Construction Of Goatpox Virus Gene Deleted Transfer Vector And Generation Of Gene Deleted Mutant

Goat and sheep pox diseases are serious infectious diseases caused by infection of capripoxvirus (CPV). In China, the attenuated living goatpox vaccine was used to prevent goat and sheep pox diseases, and there was little work developed on molecular virology of CPV.In this study, the attenuated goatpox virus vaccine strain (GTPV-TY) which was widely used in China, was regarded as the basis of the work. The new-style gene deleted goatpox vaccine was the final target. The following work were done in this study: (1)TK and P32 gene of GTPV-TY were cloned by PCR with primer pairs designed by information published on GENBANK; (2) The TK and P32 gene deleted transfer vectors were constructed, and they would be used in homologous recombinant; (3)LacZ gene was inserted into TK gene deleted transfer vector, the TK /LacZ+ /GTPV-TY mutant was screened.Thymidine kinase gene was one of the pox virus virulent genes, but it was dispensable for virus replication and infection. In this study, TK gene of GTPV-TY was amplified by PCR. TK gene was 534bp and encoded 178 amino acids. On the N terminal of TK, ATP motif was found. TK gene was conserved in CPV, the identity of GTPV-TY TK gene compared with TK gene in other CPV strains were more than 96%. Two homologous recombinant arms about 1 kb were amplified by PCR with primer pairs PRETK1/ PRETK2 and BACTK1/BACTK2 respectivly. The multiplied segments were inserted into pGEM-T easy vector; the inserted orientation was estimated by restriction enzyme. Then the two homologous recombinant arms were re-ligased as the transcript orientation in CPV genome, so the TK gene deleted transfer vector-pdTK was constructed, a 337bp DNA fragment in TK gene was deleted in pdTK. The ATP binding motif was also changed in pdTK, so that the TK activity would be absolutely deleted in the recombinant virus mutant. In the middle of the pdTK, SmaI restriction enzyme site was constructed, foreign gene could be inserted in this site.P32 gene of GTPV-TY was the homolog of Vaccinia virus (VV) H3L gene, P32 protein was one of the enveloped proteins of virion. P32 gene was amplified by PCR with primer pair designed based on CPV genome sequence published on GENBANK. P32 gene was 969bp and encoded 323 amino acids, the identity of GTPV-TY P32 gene compared with P32 gene in other CPV strains was more than 97%, showed that P32 gene was conserved in CPV as well as TK gene in CPV.P32 gene deleted transfer vector pdP32 was constucted with the methods refered to the construction of pdTK. A 525bp DNA fragment with P32 initiative code in P32 ORF was deleted, so that the recombinant virus mutant would not express P32 protein. The foreign genes could be inserted into the SmaIrestriction enzyme site as well as pdTK.In this study, a LacZ expression cassette was constructed. LacZ gene was cut from plasmid pcDNA3.1/His/LacZ by restriction enzyme. Pcmv promoter was amplified by PCR with primer pairs performed on plasmid pcDNA 5_TO. These fragments were inserted into pGEM-T easy vector, and then the recombinant plasmid pCMV-LacZ was constructed. The CMV-LacZ fragment was cut from pCMV-LacZ, and then it was inserted into the Smal site in the middle of pdTK. The homologous recombinant transfer vecter pdTK/LacZ was constructed. pdTK/LacZ was transfected fetal goat dermal cells, which was infected with GTPV-TY virus beforehand, using lepofectin transfection procedure. Virus culture was harvested when cytopathogenic effects were shown. The recombinant mutant TK-/LacZ+GTPV-TY was screened by blue plaque assay using X-gal after four cycles of plaque purification. The virions of mutant were identified by electron microscope, and the shapes of mutant virions are the same with the normal virions. The 0.4kb fragment was amplified by PCR with identified primer pair Id1/Id2 performed on DNA extracted from recombinant virus also showed that the stable recombinant virus was obtained.This study achieved goatpox virus TK gene deleted mutant, which would facilitate the further research on goatpox virus gene deleted vaccine. The double gene deleted vaccine could be constructed performed on P32 gene based on this study. The homologous recombinant plasmid pdTK and pdP32 could be used to contain foreign genes, in order to construct CPV living virus vector. This work facilited the study on CPV gene engineering vaccine and facilited the prevention of goat and sheep pox disease.