Preliminary Study On Protective Immunity Of The Outer Capsid VP4 Of Grass Carp Reovirus

Grass carp is the main species of freshwater aquaculture in China, accounting for20% of the total output of freshwater aquaculture, with highly economic value.Hemorrhagic disease of grass carp(grass carp hemorrhage) is a grass carp viral infectious disease caused bygrass carp reovirus(GCRV) and mainly popular in China’s southern aquaculture areas. Grass Carp Hemorrhage damage a variety of fish and is often outbreak epidemic in summer leading to rapid and large death of fingerling carps, thus causing serious damage and economic losses. The pathogen GCRV belongs to the genus Aquareoviridae(AQRV), which is the most virulent strain.The mature virus, highly contagious and lethal, presents a twenty- plane structure with a diameter of 70-80 nm, composed of 11 ds RNA segments.Growing evidences have supported that the virus capsid proteins involved in viral infection and had good antigenicity. In the current study, we cloned grass carp reovirus(GCRV) HZ08 strain outer capsid protein VP4 gene segment into the prokaryotic p ET32a(+) expression vectors and evaluated the potential of VP4 as an effective vaccine candidate for grass carp hemorrhagic disease. In addition, we employed spores of Bacillus subtilis(B. subtilis) as an oral vector to display the antigens VP4. Oral trials in mice and fishes further demonstrated that recombinant VP4 spores induced immune protective effects, especially mucosal immune response.Artificial infection test showed that fishes immuned by the recombinant VP4 spores vaccine have certain resistance to GCRV infection, with about30-45%lowercumulative percent mortalities(CPM) than negative control group. All efforts were in an attempt to provide a novel thought against the infection of hemorrhagic disease.Objective1 To prokaryotic express of outer capsid protein VP4 encoded by grass carp reovirus(GCRV) HZ08 strain conserved regions of S6 gene and preliminary explore its immunogenicity.2 To express VP4 protein on the surface of Bacillus subtilis capsid protein Cot C and preliminary explore its immunogenicity.3 To clarify the involved mechanisms of protective efficacy elicited by B.subtilisspore expressing VP4 in mouse model and estimate the safety of vaccines based on B. subtilis spore.4 To evaluate immune effects and protective efficacy induced by VP4 expressing by B.subtilis spore in ctenopharyngodon idellus model, provide new ideas for grass Carp hemorrhagic disease(GCHD) prevention and control Methods1 VP4 gene was cloned to prokaryotic vector p ET32a(+), expressed though E.coli BL21 / DE3 system. The recombinant VP4(r VP4) protein, purified by Ni2+affinity chromatography column, was then used to immune rats for antiserum preparation. Titer and isotype level of rat anti-r VP4 Ig G was tested and analyzed by ELISA, as well as difference between test group(recombinant protein subcutaneous immunization) and the control group(PBS immunization). The antiserum was then identified by immunofluorescence.2 Cot C-VP4 gene was cloned to Shuttle plasmid vector PEB03, expressed though Bacillus subtilis WB600 system. The expression status of recombinant VP4(Cot C-VP4) protein was analyzed through SDS-PAGE, western blotting, and immunofluorescence.3 Immune effects and investigation of mechanism following the oral vaccination of B. subtilis spore vaccine to mice3.1 Immunization of rats and sample collectionGroups of mice were immunized orally with suspensions of either spores expressing Cot C-VP4 or control Cot C spores. Mice in negative control group were treated with the same volume of normal saline. Serum and intestinal mucus samples were collected for the detection of specific antibodies and lysozyme.3.2 Detection of specific anti-VP4 antibodiesUsing ELISA, plates were coated with purified recombinant VP4 protein,after the addition of HRP-goat anti-rat Ig G/Ig A antibodies, the titers of Ig G/Ig A inserum and intestinal mucus were tested.3.3 Detection of relevant enzymatic indexesThe activities of lysozyme in serum and intestinal mucus from mice weremeasured by turbidimetry. Besides, after the third immunization, the activities of acidphosphatase(ACP), alkaline phosphatase(AKP), alanine aminotransferase(ALT),and aspartate transaminase(AST) in sera from mice were also assayed.4 Preliminary investigation of oral vaccine for fish against GCRV4.1 Immunization of fish and sample collectionGroups of 50 ctenopharyngodon idellus were immunized orally with fish feed including spores expressing Cot C-VP4 and normal feed particles respectively. The immune method was sustaining feeding until 6 week.Serum, intestine and other organs were collected for future use.4.2 Identification of recombinant spores colonized in intestines of fish3 fishes were chosed at the end of immune time, after 24 h fasting, intestine of fish were sterilely collected. The tissues were homogenized innormal saline. The homogenates were diluted with normal saline and underwent water bath at 68 °C for15 min, and then plated on LB agar plates supplemented with spectinomycin(selection marker of PEB03-Cot C-VP4). After incubation at 37 °C for 18-24 h, the colonies emerged on the plates wererandomly selected for PCR analysis using primers specific to VP4.4.3 Detection of specific anti-VP4 Ig M antibodyUsing ELISA, plates were coated with purified recombinant VP4 protein respectively, after the addition of HRP-rabbit anti-ctenopharyngodon idellus Ig M antibody, the titer of Ig M in serumwas tested.4.4 Detection of cytokinesThe total RNA was extract by TRIzol method and then quantitative analysis.Expressiong level of relevant cytokines, like IFN, Ig M, Mx, My D88, TLR22,TLR3 and TLR7 was detected by real time-q PCR in spleen and kidney of fishes.4.5 Challenge of fish with different concentration of GCRVCtenopharyngodon idellus in each group were infected with GCRV after vaccination. Heater was used to keep the water temperature at 28 