Cloning And Expression Of Canine Distemper Virus H Protein Gene Fragment In E.coli

Canine distemper caused by canine distemper virus(CDV) is a highly infectious, frequently lethaldisease with high mortality in dogs and other carnivores. CDV is belong to RNA genus morbillivirus in theparamyxoviridae family that has envelop, a single-stranded, negative sense gnome of approximately 16kb.The genome contains six non-overlapping gene regions, organized 3'-N-P-M-F-H-L-5′.Many studies haveshown that two glycoproteins (Hemagglutinin H and Fusion F protein) of CDV are major target antigensfor the host immune system. Nucleocapsid (N) protein is highly conserved immunogenicity protein. Inaddition, N protein contains T cell epitope. Hemagglutinin protein, Fusion protein and Nucleocapsidprotein play an important role in immuno-prevention of canine distemper.One pair of primers had been designed and synthesized based on the Haemagglutinin(H)protein geneof the vaccine strain of canine distemper virus(CDV). The templates were produced from the reversetranscription reaction, total RNA was isolated from the Vero cells infected with CDV vaccine strain. The549bp fragments of H gene were amplified by by polymerase chain reaction (PCR). The purified cDNAfragment was inserted into pMD18-T vector.The recombinant plasmid was identified by restrictionendonuclease analysis, PCR and DNA sequencing. The results suggest the autoploid of sequence is 100%with the vaccine strainIn the Genbank. The expression vector pGEX-4T-1 and The recombinant plasmiddigested by EcoRI and Sal I respectively.The target gene was subcloned into vector pGEX-4T-1.Positiveclones named as pGEX-H with interest gene were identified by restriction endonuclease analysis andPCR. Then the positive recombinant plasmid was transformed into E.coli RosettaTM for H expression.Theinterest gene was induced to express in E. coli by 1.0m mol/L IPTG at 37℃..The culture liquid of bacteriacontaining pGEX-H was collected at different time and was subsequently examined by SDS-PAGE..Resultsshowed that the structural protein H gene of CDV could express successfully in E.coli. The expressionproduct was identified by SDS-PAGE and found to be 46kDa as expected and confirmed by Westernblotting with CDV-vaecine strain antiserum. The results revealed that the expressed fusion protein in vitrohad the critical antigenitic epitopes of H gene of CDV. The amount of target protein in inducing productswas over 27% of the total bacteria protein.This reseach is armed at cloning and expressing H gene of CDV and obtaining the easily purifiedexpression products of H gene of CDV, it would help to study of gene vaccine and diagnostic reagent.