Cloning And Prokaryotic Expression Of Canine Distemper H Gene And Canine IL-6 Fusion Gene

Canine distemper virus(CDV)is responsible for Canine distemper,which is a highly virulent infectious disease.Canine distempe has a typical symptom-dual phase heat type,mainly to invade the nervous system,respiratory system and digestive system resulting in the inflammation of the upper respiratory tract,lung and non suppurative meningitis-myelitis with nearly 90% incidence rate,60%-80% mortality rate.What is more,Canine distempe could also lead to a secondary infection of other viruses and bacteria to bring about casualties,there nominated as "devastating infectious disease’’.At present,this disease distributes almost all over the world,as one of the most serious hazards of canine disease,resulting in huge economic losses.In recent years,with the ecological environment changes,virus evolution,CDV can infect animals from the families of canines,mustelidae,raccoon family,cat and panda,which could be a threat to the fur animal breeding industry.CDV genome could encode the nucleocapsid protein(N),thematrix membrane protein(M),phospho protein(P),protein(L),fusion protein(F)and blood coagulation protein(H).H is composed of 1947 ribonucleotides,only one open reading framework.Further more,H as a glycoprotein on membrane surface of CDV,is regarded as one of the major antigen to induce neutralizing antibodies,to identify and combine with receptors,to help make CDV F protein membrane and cell membrane fusion and invade host cells,which play an important role in anti CDV immunity.In addition,H contains at least one cytotoxic T lymphocyte(CTL)epitope to generate specific CTL activity and could be usd as a genetic engineering vaccine candidate.Interleukin-6(IL-6)is an important multifunctional cytokine,which can activate target genes and also could be used as a differentiation and growth factor for hematopoietic cells,T cells,B cells,endothelial cells and osteoclasts.IL-6 could enhan cecytotoxic activity of mononuclear cells and natural killer cells(NK)and as a kind of natural immune enhancer,have an important regulatory function in the immune response.The commercialization of human IL-6has been used in the treatment of allergic diseases,infectious diseases and cancer diseases.As IL-6 does not produce immunogenicity,autoimmune diseases doe not occur,which overcomes the disadvantages of traditional immune adjuvants.Therefore,the IL-6 gene cloned into the vector,to construct a safe,efficient and economical new immune adjuvant,which has a promising prospect.A specific pair of primers were designed according to the published gene sequence of canine distemper virus and Vero cells infected by CDV were collected.H gene fragments(1125bp)were amplified by RT-PCR and obtained.The amplified products were connected to pMD18-T vector,transformed,enzyme digested and PCR analysis,and the recombinant plasmid was sequenced.The results showed that the homology between the obtained gene sequence and H gene in GenBanK was 100%.Extracted lymphocytes from healthy canine blood,isolated canine IL-6,constructed plasmid pMD-IL-6,enzyme digestion,PCR analysis,recombinant plasmid sequencing,the results showed that IL-6 gene had a homology of 98% with the one in Genebank.The recombinant plasmid of pMD-H and pMD-IL-6 were enzyme digested at the same time,then constructed plasmid pMD-H-IL-6 and enzyme digestion.PCR analysis and plasmid sequencing were needed too.The result showed a 1758 bp gene length.More over,the recombinant plasmid was transformed into the host bacteria,bacteria liquid were collected,SDS-PAGE and Western-blot analysis were conducted.The results indicated that expression of canine distemper virus proteins were stable and also could be recognized by the positive serum.The molecular size of target protein was about 69.5KD.