Cloning And Expression Of Rat Heme Oxygenase 1 In Escherichia Coli

Heme oxygenase(HO) is a rate-limiting enzyme involved in thedegradation of heme , resulting in the generation of biliverdin , iron andcarbonmonoxide.Three HO isoenzymes, HO-1 ,HO-2 and HO-3. The HO-1is a microsomal enzyme . HO-1, which is inducible and highly expressed inbrain,liver and spleen tissues . It has been alsodefined as a novel stress stimulated protein and heat-shock protein32,which is inducible by hypoxia, inflammatory,cytokines,ethanal,heavymetals,hemolysis. HO-1has anti-inflammatory, anti-apoptoticanti-apoptotic and anti-protiferative effects.It has been proved tobe able to protect the cells formed amage. HO-1 has been shown to be effective in profecting cells whenadministered locally. But overexpressed HO-1 can injure cellmembrane. Therefore, moderation inducement and regulation of HO-1expression would be extensively studied.In my experiment ,The total RNA was extracted from rat spleenand amplified by reverse transcription polymerase chain reaction(RT-PCR).PCR products were cloned into pMD18-T (TA)vector followedby DNA sequence.The cloned RHO-1 gene is composed of 870 nucleotides,and is accordance with that reported in Genbank. RHO-1 cDNA fragmentsin TA vector were subcloned into the prokaryotic expression vectorpET28a(+). The recombinant pET28a(+)/RHO-1 (rRHO-1) plasmid wastransformed into E.coli BL-21(DE3). The rRHO-1 was induced with IPTGand characterized by SDS-PAGE.The RHO-1 protein was successfullyexpressed in E.coli. This work has laid down the basis for further study ofits activity and antibody preparation.