金葡菌腸毒素表位的串聯(lián)表達(dá)與間接競爭ELISA方法的建立
發(fā)布時間：2018-12-16 21:30
【摘要】：金黃色葡萄球菌腸毒素(Staphylococcal Enterotoxin, SE)是單肽鏈毒性蛋白質(zhì),屬于典型的細(xì)菌外毒素,微量即可引起嚴(yán)重的人類食物中毒。目前已發(fā)現(xiàn)SE有20種血清型,不同血清型的檢出率與菌株分離地域和宿主來源密切相關(guān),建立某地區(qū)優(yōu)勢SE血清型的檢測方法更具實際意義。本研究室已探明安徽合肥地區(qū)乳源金黃色葡萄球菌腸毒素的優(yōu)勢血清型為SEA和SEG。目前已有檢測SEA的國標(biāo)ELISA方法,但尚無檢測SEG的免疫學(xué)方法。本研究的總體思路是在篩選鑒定SEA和SEG兩種毒素蛋白的優(yōu)勢B細(xì)胞線性表位、設(shè)計和制備多表位串聯(lián)肽的基礎(chǔ)上,建立基于表位的間接競爭ELISA法,該方法可同時檢測SEA和SEG兩種優(yōu)勢血清型。 首先,采用表位預(yù)測結(jié)合實驗驗證方法篩選鑒定SEA蛋白和SEG蛋白的優(yōu)勢B細(xì)胞線性表位。即運用DNAstar Protean軟件綜合分析腸毒素蛋白的二級結(jié)構(gòu)、柔性區(qū)域、表面可及性、親水性和抗原指數(shù)篩選其可能的B細(xì)胞線性表位,根據(jù)可能的B細(xì)胞線性表位在蛋白三維結(jié)構(gòu)中的位置,獲得潛在的優(yōu)勢表位,并進行肽ELISA鑒定。結(jié)果發(fā)現(xiàn)SEA蛋白和SEG蛋白均有3個優(yōu)勢B細(xì)胞線性表位,它們分別是P156(SEAaa156～163)、P166(SEAaa166～173)、P211(SEAaa211～218)、P37(SEG aa37～46)、P120(SEGaa120～127)PP141(SEGaa141～149)。 其次,根據(jù)SEA蛋白和SEG蛋白優(yōu)勢B細(xì)胞線性表位序列及多表位串聯(lián)肽設(shè)計原則,優(yōu)化設(shè)計多表位串聯(lián)肽。即用AAY(丙氨酸-丙氨酸-酪氨酸)接頭將SEA蛋白和SEG蛋白優(yōu)勢B細(xì)胞線性表位按不同方式串聯(lián),應(yīng)用DNAstar Protean軟件分析各種串聯(lián)方式的可行性,結(jié)果發(fā)現(xiàn)以P156-AAY-P166-AAY-P211-AAY-P37-AAY-P120-AAY-P141方式排列的多表位串聯(lián)肽中各表位間相對獨立,抗原性參數(shù)較好,為最佳串聯(lián)方式。將此表位串聯(lián)肽序列轉(zhuǎn)化為基因序列,并在基因序列兩端添加酶切位點,終止密碼子以及替換稀有密碼子,形成一個長度為318bp的表位串聯(lián)基因(命名為AG基因),委托生物公司克隆至質(zhì)粒pUC57中。 然后,大量制備多表位串聯(lián)肽及其抗體,為建立間接競爭ELISA方法提供試驗材料。應(yīng)用基因重組技術(shù)構(gòu)建重組表達(dá)質(zhì)粒pET-32a-AG,轉(zhuǎn)化至E.coli Rosetta進行大量誘導(dǎo)表達(dá),進而使用Ni-Charged Resin親合層析柱純化重組蛋白His-SE-AG。經(jīng)SDS-PAGE分析,發(fā)現(xiàn)制備的多表位串聯(lián)肽濃度為1.7mg/mL,純度達(dá)到95%。以重組表位串聯(lián)肽免疫家兔,免疫后第28天血清抗體的ELISA效價達(dá)到1：327680,瓊擴效價達(dá)到1：16,免疫血清經(jīng)飽和硫酸銨分級沉淀和Protein G親和層析法純化后兔抗SE-AG抗體的濃度為lmg/mL,純度達(dá)到95%,ELISA效價為1：819200,滿足免疫檢測試劑的要求。 最后,建立基于表位的間接競爭ELISA方法。分別以重組表位串聯(lián)肽和天然腸毒素為競爭抗原進行間接競爭ELISA。以競爭抗原濃度的常用對數(shù)為橫坐標(biāo),以各濃度抗原競爭抑制率(A/Ao)%為縱坐標(biāo)繪制標(biāo)準(zhǔn)曲線。結(jié)果顯示2個標(biāo)準(zhǔn)曲線的線性方程具有良好的擬合度,即使用表位串聯(lián)肽建立的競爭ELISA方法可以用于天然腸毒素SEA和(或)SEG的特異性檢測。靈敏性和重復(fù)穩(wěn)定性試驗的結(jié)果顯示其最低檢測限度為5.068ng/mL,批內(nèi)與批間變異系數(shù)均小于15%,具有較好的靈敏性和重復(fù)穩(wěn)定性。 綜上所述,本研究建立的基于表位的間接競爭ELISA方法是一種快速(4h左右)、特異、靈敏和穩(wěn)定的檢測方法,可同步檢測A型和G型金黃色葡萄球菌腸毒素。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R155.5
本文編號：2383073
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R155.5
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