信号肽引导源基因在昆虫表达系统中的分泌表达

Signal Peptides Leading Secretory Expression of Foreign Genes in Insect Expression System

将杆状病来源的gp67信号肽引导的慈姑蛋白酶抑制剂(API)基因与植物来源的慈姑蛋白酶抑制剂信号肽引导的慈姑蛋白抑制剂(API)基因,分别克隆到昆虫杆状病毒转载体pBacPAK8中,通过共转染将它们分别整合到昆虫病毒表达载体Bm-BacPAK6中,将获得的重组病毒CrBK-API2和CrBK-bpAPI4分别接种家蚕(Bombyxmori)细胞、家蚕幼虫及蛹体中,慈姑蛋白酶抑制剂得到了高效表达。从家蚕细胞的表达来看,外源表达产物大多存在于细胞外的上清液中;从家蚕幼虫体内表达来看,信号肽能引导外源基因高效表达,且表达产物的90%以上能分泌到细胞外;蛹体中的表达情况与幼虫中的相似,但表达产物出了不均一的现象,从表达产物的分子量测定来看,这两种信号肽在表达过程中都没有被切除。

Fusing nucleotde sequences of signal peptides of the arrowhead proeinase inhibitor of plant ofigin and gp67 of baculoirus origin with the arrowhead proteinase inhibitor(API) gene and colning them into insect baculovirus transfer vector pBacPAK8 respectively,then integrating each into insect virus expression vector Bm-BacPAK6,we harvested recombinant virus of CrBK-API4 and CrBK-gpAPI2.Inhibitor was high-level expressed.Exogenous expression products almost existed in supernatant fluid of extracellular region in Bm cells;expression in larvae showed that the signal peptides can lead foreign genes to express efficiently and 90rercent of the expression products can secret to extracellular region;in pupae,the situation was similar to sarae,but expressing products appeared to be heterogeneous,compared with larvae,there is a surplus inferior band with molecular weight about 1KD in pupae,From molecular weight of the expressing products by SDS-PAGE,we can infer that the two signal peptides were likely not cut in the expression procedure.