Tissue Special Expression And Antibacterial Activity Assay Of Antimicrobial Peptide CAMA-syn

Recently the increasing resistance of bacterial to drug caused by the widely utility ofantibiotic has result in unexpected challenge to the therapy of bacterial inflectional disease.Antimicrobial peptides (AMP), a kind of peptide antibiotic with antibacterial activity, aredefense substance with anti-pathogen activity in animal and plant immune system, which playa role in natural immune. Combination of antibiotic and antimicrobial peptide is a novelstrategy in curingseverely bacterial infectious disease. And expression of antimicrobialpeptide in special tissue of body using transgenic technology will be an effective method incuring bacterial infection disease.CAMA-syn, a hybrid composed of N-terminal α-helical segment of Cecropin A (aminoacid1-8) and Magainin2(amino acid1-12), is a new artificial antimicrobial peptide withbroad-spectrum antibacterial properties and low cytotoxicity, which will lead the direction ofantimicrobial therapy development in the future.In this study, under the proposed strategy ofgene therapy, antimicrobial peptides CAMA-syn was highly expressed in specific tissues sothat the antimicrobial peptide could meet the infected bacterial extracellular and intracellularin "close contact", achieving the purpose of killing bacterial efficiently. This will not onlyovercome the shortcomings of not-easy reaching target of antimicrobial drugs, but alsomacrophages and lung epithelial cells can be sustained expression of antimicrobial peptides toprevent the infection of bacterial in treatment. It has not been reported that antibacterialability of CAMA-syn expressed in animal cells, especially in macrophages and lung epithelialcells. In order to increase the antibacterial ability of body and reduce the side effects ofantimicrobial peptides, it is feasible to express antimicrobial peptides CAMA-syn inmacrophages and lung epithelial cell to inhibit the activation of intracellular bacterial. Thepurpose of this study is to express antimicrobial peptide CAMA-syn in the target tissues andcells, and test the antibacterial potency in vitro and in vivo of macrophages and lung epithelialcells expressing CAMA-syn by eukaryotic and adenovirus expression vector. Following is themajor work that has been done:1. Detection of the o antimicrobial peptide expression and antibacterial activity in animalcells were carried out using plasmids transfection. Construction of a series of eukoryticexpression plasmid, including pMSP-CAMA-HA and pMSP-CAMA-GFP、 pSPC-CAMA-HA and pSPC-CAMA-GFP、 pCMV-CAMA-HA and pCMV-CAMA-GFP,consisted of CMV promoter, marcropaghes special promoter (MSP) and lung epithelial cellsspecial promoter (SPC), respectively, regulating the expression of antimicrobial peptideCAMA-syn fusion with signal peptide of INF γ on the N terminal and GFP or HA tag on theC terminal. Transfected bovine embryo fibroblast BEF, macrophages RAW264.7and lungepithelial cells A549, CAMA-GFP and CAMA-HA fusion protein were detected by GFP tag,immunofluorescent staining and western blotting technic. It was demonstrated that theartificial antimicrobial peptide CAMA-syn expression in animal cells have antibacterialactivity to most microgram, including Gram-positive bacteria Salmonella abortusovis andPasteurella anatis, and Gram-negative bacteria Staphylococcus hyicus and Streptococcus suis,in which cell lyses and cell-free supernatant were collected from bovine embryo fibroblastBEF, macrophages RAW264.7and lung epithelial cells A549that expressing CAMA-syn.2. Following recombination of CAMA-syn gene fragment into the adenovirus vector, weobtained the recombinant adenovirus particles with tissues specific expression of CAMA–synafter packaging and amplification in293cell line. The recombinant adenovirus could expressantimicrobial peptide CAMA-syn and GFP simultaneously by linking CAMA-syn gene withIRES and GFP sequence, and recombining these genes into adenovirus vector, in order toobserve and locate CAMA-syn feasibly. Meanwhile, CMV promoter, marcropaghes specialpromoter (MSP) and lung epithelial cells special promoter (SPC) were used to drive theexpression of CAMA-syn in special tissues, respectively. CAMA-syn gene was provedintegrating into recombinant adenovirus genome of original generation. Our results showedthat the test of virus titer, the number of physical particles and replicating viruses and otherindicators are qualified to carry out infection of cell and animal models under requirements,with high-purity concentrated adenovirus after several rounds of amplification.3. Afterexpression of antimicrobial peptides CAMA–syn by infection of animal cells withrecombinant adenovirus, the expression efficiency, antibacterial and anti-intracellularparasites activities were further certificated. Recombinant adenovirusAd-MSP-CAMA/GFPAd-SPC-CAMA/GFP and Ad-CMV-CAMA/GFP were used to infect macrophagesRAW264.7, lung epithelial cells A549and the control group human embryonic kidney cellsPhinex and wedetermined the optimal conditions of infection and the expression of theCAMA-syn by GFP labeling and RT-PCR. The results suggested that animal cells infected byrecombinant adenovirus possessed the function of antibacterial activity in the assay of colonyformation efficiency and bacterial growth curves, in which the cell lyses and cell-freesupernatant were collected from RAW264.7, A549and Phinex cells expressing CAMA-syn,respectively. Importantly, CAMA-syn expressed by recombinant adenovirus infection could inhibit the growth of intracellular parasites, such as salmonella and attenuated bovineMycobacterium tuberculosis BCG.4. CAMA-syn expression had therapeutic effect to the intracellular bacterial parasiteinfection in vivo, when recombinant adenovirus was used to targeted expression ofantimicrobial peptides CAMA-syn for gene therapy mold. Tracheal injection, tail Intravenousinjection and intraperitoneal injection were choosed to infect adenovirusAd-CMV-CAMA/GFP on animal model. Through observation and detection of GFP andantimicrobial peptides CAMA,-syn expression in the heart, liver, spleen, lung and kidneycells, as well as the activation of immune factors and local tissue inflammation, we foundtracheal injection is the best way to lungs infected with adenovirus gene therapy. Then, afterestablishing of intracellular bacterial parasite infection model in mouse lung by trachealinjection of BCG, recombinant adenovirus including d-MSP-CAMA/GFP、Ad-SPC-CAMA/GFP and Ad-CMV-CAMA/GFP were use to target expression ofCAMA-syn for gene therapy to infection animal, respectively. It was demonstrated thattissue-special expression of antimicrobial peptide CAMA-syn by adenovirus displayedtherapeutic effect on the intracellular bacterial parasite infection, although there was slightlydifferent in CAMA-syn expression and antibacterial activity.