Establishment of Two Purification Methods of Avian influenza virus H9N2 Subtype HA Protein from Rice (Oryza sativa)
NIU Xiang-Xiang1,2, LI Xue-Yang1,2, XU Qian-Ru3, LI Qing-Mei4, WANG Ya-Nan5, ZHANG Shen-Li1,2, MA Qiang6, ZHANG Er-Qin1,2,*, ZHANG Gai-Ping1,2,*
1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; 2 International Associated Research Center of National Animal Immunology, Zhengzhou 450046, China; 3 College of Veterinary Medicine, Northwest A & F University, Yangling 712100,China; 4 Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; 5 College of Veterinary Medicine, Jilin University, Changchun 130062, China; 6 Institution of Animal Science & Veterinary edicine, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Abstract：Hemagglutinin (HA) protein is a kind of glycoprotein on the surface of Avian influenza virus, which is a main protective antigen inducing neutralizing antibody. Avian influenza virus H9N2 HA gene was successfully expressed in rice (Oryza sativa) endosperm. In order to establish a purification method for H9N2 Avian influenza virus HA protein expressing in rice, two methods were used in this study to purify HA protein. Method 1: Rice powder expressing HA protein was added into the extract in a ratio of 1∶5 mass to volume, stirred at room temperature, centrifuged and supernatant was taken. After filtration, crude HA protein was firstly captured by Q anion chromatography. Subsequently, HA protein was purified by HA protein antibody affinity chromatography, eluent was collected and the target protein was detected. Method 2: Crude HA protein solution was obtained in the same way as method 1. According to packing property of the Butyl hydrophobic, ammonium sulfate was added to HA protein solution, which was then stirred, centrifuged and filtered for a second stage of purification. The collected eluent was concentrated and changed, and purified by SP-cationic chromatography, and the eluent was collected to obtain the HA protein. The HA protein purified by the 2 methods was tested by SDS-PAGE and Western blot, and the HA protein was sent to the company for sequencing. The results showed that the purity of HA protein was about 90% by Q anion chromatography combined with HA affinity chromatography. The method of purification was relatively simple, but monoclonal antibodies need to be cultured and purified. This method was suitable for rapid and small-scale protein purification in laboratory. The purity of HA protein was about 90% by Q anion chromatography, Butyl hydrophobic chromatography and SP cationic chromatography. This method is suitable for establishing the purification process of HA protein and is used to purify HA protein on a large scale. The HA protein obtained by these two methods was sequenced and the result was consistent with the known amino acid sequence. In this study, two methods were established for the first time to obtain high purity recombinant H9N2 subtype HA protein from rice, it will provide basic data for the preparation of avian influenza H9N2 subunit vaccine in the future.
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