Clone And Expression Of Aeromonas Caviae FlagellinA (flaA)

In this assay, a series of researches was conducted on the Aeromonas strain XL2-T, such as Bacteriology and Pathology researches. The single polar flagellin gene A(flaA) was cloned even expressed by procaryon cell and eukaryon cell. Some of the relate inspection was done. This work can provide some advantage for the research and application about bactera subunit engineering vaccine in aquatic culture.Refer to correlate essay, with the reported oligonucleotide primer, the core glycosylation reigon partial sequence of the flaA genes from several A.c strains was cloned. The sequences alignment was performeded using CLUSTAL"W"method, it showed that the N-terminal and C-terminal sequence was much conservative. Consulted from Gnebank, a number of submitted A.c flaA genes was got and the alignment was also performeded using CLUSTAL"W"method. The N&C terminal sequence was also conservative. Using the designed degenerate oligonucleotide primer, the complete flaA gene of the XL2-T was cloned and recombinanted into pET-28a and pPIC9K, thus the recombinant prokaryotic expression plasmid pET-28a+flaA and the recombinant eukaryotic secret plasmid pPIC9K+flaA was constructed.pET-28a+flaA was transformed into E.coli BL21(DE3), while the recombinant was induced to express the recombinant flaA protein. T hrough subcutaneously inoculation, the anti-rcFlaA serum was prepared,and determined it's immune titer. Using the anti-rcFlaA serum, bacteria agglutination test was also conducted.Using the restricte enzyme Bgl II & Sac I, the plasmid pPIC9K+flaA was linearized from different restriction site. The linearized plasmid was electransformed separately into Pichia pastoris strain GS115, so the MutS and Mut+ recombinant was obtained. The prokaryotic flaA gene was induced to express by eukaryotic cell.Among the bacteriology research about the A.caviae strain XL2-T, the optimal culture medium and cultivate conditions and the preservation conditions was investigated, the clone morphology and bacteria morphology was studied, the bacteria physiological and chemical characterization was identified, the 16SrDNA molecular identification, the motility assay, the correlate virulences and the drug sensitivity test was also conducted. The study was focused on the flagella research, include the silver stain and lefison stain, the polar and lateral flagellin purification. With the anti-rcFlaA serum, immunoblot was done. All the lateral and polar flagellin purified from the three tested A,c strains had cross-positive reaction. This result proved that using the flaA gene , if genetic engineering vaccine is made and applicated some day, it can provide fish protection to different A.c strains. This idea is even supported by the followed bacteria agglutination test result.