6B11卵巢癌抗独特型单链抗体表达及序列分析

目的：克隆和表达６Ｂ１１卵巢癌抗独特型抗体单链可变区基因，并进行序列分析。方法：采用逆转录ＰＣＲ和基因重组技术，扩增并构建６Ｂ１１鼠抗独特型抗体单链可变区基因，重组至噬菌体表面表达载体，在大肠杆菌表达，并利用双脱氧末端终止法进行核苷酸序列分析。结果：阳性克隆表达产物与卵巢癌单克隆抗体ＣＯＣ１６６－９特异结合；ＤＮＡ序列分析表明重链和轻链可变区分属小鼠免疫球蛋白重链第Ｉ亚群和轻链第Ⅱ亚群。结论：自６Ｂ１１小鼠杂交瘤细胞成功地克隆表达了该抗独特型单链抗体基因，为进一步人源化改造，推进卵巢癌的免疫治疗奠定了良好基础。

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

^（99m）Tc标记卵巢癌单抗的实验研究

用９９ｍＴｃ标记卵巢癌单抗，对标记方法、纯化手段、还原后抗体存放时间与标记率关系、标记物免疫活性以及用该标记物对荷瘤裸鼠放免显象进行探讨，提示９９ｍＴｃ标记卵巢癌单抗的可行性。