BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody...BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.展开更多
BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a...BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.展开更多
AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNA...AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.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
AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.METHODS: Extract total RNA from tissue of local cancer metastasis ...AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer.CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.展开更多
AIM: To establish a convenient immuncassay method based on recombinant antigen preS1(21-1l9aa) to detect anti-preS1antibodies and evaluate the clinical significance of antibodies in hepatitis B.METHODS: The expression...AIM: To establish a convenient immuncassay method based on recombinant antigen preS1(21-1l9aa) to detect anti-preS1antibodies and evaluate the clinical significance of antibodies in hepatitis B.METHODS: The expression plasmid pET-28a-preS1 wasconstructed, and a large quantity of preS1 (21-119aa)fragment of the large HBsAg protein was obtained. ThepreS1 fragment purified by Ni2+ -IDA affinity chromatographywas used as coated antigen to establish the indirect ELISAbased on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. Forfollow-up study, serial sera were collected during theclinical course of 21 HBV-infected patients and anti-preS1antibodies, preS1 antigen, HBV-DNA and other serologicalHBV markers were analyzed.RESULTS: preS1 (21-119aa) fragment was highly expressedfrom the plasmid pET-28a-preS1 in a soluble form in E. Coli(30 rog@ L-1 ), and easily purified to high purity over 90 % byone step of Ni2+ -IDA-sepharose 6B affinity chromatography.The purity and antigenicity of the purified preS1 (21-119aa)protein was determined by 150 g@ L-1 SDS-PAGE, Westemblot and a direct ELISA. Recombinant preS1 (21-119aa)protein was successfully applied in the immunoessay whichcould sensitively detect the anti-preS1 antibodies in serumspecimens of acute or chronic hepatitis B patients. Resultsshowed that more than half of 19 acute hepatitis B patientsproduced anti-preS1 antibodies during recovery of thedisease, however, the response was only found in a few ofchronic patients. In the clinical follow-up study of 11patients with anti-preS1 positive serological profile, HBsAgand HBV-DNA clearance occurred in 6 of 10 acute hepatitis Bpatients in 5-6 months, and seroconversion of HBeAg anddisappearance of HBV-DNA occurred in 1 chronic patientstreated with lavumidine, a antiviral agent.CONCLUSION: The high-purity preS1 ( 21-119aa) coatedantigen was successfully prepared by gene expression andaffinity chromatography. Using this antigen, a convenientlydetective system of anti-preS1 antibodies in sero wasestablished. Preliminarily clinical trial the occurrence of anti-preSl antibodies in acute hepatitis B patients suggests theclearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvementor recovery from the disease.展开更多
OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selec...OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.展开更多
基金This study was supported by grants from the National Natural Science Foundation of China (No. 30572213)and Student Innovation Program of Shanxi Medical University (No.200404).
文摘BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.
基金the National Natural Science Foundation of China, No. 30500573
文摘BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.
文摘AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.
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.
基金Supported by the Natural Science Foundation of Guangdong Province,China, No. 980120the Foundation of Excellent Youth Teacher,China,2001
文摘AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer.CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.
基金the grants No.KY951-Al-301 and No.KY95T-06-03 from the 9th Five Years Plan Key Research Programs of the Chinese Academy of Sciences.
文摘AIM: To establish a convenient immuncassay method based on recombinant antigen preS1(21-1l9aa) to detect anti-preS1antibodies and evaluate the clinical significance of antibodies in hepatitis B.METHODS: The expression plasmid pET-28a-preS1 wasconstructed, and a large quantity of preS1 (21-119aa)fragment of the large HBsAg protein was obtained. ThepreS1 fragment purified by Ni2+ -IDA affinity chromatographywas used as coated antigen to establish the indirect ELISAbased on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. Forfollow-up study, serial sera were collected during theclinical course of 21 HBV-infected patients and anti-preS1antibodies, preS1 antigen, HBV-DNA and other serologicalHBV markers were analyzed.RESULTS: preS1 (21-119aa) fragment was highly expressedfrom the plasmid pET-28a-preS1 in a soluble form in E. Coli(30 rog@ L-1 ), and easily purified to high purity over 90 % byone step of Ni2+ -IDA-sepharose 6B affinity chromatography.The purity and antigenicity of the purified preS1 (21-119aa)protein was determined by 150 g@ L-1 SDS-PAGE, Westemblot and a direct ELISA. Recombinant preS1 (21-119aa)protein was successfully applied in the immunoessay whichcould sensitively detect the anti-preS1 antibodies in serumspecimens of acute or chronic hepatitis B patients. Resultsshowed that more than half of 19 acute hepatitis B patientsproduced anti-preS1 antibodies during recovery of thedisease, however, the response was only found in a few ofchronic patients. In the clinical follow-up study of 11patients with anti-preS1 positive serological profile, HBsAgand HBV-DNA clearance occurred in 6 of 10 acute hepatitis Bpatients in 5-6 months, and seroconversion of HBeAg anddisappearance of HBV-DNA occurred in 1 chronic patientstreated with lavumidine, a antiviral agent.CONCLUSION: The high-purity preS1 ( 21-119aa) coatedantigen was successfully prepared by gene expression andaffinity chromatography. Using this antigen, a convenientlydetective system of anti-preS1 antibodies in sero wasestablished. Preliminarily clinical trial the occurrence of anti-preSl antibodies in acute hepatitis B patients suggests theclearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvementor recovery from the disease.
基金The project was supported by a grant from the National Natural Science Fundation of China (No. 39860078) and Xinjiang Natural Science Fundation China (No. 200221101).
文摘OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.
