目的 构建携带PrtA基因的原核表达载体,诱导表达具有活性的重组PrtA融合蛋白.方法 根据GenBank中肺炎链球菌表面蛋白细胞壁相关丝氨酸蛋白酶A(PrtA)蛋白基因序列,设计合成了一对特异性引物,扩增目的 片段,建立了RT-PCR检测方法.应用RT-RCR方法 扩增得到PrtA蛋白基因,并克隆到pMD18-T载体上,筛选、鉴定阳性重组子pMD18-T-PrtA,然后克隆到原核表达载体pET-30a(+)上,经双酶切、PCR鉴定,获得重组表达质粒pET-30a-PrtA.将含有pET-30a-PrtA的重组菌,经终浓度为0.6 mmol/L的IPTG诱导后,重组PrtA蛋白获得高效表达.通过Western blot检测结果,检测表达的重组PrtA蛋白的反应原性.结果 所获PrtA基因与GenBank的基因序列同源性为99%;重组质粒经IPTG诱导,不同浓度的IPTG均能诱导重组蛋白表达,且IPTG终浓度为0.6 mmol/L时,目的 蛋白的表达量最高,约占菌体总蛋白的18%.通过抗-His标签单克隆抗体在IPTG诱导6 h的重组菌蛋白中检测到一条大小约为40×103的特异性清晰条带,与预期结果 相符,表明表达的蛋白为肺炎链球菌重组PrtA蛋白.结论 成功地克隆和表达了肺炎链球菌表面蛋白细胞壁PrtA,为进一步研究以PrtA蛋白作为免疫原预防和治疗由肺炎链球菌引起的疾病奠定基础.%Objective To construct a prokaryotic expression vector carrying PrtA gene,and to express recombinant pneumococ-cal surface proteins cell wall associated serine proteinase A(PrtA)fusion protein with activities. Methods PrtA gene specific primers were designed and synthesized according to database in GenBank. RT-PCR, amplifying target fragment was constructed by testifying different optimized condition. Then,PrtA gene was amplified by RT-PCR from total RNA and cloned into pMD18-T vector. The positive pMD18-T-PrtA plasmid was constructed by screening and identification. Then the target gene was subcloned into the downstream of pET-30a( + ) vector,which was denominated as pET-30a-PrtA,and the insertion of target gene into pET-30a ( + ) vector was confirmed by PCR and restriction enzyme digestion. The recombinant strain E. Coli BL21 (pET-30a-PrtA) was induced by 0. 6 mmol/L IPTG and the recombinant PrtA protein was expressed at high level. It has been proved that the recombinant PrtA protein of Streptococcus pneumoniae(S. Pneumoniae) had good reactionogenicity by western-blot. Results The PrtA gene shared 99% nucleotide homology to GenBank sequences. Reconstructed pET-30a-PrtA plasmid,being induced by IPTG,showed that different concentrations of IPTG all could induce recombinant protein expression,and when the concentration of IPTG eventually was 0. 6 mmol/L,the expression of objective protein reached the highest level,accounts for about 18% of thalline total proteins. Clear specificity channel of molecular weight about 40X103 could be detected among thalline total proteins of reconstructed plasmid, when being induced by IPTG for 6 h,with monoclonal antibody labeled by Anti-His, which matched with expectation and indicated that the expressed protein was recombinant pneumococcal PrtA protein. Conclusion PrtA gene from S. Pneumoniae was cloned and expressed successfully, laying bases for further research of PrtA protein to be used as immunogen for the prevention and treatment of diseases caused by S. Pneumoniae.
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