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
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
目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)...目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)和轻链可变区(VL)基因,用一个柔性片段(Linker)连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体(Help phage)M13K07超感染,构建单链抗体片段(Single chainfragment of variation,ScFv)噬菌体抗体库。结果:经过富集筛选后,库容量达到3×106cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论:成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。展开更多
A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H an...A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.展开更多
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.
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.
文摘目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)和轻链可变区(VL)基因,用一个柔性片段(Linker)连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体(Help phage)M13K07超感染,构建单链抗体片段(Single chainfragment of variation,ScFv)噬菌体抗体库。结果:经过富集筛选后,库容量达到3×106cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论:成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。
文摘A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.
