【摘要】 分别将带有Igκ、MMP9、P37信号肽序列的Hsc70-SNCG融合蛋白序列定向插入真核表达载体pVAX1中,经酶切,及插入片段序列测定对重组质粒进行鉴定,证明成功构建了带有三种信号肽的Hsc70-SNCG真核表达质粒pVAX-Igκ-Hsc70-SNCG、pVAX-MMP9-Hsc70-SNCG、pVAX-P37-Hsc70-SNCG。真核表达质粒常规转染QM-7细胞,ELISA、Western-blot检测结果提示三种信号肽均可诱导融合蛋白Hsc70-SNCG的分泌表达。基因枪质粒免疫BALB/c小鼠,ELISA检测结果提示三种真核分泌表达质粒均可引发小鼠抗SNCG体液免疫反应,且激发体液免疫反应的强弱依赖于质粒含有信号肽诱导分泌表达的水平。更多还原

【Abstract】 Hsc70-SNCG fusion protein cDNA fragment containing signal peptide sequence of Igκ,MMP9,and P37 was inserted into the vector pVAX1 to construct recombinant plasmid pVAX-Igκ-Hsc70-SNCG,pVAX-MMP9-Hsc70-SNCG,and pVAX-P37-Hsc70-SNCG. Three eukaryotic vectors were constructed and verified by restriction enzyme digestion and sequencing. After transfection with recombination plasmids in QM-7 cells,the transient expression and secretion of three fusion proteins were detected by ELISA and Western Blot. The results suggested that Hsc70-SNCG carrying three different signal peptides could be expressed and secreted by transfected cells,and three signal peptides effectively directed secretion of fusion protein by QM-7 cells. BALB/c mice were immunized by three plasmids using gene gun system. The serum levels of anti-SNCG antibodies in mice were measured by ELISA. The results showed that three secreted plasmids could stimulate humoral immune responses to SNCG in mice,which depended on the secreted expression levels induced by signal peptides.更多还原