肠毒素大肠杆菌K88生物探针制备及其快速检测分析技术研究
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摘要
肠毒素大肠杆菌K88(ETEC K88)是一种常见的肠道微生物,也是引起仔猪腹泻和死亡的主要致病微生物。养殖业中准确、低成本、简便、快速地找出引起仔猪腹泻的原因及微生物的种类甚至是血清型一直是一个难以攻克的难题,一些很好的生物产品如疫苗、卵黄抗体等也因此在仔猪疾病预防和治疗中的不能大规模低成本应用。建立和完善针对于ETEC K88的低成本、简便、快速分析检测体系并实现在养殖业中的商业化应用是降低这一类致病微生物引起的仔猪死亡的重要手段。
本文以ETEC K88为目标模式菌,利用生物学方法筛选和制备以K88菌毛蛋白为靶标的单克隆抗体和适配体、以ETEC K88菌体为靶标的细胞适配体和适配体文库,鉴定了它们与靶标的结合性能,初步构建了基于这几种生物探针的检测体系分别用于K88菌毛蛋白和ETEC K88菌体的快速检测分析。同时,成功建立了一个新的FluMag-Cell-SELEX (FluMag Cell systematic evolution of ligands by exponential enrichment)细胞适配体筛选方法。实验结果如下：
本文优化了ETEC K88的发酵工艺,在优化的玉米浆培养基和发酵条件的基础上制备纯度和浓度满足单克隆抗体和适配体筛选要求的K88菌毛蛋白,SDS-PAGE显示了单一条带,Bradford法测定浓度为0.642mg/ml。
利用杂交瘤技术制备并生物素化K88菌毛蛋白的单克隆抗体(K88mAb),测定抗体效价为1：12800,亲和力常数Kaff测定为(1.616±0.033)×108M-1,流式细胞仪分析和荧光显微镜分析抗体与目标菌结合性能良好,荧光分光光度计分析该抗体和ETEC K88菌体有很好的结合特异性。初步建立了基于K88mAb的ABS-ELISA方法检测K88菌毛蛋白体系,确定其检测下限为12.53ng/ml。
采用多孔板法SELEX技术筛选到K88菌毛蛋白的DNA适配体,其中亲和力最高的适配体Apt37Kd值达到了25±4nmol。荧光显微镜和荧光分光光度计分析该适配体与目标菌结合性能良好而且有很好的结合特异性。初步建立了基于K88菌毛蛋白适配体Apt37的Aptamer-ABS-ELISA体系检测K88菌毛蛋白,确定其检测下限为25.1ng/ml。
分别采用SDS-PAGE和平板计数法验证了K88IMB (Immunomagnetic bead)捕获ETEC K88的可靠性,实验证明K88IMB对ETEC K88在102、103CFU/ml的低浓度下捕获率分别可达74.5%和63.8%,说明K88IMB可有效应用在接下来的实验体系中。
经典离心分离法Cell-SELEX技术与本文建立的FluMag-Cell-SELEX技术筛选ETEC K88细胞适配体得到了相同的高重复度序列,说明FluMag-Cell-SELEX技术是成功的。亲和力最高的细胞适配体测定Kd值为15+4nM。激光共聚焦显微镜和流式细胞仪分析表明适配体Apt B12、FluMag-Cell-SELEX技术中最后一轮的富集适配体文库这两种生物探针和ETEC K88的结合性能均良好。
建立了基于细胞适配体／适配体文库的直接荧光探针分析-IMS(Immunomagnetic separation)技术相结合快速检测ETEC K88体系。以细胞适配体文库作为直接荧光探针时,检测信号比单个适配体探针更强,体系对纯培养ETEC K88的检测下限为1.1×103CFU/ml,对低至2.1×103CFU/g模拟的腹泻仔猪粪便样本都可以检测到可信信号。
Diarrhea of piglets caused by enterotoxigenic Escherichia coli K88(ETEC K88) is an important factor of both mortality and reduced growth rate resulting in heavy economic losses. Swift and precise identification of ETEC K88from infected samples is critical for the subsequent treatment of infectious diseases caused by ETEC K88. K88fimbriae, expressed on the surface of K88ETEC strains, are responsible for their adhesion and colonization on small intestinal and producing enterotoxins, as well as being special detection target to ETEC K88. In fact, in clinical diagnosis, it's very hard to simply and quickly distinguish ETEC K88from other pathogens resulting in diarrhea of early weanling piglets by the commonly used methods, which makes some very good preventive or treatment products such as specific vaccine, egg yolk antibody not in widespread use at low cost and leads to abuse of antibiotics in livestock breeding. It's necessary to develop a detection platform that's precise, convenient, rapid and low cost to reduce the loss resulting from diarrhea of piglets in breeding.
