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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。
The Hsc70-SNCG fusion protein sequence with Igκ, MMP9 and P37 signal peptide sequences was inserted into eukaryotic expression vector pVAX1 respectively. The recombinant plasmids were confirmed by restriction enzyme digestion and insert fragment sequencing. Hsc70-SNCG eukaryotic expression plasmids pVAX-Igκ-Hsc70-SNCG, pVAX-MMP9-Hsc70-SNCG and pVAX-P37-Hsc70-SNCG with signal peptide. The eukaryotic expression plasmids were transfected into QM-7 cells routinely. The results of ELISA and Western-blot showed that all three signal peptides could induce the secretion of Hsc70-SNCG. BALB / c mice were immunized with gene gun plasmid. The results of ELISA indicated that all the three eukaryotic expression plasmids could induce humoral immune response against SNCG in mice, and the strength of humoral immune response was dependent on the expression of plasmid containing signal peptide Level.