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作 者:刘峰涛[1] 颜景斌[1] 任兆瑞[1] 黄淑帧[1] 曾溢滔[1]
机构地区:[1]上海交通大学医学遗传研究所,上海200040
出 处:《遗传》2004年第5期567-573,共7页Hereditas(Beijing)
基 金:国家863高技术项目(2002AA206201)~~
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 7kb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 7kb山羊β 酪蛋白启动子,催乳素基因利用2 0kb山羊β 酪蛋白启动子,分别转录。脂质体转染后,RT PCR以及ELISA分析结果证实外源基因的表达得到提高。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 6μg/L提高到18 4μg/L和37 2μg/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 7μg/L和19 9μg/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572ng/L提高到1340ng/L(P<0 05);在HC 11细胞由783ng/L提高1040ng/L(P<0 05)。结果证明催乳素可以有效的提高外源基因在哺乳类细胞的表达。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 as well 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 genes 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.
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