浙江农业学报2026，Vol.38Issue(5)：867-875,9.DOI:10.3969/j.issn.1004-1524.20250283
伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化
Expression of pseudorabies virus gD protein in rice cells and optimization of purification con-dition
摘要
Abstract
To explore the feasibility of expressing the pseudorabies virus gD protein in rice cells and to lay a founda-tion for developing a pseudorabies virus subunit vaccine,this study optimized and artificially synthesized the pseudor-abies virus gD gene based on rice codon usage bias,constructing the recombinant plant expression vector pCAM-BIA1300-gD.This vector was introduced into rice callus via Agrobacterium-mediated transformation,and resistant calluses were obtained after co-cultivation and dark culture screening.Callus genomic DNA was extracted using the CTAB method,and positive calluses were identified by PCR.Positive calluses were expanded in culture,ground,and added to liquid medium for shake-flask cultivation.Western blot was used to detect the expression of the gD pro-tein.Based on the physicochemical properties of the target protein,purification conditions including pH value,cation exchange resin,and hydrophobic resin were screened and optimized.The results showed that 110 calluses were ob-tained after hygromycin screening,with 92 identified as positive by PCR,yielding a positive rate of 83.6%.Western blot analysis of 35 cell lines revealed successful gD protein expression in 15 lines.Purification via two-step chroma-tography using cation exchange resin 650F and hydrophobic resin GE Butyl 4FF yielded gD protein of certain purity.This study established a foundation for using rice as a bioreactor to produce pseudorabies virus subunit vaccines.关键词
水稻/愈伤组织/伪狂犬病毒/植物表达载体/gD 蛋白/密码子优化/蛋白质纯化Key words
rice/callus/pseudorabies virus/plant expression vector/gD protein/codon optimization/protein puri-fication分类
农业科技引用本文复制引用
寇少康,陈恒耀,潘世源,张江楠,程文博,楚红燕,张磊,张二芹..伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化[J].浙江农业学报,2026,38(5):867-875,9.基金项目
河南省重大科技专项项目(221100110600) （221100110600）
河南省科技攻关项目(222102110210) （222102110210）
