大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究

RECONSTRUCTION AND APPLICATION OF EUKARYOTIC EXPRESSION PLASMID OF RAT PROLACTIN GENE

７３５ｂｐ的ＰＲＬｃＤＮＡ片段从质粒ＰＲＬ－ＳＰ６５＃１中回收后，用粘性末端连接法将其重组到真核表达载体ｐｃＤＮＡ３上，筛选出正向连接重组体ｐｃＤＮＡ３－ＰＲＬＳ和反向连接重组体ｐｃＤＮＡ３－ＰＲＬＡＳ。将重组体ｐｃＤＮＡ３－ＰＲＬｓ和空载体ｐｃＤＮＡ３分别转入ＮＩＨ３Ｔ３细胞系，用Ｇ４１８筛选出阳性细胞后与未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２的情况下，用原位杂交的方法，分别用ＰＲＬｃＤＮＡ探针和原癌基因ｃ－Ｈ－ｒａｓｃＤＮＡ探针进行检测，未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２时都几乎无催乳素基因的表达，同样，转入空载体的ＮＩＨ３Ｔ３细胞也无ＰＲＬ的表达，而转入重组体ｐｃＤＮＡ３－ＰＲＬＳ的ＮＩＨ３Ｔ３细胞则有大量的ＰＲＬ基因的表达，与对照组相比有显著差异（Ｐ＜０．０１）。正常和转入空载体的ＮＩＨ３Ｔ３细胞有一定程度的原癌基因ｃ－Ｈ－ｒａｓ的表达，当分别加入Ｅ２和转入重组体ｐｃＤＮＡ３－ＰＲＬＳ后，ＮＩＨ３Ｔ３细胞中的ｃ－Ｈ－ｒａｓ基因表达水平都显著升高（Ｐ＜０．０５）。

A 735bp fragment, generated by a complete digestion of PRL SP65#1 plasmid with restriction enzymes EcoRI, was inserted into the EcoRI site of pcDNA3 eukaryotic expression vector by ligation of protruding ended DNA. 11 positive colonies of 12 colonies have been determined with mapping analysis, the sense and antisense recombinant has been designated as pc DNA PRL S and pcDNA PRL AS , respectively. Recombinant plasmid pcDNA PRL S and vector pcDNA3 were transfected into NIH3T3 cell lines by lipofect AMINE transfecting method. The transfected cells were screened by G418. The transfected NIH3T3 cells and non transfected NIH3T3 cells were cultured in 24 well plate. The cells were divided into 2 groups, one group was added PRL cDNA probe, the other was added c H ras cDNA probe. In each group, there were subgroups:Blank vector transfected subgroup, Recombinant plasmid transfected subgroup, Non transfected NIH3T3 cells subgroup and Non transfected NIH3T3 cells treated with 10 -5 M E 2 subgroup. When all of groups cells have been in logarithmic growth period, the PRL cDNA and c H ras cDNA probes were used to determine the expression of prolactin gene and c H ras gene by in situ hybridization. There was almost no expression of PRL gene in either Non transfected or Blank vector transfected NIH3T3 cells, neither after E 2 treatment. After pcDNA3 PRL S was transfected, the expression of PRL gene largely increased, compared with Blank vector transfected NIH3T3 cells grourp( P <0.01). There was a little expression of c H ras gene in Non transfected subgroup and Blank vector pcDNA3 transfected cell subgroup, but when the recombinant pcDNA PRL S has been transfected into NIH3T3 cells and the nontransfected NIH3T3 cells were treated with E 2 for 24 hours, the expression of c H ras gene in these two subgroubs significantly increased, and compared with Blank vector pcDNA3 transfected cells subgroup and non transfected NIH3T3 cells subgroup( P <0.05; P <0.0 1) respectively.