Construction Of Recombinant Adenovirus Co-Express SAL-2 And HLYZ Gene And Its Evaluation Of Antibacterial

Virolysin is one of the cell wall hydrolases produced during the late infection of phage,which achieve the objective of cracking bacteria through hydrolysis of connection between the host cell wall components.SAL-2 is a kind of virolysin produced by phage SAP–2 of staphylococcus aureus?S.aureus?which belongs to podoviridae family,it can remove the outside biofilms effectively and has a broad bacteriostatic spectrum of S.aureus,S.epidermidis and S.pyosepticus.Human lysozyme?hLYZ?which also named N-acetyl acid hydrolysis enzyme is a part of the body's immune defense system,it cracks bacterias by drolyzing the?-1,4-glycosidic bond between NAM and NAG of bacterial cell walls.hLYZ has obvious bacteriostatic action to main pathogenic bacteria of dairy cow mastitis such as S.aureus,S.pneumonia and E.coli,it also can strengthen the body's immunity and becoming the research hotspotin recent years gradually.Adenovirus vector?Adv?is one of the most widely application prospect of transgenic carriers for its high degree,high efficiency,the advantage of not integrate into the host cell chromosome and low pathogenic.SAL-2 and hLYZ gene were amplified from S.aureus phage and somatic cells in human milk respectively,the target gene was cloned into pShuttle-IRES-hrGFP-1 for the recombinant shuttle vector.Screened by kanamycin selection,bacterial PCR,double enzyme digestion and sequencing,the right recombinant shuttle vector and pShuttle-IRES-hrGFP-1 were linearized by PmeI,then homologous recombinant with pAdEasy-1 in BJ5183 cells.Recombinant was screened by kanamycin and Pac I digestion to confirm,recombinant adenovirus vector with target gene were named Ad-SAL,Ad-LYZ,Ad-SAL-LYZ and Ad-GFP respectively.The recombinant adenovirus linearized by PmeI were transfered to HEK293 cells,packed to achieve recombinant adenovirus,harvest recombinant adenovirus can infect HEK293 cells and lead to CPE.Expression of recombinant adenovirus in HEK293 cells was detected by RT-PCR,using CsCl density gradient centrifugation to purify recombinant adenovirus and detect its titer by TCID₅₀,positive recombinants were named Ad-SAL,Ad-LYZ,Ad-SAL-LYZ and Ad-GFP respectively.Total protein were extracted from HEK293 cells after transfection with recombinant adenovirus,then detected the protein concentration and its bacteriostatic effect to experimental strains through paper disk method.Comparing the bacteriostasis effect of target protein at minimum inhibiting concentration?MIC?and antibiotics after detection of MIC.RT-PCR showed that recombinant adenovirus with target gene was transfered into AD293 cells successfully,Western blotting anlysis showed that the SAL-2 gene and hLYZ gene was successfully expressed in the recombinant adenovirus,fluorosence microscope showed that packed Ad-GFP possesses high infectious activity and formed viral particles in AD293 cells,viral particles were purified by Cs Cl density gradient centrifugation and their titer were detected as 10⁸.17.17 TCID₅₀/mL,10⁸.50.50 TCID₅₀/mL,10⁸.83.83 TCID₅₀/mL and 10^7.60TCID₅₀/mL,which indicated that replicate-deficiency recombinant adenovirus with target gene was constructed successfully,and reached the titer requirement for experiment in vitro.The concentration of protein extracted from HEK293 cells which transfected with recombinant adenovirus were 2.221 mg/m L?1.935 mg/mL?3.144 mg/m L respectively.All experimental strains except E.coli are sensitive to target protein and bacteriostatic effect of co-expressed protein is enhanced.MICs of co-expressed protein to experimental strains are37.5?g/m L?75?g/mL?150?g/m L?150?g/m L?300?g/m L respectively,the bacteriostatic effect of target protein at MIC can reach even better than common antibiotics.This study used the broad-spectrum antibacterial effect on S.aureus,the effective bacteriostasis characteristics of hLYZ and the advantage that will not damage animal cells,and adenovirus vector system is highly efficient and stable expression of foreign genes in eukaryotic cells,successful constructed the recombinant adenovirus co-express SAL-2 gene and hLYZ gene.Constructed adenovirus can express in HEK293 cells effectly and expressed protein showed a good antibacterial activity to experimental strains,provides new thoughts and theoretical and experimental basis for prnvention and control of clinical bacterial diseases.