目的　探讨和构建一种用于靶向性基因治疗的非病毒载体。方法　采用基因工程的方法构建H1s EGFc融合蛋白表达载体 ,在毕赤酵母表达系统中获得分泌表达的融合蛋白。使用阴离子交换柱及分子筛进行纯化。制备H1s EGFc融合蛋白与pKG杀伤基因表达重组体的复合物。选用表皮生长因子受体高表达的HeLa细胞及表皮生长因子受体不表达的Jurkat细胞进行复合物特异性杀伤活性测试。结果　构建并表达了H1s EGFc融合蛋白 ,所获融合蛋白纯度可达 90 %以上。该融合蛋白能够有效地包装杀伤基因表达载体 ,将其导入表皮生长因子受体特异性表达的肿瘤细胞中 ,达到靶向性杀伤作用 ,当融合蛋白 /杀伤基因复合物使用剂量分别是 3、6、9μg/ml时 ,杀伤率分别为30 6 %、36 2 %和 5 8 1% ;而对表皮生长因子受体不表达的细胞无杀伤作用。结论　融合蛋白H1s EGFc能够有效地用于功能基因的包装及转运 ,可以作为非病毒载体应用于恶性肿瘤的靶向性基因治疗。 Objective To investigate and construct a non-viral vector for targeted gene therapy. Methods The H1s EGFc fusion protein expression vector was constructed by genetic engineering and secreted fusion protein was obtained in Pichia pastoris expression system. Purification using anion exchange column and molecular sieve. A complex of H1s EGFc fusion protein and recombinant pKG killer gene was prepared. HeLa cells with high expression of epidermal growth factor receptor and Jurkat cells with no expression of epidermal growth factor receptor were selected for specific activity test. Results The H1s EGFc fusion protein was constructed and expressed, and the purity of the obtained fusion protein was over 90%. The fusion protein can effectively package the anti-gene expression vector and introduce it into the tumor cells specifically expressed by the epidermal growth factor receptor to achieve the target killing effect. When the fusion protein / anti-gene complex dose is 3,6 , 9μg / ml, the killing rates were 30 6%, 36 2% and 5 8 1%, respectively, while the EGFR did not express cells without killing effect. Conclusion The fusion protein H1s EGFc can be effectively used for the packaging and transport of functional genes and can be used as a non-viral vector in the targeted gene therapy of malignant tumors.