Construction And Preliminary Identification Of Yeast Two-hybrid Bait Plasmids Carrying Vesicular Stomatitis Virus Glycoprotein CDNA Fragment

Vesicular stomatitis (VS) is caused by vesicular stomatitis virus (VSV). VSV causes acute febricity. VSV can infect many animals (for example cattle, horses, pigs and deer) and sometimes people can be infected with dengue fever and acute fever flu-like symptoms (zoonosis). The clinical signs of Vesicular Stomatitis (VS) are characteristic of water vacuoles in mucosal membranes of mouth, tongue, lip, papillae and coronet. The disease first occurred in the middle of United States and North America, then spreading to South America, Africa, Europe and Asia, some countries and regions later.At present, the studies receptor for VSV remain unclear.Whether the phosphatidylserine is receptor for VSV is not clear.There is a lot of controversy.Therefore, studies the receptor for VSV will become the focus on research. in the future.Since the Human Genome Project is completed, at present,researchers proceed in researching proteomics that has become a focus of research in the field.People are more concerned with understanding the role of protein, and the current many the technologies can be applied to study proteins and protein interactions. For example, bioinformatics analysis, phage display, protein chip technology, biological mass spectrometry and yeast two-hybrid system.,etc. Among them, since the yeast two-hybrid technology is invented,its own advantages, which is very popular and has become an important method to study proteins interaction.Beacause of the traditional yeast two-hybrid protein interaction occurred in the nucleus, so cytoplasm protein study is restricted. The base on traditional yeast two-hybrid technology, it developed a derived from yeast two-hybrid system, named Sos-recruitment system(SRS). It can study the proteins interaction in the cell membrane location , overcome the traditional yeast two-hybrid system limitations, and more have high sensitivity and less false positive advantage, which extends the yeast two-hybrid applications. In this study ,used Sos-recruitment system (SRS), first of all, designed primers for the vesicular stomatitis virus glycoprotein gene , using the method of PCR amplification of fragments required.Through two twice clone, vesicular stomatitis virus glycoprotein gene expression vector was introduced into pSos vector, recombinant plasmid is named pSos-VSV-G1243. By the identification of PCR methods, restriction enzyme digestion method to identify , sequencing results of nucleotide showed that recombinant plasmid bait was successful constructed.To detect recombinant plasmid whether can be self- activation and non- toxic for yeast strains, the results indicate that the bait plasmid is not self- activation, yeast cdc25H (α) strains can grow well when are transferred bait plasmid (pSos- VSV-G1243). The results indicate bait plasmid (pSos- VSV-G1243) is non-toxic for yeast cdc25H (α) strains. Protein extract are carried out SDS-PAGE analysis, Western blot analysis, the results indicate the bait plasmid can success to express fusion protein in the yeast cytoplasm . The above results show that the recombinant plasmid bait (pSos-VSV-G1243) can be used for SRS screening system , for the further to fish the VSV receptor from the cDNA library of sensitive cell.