【摘要】 目的 :研究CPSF在真核表达载体上的克隆与瞬时表达。方法 :以质粒pBS176 1为模板扩增TAP -tag片段 ,PCR产物经纯化后克隆在真核表达载体pTRE2 -hyg上。再以pUK -CPSF 30k、73k、10 0k为模板扩增CPSF基因片段 ,将其克隆在质粒pTRE2 -hyg-TAP -tag中TAP -tag片段的下游 ,并将重组质粒转化入细胞株HelaTet -offS3细胞内。结果 :细胞抽提液经SDSPAGE电泳后进行蛋白质印迹杂交 ,胶片上出现野生型的CPSF条带和分子量较大的滞后条带。后者经分子量与分子标记对照 ,确系重组体TAP -tag-CPSF所表达的蛋白条带。结论 :重组质粒pTRE2hyg -TAP -tag-CPSF 30k ,73k ,10 0k在HelaTet -offS3内完好表达。更多还原

【Abstract】 Object:To study the clone of CPSF in the eukaryotic expression vector and transient expression.Methods:Tandem Affinity Purification tag (TAP-tag) was amplified with the plasmid pBS1761and the product of PCR was purified with agarose gel electrophoresis.Then the TAP-tag fragment was cloned in the eukaryotic expression vector pTRE2-hyg.Furthermore the genes of the cleavage and polyadenylation specificity factors (CPSF) 30k,73k,100k were amplified and cloned in the down stream of the TAP-tag.The recombinant plasmids were delivered to the cell strain of Hale Tet-Off S3 using liposome-mediated transfection.Results:The SDS PAGE and Western-blotting were carried out with the cell extracts and both the wild and recombination protein bands were presented in the X-ray film.It was confirmed that the recombinant plasmids can transient expression well in the Hela cell strain.Conclusion:The recombinant plasmids of pTRE2hyg- TAP-tag-CPSF 30k,73k,100k are well expressed in the cell strain of Hale Tet-Off S3.更多还原