Cloning Of DNase B Gene And Its Expression In E.coli

DNase B is one of the secretory proteins of Group A 8-hemolytic streptococci (GAS), and exits in almost all GAS. DNase B sequence is conservative, can be used to test the antibody of DNase B. The anti-deoxyribonuclease-B titer (anti-DNase-B, or ADB) is regarded as one of the most sensitive markers of GAS infection, and recommended by Jones standard. However, the liquid culture of GAS is more complicated, dangerous and cost much. Furthermore, purifications of DNase B are complicated too. For these reasons, the gene for GAS DNase B has been cloned and vectors incorporating the cloned DNA have been used to transform Escherichia coli, allowing efficient and rapid production of the DNase B in E. coli without the necessity of growing large quantities of S. pyogenes.A strain of GAS was collected from a clinic sample. Primers were designed based on the published sequences of DNase B gene. The genomic DNA of GAS was isolated and used as the PCR template. The DNase B gene were amplified by PCR, the fragments of DNA were cloned into T-vector and checked by sequencing. The DNase B gene, which cloned in this experiment, has 98% homology with those loaded in Genebank.Another PCR were performed, the PCR productions were digested with endonuclease Kpnl and HindIII. and ligated with the plasmids pET-41a(+) cut by the same endonucleases, then transformed E.coli BL21(DE3). The recombinants were screened on the selected plates. One of the recombinants plasmids was isolated, identified, and sequenced. The fusion expressing vector of pET-41a (+) / DNase B was successfully constructed. The DNase B protein was expressed in E coli as fusion protein with glutathione Stransferase(GST) induced by IPTG. The fusion protein was purified by Ni-NTA column. Purified fusion protein was obtained. SDS-PAGE indicates: the recombinant protein mostly was soluble, and a little portion exited in inclusion body. The supernant flows through Ni-NTA gel for affinity chromatography, and thepurity is over 91.5%. The result of Western-blotting indicated the recombinant GST-DNase B fusion protein could specificly react with serum from GAS sufferer; there was an obvious band at 55KD. ELISA test confirms the specificity of this fusion protein. These results indicate that the fusion protein possesses favorable immuno-reactivity. So, these work have laid a solid foundation for the test of anti-DNase B and the research of GAS vaccine.