抗胃癌免疫毒素表达质粒的构建、表达及对肿瘤细胞的特异杀伤

Construction of Anti carcinoma Immunotoxin Expressing Plasmid, Expression and its Killing Special to Carcinoma Cells

以含单链抗体 ( Sc Fv) 3H1 1基因全长的质粒 DNA为模板 ,利用 PCR技术扩增 3H1 1 Sc Fv基因片段 ,扩增片段及绿脓杆菌外毒素 PE38表达质粒 p YR39- 1 - PE38经 H ind /N de 酶切、连接 ,转化大肠杆菌 BL2 1,构建免疫毒素的表达质粒 p YR3H1 1 - PE38.转化菌在 IPTG诱导下 ,表达免疫毒素 3H1 1 - PE38,3H1 1 - PE38经纯化、变性、复性处理后 ,通过 MTT法检测其对胃癌细胞MGC80 3的杀伤活性 .结果表明 ,3H1 1 - PE38浓度不变 ,其杀伤率在一定的范围内随作用时间的延长而增加 ,当浓度为 5× 1 0 -8mol/L,作用时间为 60 h时 ,其对胃癌 MGC80 3细胞的杀伤率达74 .2 % ,而同等条件下抗 DNA免疫毒素 p Ig2 0 - PE38的杀伤率仅为 9.2 % ;作用时间一定 ( 60 h) ,免疫毒素浓度与杀伤率呈正相关 ,在 1 0 -10 mol/L以下 ,杀伤率几乎为零 ,而浓度高于 5× 1 0 -8mol/L时 ,杀伤率超过 70 % . 3H1 1 - PE38能够有效杀伤与之特异结合的胃癌细胞 。

The 3H11 single chain antibody(ScFv)has binding activity to tumor cells,3H11 ScFv full length cDNA was amplified by polymerase chain reaction.The amplified fragments and plasmid expressing Pseudomonas Exotoxin 38(pYR39 1 PE38)were digested with Hin d Ⅲ/ Nde l respectively,linked and cloned to E.coli BL 21 .The transformed BL 21 was induced by IPTG to express anti carcinoma immunotoxin 3H11 PE38.The killing activity of 3H11 PE38 special to carcinoma cells MGC803 was evaluated with MTT method after it was purified、denatured and renatured.Experiment results indicated that the efficiency on MGC803 was increased when reaction time was lengthened in a certain concentration rang.The efficiency ratio to kill MGC803 could reach 74.2% when 3H11 PE38 concentration was 5×10 -8 mol/L and reaction time was 60 h,but the ratio of anti DNA immunotoxin pIg20 PE38 was only 9.2%;When incubated the MGC803 with 3H11 PE38 concentration at 10 -10 mol/L for 60 h,the efficiency was not obtained,but it could exceed 70% when the concentration was over 5×10 -8 mol/L.Those results show that 3H11 PE38 can kill the cancer cells which bind with it effectively and has potential therapeutic application.