135 位点表达ORFV F1L 基因的重组山羊痘病毒的构建.

[ Objective] The ORFV FIL gene and marker genes were inserted at the 135 gene locus of goat virus by Cre/loxP system to provide a strategies for the design and development of a vaccine to prevent the infection of Orf virus in clinic. [ Method] Based on pDonor vector, about 500 bp fragments of the region sequence on both sides of 135 gene coding region were used as upper and lower recombination homologous arms. The expression cassette named pGTPV( 135 )-ORFV-FlL, which also included 135 gene, maker gene gpt and EGFP, early and late promoter, and loxp locus. PCR identification and sequencing were performed on the positive clone. The correct sequence bacterial fluid was inoculated into ampicillin LB liquid medium for overnight culture. The endotoxin was removed and the plasmid was extracted and identified by double enzyme digestion. The immortalized goat testicular cells were infected with low virulent goatpox virus at 2 hours, the recombinant plasmid was transfected into immortalized goat testicular cells by liposome method to observe the cytopathic effect and the expression of the marker gene EGFP. The virus solution was harvested 48 hours later, repeated freezing and thawing for 3 times, and then passed to 3 generations blindly. The integration of the target gene was identified by PCR. The ORFV positive serum was used as the first antibody, and the donkey anti goat IgG was used as the second antibody. The recombinant virus was identified by western blot analysis of the expression of the target gene. [Result] The recombinant plasmid of goat pox virus expressing Fl L gene of ORFV was identified by PCR and sequenced. The result of comparison with the target sequence is correct, which indicated that the recombinant plasmid was successfully constructed. The concentration of the recombinant plasmid was determined after endotoxin removal and extraction, and the results were correct. After the identified recombinant plasmid was transfected into immortalized goat testicular cells, green fluorescence was observed 48 hours later, and Fl L gene was integrated into the recombinant virus by PCR identification. The western blot analysis showed that the foreign gene Fl L inserted by the recombinant goat pox virus was successfully expressed. [ Conclusion]The FIL gene ofORFV was inserted into the 135 gene site of the attenuated strain of goatpox virus, and the recombinant virus was obtained in immortalized goat testicle cells. The target gene can be stably expressed, and a method for constructing a recombinant goatpox virus with the 135 gene site inserted into the FIL gene of goatpox virus was successfully established. [ABSTRACT FROM AUTHOR]