抗人肺腺癌单克隆抗体重,轻链可变区基因的分离克隆和序列测定

根据鼠免疫球蛋白重。轻链可变区基因ＦＲ１和ＦＲ４的序列保守性，化学合成了适于体外扩增Ｉｇ重、轻链可变区基因（Ｖ_Ｈ和Ｖ_Ｌ）的数对引物。以分泌抗人肺腺癌单抗的杂交瘤细胞株ＷＬＡ－２Ｃ４的基因组ＤＮＡ为模板，ＰＣＲ扩增Ｖ_Ｈ和Ｖ_Ｌ基因，分别克隆人ｐＵＣ１９载体。转化子经蓝、白斑筛选，酶切鉴定，双脱氧测序证实确为鼠单抗可变区基因，其中Ｖ_Ｈ基因全长为３４８ｂｐ，编码１１６ａａ，属重链ⅡＢ亚类；Ｖ_Ｌ基因全长３１８ｂｐ，编码１０６ａａ，属Ｋ轻链Ⅵ亚类。

Cloning and Sequencing of V_H and V_L Genes of an Anti-Human Lung Adenocarcinoma Monoclonal Antibody

By comparing the conserved regions at each end of the nucleotide sequences of murine germ-line genes encoding FR1 and FR4 regions of immunoglobulin heavy and light chain variable regions,we designed two sets of primer for amplification of VH and VL genes.Hybridoma cell WLA-2C4,secreting an antihuman lung adenocarcinoma McAb,was cultured and the genome DNA was extracted and used as template for PCR.After PCR the desired VH and VL fragments were amplified.The PCR products were then cloned into pUC19 vector.By screening and identification,several recombinants that had been inserted with the target fragments were obtained.Then they were sequenced with Sanger's method.It was confirmed by computer-assisted comparative sequence analysis that clones of full-length and potentially functional VH and VL genes from the hybridoma cell line WLA-2C4 were ob-tained.