大鼠酰胺化酶的信号肽引导的昆虫细胞表达外泌系统

将大鼠酰胺化酶的信号肽及前导肽编码序列引入昆虫核多角体病毒转移表达载体,构建ＰＡＢＣ ｈＧＲＦ（Ｇｌｙ）、ＰＡＢＣ ＩＧＦ Ｉ融合基因的昆虫细胞分泌表达质粒ｐＢａｃＰＡＧ２、ｐＢａｃＰＡＩ,并与经修饰的银纹夜蛾核多角体病毒ＢａｃＰＡＫ６线性化ＤＮＡ共转染秋粘虫细胞Ｓｆ２１,通过同源重组、筛选和鉴定,得到它们的重组病毒ＢａｃＰＡＧ、Ｂａｃ ＰＡＩ。将重组病毒感染Ｓｆ２１细胞,ＰＡＢＣ ｈＧＲＦ（Ｇｌｙ）和ＰＡＢＣ ＩＧＦ Ｉ均得到有效外泌表达,表达产物通过ＩｇＧ Ｓｅｐｈａｒｏｓｅ柱可获得快速纯化。

Efficient Secretion of Proteins Expressed from Insect Cells Directed by PAM Signal Peptide

Signal and leading peptide sequences of rat PAM was inserted into the baculovirus transfer vector, and secretion expression plasmids pBACPAG2 and pBacPAI for the fusion gene PABC hGRF and PABC IGF I were constructed, respectively. By cotransfection with linear genomic DNA of modified Autographa californica nuclear polyhedrosis virus (BacPAK6) and homologous recombination, the recombinant AcNPV, BacPAG and BacPAI, were obtained and identified. Fusion proteins PABC hGRF and PABC IGF I were secreted efficiently from Sf21 cells infected with BacPAG and BacPAI, respectively, and those fusion protein could be purified efficiently by IgG affinity column.