Objective To construct an immune VNAR phage display library,and develop potent VNARs targeting HtrA which is a critical virulence factor of Helicobacter pylori from the library.Methods A white-spotted bamboo shark was...Objective To construct an immune VNAR phage display library,and develop potent VNARs targeting HtrA which is a critical virulence factor of Helicobacter pylori from the library.Methods A white-spotted bamboo shark was immunized with recombinant HtrA protein,and the coding sequence of VNARs was cloned from its peripheral blood leukocytes(PBLs)and spleen tissues.VNAR phage display library was constructed,and anti-HtrA VNARs were selected from the library.The recombinant,expression and purification of VNARs were carried out by using Escherichia coli expression system.ELISA was used to detect the affinity and stability of VNARs.Results VNAR phage display library targeting HtrA was constructed,and two VNARs(G11 and 1A5)were selected,expressed in E.coli BL21(DE3)cells,and purified for further characterization.The affinity and stability of VNARs were assessed.The affinity values of G1l and 1A5 were 21.2,27.3 nmol/L,respectively,and they showed high stability in low pH solutions.A bivalent VNAR Bi-1A5 was constructed and showed a significant increase in antigen binding affinity.Conclusion VNAR molecules with high affinity targeting HtrA could be obtained by immunizing marine species whitespotted bamboo shark,which provided a new idea for the diagnosis and treatment of H.pylori infection.展开更多
Fifty whitespotted bamboo sharks(Chiloscyllium playgiosum)of both sexes were used to establish a large capacity variable domain of the new antigen receptor(VNAR)library with a total capacity of over 10^(9) colony-form...Fifty whitespotted bamboo sharks(Chiloscyllium playgiosum)of both sexes were used to establish a large capacity variable domain of the new antigen receptor(VNAR)library with a total capacity of over 10^(9) colony-forming units(CFU).It was applied to screen VNARs against human serum albumin(HSA)and human transcription factor EB(TFEB),respectively.Meanwhile,VNAR libraries specific to HSA and TFEB with capacities above 10^(8) CFU were obtained following conventional immunization.These two approaches were systematically studied in terms of VNAR yield and composition.By comparing the VNAR sequences obtained from naı¨ve and antigen-immunized libraries,we found that the complementary-determining region 3(CDR3)of the former differs in composition from that of the latter.It shares a higher degree of homology with the naı¨ve library.Meanwhile,the binding efficiency assessed by ELISA is also different between the naı¨ve and antigen-immunized libraries.The binding of VNARs from the TFEB-immunized library appeared to surpass that observed with the naı¨ve libraries,whereas the performance of VNARs from the HSA-immunized library indicated that both the immunized and naı¨ve libraries for HSA had positive binding responses in polyclonal and monoclonal ELISA.The results are useful to develop novel diagnostic and therapeutic products based on shark VNARs.展开更多
