Generation Of Novel Genotype Ⅱ GCRV-VLPs Vaccine Based On The Baculovirus-Insect Cells Expression System

Grass carp(Ctenopharyngodon idella)is one of the most important aquaculture species in China.It has been widely cultivated over the last 60 years and has a great commercial value.However,grass carp hemorrhagic disease(GCHD)caused by grass carp reovirus(GCRV)is a serious infectious disease that mainly affects fingerlings during rearing and leads to severe economic losses to aquaculture in China.GCRV belongs to the genus Aquareovirus of the family Reoviridae and was the first viral pathogen identified from aquatic animals in China in1983.It is known that GCRV strains can be divided into three distinct genotypes(Ⅰ,Ⅱ,and Ⅲ)based on sequence comparison and analysis.Recent epidemiological investigations showed that most GCRV strains isolated are GCRV genotype Ⅱ in China,which is more virulent and causes severe hemorrhagic symptoms with mortality reaching 80%.Currently,there is no specific treatment against GCHD,and the only available protection has been vaccination strategies.Virus-like particles(VLPs)serve as an ideal platform in the research of antiviral vaccines.The VLPs self-assemble structures by one or several viral structural proteins,therefore the risk of a viral replication can be fully avoided.Additionally,they allow the display of complex and native antigens in a highly repetitive form on their surface,which also can induce both cellular and humoral immune responses.Previous studies have shown that the segments S3,S4,S6,and S11 of type Ⅱ GCRV are immunogenic.Hence,we employed those segments to generate type Ⅱ GCRV-VLPs by baculovirus-insect cells expression system.The research contents and results are as follows:(1)Construction of GCRV-Ⅱ RecombinantBaculoviruses:The total RNA of Hu Nan1307was extracted and the GCRV-11 was amplified by RT-PCR.The recombinant plasmidBacmid-S11 was constructed by baculovirus-insect cells expression system.Recombinant baculovirus rBac-S3,rBac-S4,and rBac-S6 were obtained by transfecting recombinant plasmidBacmid-S3\S4\S6 which have been successfully constructed in our laboratory.And recombinant baculovirus rBac-S11 was obtained by transfecting recombinant plasmidBacmid-S11 into Sf9insect cells,The titer of recombinant baculovirus were 2.4×10~6IFU/ml,2.4×10~6IFU/ml,3.2×10~6IFU/ml and 3.2×10~7IFU/ml,respectively,usingBac-Pak rapid titration kit.Indirect immunofluorescence(IFA)was used to identify the expression of recombinant proteins,and the results showed that the recombinant baculovirus rBac-S3\S4\S6\S11 could express the target proteins.(2)Construction of genotype Ⅱ GCRV-VLPs:The recombinant baculoviruses rBac-S3,rBac-S4,rBac-S6 and rBac-S11 were co-infected according to MOI=5 to construct S3-S6-S11-VLPs and S3-S4-S6-S11-VLPs.The assembly of virus-like particles was observed through ultrathin cell section and purified VLPs under electron microscope.Indirect immunofluorescence(IFA)and Western Blot were used to identify the protein expression in VLPs.The results showed that both VLPs were successfully constructed and expressed the target proteins.(3)Immunogenicity analysis of type Ⅱ GCRV-VLPs:The purified S3-S6-S11-VLPs and S3-S4-S6-S11-VLPs were immunized with 30μg of grass carp.After immunization,the kidney and spleen of grass carp were collected and the m RNA relative expression levels of IFN,TLR-3,TLR-7 and Ig M genes were detected by q RT-PCR to evaluate the immunological effect of the VLPs.The results showed that from the 5th day of immunization,the expression levels of immune factors in each immune group were significantly increased compared with the PBS control group(P<0.001),and reached the peak on the 7th day,and then decreased.Indirect ELISA was used to detect the specific Ig M antibody levels in the serum samples.The results showed that the specific Ig M antibody levels in the immunized groups began to increase significantly at 14 dpi compared with the PBS control group(P<0.05),the OD450nm value of S3-S4-S6-S11-VLPs+adjuvant group was highest compared with other immunized groups.To evaluate the protective effectiveness of the GCRV-VLPs,each group was challenged with a lethal dose of GCRV on 21 dpi.The results showed that the S3-S6-S11-VLPS+adjuvant group exhibited the highest relative percent survival of 67.86%.