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| Construction and Verification of Neotype Expression Vector Containing Two Expression Cassettes |
| DU Shou-wen1,2, LI Chang2, WANG Yu-hang1,2, REN Da-yong1,2, LIU Cun-xia1,2, SUN Dan-dan2, ZHU Na2, LI Yi2, QIN Yan-qing2, JIN Ning-yi2 |
1. College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China;
2. Genetic Engineering Laboratory, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China |
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Abstract Objective: To construct and verify the neotype gene vaccine eukaryotic expression vector pVAX2 which contains two gene expression cassettes. Methods: The new expression cassette was designed and obtained by artificial chemical synthesis that contains eight elements—Human cytomegalovirus (CMV) promoter, T7 promoter, signal peptide sequences, Hemagglutinin A epitope genes (HA), multiple cloning sites region(MCS),c-myc epitope, Platelet-derived growth factor receptor transmembrane domain (PDGFR-TM) and Bovine growth hormone polyadenylation (BGH polyA). And the restriction enzyme NruⅠwas added to its upstream and downstream, and then it linked with cloning vector pGH to construct pGH-Ⅱ. The pGH-Ⅱ was cutted with NruⅠ,and the fragment (1 300bp) was reclaimed and cloned into the eukaryotic expression vector pVAX1 after removing phosphoryl groups with Alkaline phosphatase to construct the neotype gene vaccine eukaryotic expression vector pVAX2, which was identified with NruⅠand BglⅡ/PstⅠ. To identify the expression ability of pVAX2, the enhanced green fluorescent protein (EGFP) genes as report gene was inserted into the difference cassette of the two expression cassettes, and then transfected into BHK-21cells by liposome. The function of pVAX2 was verified by the reverse-transcriptase-polymerase chain reaction (RT-PCR) and fluorescence microscope. Result: The expression of EGFP in both expression cassettes was high efficiency in BHK-21 cells, and didn’t impact each other. Especially, the new expression cassette allowed display of proteins on the cell surface. Conclusion: The eukaryotic expression vector pVAX2 is successfully constructed, which could be used for the research of polyvalency DNA vaccines.
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Received: 05 May 2011
Published: 25 September 2011
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Cite this article:
DU Shou-wen, LI Chang, WANG Yu-hang, REN Da-yong, LIU Cun-xia, SUN Dan-dan, ZHU Na, LI Yi, QIN Yan-qing, JIN Ning-yi. Construction and Verification of Neotype Expression Vector Containing Two Expression Cassettes. China Biotechnology, 2011, 31(9): 14-20.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I9/14
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[1] Mancini B M, Fontaine H, Brechot C, et al. Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers. Vaccine, 2006, 24(21):4482-4489.
[2] Graham B S, Koup R A, Roederer M, et al. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine. J Infect Dis, 2006, 194(12):1650-1660.
[3] Richard J A, Joerg S. Plasmid DNA and viral vector-based vaccines for the treatment of cancer.Vaccine, 2007, 25(2): 24-34.
[4] 奥斯伯 F M, 布伦特 R, 金斯顿 R E, 等. 精编分子生物学实验指南, 第5版. 北京: 科学出版社, 2008. 19. Ausubel F M, Brent R, Kingston R E, et al. Short Protocols in Molecular Biology, 5 th ed. Beijing: Science Press, 2008. 19.
[5] Aparna K, Ramandeep S, Shakila H, et al. Elicitation of efficient, protective immune responses by using DNA vaccines against tuberculosis. Vaccine, 2005, 23(48-49): 5655-5665.
[6] Ma M X, Jin N Y, Liu H J, et al. Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18. Antiviral Research, 2007, 76(1): 59-67.
[7] Chen R Y, Chen H X, Lin J X, et al. In vivo transfection of pcDNA3.1-IGFBP7 inhibits melanoma growth in mice through apoptosis induction and VEGF down expression. Journal of Experimental & Clinical Cancer Research, 2010, 29(1): 13.
[8] Julie E M, Mark K L, LaSonji A H, et al. A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial. Vaccine, 26(50):6338-6343.
[9] Larry R S, Mary K W, Ming Y, et al. Phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid DNA vaccines encoding influenza A virus H5 hemagglutinin. Vaccine, 2010, 28(13): 2565-2672.
[10] Geoffrey J G, Lindsey R B, Margaret W, et al. Safety and immunogenicity of cytotoxic T-lymphocyte poly-epitope, DNA plasmid (EP HIV-1090) vaccine in healthy, human immunodeficiency virus type 1 (HIV-1)-uninfected adults.Vaccine, 2008, 26(2): 215-223.
[11] Tahara H, Zitvogel L, Storkus W J, et al. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector. J Immunol, 1995, 154(12):6466- 6474.
[12] Andersson S, Davis D L, Dahlback H, et al. Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. Biol Chem, 1989, 264(14): 8222-8229.
[13] Coloma M J, Hastings A, Wims L A,et al. Novel Vectors for the Expression of Antibody Molecules Using Variable Regions Generated by Polymerase Chain Reaction. Imm Methods, 1992, 152(1): 89-104.
[14] Kolodziej P A, Young R A. Epitope Tagging and Protein Surveillance. Methods Enzymol, 1991, 194:508-519.
[15] Evan G I, Lewis G K, Ramsay G, et al. Isolation of Monoclonal Antibodies Specific for c-myc Proto-oncogene Product. Mol Cell Biol, 1985, 5(12):3610-3616.
[16] Gronwald R G, Grant F J, Haldeman B A, et al. Cloning and Expression of a cDNA Coding for the Human Platelet-Derived Growth Factor Receptor: Evidence for More Than One Receptor Class. Proc Natl Acad Sci USA, 1988, 85(10):3435-3439.
[17] Goodwin E C, Rottman F M. The 3'-Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation. J Biol Chem, 1992, 267(23):16330-16334.
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