Cloning And Expression Of Heme Oxygenase-1(HO-1) Gene Of Pimephales Promelas

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme. HO-1, which is also known as heat shock protein32(HSP32), plays roles in anti-inflammation, antiproliferation and antiapoptosis. More and more studies have documented the cytoprotective effects of HO-1during the period that the body is infected by microbio including viruses. To our knowledge, studies on the effects of HO-1in virus infection have focused mainly on mammals.To study the effects of virus infection on fish HO-1expression and evaluate the role of fish HO-1in virus infection, which can provide a potential therapeutic approach to virus-induced diseases, the full-length of Fathead minnow Pimephales promelas HO-1cDNA sequence was cloned and isolated from Epithelioma Papulosum Cyprini (EPC) cells, and then the effects of Spring Viraemia Of Carp Virus (SVCV) infection and CoPP on fathead minnow HO-1expression were studied in vitro. The results are summarized as follows:1. The full-length sequences isolation and homologous analysis of Pimephales promelas HO-1cDNAThe Pimephales promelas HO-1cDNA was isolated from EPC cells by RT-PCR and RACE. Fathead minnow HO-1cDNA is characterized with the full-length of1110bp,52bp in the5’untranslated region,203bp in the3’untranslated region and a poly A signal sequence of AATAAA in the3’noncoding region, and an instability RNA sequence of ATTTA. The open reading frame of HO-1gene contains858bp which encodes285amino acids. Pimephales promelas HO-1gene contains a heme oxygenase signature sequence (AA129-152) and a transmembrane domain sequence (AA264-285) by predicting gene functionality. Deduced amino acid sequence analysis shows that Pimephales promelas HO-1gene were homologous to mammals, birds and other fish, as it is with a high homology to fish (58.8%-72.7%), especially to Ictalurus furcatus, Ictalurus punctatus (72.4%and72.7%, respectively), and with a low hemology to Homo sapiens, Mus musculus, Gallus gallus (44.1%、44.1%and45.4%, respectively).2. The effects of SVCV infection on Pimephales promelas HO-1expression The effects of SVCV infection on Pimephales promelas HO-1expression was studied in vitro by RT-PCR and Real-Time PCR. When EPC cells were infected with SVCV at1MOI, the expression of HO-1significantly decreased at3h post-infection and reached the minimum at6h post-infection following by a gradual increase. When EPC cells were infected with SVCV at0.1MOI, the expression of HO-1significantly decreased at6h post-infection and reached the minimum at12h post-infection, and then the expression of HO-1began to increase at24h post-infection. The results show that SVCV infection may down-regulate the expression of HO-1in EPC cells.3. The effects of CoPP on Pimephales promelas HO-1expressionIn the present study, whether Pimephales promelas HO-1expression in in vitro EPC cells could be induced by CoPP was studied by semiquantitative PCR. The results suggested that CoPP didn’t induce HO-1expression when EPC cells were exposed to CoPP at different concentrations.