【摘要】 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。更多还原

【Abstract】 Objective To screen an anti-botulinum neurotoxin serotype A light chain( Bo NT/A LC) antibody from the complete synthesis,humanized phage single chain antibody library and to express,purify and detect the complete antibody.Methods The Bo NT / A LC coding sequence was optimized according to the preference codon of E. coli,synthesized,inserted into p TIG and transformed into BL21( DE3) plys S. After being induced by IPTG,the recombinant protein was purified by Ni-NTA affinity chromatography. The specific clones against Bo NT / A LC were enriched and screened from the phage antibody library by " adsorption-elution-amplification" procedures. The variable region of antibody light and heavy chain was amplified and ligated into L293-CL and H293 vectors before the recombinant plasmids were transiently co-transfected into 293-F cells. After purification by Protein A column from culture supernatant,the antibody’s purification and specificity were analyzed by SDS-PAGE,Western blotting and ELISA. Results The soluble Bo NT / A LC expressed accounted for 36. 8% of total bacterial protein,and the final purity was up to 99% after purification. Ten antibodies were screened form the phage antibody library,among which Lab1 and Lab2 were picked out for preferable specificity and whole antibodies were prepared. The results of Western blotting and ELISA showed that the two whole antibodies could specifically combine with Bo NT / A LC. Conclusion This study provides two specific anti-Bo NT / A LC antibodies,which may be used for Bo NT detection and botulism therapy.更多还原