| Defensin is a small molecμLe peptide with broad spectrum anti-bacterial, fungal, viral and other biological activity in vivo, which is an important component of the innate immune for organism. There are fourteen kinds of defensins in the chicken body which are all β-defensins according to the related report.The activity of chicken β-defensins13 (Gal-13), which was extracted from the intestines of chickens by acid extraction method, had been confirmed according to the research. But it was a tedious and time consuming extraction process, and its extraction volume was limited. Howere, there are no reports about Gal-13 which was obtained by the method of genetic engineering. In this paper, the gene of Gal-13 mature peptide was cloned and induced in E.coli BL21 and Pichia pastoris X-33 after the prokaryotic and the eukaryotic expression vector were successfully constructed. It laid the foundation for its further bioactivity research and application in the animal husbandry production.1. The amplification of Gal-13 gene and its induced expression in E.coli.The full sequence of Gal-13 cDNA consists of 312 base pairs, encoding 60 amino acids. And it is composed of 20 amino acids signal peptides,4 amino acids leader peptides and 36 amino acids mature peptides. The primer was designed by the Primer 5.software according to the sequence of Gal-13(Login ID:DQ 858328) which had been published in NCBI. The full length sequence of Gal-13 cDNA was amplified by RT-PCR technology, and the template was the total RNA of chicken lever. Then, it was analyzed and compared with its related sequences, and the phylogenetic tree was constructed by the method of Neighbor-Joining. In addition, new primers were designed to amplify the sequence of Gal-13 mature peptide after sequenced. And the restriction endonuclease site of BamHI and XhoI were introduced into the mature peptide gene sequence. Then, the recombinant plasmid pGEX-4T-1-Gal-13 was constructed after the target gene and the plasmid were double digested, respectively. And the recombinant plasmid was induced in E.coli BL21 after the transformation. The resμLt showed that Gal-13 was highly expressed in E.coli. And its best induced expression time was 5h, optimal inducer concentration was 0.2 mmol/L. The recombinant protein was showed at a level of 31ku according to the SDS-PAGE and Western-blot analysis. It showed that the sequence of Gal-13 mature peptide was successfully expressed in E.coli, and it was expressed mainly in the form of inclusion bodies, less soluble protein was in the supernatant soluble.1. The induced expression of Gal-13 mature peptide sequence in Pichia pastoris.The new primers which included the restriction endonuclease site of EcoR Ⅰ and Kpn Ⅰ were designed according to Gal-13 sequence to amplify the mature peptide gene sequence. Then, the recombinant plasmid pPICZαA-Gal-13 was built after the target gene and the plasmid were double digested, respectively. And the correct recombinant plasmid was linearized and transformed into Pichia Pastoris strain X-33 by electroporation. The positive recombinant strain was induced by methyl alcohol afterwards. And the expression supernatant was identified by Tricine-SDS-PAGE. The results showed that the target sequence was correctly inserted into the expression plasmid. In addition, the recombinant plasmid was successfully transformed into Pichia pastoris X-33. The multi-copy recombinant strains, which were Mut+ phenotype, were obtained after the screening and identification. And Gal-13 was secreted into the microbial supernatant. The expression quantity of Gal-13 achieved a very high value in the microbial supernatant at the time of 72h and the production showed at a level of 5.9ku according to the Tricine-SDS-PAGE, it was consistent with the expected result. Gal-13 had certain inhibition effects on Salmonella gallinarum(CVCC534) and Staphylococcus aureus(ATCC25923) according to the antibacterial test. |
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