Screening And Identification Of Cellular Proteins Interacting With 3AB Protein Of Coxsackievirus A16

Coxsackievirus A16(CVA16)is one of the main causative pathogens of hand,foot and mouth disease(HFMD).Mainly infected infants under 5 years of age,symptoms mainly have hand,foot,mouth and other parts of the emergence of herpes,spot,individual cases of aseptic meningitis,encephalitis,myocarditis,etc..CVA16 genome RNA encoding an open reading frame,sub P1,P2 and P3 three regions,P2 encoding the non structural protein 2A,2B,2C,P3 encoding of non structural protein 3 A,3B,3C and 3D.Non structural proteins of the virus play many roles in the process of virus infection.In this paper,yeast two hybrid technique was used to screen proteins interesting with and precursor protein 3AB of 3 A and 3B to further understand the function of 3AB protein and the role of CVA16 protein in the process of infection of host cells.The study consists of two parts:1.Screening of Proteins Interacting with 3AB by Yeast Two-HybridPCR was performed to amplify the gene of 2A,2B,2C,3 AB,3C and 3D from the plasmid pMD18-T-F4,pMD18-T-F5,pMD18-T-F6 and pMD18-T-F7;the PCR product was cloned into yeast expression plasmid pGBKT7-BD.The reconstituted plasmid was electrotransformationed into Y2HGold yeast competent cell,screening and identifing positive clone.Cytotoxicity of the bait protein were detected by cultured in different SD.The expression of bait protein was analyzed by Western Blot.Then transcriptional activation of the bait protein were detected by cultured in different SD.Screening of Proteins Interacting with bait protein by Yeast Two-Hybrid.The diploid cells were plated on DDO/X/A,blue colonies were picked to QDO/X/A plate.The plasmid of blue colonies were transformed into DH5a,plating on Amp+ and Kan+resistant plate,sequencing the plasmids.Library plasmids and bait plasmids were co-transformed into Y2HGold and plated on QDO/X/A.The plasmids of blue colonies were sequenced and analysed by NCBI database blast and identified one target proteins:Acyl-CoA binding domain containing 3(ACBD3).2.Identification of the interaction of 3AB and ACBD3 by Co-IPThe eukaryotic expression vector of bait and target protein genes,pCDNA3.1-3AB-2 X Flag and pCDNA3.1-ACBD3-2 X HA,were constructed respectively.Two eukaryotic expression vector were transiently transfected into HEK293T cells,the expression of 3AB and ACBD3 gene were detected by Western blot.3AB and ACBD3 expressed correctly.Two vector were co-transfected into HEK293T cells,the interaction of 3AB and ACBD3 was identified by Co-IP.Based on Yeast Two-Hybrid,we found one protein ACBD3 interacted with 3AB.Reply verification were correct.3AB and ABCD3 expressed correctly in HEK293T cells.The interaction of 3AB and ABCD3 were verified by Co-IP in HEK293T cells.This is helpful for exploring the functions of 3AB and the role of ACBD3 in the CVA 16 infection process.