Clone and prokaryotic expression of staphylococcus aureus drug resistance adenylyltransferase gene / 国际检验医学杂志

ABSTRACT

Objective To clone and express Staphylococcus aureus drug resistance adenylyltransferase gene in E .coli BL21 ,and to make the foundation for its function research .Methods Primers were designed on the basis of adenylyltransferase gene in gen‐bank ,PCR was used to amplify adenylyltransferase gene using Staphylococcus aureus genomic DNA as template .The obtained PCR production was attatched with pGEX‐4t‐1(+ ) plasmid ,and transformed into E .coli BL21 (DE3) .The recombinant plasmid was di‐gested by double enzyme digestion and identified by gene sequence .The recombinant protein was induced to expression by IPTG and identified by Western‐blotting .Results Using Staphylococcus aureus genome as a template ,the target fragment about 800 bp was successful amplified .After enzyme‐cutting and DNA‐sequencing ,the target fragment showed that the ORF begin with ATG ,end with TAG ,783 bp in length ,the predicted isoelectric point and molecular weight were 7 .75 and 29 × 103 ,and it was homology 99%homology with the reported sequence gene in genbank .SDS‐PAGE and Western‐blot showed the molecular weight of recombinant fusion protein was about 55 × 103 .Conclusion Adenylyltransferase gene of Staphylococcus aureus was successfully cloned and ex‐pressed in E .coli as a fusion protein ,which makes the foundation for the research of its function .