Screening Of Proteins Interacting With Streptococcus Equi Ssp. Zooepidemicus M-Like Protein In Swine Vascular Endothelial Cells

Streptococcus equi ssp. zooepidemicus is the main pathogen in China which made substantial economic losses for pig industry. M-like protein is a major surface protein that conveys the resistance to phagocytosis and it is a virulent factor with opsonsin function. In the present study, we used a yeast two-hybrid system to screen the interacting proteins with M-like protein from swine umbilical vein vascular endothelial cells(SUVECs) full-length cDNA library in order to get new insight into the pathogenic mechanism of this bacteria.Total RNA was extracted from SUVECs and mRNA was purified.The first-strand cDNA was synthesized using switching mechanism at 5 end of RNA transcript(SMART) technology,and double strand cDNA was synthesized with LD-PCR.Then double strand cDNA was cut by SfiⅠrestriction enzyme and ligated with pGADT7 AD vector.The recombinant vector was transformed into Escherichia coli DH10B by electroporation to construct full-length cDNA library.The library quality was evaluated and the result showed that the titer of primary cDNA library were 1.01×107 CFUs,the recombination rate was nearly 100%,the inserted fragments were distributed from1.8-4.4kb,and average size was 2.7kb,the library was of high quality for cloning target genes and expressing target proteins.Interacting proteins with M-like protein from SUVECs full-length cDNA library were screened by a yeast two-hybrid system. Combining the bait strain Y2HGold[pGBKT7-szp] with SUVECs cDNA library strain Y187[pGADT-cDNA] was to form diploid yeast and blue colonies were screeed on a series of nutrition-selective plates with X-α-gal and Aureobasidin A. Plasmids in blue colonies were extracted and transformed into E. coli DH5αto amplify the cDNA library plasmid. Yeast two-hybrid retransformation experiment was applied to confirm the positive interactions. The result indicated that there is no existence protein from the full-length cDNA library interacting with M-like protein.