The Baculovirus Mediated Coexpression Of P12A And 3C Genes Of Foot-and-Mouth Disease Virus Type O In SF9 Cells And BHK Cells

Foot-and-mouth disease (FMD), which is caused by Foot-and-mouth disease virus (FMDV), is acute and severe infection disease of artiodactyls. This disease has a worldwide prevalence and causes serious losses of animal husbandry. Traditional vaccine plays a key role in preventing FMD, but it has a number of disadvantages such as high cost, possibility of spread the virus and so on. Therefore, it is very important to exploit a new and efficient type of vaccine for preventing FMD. The empty capsid of the virus has been received great attention for the same immunogenicity as the whole viral particle. In this study, we used the capsid precursor protein P12A gene and protease 3C gene of FMDV type O as heterologous gene and constructed two different recombinant baculoviruse, which one of them can express the proteins in Sf9 cells and the other can function in BHK cells. This experiment offered a foundation for further research of empty capsid vaccine and live carrier vaccine.1. The recombinant baculovirus BV-P12A-3C was constructed by inserting PCR products of P12A gene and 3C gene of FMDV type O under the control of PH promoter and P10 promoter respectively in baculovirus pFastBacTMDual vector and then infected the Sf9 cells. Indirect immunofluorescence assay showed bright green fluorescence in infected cells and no fluorescence obtained in control cells; Indirect sandwich-ELISA assay also displayed that the expressed protein had a certain degree antigenicity, which could be recognized by positive serum of FMDV type O, but the value was lower. This may relate with quantity of the expressed protein and the real reasons to this phenomena need further study.2. A recombinant baculovirus named BV-VSV·GED-CMV-P12A-3C was constructed via inserting the series gene sequence formed by P12A and 3C gene of FMD type O under the position of VSV·GED-CMV sequence, which followed the PH promoter of the baculovirus vector pFastBacTMDual and contained a series sequence of 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of vesicular stomatitis virus (VSV·GED), ployA and Cytomegalovirus promoter sequence (CMV), and then transfected into Sf9 cells. After 48 hours infection of BHK cells with the recombinant baculovirus BV-VSV·GED-CMV-P12A-3C, the green fluorescence was observed in infected cells with indirect immnofluorescence assay. Sandwich ELASA method was used to detect the infected cell lysates and showed a specific response with the positive serum against FMDV, but the ideal OD value for the lyastes was less than 1/4 of the control group; a protein band of 25 Kda equal to VP3/VP1 was also detected by western blotting assay and this result indicated that the capsid precursor protein P12A was successfully digested by 3C protease. Nevertheless, whether the targeted gene P12A-3C can express and assemble itself for the empty capsid in vivo need further reseach. In conclusion, there was no doubt that the experiment result provided a pre-work for deeper study of FMD live carrier vaccine.