| Coccidiosis, which is caused by Eimeria spp all over the world, is a very severe parasitosis to poultry. Chemotherapy has been restrained to control coccidiosis because of appearance of drug-resistant strains and drugs residues. The development of vaccine against coccidiosis becomes more and more important in recent years. The live vaccine is mainly used now, but it has potential pathogenicity. The recombinant vaccine becomes more important, especially the live vector vaccines. Fowlpox Virus(FPV), which has strict specifity and safety, is an important animal expression vector followed by pox virus and has been used widely to produce gene engineering technology live vector. In this study, E.tenella SO7 gene and Rhomboid protein family relative gene were used as candidate genes of recombinant vaccine and inserted into the downstream of complex promoter of virus vector, Fowlpox recombinant virus vectors was successfully constructed. plasmid of Fowlpox recombinant virus vectors and Fowlpox virus were co-transfected into chicken embryo fibroblast (CEF) and screened by BrdU drug. It was identified by RT-PCR and protein printing, proving that three strains of Fowlpox E.tenella recombinant virus were obtained. Seven-day chicken were vaccinated with the three strains Fowlpox recombinant virus, demonstrating that recombinant protein had immunogenicity and could induced strong immune response and immune protection ability. The details were as following:Amplification, cloning and sequencing of interested gene The SO7 gene, which was 651bp, was amplified from total RNA of purified yolk sac by RT-PCR, The amplification products of SO7 gene by us was 97.7% homology to that of SO7 gene in GenBank after comparing analysis, and the correlated gene ORF of Rhomboid protein family (Rhomboid in short) with length 774 was successfully obtained.Construction, transfection and identification of Fowlpox recombinant virus The SO7 gene and Rhomboid gene were inserted into the downstream from complex promoter ATI-P7.5×20 of PUTA2 expression vector respectively, producing recombinant virus transfer vector PUTA-SO7, PUTA-Rhomboid and PUTA-SO7-Rhomboid. Three vectors and 282E4 strain of Fowlpox virus were transfected into chicken embryo fibroblast (CEF) by lipid ,resulting to three strains of recombinant Fowlpox virus vUTA-SO7,vUTA-Rhomboid, vUTA-SO7-Rhomboid which can express E.tenella antigen gene correctly by BrdU drug and proved by RT-PCR,SDS-PAGE and protein printing.Protection effect of Recombinant Fowlpox virus After CEF amplified incubation of three recombinant virus, inoculate two times in seven-day and fourteen-day chicken with three doses, on the twenty-first day after birth, inoculate vaccinated chicken with 5 X 104 E.tenella yolk sac /chicken, on the eighth day, weighed up and anatomized the chicken, determined relative increased weight ratio.cecum pathological changes grade(OPG). The results indicated that, the main difference of testing group and control group after inoculated was the expelling quantity of yolk sac in aspect of expelling from yolk sac. Chicken in testing group expelled less yolk sac than control group every day, and expelling time was shorter. The gained weight of chicken vaccinated recombinant virus was different with the chickens in control group significantly (P<0.05) ,the highest relative gained weight was 84%. The gained weight in the group vaccinated with vUTA-SO7-Rhomboid was better than the group with vUTA-S07 and vUTA-Rhomboid. Decreasing ratio of cecum pathological changes grade(OPG) in the three groups had significant difference with the control group (PO.05) ,the highest protection ratio 54% was in the group with vUTA-SO7-Rhomboid. |
