Construction Of Yeast Two-hybrid Bait Plasmid With Pseudorabies Virus EPO Gene And CDNA Library From PK-15 Cells

Pseudorabies(PR)is an acute infectious disease characterized by Pseudorabies Virus(PRV),which causes a variety of livestock and wild animals with fever,itching,encephalomyelitis,abortion and death.Pigs are the storage host and the main source of infection,when pigs infected with PRV manifested as suckling pigs died,fattening pigs growth stagnation and pregnancy sows breeding disorders.The disease in Europe,Asia and other countries were spreading trend,China is particularly serious.PR endangering the world porcine culture and make great economic losses in the world.PRV is a double-stranded DNA virus,the size is about 150 Kb,and it can encode 70 to 100 proteins.EPO protein is PRV early protein,and it contains zinc finger structure similar to the ring finger domain,the domain is mainly a combination of DNA,RNA and protein interaction between regions.It is presumed that EPO plays an important role in protein-protein interaction terms.The yeast two-hybrid bait plasmid with pseudorabies virus EPO gene and PK-15 cells cDNA library would be a valuable resource for interaction mechanism between PRV EPO protein and host cell PK-15.The detail research contents are as follows:1.Construction of bait plasmid pGBKT7-EP0Amplification of EPO gene by PCR and cloned into the yeast two-hybrid vector pGBKT7,the recombinant bait plasmid pGBKT7-EP0 by restriction endonuclease EcoR I and Pst I double enzyme digestion and gene sequencing analysis,revealed the successful construction of bait plasmid pGBKT7-EP0.The bait plasmid was transferred into Y2HGold yeast cells,and after toxicity test,self-activating effect detection,yeast DNA extraction and Western-blot detection,the results show that the bait plasmid expressing effect on yeast cell non-toxic,no since the activation effect,while PCR and Western blot test results showed that the bait plasmid pGBKT7-EP0 was successfully transferred into yeast cells and the BD-EPO-Myc fusion protein was successfully expressed in this experiment.2.Construction and identification of PK-15 cells cDNA libraryThrough PRV host cells PK-15 cells to construct a yeast two-hybrid cDNA library.In this study,the total RNA was extracted from PK-15 cells,and the full-length double-strand cDNA was synthesized by SMART and LD-PCR,which was ligated to three-frame expression vector after nomalization and Sfi I digestion,respectively.The ligation product was transformed into E.coli Electro-cell to construct primary three-frame expression library.The titer of three frame libraries were 3.0× 106CFU,2.0×106CFU,and 2.0×106CFU.The insert the fragments are more than 500 bp,the recombination rates of three frame libraries are more than 93%.Three primary libraries were mixed and amplified,transformed into E.coli Electro-cell,extraction of plasmid library.At last,the plasmid library was transformed into Y187 yeast competent cells,the cDNA library capacity was 7.5 × 105 CFU,which was consistent with the requirement of yeast two-hybrid cDNA library construction.In conclusion,the bait plasmid pGBKT7-EP0 and the yeast cDNA library were successfully constructed would be a valuable resource for interaction between PRV EPO protein and PK-15 cells protein.