›› 2010, Vol. 30 ›› Issue (11): 1348-.doi: 10.3969/j.issn.1674-8115.2010.11.007

• Original article (Basic research) • Previous Articles     Next Articles

Preparation and identification of 190CT3-myc fusion polypeptide

YAO Yun1, JIANG Dan-dan2, SUN Qing3, LIU Hou-qi3   

  1. 1.Teaching and Research Section of Histo-embryology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China； 2.Scientific Research Section, Shanghai Da'an Medical Testing Center, Shanghai 201203, China； 3.Department of Embryology and Histology, College of Basic Medical Sciences, Research Center of Developmental Biology, Second Military Medical University, Shanghai 200433, China
  • Online:2010-11-25 Published:2010-11-29
  • Supported by:

    Shanghai Education Committee Foundation, P13910, J50301

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Abstract:

Objective To construct a myc-tagged 190CT3 polypeptide (190CT3-myc) with genetic recombination method. Methods PCR was used to amplify cDNA sequences encoding 190CT3-myc fusion polypeptide, which were cloned to T vectors. After enzyme digestive identification of the sequences, 190CT3 eukaryotic expression vector was constructed and attached with myc fusion protein tag, and was transfected into Chinese hamster ovary (CHO) cells. After screening by G418, CHO cells which stably expressed target genes were obtained, and were identified by immunofluorescence method. 190CT3-myc fusion polypeptide was purified with antigen-antibody affinity chromatography column, and Coomassie brilliant blue staining and Western blotting were conducted after SDS-PAGE. Results cDNA fragment encoding 190CT3-myc was obtained by PCR amplification, and was confirmed by enzyme digestive identification of the sequences. Immunofluorescence method revealed that CHO cells transfected with recombinant plasmid stably expressed 190CT3-myc. Coomassie brilliant blue staining indicated the expression of fusion polypeptide 190CT3myc accounted for about 10% of the total protein in cells, with the purification ≥90%. Western blotting demonstrated that the relative molecular weight of purified fusion polypeptide was 17 000, which was in accordance with target protein. Conclusion 190CT3-myc recombinant polypeptide has been successfully prepared, which may provide new ideas for the drug target research of leukemia.

Key words: fusion polypeptide, eukaryotic expression vector, transfection, protein purification