Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA...Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.展开更多
抗体是一类能特异性识别并结合抗原的免疫球蛋白,也是机体体液免疫的主要成分。随着科学技术的发展及对抗体研究的深入,抗体的应用领域不断扩展,可作为亲和配体或探针用于蛋白互相作用、蛋白与核酸相互作用、生物大分子定位与分布等研究...抗体是一类能特异性识别并结合抗原的免疫球蛋白,也是机体体液免疫的主要成分。随着科学技术的发展及对抗体研究的深入,抗体的应用领域不断扩展,可作为亲和配体或探针用于蛋白互相作用、蛋白与核酸相互作用、生物大分子定位与分布等研究,也可作为免疫抑制剂或治疗药物用于免疫性疾病、肿瘤、感染等疾病的治疗,还可结合各种标记技术用于疾病诊断、食品安全检测和环境监测。但传统抗体生产成本高、组织穿透力差、免疫排斥反应等缺点限制了其在疾病治疗等领域的广泛应用。近年来,科学家们致力于新型小分子抗体的寻找和研究,先后研制出嵌合抗体、小分子抗体、双特异性抗体等新型抗体。其中,纳米抗体(nanobody,Nb)又称重链可变区(variable heavy chain domain,VHH)抗体,是一种仅由一个重链可变区组成的单域抗体。纳米抗体保留了重链抗体完整的抗原结合能力,且具有分子质量小、组织穿透性强、抗原亲和力高、能识别隐藏表位、免疫原性低、结构稳定、水溶性好、生产成本低、易于产业化等优点,在疾病诊断、癌症和感染性疾病治疗、小分子药物及毒素残留检测等领域展现出巨大的应用前景。作者首先介绍了纳米抗体的特点,然后简述了纳米抗体的制备流程,重点综述了纳米抗体在疾病诊断、疾病治疗、食品安全和环境监测等领域的应用,最后对纳米抗体在兽医临床的应用前景进行了分析和展望。展开更多
African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in ...African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.展开更多
Pancreatic cancer stands out as a recognized intractable tumor due to its high malignancy and mortality rates,which are largely attributed to the insensitivity of current clinical chemotherapies or multidrug-resistanc...Pancreatic cancer stands out as a recognized intractable tumor due to its high malignancy and mortality rates,which are largely attributed to the insensitivity of current clinical chemotherapies or multidrug-resistance.Combinatorial chemo and gene therapy that integrates different therapeutic targets,may increase the chemosensitivity of pancreatic cancer and synergistically enhance the antitumor efficacy.However,conventional co-delivery of gene and chemo drugs is intensively dependent on complex nanoparticle delivery systems,thus would be limited by unstable drug packaging,nonspecific biodistribution,and biosafety problem.Herein,we rationally designed an epidermal growth factor-receptor(EGFR)-targeted and gemcitabine-incorporated oligonucleotide(termed as chemogene)with anti-Bcl-2 sequence,which achieves simple and precise integration of gemcitabine into a gene regulative agent,as well as the EGFR-targeted delivery for pancreatic cancer therapy.Through solid-phase synthesis,gemcitabine,as the first-line chemodrug for pancreatic cancer,is introduced to the antisense oligonucleotide to replace all cytosine nucleosides to obtain the gemcitabine-integrated chemogene(Ge-ASO^(Bcl-2)).Thereafter,Ge-ASO^(Bcl-2)is covalently coupled with EGFR nanobody to construct the final targeted chemogene without any exogenous carriers.Notably,this nanobody-conjugated chemogene exhibits remarkable tumor targeting capability and antitumor effects both in vitro and in vivo,which initiates a first step toward the application of combinatorial chemo and gene therapy for future pancreatic cancer treatment.展开更多
Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dyn...Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.展开更多
