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Journal of Zhejiang University SCIENCE A 2001 Vol.2 No.4 P.445-447

http://doi.org/10.1631/jzus.2001.0445


CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT OF THE AAV VECTOR AND HUMAN INTERFERON-GAMMA


Author(s):  ZHU Hai-hong, CHEN Zhi, ZHANG Ming-tai, DING Lie-ming

Affiliation(s):  Institute of Infectious Diseases, College of Midicine, Zhejiang University, Hangzhou 310003, China; more

Corresponding email(s): 

Key Words:  Adeno-associated virus, gene therapy, interferon-gamma


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ZHU Hai-hong, CHEN Zhi, ZHANG Ming-tai, DING Lie-ming. CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT OF THE AAV VECTOR AND HUMAN INTERFERON-GAMMA[J]. Journal of Zhejiang University Science A, 2001, 2(4): 445-447.

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Abstract: 
To construct and identify further a recombinant of adeno-associated virus and interferon-gamma for gene therapy, the full-length IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from the positive clone was then subcloned into pwpl9. After the correct recombinant was identified by digestion with SacI and BamHI, it was transfected into lymphocyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected by RT-PCR and ELISA. The result showed that the IFN-γ were expressed in the H9 cells transfected with pwp/IFN-γ. The so constructed recombinant plasmid pwpl9/IFN-γ containing the full-length IFN-γ gene was expressed in mammalian cells.

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Reference

[1] Bjorklund, A., Kirik, D., Rosenblad, C. et al., 2000, Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model. Brain Res., 886(1-2):82-98.

[2] Cottard, V., Mulleman, D., Bouille, P. et al., 2000, Adeno-associated virus-mediated delivery of IL-4 prevents collagen-induced arthritis. Gene Ther., 7(22):1930-1939.

[3] Devos, R., Cheroutre, H., Taya Y. T. et al., 1982, Molecular cloning of human immune interferon eDNA and its expression in eukaryotic cells. Nucleic Acids Res., 10(8):2487-501.

[4] Nahreini, P., Woody, M.J., Zhou, S.Z. et al., 1993,Versatile adeno-associated virus 2-based vectors for constructing recombinant virions. Gene, 124(2):257-262.

[5] Sambrok, J., Fristch, E.F., Maniatis, J., 1989. Moecular Cloning Laboratory Mannual. 2nd edition. Cold Spring Harbor, Cold Spring Harbor Laboratory Press. NY.

[6] Samulski, R. J., Chang, L. S., Shenk T. et al., 1989, Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol., 63(9):3822-3828.

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