Construction And Ldentification Of Yeast Two-Hybrid CDNA Library Of MDBK Cells

Vesicular stomatitis (VS) is a virulence infectious disease which is caused by vesicular stomatitis virus (VSV), and this kind of disease can infect people and animals.The clinical signs of Vesicular Stomatitis are characteristics of water vacuoles and ulcers on the tongue, lips, cheeks, papillae, and coronet, and human can be infectied accidentally with flu-like symptoms too. The disease first broke out in the middle part of United States and North America then gradually spread to South America, Africa, Europe and some Asian countries and regions, and our country have reported the disease as well. In order to provide the theory of preventive treatment for the disease, the research of VSV molecular pathogenesis, particularly the study of a cell membrane receptor of VSV, has become the focus of research.The yeast two-hybrid system which was regarded as a simple and effective tool for studying protein-protein interaction has been widely used in virus research. CytoTrap XR yeast two-hybrid system(Sos-recruitment system) which was used in this study was based on the traditional yeast two-hybrid system. Comparing with the traditional yeast two-hybrid system, the advantage of this system is that the detected interaction between proteins was occurred in the cytoplasm rather than nucleus. It is more suitable for the study of transcription factors and the proteins that require post-translational modification in the cytoplasm. CytoTrap XR yeast two-hybrid system provide a more accurate, simple and efficient method for studying protein-protein interaction in vivo.According to the broad cell tropism of VSV, the MDBK cells was artificial infected by VSV, meanwhile, the apparent CPE of MDBK cells was observed by inverted microscope. The typical bullet-shaped virus particles was observed by electron microscopy negative staining. These results confirmed VSV can inflect MDBK cells and also can propagate on them. The indirect immunofluorescence showed that the green fluorescence was appeared in the cytoplasm of MDBK cells VSV. It proved that VSV was proliferated in the cytoplasm of MDBK cells.We can speculate that the specific receptors of VSV exist on the surface of MDBK cells membrane according to the fact that VSV can infect MDBK cells and produce a typical CPE. In the research the total RNA was isolated from MDBK cells, and the mRNA was purified, which the cDNA with restriction enzyme cutting sites was reversed. The MDBK cells yeast two-hybrid cDNA library was also built by Using CytoTrap XR library construction kit in the study. It was identified that the original library contained 3.5×105 clones, and the amplified library contained about 6×108 clones, and approximately 95% of the library were recombinant. It was also identified that the cDNA fragmengts are larger than 500bp, and the average insert size was about 800bp. It proved that the MDBK cells yeast two-hybrid cDNA library was successfully constructed which establish a solid foundation for screening the membrane receptors and further studying the molecular pathogenesis of VSV.