Computer-aided-designing And Functional Study Of Recombinant Foot-and-Mouth Disease Virus Vaccine

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Recent FMD outbreaks in some countries and their significant economic impact have increased the concern of governments worldwide. Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus of Picornaviridae. There are seven serotypes of FMD virus, namely, O, A, C, SAT 1, SAT 2, SAT 3 and AsiaⅠ. By electron microscopy, the FMD virion appears to be a round particle with a smooth surface and a diameter of about 25 nm. The structural proteins, VP1,VP2,VP3, fold into an eight-stranded wedge-shaped ?-barrel which fit together to form the majority of the capsid structure. The VP4 protein is buried within the capsid. The virus particle contains a loop on its surface betweenβ-strands G and H of the capsid protein VP1 (G-H loop), which is often mutated in antigenic variants. The G-H loop also contains a highly conserved RGD motif that consist of three amino acid residues( Arg-Gly-Asp ),and that has been identified as a cell binding site in competition studies using synthetic peptides.Currently, FMD vaccine is inactivated whole FMDV preparation that is formulated with adjuvant prior to use in the field. The introduction of the killed FMD vaccine has been extremely successful in reducing the number of FMD outbreaks in many parts of the world where the disease is enzootic. However, there are safety concerns, which urge researchers over the past 20 to 25 years to develop alternative FMD vaccines that do not require infectious virus. However, recombination protein vaccines represent a safe alternative to the current virus-inactivated immunogens. Computers play an important role in designing recombination protein vaccine, which requires the identification and evaluation of the B epitope and T epitope of the FMDV. Computers can help immunologists to identify the epitopes which trigger immune responses. The position and conformation of the epitope should be considered in vaccine design. Computer can also help immunologists to predict 3D structures of the vaccine. Some web servers for building the 3D model are available. So we can use them conveniently in our vaccine design.In our studies, we chose a candidate FMDV by ClustalX, analyzed epitopes of the candidate FMDV by DNAStar, predicted the 3D structure of the vaccine (named RA2) by Geno3D. Then, we constructed the gene encoding RA2, which was subcloned into pET28a and expressed in BL21 successfully. The functional study indicates that RA2 can induce high titer neutralization antibody in guinea pigs and cows. This thesis includes the following parts:1. Choose candidate FMDV by ClustalX Because of the variation of FMDV, We needed to choose a consensus srtain for our vaccine design. So 29 AsiaⅠFMDV VP1's sequences were download, then analyzed by ClustalX. Those sequences were conservative, so we chose a typical one discovered in China for our vaccine design.2. Analyze epitopes of candidate FMDV by DNAstarBy DNAStar, We analyzed the second structure, coil region, hydrophilicity, surface probability and antigenic index of the candidate virus. According to the analysis, two B epitopes and two T epitopes were selected to construct our vaccine,named B1, B2, T1 and T2.3. Predict the 3D structure of RA2By connecting the four epitopes in different ways, we designed RA2 structure that is consequently predicted by Geno3D. In the structure, the RGD's location simulates that in the real FMDV, and gene A (consisting B1 and T1) and gene B (consisting B2 and T2) are included, resulting in RA2 (consisting B+B+A+B+B). As a result, three B epitopes are on the surface of RA2 where RGDs are on the top of the loop. The evaluation on tertiary structure of predicted RA2 indicates that most residues are located in favourable site.4. Construct recombinant plasmids pET28a-RA2Gene A and B were synthesized using 2 round of PCR separately and the resultant products were confirmed by agarose gel electrophorosis. Gene A and B were subsequently cloned into pMD18-T vectors, then this ligates were transformed into JM109 host bacterium. The pMD18-T-A was extracted from antibiotic resistant clone, digested by EcoRⅠand analyzed by agarose gel electrophorosis in which a gene fragment of 222 bp was released. After being sequenced, gene A was confirmed to be exactly the gene designed (provided by Haifei Xu in our lab ) . The pMD18-T-B was extracted from antibiotic resistant clone, digested by EcoRⅠand analyzed by agarose gel electrophorosis in which a gene fragment of 225 bp was released. After being sequenced, gene B was confirmed to be exactly the gene designed (provided by Zhao Cao in our lab ).The B fragment, released from pMD18-T-B by digesting with BglⅡ, was recovered from the agarose gel and was subcloned into pMD18-T-B at the site of BglⅡ. Using similar method, pMD18-T-RA2 was constructed. Then it was digested by EcoRⅠand Hind III and the gene RA2 was released and recovered from agarose gel, which was introduced into pET28a at the site of EcoRⅠand HindIII. The pET28a-RA2 was extracted from antibiotic resistant clone, digested by EcoRⅠand Hind II and analyzed by agarose gel electrophorosis in which a gene fragment of 765 bp was released. The insert is conformed by DNA sequencing.5. Express and determinate on inclusion expression of RA2The pET28a-RA2 was transformed into BL21 and positive clone was selected and cultured. After being induced by IPTG, the bacteria was collected and lysed. The lysates were characterized by SDS-PAGE. The expressed RA2 was confirmed by the presence of an additional band of 33 KD compatible with the predicted. The expressed RA2 was further confirmed by Western blot.After being induced by IPTG, the bacteria were collected and splited by ultrasound. Deposit and supernatant of the splited bacteria were collected. SDS-PAGE analysis showed that RA2 was mainly inclusion bodies in endochylema of BL21.6. Purificate and observe the stability of RA2The recombinant protein RA2 was purified through nickel chromatography and the purity of the purified RA2 was nearly 95% (done by Peiyin Zhang in our lab ).Two tubes containing purified RA2 dissolved in PBS were stored at -20 ℃and 4℃respectively. SDS-PAGE analysis showed that RA2 in the samples collected on day 15, 30, 45 and 70, begun to degradate after 30 days at 4℃, and there was no degradation after 75 days at -20℃. This result illustrates that RA2 is stable in low temperature.7. The efficacy of RA2To study the immunogenicity of RA2, Guinea pigs and cows were immunized with RA2. The serum were collected and tested by Elisa, virus neutralization using BHK-2 and suckling mice protection. The results show that high titer antibodies can be induced in guinea pigs by RA2. Antibodies can also be induced in cows by RA2 but lower than that by inactived vaccines. 3/5 of the immunized cows were protected after AsiaⅠFMDV's attack.Together, the recombinant Bovine AsiaⅠFoot-and-Mouth Disease Virus Vaccine RA2 may be developed into a safe and effective novel vaccine.