抗蓖麻毒素A链人源化单域抗体的设计、原核表达及生物活性检测

Rational design, expression and biological activity assessment of a novel peptide based on ricin toxin antibody

目的运用计算机辅助蛋白质分子设计的方法设计针对蓖麻毒素A链（RTA）的拮抗肽，实现在大肠杆菌BL21中的可溶性表达，并对其生物学活性进行评价。方法根据RTA的晶体结构、RTA-rRNA相互作用复合物模型，在CVFF（consistent-valence force field）、Amber力场下，对RTA的空间构象进行理论模拟，初步确定其生物活性功能域；然后针对该功能域设计小分子拮抗肽，并借助人抗体重链可变区骨架，在CDR3区对拈抗肽进行展示，用重叠延伸PCR全基因合成人源化的单域抗体并克隆至载体pET-32a（＋）；双酶切和DNA测序技术对构建的载体进行鉴定；IPTG诱导人源化的单域抗体表达，用镍离子亲和层析纯化，竞争ELISA和MTr法分别进行结合和中和活性检测。结果从头搭建并设计合成了人源化的单域抗体，实现了其原核表达，并进行了生物活性检测；建立了基于人源化的单域抗体的RTA和蓖麻毒素检测方法。结论研究结果为新型蓖麻毒素小分子拈抗剂的研制奠定了理论和实验基础。

Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain （RTA） and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guided molecular design method. Its coding sequence was obtained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then expressed in E. coli BL21 （ DE3 ）, identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for ricin was evaluated by competitive ELISA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coli. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel peptide based on ricin toxin antibody provides a novel method to develop new generation of ricin antagonists.