A Preliminary Study On The Biological Characteristics Of Human Astro Virus Non Structural Protein NsP1a/1

Human astrovirus(HAstV)is one of the major pathogens that causing viral diarrhea and acute gastroenteritis in infants and young children,accompanied with encephalitis and viremia in immunocompromised patients.The infection of HAstV can cause the outbreak and sporadic of diarrhea.The source of infection from the contaminated water,food and contact with objects,it is transmitted via the fecal oral route.Have symptoms of diarrhea,vomiting,loss of appetite,occasionally accompanied by fever,abdominal pain,every year,about 2100000 infants and young children die from diarrhea and its complications in the world.At present,the domestic and foreign research on HAstV mainly focus on the field of epidemiology survey,genotyping and genetic analysis,clinical detection and so on,but there are only a few studies on its molecular pathogenic mechanism,and no vaccine or therapeutic drugs for the HAstV,so is necessary and urgent for the further research.This paper selects the HAstV non structural protein nsP1a/1 and preliminary study of its biological function.The main research work of this paper includes the following three aspects:(1)The construction of HAstV non structural protein nsP1a/1 eukaryotic expression vector and its effect on IFN-beta;(2)Yeast two hybrid of HAstV non structural protein nsP1a/1;(3)The expression and purification of HAstV non structural protein nsP1a/1 and preparation of polyclonal antibody.(1)The effect of HAstV non structural protein nsPla/1 on IFN-beta;In this study,PCR amplification of HAstV nsP1a/1 gene and inserted into the eukaryotic expression vector pEGFP-N2 and pcDNA3.1,the recombinant expression vectors were comfirmed by restriction enzyme digestion and sequencing.Transfected the eukaryotic expression vector nsP 1 a/1-pEGFP-N2 and nsP 1 a/1-His-pcDNA3.1 into 293T cells,the expression of nsP1a/1 gene were detected by fluorescence microscopy(nsP1 a/1-pEGFP-N2)and Western blot(nsP 1 a/1-His-pcDNA3.1).Then,the recombinant expression plasmid nsP1a/1-His-pcDNA3.1 was transfected into 293T cells again,and using poly(I:C)to stimulate the production of IFN-beta,RT-PCR and Real-time qPCR were used to detect whether nsP1 a/1 can inhibit IFN-beta produce.The results show that nsP1a/1 can not inhibit type I interferon.(2)The transcriptional activity determination of HAstV non structural protein nsP1a/1Because there is no needed enzyme cutting sites in the multiple clone site(MCS)of bait vector pGBKT7,so we transformed the MCS of bait vector by PCR,and named pGBST7.The HAstV nsP1a/1 gene was inserted into the bait vector pGBST7 and the recombinant vector was comfirmed by restriction enzyme digestion and sequencing.Transformed the bait vector into Y2HGold yeast cells and detected the toxicity and transcriptional activity,finding that the nsP1a/1 exhibited transcriptional activity,namely autoactivation,illustrating transcription factors or transcriptional activation domain were exist in the nsP1a/1.Followed by construction of deletion mutants to identified the autoactivation region is located between the 130-522bp.Then analyzed the amino acid sequence by the software and finding the transcription factor characteristics structure.We transformed the bait vector ?388-522 nsP1a/1-pGBST7 into yeast strain Y2HGold,carrying out yeast two hybrid experiments,the experiment was repeated 3 times,not screened the target protein.(3)The expression and purification of HAstV non structural protein nsPla/1 and preparation of polyclonal antibody.The HAstV nsP1a/1 gene was inserted into the prokaryotic expression vector pET-28a,at the end of gene the His-tag was constructed to facilitate protein purification,the recombinant vector was comfirmed by restriction enzyme digestion and sequencing.The recombinant plasmid nsP1a/1-His-pET-28a was transformed into BL21 cells and induced by IPTG,nsP1a/1 was highly expressed,and mainly in the form of inclusion body.The target protein was purified by Ni NTA affinity chromatography and used to immunize SD rats.The polyclonal antibody titer was determined as 1:256000 by indirect ELISA,according to the Western blot identified the polyclonal antiserum has good specificity.This paper preliminary explored the biological function of HAstV nsPla/1,confirmed that HAstV nsP1a/1 can not inhibit host cell type I interferon signal pathway.Although there is not screened the target proteins that interact with nsP1a/1 by yeast two hybrid,but we successfully prepared the polyclonal antibody of HAstV nsP1a/1,not only laid the foundation for the later research on HAstV nsP1a/1,and expanded the new ideas for the further exploration of the molecular pathogenic mechanism of HAstV.