Immunogenic Research Of Two Strains RABV Glycoprotein Expressed By Recombinant Baculoviruses

Rabies is a kind of zoonises which mainly induces encephalomyelitis and causesneurosis. This infectious disease has been prevalent even since Ancient Egyptiantimes, and still occurred in more than90countries or areas in2012. In recent10years,about50,000people died because of rabies each year. Currently, there is no effectivetreatment for the disease and vaccination is thought to be the only possible method toprevent or even eliminate rabies. At present, most of the human or animals rabiesvaccine are inactivated vaccine. However, the high cost of inactivated vaccine makesit not an ideal choice for the less developed countries to control or even eliminate therabies effectively. Subunit vaccine is a genetically engineered vaccine, which isrelatively inexpensive and not relating to the live vector virus as recombinant livevector vaccine. Hence, it is relatively easy to get the approval of relevant departments,and in fact, a variety of subunit vaccine has been available for clinical application.Therefore, subunit vaccine is expected as one of the development of the new rabiesvaccine.In this study, in order to explore the development of rabies subunit vaccine, wecarried out the following test:Construction of the recombinant baculovirus which can expressing rabies virusglycoprotein and preparation of the expressed glycoprotein. Test content: a.Cloning ofthe glycoprotein genes from the rabies virus strains SRV9and BD06. b.WithBac-to-Bac baculovirus expression system, the two glycoprotein genes were clonedinto the shuttle plasmid Bacmid (plasmid containing the baculovirus genome), and therecombinant shuttle plasmid was identified and the correct clones were selected. c.The correct recombinant shuttle plasmids were transfected into insect cells Sf9andthe recombinant baculoviruses harboring the two target genes, respectively, wererescued and identified. d.Identification and preparation of the rabies virusglycoprotein expressed by recombinant baculovirus.Immunogenic identification of glycoprotein expressed by the recombinants. Testcontent: a.Mice were immunized with crude glycoprotein expressed by recombinantat high and low dose. b.Blood serums were separated at14and28days postimmunization; the antibody levels were tested by FAVN. c.At35days post theimmunization, mice were challenged with lethal CVS-24by intracerebral injection. d.The immunogenicity of the two glycoproteins from different RV strains was analyzedby geostatistics method. Through the above test, the following results were obtained:After transfection, viral particles with obvious baculovirus form can be observedin the preparations by electron microscopy. SDS-PAGE and western blot showed thatboth of the two recombinant baculovirus(the third generation) in Sf9cells expressed aprotein in proximity of72kDa, which could reacted with the rabies virus glycoproteinmonoclonal antibody, were expressed in Sf9cells after being treated with the tworecombinants. For visualization of RV glycoprotein, Sf9cells, infected with the tworecombinants, were stained with FITC-labeled rabies virus glycoprotein monoclonalantibody; specific green fluorescence signal was observed on the surface of theinfected cells.Results of immunization showed that at high dose, both of SRV9and BD06glycoproteins expressed by recombinants could induce100%seroconversion andprovide100%protection against lethal dose challenge of CVS-24on immunized mice.But at low dose, seroconversion only occurred in part of the mice and no protectionwas provided in SRV9glycoprotein group. While, BD06glycoprotein can still induce100%seroconversion and provide80%protection on mice.The following conclusions were obtained from the above tests:1. Both SRV9and BD06glycoprotein expressed by recombinant baculovirus hadimmunogenicity and could be used as the candidate antigens for the development ofrabies subunit vaccine.2. The immunogenicity of the BD06glycoprotein to be significantly higher thanthat of SRV9glycoprotein, which indicated that the former was probably moresuitable for the development of rabies subunit vaccine.3. Existing of significant differences between different RV strains’ glycoproteinindicated that it was meaningful to further screen for the candidate glycoprotein withhigher immunogenicity, maybe, not only in the attenuated vaccine strains but also themore pathogenic strains.