Vibrio Anguillarum K Protein Prokaryotic Expression And Monoclonal Antibody Preparation

At present,domestic ane international use of outer membrane protein K andpreparation of monoclonal antibodies had not been reported.Preparation of monoclonalantibodies against Vibrio cholerae OmpK for detection to prevent aquatic animal vibriosishas certain chinical significance.Vibrio vulnificus was one of the most important pathogenic bacteria,which causedaquaculture fish bacterial disease.It was prevalenced in a wide area and highincidence,often resulted great loss in aquaculture industry.At present,the detection ofVibrio was mainly relied on pathological observation and indentification of bacteria andthey were restricted in the accuracy,sensitivity and detection.Outer membrane protein Kwas a broad host range phage KVP40receptor and in Vibrio anguillarum had beenconfirmed,located in the cytoplasmic membrane of outer wall and widely contactenvironmental science.According to the Vibrio anguillarum outer membrane protein Kgene sequence designed1pair primer and amplified the gene sequence by PCR.Thespecific band was connected into the PET-28a vector.IPTG induced expression,Ni2+column purification.Immunized Balb/c mice and preparated OmpK monoclonalantibody.For the detection and control of Vibrio disease provides the experimental basis.There were a series of experiments and the methods and results were as follows:(1) According to the Vibrio anguillarum outer membrane protein K gene sequence waslogged on the GenBank(accession number:FJ705222.1) designed1pair primer,withedHindIII in the end5,and EcoR I in the3,.The total DNA of Vibrio anguillarum wasamplified by PCR and had a specific bands about730bp.The specific bands wasVibrio anguillarum OmpK gene sequence.(2) The specific band connected into PET-28a and formed OmpK-PET-28a recombinantplasmid. It was transfoumed into BL21E.coli and induced by IPTG.. SDSverification a protein band about27kDa had been expressed. The recombinant proteins formed inclusion body, the urea decomposted and Ni2+purified.Itsconcentration about200ug/ml.(3) Hybrioma technology was used to produce mAbs against OmpK,by fusing of mouseSp2/0myeloma cells and spleen cells of Bals/c mouse immunized by OmpK.Thepositive clones which produced mAbs OmpK were screened by enzyme linkedimmunosorbent assay.After cloning,two strains of hybridoma named respectively1G2and1E4stably secreting specific mAbs against OmpK.Indentification of IgGsubgroup showed that1G2was the IgM,1E4was the IgG2b.Hydroperitoneum of theantibodies were10-5mensurating by ELISA.Bacteria cross-reaction showed that allthe mAbs had cross-reation with Vibrio anguillraum,Vibrio parahaemolyticusHarvey vibrio,Non0-1Vibrio choearae,Vibrio algionlyticus,VibrioVulnificus, and did not have cross-reaction with Delayed Edward,s bacteria,Aeromonassobria,0707Streptococcus pneumoniae.The two strains of hybridoma were used fordetection of Vibrio and further research.