Studies on the human cytomegalovirus protein pUL23 by establishment of HELF cell line stably expressing pUL23 gene |
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| Authors: | YANG Dan-li XIAO Xiao-ping GONG Jun-yuan YUAN Yue-ming LI Yue-qin ZHANG Xin ZOU Yi ZHOU Tian-hong LI Hong-jian |
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| Institution: | Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. E-mail: tzth@jnu.edu.cn, tlihj@jnu.edu.cn |
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| Abstract: | AIM: To establish HELF cell line with stable and effective expression of human cytomegalovirus (HCMV) UL23 gene as a powerful tool to study the situation of viral proteins in host cells in vitro. METHODS: UL23 gene was amplified by PCR from HCMV and then recombinant plasmid pLEGFP-N1-FLAG-UL23 was constructed by molecular cloning techniques. Artificial retroviral viruses containing HCMV UL23 gene were obtained by transferring plasmid pLEGFP-N1-FLAG-UL23 into the AmphoPackTM-293 package cells. The HELF cells were infected by the artificial retroviral particles. The positive HELF cells which stably expressed the UL23 were obtained by G418 selection. The location of the viral proteins in the cells was observed by laser confocal microscope. RESULTS: UL23 gene was amplified by RT-PCR from the positive HELF cells, and the UL23 fusion protein was expressed correctly and confirmed by Western blotting in the positive cells, indicating that the UL23 gene was stably integrated into the chromosome of HELF cells. The viral UL23 protein expressed in HELF cells was located in the cytoplasm observed by means of laser confocal microscope. CONCLUSION: Transgene cell strain stably carrying UL23 gene is successfully constructed by retroviral gene transfer techniques. The location of HCMV pUL23 in the host cells implicates that this vial protein exerts its function in cytoplasm. |
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