Studies On Characteristic And Transient Expression Of Antimicrobial Peptides In Mammary Glands

Cecropins are a family of antimicrobial peptides that were firstdiscovered in insects due to their bactericidal activity and since then they havebeen isolated from a variety of organisms. Tracheal antimicrobial peptide(TAP) is a member of the beta-defensin family of antibiotic peptides found inthe tracheal mucosa of the cow. It functions to protect the upper airway frominfection. Cecropins and bTAP have small molecular mass and broad-spectrum antimicrobial activity; they can kill a broad range of bacteria,enveloped viruses, and tumor cells, but they have no activation againstnormal eukaryotic cells. It is possible that antimicrobial peptides may becomea kind of new antimicrobial and antitumoral drug. The present study wasconducted to test whether bactericidally active cecropinB and bovine trachealantimicrobial peptide could be expressed in animal mammary gland to treatand prevent cow mastitis. For this purpose,the gene encoding cecropinB andthe gene encoding bovine tracheal antibacterial peptide were artificiallysynthesized by overlap extension PCR respectively. The codons of the geneswere optimized by the codons recognized preferentially by eukaryotic cellsand proper restriction enzyme sites were added to them. The genes wereligated to pMD18-T vector by TA clone method respectively. The clonescontaining the anticipated gene were then screened by PCR and the accuracyof their sequences were confirmed by DNA sequencing. In order to expresscecropinB and bovine tracheal antimicrobial peptide in animal mammaryglands,mammary-tissue-specific expression vector containing 5ˊflankingregulation sequence of goat β-casein and bovine growth hormonepolyadenylation signal was constructed based on eukaryotic expression vector...