The Construction And Immunogenicity Of Genetic Engineering Vaccines Against FMDV

Foot-and-mouth disease (FMD) caused by Foot-and-mouth disease virus (FMDV) is one of the most contagious animal virus diseases. In this study, by means of molecular biology, immunology, molecular virology, biochemistry and bioinformatics et al, P1 region of FMDV Chinese isolate O/LZ were cloned, sequence analyzed and expressed, the immunogenicity of one recombinant Fowl-pox viruses (rFPV) and one DNA vaccines against FMDV was evaluated.FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae, whose genome is about 8500 bases in length. During replication, the genome is expressed as a single open reading frame (ORF) that is processed into mature polypeptide products. 4 structural proteins (encoding VP4, VP2, VP3, VP1) and 7 nonstructural proteins, VP1 is one of the most important structural protein, there is an antigen combine site in VP1 of FMDV, it can induce the mice to fight the FMDV. VP1 is the focus to investigate the FMDV vaccine for all the researcher in the world. Complete the FMDV structural proteins information would contribute to understanding the molecular characteristic of FMDV or developing new vaccines and diagnosis techniques.In experiment one, P1 fragment of FMDV Chinese isolate O/LZ was cloned by RT-PCR. And sequence information of full length of P1 gene (2223nt) regions (did not including 2A regions), were acquired. The secondary structure of capsid protein of FMDV was predicted by the methods of Garnier-Robson, Chou-Fasman and Karplus-Schulz based on FMDV genome, and hydrophilicity plot, surface probability plot and antigenic index were obtained by the methods of Kyte-Doolittle, Emini and Jameson-Wolf respectively. Combining the results according to these methods, the B cell epitopes for capsid protein of FMDV were predicted. The result s showed that VP1 contains the most of the predominant epitopes of the B-cells, which suggested that VP1 should be regarded as main candidate of new FMDV genetic vaccine. Also,The VP4, VP2, VP3 have some epitopes of the B-cells, but most of them cannot be regarded as predominant antigen epitope of the B-cells. These results would be helpful for developing new type genetic vaccine of FMDV.In experiment two, we construct the express box P1-2A-IL18 first. Using the special primers (Kozak sequence was designed in the upstream, an short amino acid Linker was designed in the downstream) to expand P1-2A gene by PCR. And then the mature protein gene of swine interleukin 18(sIL18) was inserted to the downstream of P1-2A.The box was inserted to the eukaryotic expression vector pVIR which was reconstructed by pIRESneo and pVAX1, in order to construct the recombinant DNA (rDNA) vaccine plasmid pVIR-P1-2A-IL18-3C, which containing the P1-2A gene, 3C proteinase gene and sIL18 gene. The analysis by PCR and IFA revealed that the P1-2A protein was detected in these HeLa cells transfected with plasmids carrying the structural protein P1-2A and sIL18.In experiment three, the transfering vectors pUTAL-P1-2A-3C- IL18 were constructed by inserted the gene expression box P1-2A-IL18 and proteinase 3C gene of FMDV into the FPV expression plasmid pUTAL. It was then co-transfected CEF cells by DOTAP Liposomal with 0.1 MOI of Fowlpox viruses (FPV) 282E4 strain. The rFPV-P1-2A-3C- IL18 strain was obtained by the drug BrdU selection and indirect immunofluorescence assay (IFA) assay. It indicate that the recombinant FPV was successfully designed and constructed.In experiment four, these two vaccines were tested by indicating the female Balb/c mices for induction of FMDV specific immune responses. Mice were immunized intramuscularly with the single or combined vaccines. And the FMDV specific antibody, cytotoxic T lymphocyte responses and T lymphocyte subpopulation were detected. The results indicated that the cellular immune response induced by rFPV combined with DNA vaccine was the highest one than that induced by single vaccines or inactivated vaccine groups. Although the combined vaccines could stimulate the mice to produce a weaker humoral immune response than inactivated vaccine group, there was not a significant difference about them.In conclusion, both of the vaccines have good immunogenicity, rFPV vaccines combined with rDNAs are the most idealist vaccination strategy. The study will provide valuable support for FMDV vaccine's further study.