金葡菌肠毒素表位的串联表达与间接竞争ELISA方法的建立

发布时间：2018-12-16 21:30

【摘要】：金黄色葡萄球菌肠毒素(Staphylococcal Enterotoxin, SE)是单肽链毒性蛋白质,属于典型的细菌外毒素,微量即可引起严重的人类食物中毒。目前已发现SE有20种血清型,不同血清型的检出率与菌株分离地域和宿主来源密切相关,建立某地区优势SE血清型的检测方法更具实际意义。本研究室已探明安徽合肥地区乳源金黄色葡萄球菌肠毒素的优势血清型为SEA和SEG。目前已有检测SEA的国标ELISA方法,但尚无检测SEG的免疫学方法。本研究的总体思路是在筛选鉴定SEA和SEG两种毒素蛋白的优势B细胞线性表位、设计和制备多表位串联肽的基础上,建立基于表位的间接竞争ELISA法,该方法可同时检测SEA和SEG两种优势血清型。首先,采用表位预测结合实验验证方法筛选鉴定SEA蛋白和SEG蛋白的优势B细胞线性表位。即运用DNAstar Protean软件综合分析肠毒素蛋白的二级结构、柔性区域、表面可及性、亲水性和抗原指数筛选其可能的B细胞线性表位,根据可能的B细胞线性表位在蛋白三维结构中的位置,获得潜在的优势表位,并进行肽ELISA鉴定。结果发现SEA蛋白和SEG蛋白均有3个优势B细胞线性表位,它们分别是P156(SEAaa156～163)、P166(SEAaa166～173)、P211(SEAaa211～218)、P37(SEG aa37～46)、P120(SEGaa120～127)PP141(SEGaa141～149)。其次,根据SEA蛋白和SEG蛋白优势B细胞线性表位序列及多表位串联肽设计原则,优化设计多表位串联肽。即用AAY(丙氨酸-丙氨酸-酪氨酸)接头将SEA蛋白和SEG蛋白优势B细胞线性表位按不同方式串联,应用DNAstar Protean软件分析各种串联方式的可行性,结果发现以P156-AAY-P166-AAY-P211-AAY-P37-AAY-P120-AAY-P141方式排列的多表位串联肽中各表位间相对独立,抗原性参数较好,为最佳串联方式。将此表位串联肽序列转化为基因序列,并在基因序列两端添加酶切位点,终止密码子以及替换稀有密码子,形成一个长度为318bp的表位串联基因(命名为AG基因),委托生物公司克隆至质粒pUC57中。然后,大量制备多表位串联肽及其抗体,为建立间接竞争ELISA方法提供试验材料。应用基因重组技术构建重组表达质粒pET-32a-AG,转化至E.coli Rosetta进行大量诱导表达,进而使用Ni-Charged Resin亲合层析柱纯化重组蛋白His-SE-AG。经SDS-PAGE分析,发现制备的多表位串联肽浓度为1.7mg/mL,纯度达到95%。以重组表位串联肽免疫家兔,免疫后第28天血清抗体的ELISA效价达到1：327680,琼扩效价达到1：16,免疫血清经饱和硫酸铵分级沉淀和Protein G亲和层析法纯化后兔抗SE-AG抗体的浓度为lmg/mL,纯度达到95%,ELISA效价为1：819200,满足免疫检测试剂的要求。最后,建立基于表位的间接竞争ELISA方法。分别以重组表位串联肽和天然肠毒素为竞争抗原进行间接竞争ELISA。以竞争抗原浓度的常用对数为横坐标,以各浓度抗原竞争抑制率(A/Ao)%为纵坐标绘制标准曲线。结果显示2个标准曲线的线性方程具有良好的拟合度,即使用表位串联肽建立的竞争ELISA方法可以用于天然肠毒素SEA和(或)SEG的特异性检测。灵敏性和重复稳定性试验的结果显示其最低检测限度为5.068ng/mL,批内与批间变异系数均小于15%,具有较好的灵敏性和重复稳定性。综上所述,本研究建立的基于表位的间接竞争ELISA方法是一种快速(4h左右)、特异、灵敏和稳定的检测方法,可同步检测A型和G型金黄色葡萄球菌肠毒素。
[Abstract]:Staphylococcal enterotoxin (SE) is a single-chain toxic protein, which is a typical bacterial exotoxin, which can cause serious human food poisoning. At present, there are 20 serotypes of SE, the detection rate of different serotypes is closely related to the isolation region of the strain and the host source, and the detection method of the dominant SE serotype in a certain region is more practical. The research laboratory has proved that the dominant serotypes of the staphylococcal enterotoxin of the milk source in Hefei district of Anhui province are SEA and SEG. At present, the national standard ELISA method for detecting SEA is available, but there is no immunological method for detecting SEG. The general idea of this study is to establish an indirect competitive ELISA based on the table position on the basis of screening and identifying the B-cell linear table of the two toxin proteins of SEA and SEG, and the two dominant serotypes of SEA and SEG can be detected at the same time. First, the advantage of the SEA protein and the SEG protein B-cell linear table was selected by the method of table-bit prediction combined with the experimental verification method. The potential advantage is obtained from the position of the possible B-cell linear table in the three-dimensional structure of the protein by using the DNA star protean software to comprehensively analyze the secondary structure, the flexible region, the surface and the property, the hydrophilicity and the antigen index of the enterotoxin protein. position and peptide-linked immunosorbent assay The results show that both