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目的 将抗人大肠癌单克隆抗体ND 1(mAb)的VH和VL 基因进行重组 ,构建和表达ND 1scFv ,并对其在体内外的生物学活性进行检测。方法 采用RT PCR技术 ,从能够分泌mAbND 1的鼠杂交瘤细胞中扩增VH 和VL 基因 ,通过重叠延伸拼接PCR在VH 和VL 基因间引入连接短肽 ,体外构建ND 1scFv基因 ,并在大肠杆菌中表达。采用间接免疫荧光 (IFA)及ELISA法测定ND 1scFv的免疫学活性。用99Tcm 标记ND 1scFv后 ,将偶联物给予荷瘤裸鼠 ,观察其在动物体内的显像及生物学分布。结果 SDS PAGE显示 ,重组蛋白Mr 为 30 0 0 0 ,同预期结果一致。IFA及ELISA检测表明 ,ND 1scFv保留了与亲本抗体相近的免疫学活性 ,对表达相应抗原的靶细胞具有特异结合活性。体内放射免疫实验显示 ,99Tcm ND 1scFv在荷瘤小鼠体内的生物学分布 ,呈明显的肿瘤积聚趋向 ,注入体内 1h血中T/NT即达2 .6 1。结论 获得免疫学活性良好的ND 1scFv ,对荷瘤动物体内肿瘤的定位快速、准确 ,可望成为有效的肿瘤诊断和治疗的导向载体
Objective To reconstruct the VH and VL genes of anti-human colorectal cancer monoclonal antibody ND 1 (mAb) and construct and express ND 1 scFv. The biological activity of ND 1 scFv was tested in vitro and in vivo. Methods RT-PCR was used to amplify VH and VL genes from murine hybridoma cells capable of secreting mAbND1. The joining short peptides were introduced between VH and VL genes by overlap extension splicing PCR. ND 1 scFv gene was constructed in vitro and expressed in Escherichia coli In the expression. The immunological activity of ND 1 scFv was determined by indirect immunofluorescence (IFA) and ELISA. After the ND 1 scFv was labeled with 99Tcm, the conjugate was administered to nude mice bearing the tumor to observe its imaging and biological distribution in animals. Results SDS PAGE showed that the recombinant protein Mr was 30 000, consistent with the expected results. IFA and ELISA showed that ND 1scFv retained similar immunological activity as the parent antibody and had specific binding activity to target cells expressing the corresponding antigen. In vivo radioimmunoassay showed that the biological distribution of 99Tcm ND 1scFv in tumor-bearing mice showed a tendency of tumor accumulation, and the T / NT of blood injected into the body for 1h reached 2.61. Conclusion Obtaining the immunologically active ND 1 scFv can rapidly and accurately locate the tumor in tumor-bearing animals and is expected to become an effective vector for tumor diagnosis and treatment