RGD特异性结合多肽与重组改构人肿瘤坏死因子融合基因的克隆表达及其抑瘤活性

Cloning and Expression of Fusion Gene of RGD Peptide and rmhTNF in E.coli and Tumor-inhibiting Activity of Expressed Product

目的构建RGD多肽与重组改构人肿瘤坏死因子融合基因,进一步提高rmhTNF的临床疗效。方法应用基因重组技术构建了CRGDC与rmhTNF的融合基因,在E.coliDH5α中诱导表达重组蛋白RGDrmhTNF,采用溶菌酶法裂解菌体,裂解上清经硫酸铵沉淀、阴阳离子交换层析纯化后,用S180荷瘤小鼠模型和L929细胞体外杀伤实验测定纯化的重组蛋白RGDrmhTNF的体内、外杀伤活性。结果正确构建了编码RGDrmhTNF融合基因,重组工程菌升温诱导后以部分可溶形式表达RGDrmhTNF,纯化后纯度为96.88%,体外对L929细胞杀伤作用的比活性为3.6×108IUmg,与rmhTNF相当(3.0×108IUmg)。在S180荷瘤小鼠模型中,当RGDrmhTNF剂量为12×105IUkg时,对肿瘤的生长抑制率为84.9%,显著高于相同剂量的rmhTNF(肿瘤抑制率为59.09%,P<0.01)和环磷酰胺组(肿瘤抑制率为71.21%,P<0.01)。结论重组蛋白RGDrmhTNF可以提高rmhTNF的治疗效果。

Objective To construct a fusion gene of RGD peptide and rmhTNF and further improve the therapeutic effect of recombinant mutant human tumor necrosis factor (rmhTNF) in clinic.Methods A fusion gene of CRGDC (a tumor homing peptide) and rmhTNF was constructed by gene recombination technique and transformed to E.coli DH5α for expression of recombinant protein RGD-rmhTNF under induction of temperature.The recombinant bacterial strain was lyzed with lysozyme,and expressed product was purified from the supernatant of lysate by ammonium sulfate precipitation and negative and positive ion exchange chromatography and studied for tumor-inhibiting activities in vitro and in vivo by L929 cell killing test in vitro in mouse model of S180 sarcoma.Results The fusion gene encoding RGD-rmhTNF was constructed correctly,and the recombinant protein RGD-rmhTNF was expressed in partially soluble form.The expressed protein showed a purity of 96.88% after purification,and its specific activity in killing L929 cells in vitro was 3.6×108 IU/mg,which was comparable to that of rmhTNF(3.0×108 IU/mg).In mouse model of S180 sarcoma,the inhibiting rate of RGD-rmhTNF at a dosage of 12×105 IU/kg body weight on the growth of tumor was 84.9%,which was significantly higher than that of rmhTNF(59.09%,P<0.01) or cyclophosphamide(71.21%,P<0.01) at the same dosage.Conclusion The targeted delivery of rmhTNF with expressed recombinant protein RGD-rmhTNF may improve the therapeutic effect of the former.