用于大容量人源天然噬菌体抗体库的表达载体的构建及鉴定  

Construction and identification of an expressing vector for a large human naive phage antibody library

作　　者：潘博[1,2] 付文卓[3] 王晓娜[1] 张宝中[1] 米志强[1] 安小平[1] 刘大斌[1] 李存[1] 姜焕焕[1] 陈斌[1] 童贻刚[1] 

机构地区：[1]军事医学科学院微生物流行病所国家重点实验室,北京100071 [2]中国农业大学动物医学院国家海绵状脑病实验室,北京100193 [3]首都医科大学医学实验与测试中心,北京100069

出　　处：《首都医科大学学报》2012年第5期647-652,共6页Journal of Capital Medical University

摘　　要：目的构建一个应用于大容量Fab段天然噬菌体抗体库的表达载体。方法用定点突变技术将表面呈现噬菌粒载体pDF上的BssHⅡ酶切位点改为BglⅡ酶切位点,然后分别在抗体轻链和重链位置插入自杀基因SacB,构建含自杀基因的噬菌粒载体pDF-D-SacB;利用抗乙肝表面抗原抗体的基因为模板,PCR扩增重链和轻链基因片段。将PCR扩增的轻链基因和重链基因分别插入载体pDF-D-SacB内,利用电转化的方法将其转入Trans1-Blue大肠杆菌,构建2个初级质粒,再利用初级质粒超感染BSl365菌,使其轻链与重链发生重组,获得重组质粒,进一步获得抗乙肝表面抗原抗体的噬菌体。之后利用该噬菌体感染大肠杆菌Trans1-Blue进行扩增,得到大量抗乙肝表面抗原的噬菌体抗体。最后,通过酶联免疫吸附剂测定检测所获得的抗体。结果通过向pDF重轻链区域插入SacB基因,改造抗体基因克隆位点,构建了pDF-D-SacB载体;经检测,pDF-D-SacB可以表达具有功能的Fab噬菌体抗体,可以在分泌Cre蛋白酶的细菌胞内发生预期的Cre-Loxp介导的定点重组。结论所获得的含自杀基因的噬菌粒载体pDF-D-SacB适用于构建大容量噬菌体抗体库。Objective To construct an expression vector that can be applied to the construction of large Fab fragment phage display library. Methods BssH Ⅱ restriction site in phagemid vector pDF was mutated to Bgl Ⅱ restriction sites by site-directed mutagenesis techniques, and a suicide gene SacB was inserted into both the heavy and light chain region, resulting in phagemid vector pDF-D-SacB. Heavy chain （VH） and light chain （VL） genes of anti-hepatitis B surface antigen （HBsAg） antibody were inserted into the phagemid vector pDF-D-SacB separately. The recombinant VH and VL phagemids were transformed into Transl-Blue E. coli by electroporation respectively. By co-infecting the VH and VL phagemids into bacteria BS1365 at high multiplicity of infection, the recombinant phagemid containing both the VL and VH genes was obtained. After infecting transl-Blue E. coli with the recombinant phage, the recombinant anti- HBsAg antibodies were assayed by ELISA. Results By inserting SacB gene into the light and heavy chain regions of pDF and modifying the cloning sites of the vector, a new phagemid vector pDF-D-SacB was constructed. The subsequent test showed that the constructed vector can express functional Fab phage antibodies, and can also allow the Cre-LoxP mediated recombination in bacteria cell. Conclusion A suicide gene containing phagemid vector pDF-D-SacB was applicable to constructing large phage display antibody library.

关 键 词：噬菌体 抗体库 Cre-Loxp重组系统 乙肝表面抗原 

分 类 号：Q78[生物学—分子生物学]