家蚕先天免疫基因 Bmimd 的克隆、表达及序列分析

昆虫的先天免疫应答由一组基因通过级联网络调控实现。果蝇 Drosophila 免疫缺陷(immune deficiency, imd)基因在体液免疫信号传递途径中起着重要的作用。我们利用生物信息学方法进行电子克隆，成功地找到了 imd 基因在家蚕 Bombyx mori 中的同源体，命名为 Bmimd。该基因全长1 092 bp，由 4 个外显子和 3 个内含子组成，开放阅读框(open reading frame, ORF)长 750 bp，编码 250 个氨基酸，预测蛋白质分子量为 28.6 kD。Bmimd 序列中含有一个致死结构域，经聚类分析表明该结构域与哺乳动物的受体相互作用蛋白(receptor interacting protein, RIP)相似。将该基因亚克隆到 PET-50b 载体进行原核表达，表达出了带有 2 个 6×His tag 和 1 个 Nus·Tag 标签的重组蛋白。Western blotting 结果表明 Bmimd 蛋白在 5 龄 4 天家蚕的头、脂肪体 、生殖腺、表皮和中肠中都有表达，但丝腺中没有检测到表达。

Cloning, expression and sequence analysis of Bmimd, an innate immunity gene in the silkworm, Bombyx mori

The cascade of innate immunity in organisms was regulate d by a set of genes. The gene immune deficiency of Drosophila plays an important role in the pathway of humoral immunity. We got a homolog of Drosophila imd from Bombyx mori successfully through silico cloning, and named it Bmimd. The gene is 1 092 bp in length, consisting of 4 exons and 3 introns, containing an ORF of 750 bp, encoding a protein with 250 amino acids, and the predicted molecular weight is 28.6 kD. Bmimd contains a death domain, which is similar to mammalian RIP by sequence analysis. Prokaryotic expression of the gen e recombinant with six-His tag and a Nus·Tag was successfully carried out through sub-cloning into PET-50b vector. The result of Western blotting showed that the gene was expressed in head, fat body, germen, cuticle and midgut, but its ex pression was not detected in silk glands of the silkworm.