Transgenic silkworms produce recombinant human type III procollagen in cocoons
Transgenic silkworms produce recombinant human type III procollagen in cocoons
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DOI：
10.1038/nbt771
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发表时间：
2003-01-01
影响因子：
46.9
通讯作者：
Yoshizato, K
中科院分区：
文献类型：
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作者：
Tomita, M;Munetsuna, H;Yoshizato, K
我们描述了产生含有重组人胶原蛋白的蚕茧的转基因家蚕的产生。构建了含有缺失C-前肽的人III型前胶原微链、丝素轻链(L链)和增强型绿色荧光蛋白的融合基因。将该基因连接到丝素蛋白L链启动子下游，插入到piggyBac载体中。将这些载体注射到蚕卵中，在其丝腺中产生显示EGFP荧光的蠕虫。蚕茧发出绿色荧光蛋白，表明启动子和丝素L链基因引导合成的产物分泌到蚕茧中。通过免疫印迹、胶原酶敏感性试验和氨基酸序列测定，证实了融合蛋白在蚕茧中的存在。从蚕茧中提取的融合蛋白被纯化为单一的电泳带。这项研究证明了转基因家蚕作为批量生产有用蛋白质的工具的可行性。
We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.