| Vibrio mimicus is one of the common pathogenic bacteria of vibrio genus, and it distributes widely in natural waters and bodies of aquatic animals. If water quality deteriorates or immunization of bodies decreases, the V.mimicus propagates in quantity and produces various virulence factors. It not only leads to serious vibrios of aquatic animals resulting in large losses, but infects human being by food chain, leading to seasonal prevailing intestinal infection to endanger peoples' health. So the research on its biological characteristics, pathogenesis and preparation of vaccine is of most significance. Now people have studied on pathogenesis of vibrio cholera thoroughly, and found that it firstly colonized mucosal surfaces of intestine by Toxin Coregulated Pilus(TCP), then produced various virulence factors to function on aimed cells resulting in diarrhea . As one of the most important virulence factor, TCP widely distributed in classic group, Eltor group, and O139 group, taken part in pathogenic process by mediating adherence of bacteria to tissue-cell and coregulating expression of toxic gene. It hasn't been reported whether V.mimicus expressed TCP, its functions on pathogenic process and biological characteristics. Foreign scholars mainly examined TCP of V.mimicus isolated from different origins. V.mimicus HX4 strain isolated from Eriocheir sinensis was identified as a strong virulent strain, and its exotoxin were systematically studied. In order to study comprehensively the pathogenesis of V.mimicus, provide scientific evidences for the preparation of vaccine, prevent vibrios of aquatic animals. PCR and electropherogram technology were used to identified HX4 strain as TCP+ strain, further optimized the cultural conditions on TCP expression, established the methods of purification of TCP, investigated its biological characteristics systematically in this study.Firstly, TCP of V.mimicus HX4 strain were detected by PCR and electropherogram technology. We devised a pair of specific primers based on tcpA sequence registered in gene bank. by comparing banding pattern of TCP generated with the primers with the theoretical results(617bp), we concluded that there were tcpA gene in genome of V.mimicus HX4 strain. At the same time by electropherogram technique we found there were many short thick pilus in the cell surfaces, coincided with the foreign reports about TCP. So V.mimicus HX4 strain was a TCP+ strain, which could express TCP in certain cultural condition.Secondly, cultural conditions on the TCP expression were optimized in vitro by bacteriological methods and electropherogram technique. The results demonstrated that the types of media(AKI,CFA,BHI and LB), cultural temperature(4℃,20℃,30℃and 37℃),and cultural methods all affected the TCP expression. The optimal conditions were initial pH8.0 , solid media of CFA, at 30℃ for 24h in stationary and aerobic culture. Thirdly, the purification methods of TCP were established by separate technology of pilus, precipitation and PAGE-gel extraction technique. Among four elution buffers, the highest protein percentage and best PAGE profile were observed when HX4 strain was eluted with 2mol/L urea-PBS(0.01 mol/L pH7.2).Further, TCP were precipitated with three different percentage AS(30%,50% and 70%),the highest and lowest crude protein percentage were obtained respectively when70% and 30% AS were used. But there were no obvious difference judged by PAGE profile of the three crude proteins. The total protein obtained was lower with 30%AS, however, the aimed one was relatively higher. So it was concluded that 30%AS was the best percentage of precipitating TCP. Then the position in PAGE-gel and molecular weight (20.14KD) of TCP was determined by PAGE-gel extraction and electropherogram technique.At last biological characteristics of TCP were systematically studied with cellular model, intestinal model, animal test and hemagglutinative experiment. The adhesive ability of HX4 strain was tested with HEp-2 cells and intestine of Eriocheir sinensis. The results... |
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