ret基因真核表达载体的构建及其瞬时表达

Construction and transient expression of pcDNA3.0-RET

目的:构建ret基因真核表达载体pcDNA3.0-RET,为制作转基因动物提供实验基础。方法:利用HandⅢ及NotⅠ限制性核酸内切酶,消化pSK-RET及pcDNA3.0空质粒,获得目的基因及相应载体;通过T4连接酶重组DNA;通过氨苄青霉素(Amp)抗性、酶切鉴定及DNA序列分析,确定阳性克隆。通过脂质体包裹转染NIH3T3细胞,采用Westernblot方法检测载体的瞬时表达状况。结果:Amp抗性筛选阳性;酶切片段电泳分析在5400bp及3300bp处有明显条带;DNA序列分析提示重组基因与实际序列一致;Westernblot结果显示175000处有明显条带。结论:ret基因真核表达载体pcDNA3.0-RET构建成功,并可瞬时表达。

Aim： To construct the expression vector of pcDNA3.0-RET,which is the experimental base for making transgenic animals. Methods：pSK-RET and pcDNA3.0 was digested by restrict enzyme Hand Ⅲ and Not I . The cDNA fragment of RET was inserted into pcDNA3.0 and the expression vector pcDNA3.0-RET was constructed with T4 DNA ligase. Positive strain was selected by Amp resistant, restrict enzyme digestion, and DNA sequencing. The pcDNA3.0-RET was transfected into NIH3T3, and the ret transient expression was demonstrated by Western blot. Results： The result selected by Amp resistant, restrict enzyme digesting map, and DNA sequencing was all positive. A positive protein electrophoretic result was found at 175 000. Conclusion ： The expression vector of pcDNA3.0-RET has been fulfilled and transiently expressed successfully.