| Salmonella is a class of facultative intracellular pathogens which is widely distributed in nature. Currently, more than 2500 kinds of Salmonella serotypes have been identified which can infect humans and animals. Salmonella can cause enteritis, typhoid and a series of diseases. Salmonella is often detected in a variety of livestock, poultry and wildlife. Meat, vegetables, eggs and milk can be contaminated by Salmonella. Salmonella typhimurium, Salmonella enteritidis and Salmonella choleraesuis are the most common polluting bacteria. It is reported that bacterial food poisoning which were caused by the Salmonella accounted for about 42.6% ~ 60.0%.Around the world, non-typhoid Salmonella is the most important cause of foodborne diarrhea. Salmonella contamination serious threats the food safety and public health. The rapid detection of Salmonella is very important in food safety and responding to public health emergencies. Currently, the main methods of detection of Salmonella are traditional culture, molecular and immunological detection methods. In this study, we used immunogold chromatography to develop a rapid detection reagent of Salmonella could be simple and easy to be used in folk and evaluae the sensitivity and specificity of the reagents preliminarily. The main contents of this study are as follows:Part I: The prokaryotic expression of the four surface antigens such as FimA, SefA, Ail, and PadThe selection of the target antigens: We selected the major structural subunit of type-Ⅰfimbriae and sef14 fimbriae, Ail and Pad outer membrane proteins as the target antigens for detection of Salmonella. TypeⅠfimbriae is the most common and thoroughly studied fimbriae. This fimbriae that could be served as the detected target is a relatively conservative pilus in Salmonella. The sef 14 fimbriae is an unique pili of D group of Salmonella and the Salmonella enteritidis is the most common disease-causing Salmonella bacteria. Therefore, we could determine the D group Salmonella by identifying the sef14 fimbriae. The sefA gene encodes the major structural subunit of sef14 fimbriae. Ail protein is a genetic conservative, genus-specific surface outer membrane protein of Salmonella. And the Ail protein has low level expression in all serotypes of Salmonella. The Ail protein could be chosed as the candidate target for detecting of Salmonella. The Pad protein is a kind of adhesion whose role is to bind the hosts. We compared the gene and protein sequences by sequence alignment in the database of NCBI and found that the pad gene is a genus-specific gene in Salmonella.Currently, there are no reports about using the Pad protein as the target of detecting Salmonella. The Pad protein is more likely to be used as a new testing target of Salmonella.Prokaryotic expression of the four antigens: We amplified fimA, sefA, ail, and pad genes of Salmonella by polymerse chain reaction. A total of 543bp fimA gene was inserted into the prokaryotic expression vector pET-32a(+). SefA gene was to be amplified starting from 142bp and a total of 432bp was inserted into prokaryotic expressing vector. Ail gene was to be amplified starting from 61bp and a total of 438bp was inserted into prokaryotic expressing vector. Pad gene was to be amplified strarting from 46bp and a total of 675bp was inserted into pET-32a(+). We could deterimine that the target sequences were inserted into the expression vector correctly by sequencing the DNA. The analysis of sequence alignment showed that comparing with the standard strain, the sequence homology of fimA, sefA, ail and pad is 99.45%, 99.77%, 98.86% and 100%, respectively. The four positive recombinant plasmid pET32a(+)-fimA, pET32a(+)-sefA, pET32a(+)-ail and pET32a(+)-pad were transformed into E. coli BL21 (DE3). After the induction of IPTG, we obtain high level expression of recombinant proteins.The SDS-PAGE analysis showed that the molecular weight of recombinant protein of rFimA is 39kD and the recombinant protein is expressed mainly in the form of inclusion bodies. The molecular weight of rSefA is about 35kD and the recombinant protein was expressed in soluble and inclusion bodies. The molecular weight of rPad is about 42kD and its expression is mainly in the form of inclusion bodies. The four recombinant proteins had positive reaction with the goat anti-salmonella antibody which showed those recombinant proteins have good antigenicity. To obtain the rabbit anti-recombinant protein antibodies using preliminary purified antigen.Identification of the antigenicity of the four recombinant proteins: We analysed the antigenicity of the four antigens by western blotting, and the results showed that the four recombinant proteins can react with goat anti-Salmonella sera, indicating that the four recombinant proteins have good antigenictiy.Preparation of the recombinant antigens immune serum: After purified the four recombinant proteins, the concentration of four recombinant proteins were determined by ultraviolet spectrophotometry. The rabbit anti-recombinant antigens serum were preparated by the four preliminary purified recombinant proteins. Part II: Preliminary development of colloidal gold reagents and laboratory evaluationIn this study, the optimal labeling concentrations of rabbit anti-rFimA, rSefA, rAil and rPad antibody is 25μg/ml, 25μg/ml, 30μg/ml and 30μg/ml, respectively. When dilute the antibodies to the concentrations of sensitilized, we could use the sodium tetraborate (10% sucrose) to dilute the antibodies for removing the appearance of non-specific bands.The optimal sensitized concentration of rabbit anti-rAil rFimA, rSefA, rAil and rPad antibody is 2mg/ml, 4mg/ml, 4mg/ml and 4mg/ml, respectively. Preliminary laboratory evaluation of the four colloidal gold antibodies:Salmonella typhi, Salmonella enteritidis, Salmonella arizona, Salmonella typhimurium and Salmonella choleraesuis were detected by all the four colloidal gold antibodies. The minimum detectable concentration which be detected with SefA, Ail, and FimA colloidal gold labeled antibodies were 1×108 cell/ ml. The minimum detectable concentration of Pad colloidal gold antibody was 1×107 cell/ml. In detection of the 13 non-Salmonella, there were no reaction between the four collidal antibodies with Enterococcus faecalis, Hafnia H.alvei, Staphylococcus, pasteurella multocida, Vibrio cholerae Ogawa, Vibrio parahaemolyticus, Streptococcus viridans, Pseudomonas aeruginosa, Candida albicans and the four colloidal gold antibodies. However, when the cell concentration was higher than 1×108 cell / ml, there were cross reaction between Feces enterococcus, Providencia stuartii coli, Shigella flexneri 2a, Citrobacter and the four colloidal gold labeled antibodies. |
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