Expression Of Rabbit Hemorrhagic Disease Virus(RHDV) Capsid Protein Gene In Silkworm, Bombyx Mori

Rabbit hemorrhagic disease (RHD) is a highly contagious, lethal disease of domestic and wildrabbits caused by rabbit hemorrhagic disease virus(RHDV), which can cause serious influence and largeeconomic losses to rabbit producers. Recently, most of the vaccines against rabbit hemorrhagic diseaseare the inactivated vaccine in oil-emulsion and the inactivated tissue vaccine, which are made fromexperimentally infected rabbits. But there are many defects about these commercially available vaccines.In recent years, many researchers are dedicated to develop genetic engineering vaccine. Rabbithemorrhagic disease virus capsid protein (VP60), which is the major structural protein and the mainimmune protective antigen for the disease, is of great significance in developing genetic engineeringvaccines.In this study, the vp60gene sequence(GenBank accession number:AF453761) was artificiallysynthesized after codon optimization. In order to prepare polyclonal antibody for analyzing theexpression protein in silkworm, a fragment of vp60gene was amplified with PCR and cloned into thevector pMD18-T. The recombinant plasmid pET-28a-RHDV-vp60, which was transformed into E.colistrain BL21(DE3), was induced to express by0.5mM IPTG at37°C for4h. Next, the product wasanalysed by SDS-PAGE, and the result indicated that there was a new26kDa electrophoretic bandcorresponding to the presumptive interest protein. In addition, the result of mass spectra also showedthat the recombinant protein was expressed successfully. The result of ELISA showed the antibody titerwas1:6400when the antigen concentration was2μg/mL. It could be used to analyze the products inBombyx mori.The optimized target gene were inserted into the baculovirus transfer vector pVL1393andconstructed the recombinant plasmid pVL-RHDV-vp60, with which co-transfected linearted DNA ofBm-BacPAK6virus into Bm-N cells. By homologous recombination, the recombinant virusreBm-RHDV was obtained. Then, the fifth instars of Bombyx mori were infected with the recombinantvirus and enzyme-linked immunosorbent assay was used to detect the expression of vp60. At the sametime, the virus with high expression was isolated. After the fifth instar larvae of Bombyx mori andsilkworm chrysalis was infected, we collected the hemolymph and purified it by ultracentrifugation. Thescanning electron microscope result confirmed that the polyprotein of RHDV expressed in silkworm.The vaccines made of silkworm chrysalis were used to immunize rabbits and the changes ofantiserum were assayed by ELISA. The result demonstrated that the specific antibody was induced byRHDV empty capsids antigen in all vaccinated rabbits. Pathological examination demonstrated thatthere was no obvious side effect in vaccinated animals and the self-made vaccine possessed good safety.This work pave the way to the development of genetic engineering vaccine of RHDV, the preparation ofdetection kit, the forming of virion particle, and the interaction between RHDV and the receptor,etc.