Production of human defensin 5 recombinant protein and regulation of the gene expression
| Autor: | Kung-Hao Hsu, 許恭豪 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 Human defensin 5 (HD5) exhibits high antimicrobial activity against a broad spectrum of pathogenic agents. HD5 has been reported to be involved in innate immunity and host defense. In this study, HD5 cDNA was amplified by polymerase chain reaction (PCR) using human lung large cancer cell H661 cDNA as template to produce the bioactive HD5 recombinant protein. The 96-bp DNA fragment encoding mature HD5 peptide (amino acid 63-94) was subcloned into the pGAPZαA expression vector and transfected into P. pastoris X-33 expression host by electroporation. The recombinant HD5 (rHD5) crude extract from transfected P. pastoris showed antimicrobial activities against Salmonella typhimurium, Staphylococcus aureus and pathogenic E. coli. However, rHD5 did not inhibit the growth of lactic acid bacteria such as Lactobacillus bulgaricus, Bifidobacterium bifidum, and B. longum. These results indicated that the rHD5 expressed in P. pastoris selectively inhibited the growth of specific bacteria. To investigate the mechanism of DEX effect on HD5 expression in H661 cells, real-time PCR assay was used to determine steady states of HD5 mRNA. HD5 mRNA was rapidly down-regulated by dexamethasone (DEX) or a-amanitin treatment in H661 cells. These results imply that HD5 mRNA is extremely unstable and the half-life of HD5 mRNA is estimated to be less than 2 min in H661 cells. The down-regulation of HD5 mRNA in DEX-treated cells may be mediated by the inhibition in transcription and the decrease in stability of HD5 mRNA. In addition, to elucidate the involvement of untranslated regions (UTR) on stability of HD5 mRNA, four expression vectors were constructed by inserting Full-length HD5, 5′UTR-HD5 coding region, HD5 coding region-3′UTR or HD5 coding region, respectively. The expression vectors were transfected into H661 and H520 cells, respectively. These expressions of HD5 mRNA were determined by reverse transcription-PCR (RT-PCR) assays. The results showed that there was no difference in the expression of HD5 mRNA between Full-length and HD5 cording region plasmid transfected cells. In contrast, the levels of HD5 mRNA were significantly decreased in the deletion of 5′UTR and 3′UTR plasmid transfected cells. These results implied that the deletion of 5′UTR or 3′UTR (the post-transcriptional mechanism) may change HD5 mRNA stability in H520 cells. To elucidate the promoter activity of promoter region of the HD5 gene, a 280 bp and a 580 bp fragment of HD5 promoter regions were cloned into pGL3-Basic. Plasmids were transfected into H661 cells and the promoter activity were detected by analysis of luciferase activity. Luciferase promoter-based reporter assays suggested that promoter activity of this 580 bp sequences was higher than that of 210 bp sequences (p< 0.05). These results revealed a positive-regulatory region located between nucleotides −210 and −580 that may be responsible for HD5 transactivation in H661 cells. In conclusion, 5′UTR or 3′UTR was involved in HD5 mRNA stability in H520 cells, and a positive-regulatory region located between nucleotides −210 and −580 that may be responsible for HD5 transactivation in H661 cells. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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