Molecular analysis and recombinant expression of bovine neutrophil β-defensin 12 and its antimicrobial activity.
To analyze molecular characteristics and antimicrobial activity of bovine neutrophil β-defensin 12 (BNBD12), Pro-BNBD12 gene was amplified from Chinese Holstein cow using reverse transcription polymerase chain reaction. Based on the sequence of mature BNBD12 and codon preference in E. coli, a modified mature BNBD12 cDNA was synthesized, cloned into pET32a (+) vector and expressed in E. coli BL21 as a 26 kD fusion protein after isopropyl β-D-1-thiogalactopyranoside induction. The expressed mature BNBD12 accounted for 21.4% of total protein. 0.05 mg/ml purified BNBD12 had antimicrobial activity against both E. coli and S. aureus in vitro assayed by agar diffusion method. Electron microscopy found that BNBD12 treatment of E. coli and S. aureus could induce cell content leakage. Taking together, BNBD12 protein was successfully expressed in E. coli and it has antimicrobial activity against both Gram-positive and negative bacteria and potentials in control of mastitis clinically.
Wu
Jianming
J
Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, No. 159-1, Industry North Road, 250100, Jinan, Shandong Province, People's Republic of China.
Wang
Changfa
C
He
Hongbin
H
Hu
Guixue
G
Yang
Hongjun
H
Gao
Yundong
Y
Zhong
Jifeng
J
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D052978:Disk Diffusion Antimicrobial Tests; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D005822:Genetic Vectors / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011387:Prokaryotic Cells / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism* / Q000494:pharmacology*; D020539:Sequence Analysis, Protein; D013211:Staphylococcus aureus / Q000187:drug effects; D023083:beta-Defensins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*
D000890:Anti-Infective Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D023083:beta-Defensins; C406410:bovine neutrophil beta-defensin 12