抗对硫磷单链抗体在昆虫细胞中的表达及活性鉴定

利用Bac-to-Bac杆状病毒表达系统,在Sf9昆虫细胞中表达抗对硫磷单链抗体,并评价该重组抗体scFv-4C6的分子识别活性。以分泌能特异性识别对硫磷的单克隆抗体的杂交瘤细胞株4C6为RNA来源,采用RT-PCR方法扩增抗体的重链和轻链可变区基因,经重叠延伸PCR方法串联拼接获得单链抗体基因片段(scFv-4C6)。构建包含目的片段的重组杆粒Bacmid-scFv-4C6,转染Sf9细胞表达目的蛋白,采用免疫印迹法(Western blotting)检测表达产物,间接竞争酶联免疫吸附(ic-ELISA)法评价产物的生物活性。结果表明:scFv-4C6基因片段拼装正确,成功转染Sf9细胞,并在转染后72 h表达量最高,表达的单链抗体大小为28.3 kD;表达产物能特异性识别对硫磷,IC50值为7.9 ng/mL,对甲基对硫磷和杀螟硫磷分别有12%和1.8%的交叉反应率,与亲本单克隆抗体的识别性能相似。该研究表明,具有生物活性的抗对硫磷单链抗体scFv-4C6可在昆虫细胞中成功得到表达。

Expression and characterization of single-chain variable fragment(scFv) antibody against parathion-ethyl in insect cells

In this study, scFv-coding genes were obtained from a hybridoma cell and expressed in Bac-to-Bac baculovirus expression system, and the characterizations of scFv were verified by Western blotting, ELISA and compared with monoclonal antibody. Firstly, the specific heavy and light variable chains were amplified from hybridoma cell line 4C6, which secreting monoclonal antibody(mAb) against parathion-ethyl, and then they were assembled as scFv fragments by splice overlap extension polymerase chain reaction(SOE-PCR). Thereafter, the bacmid-scFv-4C6 was constructed and transfected into Sf9 insect cells to generate the recombinant antibody which was further verified by Western blotting. The results demonstrated that the recombinant scFv-4C6, with the relative molecular mass of approximately 28.3 kD, was successfully constructed and expressed in Sf9 cells. Besides, the expression peak was reached at 72 h after the transfection. As tested by ic-ELISA, the performances of scFv-4C6 were similar to those of the parental mAb, with a high sensitivity(IC₅₀ value) of 7.9 ng/mL to parathion-ethyl, 12% cross reactivity to parathion-methyl, and 1.8% cross reactivity to fenitrothion.