抗EGFR单链抗体融合gelonin毒素的原核表达载体的构建及其生物活性研究  

The Construction of Anti-EGFR Single Chain Variable Fragment Fused with Gelonin Toxin Prokaryotic Expressing Plasmid and the Study of Its Anti-tumor Effects

作　　者：邱吉[1] 李倩[1] 李小磊[1] 周西坤[1] 黄浓郁[1] 李炯[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2011年第5期621-624,共4页Journal of Sichuan University(Medical Sciences)

基　　金："重大新药创制"科技重大专项基金(20092X09103-714)资助

摘　　要：目的构建表达抗表皮生长因子受体(EGFR)单链抗体融合gelonin毒素的原核表达载体,并初步验证其功能。方法以抗EGFR单链抗体基因为模板,通过PCR及酶切连接将该基因定向克隆入含gelonin毒素的原核表达载体pET32a中,将该质粒转入BL21(DE3)中经异丙基疏代半乳糖苷(IPTG)诱导表达得到包涵体,通过变性、复性并进行阳离子交换层析得到较纯的目的蛋白(重组免疫毒素rEG蛋白)。对纯化的rEG蛋白用Westernblot检测其免疫学特性,用细胞免疫组化实验及MTT实验测定其靶向性及生物学活性。结果经酶切及测序证实重组质粒pET32a-rEG构建成功。诱导得到的包涵体经变性、复性后使用阳离子交换层析而纯化得到rEG蛋白。纯化的rEG蛋白经Western blot鉴定,可以与兔抗gelonin血清特异性结合。经细胞免疫组化实验鉴定,rEG蛋白可以有效靶向EGFR阳性细胞,MTT实验也证实纯化的rEG蛋白能特异性抑制EGFR阳性表达的肺腺癌A549细胞的生长。结论成功制备了高纯度、具有生物学活性的rEG免疫毒素,为进一步研究其生物学功能提供了条件及基础。Objective To construct a prokaryotic expressing plasmid for recombinant immunotoxin which fused anti-EGFR scFv together with gelonin toxin,express and verify its function.Methods The gene fragments coding anti-EGFR single chain fragment were amplified with PCR and cloned into pET32a vector which contains gelonin toxin.The new plasmid was transformed into BL21（DE3） cells.The induced inclusion bodies were denatured,refolded and purified through SP Sepharose Fast Flow Column.The purified immunotoxin rEG was identified by western blot analysis,and the bioactivity was identified using cell immnuohistochemistry and MTT assay.Results The expressing vector pET32a-rEG has been constructed correctly,confirmed by restriction endonuclease digestion and sequencing.The recombinant immunotoxin rEG was purified after denaturing the inclusion bodies,refolding and cationic exchange chromatograph.The purified protein rEG had the right immunology specificity.rEG could efficiently target to EGFR positive cells identified by cell immnuohistochemistry.And the result of MTT assay showed rEG could specifically kill EGFR positive cells.Conclusion The recombinant immunotoxin rEG with high purity and biologic activity was prepared in this study,which would become the basic for the further study of the biologic function of rEG.

关 键 词：免疫毒素 单链抗体 表皮生长因子受体 GELONIN 

分 类 号：R392.1[医药卫生—免疫学]