Expression And Preliminary Application Of Nuclear Protein Of H5N1 Avian Influenza Virus

Influenza A viruses often infected human and animal,in 1997,the first person infected with the H5N1 subtype avain influenza virus and caused death in Hong Kong.The highly pathogenic H5N1 subtype avian influenza caused great damage to human health and property.In the early stages of the influenza epidemic,an accurate?rapid?convenient and efficient detection method is of great significance for the timely prevention and control of the development of the influenza virus epidemic.The nuclear protein(NP)of influenza virus is highly conserved,which is the basis of influenza typing and identification.It has been reported that NP proteins can be expressed in prokaryotic and eukaryotic systems,but each has its own advantages and disadvantages.The lateral flow immunochromatographic strip(LFICS)is currently the most widely used POCT(point-of-care testing)detection method,with low cost ? short detection time ? suitable for field diagnosis,etc.Early diagnosis of influenza virus and detection of influenza antibody production in the body.In this study,the nucleoproteins of H5N1 A/meerkat/Shanghai/SH-1/2012(clade 2.3.2.1)were expressed using prokaryotic E.coli expression system and eukaryotic baculovirus expression system,respectively,and recombinant vectors containing NP genes were constructed.Transformed or transfected E.coli or Sf9 cells,the protein was large-scale cultured and expressed,indirect immunofluorescence?ELISA?SDS-PAGE?WB and other methods to identify the expression of NP protein.The target protein can be successfully expressed by both systems,but the NP protein and antibody expressed by the eukaryoticbaculovirus expression system have a high binding activity.Using this NP protein as a marker antigen,the method of competition and the double antigen sandwich are respectively used.The colloidal gold lateral immunochromatographic test strip was established.According to the specificity and sensitivity analysis,the NP protein expressed by the baculovirus expression system is more suitable for the preparation of colloidal gold lateral immunochromatographic test strips using the double antigen sandwich method..And we use this colloidal gold lateral immunochromatographic test strip to detect different subtypes of influenza pathogenic sera,and to monitor the production of antibodies in different subtypes after influenza virus infection or vaccine immunity.Our results indicate that both the prokaryotic E.coli expression system and the eukaryotic baculovirus expression system can both express NP protein,and the NP protein expressed in the eukaryotic expression system utilizes the double antigen sandwich method to establish colloidal gold lateral immunochromatography.This study was contributed to test strips to achieve monitoring of influenza virus infection and evaluation of vaccine immunity,and to help prevent and control influenza virus infection.