鼠抗人转铁蛋白单链抗体基因的构建

THE CONSTRUCTION OF THE GENE OF SINGL E CHAIN VARIABLE FRAG-MENTS OF MOUSE'S ANTIBODIES AGAINST HUMAN TRANSFER RIN

目的为了得到可经载体连接表达、并只由抗体可变区构成的单链抗体基因而进行本实验。方法经扩增得到的鼠抗人转铁蛋白抗体轻、重链可变区基因经凝胶电泳分离、离心柱纯化及定量,同能柔性折叠的一段短连接肽基因一起进行扩增拼接反应,而后在含限切酶位点的引物指导下扩增引入限切酶SfiI和NotI的酶切位点识别序列,产物以电泳鉴定、纯化和定量,再分别用SfiI和NotI酶切消化,最后再柱纯化。结果得到了携带可与载体连接的粘性末端的鼠抗人转铁蛋白单链抗体基因。结论两个不同的基因通过两端含互补序列的第三个基因片断可只经过扩增而无需DNA连接酶的作用而连接起来。对于抗体基因可由此而产生新的轻重链基因的组合,有可能产生性能更优异的抗体。

This experiment was carried out in order to obtain the ScFv,that is the sin gle chain variable fragment,which is only composed of light chain variable region and heavy chain variable region of antibodies,and can li gate with a vector and able to express through t he vector.For the light and heavy chain variable regional genes of mouse' s anti-bodies against human transferrin ob tained by PCR amplification,the ele ctrophoresis separation in agarose gel,purifi-cation by microspin columns and quan titation by comparison with V H marker,were carried out.Suitable a mount of these two genes were again amplified along with a primer of gene of one flexible p eptide linker altogether,then the a mplified product was introduced the recognit ion sites of restriction enzymes,on e end to Sfi I and another to Not I.Afte r detected by electrophoresis,successively p urified and quantitated,the product was digested with Sfi I followed by Not I,through purification the gene of single chain variable fragments of mouse's antibodies against human transferrin wa s received,with each of two ends cohesive.The ScFv gene was obtained.Two different gene fragments can be connected not by DNA ligating enzyme but by amplificatio n reaction containing a Third gene fr agment,which has cohesive end at its each side against the two different gene fragm ents.New combinations of antibodie s'genes of light and heavy chains cou ld be e-merged through this process,therefore the antibodies with better quali ties may be born.