抗人P185^(erbB2)单抗C25的生物活性及其可变区基因的克隆

Active identification of an anti human P185^(erbB2) McAb C25 and cloning of its variable region genes

了解抗人P185erbB2单抗C25的生物学活性,并克隆其可变区基因 ,为研制抗人P185erbB2的人源化抗体奠定基础。方法 :以细胞ELISA、免疫组化等方法分析C25单抗的抗原结合特性 ,用MTT法检测其对乳腺癌细胞SKBR3、及卵巢癌细胞SKOV3 增殖的抑制作用及对化疗药的增敏作用。经RT PCR扩增C25单抗的轻、重链可变区基因 ,克隆后进行核苷酸序列分析。结果 :C25单抗能特异结合人P185erbB2胞外区抗原 ,可显著抑制SKBR3、SKOV3 细胞增殖 ,与顺铂具协同作用。抗体的轻链可变区基因327bp,属于小鼠κ轻链第Ⅲ亚群 ;重链可变区基因366bp属于小鼠IgG1第Ⅲ (A)亚群。结论 :C25单抗可显著抑制人P185erbB2过表达肿瘤细胞的增殖并与顺铂具协同作用 。

Objective: Among 10 strains of anti human P185erbB2 McAbs prepared earlier, C25 having high affinity was chosen to identify the activecharacters and clone its variable region genes, so as to construct humanized antibody Methods: The antigen recognition character of C25 was analyzed by cellular ELISA and immunohistochemistry Inhibitory effects of C25 alone and combinations with chemotherapeutic agents to proliferation of SKBR3 and SKOV3 cells were detected by MTT assay Genes of VH and VL were amplified from the total RNA ofthe C25 hybridoma by RT PCR,cloned into vectors,and then sequenced Results:C25 recognized extracellular domain of P185erbB2 specifically, inhibited proliferation of SKBR3 and SKOV3 cells significantly and had synergistic effect with CDDP The 327 bp VL gene of C25 belongs to mouse kappa light chain subclass Ⅲand rearrangement in VJκ2 type; the 366 bp VH gene of C25 belong to mouse IgG1heavy chain subgroup Ⅲ(A) and rearrangement in VDJH4 type Conclusions: McAbC25 can inhibit proliferation of cells overexpressing P185erbB2 significantly and have synergy with CDDP The nucleotide sequence analysis indicates that the cloned gene code VL and VH of McAb C25 respectively