提取北京油鸡心、肝、脾、肺、肾、脑、腿肌与胸肌等不同组织的总RNA, 利用RT-PCR方法检测a1-酸性糖蛋白基因(a1-AGP)mRNA的差异表达。将a1-AGP基因完整开放阅读框定向插入pEGFP-C1, 构建带有GFP报告基因的重组表达载体pEGFP-α1-AGP, 将其导入北京油鸡体外培养细胞中, 并进行G418药物筛选和克隆化培养。结果表明: a1-AGP基因开放阅读框长度为612 bp, 编码203个氨基酸, 在北京油鸡肝、肺、腿肌和胸肌中有表达; 转染后24、48和72 h, pEGFP- a1,AGP转染率在31.3%~47.6%之间, 绿色荧光主要集中在细胞核中, 随着表达量的增加, 绿色荧光在细胞核中聚集成团块状或颗粒状, 经药物筛选和克隆化培养, 获得表达pEGFP-a1-AGP融合蛋白的阳性克隆细胞株, 利用RT-PCR与Western blotting检测确认pEGFP-α1-AGP已整合到北京油鸡成纤维细胞的基因组中, 在优化的条件下, 阳性细胞凋亡率、形态、生长与增殖状况与对照组比较差异不显著(P >0.05)。
The specific expression of α1-AGP gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR. The full-length cDNA of α1-AGP was inserted into pEGFP-C1 multi-cloning sites to construct recombinant eu-karyotic expression vector pEGFP-α1-AGP. The lipofectin method was used to transfect the pEGFP-α1-AGP into Beijing fatty chicken fibroblast cells. The open reading frame of Beijing fatty chicken α1-AGP gene was 612 base pairs in length, which was expressed higher in liver and lung than in muscle. This gene did not express in heart and kidney. The expression efficiency ranged from 31.3% to 47.6% in 24, 48, and 72 h after transformation. The green fluorescence mainly concen-trated in the nucleus. With the increase of the expression of green fluorescence, granula was observed in the nucleus. RT-PCR and Western blotting analyses showed that pEGFP-α1-AGP had been integrated into the genome of Beijing chicken fibroblast cell with normal expression ldvel. In optimized condition, there was no significant effect (P>0.05) on apoptosis ratio, positive cell shape, growth and reduplication state comparing with the control group. This research estab-lished the foundation for further function research of α1-AGP gene and application in transgenic animal cloning.
