Host And Human Astro Virus Nsp1a/3 Protein Interaction Screening And Polyclonal Antibody Preparation

Astrovirus(Astrovirus)is a patihogen that infection mammals and birds,belonging Astroviridae(Astroviridae),and RNA genome is non-segmented,non-enVeloped.Human astrovirus(Human Astrovirus;HAstV)genome contains three open reading frame and coding for nonstructural proteins nsP1a,nsP1b and capsid protein Astrovirus infection of host through the fecal-oral route,the main cause of diarrhea in infants and children,abdominal pain and other symptoms,is the second pathogens of which can cause viral diarrhea in children,HAstV global infection rate of about 10%.According to China of seven regional perspective from 1998 to 2005,the HAstV infection rate was 5.5%,some in high season up to 36.2%while the fulminant diarrhea virus detection rate is as high as 70%.HAstV divided into eight serotypes which according to ORF2 protein area nucleotide fragment length 348bp.Currently,the advantage of epidemic strains in china is HAstV type I.Currently,due to less research on human astrovirus,viral infection and replication mechanism is unclear,that non-structural proteins in the viral infection and replication process play an important role usually.In the infection early of HAstV that nsPla protein were cut into four segments by proteases,which nsP1a/3 containing protease motif.Typically,the process of viral infection and replication,the target protein of host cell and viral proteases interactions,in such as HCV protease.Hydrolysis polyprotein precursor is a key in the process of human astrovirus replication,this process need the hydrolysis of the ORF la-encoded protease,because nsPla/3 containing a protease motif and likely play an important role in this process,but nsPla/3 interacting with the target protein of host cell is unclear.This paper is intended to study nsPla/3 with the target protein of host cell,screening protein expression at the cellular and the target protein immunized-rat,preparation polyclonal antibodies,which lay a foundation for further study the interaction of HAstV risP1a/3 with target proteins.The study consists of three parts:1,Host and nsPla/3 protein interaction screeningThe nsP1a-pEGFP-N2 as template,PCR amplification nsP1a/3 gene,the gene is inserted into yeast bait vector pGBST7.nsPla/3-pGBST7 electricity transformed into Y2HGold yeast competent The cytotoxicity of the expression products of the recombiant plasmid nsPla/3-pGBST7 and the autoactivation of the report gene in yeast transformants were examined.Then,by the way of Y187 and Y2HGold co-cultured,after the diploid cells were plated SD/-2/X/A,blue colonies on the plate were picked to SD/-4/X/A tablet,the plasmid of blue colonies transformed DH5μ,plated Amp+ and Kan+ resistant plate,sequenced the plasmids.Library and bait plasmids were co-transformed into Y2HGold and S plated SD/-4/X/A,The plasmids of blue colonies were sequenced,NCBI database blast,identified two target proteins:TMEM120Aand ZBRK1.2,Construction target protein and nsPla/3 interaction identification systemTo construct the expression vector of bait and target protein genes,nsP1a/3-pCDNA3.1-Flag,TMEM120A-pCDNA3.1-HA,ZBRKl-pCDNA3.1-HA,respectively.Transfected HEK293T cells,confirmed that the constructed vector can be stably expressed in HEK293T cells by western blot.3,polyclonal antibody preparation of nsPla/3,TMEM120A,ZBRK1To construct gene prokaryotic expression vector nsPla/3-pET-28a,TMEM120A-pET-32a,ZBRK1-pET-28a respectively.Expressed in E.coli strain BL21(DE3),rosetta expression of three proteins,then western blot protein identification After optimization of expression conditions,a large number of target protein expression,using HisTrap HP Ni2+-NTA column purified protein target,andthen gradient dialysis,protein refolding,imageJ and BCA analysis the results of SDS-PAGE which to determine the protein concentration and purity.The target protein mixed with Freund’s adjuvant after intraperitoneal injection SD rats,fourth immunization,blood of abdominal aortic.ELISA confirmed antibody titers were 1:30000,1:64000,1:256000,antibody have good specificity by western blot.In this paper,the successful use of the yeast two-hybrid screening two target protein,To construct eukaryotic expression vector of the target and bait genes,verify that it can be stably expressed in HEK293T cells.Three proteins were prepared polyclonal antibody,which lay a foundation for further study the nsPla/3 protein biological function.