Prokaryotic Expression And Purification Of Diagnostic Antigen 3ABC Of FMD For Differentiating Infection From Vaccination & Preliminary Study On Inhibition Of FMDV Replication By RNAi

Foot-and-mouth disease (FMD) caused by foot-and-disease virus (FMDV) is a highly contagious disease among cloven hoofed animals and was listed first among type-A infectious disease by Office International des Epizooties (OIE). Vaccination is a main measure to prevent and cure FMD in most countries where this disease exists, but in order to control FMD, the fist thing to do is differentiating virus infected and carrier animals from vaccinated population. The paper is divided into two parts, and the first part is about the prokaryotic expression of diagnostic antigen 3ABC of FMD and its purification; the second part is inhibition of FMDV replication in BHK cells by RNAi. In the first part, nonstructural protein of FMDV was expressed in E. coli, and purified. The objective of this part is designed to establish basis for development of diagnostic kit for distinguishing FMDV infection and vaccination. The 3ABC of FMDV type O fragment was amplified and inserted into prokaryotic expression vector, pQE30Xa. PCR, double restriction digestion and sequencing were used to show that recombinant vector pQE3ABC was constructed successfully. The recombinant vector was transformed into hosts, M15(pREP4) and SG13009(pREP4) to express the fusion protein. The fusion protein is about 40kD in size indicated by SDS-PAGE. Western-blot analysis demonstrated that the fusion protein can react specifically with antibody against standard FMDV type O. Fusion protein expression conditions were optimized and results were as follows: proper concentrations of ampicillin and IPTG were 100μg/mL and 1mM respectively; expression effect between two hosts were not distinct. The fusion protein was purified through Ni-affinity chromatography and SDS-PAGE analysis shows the product was purified. The renaturation of the fusion was performed with dilution method (ultrafilteration) and renatured fusion was quantified by BCA-protein quantification assay kit, and the concentration was at the level of 80-100μg/mL.The second part is about the preliminary studies on inhibition of FMDV replication in BHK cells. The aim of this part is to do some meaningful works for FMD control and therapy using RNAi. The genome of FMDV belongs to single-stranded, sense RNA, can be not only a template as viral replication but also be mRNA to translate protein, which makes it appropriate for the studies of antiviral research. According to the conservative flanking-sequence, two primers were designed to amplify the tandem sequence, IRES (internal ribosome entry site) and L sequence of foot-and-mouth disease virus, using RT-PCR and PCR methods. The PCR product was sequence and sequencing result shows that IRES is very conservative without mutation and seven base mutations in gene L. On the basis of sequencing, gene L was selected as target gene for it is a virulent factor of FMDV. A target sequence (21nt in length) was screened which locates 229 nt downstream from AUG of gene L. siRNA expression cassette (SEC) was synthesized using PCR method in vitro. The purified SECs were transfected into cultured cells by lipofectin reagent at 50-70% cell confluence. FMDV was inoculated 4 hours after transfection, and inhibition effect of viral replication was detected 24 hours after inoculation by indirect immuno fluorescent assay. The results show that SEC-L229 can inhibit dramatically the viral replication in BHK cells in the sequence-specific manner (>80%), and decrease the cell death rate. Inhibition between 25ng and 50ng SEC-L229 treatment groups was not distinct. Preliminary results from the second part show that SEC can be synthesized rapidly and economically using PCR method, and the SEC can inhibit specifically FMDV replication in BHK cells which maybe provide a potential therapy pathway for this disease.