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
Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa...Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.展开更多
The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes ...The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.展开更多
It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain...It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.展开更多
SINCE the first report on immunoglobulin gene expression in plant by Hiatt et al. in 1989,analogous researches have been further developed. Up to now, it has become a new method touse transgenic plants and plant cells...SINCE the first report on immunoglobulin gene expression in plant by Hiatt et al. in 1989,analogous researches have been further developed. Up to now, it has become a new method touse transgenic plants and plant cells to produce antibodies and study disease-resistance breedingof crops, plant metabolism and growth, which is an organic combination of immunologyand plant sciences at molecular level. According to recent reports, both higher and lowerplants, such as tobacco, Arabidopsis, calli of maize and unicellular green alga, can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
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.
基金supported by the National Natural Science Foundation of China,No.30360100,30760234,30860260,81160373,81360458
文摘Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
文摘The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.
文摘It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.
文摘SINCE the first report on immunoglobulin gene expression in plant by Hiatt et al. in 1989,analogous researches have been further developed. Up to now, it has become a new method touse transgenic plants and plant cells to produce antibodies and study disease-resistance breedingof crops, plant metabolism and growth, which is an organic combination of immunologyand plant sciences at molecular level. According to recent reports, both higher and lowerplants, such as tobacco, Arabidopsis, calli of maize and unicellular green alga, can
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。
