【摘要】 目的克隆淋球菌孔蛋白PIB和PIA基因并构建融合蛋白真核表达质粒pCI-PIB-PIA,了解pCI-PIB-PIA能否被小鼠骨骼肌细胞摄取并表达。方法采用PCR方法从质粒pET-PIB和pET-PIA中获得孔蛋白PIB和PIA全长DNA片段,构建表达融合蛋白PIB和PIA的真核表达载体pCI-PIB-PIA。pCI-PIB-PIA肌肉注射免疫BALB/c小鼠,ABC法检测pCI-PIB-PIA免疫小鼠肌细胞中PIB-PIA融合蛋白的表达。结果PCR可扩增出预期大小的全长PIB和PIA基因片段。与GenBank报道的PIB和PIA序列比较,重组质粒pCI-PIB-PIA中目的插入片段的核苷酸序列同源性达到99%以上。免疫小鼠的肌细胞能摄取pCI-PIB-PIA并表达PIB-PIA融合蛋白。结论本研究成功地构建了淋球菌孔蛋白PIB和PIA基因重组真核表达载体pCI-PIB-PIA;pCI-PIB-PIA接种小鼠后可见免疫小鼠肌细胞中PIB-PIA融合蛋白的表达,该结果可作为淋球菌多价DNA疫苗的研究基础。更多还原

【Abstract】 Objective Clone the genes of porin PIB and PIA of Gonococcus. To construct a recombinant eukaryotic expression vector containing both porin gene PIB and PIA, and to confirm the expression of pCI-PIB-PIA in muscule cells. Method The entire PIB and PIA genes were amplified from pET-PIB and pET-PIA by PCR. An eukaryotic expression vector pCI-PIB-PIA expressing the fusion protein PIB-PIA was then constructed. BALB/c mice were immunized with pCI-PIB-PIA by intramuscular injection. ABC assay was used to examine the expression of PIB-PIA in muscule cells after immunization. Result The entire PIB and PIA gene fragments with the expected size were amplified and obtained by PCR. The PIB and PIA had over 99% of sequence homology with the previously reported genes. The fusion protein PIB-PIA could be detected in mice after immunization. Conclusion In this study we successfully constructed a recombinant eukaryotic expression vector pCI-PIB-PIA. With the availability of this fusion protein PIB-PIA, the future development of vaccine against Gonococcus will be facilitated.更多还原