Construction Of Recombination Marek’s Disease Viruses(MDVs) Expressing H5N2-HA

Marek’s disease(MD) is a highly contagious disease virus of chickens,which has the common lymphoproliferative and immunosuppressive characteristics.MDV is divided into 3 serotypes, MDV which can cause tumors belong to MDV-1, naturally without tumors belonging to serotype 2 MDV, herpesvirus of turkeys(HVT) belong to serotype 3.A serotype 1 of field recombination MDV strain GX0101 which integrated with the long terminal repeat(LTR) of reticuloendotheliosis virus(REV) was isolated from chickens that suffered from tumour in Guangxi province of south china by our lab. In order to understand the effect of insertion on GX0101,we build GX0101 into a bacterial artificial chromosome(BAC). on the basis of cloning,we knock out REV- LTR to build a reassortant virus GX0101 Δ LTR, proved by animal, the insertion of REV- LTR resulted in a significant enhancement of the level of communication competence. Two Meq gene of GX0101(GX0101Δmeq) have been deleted in this system and proved that GX0101Δmeq can be used as MDV vaccine to protect chicken and its protective effect is better than CVI988. In this study, we successfully constructed a recombinant strain of the virus, which was based on the bacterial artificial chromosome system which was constructed from GX0101 expressing HA gene of the H5N2.1. Preparation of H5N2-HA protein polyclonal antibody and construction of H5N2-HA eukaryotic expression plasmidIn order to verify the expression of HA protein in the recombination virus expressing H5N2-HA using GX0101ΔMeq as vector infected CEF cells, polyclonal antibody anti H5N2 avian influenza HA protein was prepared from mice. The complete sequence of HA gene was obtained from H5N2 avian influenza virus JX534549 strains from NCBI, thenwhich was synthesized by the Biological Engineering(Shanghai). and it was divided into two sections which were inserted into the prokaryotic expression system of PET-32 a respectively to construct prokaryotic expression plasmid of segmental HA protein. Then the two plasmids were transformed into expression E.coli strain BL21. Through inducing the expression BL21 by IPTG, we found that only a HA protein can be expressed, and the other one was no expression. The HA protein was used as antigen to inoculate Balb/C mice. And the serum from the mice inoculated 3 times was detected weather it can be used as a polyclonal antibody against HA protein. This mice serum was used to detect CEF cells,which was transfected by the infectious clone of H5N2—HA by IFA. The IFA results showed that the serum after 50 times diluted can detect HA protein in H5N2 infected cells, which proves that the serum can be used as HA protein polyclonal antibody to detect the expression of HA in constructed recombination MDVs.In order to construct recombination MDV expressing H5N2-HA using GX0101ΔMeq as vector, we need to construct H5N2-HA HA eukaryotic expression plasmid firstly. The HA-ORF was cloned into the eukaryotic expression vector pc DNA3.1(-) that contained CMV promoter. Then the HA eukaryotic expression plasmid was transcripted into CEF cells to verify the expression of HA. The result indicated that HA protein was expressed in eukaryotic expression plasmid under CMV promoter detected by IFA using the anti-H5N2-HA chicken serum. Therefore, the HA eukaryotic expression plasmid constructed in this research can successfully expressed the HA gene, and can be used for the follow-up study that integrated into GX0101 Δ Meq.2. Construction of recombination virus r GX-CMV-HA using GX0101ΔMeq as vector with Red/ET recombinant system and Flp/FRT recombinant systemFirstly Kanrresistant gene expression cassette was inserted before the HA gene expression cassette. the HA expression cassette with resistant gene as exogenous fragment was inserted into GX0101ΔMeq vector, using Red E/T recombination system in Escherichia coli EL250. Then the Kanrresistant gene was deleted from recombinant plasmids under the induction of L-arabinose using Flp/FRT recombination system. The recombination plasmid pr GX-CMV-HA extracted from the verified E.coli EL250 that the Kanrresistant gene have completely deleted, was transfected into CEF cells to rescue recombination virus, andverification of the expression of HA gene in recombinant virus HA through IFA. The results showed that, we obtained a recombinant virus r GX-CMV-HA through the two recombination system, and the HA gene was expressed in r GX-CMV-HA infected CEF cells successfully.