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目的 :为获得高表达有活性的p16蛋白 ,构建含p16cDNA的重组转移载体。方法 :EcoRI xhoI双酶切克隆质粒pBLUESCRIPT p16 ,低融点琼脂糖回收 0 8kb的p16cDNA片段 ,插入质粒pSXIVVI+X3,构建含p16的重组载体pX3 p16 ,并对重组子以菌落原位杂交和酶切电泳两种方法进行鉴定。结果 :电泳证实低融点琼脂糖回收的片段为 0 8kb。菌落原位杂交筛选 8个阳性克隆 ,酶切电泳证实阳性克隆中p16cDNA插入方向正确。结论 :成功构建了含p16cDNA的重组转移载体 ,为p16在昆虫细胞和活体昆虫中的表达奠定了基础
Purpose : To obtain high expression of active p16 protein, construct a recombinant transfer vector containing p16 cDNA. Methods: pBLUESCRIPT p16 plasmid was digested with EcoRI xhoI, and the p16cDNA fragment of 0 8 kb was recovered from the low melting point agarose. The plasmid pSXIVVI+X3 was inserted to construct the recombinant vector pX3 p16 containing p16, and the colony was in situ and digested by the colony. Electrophoresis two methods for identification. RESULTS : The electrophoresis confirmed that the fragments recovered from the low melting point agarose were 0 8kb. Eight positive clones were screened by colony in situ hybridization. The direction of insertion of p16 cDNA in positive clones was confirmed by enzyme electrophoresis. Conclusion: The recombinant transfer vector containing p16 cDNA was successfully constructed and laid the foundation for the expression of p16 in insect cells and living insects.