Establishment And Identification Of Anti-Foot-and-Mouth Disease Transgenic Pig Fibroblasts Cell Lines

Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus is one of the most animal spirited contagions, which main infection cows, goats, sheep and cattle, pigs livestock,and it is difficult to control once the outbreak of disease trends in branding due to susceptible animals are widely distributed and dense. Major economic losses and political impact was caused after each time of FMD outbreaks. Therefore, it is one of infectious disease on the list of World Organization of Animal Disease (OIE) which must be reported, also known as the "economic and political disease". The disease is not only a serious threat to the healthy development of animal husbandry, and cause huge economic losses for countries or regions which was infected by epidemic disease , but also a social public health problem in countries of concern to a main problem of food safety.7 serotypes of FMDV including A, O, C, Asia 1, South Africa-based (SAT1, 2, 3) have been identified till now, and serotype distribution has a certain geographic regional world widely. Serotype O, A and Asia I of FMDV were major epidemic in our neighboring countries and Southeast Asia, which threaten the development of animal husbandry in our country at any time. In developed country, Take killing all infected animal can effectively purify pathogen, but its cost highly. Universal immunization method to prevention and control of FMD is mainly taken in most developing countries. Inactive incompletely and live virus escape and other security risks were main obstacles to inhibit the useful of FMDV inactive vaccine used widely in China.In deed to solve the lacks of methods on prevent and control foot and mouth disease currently, we had screened three efficient anti-foot-and-mouth disease virus-related genes: swine broad-spectrum anti-viral interferon-γ(IFN-γ) gene, anti-mouth disease virus single chain antibody gene (scFv) and swine IFN-γ/scFv fusion gene. Three strains recombinant retrovirus highly express anti-FMDV genes were constructed respectively. Then three anti-FMDV swine somatic cell lines with transgenic anti-FMDV gene were selected respectively and product transgenic pigs against foot and mouth disease with transgenic and somatic cell cloning technology.We choose to use retroviruses vectors systems constructed basis on Moloney Murine Leukemia Virus (MMLV). The recombinant retrovirus vectors pFB-IFN,pFB-scFv,pFB-Is and pFB-EGFP was constituted by inserting anti-FMD virus-related genes including IFN-γin swine ,single chain antibody gene scFv, swine IFN-γ/scFv fusion gene and contrast gene EGFP in to the vector pFB-neo derived from the MMLV system type (cis) function components (including LTR, packaging signal etc). The expression vector pVPack-GP and pVPack-10A1 was constituted by cloning express structure protein gag-pol and envelope protein env gene to pVPack expression vector respectively, the helper virus plasmid of packaging do not contain LTR and packaging signal sequence. The recombinant retroviral vector and two helper virus plasmid of packaging were co-transfected into packaging cell line HEK-293T, and then can express virus structure proteins and packaging signals, retroviruses can smooth produce. The recombinant virus containing anti-FMDV gene and contrast gene was collected and infected pig fibroblasts cells. These anti-FMDV gene and control gene was integrated into the cell genome DNA by recombinant virus infected cells with the formation of reverse transcription cDNA. The green fluorescent protein was observed under fluorescence microscope, the positive resistance cells were screened by G418. When it appears a single cell clones by screened, expand training until the growth to be in good growth to be in good condition. One part of the cells were used to extract cell genomic DNA, and then verify the integration situation for anti-FMD virus-related genes by PCR method. The other part of the cells were infected with FMD virus type A, compared with the killing rate between positive cells and positive cells and by MTT method, to verify cell function for resistance to foot-and-mouth disease.After swine fibroblast cells infected by recombinant virus which carrying control EGFP gene, green fluorescent protein was observed with fluorescence microscopy, indication successful packaged recombinant virus could enter the cells and integrated into cell genome. Genome of swine fibroblast cells with anti-FMDV gene was extracted for verify the integration of gene by specific PCR method, and then the positive results indicate construction complete of anti-FMDV swine fibroblast cells. The anti-FMDV activity of anti-FMDV swine fibroblasts cells was measured by MTT method compared with swine normal fibroblasts WMP cells, both infected by FMDV type A. Our results shown that, anti-FMDV activities were performed by WMP-IFN, WMP-scFv and WMP-Is in different levels respectively. In addition, the activity of anti-FMDV was on a reverse dose corresponding relationship with WMP-IFN, WMP-scFv and WMP-Is with increasing of FMDV infection dose in a certain period. The effective of anti-FMDV was on a period corresponding relationship with WMP-IFN, WMP-scFv and WMP-Is with prolonging of infection period in a certain infection dose. These results indicate that, anti-foot and mouth disease transgenic pig fibroblast cell line with anti foot and mouth function was established by the retroviral vector system in this study, which could inhibit the infection caused by foot and mouth disease virus in the condition of a certain infection dose and time period. That will be helpful for breed transgenic pigs that could be integrate with somatic cell cloning technology effectively, in deed against foot and mouth disease in future..