Construction Of Recombinant Adenovirus Ec-cLYZ Gene And HLTF Gene And Its Evaluation Of Antibacterial

Compared with mammals,fish have a more vulnerable acquired immune system,innate immunity plays an extremely important role against pathogen invasion.The Epinephelus coioides c type Lysozyme(Ec-c LYZ)not only can inhibit gram-positive bacteria but also inhibit gram-negative bacteria,which is one of the important nonspecific immune factors in the body.Human Lactoferrin(h LTF)has many biological function such as broad spectrum of antibacterial and immune,antioxidation,anti-inflammatory,antiviral and anti-cancer,which is a iron glycoprotein exist in milk and rich in amino acids.Because the h LTF has strong adhesion with ferric ion,which is indispensable to all bacterial growth,h LTF can prevent bacterial to use iron ion and have the effect of antibacterial sterilization.On the other hand,h LTF is a kind of protease which can degrade some bacterial virulence and reduce pathogenic microbes.This study constructed recombinant adenovirus that expressed c LYZ gene and h LTF gene and evaluated double genes synergy bacteriostatic effect.Based on the gene sequences of c LYZ and h LTF in Gen Bank,specific primers were designed.Then c LYZ and h LTF were amplified from head kidney tissues in groupers and somatic cells in human-beings.Target fragments combined with vector p MD-19 T,named p MD-c LYZ and p MD-h LTF,respectively.After restriction enzyme digestion and sequencing,the vectors of above-mentioned and plasmid p DC315-MCS-EGFP were digested by double restriction enzyme.The adenovirus plasmid p DC315-c LYZ ? p DC315-h LTF ? p DC315-c LYZ-h LTF ? empty plasmid p DC315-MCS-EGFP and framework plasmid were transfected into HEK 293 cell with Lipofiter TM,which were constructed successfully.The recombinant adenovirus that infected HEK293 cells caused cytopathic effect.Replication of recombinant adenovirus and the expression of foreign protein in cell were measured by RT-PCR and western blotting,respectively.After purification,the virus titer of recombinant adenovirus was determined by TCID50,and the obtained positive recombinant adenovirus were named Ad-c LYZ?Ad-h LTF?Ad-c LYZ-h LTF and Ad-GFP.Total protein extraction of transfected cells was performed and protein concentration was determined.Filter paper method was used to evaluate the inhibitory effect of proteinextraction solution on tested strains.Minimal inhibitory concentration(MIC)of targeted protein was detected and its antibacterial effect was compared with that of antibiotics.The results of RT-PCR showed that recombinant adenovirus with target gene were successfully transferred into HEK293 cells.Targeted proteins of packaged Ad-c LYZ ? Ad-h LTF and Ad-c LYZ-h LTF viruses were found to be expressed in HEK293 cell by western blotting.Plenty of varial particles were amplified in cells,indicating that the packaged viruses have a good infectious ability.After purification,the virus titer was determined as 109.54 TCID50/m L ? 109.25 TCID50/m L ? 109.87TCID50/m L and 109.16 TCID50/m L,respectively.High expression of foreigh protein has a stable infective ability and its viral titer meet the requirement for the follow-up study.After total protein extraction,the protein concentrations of Ad-c LYZ?Ad-h LTF and Ad-c LYZ-h LTF were measured as 2.481 mg/m L?2.675 mg/m L and 3.228 mg/m L,respectively.Sensitivity tests showed that five kinds of pathogen ware sensitive to c LYZ?h LTF and dual gene protein and antibacterial effect was enhanced by dual gene protein.MIC of Streptococcus agalactiae?Staphylococcus aureus?Staphylococcus epidermidis?Sreptococcus dysgalactiae and escherichia coli were determined as 37.5?g/m L?37.5?g/m L?37.5 ?g/m L?75 ?g/m L and 150 ?g/m L.Antibacterial effects of targeted protein at MIC are the same as or even better than common antibiotics.This study is the first time that combining broad-spectrum antibacterial effect of grouper c type lysozyme with bacteriostatic effect of human lactoferrin and recombinant adenovirus vector system which is able to express exogenous gene efficiently and steadily.The experimental results showed the target protein had good antibacterial activity to test strains.This research provided both theoretical and experimental basis for biological prevention and control of clinical bacterial disease.