非转座子载体介导的转基因家蚕表达hIL-28A

为了探讨非转座子载体介导转基因家蚕表达外源基因的可能性,将hIL-28A克隆进昆虫细胞表达载体pIZT/V5-His,构建了重组载体pIZT/V5-His-hIL-28A.利用精子介导法将该重组载体导入家蚕卵,通过绿色荧光筛选并结合PCR、DNA杂交等分子鉴定,证实成功获得了转基因家蚕.Western blotting结果显示,转基因家蚕表达重组hIL-28A的分子质量为25 ku,ELISA检测结果显示,hIL-28A在G3代转基因蚕、后部丝腺、脂肪组织冻干粉中的含量分别为0.198、0.320和0.238 ng/g.表明通过非转座子载体介导可以将外源基因导入家蚕基因组并实现外源基因的表达.

Expression of hIL-28A in transgenic silkworm mediated by non-transposon vector

To explore the possibility of foreign gene expression in transgenic silkworm mediated by non-transposon vector, a hIL-28A gene was inserted into the insect cells expression vector pIZT-V5-His to generate recombinant vector pIZT/V5-His-hIL-28A, the vector was transferred into silkworm eggs by sperm mediated gene transfer, screening for gfp gene and verified by PCR and Dot blot hybridization. Transgenic silkworms were obtained after a specific band with the molecular mass of 25 ku could be detected in transgenic silkworm by Western blotting using an goat anti-hIL-28A antibody, and the content of hIL-28A in the G3 generation transgenic silkworms estimated by ELISA was approximately 0.198, 0.32, 0.238 ng/g in freeze-dried whole bodies, posterior silk glands and fat bodies, respectively. These results suggested that a heterologous gene could be integrated into silkworm genome by non-transposon vector and expressed successfully.