遗传 ›› 2004, Vol. 26 ›› Issue (5): 567-573.

• 论文 • 上一篇    下一篇

催乳素和外源基因乳腺共表达载体可提高外源基因在哺乳细胞的表达

刘峰涛; 颜景斌; 任兆瑞; 黄淑帧; 曾溢滔LIU Feng-Tao; YAN Jing-Bin; REN Zhao-Rui; HUANG Shu-Zhen; ZENG Yi-Tao   

  1. 上海交通大学医学遗传研究所,上海 200040Institue of Medical Genetics,Shanghai Jiaotong University,Shanghai 200040,China
  • 收稿日期:1900-01-01 出版日期:2004-10-10 发布日期:2004-10-10

Mammary Gland Dual-Expression Construct Containing Prolactin and Foreign DNAs Can Promote the Expression of Foreign Proteins in Mammary Cells

  • Received:1900-01-01 Online:2004-10-10 Published:2004-10-10

摘要:

为探讨应用牛催乳素促进乳腺生物反应器高效表达外源蛋白(如人乳铁蛋白、人血小板生成素)的可行性,构建两种牛催乳素和外源基因乳腺共表达载体。一种为牛催乳素基因和外源基因共用一个6.7 kb山羊β-酪蛋白启动子启动转录,两个基因以内部核糖体进入位点(IRES)连接;另一种为外源基因利用6.7 kb山羊β-酪蛋白启动子,催乳素基因利用2.0 kb山羊β-酪蛋白启动子,分别转录。脂质体转染后,RT-PCR以及ELISA分析结果证实外源蛋白的表达得到提高。在COS-7细胞(猴胚肾细胞),两种载体分别使乳铁蛋白的表达由12.6 μg/L提高到18.4 μg/L和37.2 μg/L(P<0.05);在HC-11细胞(小鼠乳腺上皮细胞),两种载体分别使乳铁蛋白的表达由13.7μg/L提高到20.7 μg/L和19.9 μg/L(P<0.05)。利用双启动子载体,使人血小板生成素在COS-7细胞由572 ng/L提高到1340 ng/L(p<0.05);在HC-11细胞由783 ng/L提高1040 ng/L(P<0.05。本文结果证明催乳素可以有效的提高外源基因在哺乳细胞的表达。

Abstract: To investigate the feasibility in the promotion of the expression of foreign proteins, such as human lactinferrin (hLF) and thrombopoietin (TPO), in the transgenic animals-mammary gland bioreactor by bovine prolactin (bPRL), two types of mammary gland dual-expression vectors containing bPRL and foreign DNAs were constructed. In one type of vector, both foreign and bPRL DNAs were linked by internal ribosome entry site (IRES) which shared one goat 6.7 kb of β-casein promoter; while in the other type of vector, the transcriptions of the foreign gene aswell as bPRL were respectively directed by 6.7 kb goat β-casein promoter and 2.0 kb of goat β-casein promoter. After transfection of the vectors with lipofectin method, the expression of foreign proteins were measured by RT-PCR as well as ELISA assay. The results showed that bPRL could obviously promote the expression of foreign proteins (hLF and TPO) in cultured cells. The hLF expression level was increased from 12.6 μg/L to 18.4 μg/L and 37.2 μg/L in the COS-7 cells (African green monkey kidney cell) (P<0.05), and from 13.7 μg/L to 20.7 μg/L and 19.9 μg/L in the HC-11 cells (epithelial cell of mouse mammary gland) (P<0.05) by two vectors, respectively. TPO expression level was increased from 572 ng/L to 1340 ng/L in the COS-7 cell (P<0.05), and from 783 ng/L to 1040 ng/L in the HC-11 cell (P<0.05) by the vector which have two promoters. This work indicate that bovine prolactin can effectively increase the expression of foreign genes in mammal cells.

关键词: 血小板生成素, prolactin, 表达
Key words,
催乳素, 乳铁蛋白