Study On Construction And Immunity Of Recombinant DNA Vaccine And Fowlpox Virus Vaccine Against Foot-and-Mouth Disease Type Asia 1

Foot-and-mouth disease (FMD) caused by FMD virus (FMDV) is one of the most contagious animal virus diseases. It is on the A list of infectious disease of the World Organization of Animal Disease (OIE). At present, FMD is prevalence in all at of the world and it is also one of the most important infectious animal diseases in China. The FMD virus exists in the form of seven different serotypes: O, A, C, Asia1, and South African Territors1 (SAT1), SAT2 and SAT3. Historically, FMD Serotype Asia1 had been spread in part of China. Now it is still prevalence in the many countries around China and Southeast Asia. In March 2005, the first outbreak of FMDV type Asia 1 in China was reported in Hong Kong SAR. Subsequently, disease was spread to mainland of China in April 2005. Up to September of 2006, there were more than 15 outbreaks in mainland of China. The disease poses a serious threat for animal health and exacts an economic toll on the livestock Industry.Vaccination is the most reliable and effective approach for prevention of FMD. Nowadays widely used in the prevention of FMD vaccine is inactivated vaccine. However, researchers found that there were serious security risks in inactivated vaccination. Therefore, the development of a safer, effective FMD vaccine has become a new focus on growing attention by researchers.In this study, a FMDV type Asia 1 was isolated from vesicular liquid and vesicular epithelium and designated as Asia1/JL/05, and the P1-2A gene of Asia1/JL/05 was cloned and sequenced. Then a plasmid (pVAX1P1-2A-3C ) was constructed, which contains all of the structural and non-structural proteins necessary for the formation of FMD type Asia 1/JL/05 empty capsids, including the P1 sequence plus the 2Apro and 3Cpro proteases necessary for processing of the P1 polyprotein into VP1, VP3 and VP0. Furthermore, recombinant fowlpox virus of Asia 1 FMDV (rFPV-vUTAL-P1-2A-3C-IL18) and recombinant fowlpox virus, vUTAL-PI-2A-IL18-P1-2A-3C, contained gene of P1-2A-3Cof FMD type Asia 1 and gene P1-2A of FMD type O and interleukin 18 of swine were also obtained, respectively. At last, animal experiments was employed to evaluate the immunogenicity of pVAX1P1-2A-3C and vUTAL-P1-2A-3C-IL18 and vUTAL-P1-2A-IL18-P1-2A-3C in mice and guinea pigs. To describe is below.At first, a FMDV serotype Asia1 named Asia 1/JL/05, in vesicular liquid and vesicular epithelium of the tongue was identified by indirect hemagglutination assay and RT-PCR, and isolated by culture in suckling mice. This FMDV Asia 1/JL/05 strain was carried out serial passage culture by suckling mice continuously 4 times .The results indicated that the FMDV Asia 1/JL/05 strain was adapted in suckling mice. The titer of FMDV Asia 1/JL/05 strain was above LD50108.0. And the FMDV Asia 1/JL/05 strain was also cultured on baby hamster kidney cell(BHK-21)by serial passage. The results indicated that it could adapted well on BHK-21 cells and the titer could reach TCID5010-5.9/0.1mL。The viral structural protein precursor P1–2A of 2223bp and the non-structural proteins 3C (proteinase) fragments of 639bp of FMDV type Asia 1/JL/05 were amplified by RT-PCR from vesicular fluid, respectively. The phylogenetic tree shows that JL/05 has the closest relationship to Jiangsu/05. The 3C amino acids sequence of FMDV type Asia 1/JL/05 has above 97% sequence homology with FMDV type O and sub-serotype Asia 1.The plasmid of pVAX1P1-2A-3C, which contained genome of the structural precursor protein P1 and non-structural protein 2A and protease 3C of FMD type Asia 1/JL/05 empty capsids was constructed. The expression of P1-2A in HeLa cell was