为构建特异性犬细小病毒(Canine parvovirus,CPV)的纳米抗体库,并获得特异性抗CPV的重链单域抗体(Single variable domain on a heavy chain antibodies,VHH抗体),以分离鉴定的一株CPV-2c毒株(TS02株)作为免疫原免疫羊驼,四免后采集外周...为构建特异性犬细小病毒(Canine parvovirus,CPV)的纳米抗体库,并获得特异性抗CPV的重链单域抗体(Single variable domain on a heavy chain antibodies,VHH抗体),以分离鉴定的一株CPV-2c毒株(TS02株)作为免疫原免疫羊驼,四免后采集外周血,分离淋巴细胞,利用巢式PCR扩增VHH序列,通过噬菌体展示技术成功构建了纳米抗体的CPV免疫文库,进一步通过ELISA特异性筛选具有高亲和力的抗CPV纳米抗体序列。将筛选出的抗体序列插入pcDNA3.1真核表达载体构建pcDNA3.1-VHH重组质粒,采用PEI转染高密度的HEK293F悬浮细胞,瞬时表达、纯化VHH,进行鉴定。结果表明,四次免疫后羊驼血清效价高达1∶25000,达到了建库要求,构建的纳米抗体的CPV免疫文库,库容达2×10^(6)CFU/mL,文库多样性良好。通过特异性筛选获得了4株具有高亲和力和特异性的抗CPV纳米抗体序列,采用HEK293F细胞瞬时表达并纯化获得4个重组VHH,分别是CPV-VHH-H1、CPV-VHH-D4、CPV-VHH-F5和CPV-VHH-E3。ELISA和IFA试验结果表明,表达出的4株重组纳米抗体均能够特异性结合CPV。本研究成功构建了CPV特异性纳米抗体文库,并筛选出4株特异性结合CPV的VHH抗体,为VHH抗体在犬细小的诊断和治疗方面的应用进行了初步探索。展开更多
从谷氨酸棒杆菌CGMCC1.15647基因组中鉴定蛋白丰度最高基因的5′UTR(5′-untranslated region)及其下游序列,并利用这些5′UTR及其下游序列与两种高强度启动子P H36和P tac组合分别构建一系列单顺反子和双顺反子表达载体。5′UTR及其下...从谷氨酸棒杆菌CGMCC1.15647基因组中鉴定蛋白丰度最高基因的5′UTR(5′-untranslated region)及其下游序列,并利用这些5′UTR及其下游序列与两种高强度启动子P H36和P tac组合分别构建一系列单顺反子和双顺反子表达载体。5′UTR及其下游序列显著提升启动子的表达强度,其中表达强度最高的pTac-B2826-EGFP荧光强度是阳性对照pTac-Positive的3.6倍。使用筛选的增强型表达载体在谷氨酸棒杆菌中成功地表达VHH蛋白(variable domain of heavy chain of heavy-chain antibody),在摇瓶发酵水平,VHH的分泌表达量达到85.4 mg/L。筛选的双顺反子载体可成为谷氨酸棒杆菌内源基因过表达和重组蛋白生产的有利工具。展开更多
Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high p...Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.展开更多
Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original wh...Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD<sub>280</sub> nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">−</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.展开更多
文摘为构建特异性犬细小病毒(Canine parvovirus,CPV)的纳米抗体库,并获得特异性抗CPV的重链单域抗体(Single variable domain on a heavy chain antibodies,VHH抗体),以分离鉴定的一株CPV-2c毒株(TS02株)作为免疫原免疫羊驼,四免后采集外周血,分离淋巴细胞,利用巢式PCR扩增VHH序列,通过噬菌体展示技术成功构建了纳米抗体的CPV免疫文库,进一步通过ELISA特异性筛选具有高亲和力的抗CPV纳米抗体序列。将筛选出的抗体序列插入pcDNA3.1真核表达载体构建pcDNA3.1-VHH重组质粒,采用PEI转染高密度的HEK293F悬浮细胞,瞬时表达、纯化VHH,进行鉴定。结果表明,四次免疫后羊驼血清效价高达1∶25000,达到了建库要求,构建的纳米抗体的CPV免疫文库,库容达2×10^(6)CFU/mL,文库多样性良好。通过特异性筛选获得了4株具有高亲和力和特异性的抗CPV纳米抗体序列,采用HEK293F细胞瞬时表达并纯化获得4个重组VHH,分别是CPV-VHH-H1、CPV-VHH-D4、CPV-VHH-F5和CPV-VHH-E3。ELISA和IFA试验结果表明,表达出的4株重组纳米抗体均能够特异性结合CPV。本研究成功构建了CPV特异性纳米抗体文库,并筛选出4株特异性结合CPV的VHH抗体,为VHH抗体在犬细小的诊断和治疗方面的应用进行了初步探索。
文摘从谷氨酸棒杆菌CGMCC1.15647基因组中鉴定蛋白丰度最高基因的5′UTR(5′-untranslated region)及其下游序列,并利用这些5′UTR及其下游序列与两种高强度启动子P H36和P tac组合分别构建一系列单顺反子和双顺反子表达载体。5′UTR及其下游序列显著提升启动子的表达强度,其中表达强度最高的pTac-B2826-EGFP荧光强度是阳性对照pTac-Positive的3.6倍。使用筛选的增强型表达载体在谷氨酸棒杆菌中成功地表达VHH蛋白(variable domain of heavy chain of heavy-chain antibody),在摇瓶发酵水平,VHH的分泌表达量达到85.4 mg/L。筛选的双顺反子载体可成为谷氨酸棒杆菌内源基因过表达和重组蛋白生产的有利工具。
基金supported by a grant from the National Basic Research Program of China(2013CB127804)the National Natural Science Funds(31171696,China)the Research Program of the State Key Laboratory of Food Science and Technology,Nanchang University(SKLF-MB-201002)
文摘Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.
文摘Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in <em>E. coli</em> WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD<sub>280</sub> nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and <span style="white-space:nowrap;">−</span>0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.
