【摘要】 目的 :克隆抗人CD1 54抗体轻重链可变区基因 ,并分析其核苷酸序列 ,为基因工程抗体的构建奠定基础。方法 :从分泌能抑制免疫反应的抗人CD1 54单克隆抗体杂交瘤细胞株 7E8中提取总RNA ,合成cDNA第一链后 ,经PCR扩增获得抗人CD1 54单抗轻链可变区 (VL)和重链可变区 (VH)基因 ,分别克隆入pUC1 8载体 ,并进行序列分析。结果 :①抗体的轻链可变区基因全长为 341bp ,编码 1 1 3个氨基酸 ,归属于Ig的Vκ2基因 ,氨基酸序列分析结果显示轻链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区 ,在第 2 3位和第 93位氨基酸为半胱氨酸 ,是与抗体二硫键形成有关的两个特征性氨基酸 ;②抗体的重链可变区基因全长为 354bp ,编码 1 1 8个氨基酸 ,归属于小鼠IgVH基因 ,D、J区基因分别属于DSP2 .9和JH2 ,氨基酸序列分析结果显示 ,重链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区 ,在第 2 3和第 97位氨基酸为半胱氨酸 ,是与抗体二硫键形成有关的两个特征性氨基酸。结论 :经核苷酸序列分析证明所克隆的基因分别为抗体的轻、重链可变区基因更多还原

【Abstract】 AIM:To obtain the light chain region(VL) and heavy chain region(VH) genes from the hybridoma cell line and analyse their sequence for construction of the engineering antibody against hCD154. METHODS: In this research ,total RNA was extracted from the hybridoma cell line 7E8, which secretes McAb against hCD154, and subjected to reverse transcription. The VL gene and VH gene were amplified by PCR, cloned into puc18 vector and sequenced by Sanger’s dideoxymediated chain-termination method. RESULTS: The VL cDNA of 7E8 McAb consists of 341 bp encoding 113 amino acid residues. Compared with mouse Ig database, the VL region is in accord with the characterization of DNA sequence present in the mouse Ig Vk region , it belong to mouse Vк2 light chain. The VH cDNA of 7E8 McAb consists of 354 bp encoding 118 amino acid residues. Compared with mouse Ig database, the VH region is in accord with the characterization of DNA sequence present in the mouse Ig VH region. CONCLUSION: The DNA squence analysis showed that the cloned genes code the light and heavy chain variable region of mouse respectively.更多还原