In this study, ETEC K88was served as target bacteria for the selections and preparations of monoclonal antibody (mAb) and DNA aptamer against K88fimbrial protein, DNA aptamer and aptamer library against ETEC K88cell. After the studies on characteristics of these bio-probes, they were used to establish rapid detection platforms for ETEC K88and its K88fimbrial protein. In the meanwhile, a technique named "FluMag-Cell-SELEX"(FluMag Cell systematic evolution of ligands by exponential enrichment) was constructed based on Cell-SELEX combined with IMS technology for the selection of DNA aptamer against cell. The methods and experiment conclusions are listed as the followings:
The fermentation conditions of ETEC K88were optimized by the means of single factor experiment and orthogonal experiment to produce K88fimbrial protein as more as possible. Suitable purity (only one band on SDS-PAGE gel) and concentration (0.642mg/ml) of K88fimbrial protein were obtained to demand the need of selection of mAb and aptamer.
K88mAb was prepared by hybridoma technique and biotinylated. Kaff of the mAb was determined as (1.616±0.033)×108M-1and titer as1:12800by typical avidin-biotin system ELISA (ABS-ELISA). The ability of K88mAb binding to ETEC K88was identified by fluorescence microscopy and flow cytometry. Fluorescence spectrophotometry was used to verify the specificity of mAb binding to ETEC K88. In addition, ABS-ELISA system was prelimilarily established based on K88mAb for the the rapid detection of fimbrial protein, the sensitivity of which was determined to be12.53ng/ml.
Aptamers against K88fimbrial protein were obtained by plate SELEX as well. Apt37had the best affinity with K88fimbrial protein and its Kd (dissociation constant) was found as25±4nM via fluorescence analysis. The same as K88mAb, the binding ability between mAb and ETEC K88was identified by fluorescence microscopy. Fluorescence spectrophotometry was used to verify the specificity of mAb binding to ETEC K88. Aptamer-ABS-ELISA system was prelimilarily established based on Apt37for the the rapid detection of K88fimbrial protein, the sensitivity of which was determined to be25.1ng/ml.
IMBs (Immunomagnetic beads) were formed by the incubation of biotinylated K88mAb and streptavidin modified MB (magnetic beads) and verified their capture efficiency to ETEC K88cell by the mean of plate count. The result revealed that74.5%,63.8%of the initial bacteria is respectively captured when the dilutions of ETEC K88at102,103CFU/ml.
Classic Cell-SELEX and IMS-Cell-SELEX (stablished here) were done to select DNA aptamers against ETEC K88cell. From both of them, the same aptamers with high reproducibility among more than40clones were gained, indicating that the new IMS-Cell-SELEX technology for the selection of aptamer against cell was successful. Apt B12was selected to determine Kd as15±4nM and identified its affinity with ETEC K88by confocal imaging and flow cytometry. The last selection of aptamer pool during FluMag-Cell-SELEX procedures, which is so called "DNA aptamer library" in this study, was cloned by PCR and also identified its binding ability with ETEC K88. Then, both the probes were successfully used to be direct fluorescence reporter to establish a rapid detection system for ETEC K88combined with IMS technology, which yielded a detection limited as1.1×103CFU/ml for pure culture cells and2.1×103 CFU/g for artificially contaminated fecal sample when aptamer library acted as probe, whereas individual aptamer could not..
本文以ETEC K88为目标模式菌,利用生物学方法筛选和制备以K88菌毛蛋白为靶标的单克隆抗体和适配体、以ETEC K88菌体为靶标的细胞适配体和适配体文库,鉴定了它们与靶标的结合性能,初步构建了基于这几种生物探针的检测体系分别用于K88菌毛蛋白和ETEC K88菌体的快速检测分析。同时,成功建立了一个新的FluMag-Cell-SELEX (FluMag Cell systematic evolution of ligands by exponential enrichment)细胞适配体筛选方法。实验结果如下：
本文优化了ETEC K88的发酵工艺,在优化的玉米浆培养基和发酵条件的基础上制备纯度和浓度满足单克隆抗体和适配体筛选要求的K88菌毛蛋白,SDS-PAGE显示了单一条带,Bradford法测定浓度为0.642mg/ml。
利用杂交瘤技术制备并生物素化K88菌毛蛋白的单克隆抗体(K88mAb),测定抗体效价为1：12800,亲和力常数Kaff测定为(1.616±0.033)×108M-1,流式细胞仪分析和荧光显微镜分析抗体与目标菌结合性能良好,荧光分光光度计分析该抗体和ETEC K88菌体有很好的结合特异性。初步建立了基于K88mAb的ABS-ELISA方法检测K88菌毛蛋白体系,确定其检测下限为12.53ng/ml。
采用多孔板法SELEX技术筛选到K88菌毛蛋白的DNA适配体,其中亲和力最高的适配体Apt37Kd值达到了25±4nmol。荧光显微镜和荧光分光光度计分析该适配体与目标菌结合性能良好而且有很好的结合特异性。初步建立了基于K88菌毛蛋白适配体Apt37的Aptamer-ABS-ELISA体系检测K88菌毛蛋白,确定其检测下限为25.1ng/ml。
分别采用SDS-PAGE和平板计数法验证了K88IMB (Immunomagnetic bead)捕获ETEC K88的可靠性,实验证明K88IMB对ETEC K88在102、103CFU/ml的低浓度下捕获率分别可达74.5%和63.8%,说明K88IMB可有效应用在接下来的实验体系中。
经典离心分离法Cell-SELEX技术与本文建立的FluMag-Cell-SELEX技术筛选ETEC K88细胞适配体得到了相同的高重复度序列,说明FluMag-Cell-SELEX技术是成功的。亲和力最高的细胞适配体测定Kd值为15+4nM。激光共聚焦显微镜和流式细胞仪分析表明适配体Apt B12、FluMag-Cell-SELEX技术中最后一轮的富集适配体文库这两种生物探针和ETEC K88的结合性能均良好。
建立了基于细胞适配体／适配体文库的直接荧光探针分析-IMS(Immunomagnetic separation)技术相结合快速检测ETEC K88体系。以细胞适配体文库作为直接荧光探针时,检测信号比单个适配体探针更强,体系对纯培养ETEC K88的检测下限为1.1×103CFU/ml,对低至2.1×103CFU/g模拟的腹泻仔猪粪便样本都可以检测到可信信号。
