抗幽门螺杆菌UreB重组全人源化单域抗体的基因工程表达及分子改造  

Genetic engineering and molecular modification of recombinant fully humanized single-domain antibody against Helicobacter pylori UreB

作　　者：王雪芳 赵阳 刘竹青 郭乐[3] 仲飞亮 罗学刚 WANG Xuefang;ZHAO Yang;LIU Zhuqing;GUO Le;ZHONG Feiliang;LUO Xuegang(Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education and Key Laboratory of Industrial Microbiology of Tianjin,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457;National Demonstration Center for Experimental Bioengineering Education,Tianjin 300457;Ningxia Key Laboratory of Clinical Pathogenic Microorganisms,School of Laboratory Medicine,Ningxia Medical University,Yinchuan,750004,China)

机构地区：[1]天津科技大学生物工程学院工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室,天津300457 [2]生物工程国家级实验教学示范中心,天津300457 [3]宁夏医科大学检验学院宁夏临床病原微生物重点实验室,银川750004

出　　处：《中国药科大学学报》2024年第5期666-672,共7页Journal of China Pharmaceutical University

基　　金：宁夏回族自治区重点研发计划项目(2020BFG02012);国家重点研发计划项目(2018YFA0901700,2017YFD0400303)。

摘　　要：为了构建针对幽门螺杆菌(Helicobacter pylori,Hp)脲酶的单域抗体的基因工程表达系统。首先利用Pymol、ITASSER和ClussPro2等AI辅助工具分析比较了不同抗体与Hp脲酶亚单位B(UreB)之间的分子间作用力,确定了VL全人源化单域抗体UreBAb作为重点研究对象。根据大肠埃希菌密码子偏好性优化了UreBAb基因序列,并将其分别插入pET28a、pE-SUMO等表达载体,转化大肠埃希菌Rosetta(DE3)获得了重组表达菌株,通过IPTG诱导制备重组抗体蛋白,并利用提取的Hp脲酶作为抗原,分析验证了重组表达抗体的活性。SDS-PAGE检测结果显示UreBAb和SUMO-UreBAb均获得了正确的表达,纯化后的蛋白产率分别为0.34和0.41 mg/mL。单向免疫扩散实验结果证实这两种重组表达抗体均与Hp UreB抗原具有良好的亲和力,对脲酶的抑制率分别为51.27%和74.07%。进而,结合AlphaFold2、cluspro2、mCSM-AB、OSPREY和FoldX等人工智能工具,评估分析了影响抗原-抗体复合物稳定性的关键位点及其突变策略,随后进一步构建了9个UreBAb进化突变体表达菌株,活性分析结果显示,这些突变体与抗原的结合活性均得到了提高,其中以I107W突变体的活性提升最为显著,相较于野生型UreBAb,提高了24.95%。本研究为Hp全人源化单域抗体的开发奠定了良好的基础。To construct a recombinant expression system for a single-domain antibody targeting the urease of Helicobacter pylori(Hp),this study employed several strategies.First,using artificial intelligence(AI)auxiliary tools such as Pymol,I-TASSER,and ClussPro2,the molecular interactions between different antibodies and Hp urease subunit B(UreB)were analyzed.The fully humanized single-domain antibody UreBAb was identified as the primary research target.Next,the UreBAb gene sequence was optimized based on Escherichia coli codon preferences,and was inserted into expression vectors such as pET28a and pE-SUMO.The resulting recombinant expression strains were obtained by transforming Escherichia coli Rosetta(DE3).Recombinant antibody proteins were prepared through IPTG induction,and its activity was detected using extracted Hp urease as the antigen.SDS-PAGE analysis confirmed the correct expression of both UreBAb and SUMO-UreBAb,with protein yields of 0.34 mg/mL and 0.41 mg/mL,respectively.Unidirectional immunodiffusion experiments further confirmed that both recombinant antibodies exhibited strong affinity for Hp UreB antigen,with inhibition rates of 51.27%and 74.07%,respectively.Additionally,leveraging artificial intelligence tools such as AlphaFold2,cluspro2,mCSM-AB,OSPREY,and FoldX,the study evaluated and analyzed key binding sites and mutational strategies affecting the stability of the antigen-antibody complex.Subsequently,nine UreBAb evolution mutants were constructed,and their binding activities with the antigen were enhanced.Among these,the I107W mutant showed the most significant improvement,achieving a 24.95%increase compared to the wild-type UreBAb.This research lays a solid foundation for the development of fully humanized single-domain antibodies against Hp.

关 键 词：幽门螺杆菌 脲酶 全人源化单域抗体 基因工程 AI辅助分子改造 

分 类 号：Q786[生物学—分子生物学]