Study On Expression Of Foreign Genes Inserted Into Locus Of "GTPV_gp024" In Goatpox Virus

Goatpox virus (Goatpox virus, GTPV) vaccine research of Goatpox (Goatpox, GTP) live poxvirus vector has becoming hot. Because of genome had a large number of nonessential regions which are useful in inserting large exogenous genes and with infection-specific,the GTPV can insert a variety of immunogenic genes, and become a good choice as vaccine vector. Now researches are mainly focused on thymidine kinase (TK) gene of GTPV for insertion site, however, the main challenge in construct of capripoxvirus vectors is identification of more non-essential regions.In this paper, the first template is attenuated vaccine strain AV41 of GTPV in china, partial gene of palmitylated EEV envelope protein in locus"GTPV_gp024"had been cloned and sequenced. Sequence analysis reveals that full length of the GTPV_gp024 gene is 1113bp, which can be translated as 371 amino acids. Identity of nucleotides sequences up to 99.8% relative to that of strain pellor of GTPV, and homology amino acid sequences is more than 50% to that of other related genes of Poxvirus. The study found that the function like p37 protein is similar to the F13L gene of poxvirus, its deletion did not affect replication and dissemination of the virus, and can be used as nonessential region to insert foreign genes.The primers of homologous arms had been designed for missing part of GTPV_gp024 gene, and for inserting of LacZ gene, vaccinia virus back to back promoter like P11-P7.5 and GPT gene expression cassette. A new transfer vector of GTPV had been achieved through homologous recombination and cotransfection to Lamb testes (LT) cells with its parental virus. There had blue plaques in three rounds of blue plaque purification after the X-gal staining. It proves that a new GTPV_gp024 recombinant virus has been obtained. Then, the length of homologous arms had been altered, LacZ gene replaced by EGFP-GPT report genes for another transfer vector.Recombinant virus had been obtained as above procedure did, pressure screened with GPT complementarily, investigated by fluorescence microscope, and target gene was amplified by PCR. Analysis data revealed that the recombinant virus is positive, there was not parental pollution with GTPV virus after fifth rounds screen of recombinant virus, however, TCID50 of the recombinant virus had decreased slightly.The conclusion shows that, deletion of the palmitylated EEV envelope protein and insertion of foreign genes into the locus of strain AV41 cannot interfere with replication of GTPV, and foreign genes can be allowed to express in the recombinant virus. This builds a basis for further study to GTPV as live vector for recombinant vaccine.