Bioinformatics Analysis of the Extracellular Region of NA Protein in Novel H7N9 Avian Influenza Virus and Preparation of Polyclonal Antibodies

doi:10.13560/j.cnki.biotech.bull.1985.2019-0762

Abstract

Abstract: This work aims to express and purify the extracellular region of NA（neuraminidase）protein in novel H7N9 avian influenza virus（Anhui Strain）in prokaryotic expression system and to prepare its polyclonal antibodies. Firstly,bioinformatics analysis of the extracellular region of NA protein in novel H7N9 avian influenza virus was conducted. The codon optimization was performed according to the codon preferences in the Escherichia coli expression system,and the gene encoding this protein was synthetized. Subsequently the recombinant plasmid pET28b-tN9（truncated N9）carrying chemically synthesized extracellular region of NA gene was transformed into E. coli BL21（DE3）,E. coli Rosetta and E. coli Arctic Express（DE3）and their expressions were induced with IPTG,and the SDS-PAGE identification was conducted. The recombinant E. coli BL21（DE3）was cultured for more mass,induced by IPTG,the induced products were purified by Ni column,analyzed using SDS-PAGE,and identified by mass spectrometry. The purified protein was used to immunize rabbit to prepare polyclonal antibodies. Western blotting and an indirect ELISA assay were performed to determine specificity and titer of the polyclonal antibody. As results,the recombinant protein was successfully expressed and purified. Western blotting analysis showed that the prepared polyclonal antibody specifically recognized the recombinant protein and H7N9 avian influenza virus（Shanghai Strain）. The titer of the antibody was about 1：256000 detected by indirect ELISA. In conclusion,the specific polyclonal antibodies with high titer were successfully prepared by immunizing the rabbit using the purified tN9 protein in in prokaryotic expression system,laying a foundation for further studying the structure and function of the NA protein,pathogenesis and building rapid detection methods of H7N9 avian influenza virus.

Key words: H7N9 avian influenza virus, bioinformatics, extracellular region of NA, affinity purification, polyclonal antibody

QIU Shu-xing, YIN Xing, SU Shu-juan, YIN Jun-lei, ZHANG Jia-you, LIU Xue-he, JIA Kun-yi, YANG Xiao-ming. Bioinformatics Analysis of the Extracellular Region of NA Protein in Novel H7N9 Avian Influenza Virus and Preparation of Polyclonal Antibodies[J]. Biotechnology Bulletin, 2019, 35(12): 85-93.

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