| A serotype1of field recombination Marek’s disease virus (MDV) strain GX0.101which integrated with the long terminal repeat (LTR) of reticuloendotheliosis virus(REV) was isolated from chickens that suffered from tumour in Guangxi provinceofsouth china by our lab in2001. It is a very virulent virus. It was constructedintobacterial artificial chromosome (BAC) system and carried out a series of researchbased on it. Both of the Meq genes of GX0101(GX0101Δmeq) have been deletedusing this system and proved that GX0101Δmeq can be used as MDV vaccine toprotect chicken and its protective effect is better than CVI988. During this research,we used the GX0101Δmeq strian as vector to express the H9N2-HA of Avianinfluenza virus. Different recombination viruses were constructed and studied on theirbiologic activity both in vitro and in vivo.1Preparation of H9N2-HA protein polyclonal antibody and construction ofH9N2-HA eukaryotic expression plasmidIn this study, different recombination viruses expressing H9N2-HAusing GX0101ΔMeq as vector were constructed. In order to verify the expression ofHA protein in recombinant viruses infected CEF cells, polyclonal antibody anti H9N2avian influenza HA protein was prepared from mice The IFA proves that the serumcan be used as HA protein polyclonal antibody to detect the expression of HA. Inorder to construct recombination MDV expressing H9N2-HA using GX0101ΔMeq asvector, we need to construct H9N2-HA HA eukaryotic expression plasmid firstly. TheHA-ORF was cloned into the eukaryotic expression vector pcDNA3.1(-) thatcontained CMV promoter. Then the HA eukaryotic expression plasmid wastranscripted into CEF cells to verify the expression of HA. The result indicated thatHA protein was expressed in eukaryotic expression plasmid under CMV promoter detected by IFA using the anti-H9N2-HA chicken serum. Therefore, the HAeukaryotic expression plasmid constructed in this research can successfully expressedthe HA gene, and can be used for the follow-up study that integrated into GX0101ΔMeq.2Construction of recombination virus rGX-CMV-HA using GX0101ΔMeq asvector with Red/ET recombinant system and Flp/FRT recombinant systemThere are mainly two methods that are commonly used to construct recombinationMDV viruses. One is homologous recombination in eukaryotes (Darteil et al.,1995).The other method is based on the bacterial artificial chromosome (BAC) system in aprokaryotic cell using the Red/ET recombinant system. Extracts from recombinantplasmids are transfected into eukaryotes to rescue the recombinant viruses. And thereare also many different selectable strategies based on the Red/ET recombinant system.The research of this part is mainly to solve problem of the method for constructing therecombinant MDV. Our research has tried different methods for the construction ofrecombinant MDV, these methods include the classic homologous recombination ineukaryotic cell, RedE/T recombination in BAC system (Application ofCounter-Selection BAC Modification Kit), using RedE/T recombination and Flp/FRTrecombination in BAC at the same time. Finally, after nearly one and a half years ofstudy on the method for construction of the recombinant MDV, we established a veryconvenient way to construct MDV.Firstly Kanrresistant gene expression cassette was inserted before the HA geneexpression cassette. the HA expression cassette with resistant gene as exogenousfragment was inserted into GX0101ΔMeq vector, using RedE/T recombination systemin Escherichia coli EL250. Then the Kanrresistant gene was deleted fromrecombinant plasmids under the induction of L-arabinose using Flp/FRTrecombination system. The recombination plasmid prGX-CMV-HA extracted from theverified E.coli EL250that the Kanrresistant gene have completely deleted, wastransfected into CEF cells to rescue recombination virus, and verification of theexpression of HA gene in recombinant virus HA through IFA. The results showed that,we obtained a recombinant virus rGX-CMV-HA through the two recombination system, and the HA gene was expressed in rGX-CMV-HA infected CEF cellssuccessfully.3Comparative transcriptional activity of five promoters in BAC-cloned MDVfor the expression of the hemagglutinin gene of H9N2avian influenza virusDuring the construction of rMDV, the activity of promoters to transcribe foreigngenes is one of the major factors that can affect protective efficacy. To investigate thetranscription activity and efficacy of five different promoters, the advantage of anexisting rMDV BAC infectious clone that had been previously constructed was usedto construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from alow pathogenic Avian Influenza virus (LPAIV) H9N2strain was inserted into the US2region under five selected promoters. These five promoters included three MDVendogenous promoters (the promoter for the gB gene and a bi-directional promoter inboth directions for pp38(ppp38) and1.8kb RNA transcripts (p1.8kb)), and twoexogenous promoters (CMV and SV40). Among these five promoters, the CMVpromoter demonstrated the highest activity, followed by p1.8kb and SV40, which hada similar transcriptional activity level. Two of the MDV endogenous promotersshowed much lower transcriptional activities, particularly the promoter ppp38, whichhad the lowest activity. Although the entire study was performed in vitro, the dataobtained provided useful criteria for the selection of appropriate promoters during theconstruction of rMDV vectors. Thus, evaluation of gene transcription and subsequentprotein expression under different promoters in animal experiments is valuable. Thevivo experiment proved that the recombination viruses rGX-CMV-HA, rGX-SV40-HA andrGX-p1.8kb-HA were without any protective efficacy on SPF chicken. While the recombinationvirus rGX-pPP38-HA provided50%protection against the challenge with H9N2and25%chickens in the group of rGX-gB-HA were protected.4Transcriptional activity comparison of different sites in recombinant Marek’sdisease virus for the expression of the H9N2avian influenza virus hemagglutiningeneTo compare the transcriptional activity of different sites of rMDV, an H9N2avian influenza virus hemagglutinin gene (AIV-H9N2-HA) expression cassette that used thebi-directional promoter of serotype1MDV (MDV1) in the1.8kb RNA transcriptdirection (p1.8kb) as a promoter was inserted into4different regions of MDV usingthe bacterial artificial chromosome (BAC) vector based on the previous research. Theinsertion regions included3of its own sites (US2, US10and one of Meq genes) in theMDV genome and a foreign site (gpt gene) in the BAC vector. Quantitative PCR andenzyme-linked immunosorbent assay (ELISA) were used to analyze and compare theH9N2-HA expression levels of these different rMDVs both at the mRNA level and atthe protein level. The results indicated that among the four tested insertion regions,the HA expression cassette in the US2region demonstrated the highest activity,followed by that in the Meq region, which was almost equal to that of US10. Further,the expression cassette had the lowest activity in the foreign region gpt gene. Theabove data could be useful for choosing proper recombinant insertion regions in theconstruction of rMDV to express different foreign genes, and it is a prerequisite fordeveloping effective MDV-vectored recombinant vaccines. The vivo experiment provedthat the recombination viruses rGX-US2-HA, rGX-US10-HA and rGX-gpt-HA were without anyprotective efficacy on SPF chicken. While the recombination virus rGX-meq-HA provided60%protection against the challenge with H9N2. |
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