蜘蛛拖牵丝蛋白基因转家蚕表达质粒的构建

Construction of Silkworm Transgenic Spider Draggling Silk Gene Expression Vector

利用DNA重组技术对络新妇蛛(Nephila clavipes)拖牵丝蛋白基因MaSp1高度重复序列进行多次重组,人工构建成1.6 kb的蜘蛛拖牵丝蛋白人工基因Sil-E,DNA序列分析证明了人工基因序列的正确性.将家蚕L链基因启动子片段、L链cDNA、L链基因终止子融合在一起,构建成丝腺特异性表达单元.再与Sil-E融合构建成蜘蛛拖牵丝蛋白基因家蚕丝腺特异表达单元.将该表达单元克隆到转座子piggyBac的转基因载体中,获得了蜘蛛拖牵丝蛋白转基因表达载体.采用显微注射法将其与辅助质粒共导入到家蚕蚕卵中.筛选转基因阳性个体,经PCR和Southern杂交鉴定,结果表明目的基因整合到家蚕基因组中,为进一步研究家蚕作为生物反应器表达蜘蛛拖牵丝蛋白基因奠定了基础.

According to the repetitive protein sequence and partial cDNA sequence of Nephila clavipes spider＇ s major ampullate spidroin 1 gene, a synthetic gene repetitive monomer of 105 bp encoding 33 repetitive amino acid of spider silk protein was designed and synthesized, and the DNA monomer sequences were multimerized to a fragment of 1.6 kb （named synthesized Sil-E gene）, which encoded high molecular weight synthetic spider silks using a＂head to tail＂construction strategy in Escherichia coli. Then, promoter and terminator of L chain gene, L chain cDNA and the synthesized Sil-E gene were fused by DNA recombinant to construct an expressed unit with silkworm post-gland special expression. The expressed unit was inserted into the piggyBac transposon expression plasmid, and an expression vector system of the synthesized Sil-E gene was obtained. Using spermmediated method, the expression plasmid and helper plasmid DNA were introduced into eggs of silkworm, and the results of PCR and Southern blotting indicated the vector system was an effective transgenic vector of silkworm.