Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,...Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.展开更多
文摘Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.
