H5N1亚型禽流感病毒HA蛋白在水稻胚乳中的表达及其纯化

Expression and Purification of H5N1 Subtype Avian Influenza Virus HA Protein in Rice Endosperm

为制备H5N1亚型禽流感病毒HA蛋白并评估其免疫原性,利用水稻表达系统表达H5N1亚型禽流感病毒重组HA蛋白。首先构建了重组植物表达载体pCAMBIA1300-HA,该载体具有GT13特有启动子、信号肽SP和终止子,有利于外源基因在水稻中表达。之后将重组质粒pCAMBIA1300-HA通过电转化法导入根癌农杆菌EHA105中,筛选出阳性菌落并侵染水稻愈伤组织。经过暗培养、潮霉素筛选、分化、生根、成苗,采用CTAB法提取水稻叶片DNA,PCR鉴定结果显示,HA基因已插入到水稻基因组中,重组HA基因大小为4660 bp。将阳性植株移植到大田中,4个月后收获水稻种子,提取水稻胚乳蛋白,Western blot鉴定结果表明,HA蛋白在水稻胚乳中成功表达。重组HA蛋白通过Q阴离子层析、疏水层析和凝胶过滤层析三步分离纯化,其纯度达到90%以上。最后将纯化后的重组HA蛋白与ISA 50V佐剂混合并乳化制成疫苗,免疫小鼠,小鼠血清具有较高的特异性抗体水平,表明重组HA蛋白免疫原性较好。综上,成功构建了H5N1亚型禽流感病毒HA重组蛋白的水稻表达系统,并分离纯化获得了高纯度和免疫原性良好的重组HA蛋白。

In order to prepare the HA protein of H5N1 subtype avian influenza virus and evaluate its immunogenicity,the recombinant plant expression vector pCAMBIA1300‐HA was constructed to express the recombinant HA protein of H5N1 subtype avian influenza virus by using a rice expression system,which had a GT13‐specific promoter,signal peptide SP and terminator,and was conducive to the expression of exogenous genes in rice.The recombinant plasmid pCAMBIA1300‐HA was introduced into Agrobacterium tumefaciens EHA105 by electroconversion,and the positive colonies were screened to infect rice calli.After dark culture,hygromycin screening,differentiation,rooting,they grew into seedlings.The DNA of rice leaves was extracted by CTAB method,and the results of PCR identification showed that the HA gene had been inserted into the rice genome,and the size of the recombinant HA gene was 4660 bp.The positive plants were transplanted into the field,and the rice seeds were harvested 4 months later,and then the rice endosperm protein was extracted.Western blot identification results showed that HA protein was successfully expressed in the rice endosperm.The recombinant HA protein was separated and purified by Q anion chromatography,hydrophobic chromatography and gel filtration chromatography,and its purity reached more than 90%.The purified recombinant HA protein was mixed with ISA 50V adjuvant and emulsified to make a vaccine,which was used to immunize mice.The mice serum had a high level of specific antibodies,indicating that the immunogenicity of the recombinant HA protein was better.In summary,a rice expression system for the recombinant HA protein of H5N1 subtype avian influenza virus was successfully constructed,and the recombinant HA protein with high purity and good immunogenicity was isolated and purified.