鸡IL-2基因在输卵管上皮细胞中的表达及其活性检测

Expression of Chicken IL-2 cDNA in Oviduct Cells in Vivo and Test of the Biological Activity

探讨鸡IL-2基因cDNA在输卵管上皮细胞中的定位表达和生物学活性。利用卵清蛋白和卵清溶菌酶基因5′端调控区序列构建输卵管定位表达盒,将鸡IL-2基因cDNA分别置于表达盒的调控序列下游,构建定位表达载体pcDNA3-OVP-IL2和pcDNA3-LGP-OVP-IL2。表达载体转染鸡输卵管上皮细胞,激素诱导48h。经Western blot和ELISA检测,转染pcDNA3-OVP-IL2和pcDNA3-LGP-OVP-IL2表达载体的细胞培养液中均有Mr22 ku的蛋白,96 h细胞上清液中IL-2的表达量分别为2.659μg/L和5.724μg/L;淋巴细胞转化试验检测显示,所表达的重组蛋白具有促进T淋巴细胞转化和促进淋巴母细胞成熟的活性。实验表明融合调控序列能够调控IL-2基因在鸡输卵管上皮细胞中表达具有生物学活性的IL-2。

To recover easily and rapidly chicken IL-2 gene products from oviduct cells in vivo and to test the content and the biological activity of IL-2. Two vector cassettes carrying chicken ovalbumin promoter（OVP） and chicken lysozyme gene 5′-flanking glucocorticoid/progesterone hormone receptor sites （LGP）were constracted, and IL-2 encoding DNA fragment was Iigated into the vector cassettes. When the resultant construct oviduct-specific expression plasmids pcDNA3-OVP-IL2 and pcDNA3- LGP-OVP-IL2 were transfected into the chicken oviduct cells, IL-2 expression at 48h after being induced by Progesterone and Glucocorticoid as well as Insuline was confirmed by Western blot, ELISA test for the content and MTT colorimetry test for the biological activity. The results showed that the plasmids pcDNA3-OVP-IL2 and pcDNA3-LGP--OVP-IL2 could express IL-2 in oviduct cells. SDS- PAGE and Western blot confirmed that the expressed protein was about 22 ku, ELISA test for the expression yield was 2. 659 μg/L and 5. 724 μg/L, respectively. MTT colorimetric assay showed that the recombinant protein could induce chicken T lymphocytes to proliferate in vivo. This study shows that the recovery of pharmaceutical proteins from the egg white protein is an effective method in transgenic chicken bioreactors.