Construction And Expression Of Seven Deletion Mutants Of Human Astrovirus C-terminal NsP1a/4Protein

ObjectiveHuman astrovirus (HAstV) are one of the leading causes of actue virualdiarrhea in infants.Our previously studies indicated that human astrovirusnon-structural protein nsP1a induced intestine epithelial cells apoptosis andplays a major role in the events leading to diarrhea. NsP1a C terminal proteinnsP1a/4was the major functional protein and probobly have the main apoptosisdomains. In the present work, nsP1a/4and six deletion mutants of nsP1a/4were cloned into Green Fluorescent protein (GFP) vector pEGFP-N3toconstructed recombinant plasmids. These deletion mutants of nsP1a/4Proteinsuccessfully expression are plays important foundation to gain further insightsinto the mechanisms of astrovirus nsP1a/4protein induced apoptosis and it willalso provided research platform to further confirmed the molecule mechanismsof nsP1a/4protein function and pathogenic mechanism of human astrovirusviral diarrhea. Methods1.Construction of plasmid pEGFP-N3-nsP1a/4: The nsP1a/4gene wasamplified by PCR from a temple of plasmid pEGFP-N3-nsP1a.The amplifiedDNA and pEGFP-N3vector were digestion by restriction enzyme BamH I andXho I, respectively. Then digested DNA and vector were conjuncted by T4ligase, transformation and sequencing,respectively.2.Construction of six deletion mutant of human astrovirus nsP1a/4protein:According to human astrovirus nsP1a/4protein death domain, Phosphorylationsites and protein processing sites information, we selected the sites of deletionmutants, Δ1-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304.3.A large-scale plasmid was prepared by Endotoxin-free Plasmid Extractionkit,then transfected BHK21cell with the recombination plasmidPEGFP-N3-nsp1a/4、pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、pEGFP-N3Δ1-209、pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304by transfectionreagent Lipofectamine2000.After48-72hours, transfected cells were detectedby RT-PCR. The recombinant protein expression in cells was analysis byfluorescence microscope analysis GFP tag protein and western blot.Results1.nsP1a/4and six deletion mutant Δ1-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304were successfully amplified.2.The result of sequencing confirmed that the recombination plasmidPEGFP-N3-nsp1a/4、pEGFP-N3-Δ1-88、pEGFP-N3Δ1-176、pEGFP-N3Δ1-209、pEGFP-N3Δ53-174、 pEGFP-N3Δ225-304、 pEGFP-N3Δ273-304were successfully constructed.3.The result of western blotting and immunofluorescent show that the GFPfusion protein nsP1a/4-EGFP, Δ1-88-EGFP、Δ1-176-EGFP、Δ1-209-EGFP、Δ53-174-EGFP、Δ225-304-EGFP and Δ273-304-EGFP expressed successfullyin BHK21cells after transfected48-72hours.ConclusionsThe result indicated seven recombinant plasmids (pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、pEGFP-N3Δ1-209、pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304)were successfully express in BHK21cell. Thesedeletion mutants of nsP1a/4Protein successfully expression are plays importantfoundation to gain further insights into the mechanisms of astrovirus nsP1a/4protein induced apoptosis and it will also provided research platform to furtherconfirmed the molecule mechanisms of nsP1a/4protein function andpathogenic mechanism of human astrovirus viral diarrhea.