Development Of Genetic Engineering Subunit Vaccine Against Rabies

Rabies is an enzootic viral disease caused by the rabies virus, once infected, it is difficult to cure. Vaccination is the most effective means to prevent and control rabies at present. The current used rabies vaccine for animals in China is mainly traditional attenuated vaccine and inactivated vaccine. Attenuated vaccine often causes side-effects and potential security risks, inactivated vaccine derived from cell culture are too expensive to be used for immunization and treatment of animals in our country. In this study, rabies virus nucleoprotein and glycoprotein of CVS strain expressed at highly level in silkworm pupae by using the silkworm baculovirus expression system. Meanwhile, nucleoprotein and glycoprotein of rabies virus were co-expressed at highly level in silkworm pupae by using recombinant DNA technology,and immunogenicity of the expression products were conducted respectively.1. In this study we describer a new silkworm-baculovirus expression system to express the nucleoprotein (N) gene of rabies virus and evaluation of immune response in BALB/c mice. N gene was cloned by reverse transcriptional polymerase chain reaction (RT-PCR) from brain tissue of mice that infected of rabies virus CVS strain. The N gene fragment obtained by EcoRI/XbaI-digested was inserted into the baculovirus transfer vector pVL1393 to generate plasmid pVL-N. The recombinant silkworm baculovirus rBmNPV(N) containing the N gene of RV was obtained after co-transfection and plaque screening. Indirect immunofluorescence assay (IIFA) showed that N protein was successfully expressed in Bm-N cells. The results of Western blot showed that N protein to be at 54KD, just as expected. The titers of N antigen expressed in silkworm were about 1:50,000 detected by ELISA. High level of the anti-rabies virus IgG antibody were induced in mice that immunized by intramuscular with N antigen and N vaccine. Antibody subtype analysis showed that N antigen can induce cellular immune response. Mice were challenged 28 days after immunization. The survival rate of mice immunized intramuscularly with N antigen was 50%, whereas intramuscularly immunized with N antigen in oil adjuvant showed 80% protection.2. The recombinant silkworm baculovirus rBmNPV(G) containing the G gene of RV was obtained by using the silkworm baculovirus expression system. IIFA was used to verify that rBmNPV(G )could validly express the G protein in Bm-N cells. The results of Western blot analysis showed that the G protein to be at 63KD, just the same as the mature G protein expected. The titers of G antigen expressed in silkworm were about 1:8192 detected by ELISA. High level of the anti-rabies virus IgG antibody were induced in mice that immunized by intramuscular with G antigen and G vaccine. Antibody subtype analysis showed that G antigen can induce humoral immune response. Mice were challenged 28 days after immunization. The survival rates of mice immunized intramuscularly with G antigen and G vaccine was 100%, with no clinical signs or discomfort from the G antigen regardless of the route of administration or the amount of G antigen.3. In this experiment, G and N protein of the rabies virus were co-expressed in silkworm pupae by using silkworm baculovirus expression system and evaluation of immune response in BALB/c mice. The recombinant baculovirus transfer vector pVL-G-IRES-N that containing the G-IRES-N cassette was constructed by inserted a fragment of IRES sequence between two genes. The recombinant silkworm baculovirus rBmNPV(G-IRES-N) was obtained after co-transfection and plaque screening. The results of Western blot analysis showed that the G protein to be at 63KD and N protein to be 54 KD in the same band, just the same as the mature G protein and N protein expected. ELISA assay indicated that G and N antigen were co-expressed at high level in silkworm. Time course study for the expression of glycoprotein in silkworm pupae indicated that the highest expression level of antigen to be obtained about 4-5d after silkworm pupae infected by recombinant virus. High level of the anti-rabies virus IgG antibody were induced in mice that immunized by intramuscular with G /N antigen and G/N vaccine. Antibody subtype analysis showed that G/N antigen can induce both humoral immune response and cellular immune response. 28 days after immunization, mice were challenged with 20LD₅₀ of CVS virus. The survival rates of mice immunized intramuscularly with G/N antigen and G/N vaccine was 100%, with no clinical signs or discomfort in immunized mice and dogs from the G/N antigen regardless of the route of administration or the amount of G/N antigen.