Expression And Immunogenicity Analysis On The Fusion Protein OmpA From Aeromonas Hydrophila Strain J-1 And Construction Of Expressing Vector With The Fusion Gene Suitable To Tetrahymena Thermophila

Aeromonas hydrophila is the causative agent of fatal hemorrhagic septicemia in fish and can cause a wide variety of human infections. Outer membrane protein(OMP) has been proved to be an important protective antigen against A. hydrophila.1. Primers were designed based on the outer membrane protein A(OmpA) gene sequence published in GenBank. A 960 bp DNA fragment was amplified from 15 of 18 different strains of Aeromonas hydrophila by PCR.The PCR products of J-1, Y-65 and SPS-104 were inserted into pMD18-T vector respectivelyly, and the positive recombinants were identified by endonuclease digestion, PCR and DNA sequencing. The ompA sequences of J-1, Y-65 and SPS-104 had 93.1% to 95% homology to those from other strains of A. hydrophila at NCBI, and all lacked 4 acid residues in two sites. The homologies ompA gene and amino acid sequences between J-land SPS-104 were 99.2% and 98.1%, and the sites for lacking 4 acid residues were same. But the homologies between J-1 and Y-65 were 88.7% and 89.6%, and the lacking sites were a little different. The forecasts of antigenicity and surface structure with DNAStar Protean software showed that compared with 5 reference strains of A. hydrophila, the distribution of antigen sites was identity. It suggested that OmpA might be a common protection antigen in Aeromonas.2.With the specific primers, the coding region of mature protein ompA gene from Aeromonas hydrophila strain J-1 was amplified by PCR and cloned into pET-32a(+). The recombinant plasmid pET32-ompA were transformed into E.coli BL21(DE3) and induced with IPTG. SDS-PAGE and Western blot analysis showed that the target recombinant protein with the molecular weight of 57.4kD was hyperexpressed in the form of inclusion body. After purified by Ni²⁺-affinity chromatography the protein was used to immunize the rabbit. Antiserum was collected and used to detect the specificity of recombinant protein. ELISA showed that the titer of antiserum was above 1:12800. Moreover, the antiserum reacted not only to specifically with the purified recombinant protein but also to OMPS of about 35kD from 8 typic Aeromonas isolations from 3 domestic major serotypes and 1 Aeromonas salmonicida in Western blot. The result indicates that the recombinant OmpA has the same immunity epitope as the nature one and maybe the common protective antigen in Aeromonas hydrophila. 3. Two pairs of primers PN1 and PN2, PM3 and PM4 were respectively designed based on the sequences of neomycin resistance protein gene(neo) and ompA gene of A. hydrophila J-l. The vector pSK-H4-I-neo and genomic DNA of A. hydrophila J-l were used as PCR templates for neo gene and ompA, respectively. Then neo and the partial ompA fragmetnts were purified by DNA agarose gel purification Kit. With these two fragments mixed together as the template, one target fragment about 1.5 kb was obtained with primer PN1 and PM4 by the second step PCR amplification. A linker,(Gly4Ser) 3, was inserted between these two genes. The 1.5 kb fragment was digested by EcoRI and BamHI and ligated into EcoR and BamHI linearized plasmid pSK-H4-I-neo. pSK-H4-I-neo-GS-ompA, an expression vector with the fusion fragment was then successfully constructed. The vector will further be transformed into T. thermophila and then be integrated by precise homologous recombination into the genome, based on the presence of homologous arm flanking the targeted neo-GS-ompA gene in the insert.4. The proper time for electroporation was firstly determined and then under different buffers, voltages and capacitors, the gene targeting vector pSK-H4-I-neo-GS-ompA and the linear fragment containing whole coding region were introduced by allelic exchange into the conjungants of T. thermophila in order to co-express neomycin and OmpA. The cells of mating type, T. thermophila BF₁ and BF5, were hungry for 22 hours and then mixed. The conjugation started 6 hours after mixation and the number of conjugation cells reached the highest ratio after 7～8 hours. But the transformants with neomycin resistance were not obtained. There might be due to a wide difference in the usages of synonymous condons between bacteria and eukaryotics Tetrahymena.