The genetic engineering subunit vaccine of FMDV mainly uses various expression systems to express VP 1 protein and make vaccines. Kupper et al. (198 1) cloned the FMDVVP 1 gene and inserted it into the downstream of the PL promoter, thus realizing the prokaryotic expression of the VP 1 gene. Indirect ELISA and radioimmunoassay confirmed the antigenicity of the expressed product, which provided a theoretical basis for the development of FMDV genetic engineering subunit vaccine. In the same year, Kilde immunized pigs and cattle with FMDVVP 1 Protein A expressed in E.coli, which could induce the production of neutralizing antibodies. Cows can resist the attack of FMDV by using high concentration of VP 1 protein or repeatedly inoculating cows. Morgan confirmed that pigs inoculated with A 12-32 dimer can also produce high levels of neutralizing antibodies, which can protect pigs from virulent viruses, but this technology is not suitable for O-type FMDV. It has been found that the tandem expression of structural genes and nonstructural genes 2A and 3C of FMDV can produce 76S virus-like particles. The purified virus particles can be used to immunize animals, and its immune effect is similar to that of the whole virus, and it can produce high-level neutralizing antibodies, which can resist the attack of virulent viruses and completely solve the virus spread risk of conventional FMDV vaccines, showing its good prospects for many years and months. In addition, VP 1 protein was expressed by yeast and baculovirus system, which solved the problem that VP 1 protein was not modified in prokaryotic expression system to improve its immunogenicity. Edible vaccine is to introduce immunogenic genes into plants by using Pseudomonas aeruginosa or gene gun technology to obtain plants expressing immunogenic proteins.
FMD transgenic plants can be fed with vaccines, which is one of the early studies and good examples. As early as 1998, Carrillo et al. obtained transgenic Arabidopsis thaliana with the main protective antigen gene VP 1 of FMDV. Mice immunized with leaf extract can induce specific antibodies, and all immunized mice can resist the attack of lethal dose FMDV. This is the first report to protect all immunized animals with virus antigen expressed by transgenic plants, and the primary immunization dose is only 25-50 mg of leaves. 1999, Wigdorovitta et al. successfully obtained transgenic alfalfa with alfalfa as recipient material. Mice immunized with 15-20mg can resist the attack of lethal virulent virus 100%. Carrillo et al. successfully obtained transgenic potato expressing VP 1 with potato as recipient material, and animal experiments confirmed that immunized mice could also resist virulent virus. The successful research of FMDV transgenic plant feed vaccine will bring bright prospects for the prevention and treatment of FMD in herbivores such as cattle and sheep. Synthetic peptide vaccine is a vaccine made of epitope peptide synthesized according to the amino acid sequence of immune epitope. Generally, the amino acid sequence of the main epitope of protein immunity is deduced from the analysis of the primary structure of protein and monoclonal antibody, and then the peptide is synthesized or expressed as antigen by genetic engineering.
The 40 amino acid peptides (cysteine-cysteine-200-211-158 and 200-2 13- proline-) synthesized by Dimarch from the fragment of O 10K virus. Cysteine-glycosidic acid), in which two prolines and one serine are added, and the polypeptide is folded into a three-dimensional configuration, which improves the reaction level of single-segment peptide of guinea pig (14 1- 158- proline-cysteine-glycosidic acid) and proposes N-terminal amino acid (cysteine-cysteine). Brown et al. synthesized FMDVVP 1 encoded 140- 160 and 200-2 13 peptide gene fragments by chemical method, and expressed them in Escherichia coli, and obtained good immunity by immunizing cattle and pigs with them. Doel used 40 aa synthetic peptides composed of 14 1- 158 and 200-2 13 peptides of serotypes A, O and C FMDVVP 1, respectively, and tested them in cattle and guinea pigs. As a result, each peptide produced a high level of antiviral neutralizing antibody. In guinea pig, O type and A type, the full-length cDNA of FMDV was cloned by genetic engineering, and infectious clones were constructed. RNA can be obtained at DNA level, and its virulence can be weakened without losing its immunogenicity by deleting genes related to virulence.
