鼠抗人转铁蛋白单链抗体基因序列测定及分析

从携带人转铁蛋白单链抗体基因载体转化的大肠杆菌菌株中提取质粒,经电泳分析及筛选、纯化出线状质粒DNA,定量后以载体上人转铁蛋白单链抗体基因插入子两边的已知DNA序列经合成后作为引物进行测序扩增凝胶电泳,将得到的人转铁蛋白单链杭体基因序列重轻链可变区部分同基因库中鼠抗体重轻链进行同源比对,证明人转铁蛋白单链抗体基因确由鼠抗体重轻链可变区基因构成,在此基础上,进一步对基因核苷酸序列的可变区结构特征作推断分析,并推导出其对应的氨基酸序列.

THE SEQUENCING AND ANALYSIS ON THE GENE OF SINGLE CHAIN VARIABLE FRAGMENTS OF MOUSE'S ANTIBODY TO HUMAN TRANSFERRIN PREPARATION OF GENE ENGINEERING ANTIBODY TO HUMAN TRANSFERRIN

From the cells of E. coli strain transformed by the vector with the gene of single chain variable rragments of mouse' s antibody to human transferrin, the plasmid DNA was extracted. Through electrophoresis analysis and purification by mierospin columns, the linear DNA was obtained. We used the DNA sequences of the vector pCANTAB 5E, which is adjacent and at the just two sides of the inserted gene of single chain variable fragments of antibody to human transferrin, as primers, the quantitative plasmid DNA and primers (Presynthesis) were subjected to sequencing amplified PAGE. By comparison of the obtained DNA sequence of gene of single chain variable fragments of antibody to human transferrin with that of light and heavy chain of mouse's antibody in the gene database on immunoglobulin from the Internet, we found that the obtained DNA sequence was homologous to the variable region of the light or heavy chain gene of mouse' s antibody, one to heavy chain and the other to kappa light chain, it was illustrated that the gene of single chain variable fragments of antibody to human transferrin was indeed made from the variable regions of light and heavy chain of mouse's antibody. Furthermour we analysed the structures and characters of the obtained DNA sequence according to the defined principle on the variable region both light and heavy chain and deducted the relevant amino acid sequence.