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
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
构建双峰驼天然重链抗体可变区(VHH)酵母双杂交文库,并对文库质量进行鉴定。采集未经免疫的6月龄雌性双峰驼外周血、骨髓、脾和淋巴结,并从外周血中分离淋巴细胞。抽提总RNA,反转录为cDNA,用2对引物经过2轮PCR扩增得到400bp左右的双峰驼...构建双峰驼天然重链抗体可变区(VHH)酵母双杂交文库,并对文库质量进行鉴定。采集未经免疫的6月龄雌性双峰驼外周血、骨髓、脾和淋巴结,并从外周血中分离淋巴细胞。抽提总RNA,反转录为cDNA,用2对引物经过2轮PCR扩增得到400bp左右的双峰驼VHH片段。根据Clontech公司Mate&PlateTMlibrary construction system使用手册,将带有同源臂的VHH片段与线性化的pGADT7-Rec质粒共转化Y187酵母感受态细胞,转化产物涂布SD/-Leu平板,30℃倒置培养3~5d后,收集平板上的所有克隆,即为双峰驼天然重链抗体可变区酵母双杂交文库。对文库的滴度、重组效率及多样性进行鉴定。结果显示,本研究构建的双峰驼天然重链抗体可变区酵母双杂交文库库容为2.07×107,滴度为7.6×108cfu·mL-1。随机挑取47个独立克隆进行菌落PCR鉴定,43个含有VHH片段,重组率为91.5%;选取其中19个测序,均为独立克隆,且多样性好。该研究成功构建双峰驼天然重链抗体可变区酵母双杂交文库,且文库质量满足要求,为进一步获得特定抗原的VHH奠定了基础。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.
文摘构建双峰驼天然重链抗体可变区(VHH)酵母双杂交文库,并对文库质量进行鉴定。采集未经免疫的6月龄雌性双峰驼外周血、骨髓、脾和淋巴结,并从外周血中分离淋巴细胞。抽提总RNA,反转录为cDNA,用2对引物经过2轮PCR扩增得到400bp左右的双峰驼VHH片段。根据Clontech公司Mate&PlateTMlibrary construction system使用手册,将带有同源臂的VHH片段与线性化的pGADT7-Rec质粒共转化Y187酵母感受态细胞,转化产物涂布SD/-Leu平板,30℃倒置培养3~5d后,收集平板上的所有克隆,即为双峰驼天然重链抗体可变区酵母双杂交文库。对文库的滴度、重组效率及多样性进行鉴定。结果显示,本研究构建的双峰驼天然重链抗体可变区酵母双杂交文库库容为2.07×107,滴度为7.6×108cfu·mL-1。随机挑取47个独立克隆进行菌落PCR鉴定,43个含有VHH片段,重组率为91.5%;选取其中19个测序,均为独立克隆,且多样性好。该研究成功构建双峰驼天然重链抗体可变区酵母双杂交文库,且文库质量满足要求,为进一步获得特定抗原的VHH奠定了基础。
