Identification Of Orf Virus Isolated From Nong An Area And Expression And Purificaiton Of Its Immunogenic Proteins

Orf disease, also known as Orf, is a highly contagious zoonotic infectious diseasecaused by Orf virus(ORFV) primally affecting sheep, goats and other small ruminants,occasionally infecting other wild ruminants such as camel, deer, oxen, and so on.Thelamb is the most susceptibility animal. ORFV belongs to the parapoxvirus genus ofpoxviridae family, it is highly epitheliotropic. Clinically, the formation of papules,pustules, ulcerate and scabs, around the lip, nose, breasts, four limbs, tail and otherparts of the skin are the characteristic features of orf infection mainly causingproliferative lesions of the skin and mucous membranes. The incidence of this diseaseis very high. And the disease is often caused in groups. The existing survey datashows that the incidence of the disease is nearly50%, but the incidence of sensitivesheep is close to90%. Therefore, once the disease occurs, it causes considerableimpacts on the economic income of farms, and affects the welfare of farmers.And the occurrence and prevalence of this disease have been reported in recentyears around the world. It has become one of the common disease in the flock ofsheep, and a serious threat to the healthy development of the sheep industry. In viewof the cost caused by this disease on the sheep industry, the disease has graduallyattracted the attention of people. In this study, a suspected ORFV infection case wasanalyzed, which came from a sheep farmer in Nong an area in September2011. AnOrf virus isolate, named as Orf virus Jilin-Nongan(ORFV JL-NA isolate), wasobtained from scab materials of3-month-old lamb with clinical sore mouth symptomby passaging in primary ovine fetal turbinate (OFTu) cells. The virus was identified asOrf virus based on morphology, physicochemical property and PCR. On this basis, theOrf059(F1L) and Orf011(B2L) gene was amplified by PCR using primers designed.and these two genes are the immunogen genes of the virus isolate. Then, prokaryoticexpression vetor pGEX-4T-1was used to build the recombinant plasmid ofpGEX-4T-1-Orf011(B2L), and prokaryotic expression vetor pET28a was used tobuild the recombinant plasmid of pET28a-Orf059(F1L). The expected size of the target bands approximately exist in67KD and39KD positions detected bySDS-PAGE and Western-blotting. And the results showed that the recombinantplasmids were successfully expressed in E.coli. Further, the target proteins werepurified using affinity chromatography, and the purified proteins exited intended beltsby SDS-PAGE electrophoresis and Western-blotting, which showed that targetproteins were successfully purified. The concentration of the target proteins were1.147mg/mL and1.777mg/mL respectively detected by BCA. All the resultssuggested that this study could not only provide potential immunogenic proteins forthe preparation of monoclonal antibodies, but also lay the foundation for thedevelopment of protein function, genetic engineering vaccine, clinical diagnosis andso on.