激光生物学报, 2010, 19 (3): 339, 网络出版: 2015-10-08  

北京油鸡purH基因结构与表达特征研究

Characterization of Expression of purH gene in Beijing Fatty Chicken (Gallus gallus)
作者单位
1 中国农业科学院北京畜牧兽医研究所, 北京 100193
2 蚌埠医学院检验系, 安徽 蚌埠 233030
3 蚌埠医学院生物科学系, 安徽 蚌埠 233030
摘要
提取北京油鸡8种组织的总RNA, 利用RT-PCR检测purH基因mRNA的差异表达。将purH基因完整开放阅读框定向插入pEGFP-N3, 构建带有GFP报告基因重组表达载体pEGFP-purH, 利用脂质体介导法将其导入北京油鸡体外培养细胞中, 进行G418药物筛选和克隆化培养。结果表明: 北京油鸡purH基因(GenBank 登录号: EU334506) 编码区为1 782 bp, 编码593 aa的多肽, 转染后24 h、48 h和72 h, pEGFP-purH转染率在10.3 %~53.2 %之间, 绿色荧光均匀分布于细胞质与细胞核中, 随表达量的增加, 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-purH融合蛋白的克隆细胞株, RT-PCR与Western blotting检测确认pEGFP-purH已整合到北京油鸡成纤维细胞基因组中, 在优化的条件下, 阳性细胞凋亡率、生长与增殖状况与对照组比较差异不显著(P>0.05)。
Abstract
The specific expression of purH gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR. The full-length cDNA of purH was inserted into pEGFP-N3 multi-cloning sites to construct recombinant vector pEGFP-purH. The lipofectin method was used to transfect the pEGFP-purH into fibroblast cells. Bioinformatic analysis be transfected showed CDS region of the Beijing fatty chicken purH gene was 1 782 bp, which encoded a 483-amino-acids-long peptide. The recombinant pEGFP-purH was constructed with GFP as reporter gene to transfected into Beijing fatty chicken fibroblasts. The expression efficiency ranged from 10.3 % to 53.2 % in 24, 48, and 72 h after transformation. RT-PCR and Western blotting analyses showed that pEGFP-purH had been integrated into the genome of Beijing chicken fibroblast cell with normal expression level. In optimized condition, there was no significant effect (P>0.05) on apoptosis ratio, growth and reduplication state comparing with the control group.

郭俣, 刘长青, 陆涛峰, 包阿东, 刘帅, 关伟军, 马月辉. 北京油鸡purH基因结构与表达特征研究[J]. 激光生物学报, 2010, 19(3): 339. GUO Yu, LIU Chang-qing, LU Tao-feng, BAO A-dong, LIU Shuai, GUAN Wei-jun, MA Yue-hui. Characterization of Expression of purH gene in Beijing Fatty Chicken (Gallus gallus)[J]. Acta Laser Biology Sinica, 2010, 19(3): 339.

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