摘要
目的 构建表达鼠源化抗肝癌重组免疫毒素 m sc Fv2 5- TNFα(鼠源化单链抗体 m sc Fv2 5融合人突变型 TNFα) ,对荷肝癌 (SMMC- 772 1 )裸鼠进行体内杀伤活性实验。方法 运用 PCR方法扩增 TNFα并在其 5’和 3’端增添相应酶切位点 ,经限制性内切酶酶切后 ,构建成 PGEX4 T- 1 / msc Fv2 5- TNFα融合蛋白表达载体 ,转化大肠杆菌 JM1 0 9,通过 IPTG诱导进行表达 ,在经过蛋白纯化及 Western bolt鉴定之后 ,将重组免疫毒素经尾静脉注射治疗荷肝癌裸鼠 ,2周后处死裸鼠 ,观察肿瘤的体积、质量 ,并对瘤组织进行 TNFα免疫组化染色。结果 m sc Fv2 5- TNFα治疗组对荷肝癌裸鼠的有效率为 5 / 5 ,其中 2 / 5为完全缓解 ,3/ 5为部分缓解 ,与 TNFα对照组相比 ,疗效明显高于对照组 (χ2 =4 .6 2 ,P<0 .0 5 ) ,经 m sc Fv2 5- TNFα治疗后的瘤组织 ,TNFα呈弥漫性阳性反应 ,阳性颗粒主要存在于瘤组织的胞质中。结论 鼠源化抗肝癌重组免疫毒素 msc Fv2 5-
Objective To construct and express mouse recombinant immunotoxin mscFv 25 TNFα(mouse scFv 25 and their fusion to human mutant TNFα.Methods Two relevant sites of enzymatic digestion were added to the TNFα by PCR.The TNFα was linked to the 3' end of mscFv 25 in pGEX4T 1 vector.The anti HCC recombinant immunotoxin mscFv 25 TNFα was identified by electrophoresis and purified.The HCC xenografts were injected the recombinant immunotoxin through tail vain and executed after two weeks.The bulk and weight of tumor were observed.The tumor tissues were stained by immunohistochemical with TNFα.Result The prokaryotic expression vector pGEX4T 1/mscFv 25 TNFα was constructed successfully.After induction and expression by IPIG,the expression of fusion protein is 15% of total bacteria proteins.The tumor regression trials of mscFv 25 TNFα showed 5/5 effective.It had 2/5 completely remission and 3/5 partly remission.The therapeutic result of mscFv 25 TNFα better than that of TNFα(χ 2=4.62,P<0.05).The HCC tissue which treated by mscFv 25 TNFα expressed positive reaction.The positive granule mainly existed in HCC cytoplasm.Conclusion Recombinant immunotoxin mscFv 25 TNFα has the better therapeutic effect.It can regress the growth of HCC with greatly potential.
出处
《肝胆外科杂志》
2003年第3期223-226,共4页
Journal of Hepatobiliary Surgery
基金
军队 95重点补充课题
NO.98M0 98
