靶向幽门螺杆菌UreB鲨鱼纳米抗体的筛选与鉴定

Screening and characterization of shark VNARs targeting Helicobacter pylori UreB

目的 开发靶向幽门螺杆菌(H. pylori)尿素酶B亚基(UreB)的鲨鱼纳米抗体。方法 利用大肠杆菌表达系统对UreB进行重组表达,纯化后对条纹斑竹鲨进行免疫,通过间接ELISA检测免疫后鲨鱼血浆的抗体效价。提取鲨鱼外周血淋巴细胞及脾脏组织的总RNA,即鲨源新抗原受体可变区(variable domain of immunoglobulin new antigen receptor,VNAR)构建鲨鱼纳米抗体噬菌体展示文库。扩增噬菌体文库,采用固相淘选,通过噬菌体多克隆ELISA检测特异性噬菌体的富集程度。通过单克隆ELISA鉴定阳性噬菌体,并通过序列比对,确定靶向UreB的鲨鱼纳米抗体序列。构建鲨鱼纳米抗体重组表达质粒,利用大肠杆菌表达系统制备重组鲨鱼纳米抗体。通过ELISA检测鲨鱼纳米抗体对重组UreB和H. pylori菌株的结合能力。结果 成功制备了重组UreB蛋白,用于免疫鲨鱼,经6次免疫后构建了库容量为1.28×10^(8) CFU的鲨鱼纳米抗体噬菌体展示文库。淘选到的2株靶向UreB的鲨鱼纳米抗体,具有较高的亲和力,其中1E3可特异性结合H. pylori。结论 成功筛选到靶向UreB鲨鱼纳米抗体,为H. pylori感染的诊断和治疗药物的开发奠定了基础。

Objective Development of shark VNARs targeting Helicobacter pylori(H.pylori)UreB.Methods The H.pylori urease B subunit(UreB)was recombinantly expressed using the E.coli expression system and purified for immunization of bamboo sharks.After indirect ELISA detection of?antibody titers in immune shark plasma,total RNA was extracted from shark peripheral blood lymphocytes and spleens to construct a shark VNAR phage display library.The phage library was amplified and then selected using solid-phase selection.The enrichment level of specific phages was detected by phage polyclonal ELISA.Positive phages were identified through phage monoclonal ELISA.The sequence of the shark VNARs targeting UreB was determined by sequence alignment.Recombinant expression plasmids for the shark VNARs were constructed and the recombinant VNARs were produced using the E.coli expression system.The affinity of the shark VNARs to recombinant UreB and H.pylori strains was measured by ELISA.Results In the present study,recombinant UreB protein was successfully prepared and used to immunize sharks.A shark VNAR phage display library with the capacity of 1.28×10^(8) CFU was constructed after six immunization.Two shark VNARs targeting UreB with high affinity were identified,of which VNAR 1E3 specifically binds H.pylori.Conclusion The present study successfully screened shark VNARs against UreB,providing a foundation for the development of diagnostic and therapeutic drugs targeting H.pylori.