克百威纳米抗体的结构解析与体外进化研究

Structural analysis and in vitro evolution study of anti-carbofuran-nanobody

克百威曾是一种广泛用于农业虫害防治领域的氨基甲酸酯类农药,由于其对哺乳动物具有高毒性,对人畜安全及环境造成较大威胁,因此各国陆续出台了克百威禁限用政策,但其在农产品中被检出超标的问题仍时有报道,故对其加强监测十分必要。纳米抗体是一种新型的基因工程片段抗体,具有高稳定性、结构简单和高表达量等优势。本研究通过体外亲和力成熟技术,对实验室已有的克百威纳米抗体Nb5进行改造,进一步提升其灵敏度。首先,基于AlphaFold系统,预测纳米抗体三维结构并验证模型合理性;以分子对接结合丙氨酸扫描技术,研究克百威与纳米抗体的识别机制,并验证关键结合位点。结果表明:克百威分子进入了由CDR2、CDR3及FR3区组成的活性口袋腔,纳米抗体通过氢键和疏水作用力识别克百威,所涉及的关键氨基酸分别为Arg56、Trp58、Thr101、Val102、Ala103、Asp104和Cys105。基于噬菌体展示技术,构建了克百威纳米抗体的定点饱和突变库,并筛选到一株突变体H10,其IC_(50)值为17.6 ng/mL,其灵敏度比野生型Nb5提升了1倍,亲和力常数为1.54×10^(6)L/mol。该研究结果为开发针对小分子药物的纳米抗体体外亲和力成熟技术提供了一种有效的新思路。

Carbofuran was once a highly toxic carbamate pesticide widely used in the prevention and control of pests in the agricultural field.In recent years,the problem of excessive residues of carbofuran in agricultural products has been reported,posing a great threat to human beings,mammals,and the environment.The maximum permitted levels of carbofuran in food have been established under different legislations worldwide.Therefore,it is necessary to strengthen supervision and rapid detection of carbofuran residues in food.Due to its high solubility and high yield,nanobody has been paid more attention in recent years.To further improve their sensitivity and stability to meet the demands of actual detection,the Nb5 was selected for in vitro evolution research.The structure models of Nb5 were constructed based on AlphaFold and verified by Ramachandran Plot,ERRAT,and Verify 3D.The key amino acid sites of Nb5/carbofuran were determined by LeadIT software and alanine scanning.The result showed that the carbofuran molecule was inserted into the pocket composed of CDR2,CDR3,and FR3.Nb5 and carbofuran were driven by hydrogen bonds and hydrophobic forces.Based on the confirmed mutation sites(Arg56,Trp58,Thr101,Val102,Ala103,Asp104,Cys105),a phage display saturation mutation library was established to screen mutants,and their performance was characterized.The IC50 of H10 was 17.6 ng/mL,the sensitivity was twice compared with that of the wild type Nb5,and the affinity constant was 1.54×10^(6) L/mol.This study provides an effective idea for the affinity maturation of nanobodies against small molecule drugs in vitro.