›› 2018, Vol. 45 ›› Issue (1): 131-139.doi: 10.16431/j.cnki.1671-7236.2018.01.017

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Study on Construction of DKK1 Gene Recombinant Plasmid in Aohan Fine Wool Sheep and Its Expression in Fibroblasts

ZHANG Mengyao1, YANG Feng1, LIU Kaidong2, CHENG Ming2, WANG Guoyi3, LIU Jifeng1, LIU Nan1, HE Jianning1   

  1. 1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China;
    2. Qingdao Institute of Animal Science and Veterinary Medicine, Qingdao 266100, China;
    3. Aohan Banner in Chifeng, Chifeng 024300, China
  • Received:2017-07-13 Online:2018-01-20 Published:2018-01-20


This study was aimed to constract the recombiant plasmid of DKK1 gene in Aohan Fine Wool sheep and detect its expression in fibroblasts. The genomic DNA of biceps brachii in Aohan Fine Wool sheep was extracted, a pair of primers was designed according to DKK1 gene sequence in GenBank, and the DKK1 gene fragment was amplified by PCR method. DKK1 gene was ligated into pEASYTM-T1 vector to construct pEASYTM-T1-DKK1 recombinant plasmid and transformed into E.coli DH5α competent cell, the plasmid was identified by restriction enzyme digestion. The recombinant plasimd PmaxGFP-DKK1 was constructed and transformed into E.coli DH5α competent cell. Then PmaxGFP-DKK1 plasmid was transfected into fibroblasts, and Real-time quantitative PCR was used to detect the expression level of DKK1 gene. The results showed that the PmaxGFP-DKK1 plasmid was constructed successfully (3 956 bp), and was identified by enzyme and sequencing. The expression level of DKK1 gene in fibroblasts was extremely significantly higher than control group (P<0.01). The plasmid was constructed and transfected into fibroblasts successfully, the results would lay a foundation for the further research.

Key words: Aohan Fine Wool sheep; DKK1 gene; fibroblast; transfection; Real-time PCR quantitative; expression

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