TROP-2, a tumor-associated antigen, has been implicated in the progression of various epithelial tumors. Due to its favorable expression profile, TROP-2 has emerged as a promising target for antibody-drug conjugates (...TROP-2, a tumor-associated antigen, has been implicated in the progression of various epithelial tumors. Due to its favorable expression profile, TROP-2 has emerged as a promising target for antibody-drug conjugates (ADCs) based anti-tumor therapies. Although ADCs have shown efficacy in cancer treatment, their application in solid tumors is hindered by their high molecular weight, poor tumor penetration, and release of cytotoxic molecules. Therefore, a recombinant immunotoxin was developed based on a shark-derived variable domain of immunoglobulin new antigen receptor (VNAR) antibody. VNARs are only one-tenth the size of IgG antibodies and possess remarkable tissue penetration capabilities and high stability. In this study, a shark VNAR phage display library was created, leading to the identification of shark VNAR-5G8 that targets TROP-2. VNAR-5G8 exhibited a high affinity and cellular internalization ability towards cells expressing high levels of TROP-2. Epitope analysis revealed that VNAR-5G8 recognizes a hidden epitope consisting of CRD and TY-1 on TROP-2. Subsequently, VNAR-5G8 was fused with a truncated form of Pseudomonas exotoxin (PE38) to create the recombinant immunotoxin (5G8-PE38), which exhibited significant anti-tumor activity in vitro and in vivo. Overall, this study highlights the promise of 5G8-PE38 as a valuable candidate for cancer therapy.展开更多
目的开发靶向转铁蛋白受体1(transferrin receptor 1,TfR1)且具有跨血脑屏障转运功能的鲨鱼纳米抗体。方法提取未经靶抗原免疫的条纹斑竹鲨外周血单个核细胞总RNA,通过PCR技术扩增获得鲨鱼抗体的重链可变区(variable domain of immunogl...目的开发靶向转铁蛋白受体1(transferrin receptor 1,TfR1)且具有跨血脑屏障转运功能的鲨鱼纳米抗体。方法提取未经靶抗原免疫的条纹斑竹鲨外周血单个核细胞总RNA,通过PCR技术扩增获得鲨鱼抗体的重链可变区(variable domain of immunoglobulin new antigen receptor,VNAR)基因,将VNAR构建至噬菌体展示载体中,完成鲨鱼纳米抗体天然文库的构建。扩增天然噬菌体展示文库,采用抗原固相淘选策略,通过三轮“结合-洗脱-扩增”的淘选流程完成噬菌体展示文库的富集,通过噬菌体ELISA鉴定阳性克隆,并进行抗体序列比对,以确定靶向TfR1的鲨鱼纳米抗体序列。构建鲨鱼纳米抗体重组表达载体,利用大肠杆菌原核表达系统进行鲨鱼纳米抗体的诱导表达,ELISA检测鲨鱼纳米抗体与TfR1重组蛋白的结合能力,竞争性ELISA检测鲨鱼纳米抗体与转铁蛋白(transferrin,Tf)竞争受体结合表位的情况,免疫荧光技术检测鲨鱼纳米抗体与脑微血管内皮细胞bEnd.3的结合能力,血脑屏障体外实验模型检测鲨鱼纳米抗体的跨血脑屏障转运能力。结果成功构建了库容量为2.35×10^(9) CFU的鲨鱼纳米抗体天然文库,该文库具备良好的多样性。经淘选获得2株靶向TfR1的鲨鱼纳米抗体,均具有亲和活性,其中D1具有跨血脑屏障转运功能。结论本研究成功筛选出靶向TfR1且具有跨血脑屏障转运功能的鲨鱼纳米抗体,为中枢神经系统疾病治疗提供了新的跨血脑屏障药物递送载体。展开更多
文摘Objective To construct an immune VNAR phage display library,and develop potent VNARs targeting HtrA which is a critical virulence factor of Helicobacter pylori from the library.Methods A white-spotted bamboo shark was immunized with recombinant HtrA protein,and the coding sequence of VNARs was cloned from its peripheral blood leukocytes(PBLs)and spleen tissues.VNAR phage display library was constructed,and anti-HtrA VNARs were selected from the library.The recombinant,expression and purification of VNARs were carried out by using Escherichia coli expression system.ELISA was used to detect the affinity and stability of VNARs.Results VNAR phage display library targeting HtrA was constructed,and two VNARs(G11 and 1A5)were selected,expressed in E.coli BL21(DE3)cells,and purified for further characterization.The affinity and stability of VNARs were assessed.The affinity values of G1l and 1A5 were 21.2,27.3 nmol/L,respectively,and they showed high stability in low pH solutions.A bivalent VNAR Bi-1A5 was constructed and showed a significant increase in antigen binding affinity.Conclusion VNAR molecules with high affinity targeting HtrA could be obtained by immunizing marine species whitespotted bamboo shark,which provided a new idea for the diagnosis and treatment of H.pylori infection.
基金supported by research grants from Sanya Yazhou Bay Science&Technology City Administration(SKJC-2022-01-002 to Ming-Wei Wang and SKJC-2024-03-001 to Hao Li,China)Special Project for the Promotion of Deep-Sea Technology Industry,Hainan Deep-Sea Technology Innovation Center(DSTIC-CYCJ-2023012 to Ming-Wei Wang,China).
