Stable and Efficient Expression of HA Protein of Avian influenza virus H9N2 Subtype in Rice Endosperm and Identification of Activity

doi:10.7668/hbnxb.20191161

Abstract

Abstract: In order to obtain HA protein of H9N2 subtype with high expression,easy storage,low cost and high immunoactivity in vitro,rice endosperm expression system was used to express HA protein. First,the HA gene was optimized according to rice preference codons and synthesized into the pUC57 vector. The HA gene was constructed on the intermediate vector pMP3 and the plant expression vector pCAMBIA1300 successively by double enzyme digestion and ligation. Then,pCAMBIA1300-HA was introduced into Agrobacterium tumefaciens EHA105 by electrode method. And then,TP309 rice callus was infected with Agrobacterium. Finally selected by hygromycin,differentiated,rooted,and transplanted to the field for planting. The CTAB method was used to extract the genomic DNA of the leaves,and the T₀ positive plants were identified by PCR. The expression of HA protein in rice endosperm was detected by Dot Blot and Western Blot. The positive T₁ leaf DNA was extracted,and the homozygous plants were screened by fluorescence quantitative PCR. The TP309 containing HA protein was crossed with low gluten rice using the method of pollination by killing male and shearing glumes in warm soup. The protein expression of T₃ plants after hybridization was detected by Western Blot,and the activity of HA protein expressed by rice and insects was compared by double-antibody sandwich ELISA. The results showed that the intermediate vector pMP3-HA and the plant expression vector pCAMBIA1300-HA were successfully constructed,and pCAMBIA1300-HA was successfully introduced into Agrobacterium tumefaciens EHA105. PCR detected the HA genes of 66 plants successfully integrated into the rice genome. Dot Blot and Western Blot detection results showed that HA protein was successfully expressed in rice endosperm. Using fluorescent quantitative PCR,31 homozygous plants were screened from 107 T₁ plants plants. Western Blot detection results showed that the expression of HA protein after hybridization was greatly increased. Double-antibody sandwich ELISA results confirmed that the activity of HA protein expressed in rice endosperm was higher than that of baculovirus-insect cells.

Key words: Avian influenza virus, H9N2, HA protein, Homozygote, Rice endosperm, Activity identification

CLC Number:

S851.3

ZHAO Xiangyue, ZHANG Erqin, XU Qianru, MA Fanshu, WANG Ya, NIU Xiangxiang, LI Xueyang, ZHANG Shenli, ZHANG Tongxu, DENG Ruiguang, ZHANG Yifang, ZHANG Gaiping. Stable and Efficient Expression of HA Protein of Avian influenza virus H9N2 Subtype in Rice Endosperm and Identification of Activity[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 230-238. doi: 10.7668/hbnxb.20191161.

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