| Food-borne disease has become a common worldwide public sanitation problem. Bacteria and its Toxins are the most important alogen of Food -borne disease. There are many ways to examine Food Poisoning Bacteria and its Toxins nowadays, but there has not been a way which could examine Staphylococcal Enterotoxin, EHEC O157: H7, and Clostridium Bolutinum-the three kinds of Food Poisoning Bacteria and its Toxins at the same time. Focusing on the three kinds of Food Poisoning Bacteria, which often cause Food-brone disease, and the five Toxins produced by them, this project set up a ELISA way, worked out immunity colloid gold dipsticks, and meanwhile had a elementary applying experiment.Strain of this experiment is Recombinant Toxins genetic engineering bacteria T2 of the five Toxins gene of the three kinds of bacteria- Staphylococcal Enterotoxin, EHEC O157: H7 and Clostridium Botulinum which are the common Food Poisoning Bacteria. This experiment enlarged ferment, induced and expressed using IPTG, and made SDS-PAGE electrophoresis analysis. The result showed that amounted was highest under 37℃after 5 hours, Recombinant Toxins genetic engineering T2 appeared obvious the expressed protein between 97-66 kDa, and its size was similar with the predicted molecular weight (88.899 kDa). By thin-layer scanning and to was 20.44% of total protein,the most expressed protein existed in the form of inclusion body protein(17.6%), and only a small amount was soluble protein (2.84%).The Recombinant toxins T2 inclusion body protein was denatured and renatured.The inclusion expressed protein was purified by SDS-PAGE and protein extracting liquids. After examination for protein density and quantification ,"JiRong"rabbits was immuned four times at the intervals of seven days. Blood was taken from the heart after the immunity and anti-serum was made. ELISA examination showed that different Toxins produced different anti-serum value, and when the density of the natural Toxins VT1, VT2, SEA, SEB, BoNTa was 0.5, 1, 2, 4, 4μg/mL, the responding best antibody diluting-multiple was VT1 51200×, VT2 25600×, SEA 1600×, SEB 1600×, BoNTa 1600×. By Western-blotting examination, Recombinant Toxins protein and anti-serum had a good reactionogenicity,which showed that Recombinant Toxins expressed protein could effectively stimulate the organism to produce antibody which had a good immune- ogenicity and identify the five natural Toxins of the kinds of bacteria. The experiment also extracted and prepared the two natural Toxins-BoNTa and VT1 which shared the same size with the original Toxins by SDS-PAGE examination.The experiment via optimized condition for ELISA and set up the indirect ELISA operating program. By ELISA examination, the experiment determined the best cover density for Recombinant Toxins protein antigen was 0.45μg/mL, the best diluting multiple for serum-antibody was 51200×. Doing immunity experiment by using antibody got by immunity and the five natural Toxins, we got obvious positive react from the five toxins by ELISA examination. The result of antibody specificity ELISA did not demo- nstrated obvious positive reaction with 27 kinds of bacteria such as Listeria et al., However we could see positive reaction if we took place with objective bacteria or that five natural Toxins, which explained that Recombinant Toxins antibody had a good specificity. We got the examining sensitivity degree for the five natural Toxins-VT1, VT2, SEA, SEB, BoNTa, which were 3.75 ng/mL, 7.5 ng/mL, 31.25 ng/mL, 15.6 ng/mL, 15.6 ng/mL.Using citric acid reducting process, we prepared colloid gold particles which showed nearly the same size under electron microscope, and scattered evenly. The average diameter of colloid gold grain is 20nm shown by the identification of electron microscope and ultraviolet scanning appraisal. Using colloid gold immunity chromatographic assay, we made colloid gold dipsticks and did experiment for sensitivity,specificity, and stability, which showed that the best coating density for serum-antibody and colloid gold was 25μg/ml, and the colloid gold best coating pH value was 8.5.Using purified the the recombinant toxins T2 inclusion body protein as the coating antigen,with antibody to take the gold sign protein, finally asse- mble the dipsticks. We got the dipstick lowest examination limits respectively for the five natural Toxins-VT1, VT2, SEA, SEB, BoNTa, which were 3.75 ng/mL, 7.5 ng/mL, 31.25 ng/mL, 15.6 ng/mL, 15.6 ng/mL. Colloid gold dipsticks have a good specificity, no reaction with 27 kinds of bacteria such as Listeria et al., and a good repetitiveness, and the time for guarantee the quality is 6 weeks at 4℃. |
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