鼠源E型肉毒毒素免疫噬菌体单链抗体库的构建及筛选

Construction and Screening of Murine BoNT/E Immunized Single Chain Antibody Phage Display Library

目的：构建鼠源E型肉毒毒素（BoNT/E）免疫噬菌体单链抗体库,筛选BoNT/E特异性抗体。方法：从E型肉毒类毒素免疫小鼠的脾细胞中提取总RNA,反转录成cDNA,分别扩增出小鼠重链可变区基因和轻链可变区基因;通过重叠延伸PCR将重链可变区基因和轻链可变区基因组装成scFv基因,重组于噬粒pS100中,电转化大肠杆菌TG_1,合并所有克隆成初级库;随机挑取克隆进行核苷酸序列测定,对初级库序列多样性进行分析;在辅助噬菌体M_（13）K_（07）的拯救下,构建成scFv噬菌体抗体库;用纯化的BoNT/E对鼠源BoNT/E免疫噬菌体单链抗体库进行3轮富集筛选,制备单克隆的噬菌体抗体颗粒进行酶联免疫吸附试验,阳性克隆进行核苷酸序列测定。结果：鼠源BoNT/E免疫噬菌体单链抗体库的库容为7.09×10^7,随机挑取的20个克隆序列各不相同,序列正确率为85%,基本覆盖了IgHV、IgKV、IgLV的优势家族;纯化的BoNT/E作为抗原通过3轮筛选,噬菌体抗体富集了66倍,第3轮筛选后随机挑取90个克隆制备噬菌体抗体颗粒,酶联免疫吸附试验分析有88个呈现阳性反应,序列比对得到了24个不同序列的BoNT/E特异性抗体。结论：构建了库容量达7.09×10^7的鼠源BoNT/E免疫噬菌体单链抗体库,筛选得到了24个不同序列的BoNT/E特异性抗体。

Objective： To construct a murine botulinum neurotoxin type E（BoNT/E） immunized single chain antibody phage display library, and screen high specificity antibody for BoNT/E. Methods： Total RNA was extracted from mouse spleen cells immunized with botulinum type E toxoid, and the RNA was used as template for cDNA synthesis reaction. The VH and VL genes were amplified using specific primers separately. Each VH and VL DNA was combined by SOE-PCR. The scFv fragments were connected to the phagemid pS100 and then transformed into E.coli TG1. The clones were picked randomly, its nucleic acid sequences were determined and sequence diversity was analyzed. Rescued by M13K07 helper phages, scFv phage display library was constructed. The purified BoNT/E were used for screening BoNT/E specific phage antibodies. All positive clones tested by ELISA were sequenced. Results： A murine BoNT/E immunized single chain antibody phage display library was generated, which had 7.09×10^7 individual clones. The sequences of randomly picked 20 clones were different, and its accuracy rate was 85%. This murine BoNT/E scFv phage display library had the highest diversity of variable gene repertoires, and basically covered dominant family of IgHV, IgKV and IgLV. After 3 rounds of screening, the phage antibodies had 66 times enrichment by the purified BoNT/E coated immunotubes. After the third round of screening, the positive rate was 97.78%. Sequence analysis revealed that there were 24 different clones. Conclusion： A murine BoNT/E scFv phage display library of 7.09×10^7 individual clones were successfully constructed, and won 24 BoNT/E specific antibodies.