Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.
Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.
Tanaka
Tetsuya
T
Graduate School of Agriculture, Hokkaido University, Sapporo, Japan. tetsuya@anim.agr.hokudai.ac.jp
Nakamura
Ichiro
I
Lee
Nai-Yuan
NY
Kumura
Haruto
H
Shimazaki
Kei-ichi
K
D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation / Q000187:drug effects; D007781:Lactoferrin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011525:Prototheca / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000890:Anti-Infective Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014415:Tunicamycin; D007781:Lactoferrin