摘要： 为了在体外获得表达量高、储存容易、成本低且免疫活性高的H9N2亚型HA蛋白，利用水稻胚乳表达系统表达HA蛋白。首先，将HA基因根据水稻偏好密码子进行优化，合成到pUC57载体上，通过双酶切、连接将HA基因先后构建到中间载体pMP3和植物表达载体pCAMBIA1300上；再通过电极法将pCAMBIA1300-HA导入到根癌农杆菌EHA105中；然后用农杆菌侵染TP309水稻愈伤组织；最后经潮霉素筛选、分化、生根、移栽至田间种植。利用CTAB法提取叶片基因组DNA，PCR鉴定T₀阳性植株；利用Dot Blot和Western Blot检测HA蛋白在水稻胚乳中的表达；提取阳性的T₁叶片DNA，通过荧光定量PCR方法筛选纯合植株；利用温汤杀雄剪颖授粉法，将含有HA蛋白的TP309与低谷蛋白水稻杂交；利用Western Blot检测杂交后的T₃植株蛋白表达量；利用双抗夹心ELISA方法比较水稻源和昆虫表达HA蛋白的活性。结果显示，成功构建了中间载体pMP3-HA和植物表达载体pCAMBIA1300-HA，且pCAMBIA1300-HA成功导入到根癌农杆菌EHA105中；PCR检测到有66株植株的HA基因成功整合到水稻基因组；Dot Blot和Western Blot检测结果表明，HA蛋白在水稻胚乳中成功表达；利用荧光定量PCR方法，107株T₁中筛选到31株纯合植株；Western Blot检测结果表明，杂交后的HA蛋白表达量大幅度提高；双抗夹心ELISA结果证实，水稻胚乳表达的HA蛋白的活性高于杆状病毒-昆虫细胞。

关键词: 禽流感病毒, H9N2, HA蛋白, 纯合子, 水稻胚乳, 活性鉴定

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

中图分类号: 

• S851.3