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| Cloning of PBD1 Gene and Its Expression in Escherichia coli |
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Abstract Porcine (Sus scrofa) β-defensin 1 (PBD1) is a cationic antimicrobial peptide with 3 pairs of disulfide bonds , and has very strong antibacterial activity, and thus could be a good candidate as a bactericidal agent for pigs. Two pairs of specific primers (P1/P2 and P3/P4) were designed and synthesized according to the nucleotide sequence of the full porcine β-defensin 1 (PBD1) gene published in GenBank (No. NM213838). The fragment containing the full PBD1 gene was amplified by P1/P2 and reverse transcription polymerase chain reaction (RT-PCR) from total RNA extracted from porcine tongue tissue samples, PCR product was cloned into pMD18-T vector for sequencing. The result revealed that the nucleotide sequence of PBD1 gene was consisted of 310 bp in length. The PBD1 gene of mature protein was amplified using primers P3/P4. A prokaryotic plasmid of pET28a-SUMO-PBD1 was obtained by subcloning the encoding region of the mature peptide of PBD1 gene into pET28a-SUMO vector and transformed Escherichia coli Rosetta (DE3) competent cell. The expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot test after optimizing the various expression conditions such as concentration of isopropy-β-D-thiogalactoside (IPTG) and its cultured time and temperature. Fusion protein SUMO-PBD1 was presented as a form of soluble and its expression was highest when it was induced by addition of IPTG to a final concentration of 0.7 mmol/L for 6 h at 20 ℃. The recombinant PBD1(about 4.3 kD) was successfully obtained by small ubiquitin-like modifier (SUMO) protease digestion and purified. This study will lay a foundation for further study of the antimicrobial activity of defensins.
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Received: 01 September 2017
Published: 25 March 2018
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