Construction And Expression Of The Fusion Gene 3a-HSD/CR And AiiA

Gram-negative bacterium Comamonas testosteroni has the ability to utilize various steriods as a sole carbon and energe source.3a-Hydroxysteroid dehydrogenase / carbonyl reductase(3a-HSD/CR)from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic. C. testosteroni strains are able to use steroids (testosterone, progesterone, bile acid) as sole carbon and energy sources. C. testosteroni therefore play a significant role in the bioremediation of these stablely hormonally active compounds in the environment. However the catabolic enzymes for these compounds metabolism in C. testosterone are not constitutively expressed but are induced by their respective substrates. This limits its wide use in environmental bioremediation.Bacillus thuringiensis is a gram-positive,sporo-forming bacterium capable of producing a number of toxins,including insecticidal endotoxins, exotoxins,chitinase,vegetative insecticidal proteins and proteases,with toxicity to several insect orders,nematodes,mites and protozoa.The aiiA gene encoded an enzyme involved in the degradation of N-acyl-homoserine lactones (AHLs) which decrease the virulence of bacterial pathogens.A pair of PCR primers was designed according to the sequence of 3α-HSD/CR (774 bp) published in GenBank. That the fused expression vector of 3α-HSD/CR gene was suecessfully constructed and induced in E.coli. Then expression vector pET29a-3α- HSD/CR was expressed in E. coli BL21.A pair of PCR primers was designed according to the sequence of aiiA(753 bp) published in GenBank.Then the aiiA gene was sucloned into plasmids pET29a-3α- HSD/CR yield plasmids pET29a-3α-HSD/CR–aiiA. That PCR and DNA sequencing analysis and the recombinant plasmid 3α-HSD/CR–aiiA was constructed and detected its expression in prokaryotic cell successfully with SDS-PAGE. The recombinant protein was purified by its His-tag sequence using Ni-NTA Spin Column supplied by Qiagen. Protein expression levels and its degradation ability were determined by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC). The fusion protein alleviated the pathogenesis of Eriwinia carotovora causing soft rot for carota.It was found that PCR and DNA sequencing analysis showed that the 3α-HSD/CR gene of 774 bp and the aiiA gene of 753 bp were amplified, and the recombinant plasmid pET29a-3α-HSD/CR–aiiA was constructed and detected its expression in prokaryotic cell successfully with SDS-PAGE. The fused expression vector of the recombinant plasmid pET29a-3α-HSD/CR–aiiA was suecessfully constructed The fusion protein greatly alleviated the pathogenesis of Eriwinia carotovora causing soft rot for carota.The 3α-HSD/CR–aiiA gene was obtained from DNA of pET29a-3α-HSD/CR–aiiA by PCR, and was cloned into the pCMBIA1301 mammalian expression vector. gene.The recombinant plasmid (pCMBIA1301-3α-HSD/CR–aiiA)was analyzed with restriction endonuclease digestion and PCR. It was identified that the gene fragment of 3α-HSD/CR–aiiA at length of 1533 bp were amplified andcloned into the vector pCMBIA1301 successfully.