摘要
目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。
Aim To clone and sequence variable region genes of monoclonal antibody against GSH with GPX activity from hybridoma 3G5. Methods Total RNA was isolated, purifing poly(A+)RNA (mRNA) was purified through magnesphere technology and cDNA was synthesized by reverse transcription. Both VH and VL genes were amplified by PCR. VH and VL gene were inserted into vector pGEM T. The recombinant plasmids were identified by restriction enzyme digestion and sequences were analysed. Results The recombinant plasmids pGEM T VH and pGEM T VL were constructed. VH and VL genes were categorized into mouse heavy chain subgroup III and mouse light chain subgroup V respectively. The VH and VL genes were revealed to be 372 and 324 bp, encoding 124 and 108 amino acids containing 4 and 2 Ser in their high variable domains, respectively.Conclusion The VH and VL genes have the character of mice antibody variable genes. These genes will be used for expression of single chain Fv with GPX activity.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
2000年第2期116-119,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金!资助
No.39770174
