Prokaryotic And Eukaryotic Expression Of OmpK Of Vibrio Harveyi

Vibrio harveyi is a common causative agent of Vibriosis that infects cultured marine fish. Its special outer membrane proteins (OMPs) in the cell wall play important roles in the function of cell structure stability, substances exchange and pathogenicity. Recent studies emphasized the role of the OMP of pathogenic bacteria in protective antigenicity. A target of protective immunity, OmpK, is the receptor for KVP40 which is a broad-host-range vibriophage. It exists in many kinds of vibrios and has much of chance to interact with the external environment.According to OmpK gene sequence published in GenBank (Accession AY332563), primers were designed and a piece of DNA sequence about 750 bp was amplified by PCR from genomic DNA of Vibrio harveyi. The gene was cloned into pMD18T vector and sequenced, then submitted to GenBank with the accession number HQ395754. Blast result showed that the sequence shared identity of 99.0% with OmpK of Vibrio harveyi. The restriction enzyme BamHⅠand HindⅢwas applied to digest the recombinant plasmid pMD18T-OmpK in order to obtain the OmpK gene, then the OmpK gene was subcloned into pET30a and transformed into E.coli BL21(DE3) competent cells. After all, the prokaryotic project strain E.coli/pET30a-OmpK was constructed. Induced by IPTG, the protein was expressed and formed undissolving inclusion body in the cells. SDS-PAGE profiles showed the MW of recombinant OmpK was about 33kD. After several times scrubbing by EDTA, Triton and low concentration urea, the recombinant protein was dissolved into high concentration urea, then it was renaturated by way of dialysis. Using the well-renaturated recombinant protein to immunize the New Zealand rabbit, the antiserum with high antibody value was acquired.Based on the characteristics of secreted expression vector pPIC9K, primers were designed and the mature peptide coding sequence of OmpK was amplified by PCR with pET30a-OmpK as the template, then the gene was inserted into vector pPIC9K. After all, the recombinant expression vector pPIC9K-OmpK was well constructed. The recombinant plasmid was confirmed by colony PCR, double-enzyme digestion and sequence analysis. After extracting enough construct and parent vector, the enzyme Sac I was choosed to linearize them, then they were transformed into Pichia pastor is competent cells by electroporation. Transformants GS115/pPIC9K-OmpK and GS115/pPIC9K were selected by geneticin to acquire the colonies that can resiste high concentration G418 (4.0mg/mL). Screened by MM/MD plates, phenotype of this two kinds of transformants were almost Mut+. The result of colony PCR showed that the OmpK gene was integrated into the Pichia genome. Induced by methanol, the positive transformants began to secretorily expressed the recombinant protein OmpK. SDS-PAGE analysis indicated that the recombinant protein was hyperglycosylated with a MW about 37KD, more than the expected size 29KD. Western-blotting result indicated that the recombinant protein could be specifically recognized by antiserum of OmpK expressed in the E.coli BL21(DE3). The result showed that the recombinant protein was successfully expressed in the Pichia pastoris and the immunogenicity of it was not affected.Based on the characteristics of intracellular expression vector pPIC3.5K, primers were designed and the mature peptide coding sequence of OmpK was cloned, which was inserted into the plasmid pPIC3.5K with the result of shuttle vector pPIC3.5K-OmpK being constructed. Linearizing by restriction enzyme SalⅠ, the construct and parent vector were transformed into Pichia pastoris GS115 by way of Lithium Chloride transformation. Screened by YPD geneticin plates, the transformants GS115/ pPIC3.5K-OmpK and GS115/pPIC3.5K that can resist 2.0mg/mL G418 were acquired. After Screening Phenotype by MM/MD plates, the genome of transformants were extracted and the transformants were confirmed by two pairs of primers. The result showed that transformants GS115/pPIC3.5K-ompk and GS115/pPIC3.5K were well constructed. Induced by methanol, the positive transformants began to intracellularly expressed the heterologous protein OmpK. SDS-PAGE analysis showed the recombinant protein OmpK formed inclusion body in the Pichia cells with a MW about 28.7KD. Western-blotting result showed that the unglycosylated recombinant protein can react to the anti-recombinant OmpK serum. After all, the heterologous protein OmpK was successfully expressed in the cells of Pichia pastoris.