Characterization Of Conservative B-cell Epitopes On The Nucleoprotein Of Influenza A Virus

Influenza A Viruses are enveloped, negative stranded RNA viruses in the family. The history of human influenza pandemic has brought serious economic loss to the society, a seasonal epidemic HIN1and H3N2can cause25000-50000people death every year. Humans are faced with the threat of highly pathogenic avian influenza, H7N9and highly pathogenic H5N1avian influenza virus lead to some human infectious diseases. Nucleoprotein (Nucleoprotein, NP) highly conservative, which has antigenic specificity between influenza A, B and C virus, Nucleoprotein is the basis of the classification and diagnosis of influenza virus, It is reported that NP has important influence to virus infection, copying, reproduction in recent years, and NP is the antiviral target, but due to the NP is polymer, protein conformation is complex, so we know very little about B cell conserved epitope of NP at present.The purpose of research is to find out B cell conservative epitopes of NP in order to provide references for the prevention and treatment of influenza A virus, provide effective research tool for monitoring the influenza virus.First of all, according to result of reaction of indirect ELISA with human rNP-HK/212and avain rNP-CA/04, the Mabs can be divided into three categories (Class A, Class B and Class C), the Class A Mabs have good reaction with two kinds of NP recombinant protein, the Class B Mabs have good reaction with of rNP-HK/212,but not reaction with rNP-CA/04, the Class C Mabs has good reaction with of rNP-CA/04,but have weak reaction with rNP-HK/212.Secondly, according to result of Western bloting reaction of with the three rNP segmented gene cloning of recombinant antigen, respectively Fl(1-120aa)、F2(110-301aa)、F3(293-498aa)and the full length of rNP, Class A mabs can be divided into Ⅰ, Ⅱ, Ⅲ class.Of which class I respond to F2and full length of rNP, class Ⅱ antibody is respond to full length of rNP, but not Western bloting reaction of the segments of F1, F2and F3, while class Ⅲ have no Western bloting reaction of F1, F2and F3fragment and full length of rNP.Thirdly, Identification of conservative NP linear epitope. Because of class I antibody has Western bloting reaction with full length of rNP and F2,select4D2as a representative of class I Anti-NP mAbs,then verified4D2response to the natural virus in Western blot, IFA, Co-IP, and the IHC level, and finally identified one of the conservative NP linear epitope is "RESRNPGNA",which belong to the251-243amino acids of NP, which is expected to be a useful target for protein expression and purification.Fourthly, Identifing the key amino acids of conformative epitope of Class II mAb, and explore its potential function.Ⅱ class of antibody has Western bloting reaction to the full length of rNP, but not to F1, F2and F3fragment, suggest group II antibody may recognize conformational epitope of NP, select19C10as a representative of group Ⅱ antibody, integrate the method of antigen antibody union and molecular docking, finally proved D88is the key site of conformational epitope by site mutation. prediction of the epitope recognized by mAb19C10in NP based on the3-D complex structure of NP-19C10, results provide evidence supporting the role of D88in the interaction between NP and PB2and suggest that D88is a key amino acid for RNP formation.Finally, Identification of avian host specificity of Class B mAbs.In view of the Class B prediction a key site through amino acid blastn and molecular simulation, and further verify by the method of point mutation, then successful found that the NP100th amino acid as the key point of the difference of human and avian host, and further developing a NP diagnostic kit for avian host.In summary, this study identified one of the conservative NP linear epitope is RESRNPGNA,moreover, proved D88is the key site of conformational epitope by sequence alignment and molecular simulation, further comfirmed the effect of D88on virus replication involves interaction of NP with PB2but not PB1during virus infection and suggested that D88is a key amino acid for RNP formation. Moreover,we found the NP100th amino acid as the key point of the difference of human and avian host,It is important for diagnosis and antiviral treatment of influenza A virus.