demonstrated by western blotting, IFA and RT-PCR. The mice were inoculated with the plasmid of pVAX1P1-2A-3C and produced specific humoral and cellular immune responses.The fowlpox virus shuttle plasmid pUTAL, which was composed of the combined promoter ATI-P7.5 (ATI promoter of cowpox virus and 20 tandemly repeated mutant P7.5 early promoters of vaccinia virus), and the LacZ gene controlled by the single promoter (16 tandemly repeated mutant P7.5 early promoters of vaccinia virus) as a reporter gene, was used in this study. First, an IL-18 gene was constructed replacing the LacZ reporter gene of pUTAL. Subsequently, the P1-2A precursor protein coding region of FMD type Asia 1/JL/05and 3C protease cassette P1-2A-3C was cloned into the multiple cloning sites under the combined promoter ATI-P7.5 of pUTAL-IL-18, and the shuttle plasmid pUTAL-P1-2A-3C-IL18 was obtained. Before the above cassettes were constructed, amplified regions of IL-18 and P1-2A-3C were sequenced to verify their integrity and validity. Then, the fowlpox virus shuttle vector pUTAL-P1-2A-3C-IL18 was cotransfected with fowlpox virus 282E4 into the CEF. The recombinant fowlpox virus (vUTAL-P1-2A-3C-IL18) was selected 3 passages by culturing in CEF with MEM medium containing BrdU. Experimental immunization in mice and guinea pigs showed that, the mice inoculated by vUTAL-P1-2A-3C-IL18 produced anti-FMD type Asia 1/JL/05 specific humoral and cellular responses, and the guinea pigs inoculated by vUTAL-P1-2A-3C-IL18 also produced the non-special (Stimulated by ConA) and specific (Stimulated by virus) T lymphocyte proliferation and special neutralizing antibody of anti-FMD type Asia 1/JL/05. The three of four guinea pigs inoculated with vUTAL-P1-2A-3C-IL18 was completely protected after challenged with homology virulent FMDV type Asia 1/JL/05.The cassette P1-2A-IL18, including structure precursor protein P1 encoding gene and non-structure 2A encoding gene of FMDV type O/LZ and swine interleukin 18 encoding gene, was constructed replacing the LacZ reporter gene of pUTAL. Subsequently, the P1-2A precurosor protein coding region of FMD type Asia 1/JL/05and 3C protease cassette P1-2A-3C was cloned into the multiple cloning sites under the combined promoter ATI-P7.5 of pUTAL-P1-2A-IL-18, and another shuttle plasmid pUTAL-P1-2A-3C-P1-2A-IL18 was obtained. pUTAL-P1-2A-3C-P1-2A-IL18 was also cotransfected with fowlpox virus 282E4. Another recombinant fowlpox virus (vUTAL-P1-2A-3C-P1-2A-IL18) was obtained. The expressions of P1-2A of FMDV type Asia 1/JL/05 and FMDV type O/LZ in CEF were indentified, respectively. The mice were inoculated with vUTAL-P1-2A-3C-P1-2A-IL18 and produced specific humoral and cellular immune responses to FMDV type Asia 1 and type O.The vUTAL-PI-2A-IL18-P1-2A-3C was administered into quadriceps femoris of mice with alone or with another vUATL3CP1 together, which expressing P1-2A and 3C genes of FMDV O/NY00. The anti-FMDV type O and anti-FMDV type Asia 1 of specific antibody of mice was detected by I-ELISA from mice inoculated with vUTAL-P1-2A-IL18-P1-2A-3C or vUTAL-P1-2A-3C-IL18 plus vUATL3CP1. The anti-FMDV type O and anti-FMDV type Asia 1 of specific splenocytes proliferative activity of mice inoculated was also detected by MTT and ELISPOT. The results indicated that the mice was inoculated with vUTAL-PI-2A-IL18-P1-2A-3C, alone, producing higher levels of specific humoral immune and cellular immune against FMDV type Asia 1/JL/05 and FMDV O/LZ, than the ones inoculated with vUTAL-P1-2A-3C-IL18 and vUATL3CP1together.The results of immunity experiment indicated that plasmid pVAX1P1-2A-3C, recombinant fowlpox vUTAL-P1-2A-3C-IL18 and recombinant fowlpox vUTAL-P1-2A-3C-P1-2A-IL18 would be potential multivalent vaccine candidates.