Cloning And Expression Of F, H And N Gene Of Canine Distemper Virus

Canine distemper caused by canine distemper virus (CDV) is a highly infectious, frequently lethal disease with high mortality in dogs and other carnivores. CDV is belong to RNA genus morbillivirus in the paramyxoviridae 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 have shown that two glycoproteins (Hemagglutinin H and Fusion F protein) of CDV are major target antigens for the host immune system. Nucleocapsid (N) protein is highly conserved immunogenicity protein. In addition, N protein contains T cell epitope. Hemagglutinin protein, Fusion protein and Nucleocapsid protein play an important role in immuno-prevention of canine distemper.This reseach is amied at cloning and expressing N?F and H gene of CDV and obtaining the easily purified expression products of N?F and H gene of CDV, it would help to study of gene vaccine and diagnostic reagent.1.Cloning and expression of CDV NcCDV RNA was extracted from CDV-Onderstepoort strain. The full length N gene was amplified by RT-PCR and the conserved gene of N (Nc) was amplified by PCR with two specific primers. This sequence was cloned into plasmid pGEM-Teasy. Both the DNA sequence of the cloned gene and amino acid sequence have identity of 99.7%, 99.3% and 100%, 100% with CDV Onderstepoort strain. The two Nc genes were cloned into GST fusion-expression plasmid pGEX-5x-3 and MBP fusion-expression plasmid pMAL-C2, respectively, to construct recombinant plasmid pGEX-5x-3-Nc2 and pMAL-C2- Ncl. The recombinant plasmid were verified by PCR and restriction endonuclease analysis. The recombinant plasmid pGEX-5x-3-NC2 and pMAL-C2- Ncl were transformed into the BL21(DE3)plys and DH5?respectively, and induced by IPTG. As a result, recombinant protein of pMAL-C2- Ncl was obtained, however, the pGEX-5x-3-NC2 recombinant protein could not expressed.2.Cloning and expession of CDV H geneThe full length H gene of CDV was amplified from CDV RNA by using RT-PCR with apair of primers, then this fragment was cloned into pGEM-Teasy. The DNA sequence of the cloned gene and the deduced amino acid sequence showed an identity of 98.8% and 98.2% with Onderstepoort strain, respectively. The recombinant expression plasmid pQE-32-H was constructed by using pQE-32 signed with 6 X His. The recombinant expression plasmid was verified with PCR and restriction endonuclease analysis before transformed into E.coli Ml5. The plasmid was induced with IPTG, however, the intrerest protein was not obtained.3.Cloning and expressin of CDV F geneThe full length F gene of CDV was amplified from CDV RNA by using RT-PCR with a pair of specific primers, then using the full length F gene as template, the deleted hydrophobic domains of F gene (dF) was amplified by overlap extension -PCR(OE-PCR)with four specific primers and this fragment was cloned into pGEM-Teasy and sequenced.Sequence analysis showed that the DNA sequence of cloned gene and the deduced amino acid sequence has identity of 99.1% and 98.4%,respectively.The dF fragment was cloned into pQE-30 to construct a recombinant expressing plasmid pQE-30- dF. The recombinant plasmid was verified by PCR and restriction endonuclease analysis, then it was transformed into E.coli strain Ml5 and induced by IPTG and obtained high level expression of aimed protein. The expression product dF gene was identified by SDS-PAGE and western-blotting, and found to be 47000 in size as a fusion protein. The protein was purified by Ni-NTA agarose and recovered. The purified product of dF protein was injected into the mice every two weeks and for four times. The antiserum was collected from the immunized mice and used for immunity fluorescence assay (IFA) with Vero monolayer infected by CDV. The positive staining was found and localized on the infected cells.4. Application of CDV F proteinAn indirect ELISA method for measuring antibody of canine distemper virus (CDV) was established with recombinant fusion protein (F) of CDV as antige...