利用非转座子载体介导的转基因家蚕表达人胰岛素样生长因子Ⅰ

为了实现利用非转座子载体介导的转基因家蚕整体表达人胰岛素样生长因子Ⅰ(hIGF-Ⅰ),将hIGF-Ⅰ基因克隆进非转座子类型的昆虫细胞表达载体pIZT/V5-His,构建转基因载体pIZT/V5-His-hIGF-Ⅰ。利用精子介导法将该转基因载体导入家蚕卵,通过绿色荧光筛选并结合PCR和Dot blotting检测鉴定,表明已成功获得hIGF-Ⅰ转基因家蚕。对培育至G2代的转基因家蚕5龄幼虫蛋白质样品进行Western blotting分析,结果显示hIGF-Ⅰ在转基因家蚕中获得表达,重组hIGF-Ⅰ的分子质量约12.5 kD;ELISA检测hIGF-Ⅰ在G2代转基因家蚕5龄幼虫全蚕以及后部丝腺、脂肪体冻干粉中的质量比分别为65、411、469 ng/g。试验结果再次证实通过非转座子载体pIZT/V5-His介导可以将外源基因导入家蚕基因组,并实现外源基因在转基因家蚕中整体表达。

Expression of Human Insulin-like Growth Factor Ⅰ in Transgenic Silkworm Mediated by Non-transposon Vector

To realize the expression of hIGF-Ⅰ(human insulin-like growth factorⅠ) in whole body of transgenic silkworm mediated by non-transposon vector,hIGF-Ⅰ gene was cloned into the non-transposon vector pIZT/V5-His to construct transgenic vector pIZT/V5-His-hIGF-Ⅰ.pIZT/V5-His-hIGF-Ⅰwas then transferred into silkworm eggs by sperm-mediated gene transferring method.By screening of green fluorescence and verification of PCR and Dot blotting hybridization,the results showed that hIGF-Ⅰtransgenic silkworm was successfully obtained.Western blotting analysis on the protein sample from the 5th instar transgenic silkworm larvae of G2 generation demonstrated that the recombinant hIGF-Ⅰ with a molecular weight of 12.5 kD was expressed in the transgenic silkworm.ELISA assay showed that the mass ratio of hIGF-Ⅰin freeze-dried powders of whole body,posterior silk gland and fat body from the 5th instar transgenic silkworm larvae of G2 generation was 65,411,469 ng/g,respectively.These results reconfirmed that exogenous genes could be integrated into silkworm genome with the mediation of non-transposon vector pIZT/V5-His and expressed successfully in whole body of the transgenic silkworm.