Recombinant Expression And Immunogenicity Of OmpA Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a common main pathogenic bacterium in aquaculture and seafood products.Rapid and accurate immunoassay is still needed.Bacterial outer membrane protein has good specificity and surface exposure and is a good choice as an antigen.OmpA is one of the most important outer membrane proteins of Gram-negative bacteria with high abundance.Studies have shown that the OmpA protein of Vibrio parahaemolyphus can prepare antiserum to recognize bacteria,but the cross-reaction of serum is not clear.In this thesis,the OmpA protein of Vibrio parahaemolyticus was used as the research object,and the recombinant OmpA protein and OmpA polypeptide were prepared as the immunogen,and their immunogenicity was studied.According to the gene sequence(gi＿28897538)of the outer membrane protein OmpA of Vibrio parahaemolyticus published by GenBank,the corresponding primers were designed,and the enzyme cutting sites Bam HI and Xho I were inserted at both ends of the primers.The plasmid p ET32 a was used as the construction vector othe f recombinant plasmid.The target fragment(977bp)amplified by PCR from Vibrio parahemolyticus(CICC 21618)was digested with plasmid p ET-32 a,and the recombinant plasmid p ET32a-OmpA was constructed by ligase T4.The recombinant plasmid was transferre Escherichiaichia coli DH5α for amplification.The recombinant plasmid was then transfected into Escherichia coli BL21(DE3)for expression.The recombinant protein OmpA was expressed in soluble form and purified by affinity chromatography to obtain the recombinant protein His-OmpA(about 40 k Da).NCBI BLAST was used to compare and analyze the amino acid sequences of Parahaemolyticus OmpA with those of Vibrio strains,and the sequence similarity of some vibrio strains was more than 80%.Then,Meg Align,Prote,an and Py MOL software were combined to analyze the homology,specificity,hydrophobicity and peptide sites on the protein,and a hydrophilic peptide sequence(SNNVSKAAG)located in the outer membrane region of OmpA protein was obtained.Sulfo-smcc coupling agent was used to conjugate the peptide with BSA protein,and the electrophoretic characterization showed that the protein band moved up obviously,and the peptide immunogen was successfully prepared.Mice were immunized with recombinant protein and polypeptide immunogen,and serum recognition and cross reaction were studied by ELISA and western blot.The results showed that the recombinant protein OmpA had the highest titer(1:12 1500)with vibrio parahaemolyticus(CICC 21618),while the titer of the recombinant protein OmpA against other 5 vibrio parahaemolyticus and 11 Vibrio strains was in the range of 1:45 00-1:13 500,and there was a difference.The titer of Escherichia coli was the highest among non-vibrio bacteria,reaching1:121500.Western blotting results showed that the serum could react specifically with his-OmpA protein and the whole protein of six strains of Vibrio parahemoleuticus(CICC 21618,CICC21617,CICC 21619,CICC 10552,CICC 21528,CICC 1.1616).There was only slight cross-reaction with other vibrio strains,but there were significant reaction bands for Citrate malonate,cloacae and Escherichia coli.The above results can indicate that the outer membrane protein OmpA of Vibrio parahaemolyticus has good immunogenicity and surface exposure,but whether the monoclonal antibody can be prepared uniformly to recognize Vibrio parahaemolyticus remains to be further studied.