Grass Carp Reovirus Coat Shell Protein Vp7 Nucleic Acid Vaccine Research

Grass carp(Ctenopharyngodon idellus), is one of main variety of freshwater fish. But grass carp diseases are various, and the most serious one among them is hemorrhagic disease of grass carp. The mortality rate of grass carp is very high after GCRV infection, and sometimes leading to a high mortality of about eighty percent or even above that. Grass carp reo virus (GCRV) was confirmed as the major pathogen of hemorrhagic disease of grass carp.GCRV, as a member of genus Aquareovirus in the family Reoviridae, was the first viral pathogen to be identified from aquatic animals in China and has been recognized as the most pathogenic among all the isolates of aquareoviruses reported to date. GCRV mainly infects living tissue cells of grass carp’s liver, heart muscles, kidneys, spleen gill, and bowel and so on. That’s why chemical drug is difficult to achieve the treatment effect and it becomes the "bottleneck" problems of grass carp breeding in the development. GCRV not only causes severe acute hemorrhagic disease in fingerling and yearling grass carp, but also can infect the Mylopharyngodon piceus, Pseudorasbora parva and Hemi-eulter leueieulus Bas, et al. These show that GCRV constitute serious harm to our country’s freshwater aquaculture industry.GCRV is three-dimensional icosahedral symmetry, and composed of core RNA and Multilayer capsids (outer-capsid, middle-capsid and inner-capsid). GCRV is a nonenveloped icosahedral particle comprising11double-stranded RNA genome segments surrounded by multiple concentric protein capsids. In recent years, genome sequence and3D structure reconstruction of GCRV indicate that the11genome segments encode seven structural proteins (VP1-VP7) and five nonstructural proteins, Among the structural proteins of GCRV, VP7and VP5are the outer capsid proteins of the virus. VP7is encoded by the S10gene fragment, revealed to play an irreplaceable role in the virus infection and pathogenic process, and has neutralization ability. Both VP5and VP7were capable of neutralizing viral infectivity. Particularly, the neutralizing activity of VP7was3times higher than that of VP5, suggesting that VP7might be a dominating epitope. In addition, the recombinant plasmid containing VP7gene can be transferred into eukaryotic cells to be expressed successfully, and it is suggested that VP7had the potential to be used as DNA vaccine.Nucleic acid vaccine, genetic vaccine or DNA vaccine, is a new vaccine for protecting an organism against disease by injecting it with genetically engineered DNA to produce an immunological response. Nucleic acid vaccines have stronger antigenicity, longer minimum protection time, higher safety, and reparation and transportation are more convenient. These chief advantages greatly overcome the drawbacks existing in inactivated vaccine, attenuated vaccine and subunit vaccine, and have caused extensive concern within the industry.VP7genes and (3-actin promoter from Megalobrama amblycephala were cloned into pFastBacTM ual vector. Meanwhile, another VP7gene was amplified and cloned in control of baculoviral polyhedron promoter (polh) to obtain recombinant plasmid pFastBac-β-VP71-VP72. Immune experiment and immunological effect were studied. The recombinant plasmids were divided into three groups based on doses:10μg,30μg,60μg.30μg pFastBacTM ual group and control group were designed in addition. Immune efficacy was determined by means of transcriptions of VP7, antibody detection by indirect agglutination reaction and challenge experiment on the14th,21th,28th, and49th day after immunization. The results showed that pFastBac-β-VP71-VP72was transformed into body cell, VP7gene could be controlled for expression constantly by P-actin promoter, and transcription of VP7was still in process on the49th day. Antibody was produced and the titers reached their peak on the21th day after immunization. The mortality rates were0%,0%and5%respectively in10μg,30μg and60μg vaccine immunization groups,30%in pFastBacTM ual group, and100%in control group after GCRV injection.All results suggested that pFastBac-β-VP71-VP72had good immuno-protective efficacy as DNA vaccine against GCRV. Therefore, the study provided important academic foundation for research, development and application of the GCRV genetic vaccine.