J4 ›› 2011, Vol. 10 ›› Issue (6): 21-24.

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Human Cyclin E Cloning and Eukaryotic Expression Vector Construction

Objective：To construct eukaryotic expression vector with FLAG epitope highly expressing human cyclin E gene．Methods：Amplify the cDNA of cyclin E gene from total RNA isolated from HeLa cells using RT-PCR．After sequenced，the open reading frame(ORF)of cyclin E cDNA was cloned into eukaryotic expression vector p3XFLAG—CMVTM-14 to form a recombinant plasmid naIned as p3XFLAG- cyclin E．Then lipofectamine 2000 was used to transfected eukaryotic expression vectors into HeLa cells．Finally，Western—blot was applied to detect the expression of human cyclin E in HeLa cells．Results：Restriction enzyme digestion and nucleotide sequencing results confirmed that the recombinant plasmid was successfully constructed．By W estern-blot，we found that the human cyclin E were expressed in HeLa cells．Conclusion： W e cloned human cyclin E and constructed eukaryotic expression vector，which enable US do further research with cyclin proteins．   

  1. 1．College ofLife Science，Yunnun University，Kunming 650031，China；
    2．Pre-clinicalCollege，DaliUniversity，Dali，Yunnan 671000，China
  • Online:2011-06-01 Published:2011-06-01

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Abstract:

Objective：To construct eukaryotic expression vector with FLAG epitope highly expressing human cyclin E gene．Methods：Amplify the cDNA of cyclin E gene from total RNA isolated from HeLa cells using RT-PCR．After sequenced，the open reading frame(ORF)of cyclin E cDNA was cloned into eukaryotic expression vector p3XFLAG—CMVTM-14 to form a recombinant plasmid naIned as p3XFLAG- cyclin E．Then lipofectamine 2000 was used to transfected eukaryotic expression vectors into HeLa cells．Finally，Western—blot was applied to detect the expression of human cyclin E in HeLa cells．Results：Restriction enzyme digestion and nucleotide sequencing results confirmed that the recombinant plasmid was successfully constructed．By W estern-blot，we found that the human cyclin E were expressed in HeLa cells．Conclusion： W e cloned human cyclin E and constructed eukaryotic expression vector，which enable US do further research with cyclin proteins．

Key words: cyclins, plasmid vector construction, eukaryotic expression

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