Applying The Insect-baculovirus Expression Vector Systems To Produce Foot-and-mouth Virus-like Particles And Detecting The Immunogenicity Of The Product

Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease,which is caused by foot-and-mouth disease virus (FMDV) and commonly occurs incattle, sheep, pigs and other cloven-hoofed animals. Once animals are infected, themucous membrane of mouth, the hooves, and the skin of mammary gland will festerand appear vesicular exanthema. All of the infected animals will show clinical symptom,inducing sharp decrease of livestock product. Because of extremely high efficience oftransmission, FMD might be spread around the world rapidly and expansively. Once itoccurs, many developed countries will kill and then bury the infected animals deeply.Considering the severe destructive loss and the extensive influence, the OfficeInternational des Epizooties (OIE) ranks the FMD in the first pace of Class I deadlycontigious diseases.At present, the effective precautionary measure for FMDV is to implement vaccineinjection. However, during the process of large scale inactived vaccine production, it ispossible that some active FMDVs escape from the production workshop, consequently,lead to extensive epidemic. Meanwhile, the application of vaccine can add moredifficult into the epidemiological investigation of FMD. Therefore, preparation ofsubunit vaccine is one of the important ways to solve these problems. Nevertheless, thecommon kinds of subunit vaccine can not contain all of the antigenic epitopes, and theimmunogenicity of this vaccine is not good enough for an effective vaccine. In contrast,the capsid of FMDV has the whole antigenic epitopes, and has no genetic material in itat all. When the capsid is transmitted into the body of animal, it can cause not only thehumoral immunity but also the cellular immunity with high immunogenicity.Additionally, the capsid cannot replicate and proliferate in the body of animal. In this study, we aim to produce foot-and-mouth virus-like particles by applying theBac-to-Bac insect-baculovirus expression vector systems, as a new pattern of FMDVcandidate vaccine.The FMDV is a kind of virus with an about8.5kb positive single strand RNAgenome. The capsid proteins are encoded by P1gene, including VP4, VP2, VP3andVP1. In this study, we aligned the genes we already had and the full length DNAsequence of DQ989304.1from GenBank, and made out the sequence information ofeach structural protein. The length of each structure protein gene:255bp,654bp,657bp and624bp. The amino acid information:85aa,218aa,219aa and208aa. Thelength of2A gene is54bp, encoding18aa. The length of3C protease gene is639bp,encoding213aa. In this study, we jointed the structural gene P1, and non-structuralgenes2A,3C and partial2B,3B in orderly. Then we inserted the jointed genes into thepFastBac1plasmid, and constructed the recombination Donor pasmid successfully. Wegot recombination baculovirus plasmid, rBacmid, including FMDV P12A3C gene bymeans of site-specific recombination between the recombination Donor plasmid and thebaculovirus plasmid in E.coli. Then the rBacmid was transfected into the Sf9insect cellline by lipidosome-mediated method. In the subsequent study, we proved that theP12A3C complex gene expressed in the insect cell line and the protein can self-spliceand self-assemble by testing the level of cell morphology, molecular of DNA, RNA andprotein, and the morphology of FMDV VLPs. In order to obtain enough FMDV VLPs,we cultivated the Sf9cell in large scale. We select guinea pigs as the experimentalanimal in this study. Then, according to the plan we immunized the guinea pigs for3times, and we got4batches of serum samples by cardiac blood collection method. Wedesigned indirect ELISA test, assayed the titer of antibody of experimental group andverified that the FMDV VLPs we produced from the insect cell can induce highantibody titer. This study can provide some basis on the development of FMD vaccine.