生长激素释放因子在CHO细胞的表达

Expression of Growth Hormone Releasing Factor in CHO Cells

在对生长激素释放因子 (GRF)基因改造和化学合成 ,并构建了其真核表达载体 pc DNA3- GRF(1- 32 )的基础上 ,用 L ipofectin将上述载体转染 CHO细胞进行瞬时表达。提取转染细胞总 RNA,用 RT- PCR和 Dot blotting检测 GRF基因的表达情况 ,用 Western blotting检测转染细胞上清液的表达产物 ,均得到了阳性结果。制备大鼠垂体单层细胞 ,测定表达产物的生物学活性 ,结果表达产物可刺激生长激素释放 ,并且比对照组提高 3.8倍。试验结果表明 ,已构建的真核表达载体 pc DNA3- GRF(1- 32 )能表达出有生物学活性的 GRF。

Chemical synthesis of the GRF(1 32) gene has been finished in our former work.This GRF(1 32) gene has been inserted into a plasmid, pcDNA3, to construct an expression vector.In this study, pcDNA GRF(1 32) is expressed in CHO cells.The expression vector was purified with a High Pure Plasmid Extraction Kit and then transfected into prepared CHO cells in 60% confluent.The expression of GRF(1 32) was detected by RT PCT with a pair of 23 bp primers, followed by Dot blotting with a synthesized (83 nt) probe, which had been radioactively labelled with 32 P with T4 polynucleotide kinase.Positive results were obtained.Supernatant of transfected CHO cells was analyzed with Western blot, whose result showed a special GRF band.Dispersed rat pituitary were used to determine the activity of expressed GRF(1 32).Results showed that the expressed products stimulated Growth Hormone(GH) secretion by pituitary cells at levels which were 3 8 folds higher than the control group.The results in this study indicate that we have constructed GRF eukaryotic expression vector successfully.