SEA and SEG proteins have three dominant B-cell linear table bits, which are P156 (SEAaa156-163), P166 (SEAaa166-173), P211 (SEAaa211-218), P37 (SEG aa37-46), P120 (SEGaa 120-127) PP141 (SEGaa 141-149), respectively. and secondly, designing the multi-table bit according to the SEA protein and the SEG protein dominant B cell linear table bit sequence and the multi-epitope serial peptide design principle, The serial peptide is connected in series by using the AAY (Alanine-Alanine-Tyrosine) linker in series with the linear table bits of the B cell linear table of the SEG protein, and the feasibility of various serial modes is analyzed by using the DNAstar protean software. The results show that the inter-table phase of each table in the multi-table-position series peptide arranged in the P156-AAY-P166-AAY-P211-AAY-P37-AAY-P120-AAY-P141 mode The independent and antigenic parameters are better and the best series The method comprises the following steps of: converting the sequence peptide sequence of the table into a gene sequence, adding enzyme cutting sites at both ends of the gene sequence, stopping the codon and replacing the rare codons, and forming a table-position series gene (named AG gene) with a length of 318bp, and the biological company is entrusted to clone to the plasmid pUC 57. Then, a large number of multi-epitope tandem peptides and their antibodies were prepared, to establish an indirect competitive ELISA method. The recombinant expression plasmid pET-32a-AG was constructed by a gene recombination technique and transformed into E. coli Rosetta for a large number of induction expression, and the recombinant protein His-SE-AG. SDS-PAGE analysis found that the prepared multi-epitope tandem peptide concentration was 1. 7mg/ mL, purity The titer of the anti-SE-AG antibody was 1: 327680, the titer of the anti-SE-AG was 1: 16, the concentration of the anti-SE-AG antibody was lmg/ mL, the purity reached 95%, and the ELISA titer was 1: 8. 19200, to meet immune detection Reagent requirements. Finally, an indirect competition based on table bits is established in that method, the recombinant epitope serial peptide and the natural enterotoxin are respectively used for indirectly and indirectly, Competitive ELISA. The constant logarithm of the concentration of the competitive antigen is the abscissa, and the inhibition rate of the antigen of each concentration (A/ Ao)% is the vertical position. The results show that the linear equation of the two standard curves has a good fit, that is, the competitive ELISA method established by using the table-position series peptide can be used for the natural enterotoxin SEA and/ or the SE The results of sensitivity and repeated stability test show that the minimum detection limit is 5.068ng/ mL, the coefficient of variation between the batch and the batch is less than 15%, and it has a good sensitivity. To sum up, the indirect competitive ELISA method based on the table bits established in this study is a rapid (left-and-right), specific, sensitive and stable detection method, which can be used to detect the type and G-type gold synchronously.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

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