Diarrhea of piglets caused by enterotoxigenic Escherichia coli K88(ETEC K88) is an important factor of both mortality and reduced growth rate resulting in heavy economic losses. Swift and precise identification of ETEC K88from infected samples is critical for the subsequent treatment of infectious diseases caused by ETEC K88. K88fimbriae, expressed on the surface of K88ETEC strains, are responsible for their adhesion and colonization on small intestinal and producing enterotoxins, as well as being special detection target to ETEC K88. In fact, in clinical diagnosis, it's very hard to simply and quickly distinguish ETEC K88from other pathogens resulting in diarrhea of early weanling piglets by the commonly used methods, which makes some very good preventive or treatment products such as specific vaccine, egg yolk antibody not in widespread use at low cost and leads to abuse of antibiotics in livestock breeding. It's necessary to develop a detection platform that's precise, convenient, rapid and low cost to reduce the loss resulting from diarrhea of piglets in breeding.
In this study, ETEC K88was served as target bacteria for the selections and preparations of monoclonal antibody (mAb) and DNA aptamer against K88fimbrial protein, DNA aptamer and aptamer library against ETEC K88cell. After the studies on characteristics of these bio-probes, they were used to establish rapid detection platforms for ETEC K88and its K88fimbrial protein. In the meanwhile, a technique named "FluMag-Cell-SELEX"(FluMag Cell systematic evolution of ligands by exponential enrichment) was constructed based on Cell-SELEX combined with IMS technology for the selection of DNA aptamer against cell. The methods and experiment conclusions are listed as the followings:
The fermentation conditions of ETEC K88were optimized by the means of single factor experiment and orthogonal experiment to produce K88fimbrial protein as more as possible. Suitable purity (only one band on SDS-PAGE gel) and concentration (0.642mg/ml) of K88fimbrial protein were obtained to demand the need of selection of mAb and aptamer.
K88mAb was prepared by hybridoma technique and biotinylated. Kaff of the mAb was determined as (1.616±0.033)×108M-1and titer as1:12800by typical avidin-biotin system ELISA (ABS-ELISA). The ability of K88mAb binding to ETEC K88was identified by fluorescence microscopy and flow cytometry. Fluorescence spectrophotometry was used to verify the specificity of mAb binding to ETEC K88. In addition, ABS-ELISA system was prelimilarily established based on K88mAb for the the rapid detection of fimbrial protein, the sensitivity of which was determined to be12.53ng/ml.
Aptamers against K88fimbrial protein were obtained by plate SELEX as well. Apt37had the best affinity with K88fimbrial protein and its Kd (dissociation constant) was found as25±4nM via fluorescence analysis. The same as K88mAb, the binding ability between mAb and ETEC K88was identified by fluorescence microscopy. Fluorescence spectrophotometry was used to verify the specificity of mAb binding to ETEC K88. Aptamer-ABS-ELISA system was prelimilarily established based on Apt37for the the rapid detection of K88fimbrial protein, the sensitivity of which was determined to be25.1ng/ml.
IMBs (Immunomagnetic beads) were formed by the incubation of biotinylated K88mAb and streptavidin modified MB (magnetic beads) and verified their capture efficiency to ETEC K88cell by the mean of plate count. The result revealed that74.5%,63.8%of the initial bacteria is respectively captured when the dilutions of ETEC K88at102,103CFU/ml.
Classic Cell-SELEX and IMS-Cell-SELEX (stablished here) were done to select DNA aptamers against ETEC K88cell. From both of them, the same aptamers with high reproducibility among more than40clones were gained, indicating that the new IMS-Cell-SELEX technology for the selection of aptamer against cell was successful. Apt B12was selected to determine Kd as15±4nM and identified its affinity with ETEC K88by confocal imaging and flow cytometry. The last selection of aptamer pool during FluMag-Cell-SELEX procedures, which is so called "DNA aptamer library" in this study, was cloned by PCR and also identified its binding ability with ETEC K88. Then, both the probes were successfully used to be direct fluorescence reporter to establish a rapid detection system for ETEC K88combined with IMS technology, which yielded a detection limited as1.1×103CFU/ml for pure culture cells and2.1×103 CFU/g for artificially contaminated fecal sample when aptamer library acted as probe, whereas individual aptamer could not..
引文
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