| Turkey herpesvirus(HVT)is a ubiquitous,nonpathogenicvirus of domestic turkeys,and it is classified as the third serotype within the Marek's disease virus(MDV)group of antigenically and genetically related lymphotropic avian herpesviruse.HVT is nonpathogenic in chickens,has been used extensively and effectively as vaccines against virulent MDV1.The genome regions containing these non-essential open reading frames(ORFs)often have been used for the insertion of foreign genes when constructing recombinant viruses HVT and combinations of HVT.Therefore,HVT is an ideal chicken live vaccine virus vector.This study established a TaqMan probe fluorescent quantitative PCR method to detect rHVT-VP2 DNA.Simultaneously,the aim of this study was to investigate the differences of virus replication efficiency between chick embryo fibroblasts and feathers,and the copies of virus in different tissue sample,and relationship between the copies of virus and antibody level after infected rHVT-VP2,laid the foundation for the development of Recombinant live vector vaccine vaccine and the evaluation of immunogenicity.Based on selected sequences of SORF1 of HVT in Gen Bank,one pairs of specific primers and one TaqMan fluorogenic probes were designed by invitrigen vector NTI.The fragments were cloned into T-VectorpMD19(Simple)and transformed into DH5a.Then,positive clone selection and identification,determination of nucleotide sequence.The screened positive recombinant plasmids were purified and by 10 times gradient dilution method as standard quantitative template to develop FQ-PCR.Simultaneously,by optimization of concentration of primer and TaqMan probe,annealing temperature,the optimal reaction system and reaction conditions were established.The results showed this method was specific for HVT,no positive signal was amplified from infectious bronchitis virus,infectious bursal disease virus,Marek's disease virus,Newcastle disease virus and no specific pathogenchick embryo fibroblast.The stability and reproducibility of the same recombinant plasmids were quantitatively detected one month interval,and the data from two experiments was not significant statistically.The coefficient of variation of inter-assay and intra-assay for HVT was 0.11%?2.13%.The datas show that the method has an excellent reproducibility and stability.The constructed T-SORF1 recombinant plasmid was diluted 10 times with double distilled water,and routine PCR and TaqMan probe FQ-PCR were used for detection.The resuts showed that TaqMan probe FQ-PCR had a higher sensitivity compared with regular PCR methods.Secondly,the replication efficiency of recombinant herpesvirus of turkey rHVT-VP2 and HVT FC126 in CEF and chicken feather follicle were quantitatively detected by optimized quantitative PCR.The results showed that the replication efficiency of recombinant herpesvirus of turkey rHVT-VP2 in vivo and in vitro was lower than that of HVT FC126.In different tissue sample of of Immunized Chicken,the feather follicle has the most number of virus,and the value of the virus in the feather follicle was quantitatively detected.It was found that the copies of the virus reached the peak at the 4?5th weeks after immunization.In order to determine the correlation between rHVT replication efficiency in chickens and the serum antibody responses to VP2 antigen,the average titers of rHVT-VP2(virus copies/milliliter)in each chicken from 2 to7 weeks of age were calculated,as well as those of the AGP antibodies to the VP2 antigen.There was a significant correlation between the rHVT-VP2 replication efficiency in chickens and the induced AGP antibody titers with both rHVT-VP2. |
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