Construct Recombinant Adenovirus Co-express Triple Epitope Gene Of The Main Cow Mastitis Pathogens And Epinephelus Coioides C Type Lysozyme Gene And Antibacterial Evaluation

Dairy cow mastitis is an important disease that seriously affects the production of dairy cows.This disease is mainly caused by the three pathogens,Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae cross or co-infection,plague the healthy development of world dairy industry.At this stage,antibiotics is mainly relied on as a prevention and treatment.However,frequent use antibiotics can lead to bacterial resistance and drug residues,it is not a long-term dependency approach.Select the main pathogens causing dairy mastitis specific proteins,Ebps and Clf A of Staphylococcus aureus,Omp A and Omp C of Escherichia coli,SIP and PGK of Streptococcus agalactiae.By analysising of three common pathogens in B cells,Th cells,CTL cells dominant epitopes,predicting and evaluating its antigenicity,we designed and synthesised the triple recombinant epitopes sequence（TRE）.The Epinephelus coioides c type Lysozyme（Ec-c LYZ）sequence was obtained on Gene Bank and subjected to mammalian cell-preferred codon optimization.Both were co-expressed in the Ad Max system of adenovirus through high efficient self shearing peptide Thoseaasigna virus 2A（T2A）.Application of adenovirus Ad-Max system,connected the recombinant epitopes sequence,T2 A sequence and Ec-c LYZ sequence to p DC315-MCS-EGFP,and used Lipo Fiter^TM liposome transfection method HEK293 cells were co-transfected with p BHGlox E1,3Cre realization of homologous restructuring.Acquisition recombinant adenovirus Ad-EGFP、Ad-TRE-EGFP 、 Ad-c LYZ-EGFP and Ad-TRE-T2A-c LYZ-EGFP.In this study,Ad Max recombinant adenovirus system co-express Triple Epitope gene and Ec-c LYZ gene was first time constructed.It protect the cow from body’s own immune system and lysozyme protein two aspects.SD rats were immunized every two weeks during pregnancy,four immunizations were conducted.The results of ELISA showed that Ad-TRE-EGFP and Ad-TRE-T2A-c LYZ-EGFP groups content of antibody in the serum were significantly higher than PBS,Ad-EGFP and Ad-c LYZ-EGFP groups（P < 0.05）.The amount of specific antibodies against Staphylococcus aureus,Escherichia coli and Streptococcus agalactiae in peripheral blood of rats increased with time,and one week after the fourth immunization the content of antibodies in serum reached the highest level and significantly higher than pre-immune level of antibodies（P < 0.01）.After the fourth immunization the T lymphocyte typing test experiment showed that the positive rate of CD3⁺CD⁴+ and CD3⁺CD8⁺ cells in the Ad-TRE-EGFP and Ad-TRE-T2A-c LYZ-EGFP groups were significantly higher than that in the PBS,Ad-EGFP and Ad-c LYZ-EGFP groups（P < 0.05）.100 u L of 105cfu/m L of the three kinds of bacterial fluid were respectively introduced into the fourth breast of the three treatments rats corresponding to each of the immunized groups by using a glass catheter.The specimens were dissected and observed by histological section.The results showed that the protective rates of the vaccines in the immunized groups were 63% of Ad-TRE-EGFP group,70% of Ad-c LYZ-EGFP group,93% of Ad-TRE-T2A-c LYZ-EGFP group,and 96% of Ad-TRE-EGFP+Ad-c LYZ-EGFP group and protected SD rats’ breast tissues were not obvious pathological changes.The above results showed that the recombinant adenovirus constructed in this study could affect the immunization of immunized rats.Significantly improve the level of serum antibody for further exploring the effect of recombinant adenovirus in the immune system and the functions of co-expression proteins provide theoretical supports.