利用细胞融合、克隆和筛选等技术,获得1株抗SsMps重组蛋白的单克隆抗体1D9。Western blot结果显示该单克隆抗体能够特异识别SS2菌体组分中的SsMps蛋白,并通过亚型鉴定试剂盒鉴定亚型,结果显示1D9重链为IgG1,轻链为K链,可用于后期对SsMps蛋白功能及应用研究。最后为了鉴定SsMps蛋白的单抗1D9的抗原表位,通过对随机十二肽噬菌体展示文库,进行3轮淘选,得到18株能够有效结合单抗1D9的阳性噬菌体。测序结果显示：7株噬菌体展示的十二肽序列为LPQRRQTRIMII,2株序列为RRHTTRKRMLKT,分别与SsMps蛋白的氨基酸序列162～183位和445～466位氨基酸有一定的相似度。合成这两段多肽,并经过与单抗的结合实验以及与SsM ps蛋白竞争结合单抗1D9实验,证实这两段多肽均和天然抗原表位相似,因此推断单抗1D9识别的抗原表位存在于162～183位和445～466位。
[Abstract]:Streptococcus suis serotype 2 (Streptococcus suis serotype 2, referred to as SS2) is an important zoonotic pathogen, the infection occurred in many countries around the world, cause meningitis, human and animal septicemia, arthritis, endocarditis and toxic shock syndrome and other diseases, a mass or sporadic, causing huge the economic losses to the pig industry, but also pose a serious threat to human health. In order to prevent SS2 infection, the researchers have been devoted to the pathogenic mechanism. It was found that a variety of virulence factors including SS2, capsular polysaccharide, lysozyme release protein, hemolysin, extracellular factor protein and surface LPXTG protein which exists in the. Cell surface LPXTG protein on cell surface by the N anchor, resulting in the infection process and the first direct contact with host cells or proteins that play an important role in pathogenicity. Because of this, the research of LPXTG protein The function is an important way to understand the pathogenicity of SS2. After the 05ZYH33 genome sequence of SS2 strain genome analysis found that the number SSU05-0474 of the product is a kind of LPXTG protein, the protein and pili cytoskeletal proteins with high homology, so in this paper referred to as SsMps (S. suis Major Pili Subunit protein protein). According to reports of pili cytoskeletal proteins in interactions with host plays an important role in other research we found that SsMps is capable of binding to host an important immune factor, so it may play a role in the pathogenicity of SS2, research its function is of great significance to understand the pathogenic mechanism of SS2. In this study, the genomic DNA of Streptococcus suis serotype 2 standard strain 05ZYH33 as template, ssMps gene was amplified by PCR 1443bp, I and Nde by restriction endonuclease Xho I digested and cloned into the expression vector pET22b, construct pET22b The recombinant plasmid /ssMps. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) after recombinant bacteria by adding a final concentration of 0.8mmol/L IPTG, the temperature of 22 DEG C induced expression of SsMps protein, obtained soluble expression, expression of Escherichia coli BL21 accounted for about 10% of total protein (DE3), purified by SsMps protein affinity chromatography analysis by SDS-PAGE electrophoresis, showing a protein with a molecular weight of 45 kDa, consistent with the expected size protein. And then by Western-Blot analysis, proved the successful expression and recombinant protein with His tag was purified. The recombinant protein as immunogen, immune BALB/C mice, using cells fusion, screening and cloning technology, monoclonal antibody 1D9.Western blot results of recombinant anti SsMps protein of 1 strains showed that the monoclonal antibody could specifically recognize SS2 cell components in SsMps protein, and the subtype identification Kit for identification of subtypes, the results showed that 1D9 heavy chain IgG1 and light chain of K chain, can be used for further research of SsMps protein function and application. Finally, in order to epitope identification of SsMps protein monoclonal antibody 1D9, through twelve random peptide phage display library, 3 rounds of panning, 18 strains can be effectively combined positive phage antibody 1D9. The sequencing results showed that twelve strains of phage display peptide sequence 7 for LPQRRQTRIMII, 2 strains of the RRHTTRKRMLKT sequence, and the amino acid sequence of SsMps protein were 162 ~ 183 and 445 ~ 466 amino acids have a certain similarity. Combined these two peptides, and with monoclonal antibody binding experiments and SsM PS and protein binding of monoclonal antibody 1D9 experiments confirmed that these two peptides are natural and epitopes similar, therefore infer mAb 1D9 recognized epitopes present in 162 ~ 183 and 445 ~ 466.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.611

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