Construction, Identification And Expression Of Recombinant Baculovirus Of M, F And H Genes Of Canine Distemper Virus

In recent years, virus-like particles(VLPs) were concerned as a kind of the vaccine with the good safety and immunogenicity in the virus subunit vaccine research. The researches has shown that the paramyxovirus matrix protein(M) is the key factor for the formation and budding of the VLPs. VLPs had the similar size of virus particle, could be formed based on M co-expression with the surface glycoprotein(F, H). The VLPs show similar antigenicity as virus. Therefore, M, F and H proteins with the native structure are the prerequisites for the assembly of canine distemper virus VLPs.Based on baculovirus-insect cell expression system, we have constructed recombinant baculovirus expressing M, F and H proteins of canine distemper virus and prepared polyclonal antibodies of mouse anti-M, F, H protein and rabbit anti-H protein. We identified the target protein of M, F and H in the insect cells infected with recombinant baculovirus respectively by polyclonal antibodies. Our studies laid the material foundation for the assembly of canine distemper virus VLPs.The main research contents are as follows: 1. Prokaryotic expression of M, F, H genes of canine distemper virus and preparation ofpolyclonal antibodiesTaking canine distemper virus lesser panda attenuated strain(CDV-LP) as the study object, the research designed the primers for the major epitope region of M, F and H proteins by amplifying the CDV M, F, H gene segment of the size of 1005 bp, 1050 bp and 1104 bp with the major epitope region coding sequence except transmembrane region and signal peptide sequence with the method of RT-PCR(Reverse Transcription-Polymerase Chain Reaction), cloned to prokaryotic expression vector of pET-30a(+), constructed the recombinant plasmid of pET-30a(+)-M, pET-30a(+)-F and pET-30a(+)-H, and transformed the expression plasmids into Escherichia coli BL21(DE3) strain. With 4 hours induction expression in 0.4 mM IPTG at 37℃, the SDS-PAGE result shows that the molecular weight of expressed M, F and H proteins were identified as 43 ku, 40 ku and 46 ku, which are consistent with the expected value. Recombinant proteins mainly were expressed in the form of inclusion body and they can be recognized with dog anti-CDV polyclonal antibody by Western bolt. Purified recombinant M, F and H proteins by Ni-NTA affinity chromatography were immunized BALB/c mice and rabbit by subcutaneous injection. Blood were collected and produced the polyclonal antibodies of mouse anti-M, F, H protein and rabbit anti-H protein after three times’ immunization by three weeks intervals. 2. Construction, identification and expression of recombinant baculovirus of M, F and Hproteins of canine distemper virusTo express the native structure proteins of M, F and H of canine distemper virus, the M, F, and H ORF of CDV LP were cloned into pFastBac?1 vector and then transformed into competent E.coli cells DH10Bac? after sequencing, acquired shuttle plasmids of rBacmid-M, rBacmid-F and rBacmid-H by homologous recombination. The recombinant plasmids were transfected into Sf9 cells and obtained recombinant baculoviruses of rpFB-M, rpFB-F and rpFB-H. The recombinant baculoviruses were identified by IFA using the above polyclonal antibodies of mouse anti-CDV M, F, H and rabbit anti-H protein, the results shows that the specific fluorescence response was visible on the infected cell membrane of each recombinant baculoviruses. Meanwhile, the recombinant baculoviruses were analyzed by Western blot, rM, rF and rH proteins were detected with the molecular weight of 37 ku, 63 ku and 68 ku, which indicated the successful construction of each recombinant baculoviruses and expression of matrix protein, envelope glycoproteins F and H proteins in baculovirus-insect cell system.In summary, this research successfully expressed CDV M, F and H in baculovirus-insect cell system, the recombinant proteins had a good reactionogenicity, reacted specifically with the mouse and rabbit polyclonal antibody of M, F and H, which laid the foundation for the VLPs construction and vaccine development for CDV.