人生长激素基因逆转录病毒载体的构建及其对3T3和ES细胞的转化与表达

为了研究生长激素基因在异源细胞中的表达，构建了携带人生长激素基因（ｈＧＨ）的逆转录病毒载体ｐＩＮＳ－ＧＨ，并将其导入小鼠成纤维细胞３Ｔ３和小鼠胚胎干细胞（ＥＳ细胞）ＣＣＥ中。ｐＩＮＳ－ＧＨ转化３Ｔ３细胞，获得了高效表达的克隆，即用放射免疫法检测到克隆培养基中有大量的人生长激素蛋白富积，如ＧＨＳＮＣ２０，富积率高达３７８４ｎｇ／ｍｌ，而且外源基因的整合很稳定。不同的３Ｔ３转化克隆的表达率有显著的差异，Ｓｏｕｔｈｅｒｎ杂交的结果表明，这种差异与外源基因整合的拷贝数无关。ｐＩＮＳ－ＧＨ转化ＣＣＥ细胞没有获得明显表达的克隆，而且外源基因的整合也不稳定。转化的ＥＳ细胞克隆总ＤＮＡ的Ｓｏｕｔｈｅｒｎ杂交结果表明，ｈＧＨ基因的确整合到细胞基因组中。目前尚未发现明显的外源ＤＮＡ重排的迹象。

Construction of A Retroviral Vector Carrying Human Growth Hormone Gene and Its Introduction and Expression in 3T3 and ES Cells

In order to study the expression of growth hormone gene in heterologous cells,the retroviral vector pINS was used to construct the recombinant plasmid carrying the human growth hormone gene(hGH),and then it was introduced into mouse fibroblast cell line 3T3 and mouse embryonic stem cell line CCE,Levels of accumulation of hGH secreted into medium was quantified by a radioimmunoassay. Expression level of hGH gene varied considerably from one clone to another. Several 3T3 clones expressing hGH gene at high level were obtained,such as GHSNC 20,in which hGH concentration in medium was 3784n/ml,Integration of exogenous gene was stable. Southern blot result showed that such kind of difference was not related to the number of integrated copies of the foreign gene. The expression of ES clones transfected with pINS-GH was undetectable, and integration of the foreign gene was unstable.Southern blot result showed that hGH gene really integrated into the cellular genome, and no rearrangement of foreign gene is found at present.