Subcloning And Site-directed Mutagenesis Of DNase B Gene And High-level Expression And Purification Of The Target Protein

DNase B is one of the secretory proteins of Group Aβ-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) are regarded as one of the most sensitive signals of GAS infection.However,purifications of DNase B are complicated and expensive.So it is very difficult to suffice the clinical demands.In order to obtain efficient and inexpensive DNase B protein,recombinant expression vector of DNaseB in Escherichia coli was constructed by genetic engineering.Two experimental ways was studied.The first experimental way:A 0.5 kb dock-tailed DNase B gene fragment was amplified by PCR from plasmid pMD18-T-DNase B.Then it was subcloned into pET-28a(+)resulting pET-28a(+)-DNase B.The recombinant plasmid DNA was studied in detail by restriction endonuclease and nueleotide sequencing.The result showed that the nucleotide sequence of the inserted DNA fragment was identical to that of DNase B gene.The recombinant plasmid was transformed into E.coli BL21(DE3).The target protein(about 22 kd)was expressed by IPTG induction.The result of western-blotting indicated that the recombinant dock-tailed His-DNase B fusion protein could specifically react with serum from GAS sufferer.However,the quantity of DNase B fusion protein was not enough.The second experimental way:In order to mutate the enzyme activity sites of DNase B.The two-step polymerase chain reaction(PCR)was used for the site-directed mutagenesis.The mutated fragment of DNase B gene was cloned into T-vector and checked by sequencing.The PCR production was digested with endonuclease EcoRâ… and Hindâ…¢,and ligated with the plasmids pET-28a(+)cut by the same endonucleases,then transformed into E.coli BL21(DE3).The DNase B protein was expressed in E.coli as fusion protein(about 29 kd)with His.Tag induced by IPTG.The DNase B protein was purified by Ni-NTA column.Purified fusion protein was obtained.SDS-PAGE indicated: the recombinant protein mostly was exited in inclusion body.The inclusion body was washed by different concentration urea solution,then was dissolved in 8mol/L urea solution.The protein solution flows through Ni-NTA gel for affinity chromqtography, and the purity was high.The concentration of DNase B protein was quantitative analysis by BCA way.The immunity of recombinant His-DNase B fusion protein was studied.The results of western-blotting indicated that the recombinant His-DNase B fusion protein could specifically react with serum from GAS sufferer.ELISA and latex agglutination test confirmed the immunoreactivity and specificity of this fusion protein.In order to investgate fermentation conditions of gene engineering E.coli strain for DNase B.Orthogonal test was studied.Finally,the optimal combination of fermentation conditions that A₂B₁C₁D₃ was found.That was inoculum volume 2%,induction OD 0.5, concentration of IPTG 0.6mM and induction time 5h.At the same time,the fermentation conditions of DNase B was validated by full automatic fermentor.These results would provide scientific basis for the production of DNase B protein of group A Streptococcus pyogenes.