免疫球蛋白变区基因的体外扩增及人－鼠嵌合抗体的表达

应用聚合酶链反应法体外扩增抗人小细胞肺癌单克隆抗体的变区基因，经核苷酸序列分析，轻链变区基因为３２４ｂｐ，编码１０８个氨基酸；重链变区基因为３５１ｂｐ，编码１１７个氨基酸。将重链变区基因克隆至ｐＳＶ_２△ＨｇｐｔＨｕγ_３质粒，经一系列克隆、筛选，得一插入方向正确的表达人－鼠嵌合重链抗体的载体－ｐＳＶ_２△Ｈｇｐｔ２Ｆ７ＶＨＨｕγ_３。用电穿孔方法将该质粒导入鼠源骨髓瘤细胞Ｊ５５８Ｌ，用霉酚酸进行初步筛选，最终用兔抗人免疫球蛋白ＩｇＧＦｃ片段的抗体筛选得七株阳性克隆，免疫印迹法结果表明在与人ＩｇＧ相同位置上有一阳性条带。

Cloning of Immunoglobulin Variable-region Genes by the Polymerase Chain Reaction and Expression of Heavy-Chain Chimeric Antibody in J558L Cell

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction.Here we applied the technique to clone and sequence the variable domains of monoclonal antibody which reacts with human small cell lung carcinoma line NCIH128. We have constructed recombinant plasmid──pSV_2△Hgpt2F7VHHuγ_3, transfected the plasmid into J558L cell by electroporation. Seven positive clones are acquired by rabbit-anti-human IgG Fc fragment antibody, the result of Western blot has shown a 56kD positive band, which is identical to that of human IgG.