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The Chinese Journal of Dental Research

Year 2001
Volume 4 , Issue 2

Pages: 35 - 39

Pyruvate Oxidase Gene from Streptococcus oralis: Molecular Cloning and Sequence Analysis of the Gene

Jincai Zhang, DDs, PhD/Benxiang Hou, DDS, PhD/Rong Zhang, DDS/Wei Qian, DDS/Yunhui Zhang, DDS

Objective: Production of hydrogen peroxide is the main mechanism through which Streptococcus sanguis antagonizes the putative periodontal pathogens. To elucidate the regulation effect on hydrogen peroxide production of S sanguis in vivo, we cloned and sequenced the gene of pyruvate oxidase (Sopox) from Streptococcus oralis (S sanguis Type II). Methods: The PCR primers for Sopox gene were designated and synthesized according to the sequence of pyruvate oxidase gene (spxB) of Streptococcus pneumonia. The PCR product was cloned into pUC18 and subcloned into M13mp18 and M13mp19 for DNA sequencing. After getting the full nucleotide sequence, Sopox was recombined with expression vector pBV220 and the expression of Sopox in Esherichia coli JM105 was observed. Results: The nucleotide sequence of the full gene was revealed to be 1788 bps with one open reading frame coding pyruvate oxidase with 591 amino acid residuals. Homology of nucleotide sequence between spxB and Sopox is 94%. After cloning into pBV220, pBV220/Sopox/JM105 was successfully expressed as a protein with molecular weight of 65 kDa on SDS-PAGE after induction and the expression reached the maximal amount with induction at 42C for 4 hours. Conclusion: Sopox gene was successfully cloned and sequenced from S oralis. Further studies are underway to investigate the upstream structure of Sopox gene and to elucidate the regulatory mechanism of hydrogen peroxide production.



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