| According to the data concerned and with the aids of DNAstar software, the 250-460 amino acids of the glycoprotein B of BamHI-I3 domain of Marek's disease virus GA strain was selected as target fragment for tandem expression. With the help of Primer5.0 software two pairs of primers, Primer(U1) and Primer(U2), Primer(D1) and Primer(D2), were designed according to the sequence published in the GenBank. And an ATG GCG was added to the 5' tern of Primer(U1), a TAA was added to the 3' tern of Primer(D2) and a linker of nine amino acids was designeo at the 5' tern of Primer(D1) .Thus a complete ORF formed when upstream fragment gB(U) linkered with downstream fragment gB(L/D).The gB(U) and gB(L/D) were amplified respectively based on pairs of Primer(U1) and Primer(U2) , Primer(D1) and Primer(D2) by polymerase chain reaction (PCR). PCR products, gB(U) and gB(L/D) were individually cloned into pBluescriptSKII+ plasmid, pSKgB(U) and pSKgB(L/D) were constructed. Then the pSKgB(U) was digested with XbaI+BamHI and g(U) was cloned into pSKgB(L/D) , accordingly pSKgB(U/L/D) were constructed. Finally pSKgB(U/L/D) was digested with XbaI+PstI, and then gB(U/L/D) were cloned into pEFgpt12S, pEFMDgB(U/L/D) recombinant fowlpox virus transfer vector were constructed.The quantity of parental FPV used in transfection and the optimal titer of Mycophenolic Acid (MPA) in selectable culture were determined. The recombinant fowlpox virus transfer plasmid vector pEFMDgB(U/L/D) was cotransfected with fowlpox virus 282E4 strain at CEF by calcium phosphate-DNA coprecipitation method and the target linked fragment was inserted into ORF1 domain of FPV. Then the recombinant virus was selected with selectable culture containing MPA and was purified after 10 passages. Finally, Us specificity was identified positive on its infected CEF through Indirect Immunofluorescence Assay (IFA), this result shows that the recombinant fowlpox virus has successfully expressed the foreign genesand the tandem espressed products have antigenicity; Comparing the TCID50 of the recombinant fowlpox virus (rFPV-MDgB(U/L/D) ) with that of parental FPV, there was no obvious differences between them. The result shows that insertion of foreign genes into ORF1 domain of FPV doesn't affect the propagation and biological characteristics of this virus.The selected recombinant fowlpox virus tandem espressing the linked fragment gB(U/L/D) was cultivated for 5 passages in normal culture (2% MEM) and then the tilers of the recombinant in the selectable culture (2% MXHAT) and normal culture were tested and compared to the titer of parental FPV in normal culture. Three values were same on the whole, which show that the recombinant was purified and its replication was stable relatively. One-day old SPF chickens were vaccinated with rFPV-MDgB(U/L/D) + CVI988 (commercial vaccine) bivalent vaccine and CVI988(commercial vaccine) respectively. All chickens, experimental groups and the control group, were challenged with RB1B strain very virulent MDV at 7 days post vaccination and housed in isolation for 9 weeks. During this course, 2-3 chickens of every group were sampled 6 feathers of wings and blood to prepare serum and if chickens died they were examined for gross lesions of MD. At the end of the ninth week, all the survivors were killed and were examined for lymphoid tumors and other lesions of typical of MDV. The result of AGP assay of effect of vaccines on level of challenge virus shedding from feather follicles of chickens shows that both vaccines can inhibit replication of RB1B strain MDV in feather follicles of chickens. And AGP test of antibodies in blood from chickens challenged with RB1B strain shows that antibodies in both experimental groups gradually raised 2 weeks post vaccination. Finally, the morbidity of experimental groups was significant lower than the control group, but the morbidity of these two experimental groups has no significant differences. This result demonstrates that both vaccines can protect chickens from infected by RB1B strain MDV.
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