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Expression of Recombinant Human Beta-Defensin-2 in Mouse Fibroblast Cells and Its Antiseptic Activity

Author: LiJuan
Tutor: ChenXinNian
School: Lanzhou University
Course: Pathology and Pathophysiology
Keywords: Human β- defensin-2 Gene transfection Mouse fibroblasts Gene Expression Antibacterial activity
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 76
Quote: 0
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Abstract


Objective: To obtain human β- defensin -2 (hBD-2) gene and construct its eukaryotic expression vector transfected mouse fibroblast cells ( L929 ) to detect recombinant hBD-2 expression vector expression in L929 cells , preliminary determination hBD-2 in vitro antibacterial activity of hBD-2 gene transfection and expression of the foundation . Methods : PCR primers were designed according to the Genebank in the hBD-2cDNA nucleotide sequence . By RT-PCR from human cervical tissue for human β- defensin-2 gene , and then cloned to construct the eukaryotic expression vector pCAGG - hBD- 2 enzyme digestion and sequencing . Using calcium phosphate coprecipitation pCAGG-hBD-2 Import L929 cells , hBD-2 mRNA expression was detected by RT-PCR and Western-blot analysis identified the protein expression of hBD-2 gene , and finally using the Kirby-Bauer paper sheet diffusion method reorganization of hBD - 2 antibacterial activity . Results: 1 . RT-PCR amplification of a fragment of about 300bp , sequencing analysis showed that from Genebank LOG the hBD-2cDNA sequence , the fragment of the whole sequence of hBD-2 encoding . 2. Constructed the eukaryotic expression vector pCAGG-hBD-2 by restriction endonuclease and sequencing results showed that hBD-2 gene expression vector direction of the connection correctly, can be transfected . 3. L929 cells pCAGG-hBD-2 after transfection total RNA as a template for RT-PCR reaction amplified specific bands ; positive Western blot . That the constructed expression vectors pCAGG - hBD- 2 transfected L929 cells can correct expression of hBD - 2 4 . The KB paper method detection results transfected cell supernatant pCAGG-hBD-2 significant killing effect against Staphylococcus aureus ( ATCC 25923 ) , Pseudomonas aeruginosa (ATCC 27853) . Conclusion: We successfully constructed the eukaryotic expression vector of hBD-2 pCAGG-hBD-2, transfection pCAGG-hBD-2 mouse fibroblast cells highly expressed hBD-2, the supernatant of the transfected cells of Staphylococcus aureus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) have a role in the killing .

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