Study On Immunofluorescence Technique Detection Method Of Clam Perkinsus Infection

Perkinsus infection of oysters can have severe pathogenic effect on its hosts and destroyed the oyster industries in several countries. Ray Fluid Thioglycolate medium (RFTM) culture assay as the Classical dection method may have time-consuming and culture complex, still need to other auxiliary method can determine the positive Perkinsus. To meet the Fast and accurate, this study was produced specific antigen and its corresponding specific monoclonal antibodies, and on this basis. The immunofluorescence technology was selected to detect Perkinsus sp. in clams and oysters.According to published Genbank Perkinsus marinus CB5D4cell-wall protein (MOE) gene sequence designed and synthesized primers, CB5D4gene was amplified from Perkinsus marinus genomic DNA. A CB5D4-pMD19Tsimple plasmid for the detection of Perkinsus marinus based on CB5D4has been developed. CB5D4was ligated to expression vector pET32a and transformed into BL21(DE3) Escherichia coli, and induced to express by inducing with IPTG.by means of SDS-PAGE and Western Blot analysis. The results indicated that the protein was expressed as a soluble protein. The purified protein was used to immunize the mouse and cell fuse, detecting, selecting, colonizing and so on, we produced6specific monoclonal antibodies against Perkinsus marinus and research their characteristics. They were named respectively1E11,1B5,1B6,2A10,3B5and4E5.these results had laid the foundation for the serological assay detecting Perkinsus marinus.We marked the FITC fluorescent on purified monoclonal antibody, using marked the FITC fluorescent monoclonal antibodies to testing clam tissue section, established the immunofluorescence test method of Perkinsus marinus.