Construction And Bioactivity Analsis Of Recombinant Eukaryotic Plasmid For A Novel Bovine Lysozym Gene

Lysozyme, which was recognized as bacteriolytic agent having an ability to hydrolyze bacterial cell walls, is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears and egg white. It lyses certain bacteria by hydrolysing theβ-linkages between the muramic acid and N-acetylglucosamine of the mucopolysaccharides which are present in the bacterial cell wall. It has become one of the most intensively studied proteins. The action of Lysozyme on bacteria works cooperatively and synergistically with antibiotics, which has a very important practical value in medicine area. In present, the research of lysozyme develops greatly because of the development of the technology of biology and immunology. And it becomes very import that using the technology of DNA recombination to boost the antibacterial effect of lysozyme.There are about ten kinds of bovine lysozyme that are potentially produceded in neutrophil and macrophage in mammary gland, stomach and tracheal tissue, and secreted into saliva, tears fluid and milk which is an important food for human beings. In order to construct a novel lysozyme gene to improve its antibacterial activity and develop effective medicine to overcome the increasing infection of animals by antibiotic resistant bacteria, the experiment was carried out to reconstruct the eukaryotic expression vector for bovine milk lysozyme gene (Lys) by inserting theLys cDNA fragment into VR1020 plasmid. The VR1020 was digested by BamHⅠand BglⅡ, and then ligated with Lys cDNA digested wit hthe same double endoenzymes at 16℃overnight. The ligated products were used to transform E. coli DH5αcompetent cell, and the positive recombinant was screend out by inoculation of transformed cells in Lurea Bertani (LB) broth with kanamycin at 100μg/ml and shaked for 16 hours at 37℃. Bacterial cells were pelleted by centrifugation and plasmid DNA extracted following large-scale alkine lysis. Then the recombinant plasmid was digested by BamHⅠand BglⅡand plasmid PCR to identify the cloned Lys gene. Through DNA sequencing, it was proved that the Lys cDNA fragment was successfully inserted into the eukaryotic plasmid VR1020 in correct orientation.The ability of VRL to express Lys in vitro was assayed by RT-PCR in eukaryotic cells HEK293 that were transiently transfected with VRL wraped in JetPEI^TM Cationic polymer transfection reagent and chitosan nanoparticles (CNP). The result was found that the mRNA was detected in the the total RNA of HK293 cells transfected after 24, 48, 72h; It was also showed that the expression effect of the gene enwrapped by CNP was the same with or better than that of JetPEI^TM Cationic polymer, indicating CNP is practicable package to facilitate the plasmid transfectection of eukaryotic cells. The expression products in the supernatant of transfected cell medium manifested significant inhibitary effect to suppress the growth of Streptococcus pneumoniae and Staphylococcus aureus bacteria in vitro(P＜0.05), and this suggest that the recombinant eukaryotic plasmids of novel bovine lysozyme gene could be further developed into effective antibacterial reagent to control animal infection.