Construction And Identification Of Recombinant ORFV F1L Gene Adenovirus

Contagious ecthyma, commonly known as Sore mouth disease, is an acute, highly contagious, zoonotic disease caused by orf virus. The virus mainly attacks the lambs aged3-6months and causes blisters, pustules, papules and verrucous crusts in the infected skin and mucosa on animals’ lips, gingival, tongue, oral cavity mucosa, skin around the nostrils, breast and base of tail. The disease has very high incidence and infectious. Usually more than50%of the sheep get infected and this incidence rates up to90%in the sensitive sheep. However, the mortality is rare and usually about1%～25%, sometimes up to50%in the condition of infected following by Bacillus necrophorus or other pathogens. At present, there is no effective drug for prevention and treatment of contagious ecthyma, conventional attenuated vaccines and inactivated vaccines play a role in the prevention of the disease. As the rapid development of the sheep industry, how to reduce vaccine production cost, improve immune protection efficiency, simplified immunization program in the prevention and treatment process of contagious ecthyma, becomes a problem calls for immediate solution. It has major significance to develop a safe, efficient, economic, practical novel vaccine against contagious pustular diseases. We can design the ORFV gene engineering vaccine to deal with this problem. This topic mainly by orf virus envelope protein gene (F1L) construction of a recombinant adenovirus vector, for further carrying out the effective prevention of contagious ecthyma provides candidate vaccine.In order to get a secure and efficient new generation vaccine, this subject carried on preparation of DNA vaccine against ORFV by using the good immunogenicity gene, ORFV059(F1L). A pair of primer was designed according to the complete F1L gene sequence based on the genomic sequence published on GenBank to amplify contagious ecthyma virus ORFV/JLSY04strains. The F1L gene was obtained by cloning and sequencing, and the length was1011bp. Sequence results were analyzed using BLAST and a phylogenetic tree was constructed using N-J method of MEGA. The phylogenetic studies of the F1L genes showed that JLSY04was closest to the Jilin isolate and furthest to the OV/Torino isolate. Sequence analyses of the nucleotide showed that the JLSY04isolate shared relationship with other ORFV isolates from different regions (97.2%～99.7%), and shared the highest homology with ORFV Jilin isolate, which is99.7%. These results show that there is little difference in the different regions strains of FIL gene.F1L gene was amplified by PCR, subcloned into the vector pShuttle2and the shuttle plasmid F1L-p was constructed. Then the plasmid was digested with PI-Sce I/I-Ceu I to connect with the Adeno-X Viral-DNA digested with the same enzyme and electrotransformated into DH10B cell. After the bacteria liquid PCR, positive clones screened, digestion and DNA sequencing, the recombinant ORFV F1L gene adenovirus plasmid Adeno-F1L was succefully made up. Recombinant adenovirus vector virus the plasmid Adeno-FIL was transfected the293cells, packing reorganization and amplified to get orf Virus FIL recombinant adenovirus. The recombinant adenovirus was purificated with the ion exchange technique, identified by polymerase chain reaction and the virus titer is5X106PFU/ml. The PCR detection results showed that the orf Virus FIL recombinant adenovirus was constructed successfully. These results lay the foundation for the immune characteristic clarification of Orf virus, also to provide the effective prevention of the disease of sheep contagious pustular candidate vaccine.