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目的从全合成人源性噬菌体单链抗体库中筛选抗A型肉毒神经毒素轻链(Bo NT/A LC)特异性单抗,并进行全抗的表达、纯化与鉴定。方法根据大肠杆菌密码子偏好性优化并合成Bo NT/A LC序列,克隆至p TIG质粒,转化BL21(DE3)plys S感受态细胞,IPTG诱导表达后Ni-NTA亲和层析纯化目的蛋白。利用纯化的轻链蛋白从噬菌体抗体库中经“吸附-洗脱-扩增”程序淘选富集抗体并进行特异性筛选。PCR扩增抗体轻重链可变区分别连入L293-CL和H293载体,瞬时共转染293-F细胞,培养上清经Protein A柱纯化,通过SDS-PAGE、Western印迹和ELISA检测抗体纯度和特异性。结果可溶性表达的Bo NT/A LC占全菌蛋白的36.8%,纯化后蛋白纯度达99%。从噬菌体抗体库中筛选到10株单抗,选择其中特异性最好的两株单抗Lab1、Lab2制备了全抗,Western印迹和ELISA检测结果显示其可特异性识别结合Bo NT/A LC蛋白。结论该研究得到了两株特异性的Bo NT/A LC抗体,可能用于A型肉毒毒素检测和肉毒中毒治疗。
OBJECTIVE: To screen BoNT / A LC specific monoclonal antibodies against human botulinum neurotoxin type A (light chain) and to express, purify and identify the full anti-human phage scFv library. Methods The BoNT / A LC sequence was optimized and synthesized based on the codon preference of E. coli. The recombinant plasmid was cloned into pTIG plasmid and transformed into BL21 (DE3) plys S competent cells. After induced by IPTG, the target protein was purified by Ni-NTA affinity chromatography. The purified antibody is screened by phage antibody library using purified light chain protein by “adsorption-eluting-amplification” procedure and screened for specificity. The PCR products were ligated into L293-CL and H293 vector respectively and transiently co-transfected into 293-F cells. The culture supernatant was purified by Protein A column. The purity of antibody was detected by SDS-PAGE, Western blot and ELISA. Specificity. Results The soluble expressed BoNT / A LC accounted for 36.8% of the total bacterial protein and the protein purity was 99% after purification. Ten monoclonal antibodies were screened from the phage antibody library and the two McAbs Lab1 and Lab2 with the best specificities were selected. The results of Western blotting and ELISA showed that they could specifically recognize the protein bound to Bo NT / A LC . Conclusions Two specific BoNT / A LC antibodies were obtained in this study and may be used in botulinum toxin type A testing and botulism.