摘要
目的:通过基因工程抗体改造技术构建并表达抗人CD40人-鼠嵌合抗体。方法:从分泌鼠抗人CD40单克隆抗体(mAb)的杂交瘤细胞株(5C11)中提取总RNA,用一对特异性引物通过RT-PCR扩增mAbVH和VL的DNA编码区基因。根据序列分析的结果,设计引物扩增VH和VL基因相应的信号肽序列。利用基因重组技术,将mAb5C11的VH、VL基因及其相应信号肽序列与人IgG1的CH基因、Cκ链基因进行拼接,构建人-鼠嵌合抗体基因的表达质粒pIRES/h5C11。用脂质体法将其导入293T细胞株中进行瞬时表达。利用流式细胞术对表达产物进行鉴定。结果:NCBI基因数据库Blast的结果显示,克隆的基因序列符合小鼠VH、VL基因及其信号肽序列的特征。表达质粒pIRES/h5C11的构建正确,并在293T细胞株中获得瞬时表达。表达的抗人CD40的人-鼠嵌合抗体和mAb5C11具有相同的抗原结合位点。结论:成功地克隆了鼠抗人CD40mAbV区编码基因,并实现了可溶性抗人CD40的人-鼠嵌合抗体在293T细胞中的瞬时表达。
AIM： To construct and express a chimeric antibody against human CD40 molecule by genetic engineering antibody transformation. METHODS： Total RNA was extracted from the murine hybridoma cell 5C11 which secreted anti-CD40 monoclonal antibody （mAb）. The genes encoding VH and VL of mAb 5C11 were amplified by RT-PCR. According to sequence analysis, the primer was designed to amplify signal peptide sequences relative to VH and VL genes. The VH and VL genes of mAb 5C11 and relative signal peptide sequences were spliced with CH and C, genes of human IgG1 to construct expression plasmid pIRES/h5C11 of human-mouse chimeric antibody gene and the plasmid was transfected into 293T cells under Lipofectamine mediation for transient expression. Expressed product was analyzed by flow cytometry. RESULTS： The result of NCBI gene data bank blast revealed that cloned gene sequence accorded with mice＇ VH and VL genes and feature of signal peptide sequence. The constructed plasmid pIRES/h5C11 was correct. The restriction endonuclease digestion analysis and PCR showed that the recombinant genes were subsequently cloned into vector plRES. FACS analysis showed that human-mouse chimeric antibody against human CD40 maintained the binding activity and specificity to human CD40 molecule. CONCLUSION： The gene encoding variable region of human-mouse chimeric antibody against CD40 is cloned successfully and the human-mouse chimeric antibody against CD40 is expressed transiently in 293T cells,
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期189-192,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助(No.2001AA215341)
江苏省自然科学基金资助(No.BK2001211)
国家自然科学基金重点项目资助(No.30330540)
