Expression Of Porcine Aminopeptidase N And Its Effect On Porcine Deltacoronavirus Proliferation On The Cell

Porcine deltacoronavirus(PDCo V),a novel swine enteropathogenic coronavirus,could causes enteritis,diarrhea and mortality in piglets.It belongs to Coronaviridae and deltacoronaviruses.Coronavirus members.PDCo V has a higher infection rate in neonatal piglets.The necrotomy of the diseased pig shows severe atrophy of the small intestine villi,and the virus is mainly detected in the small intestine contents and feces.The epidemic has been found in many countries and regions including the United States,China,Vietnam and Laos.The clinical pathological conditions and anatomic pathological changes of PDCo V are similar to those of porcine epidemic diarrhea(PED)and porcine transmissible gastroenteritis(TGE),and they are often mixed infections.Reports show that porcine aminopeptidase N(p APN)is an infectious receptor of porcine transmissible gastroenteritis virus(TGEV).The addition of a certain amount of soluble p APN to Vero cells can promote the replication of PEDV.In order to determine whether p APN is a cellular infectious receptor for PDCo V,the entire p APN gene was amplified and cloned in this study.The p APN extracellular domain p APN-C protein was expressed in BHK-21,293 T cells and E.coli,respectively.The polyclonal serum of p APN was prepared after rabbits;293T and BHK-21 cells expressing p APN were infected with PDCo V,and TGEV was set as a positive control.The results showed that 293 T and BHK-21 cells expressing p APN were capable of infecting PDCo V,and this infection could be inhibited by p APN antiserum;the antisera were exposed to PDCo V-sensitive ST cells and then administered to the cells,3 h,12 h,and 24 The relative content of h virus and TCID50 were significantly reduced.Thus,it is speculated that p APN is an infectious receptor for PDCo V.The specific test content is as follows:1.Construction and in vitro expression of p APN eukaryotic expression plasmidAccording to the sequence of p APN gene in Gen Bank(ID: HQ824547.1),p APN detection primers and primers for amplification of whole genes were designed.In order to determine the expression of p APN in pigs and cells,the assay was performed with real-time fluorescence quantitative RT-PCR and ordinary RT-PCR.The results showed that p APN was highly expressed in porcine kidney,small intestine tissues,and ST and LLC-PK cells;then the whole p APN gene was extracted from porcine intestine tissue and cloned into p MD18-T vector,which was correctly sequenced and named p MD-18T-p APN;then used as a template to amplify p APN extracellular domain p APN-C,constructed p EGFP-N1-p APN-C eukaryotic expression vector,and sequenced correctly,transfected into 293 T and BHK-21 cells,in 24 A large amount of green fluorescence was observed at h.After 24 h,cells were harvested and detected by real-time fluorescence quantitative PCR.The m RNA level of p APN in the transfection group was 15,000 times that of the empty cell and empty vector group.WB assay was performed on the samples.The results showed that: The dye group was successfully expressed.2.Expression of p APN prokaryotic protein and preparation of polyclonal antibodiesTo obtain p APN recombinant protein and prepare polyclonal serum of p APN.In this study,a pair of specific primers for amplifying p APN-C was designed,and p APN-C was amplified and cloned into prokaryotic expression vector p ET-28 a to construct a recombinant expression plasmid.After double digestion and correct sequencing,it was named: p ET-28 a.-p APN-C.Subsequently,it was transformed into E.coli BL21 and a recombinant protein p APN-C with a size of approximately 53 k Da was successfully expressed.After optimization of the concentration of IPTG,the expression level was highest at 0.6 m M IPTG.The solubility analysis of the protein showed that: The recombinant protein was mainly in the form of inclusion bodies;it was denatured with 6 mol urea solution and renatured with 2 mol urea solution.After purification,the recombinant protein concentration was 2.6 mg/m L and the purity was high;the purified p APN-C was purified.After emulsifying the recombinant protein with Freund's adjuvant and immunizing New Zealand white rabbits four times,the serum was isolated to obtain rabbit anti-p APN polyclonal serum.The serum was 5,000-fold diluted and specifically bound to the prepared p APN-C recombinant protein;A 100-fold dilution of the p APN antibody was prepared to detect the native receptor protein on ST and LLC-PK1 cells.The indirect immunofluorescence method recognized the expression of p APN on the cell membrane.It shows that the prepared p APN polyclonal antibody has better reactogenicity.3.The proliferation of PDCo V on cells by p APN and polyclonal serumTo screen PDCo V non-susceptible cells,explore whether BHK-21 and 293 T cells can be infected by PDCo V.We inoculated PDCo V(CH-01 P4)(MOI=1)on BHK-21 cells that were overlaid with monolayers to observe cytopathic effect.Cells were harvested at 72 h,and the collected samples were continued on BHK-21 cells. After 3 passages,the infection of PDCo V on BHK-21 cells was detected by RT-PCR.The results showed that PDCo V could not be detected in 3 generations of continuous culture medium,and 293 T cells with a monolayer were infected with PDCo V(MOI=1).After 48 h of infection,PDCo V was detected by indirect immunofluorescence assay.The primary antibody was: rabbits.Anti-PDCo V N polyclonal antibody;Secondary antibody: FITC-labeled goat anti-rabbit Ig G.The results showed that 293 T cells could not detect the PDCo V pathogen after receiving the virus.This indicates that both BHK-21 and 293 T cells are non-susceptible cells of PDCo V.To determine the optimal pancreatic enzyme concentration for PDCo V infection in BHK-21 and 293 T cells.0,0.1%,0.3%,0.7%,0.9%,1% Pancreatin and 0,1,2,3,4,5 ?g/m L trypsin cultured cells were used to observe cell morphology.The results showed that the optimal pancreatin concentration for BHK-21 and 293 T cells was 0.5% Pancreatin.To determine the effect of p APN on the proliferation of PDCo V on the cells.In this study,PDCo V(MOI=2)was used to infect BHK-21 and 293 T cells transfected with p APN.At the same time,TGEV was used as the indicator virus.Mouse anti-PDCo V N polyclonal sera and rabbit anti-p APN polyclonal antibody were used as primary antibodies.Alexa Fluor The 594-labeled goat anti-mouse Ig G and FITC-conjugated goat anti-rabbit Ig G were used as secondary antibodies to detect PDCo V.The results showed that PDCo V was infected on 293 T and BHK-21 cells expressing p APN.Cells not expressing p APN did not infect PDCo V.;Description: p APN mediates PDCo V infection in BHK-21 and 293 T cells.To further determine the effect of p APN antiserum on the proliferation of PDCo V on ST cells.Incubate ST cells with monolayers with p APN antiserum for 1 h and PDCo V with MOI=1.Incubate at 37 °C with 5% CO2 for 1 h and then replace culture medium containing p APN antiserum for 3 h.Samples were collected after 12 h and 24 h,frozen and thawed three times,and the content of PDCo V was detected by q RT-PCR.At the same time,TCID50 was measured.The results showed that the anti-p APN antiserum had inhibitory effect on virus replication at 12 h and 24 h.More obvious.In summary,the p APN protein was expressed in 293 T,BHK-21 cells and E.coli,and rabbit p APN polyclonal serum was prepared.293 T and BHK-21 cells expressing p APN were infected with PDCo V,respectively,and p APN was found to be able to Mediate viral infection;anti-p APN serum can compete with PDCo V for p APN protein molecules to inhibit PDCo V proliferation.It is speculated that p APN is a cellular infection receptor for PDCo V.This result provides the research basis for PDCo V receptor research and also provides some ideas for PDCo V antiviral drug screening.