Article

The Chinese-German Journal of Clinical Oncology

, Volume 5, Issue 5, pp 358-361

Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1

  • Wenhua XiongAffiliated withDepartment of Orthopedics, Tongji Hospital, Huazhong University of Science and Technology
  • , Anmin ChenAffiliated withDepartment of Orthopedics, Tongji Hospital, Huazhong University of Science and Technology
  • , Fengjing GuoAffiliated withDepartment of Orthopedics, Tongji Hospital, Huazhong University of Science and Technology
  • , Tao HuangAffiliated withDepartment of Orthopedics, Tongji Hospital, Huazhong University of Science and Technology

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Abstract

Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPin1) genes. Methods: Total RNA was extracted from MG-63 cells, then the hPin1 cDNA was amplified by RT-PCR. The same time the sense and antisense hPin1 genes were formed by binding BamH I and Hind III in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamH I and Hind III. Results: The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH I and Hind III, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion: Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.

Key words

Pin1 isomerase antisense gene eukaryotic expressing vector