目的 构建国产沉香基原植物白木香AsMAPK3基因的原核表达载体，诱导重组蛋白正确表达，并研究AsMAPK3的亚细胞定位情况，为抗体制备准备材料，为筛选其互作蛋白及进一步研究其功能奠定基础。方法 通过PCR方法克隆AsMAPK3基因的部分编码序列，构建重组原核表达载体pET-28a-AsMAPK3，并诱导蛋白表达。克隆AsMAPK3基因全长编码区，构建绿色荧光蛋白（GFP）瞬时表达载体pAN580-AsMAPK3，通过基因枪法转化洋葱表皮，荧光显微镜观察GFP的发光情况。结果 含有重组表达载体的大肠杆菌BL21（DE3）在37℃经0.5 mmol/L异丙基硫代β-D-半乳糖苷（IPTG）诱导4 h后，可获得相对分子质量约为39 000的融合蛋白，该融合蛋白在上清和包涵体中均有表达。瞬时转化洋葱表皮的GFP荧光观察发现，AsMAPK3主要在细胞核和质膜表达。结论 成功实现了AsMAPK3的体外表达和纯化，明确了AsMAPK3的亚细胞定位，为后续开展其功能研究奠定了基础。

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3.This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions.Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein.The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector.The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope.Results The Escherichia coli BL21(DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies.The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane.Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed.This work provides a substantial foundation for follow-up function study of AsMAPK3.

国家自然科学基金资助项目（81573525，81673549）；中国医学科学院医学与健康科技创新工程——重大协同创新项目（2016-I2M-2-003）；海南省自然科学基金项目（20163150）