利用家蚕丝腺表达人干扰素-W的研究

Research on Silkworms Silk Gland Bioreactor Expressing hulFN-w

目的建立家蚕丝腺生物反应器表达外源蛋白的平台，并评估家蚕中表达的外源蛋白的实用性。方法将人干扰素-w基因（huIFN-w），构建到来源于鳞翅目昆虫粉纹夜蛾（Trichoplusia ni）的piggyBac转座子基础载体中，以家蚕中部丝腺特异表达基因Serl为启动子，眼睛和神经特异表达的增强型绿色荧光蛋白基因（EGFP）作为报告基因，构建家蚕中部丝腺特异表达载体pBac[ser 1-huIFN-w,3xp3-EGFP]。经显微注射入产后1—3h的大造（蚕种系大造P50）早期胚胎，筛选阳性个体。并对阳性个体进行分子和蛋白水平检测。结果获得了家蚕转基因阳性个体，huIFN-w基因在家蚕中部丝腺高量转录表达，重组蛋白huIFN-w具有一定的抗细胞增殖活性。结论利用家蚕丝腺生物反应器表达的huIFN-w蛋白正确并具有生物活性，表明可将家蚕丝腺作为“生物工厂”，大量和高效生产高附加值外源蛋白，同时也为生物制品的大量推广和应用奠定理论基础。

Objective To establish the silk gland bioreactor platform which expresses foreign proteins, and to assess practicality of the foreign proteins. Methods Using human interferon-wgene as the target gene, silkworm （Bombyx mori） middle silk gland specific gene Serl as promoter, enhanced green fluorescent protein （EGFP）which expresses specially in the eyes and Nervous system as a reporter, then they are built into the piggyBac -based vector derived from Trichoplusia ni Lepidoptera （Trichoplusia ni） for the silkworm middle silk gland-specific expression vector pBac [serl-hulFN -w, 3xp3-EGFP]. We injected the plasmid into post-natal 1-3h of early embryos （P50） and screened positive individuals. By PCR, RT-PCR and other methods for detection of positive individuals, and silk gland total protein was analysed by western blotting and cytotoxicity testing. Results We got the transgenic silkworm individuals, hulFN-w gene had a high level of transcription in th middle silk gland, the recombinant protein hulFN-w has a certain anti-proliferative activity. Conclusion The use of the silk gland bioreactor express biologically active protein hulFN-w is correct and that means the silk gland can be as a ＂biological factory＂ to product a large scale and efficient high value-added foreign proteins, it＇s also supply the theoretical basis for promotion and application the biological products.