Both FMDV virus and Mengo virus are small RNA viruses. People shortened polly(C) fragment of Mengo virus by DNA recombination technology, and formed a mutant. This mutant is harmless to mice. It can produce high-level antibodies in mice and protect them from the attack of virulent viruses. Therefore, the researchers tried to learn from the experience of Mengo virus and shorten the FMDVpoly(C) fragment. Although the virulence of FMDV mutant with shortened poly(C) fragment has not been weakened, it has given some enlightenment to people, and it is very possible to construct a weak FMDV strain with specific site deletion in FMDV genome.
X-ray crystal analysis shows that FMDV has a certain three-dimensional conformation, and its surface is composed of VP 1-VP3. In a highly variable region of VP 1, a highly conserved G-H-loop amino acid sequence is exposed on the surface of virus particles, including arginine-glucoside-aspartic acid (RGD) sequence, which constitutes the cell adsorption site of the virus. Ochoa et al. found that the monoclonal antibody against VP 1 could produce a certain virus agglutination process and strongly inhibit the adsorption between virus and cells when studying the crystal structure of the peptide synthesized by G-H loop, the main antigenic site of FMDVVP 1 gene fragment. Acharya et al. also found an infectious cDNA clone containing RGD sequence, which made it possible to prepare virus particles with RGD deletion or mutation. Mei Sen constructed FMDV mutant by replacing amino acids in RGD sequence of virus, which made the virus unable to adsorb and infect cells. All the above studies have confirmed that RGD sequence is necessary for virus to adsorb host cells. Mckenna et al. replaced the RGD coding sequence of wild-type SGSGVRGDFGSL with SGSNPGSL sequence to construct RGD deletion virus. This virus particle lacking RGD sequence will not adsorb and infect cells. Animal experiments with deleted virus in mice and pigs show that the wild-type virus control group has typical FMD symptoms, while the experimental group has no symptoms. After 28 days of inoculation, the blood samples of these animals were monitored for immunoprecipitation. The control group showed strong structural protein activity, but no unstructured protein activity was detected, which proved that wild-type virus could replicate in animals, while constructed virus could not replicate. Compared with the oil made from Hereford cattle, it is proved that it is consistent with the inactivated vaccine in producing serum neutralizing antibodies, stimulating immune response and animal protection, and some of it is better than the inactivated vaccine. Nucleic acid vaccine, also known as gene vaccine, is to put the gene encoding the immune protective antigen protein of pathogen under the control of true expression elements, introduce it into animals, and synthesize the antigen protein through the transcription system of host cells, thus inducing the host to have an immune response to the antigen protein. Because there are many serotypes of FMDV, there is no cross protection between them, which brings great difficulties to the imitation of FMD. In 1990s, with the emergence and perfection of the concept of genetic immunity, it brought opportunities for FMDV immunization. Benvenisti connected the complete structural gene P 1 of FMDV with the nonstructural genes 2A and 3CD, and added the internal ribosome entry (IRES) of encephalomyocarditis virus (EMCV). It was detected in pig skin by immunofluorescence and western blot, and some pigs gained resistance to the virulent attack of FMDV. Shieh did not overcome the problem that subunit vaccine could not produce lasting immune protection. The combined immunization of gene immunization and subunit vaccine enhanced the immune effect. First, mice were immunized with a plasmid containing VP 1, and then stimulated with VP 1 polypeptide conjugate (P29-KLH). Immunized mice produce high titer antibodies, which have the activity of neutralizing FMDV.
In short, the ideal vaccine must be safe, effective, cheap and easy to popularize. Although inactivated FMDV vaccine has good immunogenicity, its potential insecurity affects its use. In addition, due to the high preparation cost and high price of inactivated vaccine, the popularization of vaccine is limited. The gene vaccine has the advantage of high safety, and Togo component of the same virus or multivalent virus vaccine can be prepared as required, thus greatly saving the production cost. Therefore, FMDV memory engineering vaccine is the future development direction.