Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (23): 4284-4289.doi: 10.3969/j.issn.1673-8225.2012.23.021

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Construction of eukaryotic expression vector of rat epidermal growth factor and ciliary neurotrophic factor and their expression in cos-7 cells

He Jian1, Yin Zong-sheng1, Gao Wei-lu1, Luo Qing-li2, Zhang Sheng-quan1   

  1. 1Department of Orthopedics, First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China;
    2Key Lab of Gene Resource Utilization for Severe Hereditary Diseases of Ministry of Education of Anhui Province, Anhui Medical University, Hefei 230032, Anhui Province, China;
    3Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei 230032, Anhui Province, China
  • Received:2011-12-07 Revised:2012-01-30 Online:2012-06-03 Published:2013-11-06
  • Contact: Yin Zong-sheng, Professor, Chief physician, Doctoral supervisor, Department of Orthopedics, First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China yinzs1961@163.com
  • About author:He Jian★, Studying for master’s degree, Physician, Department of Orthopedics, First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China na520688@sina.com

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Abstract:

BACKGROUND: After activation, astrocytes exhibit the characteristics of neural stem cells and overexpress the receptor of epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), which improves complex internal environment and therefore benefits for neuronal differentiation of neural stem cells.
OBJECTIVE: To construct the eukaryotic expression vectors of pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF, and detect EGF and CNTF expression in cos-7 cells so as to provide experimental evidence for gene therapy on spinal cord injury.
METHODS: The cDNA fragments of EGF and CNTF genes were amplified from total RNAs respectively. The amplified fragments were respectively inserted into eukaryotic expression vector pSecTag2/Hygro B to construct the recombined plasmid that encoded EGF and CNTF cDNA. The plasmids carrying EGF and CNTF genes were transfected alone respectively or cotransfected into cos-7 cells by liposome method. Then the protein expressions were detected by western blot method.
RESULTS AND CONCLUSION: RT-PCR results confirmed that EGF and CNTF cDNAs were successfully cloned. DNA sequence analysis confirmed that EGF and CNTF cDNAs in the constructed vectors were consistent with target sequences in the GenBank. Then two recombinant plasmids were cotransfected into cos-7 cells by liposome reagent. At 48 hours after transfection, EGF and CNTF protein expressions in cos-7 cells with the molecular weight of Mr6 000, 22 000 were identified by western blot analysis. These findings suggest that the eukaryotic expression vectors of pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF were successfully constructed and they co-express EGF and CNTF after transfected into cos-7 cells.

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