家蚕溶茧酶基因的真核表达及其产物的生物活性检测

为了研究家蚕 Bombyx mori 溶茧酶基因 （cocoonae）真核表达及其产物的生物活性，将溶茧酶基因（GenBank 登录号 EF428980）克隆至杆状病毒转移载体 pFastBac^TM 1 中获得 pFast-cocoonase，将其转化 DH10Bac 感受态细胞后，PCR 方法检测证实所分离的重组病毒 DNA 中含有目的片段溶茧酶基因。用脂质体法 转染家蚕 BmN 细胞，获得重组病毒。SDS-PAGE 分析显示，感染重组杆状病毒 Bac-cocoonase 的细胞表达产物在约为 27.6 kD 处出现特异性条带，这与预测的蛋白大小相符。用该表达产物与茧丝反应后，电镜下观察茧丝的形态，结果表明表达产物对茧丝的丝胶层有一定的水解作用。

Eukaryotic expression of the cocoonase gene from Bombyx mori and biological activities of the expressed product

In order to research the biological activity of cocoonase expressed through eukaryotic expression system,cocoonase gene(GenBank accession number:EF428980)was cloned into baculovirus transfer vector pFastBac TM 1 to obtain pFast-cocoonase,and this recombinant vector was then transformed into DH10Bac competent cells.Cocoonase gene was verified to have been successfully transferred to recombinant bacmid by PCR.BmN cells were transfected with the recombinant bacmid through lipofectin-mediated method to obatin recombinant baculovirus.SDS-PAGE analysis showed that a specific band of about 27.6 kD existed on the lane of expression products of recombinant baculovirus,which was consistent with predicted size of the target protein.SEM micrographs were taken after treating silkworm silk with the expressed protein,and the results showed that the expressed protein could hydrolyze sericine to some extent.