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Preparation of recombinant baculovirus based on the human sodium iodide symporter which participates radiosensitive positive feedback system

GUO Rui, LI Biao   

  1. Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2013-12-28 Published:2014-01-02
  • Supported by:

    National Natural Science Foundation of China, 81101071; Shanghai Outstanding Academic Leaders Program, 11XD1403700; Program of Training Youth Scholars of Shanghai Institutions of Higher Learning, 2011

Abstract:

Objective To prepare and verify a recombinant baculovirus harboring the human sodium iodide symporter (hNIS) under control of the early growth response-1 (Egr-1) promoter, and to provide the foundation for the research on radionuclides gene targeted treatment of malignant tumors. Methods The vector pFB was cut from plasmid pFB-CMV-EGFP; Egr1 was cut from plasmid PGL3-Egr1-luc; and hNIS was cut from plasmid pcDNA3.1-CMV-hNIS. Then recombinant baculovirus plasmid pFB-Egr1-hNIS was constructed. In addition, a positive control plasmid pFB-CMV-hNIS and a control plasmid pFB-0 were constructed. Baculovirus Bac-Egr1-hNIS and its controls were prepared, verified, amplified, and collected for titer determination. After glioma cell line U87 was infected with Bac-Egr1-hNIS and exposed to 131I, the presence and enhanced expression of hNIS protein was tested by immunofluorescence using anti-hNIS antibody. To verify the function of hNIS after 131I stimulation, the uptake of iodide was determined after incubation of the processed cells with 125I. Results Recombinant plasmid pFB-Egr1-hNIS, pFB-CMV-hNIS, and pFB-0 were correctly constructed and confirmed by DNA sequencing. Transposition was correct and recombinant Bacmid was extracted. The recombinant baculovirus were packed, verified, and its titer was about 1×1010 Pfu/mL. After incubation with 3 MBq/mL 131I, immunofluorescence showed that the hNIS expression level of Bac-Egr1-hNIS infected U87 cells increased dramatically. In another side, 131I stimulated U87 cells accumulated more 125I than did non stimulated cells. Conclusion The recombinant baculovirus Bac-Egr1-hNIS and its controls were constructed and verified, and high titer was obtained. This study provides a foundation of radiosensitive positive feedback system for further study.

Key words: human sodium iodide symporter, early growth response-1, recombinant baculovirus, positive feedback