Cloning, Expression Of Aeromonas Hydrophila Hemolysin Fragment A Gene And The Immunological Characterization

Aeromonas hydrophila, a normal inhabitan of the aquatic enviroment, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. It causing soft tissue wound infectious and diarrhea in the fomer and fatal hemorragic septicemia in the latter. Aeromonas hydrophila is receiving increasing attention at present. Generally, Aeromonas hydrophila pathopoiesis is close concerned with some virulence factors, especially hemolytic toxin. Some researches have proved that hemolytic toxin itself have characteristic of haemolytic, enterotoxic and cytotoxic. Hemolytic toxin has pathopoiesia independent effection on experimental animals such as Eels, cricians and Mus musculus albus. Hemolytic toxin is important common protective antigen to fishes. T-chakraborty et al suggested that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.In this test, we mainly concerned with the hemolysin gene of Aeromonas hydrophila, expressed in prokaryote Escherichia coil. We have successfully constructed the clone vector pGEMtTHA and the expression vector pETTHA of Aeromonas hydrophila hemolysin gene A fragment (hlyA), respectively. The expressed products in vitro have detected by western bolt kit ,as a result, showed that the recombinant hemolysin could be recognized by anti-hemolysin sera.1. Cloning, Expression of Aeromonas hydrophila hemolysin Fragment A gene in E. coli.Using Aeromonas hydrophila TPS30 genome DNA as template, a pair of primers was designed according to the gene sequences of Aeromonas hydrophila AHTPS30HEM (accession.NO.AB021152). Aeromonas hydrophila fragment A gene, which the length is 1482bp, was amplified by PCR and cloned into pGEM-T vector, the recombinant vector was designated as pGEMtTHA. The sequence comparision revealed a 98% homology with AHTPS30HEM (accession.NO.AB021152). This fragment was cloned into pET32a(+) vector and successfully got the expression vector pETTHA, clutured at 37℃for 2h and induced with 0.1mM IPTG for 3h. SDS-PAGE test showed that the positive band was required. By SDS-PAGE analysis, the interest protein were successfully high-expressed in inclusion bodeis and the size was about 68 kD.2. Purification and the immunogenicity of the recombinant expressed protein.The best treatment condition of inclusion body was to with 2M urea wash and dissolve with 8M urea. Using the His·Bind protein purification kit to purificate the fusion protein and the sizes of the purified protein whth a His-tag was about 68 kD. The expressed products in vitro were detected by Western bolt kit showed that the recombinant hemolysin could be recognized by anti-hemolysin sera. The biology characteristic of hlyA and the function of hlyC inducing expression of hlyA will be carried out in the future.