The Research Of Molecular Interaction Among PIFs Of ClanGV

Baculovirus has two different morphological and functional virus particle: Budded virus(BV)and Occlusion derived virus(ODV).Early studies indicate ODV invades into the midgut epithelial cells by membrane fusion,implying that there is a kind of membrane fusion protein on the surface of ODV.Present study confirmed oral infection of insect virus was related with special proteins,whose deletion or mutation will lead to a decrease of oral infection.So far it has identified seven core protein(pif0?pif1?pif2?pif3?pif4?pif5 ?pif6)closely related to oral infection in baculovirus family,which function as a complex during infection of intestinal epithelial cells in NPV family.As an important member,owing to no appropriate cell lines,the molecular biology of GV is far behind NPV.The study of molecular biology of GV contribute to understand the infectious mechanism of Baculovirus comprehensively so as to serve the life of human better.Clan GV PIFs is the object of this study,and yeast Two-hybrid systerm was used to verified interaction among PIFs.In this study,Bi-directional interaction was conducted,which means that two proteins was used as bait and prey respectively.In order to verify whether each PIF interacts with itsself by forming a dimer or polymer on the surface of virus or not,the interaction was conducted with each protein used as bait and prey simultaneously.Details are as follows:One: Primers were designed using PIF0 ~ PIF6 sequence as a template cutting off the transmembrane region.the target gene fragment was amplified by PCR.PGADT7(prey)and PGBKT7(bait)recombinant vectors of seven target gene fragment were constructed by homologous recombianation.Two:Self-activation of every bait was conducted.The result indicate that PGBKT7-PIF5 of seven bait has self-activation.when we study the interaction between PIF5 and other PIFs,the interaction was conducted merely with PIF5 as the bait.Three:PGADT7 and PGBKT7 recombinant vectors with different target gene fragment were co-transformed into yeast AH109,then applying them on SD / Trp-Leu,SD / Trp-Leu-His-Ade medium.Deficient medium SD / Trp-Leu was used to verify the efficiendy of co-transformation.TO further validate the results,positive colonies on SD / Trp-Leu-His-Ade was conducted a PCR and X-?-Gal chromogenic verification.The results demonstrated that PIF1~ PIF4 was interact with each other Bi-directionally by yeast two-hybrid,and they can interact with theirselves by forming a dimer or polymer.Unidirectional interaction are as follows:PGBKT7-PIF0+PGADT7-PIF3,PGBKT7-PIF0+PGADT7-PIF5,PGBKT7-PIF6+PGADT7-PIF3,PGBKT7-PIF6+PGADT7-PIF4,PGADT7-PIF5+P GBKT7-PIF1,PGADT7-PIF5+PGBKT7-PIF3,PGADT7-PIF5 + PGBKT7-PIF4.In summary,we hypothesize that PIF1 ~ PIF4 of GV may form a stable core of protein complex,and each PIF may form a dimer or polymer in the complex.A preliminary understanding of protein complexes of GV was gained,laying a solid foundation for the further study of intestinal infectious mechanism of GV ODV mediated by PIFs...