抗人大肠癌单链抗体—胞嘧啶脱氨酶融合基因的构建及表达

Construction and expression of fusion gene of single chain Fv against human colorectal carcinoma

目的:构建抗人大肠癌重组单链抗体ND-1scFv与酵母胞嘧啶脱氨酶CD的融合基因,并在大肠杆菌中表达.方法:采用基因重组技术借助GSGGSGLinker序列将酵母胞嘧啶脱氨酶基因融合于ND-1scFv基因的3'端,构建pET-28a(+)ND-1scFv-CD载体,转化E.coliBL21,经IPTG诱导,表达二者的融合蛋白.表达产物用Ni-NTAresin亲和层析方法纯化,并采用ELISA检测其免疫活性,MTT法测定由ND-1scFv-CD/5FC构成的ADEPT系统对大肠癌细胞的体外杀伤作用.结果:序列测定表明:ND-1scFv-CD基因全长1269bp,包含了732bp的scFv基因和477bp的CD基因.SDS-PAGE检测显示,融合蛋白相对分子质量47KDa,与预测值一致.光密度扫描结果表明,重组蛋白占菌体蛋白总量的27%,ELISA检测证实,ND-1scFv-CD保留了与ND-1scFv相近的免疫活性.MTT实验检测结果显示,由scFv-CD/5FC构成的ADEPT体系对大肠癌细胞具靶向杀伤作用.结论:成功地构建了抗人大肠癌单链抗体ND-1scFv与酵母胞嘧啶脱氨酶CD的融合蛋白,并在大肠杆菌中高效表达.融合蛋白具有良好的免疫活性和一定的酶活性.

AIM:To explore the construction of the fusion gene of recombinant ND-1scFv against human colorectal carcinoma and yeast cytosine deaminase and the expression of the fusion protein in E.coli. METHODS:Yeast cytosine deaminase gene was fused with the 3’terminus of gene of ND-1scFv against human colorectal carcinoma by a 18 bp linker with sequences encoding GSGGSG.To construct ND-1scFv-CD gene,plasmid pET 28 a (+)-ND-1scFv-CD was transformed into E. coli BL-21, and induced by IPTG to express the ND-1scFv-CD fusion protein. The expressed product was purified by affinity chromatography using NI-NTA resin, and its immunity was analyzed by ELISA. The cytotoxic activity of the ADEPT system containing ND-1 scFv-CD/5FC against human colon carcinoma cell line was evaluated by MTT assay. RESULTS:Sequencing results showed that the ND-1scFv- CD gene consisted of 1 269 bp,including ND-1scFv 732 bp and CD 477 bp. SDS-PAGE analysis showed that the expected molecular weight of fusion protein was 47 Kd. Optical density scanning showed that fusion protein expressed in E. coli accounted to 27 % of the total bacterial proteins. ELISA analysis revealed that ND-1scFv-CD reserved similar immunity of ND-1scFv. MTT assay showed scFv-CD/5FC was cytotoxic to human colorectal carcinoma cells. CONCLUSION:ND-1scFv-CD gene against human colorectal carcinoma was constructed and expressed successfully in E. coli. The fusion protein exhibited good immunity andenzymatic activity.