AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL ...AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.展开更多
Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized c...Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.展开更多
The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9...The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.展开更多
Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was...Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.展开更多
Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were a...Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS-PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E.coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E.coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.展开更多
Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ at...Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ attenuated AD-like the PDAPP mouse, and developed a new approach to AD. Such vaccines were successfully tested in mouse models of AD for the reduction of Aβ plaque burden and the improvement of cognitive performance. However, 6% of AD patients developed symptoms of brain inflammation after vaccination that resembled enceohalitis or meningitis.展开更多
A modified selectively infective phage (SIP) is developed to facilitate the selection of interacting antibody antigen pairs from a large single chain antibody (scFv) library in vivo. The system is constructed with a m...A modified selectively infective phage (SIP) is developed to facilitate the selection of interacting antibody antigen pairs from a large single chain antibody (scFv) library in vivo. The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5 E. The antigen fused to the C terminal of N1 N2 domain and the scFv to the N terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively. The phages produced by co transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm. In a model system, the scFv fragment of the anti hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 10 5 fold excess of a non interacting control pairs, which demonstrates this system to be very sensitive and facile to screen a large single chain antibody library.展开更多
基金Supported by Natural Key and Basic Research Development Program,No.2002CB513109
文摘AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.
基金Supported by the National Natural Science Foundation of China(Nos 20072010 and 20572035) and the Science Foundation ofJilin University(Nos419070100087 and 01208006)
文摘Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.
文摘The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.
文摘Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.
文摘Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS-PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E.coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E.coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.
基金the grants from National Natural Science Foundation of China (No. 3040052)Natural Science and Technology Foundation of Guangdong Province (No. 04300218, 2005B10401047, 2006B36004001)Foundation of Science and Technology Bureau of Guangzhou City (No. 2004Z3-E0151, 2005Z3-E4021, 2006J1-C0101)
文摘Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ attenuated AD-like the PDAPP mouse, and developed a new approach to AD. Such vaccines were successfully tested in mouse models of AD for the reduction of Aβ plaque burden and the improvement of cognitive performance. However, 6% of AD patients developed symptoms of brain inflammation after vaccination that resembled enceohalitis or meningitis.
文摘A modified selectively infective phage (SIP) is developed to facilitate the selection of interacting antibody antigen pairs from a large single chain antibody (scFv) library in vivo. The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5 E. The antigen fused to the C terminal of N1 N2 domain and the scFv to the N terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively. The phages produced by co transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm. In a model system, the scFv fragment of the anti hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 10 5 fold excess of a non interacting control pairs, which demonstrates this system to be very sensitive and facile to screen a large single chain antibody library.
