CPSF在真核表达载体上的克隆与瞬时表达

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pBS1761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-CPSF30k、73k、100k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Hela Tet-offS3细胞内。结果：细胞抽提液经SDS PAGE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-CPSF30k，73k，100k在Hela tet-offS3内完好表达。

Clone of CPSF in the Eukaryotic Expression Vector and Transient Expression

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.