Selection Of Ribozymes Against FMDV And Construction Of Recombinant Fibroblast Strains

Foot-and-mouth disease (FMD) is a highly contagious disease in the world wide. This disease has many characteristics including the very fast propagation, strong infection, high contagion of cloven-hoofed animals, and so on. The foot-and-mouth disease virus (FMDV) is divided into seven immunologically distinct serotypes and more than 80 subtypes. As these various types have no cross-immunity and mutated frequently, the disease often breakouts and results in a great loss in animal husbandry. At present, besides searching for new type vaccines, researchers are exploring other effective strategies against FMDV, such as transgene technology which can be used to improve the resistance of animals to the epidemic. Ribozyme is a kind of RNA enzyme, it can recognize RNA viruses specifically, inactive the virus and maintain the host cell mRNA. Because of these advantages, it makes ribozyme as a kind of safe and effective enzyme. Hammerhead ribozyme, as a member of ribozymes family, compares with other ribozymes, which has lower molecular weight, simpler structure and specific bonding site. Therefore, it has been used in gene therapy by many researchers and provided a new method for anti-FMDV. Maybe the animals could express the specific ribozyme gene for anti-FMDV by transgenetic method so that those could product the specific ribozyme by themselves to get the aim of defending the disease.According to the 2B, 3D gene of O-FMDV CHA/99 RNA secondary structure and the designing principles of hammerhead ribozyme, specific hammerhead ribozyme, which targets six catalytic locus in 2B and 3D gene fragments, were designed and synthesized. Recombinant expression plasmids pEGFP-N₁-2B and corresponding 2B ribozyme, pEGFP-N₁-3D and corresponding 3D ribozyme were cotransfected into BHK-21 cells respectively and the ribozymes which could effectively catalyse 2B or 3D were screened with these methods of fluorescence microscopy and flow cytometry. Detect the effection of anti-FMDV at the cellular level. The ribozyme screened was inserted into pGPU6/GFP/Neo plasmid and positive clone was named as pGPU6/GFP/Neo-D42. The goat fibroblasts cells transfected with pGPU6/GFP/Neo-D42 were screened by G418. After expanded culture, RT-PCR was used to detect the monoclonal cell strains.The transfected BHK-21 cells was observed by fluorescent microscope and results showed that D42 hammerhead ribozyme could abate fluorescence, which means that D42 could inhibit expression of pEGFP-N₁-3D. The catalytic efficiency of D42 reached 71% by identification of flow cytometry. Furthermore, at the same time, the number of abnormal cells in D42 transfected group is less than the control, which proves that D42 hammerhead ribozyme could inhibit the FMDV on the level of cells. Goat fibroblasts cells (GFC) were transfected with recombinant plasmid pGPU6/GFP/Neo-D42 and screened with 800μg/mL G418. After 12 days, a monoclonal cell was obtained, picked and subcultured. It is verified that D42 has been integrated into GFC successfully with RT-PCR.This paper used the principle of ribozyme catalysis to inhibit FMDV replication, and the positive cell strains, which can express D42 ribozyme, was screened by G418. The obtained cell strains provide the donor cell nucleuses for the nuclear transfer, and laid the foundation of cultivating anti-FMDV transgenic goat.