Expression Of Antimicrobial Peptides In Mammary Glands And Their Bacteriostasis Activity On The Main Pathogenic Bacteria Of Mastitis

Mastitis is a highly prevalent disease in dairy cow,and one of the most importantdiseases affecting the world dairy industry.It places a heavy economic burden on milkproducers all over the world.Mastitis is an inflammation of the mammary gland,usually caused by microbial infection.Mastitis-causing pathogens include bacteria andnon-bacterial pathogens,like mycoplasms,fungi,yeasts,and chlamydia.Staphyloco-ccus aureus,Streptococcus uberis and Escherichia coli are the main pathogensresponsible for the bulk of bovine mastitis cases.Practices currently used to control mastitis are mainly based on therapy andprevention with antibiotics.Systematic application of antibiotic therapy cures anumber of infections and plays a pivotal role in prevention of new infection during thissusceptible period.The problem is,however,that there are antibiotic residues andappearance of antibiotic-resistant bacterial strains when using antibiotics.Chinesetraditional medicine has played an important role in the treatment of mastitis.Its effectis similar to,or to some extent,lower than antibiotics.But the dosage is huge and thespending is high.Related vaccines to mastitis,such as vaccines against streptococcusand staphylococcus,are under investigation.The novel approaches of mastitis controlare still challenges in mastitis therapy and prevention.Antimicrobial peptides (AMPs) have been isolated from many differentorganisms during the last decades.These peptides exhibit broad-spectrum activityagainst a wide range of microorganisms including Gram-positive and Gram-negativebacteria,protozoa,yeast,fungi and viruses.Antimicrobial peptides represent a novelclass of antibiotics with potential antimicrobial activity against a range of infectiousbacteria including multi-resistant isolates.But they have no effect on normaleukaryotic cells.Bovine lactoferricin (LfcinB) is a peptide released from bovinelactoferrin upon gastric pepsin cleavage,LfcinB consists of 25 amino acid residues(17–41) from the N-terminal region of lactoferrin.Tracheal antimicrobial peptide(TAP) is a member of the beta-defensin family of antibiotic peptides found in thetracheal mucosa of cow.Its functions is to protect the upper airway from infection.cecropin B was shown to disrupt the bacterial outer membrane.CecropinA -magaininⅡhybrid peptides(CA-MA) is composed of cecropinA (1-8) and magaininⅡ(1-12),having an improved antimicrobial activity with low hemolytic effect.We are attempting to develop new strategies to prevent mastitis by enabling thecells of the mammary gland to produce novel antibacterial proteins.This wouldprovide a safe,effective,no residues and easy to use method for enhancing mastitisresistance.As a step toward mastitis prevention,an gene transfer technique would beused to enable mammary cells to synthesize and secrete antibacterial peptides.For thispurpose , series of tissue specific expression vector in mammary gland wereconstructed with 5′-regulatory region of the goat β-casein gene.The genes encoding lactoferricinB,cecropinB,cecropinA-magsininⅡ and bovinetracheal antibacterial peptide were artificially synthesized by overlap extension PCRmethod.Proper restriction enzyme sites were designed and signal peptides were addedto their 5′ ends.The genes were cloned into pMD18-T vector and the clonescontaining the interested genes were then identified by PCR and restriction enzyme,the accuracy of their sequences were confirmed by DNA sequencing.In order to express antimicrobial peptides in animal mammary glands ,mammary-tissue-specific expression vector containing 5ˊ flanking regulationsequence of goat β-casein and bovine growth hormone polyadenylation signal wasconstructed based on eukaryotic expression vector pIRES-neo . Then themammary-tissue-specific expression vector containing the antimicrobial peptide genesdissolved in physiologic saline were injected directly into the lactating mammaryglands of goats.The milk samples after injection were checked by Tricine-SDS-PAGEand inhibition zone assay . The results of these tests showed that themammary-tissue-specific expression vector driven by the goat beta-casein genepromoter could efficiently direct the expression of antibacterial peptides in goat milk,the expression of antibacterial proteins last for 2-3 days.All the milk samples injectedwith different vectors exhibited bacteriostatic activity to different kinds of bacterialpathogens.Two of the mammary-tissue-specific expression vectors containing lactoferricinBand bTAP gene were respectively injected into the lactating mammary glands of cowsby mammary ducts . The milk samples after injection were checked byTricine-SDS-PAGE,RP-HPLC and inhibition zone assay.The results of these testsshowed that the mammary-tissue-specific expression vector driven by the goatbeta-casein gene promoter could efficiently direct the expression of antibacterialpeptide in cow milk also and the milk samples showed bacteriostatic activity tobacterial pathogens..In order to demonstrate the application potency of these antimicrobial peptides inprevention of mastitis,we constructed the recombinant retroviral vector including goatβ-casein promoter (bCP),antimicrobial peptides genes and bovine growth hormone(BGH) polyA based on retroviral vector pLNCL.The recombinant retroviral vectorwas transfected into PA317 cells for package by liposome transfection,after 2-weeksof G418 selection,G418-resistant PA317 colonies were obtained and amplified.Thesupernatant of cell culture was harvested and used to infect NIH3T3 cells to measurethe viral titer of recombinant retrovirus.Results showed that the highest titer of viralsupernatant was 1×104CFU/ml.The supernatant of cell culture was injected into themammary glands of pregnant cows in the midanaphase for 2-3 times,the result ofTricine-SDS-PAGE and inhibition zone assay showed that antimicrobial peptidesgenes had been expressed continuously in milk of the post partum cow,the expressionof antibacterial proteins persisted for 30-45 days.Inhibition zone assay suggested thatthe milk samples also showed bacteriostatic activity.In order to provide some evidence for the application of the new methods inmastitis,the antibacterial activity of antimicrobial peptides expressed in milk wastested against main pathogenic bacterium isolated and identified from milk sample ofclinical mastitis.The results indicated that antibacterial proteins expressed in milkcould obviously inhibit the main pathogenic bacteria of mastitis.In this paper,antimicrobial peptides can be expressed effectively in mammaryglands with 5ˊ flanking regulation sequence of goat β-casein.The products in milkcould inhibit Staphylococcus aureus,Streptococcus uberis and Escherichia coli whichwere mastitis main pathogenic bacteria.The results indicated that they were candidateantibacterial proteins for mastitis prevention and the expression of these antibacterialproteins in bovine mammary glands may be used as a new way for the prevention tothe dairy cow mastitis.