Construction Of Two Recombinant Herpesviruses Of Turkey RHVT-gB1/EGFP And RHVT-EGFP

The objective of this paper is to construct recombinant Herpesviruses of Turkey .On designing construction strategy, the research focuses on the following. To obtain the strictly non-essential DNA fragment of HVT, on the basis of the sequence data of the Us region of HVT genome DNA, a pair of specific primers were designed and synthesized. Using the UVT infected CEF total DNA as template, a DNA fragment of 2014 bp was amplified by using PCR technique. The DNA fragment was then cloned into the pGEM-T easy vector and a recombinant plasmid designated as pTus 10 was obtained. The result of sequencing the cloned DNA fragment indicated that the nucleic acid sequence of the amplified 2014 bp fragment is highly homologous to the published sequence. of the corresponding fragment within the HVT US region. The non-essential fragment is composed of the ORFs of US 1 and Us2 genes as well as partial sequence of SORF3 .The cloned plasmid pTus 10 was to be used for construction of transferring plasmid vectors. To construct the gene expression cassette vector, a 1.8kb DNA fragment associated with the major antigenic epitope coding region of MDV g13 gene was inserted into the multi-cloning sites ofpEGFP-Cl and a recombinant plasmid named pGgBl was generated. The plasmid was then digested with the Hind!! I and the cutting ends were blunted and ligated again giving rise up a plasmid with Hind!!! site deleted. Deletion of the HindlJl site allows that the inserted DNA fragment of MDV gB gene is in the same ORF with the FGFP gene .The plasmid without Hind!!! site was named pGH-gB 1 which could provide the element of gene expression cassette for conveniently construction of related recombinant HVT. Based on the constructed non-essential fragment plasmid pTus 10 and the eukaryotic gene expression cassette vector pGH-g13 1, an isolated gene expression cassette fragment of about 3.8Kb was inserted into the BgilI site of HVT USIO gene through blunting ligation and a recombinant plasmid of about 8.8Kb designated as pTuGH-gBl was generated. Taken the advantage of the features of restriction sites in the plasmid, the 1.8kb DNA fragment of MDV gB gene was removed by digestion with B gill and BamHl and the left vector fragment was ligated generating another transferring plasmid vector of about 7kb named pTuGFP. The constructed transferring plasmid pTuGH-gB 1 was transfected into secondary CEFs several times by using the transfection methods of Calcium phosphate and liposome 2 ABSTRACT respectively. In liposomal transfections, transfection conditions including plasmid extraction method, the amount of liposome used, the amount of diluted vaccine viruses inoculated into secondary CEF and the incubation time after inoculation were tested. In screening recombinant viruses, the different batches of transfected CEFs with green florescent light signal were screened by using the methods of limiting dilution and Metal chain picking plaque respectively. The constructed transferring plasmid pTuGFP was also transfected into the secondary CEFs by using the optimal transfection and screening methods. The results demonstrated that Calcium phosphate transfection method was of low efficacy, that preparing plasmid with extraction Kit, adding 25u1 liposome in transfection, inoculating O.5ml diluted vaccine viruses and incubating cells 8 hours after inoculation were the optimal transfection conditions for liposome transfection method, a...