Tessier D C, Thomas D Y, Khouri H E, Laliberté F, Vernet T
Genetic Engineering Section, National Research Council of Canada, Montréal Québec.
Gene. 1991 Feb 15;98(2):177-83. doi: 10.1016/0378-1119(91)90171-7.
The baculovirus/insect cell system has been remarkably successful in yielding high levels of synthesis of many proteins which have been difficult to synthesize in other host/vector systems. The system is also capable of secreting heterologous proteins, but with generally low efficiency. We have increased the efficiency of secretion of the system by using signal peptides of insect origin to direct the secretion of a foreign protein. The precursor of the plant cysteine protease papain (propapain) has been used as a report enzyme to compare secretion efficiency. Insect cells infected with a baculovirus recombined with the gene encoding propapain fused to the sequence encoding the honeybee melittin signal peptide secreted over five times more papain precursor than the wild-type prepropapain which used the plant signal peptide. Based on these results, we have assembled pVT-Bac, an Autographa californica nuclear polyhedrosis virus transfer vector that may enhance secretion of other foreign proteins from insect cells. The vector incorporates a number of features: phage f1 ori to facilitate site-directed mutagenesis, the strong polyhedrin promoter upstream from the melittin signal peptide-encoding sequence, and eight unique restriction sites to facilitate fusion of heterologous genes.
杆状病毒/昆虫细胞系统在大量合成许多难以在其他宿主/载体系统中合成的蛋白质方面取得了显著成功。该系统也能够分泌异源蛋白，但效率通常较低。我们通过使用昆虫来源的信号肽来指导外源蛋白的分泌，提高了该系统的分泌效率。植物半胱氨酸蛋白酶木瓜蛋白酶（前木瓜蛋白酶）的前体已被用作报告酶来比较分泌效率。用与编码与蜜蜂蜂毒肽信号肽序列融合的前木瓜蛋白酶基因重组的杆状病毒感染的昆虫细胞分泌的木瓜蛋白酶前体比使用植物信号肽的野生型前体木瓜蛋白酶多五倍以上。基于这些结果，我们构建了pVT-Bac，一种苜蓿银纹夜蛾核多角体病毒转移载体，它可能增强昆虫细胞中其他外源蛋白的分泌。该载体具有许多特点：噬菌体f1 ori便于定点诱变，在蜂毒肽信号肽编码序列上游有强多角体蛋白启动子，以及八个独特的限制性酶切位点便于异源基因的融合。