文摘Fifty whitespotted bamboo sharks(Chiloscyllium playgiosum)of both sexes were used to establish a large capacity variable domain of the new antigen receptor(VNAR)library with a total capacity of over 10^(9) colony-forming units(CFU).It was applied to screen VNARs against human serum albumin(HSA)and human transcription factor EB(TFEB),respectively.Meanwhile,VNAR libraries specific to HSA and TFEB with capacities above 10^(8) CFU were obtained following conventional immunization.These two approaches were systematically studied in terms of VNAR yield and composition.By comparing the VNAR sequences obtained from naı¨ve and antigen-immunized libraries,we found that the complementary-determining region 3(CDR3)of the former differs in composition from that of the latter.It shares a higher degree of homology with the naı¨ve library.Meanwhile,the binding efficiency assessed by ELISA is also different between the naı¨ve and antigen-immunized libraries.The binding of VNARs from the TFEB-immunized library appeared to surpass that observed with the naı¨ve libraries,whereas the performance of VNARs from the HSA-immunized library indicated that both the immunized and naı¨ve libraries for HSA had positive binding responses in polyclonal and monoclonal ELISA.The results are useful to develop novel diagnostic and therapeutic products based on shark VNARs.
基金Qingdao Marine Science and Technology Center(No.2022QNLM030003-4 and 8-01,China)Program of National Natural Science Foundation of China(No.82273846)Taishan Scholars Program(No.tsqn202211058,China).
文摘TROP-2, a tumor-associated antigen, has been implicated in the progression of various epithelial tumors. Due to its favorable expression profile, TROP-2 has emerged as a promising target for antibody-drug conjugates (ADCs) based anti-tumor therapies. Although ADCs have shown efficacy in cancer treatment, their application in solid tumors is hindered by their high molecular weight, poor tumor penetration, and release of cytotoxic molecules. Therefore, a recombinant immunotoxin was developed based on a shark-derived variable domain of immunoglobulin new antigen receptor (VNAR) antibody. VNARs are only one-tenth the size of IgG antibodies and possess remarkable tissue penetration capabilities and high stability. In this study, a shark VNAR phage display library was created, leading to the identification of shark VNAR-5G8 that targets TROP-2. VNAR-5G8 exhibited a high affinity and cellular internalization ability towards cells expressing high levels of TROP-2. Epitope analysis revealed that VNAR-5G8 recognizes a hidden epitope consisting of CRD and TY-1 on TROP-2. Subsequently, VNAR-5G8 was fused with a truncated form of Pseudomonas exotoxin (PE38) to create the recombinant immunotoxin (5G8-PE38), which exhibited significant anti-tumor activity in vitro and in vivo. Overall, this study highlights the promise of 5G8-PE38 as a valuable candidate for cancer therapy.
文摘目的开发靶向转铁蛋白受体1(transferrin receptor 1,TfR1)且具有跨血脑屏障转运功能的鲨鱼纳米抗体。方法提取未经靶抗原免疫的条纹斑竹鲨外周血单个核细胞总RNA,通过PCR技术扩增获得鲨鱼抗体的重链可变区(variable domain of immunoglobulin new antigen receptor,VNAR)基因,将VNAR构建至噬菌体展示载体中,完成鲨鱼纳米抗体天然文库的构建。扩增天然噬菌体展示文库,采用抗原固相淘选策略,通过三轮“结合-洗脱-扩增”的淘选流程完成噬菌体展示文库的富集,通过噬菌体ELISA鉴定阳性克隆,并进行抗体序列比对,以确定靶向TfR1的鲨鱼纳米抗体序列。构建鲨鱼纳米抗体重组表达载体,利用大肠杆菌原核表达系统进行鲨鱼纳米抗体的诱导表达,ELISA检测鲨鱼纳米抗体与TfR1重组蛋白的结合能力,竞争性ELISA检测鲨鱼纳米抗体与转铁蛋白(transferrin,Tf)竞争受体结合表位的情况,免疫荧光技术检测鲨鱼纳米抗体与脑微血管内皮细胞bEnd.3的结合能力,血脑屏障体外实验模型检测鲨鱼纳米抗体的跨血脑屏障转运能力。结果成功构建了库容量为2.35×10^(9) CFU的鲨鱼纳米抗体天然文库,该文库具备良好的多样性。经淘选获得2株靶向TfR1的鲨鱼纳米抗体,均具有亲和活性,其中D1具有跨血脑屏障转运功能。结论本研究成功筛选出靶向TfR1且具有跨血脑屏障转运功能的鲨鱼纳米抗体,为中枢神经系统疾病治疗提供了新的跨血脑屏障药物递送载体。