目的:通过噬菌体展示技术制备抗人IgE纳米抗体,将其用于磁微粒化学发光平台建立一种猫皮屑特异性IgE抗体超敏检测方法。方法:在WHO/IUIS过敏原数据库中检索猫致敏蛋白信息,合成序列后在原核表达系统中表达和纯化猫皮屑致敏蛋白Fel d 1...目的:通过噬菌体展示技术制备抗人IgE纳米抗体,将其用于磁微粒化学发光平台建立一种猫皮屑特异性IgE抗体超敏检测方法。方法:在WHO/IUIS过敏原数据库中检索猫致敏蛋白信息,合成序列后在原核表达系统中表达和纯化猫皮屑致敏蛋白Fel d 1。使用人源IgE免疫新疆单峰驼提取淋巴细胞中的RNA建立噬菌体展示纳米抗体库,分析库容、多样性和插入率,通过筛选和鉴定获得抗人IgE纳米抗体。使用重组过敏原偶联磁微粒和吖啶酯标记纳米抗体,建立磁微粒化学法猫皮屑特异性IgE抗体检测方法。结果:噬菌体展示库的库容为1.88×108 cfu/ml,插入率为93.6%,纳米抗体纯度>95%。检测方法的线性范围为0.1~100 U/ml,与ImmunoCAP检测系统临床对比有较好的一致性。结论:成功建立了基于纳米抗体的猫皮屑特异性IgE抗体超敏检测方法,为猫过敏性疾病辅助诊断提供了技术基础。展开更多
Single-domain antibodies(sdAbs),initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs,have emerged as a promising alternative to conventional therapeutic antibodies.These sdAbs hav...Single-domain antibodies(sdAbs),initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs,have emerged as a promising alternative to conventional therapeutic antibodies.These sdAbs have many superior physicochemical and pharmacological properties,including small size,good solubility and thermostability,easier accessible epitopes,and strong tissue penetration.However,the inherent challenges associated with the animal origin of sdAbs limit their clinical use.In recent years,various innovative humanization technologies,including complementarity-determining region(CDR)grafting or complete engineering of fully human sdAbs,have been developed to mitigate potential immunogenicity issues and enhance their compatibility.This review provides a comprehensive exploration of sdAbs,emphasizing their distinctive features and the progress in humanization methodologies.In addition,we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment,such as sdAbedrug conjugates,multispecific sdAbs,sdAb-based delivery systems,and sdAb-based cell therapy.展开更多
基金supported by the National Natural Science Foundation of China(32273041)the Key R&D Program of Shaanxi Province,China(2022NY-104)the Natural Science Foundation of Shaanxi Province,China(2022JC-12)。
文摘Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.
文摘抗体是一类能特异性识别并结合抗原的免疫球蛋白,也是机体体液免疫的主要成分。随着科学技术的发展及对抗体研究的深入,抗体的应用领域不断扩展,可作为亲和配体或探针用于蛋白互相作用、蛋白与核酸相互作用、生物大分子定位与分布等研究,也可作为免疫抑制剂或治疗药物用于免疫性疾病、肿瘤、感染等疾病的治疗,还可结合各种标记技术用于疾病诊断、食品安全检测和环境监测。但传统抗体生产成本高、组织穿透力差、免疫排斥反应等缺点限制了其在疾病治疗等领域的广泛应用。近年来,科学家们致力于新型小分子抗体的寻找和研究,先后研制出嵌合抗体、小分子抗体、双特异性抗体等新型抗体。其中,纳米抗体(nanobody,Nb)又称重链可变区(variable heavy chain domain,VHH)抗体,是一种仅由一个重链可变区组成的单域抗体。纳米抗体保留了重链抗体完整的抗原结合能力,且具有分子质量小、组织穿透性强、抗原亲和力高、能识别隐藏表位、免疫原性低、结构稳定、水溶性好、生产成本低、易于产业化等优点,在疾病诊断、癌症和感染性疾病治疗、小分子药物及毒素残留检测等领域展现出巨大的应用前景。作者首先介绍了纳米抗体的特点,然后简述了纳米抗体的制备流程,重点综述了纳米抗体在疾病诊断、疾病治疗、食品安全和环境监测等领域的应用,最后对纳米抗体在兽医临床的应用前景进行了分析和展望。
基金supported by the Natural Science Foundation of China (grant no. 31941016 and 31972676)the Natural Science Foundation of Shaanxi Province of China (grant no. 2022JC-12)the Key R&D Program of Shaanxi Province (grant no. S2022-YF-YBNY0673)
文摘African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.
