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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 for large-scale Fab phage antibody library. Methods Site-directed mutagenesis was used to change the BssHⅡ restriction site on the surface of pDF, which was displayed on the surface of the phagemid vector, into BglⅡ restriction sites. Then, the suicide gene SacB was inserted into the light and heavy chains of the antibody to construct the phage containing suicide genes The vector pDF-D-SacB was used to amplify the heavy and light chain gene fragments by using the gene of anti-hepatitis B surface antigen antibody as a template. The PCR amplified light chain and heavy chain genes were inserted into the vector pDF-D-SacB respectively and transformed into Trans1-Blue E.coli by electrotransformation to construct two primary plasmids. The primary plasmid was used to super-infect BSl365 Bacteria, light chain and heavy chain recombination to obtain a recombinant plasmid, further anti-hepatitis B surface antigen antibody phage. After that, the phage was used to infect E. coli Trans1-Blue to amplify a large amount of phage antibody against hepatitis B surface antigen. Finally, the antibodies obtained are tested by enzyme-linked immunosorbent assay. Results The gene of Saccharomyces cerevisiae was inserted into the pDF heavy and light chain region to construct the pDF-D-SacB vector. The pDF-D-SacB gene could express the functional Fab phage antibody, The expected Cre-Loxp-mediated site-directed recombination occurred in the bacterial cells. Conclusions The obtained suicide gene-containing phagemid vector pDF-D-SacB is suitable for constructing a large-scale phage antibody library.