The Selection And Preparation Of ETEC K88Bio-probes And Rapid Detection Of ETEC K88Based On The Probes

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..