›› 2009, Vol. 29 ›› Issue (7): 821-.

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

Construction and identification of recombinant baculovirus vector to coexpress GDNF and EGFP gene

CHEN Yan-chun1, WANG Jun1, WANG Shi-li1, CAI Chang-ping1, LI Biao2, ZHANG Yi-fan2, GUO Rui2   

  1. 1. Department of Otolaryngology, 2. Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2009-07-25 Published:2009-09-16
  • Supported by:

    Shanghai Education Committee Foundation (06BZ036)

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Abstract:

Objective To construct a novel enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) recombinant baculovirus. Methods The target gene（EGFP and GDNF）was cloned into baculovirus transfer vector pFastBacDual, pFB-EGFP-GDNF was constructed and restriction enzyme analysis was conducted. pFB-EGFP-GDNF was transposited with baculovirus shuttle vector (Bacmid) into DH10Bac competent cells, and recombination baculovirus vector Bacmid-EGFP-GDNF was constructed. The plasmid was extracted and PCR was performed for identification. Bacmid-EGFP-GDNF was transfected with Sf9 insect cell package virus by liposomal transfection method. Immunofluorescent staining was employed to detect the expression of EGFP and GDNF protein in Sf9 cells. Results The target gene fragment was correctly cloned into pFastBacDual vector, and recombinant Bacmid was constructed. Bacmid-EGFP-GDNF was successfully transfected, and higher virus titer was obtained. The coexpression of GDNF and EGFP protein in Sf9 cells was identified by immunofluorescent staining. Conclusion The recombinant baculovirus Bacmid-EGFP-GDNF can be successfully constructed, and the protein of EGFP and GDNF is coexpressed in Sf9 cells, which paves a way for the research of GDNF gene therapy.

Key words: glial cell line derived neurotrophic factor, enhanced green fluorescent protein, baculovirus, protein expression