抗人大肠癌单链抗体的构建、表达及其体内外生物学活性的检测

目的 将抗人大肠癌单克隆抗体ND-1(mAb)的VR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。采用RT－PCR技术，从能够分泌mAb ND-1的鼠杂交瘤细胞中扩增VH和RL基因，通过重叠延伸拼接PCR在VH和VL基因间引入连接短肽，体外构构建ND－1scFv基因，并在大肠杆菌中表达。采用间接免疫荧光（IFA）EY IF工IMPG-1scFv的免疫学活性。用^99Tc^m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－PAGEW显示，重组蛋白Mr为30000，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行异结合活性。体内放射免疫实验显示，^99Tc^m-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即在2．61。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。

Construction, expression and characterization of single chain Fv antibody against human colorectal carcinoma

Aim To construct and express recombinant single chain antibody (ND 1scFv) from the variable regions of light chain and heavy chain of monoclonal antibody (mAb)ND 1 against human colorectal carcinoma and detect its biological activities in vitro and in vivo . Methods V H and V L genes were amplified from hybridoma cell IC 2 secreting mAb ND 1 by RT PCR. A linker peptide (Gly 4Ser) 3 was connected between V H and V L genes by extension overlap splicing PCR to obtain the ND 1scFv gene. Then ND 1scFv gene was cloned into pET28a(+)vector and expressed in E.coli BL 21. The immunoactivity of expressed product was analyzed by ELISA and indirect immunofluorescence technique. After ND 1scFv was labeled with 99 Tc m, 99 Tc m ND 1scFv was injected into mice bearing human colorectal carcinoma xenograft for in vivo imaging and biodistribution study. Results SDS PAGE analysis showed that the relative molecular mass( M r) of fusion protein ND 1scFv was 30 000, which was identical with the theoretically predicted value. Indirect immunofluorescence assay and ELISA analysis revealed that ND 1scFv was equal to the parent ND 1 antibody in immunoreactivity and had specific binding activity to corresponding antigen expressing target cells. In vivo radioimmunoassays showed that 99 Tc m ND 1scFv gathered significantly round tumors in mice bearing human colorectal carcinoma, and at one hour after injection, the T/NT radio in blood reached 2.61. Conclusion ND 1scFv has excellent immunoactivity and may localized accurately and rapidly on tumors, which is promising as a targeting vector for diagnosis and therapy of tumors.