基金supported by the National Key Research and Development Program of China(No.2018YFA0902601)the National Natural Science Foundation of China(Nos.52225302,52103265,and 22175116)+2 种基金the Shanghai Sailing Program(No.21YF1434300)the Natural Science Foundation of Shanghai(No.23ZR1448000)the Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(No.21TQ1400219).
文摘Pancreatic cancer stands out as a recognized intractable tumor due to its high malignancy and mortality rates,which are largely attributed to the insensitivity of current clinical chemotherapies or multidrug-resistance.Combinatorial chemo and gene therapy that integrates different therapeutic targets,may increase the chemosensitivity of pancreatic cancer and synergistically enhance the antitumor efficacy.However,conventional co-delivery of gene and chemo drugs is intensively dependent on complex nanoparticle delivery systems,thus would be limited by unstable drug packaging,nonspecific biodistribution,and biosafety problem.Herein,we rationally designed an epidermal growth factor-receptor(EGFR)-targeted and gemcitabine-incorporated oligonucleotide(termed as chemogene)with anti-Bcl-2 sequence,which achieves simple and precise integration of gemcitabine into a gene regulative agent,as well as the EGFR-targeted delivery for pancreatic cancer therapy.Through solid-phase synthesis,gemcitabine,as the first-line chemodrug for pancreatic cancer,is introduced to the antisense oligonucleotide to replace all cytosine nucleosides to obtain the gemcitabine-integrated chemogene(Ge-ASO^(Bcl-2)).Thereafter,Ge-ASO^(Bcl-2)is covalently coupled with EGFR nanobody to construct the final targeted chemogene without any exogenous carriers.Notably,this nanobody-conjugated chemogene exhibits remarkable tumor targeting capability and antitumor effects both in vitro and in vivo,which initiates a first step toward the application of combinatorial chemo and gene therapy for future pancreatic cancer treatment.
文摘Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.
文摘目的:通过噬菌体展示技术制备抗人IgE纳米抗体,将其用于磁微粒化学发光平台建立一种猫皮屑特异性IgE抗体超敏检测方法。方法:在WHO/IUIS过敏原数据库中检索猫致敏蛋白信息,合成序列后在原核表达系统中表达和纯化猫皮屑致敏蛋白Fel d 1。使用人源IgE免疫新疆单峰驼提取淋巴细胞中的RNA建立噬菌体展示纳米抗体库,分析库容、多样性和插入率,通过筛选和鉴定获得抗人IgE纳米抗体。使用重组过敏原偶联磁微粒和吖啶酯标记纳米抗体,建立磁微粒化学法猫皮屑特异性IgE抗体检测方法。结果:噬菌体展示库的库容为1.88×108 cfu/ml,插入率为93.6%,纳米抗体纯度>95%。检测方法的线性范围为0.1~100 U/ml,与ImmunoCAP检测系统临床对比有较好的一致性。结论:成功建立了基于纳米抗体的猫皮屑特异性IgE抗体超敏检测方法,为猫过敏性疾病辅助诊断提供了技术基础。
基金supported by grants from the National Key R&D Program of China(2019YFA0904400)National Natural Science Foundation of China(32270984)+1 种基金Science and Technology Commission of Shanghai Municipality(23XD1400800,China)Shanghai Municipal Health Commission(GWVI-11.2-YQ46,China).
文摘Single-domain antibodies(sdAbs),initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs,have emerged as a promising alternative to conventional therapeutic antibodies.These sdAbs have many superior physicochemical and pharmacological properties,including small size,good solubility and thermostability,easier accessible epitopes,and strong tissue penetration.However,the inherent challenges associated with the animal origin of sdAbs limit their clinical use.In recent years,various innovative humanization technologies,including complementarity-determining region(CDR)grafting or complete engineering of fully human sdAbs,have been developed to mitigate potential immunogenicity issues and enhance their compatibility.This review provides a comprehensive exploration of sdAbs,emphasizing their distinctive features and the progress in humanization methodologies.In addition,we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment,such as sdAbedrug conjugates,multispecific sdAbs,sdAb-based delivery systems,and sdAb-based cell therapy.
