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目的:制备基因工程抗体,减少或消除鼠源性单抗在人体内的免疫原性,保留其对人体抗原配体的高度特异性,发展临床导向诊断和治疗。方法:从体外分泌抗转铁蛋白受体单抗杂交瘤细胞系7579中,对其单抗可变区基因进行克隆和序列分析。利用逆转录PRG技术扩增轻、重链可变区基因,再分别与pGEM-T载体连接、并克隆于JM109受体菌之中。利用荧光染色链终止法测定其序列,采用DNASIS7分析软件和与NIH基因库比较分析。结果:轻、重链可变区分别由139和128氨基酸残基组成,包括信号作、互补决定区和骨架氨基酸残基等功能序列。分别属于鼠免疫球蛋白重连Ⅱc和k链Ⅵ家族。结论:来自单抗的可变区的基因是完整的和具有潜在功能性的,为体外获得抗转铁蛋白受体基因工程抗体奠定了基础。
OBJECTIVE: To prepare genetically engineered antibodies to reduce or eliminate the immunogenicity of murine monoclonal antibodies (McAbs) in humans and to retain their high specificity for human antigen ligands and to develop clinical-oriented diagnosis and treatment. Methods: The monoclonal antibody (mAb) monoclonal antibody (McAb) was cloned and sequenced from the in vitro anti-transferrin receptor monoclonal antibody 7579 cell line. The light and heavy chain variable region genes were amplified by reverse transcription PRG technique and ligated with pGEM-T vector respectively and cloned into JM109 recipient bacteria. The sequence was determined by fluorescent chain termination method, and the DNASIS7 analysis software and comparative analysis with NIH gene bank were used. Results: The variable regions of light and heavy chains consisted of 139 and 128 amino acid residues, respectively, including signal sequence, complementarity determining region and backbone amino acid residues. Belong to murine immunoglobulin reassociation Ⅱ c and k chain Ⅵ family. CONCLUSION: Genes from the variable region of the mAb are intact and potentially functional and lay the foundation for obtaining genetically engineered antibodies against transferrin receptor in vitro.