| For the past 30 years, the vaccines against Marek's Disease (MD) have been developed effectively. At present monovalent vaccine and polyvalent vaccine are maily used against MD. The monovalent vaccines include serotype 3 (HVT), serotype 2 (SB-1) and attenuated serotype 1 ( CVI988). The polyvalent vaccines include bivalent vaccine (serotype 1 + serotype 2, serotype 1 + serotype 3 and serotype 2 + serotype 3) and trivalent vaccine. The forgoing succession of MD vaccines paralleled the continued evolution of field strains of serotype 1 Marek's disease virus (MDV) to greater virulence. The conventional vaccines are more difficult to control very virulent and vv+ MDV. For the past 10 years, by research on molecular biology of MDV, scientists have tried to develop more effective and more convenient recombinant vaccines. The genomes of several MDVs were completely sequenced, some of important genes of MDV have been identified and localized, and the progresses of research on structure and function of these gene have been going well, all of these promoted the development of recombinant MD vaccines. MDV-gB is the main protective antigen of MDV. rFPV-gB and rHVT-gB have showed good protection. But one target antigen is difficult to induce complete protective immunity. Apart from gB, scientists are looking for other target antigens which can induce protection. In glycoproteins of herpesvirus, experiments suggested that gD, gB, gI, gC, gE, and gG could induce different levels of protection. When these glycoproteins were used in mixture, significant protective synergism was observed. Glycoprotein I is an important one among glycoproteins in herpesvirus, it can provide protective synergism when it was used with other glycoproteins. In addition, gI homologue of herpesviruses is required to mediate efficient cell-to-cell spread, involved in virulence, and functions as a receptor for the Fcdomain of immunoglobulin G. Up to now, there are no reports about MDV gI in protective experiments. In this program, we focussed on MDV gI. First we constructed a recombinant fowlpox virus (rFPV) expression gI of MDV, then explored immunogenecity and stability of the rFPV, finally investigated its immune protection.According to gI sequences of MDV 648A strain, two primers were designed, and MDV gI gene was amplified by PCR. PCR products were identified by agarose gel electrophoresis. A DNA band of 1060 bp was obtained, which was corresponded to that of MDV gI gene, and purified from gel with Qiagen gel extract kit. The PCR products were ligated into plasmid pFG1175-1 and transformed into E.coli DH5 α. After screening, one postive clone was got. The clone DNA was extracted by Qiagen midi plasmid kit and sequenced by automatic sequencer. The sequence of inserted DNA was exactly same as that of MDV-648A-gI gene. The transferring plasmid carrying gI gene was named pFGgI1175-l. Chick embryo fibrobast (CEF) cells infected FPV were transfected with pFGI1175-l using lipofectin reagent. After 3 days of incubation, the recombinant FPV were identified by blue plaque assays and purified by 4 passages of singer plaque. By PCR, the recombinant FPV was confirmed of containing gI gene of MDV. 96 well cultures of CEF cells infected with parental FPV and recombinant FPV were washed in PBS and fixed with acetone. The fixed cells were then incubated with MDV gI monoclonal antibody for 30 min at 37℃ and washed extensively in PBS. Bound antibodies were detected with fluorescein-conjugated anti-mouse IgG. The results showed that positve staining were observed only in cells infected with the recombinant FPV, but not in cells infected with parental FPV. These indicated that the recombinant FPV expressed gI gene of MDV in CEF. The recombinant FPV expressing gI gene of MDV was named rFPV-MDV648gI.The recombinant virus can replicate stably after 20 passages when it was used to infect CEF. Two groups of one-day-old specific pathogen free (SPF) chickens kept in isolators were injected subcutaneous with 104 PFU of the recombinant FPV and the parental FPV... |
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