Primary Exploration On The Functions Of Duck Hepatitis Virus 2A Protein

Duck hepatitis A virus (DHAV) was classified into the Avihepatovirus of Picornaviridae family recently. It can cause highly lethal, rapidly spreading disease in ducklings. In order to explore the potential functions of 2A protein, the bioinformatics analysis, prokaryotic expression and purification of 2A2 and 2A3 protein, polyclonal antibody of 2A1/2A2/2A3 prioteins were prepared for this study. Then, the self cleavage function of 2A1 protein, integrity and independence of 2A2 protein and 2A3 protein, the DEF apoptosis induced by 2A2 protein and its'GTPase activity were also identified in this research. Following were the results.1. Analysis the bioinformatics features of 2A proteinThrough the NetPicoRna V1.0 online server, the predicted polyprotein processing cleavage sites of 2A protein are:NPG/P at 2A1/2A2, S/H at 2A2/2A3. Alignment of 2A protein between DHAV and other picornavirus was performed with the Clustal W program and demonstrated using the GeneDoc program. The results indicate that DHAV-12A containing a NPGP-like 2A1 protein, a GTPase-like 2A2 and a H-NC-like 2A3. Phylogenetic tree was constructed by Mega 6.0 with the neighbour-joining (NJ) method and 1000 bootstrap analysis. Online homologous modeling software of SWISS-MODEL Template Library (SMTL) was used to constructed the structure of DHAV 2A2 protein. We predicted that DHAV-12A2 protein was a GTPase-like protein based on the results of phylogenetic analysis, multiple sequence alignment and homologous modeling analysis.2. prokaryotic expression, purification and western blot of 2A2 and 2A3 proteinsDHAV genome was extracted from duck embryo allantoic fluid. Two pairs of primers P1P2 and P3P4 were designed to amplify the 2A2 and 2A3 gene sequences. The gene fragments of 2A2 and 2A3 genes were firstly ligated into a pMD19-T vector, and then cloned into pET-32(a)+expression vector to obtain 2A2 and 2A3 proteins. The recombinant expression plasmid pET-32(a)+/2A2/2A3 were expressed in BL21 expression system. Following are the perfered conditions. The 2A2 and 2A3 proteins were all acquired as follows:0.4 mmol/L IPTG inducing for 4 h at 37 centigrade degree.2A2 protein can be purified by washing inclusions while 2A3 protein can be purified thro?gh the Ni2+-NTA. It confirmed that the two expressed proteins would be identified in serum against DHAV-1.3. Prepared the mouse or rabbit Polyclonal antibody of 2A1/2A/2A3 proteinsMouse were immuned by synthetic 2A1 protein, the mouse serum titer was 1:6400 by the method of ELISA. Polyclonal antibody of purificated 2A2 protein was prepared by immunization of mouse and rabbits which the agar fiffusion tiler were 1:16. Polyclonal antibody of purificated 2A3 priotein was prepared by immunization of rabbits which agar fiffusion tiler was 1:32.4.The self cleavage experiment of 2A1 proteinOne pair of primers P1P2 was designed according to the RFP gene sequence, and pDsRED served as template to amplify the RFP gene sequence. We achieved reverse primer P3 by inserting the reverse complement of 2A1 gene into 5 terminus of P2. Based on the RFP gene sequence, primers P1P3 was designed to amplify the RED-2A1 gene. The PCR product was resolved by electrophoresis in a 1%(W/V) agarose gel and then purified and ligated into pEGFP-N1 to generate the recombinant plasmid pRED-EGFP and pRED-2A1-EGFP.Cells transfected with those plasmids sent out red and green fluorescence. The lysates were analysed by western blot using mouth anti-RED/GFP/2A1 antibodys. The results showed that 2A1 protein has the function of self cleavage.5. Research about the integrity and independence of 2A2 and 2A3 proteinDEF infected with DHAV were collected by centrifugation post infection 72 h, and then lysated by cells RIPA. The cell lysates were incubated with the rabbit anti-2A2/2A3 antibodys separately to precipitated out 2A2 or 2A3 protein. It confirmed that the two expressed proteins would be identified in serum against DHAV-1. Following the 48 h incubation at 37? after infection, the cells were fixed with 4% paraformaldehyde. The primary antibodies, Rabbit anti-2A3 and mouse anti-2A2 polyclonal antibody, were incubated with cells. Then FITC-goat-anti-rabbit IgG and TRITC-goat-anti-mouse IgG were incubated with the cells. It suggests that 2A2 protein mainly engaged in the nucleus, while 2A3 protein mainly located in the cytoplasm. The results shows that the 2A2 and 2A3 protein are two different proteins and located separately in DEF.6.Research of the DEF apoptosis induced by 2A2 proteinpMD19-T/2A2 was digested with BamH ? and Xho ? and then subjected to electrophoresis in a 1.0% agarose gel. The DNA fragments of 2A2 gene was purified and ligated into the previously BamH ? and Xho ? digested expression vector pcDNA3.1(+) to generate the recombinant expression vector pcDNA3.1(+)/2A2. DEF cells transfected with pcDNA3.1(+)/2A2 were harvested 36 h later. We demonstrated that 2A2 protein expressed in cells by IFA and western blot assay.After transfected with pcDNA3.1(+)/2A2 48 h later, the cells displays fragmented nuclei, chromatin condensation by HE and DAPI staining, oligo nucleosome-size DNA ladder, and the specific brown signals formed in the nucleus of transfected cells with TUNEL staining. Hence, their death can be characterized as apoptosis. By staining cells combinated with fluorescein Annexin V-FITC and PI, we found the percentage of apoptotic cells gradually increased and reached a maximum at 48 h after transfection with FCM. The results shows that DHAV 2A2 protein could induced the DEF apoptosis.7. GTPase activity research of 2A2 proteinThe recombinant expression plasmid pET-32a(+)/2A2 was expressed in BL21. Soluble 2A2 protein was purified through the Ni2+-NTA agarose gel affinity chromatography. Using the GTPase Activity Assay kit to verify whether the 2A2 protein possess GTPase activity or not. This kit uses a single reagent formulation to accurately determine enzyme activity in 30 min at room temperature. The malachite green reagent forms a stable dark green color with free phosphate liberated. The value of OD620 detected from 2A2 protein group (0.627), empty vector group (0.124) and without GTP substrate control group (0.035) had a very significant difference P<0.01. Results indicate that 2A2 protein possess GTPase activity.