The Research On Isolation And Identification Of Pathogenic Bacteria And Prevention Of Group Died In Pelodiscus Sinensis

Aeromonas is one of the pathogens which influence individuals and water-living economic animal. Due to the swift progress of high density turtle-cultivating enterprise, diseases, which make severe economic damage, come up in a large scale. A colony of turtles died for unsure reasons in Shijiazhuang in 2008, and then similar cases occurred in all areas in Hebei province.The research got pathogen HBJY01 from the lesion site of the turtles and used traditional technology and molecule biology technology to identify what category the pathogen belongs to. Establish phylogenetic tree with gene 16SrRNA and gene gyrB, and calculate the genetic distance of every two sequences to query the evolution status. It indicated that both the results are the same, the gene 16SrRNA and gyrB enjoyed high degree homology with corresponding genes of Vickers Aeromonas. Artificial injection with HBJY01 caused a death rate of 90%, the symptom was similar to the soft-shelled turtles in a large scale. We got the same pathogen in the liver. Based on gene ompA reported by GenBank and gene sequence hly of Aeromonas hydrophila, we designed two pairs of primers, and then got gene ompA and gene hly using PCR. Sequence analysis indicated that the sizes of the segments were 1000bp and 1500bp.The homology to consulting sequence were 97.45% and 96.58%.We established recombined plasmid pET-ompA and pET-hly with the directional clone to prokaryotic expression carrier pET-28a of gene ompA and gene hly. Then we erected gene hly in the downstream of gene ompA and obtained fusion plasmid pET-ompA-hly. Transform the three types of recombined plasmid into recipient bacterium BL21 respectively. The positive recombined strain BL21(DE3)(pET-ompA), BL21(DE3)(pET-hly)and BL21(DE3)(pET-ompA-hly),after the inducement of IPTG,the result of SDS-PAGE showed there were expressions and the result of Western blot indicated the expressed protein was specific production.Optimize the induction conditions in temperature, induce time and the consistence of IPTG of successfully-constructed genetic engineering strain BL21(DE3)(pET-omp-hly). The condition of the largest amount of expressed protein was 37℃,induce time 2.5h and the consistence of IPTG at 0.2mg/mL.Conduct animal protection experiment with genetic engineering strain BL21(DE3)(pET-omp-hly).The protective immunity of the genetic engineering strain was pretty qualified according to the results of ELISA and issue slice.To find out practicable immune way, we used biodegradable macromolecule material PLGA to prepare sustained-release microspheres making use of W/O/W. Next we made the preparation initially optimized. The encapsulation efficiency of the good-appearance microspheres was 68.8%. The result of the immune animal experiment showed a high protection rate. And after the oral immune experiment ,a high antibody level showed up and there was also a long immunization period.To sum up, our study laid the foundation for the prevention of the disease.