Expression, Purification And Identification Of Hemagglutinin (HA1) Protein Of Subtype H5N1 Avain Influenza Virus

Avian Influenza(AI), since the first time appeared, had brought tremendous harmfulness and disastrous effects. The most significant characteristic of Highly pathogenic avian influenz (HPAI) can be concluded its high pathogenicity and mortality rate, the most common subtype of the HPAI virus is H1 and H7. Hemagglutinin(HA) protein is the most important structural protein for Avian Influenza Virus(AIV), also the main target antigen which could induce the humoral immunity and the function of cytotoxicity, possesses greatly valuable for research. Objective: HA1 gene of subtype H5N1 AIV was amplified by molecular biology methods, and then subcloned to procaryon expression vector; positive recombinant plasmid was chosen, and the later was transformed into E.coli, then fusion protein successfully expressed; After the optimization the circumstance of protein expression, purification and biological activity were taken, the later could provided reference to the downstream application of protein. Namely, laid solid foundation to the preparation of anti-subtye H5N1 HA1protein, and the following research, such as the manufacture of near patient testing kit. Which occupied great significance in early diagnosis and prevention of AI.Methods and Results:(1) According to the cDNA sequence about subtype H5N1 AIV in GenBank, and combined with the features of expression vector pET-28a(+), one pair of primers was designed; H5N1 AIV HA1 gene was amplified by PCR, then the amplified gene sequence was inserted into pET-28a(+)factor, after the identification, which included PCR double enzyme cut nucleotide sequence test of recombinant plasmid, eventually the pET-28a(+)/HA1 positive recombinant plasmid was obtained.(2) pET-28a(+)/HA1 positive recombinant plasmid was transformed into E.coli competent cell, and induced by IPTG, target protein expressed, by Western-blot identification, the expressed protein was found same as the designed in molecular weight, it was obviously showed that pET-28a(+)/HA1 fusion protein could successfully express in prokaryocyte, pET-28a(+)-HA1-BL21(DE3) E.coli strain, express fusion protein was acquired, that strain could stably and high efficiently express target protein. As the expressed protein possessed poly-histidine label(His-Tag label), minority dissoluble protein was purified by Ni column affinity chromatography, then took activity identification with hemagglutination test (HA test), based on the characteristic of fusion protein. (3) Owing to the majority pET-28a(+)/HA1 fusion protein was inclusion body, the amount could not meet the need of monoclonal antibody(McAb), by the purification of target protein inclusion body, then denaturation and renaturation of inclusion bodies were poformed, high purity and great amount of fusion protein was obtained.Conclusion:(1) H5N1 AIV HA1 gene was successfully got by PCR amplification;(2) Amplified gene sequence was inserted into pET-28a(+)factor, pET-28a(+)/ HA1 positive recombinant plasmid was obtained;(3) pET-28a(+)/HA1 positive recombinant plasmid was transformed into E.coli compETent cell, target protein successfully expressed;(4) Target protein was got by affinity chromatography and inclusion body purification, target protein activation test was taken, which showed that high activity of target protein; high purification of target protein indicated that target protein was suit for preparation of McAb, which laid solid foundation for manufacture of diagnosis Kit of AI.