Construction Of Recombinant Herpesviruses Of Turkey Containing Glycoprotein B And Interleukin-2 Of Marek's Disease Virus And Its Immune Protection Effect

Marek's disease (MD) is a highly contagious, malignant, MDV-induced lymphomatous disease of chickens. Marek's disease has been controlled efficiently since the early 1970s by vaccination with live, non-oncogenic strains of MDV and/or herpesvirus of turkeys (HVT), but the virulence of MDV is increasing, which reduce the protective effect of the vaccine. In this report, a recombinant virus recombinated glycoprotein B of MDV and interleukin-2 was constructed and its ability to protect against mortality and development of lymphoid tumors caused by virulent MDV in SPF chickens was also studied.According to GenBank data and analysis of DNAstar software, the amino acids of the glycoprotein B of MDV and interleukin-2 gene were selected as target fragment for fused expression. By means of Primer5.0 software, two pairs of primers, Primer(G₁) and Primer(G₂), Primer(I₁) and Primer(I₂), were designed according to the sequence published in the GenBank. And a BglII was added to the 5' tern of Primer(G₁), a HindIII was added to the 5' tern of Primer(G₂) and a Hindlll as designed at the 5' tern of Primer(I₁), a SalI was added to the 5' tern of Primer(I₂) . Thus a fused gene combined the glycoprotein B of MDV and interleukin-2 gene was formed. The gB and IL-2 were amplified respectively based on pairs of Primer(G₁) and PrimerG₂), Primer(I₁) and Primer(I₂) by polymerase chain reaction (PCR). The PCR products, gB and IL-2, were individually cloned into pEGFP-C1 plasmid, and pC1-gB-IL was constructed. Then the pC1-gB-IL was digested with NaeI+EcoO109I and gene experession cassette fragment was cloned into pTK2B, accordingly pT-C-gB-IL were constructed.The quantity of parental HVT used in transfection was determined. The recombinant Herpesviruses of Turkey transfer plasmid vector pT-C-gB-IL was cotransfected with Herpesviruses of Turkey at CEF by calcium phosphate-DNA coprecipitation method and the target fragment was inserted into TK domain of HVT. Then the recombinant virus was selected with flouresence and was purified after 10 passages. Finally, its specificity was identified positively in infected CEF by Western-blotting, the result showed that therecombinant HVT successfully expressed the foreign genes and the expressing products have antigenicity; There was no obvious difference between the TCID50 of the recombinant HVT (rHVT-gB-IL) and that of parent HVT. The propagation and the biological characteristics of the recombinant HVT (rHVT-gB-IL) did not be affected because of the insertion of foreign genes into TK domain of HVT.One-day old SPF chickens were vaccinated with rHVT-gB-IL recombined vaccine and HVT (commercial vaccine) respectively. The experimental groups and the control group were all challenged with RBIB strain very virulent MDV (vvMDV) at 7 days post vaccination. During 9 weeks detection period, 6 feathers of wing samples and blood from 2-3 chickens of every experimental and control group were collected from died chickens. At the end of the ninth week, all the survivors were killed and examined for lymphoid tumors and other typical lesions of MD. AGP test showed that the serum antibodies of chickens challenged with RBIB strain were gradual induction in two experimental groups at the third week after vaccination. The AGP assay of feather follicles of chickens showed that both rHVT-gB-IL recombined vaccine and HVT vaccine can inhibit the replication of RBIB strain MDV in feather follicles of chickens. Although the morbidity of these two experimental groups challenged with RBIB strain MDV has no significant difference, the morbidity of experimental groups was significantly lower than that of the control group. The result demonstrated that two vaccines, rHVT-gB-IL recombined vaccine and HVT vaccine, effectively protected chickens from being infected by RBIB strain MDV.