Construction And Expression Of Recombinant Eukaryotic Plasmid Of PEDV N Gene And Its Effect On Vero Cells

At present,porcine epidemic diarrhea?PED?occupies an absolute status in diseases which are prevalent worldwide and cause serious economic loss to many swineherds.It is caused that a swine of acute,heat,highly exposed intestinal a pathogenic disease by porcine epidemic diarrhea virus?PEDV?.PEDV is?coronavirus in the coronavirus,swines are infected with the disease often show characteristics of normal temperature,severe vomiting,watery diarrhea,etc.,the piglets and fattening pigs infected with the incidence of close to 100%,and piglets mortality rate is usually more than 50%,adult sows generally about 20%,while can be resistance generally.PED spread slowly,it is generally throughout the audience within a month in the case of less control.Almost in all coronaviruses,N protein plays an important role in the diagnosis and prevention of disease,pathogenesis research and so on.Because of the coronavirus N protein has some special functions,such as can cause the body's early immune response,and the body infected can produce high levels antibodies of anti-N protein in the early stage,it would be provided favorable conditions for the diagnosis of the disease and the development of the vaccine;The amino acid sequence of N protein contains a special subcellular localization signal,and the sequence is different among other virus,we can research the localization characteristics of N protein through this signal in the host cell,it can provided the possibility for studying the coronavirus pathogenesis.In this experiment,PEDV N protein was used as the research subject to construct recombinant eukaryotic plasmid of N gene,the expression of recombinant N protein was detected,and studied the N protein subcellular localization characteristics,it will laid the foundation to developing the early diagnosis and study pathogenesis of PEDV.The study aimed to carry out the following four aspects,and better experimental results were obtained.?1?Construction of recombinant eukaryotic plasmid expressing PEDV N gene.In this study,primers were designed according to the whole sequence of the N gene of PEDV CV777 strain.After the target gene was amplified,the recombinant eukaryotic expression plasmid pPM-C-His-N was constructed through ligated with eukaryotic vector pPM-C-His;By microbial PCR,restriction enzyme identification and sequence analysis,the recombinant eukaryotic expression plasmid was tested;The results were with the same of anticipation,it showed that the recombinant plasmid was constructed successfully and it provided material to use the recombinant plasmids for subsequent experiment.?2?Identification of recombinant plasmids expression in Vero cells.The recombinant plasmids were transfected into Vero cells,and empty vector control group and negative control group were added.The total RNA was extracted from the cells at 24h,and extracted total cell protein after 24h,48h and 60h.The recombinant plasmids were detected at the gene and protein levels.The distribution of N protein in the cells was detected by indirect immunofluorescence assay?IFA?after transfection 48h.The Agarose gel electrophoresis and SDS-PAGE showed that the cells transfected with the recombinant plasmid group appeared the target fragment,and the specific green fluorescence appeared in the whole cell,and the control group did not appear,it indicated that the recombinant plasmids were successfully expressed at the gene and protein level,and were transiently expressed in Vero cells.?3?Impact study of recombinant plasmid on immune level in mice.The eukaryotic plasmids were immunized with Kunming mice,and the empty vector control group and the negative control group were added.The immune serum was collected after three immunizations.The antibody titer was detected by ELISA and the reactivity of recombinant plasmid was detected by Western blotting.Then carried out the lymphocyte transformation experiment through prepare splenic lymphocyte suspension in sterile situation,and OD_570nm70nm was determined,and the effect of eukaryotic plasmids on the cellular immunity of mice was analyzed by SPSS23.0 software.The results of ELISA showed that the titer of third mice was 1:51,200,Western blotting showed that it appears the blot at the target band.The lymphocyte transformation test showed that the lymphocyte proliferation rate was significantly higher in the experimental group than in the blank control group and the negative control group,while there was no significant difference between the blank control and the negative control group,which indicated that the recombinant eukaryotic plasmid pPM-C-His-N had good immunogenicity and reactivity,and could improve the cellular immunity of mice significantly,it provides theoretical basis and data support for diagnose of disease and development of vaccines.?4?Effection of N protein on Vero cells.In order to study the characteristics of N protein subcellular localization and the effect on host cells,we used the bioinformatics software to analyze the PEDV N protein NLS sequence according to the report of other coronavirus N protein subcellular localization.We found a potential nuclear motif of pat7is located in the alkaline amino acid enrichment region,and exhibits the same high hydrophilicity as other coronaviruses,indicating that the motif is a potential subcellular localization signal,and make it possible to study localization of the PEDV N protein.Then we stained the nucleus with DAPI according to N protein localization characteristic.After incubation,the N protein was observed and analyzed under laser confocal microscopy.At the same time,the cells were stained with PI after transfection 48h,and analyzed it influenced to cell cycle by flow cytometry.The results showed that the N protein was mainly located in the cytoplasm and nucleus,and the G2-M phase was prolonged significantly,indicating that the PEDV N protein mainly through acting on the cytoplasm and nucleus of Vero cells,which effect on the cell cycle by effect on cell mitotic function,so creating conditions conducive to viral replication.In conclusion,the recombinant eukaryotic expression plasmid pPM-C-His-N constructed in this study has good reactivity and immunogenicity,and the localization of N protein in Vero cells has been studied,its mechanism of the action has been defined in host cells,laid a solid foundation for the methed of diagnostics and prevention of PEDV.