degrees Celsius,which ensures all fishwill be challenged at the same environment. Cumulative mortality rates were calculated for the next two weeks.Detection of viral load in different organs was to determine the status of infection in infected fishes.Results1PCR, double enzyme digestion and DNA sequencing results showed that recombinant plasmid p ET32a-VP4 was successfully constructed; SDS-PAGE results showed that the purpose gene, as fusion protein molecular weight was about 43 k Da,has efficient expression in E. coli BL21 / DE3, and on 37 ℃, 1 mmol/L IPTG conditions the expression was most effective; the titer of rat anti-r GCRV-HZ08 VP4 Ig G was up to 1:819,200 at 6 weeks after the immunization, and Ig G1, Ig G2 a level showed no statistical difference(P > 0.05) from 2 to 6 week all along;immunofluorescence was showed distinctly in test group, while not in the negative control group.2 Shuttle plasmid vector PEB03-Cot C-VP4 was of successful construction.Results of SDS-PAGE, western blotting showed that recombinant protein Cot C-VP4 was displayed on surface of spore coat protein. And recombinant spore carried target gene could be recognized by anti-VP4 serum.3 Immune effects and investigation of mechanism following theoral vaccination of B. subtilis spore vaccine to mice3.1 Immunization of rats and sample collectionAfter oral administration of recombinant spores, the serum VP4 specific Ig G and Ig A levels were significantly higher than that of the control group(p<0.05), and the antibody titer of serum Ig G and Ig A increased with increased frequency of immunity.After oral administration of recombinant Bacillus subtilis, there was no statistical difference in intestinal mucus for VP4 specific Ig G(p>0.05), but VP4 specific Ig A level in intestinal mucus was significantly higher than the control group(P<0.05). Theresult shows that the recombinant spores has stimulated the generation of serum and intestine in mice s Ig A.3.2 Detection of relevant enzymatic indexesCompared with the negative control group, the levels of lysozyme in the serum of mice were significantly higher, and there was no significant difference between the Cot C-VP4 spore group and the Cot C group. After oral immunization, the level of intestinal mucus of Cot C-VP4 mice was higher than that of negative control group(P>0.05). The results suggested that oral administration of recombinant spores enhanced nonspecific immunity in mice. ACP, AKP, ALT and AST levels were not statistically different(p>0.05) in serum of each group after third immunization. The oral dose of 1x109 CFU spores had no adverse effect on the physiological function of the liver in mice.4 Preliminary investigation of oral vaccine for fish against GCRV4.1 Identification of recombinant spores colonized in intestines offishAfter 6 weeks immunization, grass carp intestinal abrasive coated liquid solid LB culture plate, monoclonal colony was selected for shaking bacteria. PCR identification showed that spores containing VP4 gene in the successful colonization of the intestinal tract of grass carp.4.2 Detection of specific anti-VP4 Ig M antibodyCompared with the negative group, the injected and oral immunization group Ig M increased significantly. These results suggest that oral immunization of recombinant Cot C-VP4 spores can stimulate the grass carp generating humoral immune response, but the reaction degree was lower than injected group.4.3 Detection of cytokinesResults show that, in the grass carp spleen, cytokines of injected group was significantly higher, while oral vaccine group had no significant changes than negative group; renal cell factor of both oral vaccine group and injected group were significantly increased, which Ig M, My D88, TLR22 have statistical difference with negative group(P<0.05). It is suggested that oral immunization can activate Toll like receptor signaling pathway, which lead to more immune protective effect.4.4 Challenge of fish with different concentration of GCRV103LD50 of GCRV and 104LD50 GCRV respectively challenged the vaccine grass carps. Negative group’s cumulative mortality rate was as high as 90%, the injected group’s cumulative mortality was only 10% which showed significantprotection during twice attack toxicity experiments.The oral vaccine group’s cumulative mortality rate was 45% and 60%, which suggest that oral vaccine can make fish body produce a certain anti-virus protection. The specific Taq Man probe detection results show that in different organs of dead grass carps GCRV wasdetacted.Thus these carps infected with the virus, were diagnosed with grass carp hemorrhage.And intestinal virus load was bigger than other organs, in consistent withthan the intestinal bleeding symptoms were most pronounced.Conclusions1. This study successfully constructed the prokaryotic expression recombinant plasmid pet-32a(+)-VP4, get the fusion protein r VP4 and antiserum; recombinant protein VP4 has strong antigenicity and immunogenicity and antiserum has a specific recognition for virus.2. This study successfully constructed in Bacillus subtilis / E.coli shuttle expression plasmid PEB03-Cot C-VP4 and transformed into strain WB600 induced expression and fusion protein was successfully displayed on the surface of the spore coat; recombinant bacillus can be recognized by r VP4 antisera.3. Oral administration of recombinant spores of Cot C-VP4 stimulated the local mucosal immune response and humoral immune response in mice, and there was no damage to the liver function of the mice.4. Oral immunization Cot C-VP4 recombinant Bacillus subtilis successfully colonized in the fish intestine, and can stimulate the production of humoral immunity of grass carp. Attack the virus infection experiments verify oral vaccined fish had some protective effect to virus invasion, the cumulative mortality than those in negative control group reduced about 30-45%, which provide new ideas for the prevention and treatment of hemorrhage of grass carp.