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
Multiple sequence alignments can be used in the template-based modelling of protein structures to build fragment-based assembly models. Therefore, useful functional information on the 3D structure of the anti-MCF-7 sc...Multiple sequence alignments can be used in the template-based modelling of protein structures to build fragment-based assembly models. Therefore, useful functional information on the 3D structure of the anti-MCF-7 scFv protein can be obtained using available bioinformatics tools. This paper utilises several commonly-used bioinformatics tools and databases, including BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Data Bank), KABAT numbering and SWISS-MODEL, to gain specific functional insights into the anti-MCF-7 scFv protein and the assembly of single-chain fragment variable (scFv) antibodies, which consist of a variable heavy chain (VH) and a variable light chain (VL) connected by the linker (Gly4-Ser)3. The linker has been built as a loop structure using the Insight II software. The accuracy of the loop structure has been evaluated using Root Mean Square Deviation (RMSD). The accuracies of the VL and VH template-based structures are enhanced by using the evaluation methods Verify3D, ERRAT and Ramchandran plotting, which measure the error in the residues. In the results, 100% of the light-chain residues scored above 0.2, whereas 88.5% of the heavy-chain residues’ scored above 0.15 in the Verify3D evaluation method. Meanwhile, using ERRAT, the alignments of both chains scored more than 70% in space. Additionally, the Ramchandran plot evaluation method showed large numbers of residues in the favoured areas in both chains;these findings demonstrated that all of the chosen templates were the best candidates.展开更多
In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human an...In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.展开更多
In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage dis...In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.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
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
Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strat...Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse na ve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.展开更多
Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies a...Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT 8 were screened and identified. Human T1 D single-chain variable fragment(sc Fv) phage display library consists of approximately 1í10~8 clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F’ and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1 D.展开更多
比较表达于大肠杆菌中的人源性抗细胞毒性T淋巴细胞抗原4单链抗体(A n ti-CTLA 4 scFv)的三种体外复性方法的复性效率。稀释、透析和亲和柱上三种复性方式复性A n ti-CTLA 4 scFv,采用B rad ford法分析蛋白复性得率,采用间接细胞EL ISA...比较表达于大肠杆菌中的人源性抗细胞毒性T淋巴细胞抗原4单链抗体(A n ti-CTLA 4 scFv)的三种体外复性方法的复性效率。稀释、透析和亲和柱上三种复性方式复性A n ti-CTLA 4 scFv,采用B rad ford法分析蛋白复性得率,采用间接细胞EL ISA法检测所得蛋白的活性。透析复性蛋白得率最高,稀释复性蛋白得率其次,亲和柱上复性蛋白得率最低;透析复性所得蛋白结合活性是稀释复性的1.95倍,是亲和柱上复性所得蛋白活性的4.13倍(无谷胱甘肽氧化还原对G SH/G SSH)及3.63倍(有G SH/G SSH)。结论:采用含0.15 m o l/L N aC、l1 mm o l/L G SSH和3 mm o l/L G SH的50 mm o l/L T ris-HC l(pH 8.0)缓冲液作为A n ti-CTLA 4 scFv的复性液,4℃下透析复性48 h,可获得较高复性得率和结合活性。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.
文摘Multiple sequence alignments can be used in the template-based modelling of protein structures to build fragment-based assembly models. Therefore, useful functional information on the 3D structure of the anti-MCF-7 scFv protein can be obtained using available bioinformatics tools. This paper utilises several commonly-used bioinformatics tools and databases, including BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Data Bank), KABAT numbering and SWISS-MODEL, to gain specific functional insights into the anti-MCF-7 scFv protein and the assembly of single-chain fragment variable (scFv) antibodies, which consist of a variable heavy chain (VH) and a variable light chain (VL) connected by the linker (Gly4-Ser)3. The linker has been built as a loop structure using the Insight II software. The accuracy of the loop structure has been evaluated using Root Mean Square Deviation (RMSD). The accuracies of the VL and VH template-based structures are enhanced by using the evaluation methods Verify3D, ERRAT and Ramchandran plotting, which measure the error in the residues. In the results, 100% of the light-chain residues scored above 0.2, whereas 88.5% of the heavy-chain residues’ scored above 0.15 in the Verify3D evaluation method. Meanwhile, using ERRAT, the alignments of both chains scored more than 70% in space. Additionally, the Ramchandran plot evaluation method showed large numbers of residues in the favoured areas in both chains;these findings demonstrated that all of the chosen templates were the best candidates.
基金Supported by the National Natural Science Foundation of China(No.30970608)the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045)+1 种基金the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948)the Innovation Method Fund of China (No.2008IM040800)
文摘In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.
文摘In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.
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.
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 National Basic Research Program of China (2009CB118701,2009CB118704)the National Natural Science Foundation of China (31072233,31172434)
文摘Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse na ve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.
基金supported by National Natural Science Foundation of China (81070622, 81370939)Provincial Six Talent Foundation of Jiangsu (2010-022)+1 种基金Municipal Science and Technology Foundation of Nanjing (009010684)A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801)
文摘Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT 8 were screened and identified. Human T1 D single-chain variable fragment(sc Fv) phage display library consists of approximately 1í10~8 clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F’ and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1 D.
文摘比较表达于大肠杆菌中的人源性抗细胞毒性T淋巴细胞抗原4单链抗体(A n ti-CTLA 4 scFv)的三种体外复性方法的复性效率。稀释、透析和亲和柱上三种复性方式复性A n ti-CTLA 4 scFv,采用B rad ford法分析蛋白复性得率,采用间接细胞EL ISA法检测所得蛋白的活性。透析复性蛋白得率最高,稀释复性蛋白得率其次,亲和柱上复性蛋白得率最低;透析复性所得蛋白结合活性是稀释复性的1.95倍,是亲和柱上复性所得蛋白活性的4.13倍(无谷胱甘肽氧化还原对G SH/G SSH)及3.63倍(有G SH/G SSH)。结论:采用含0.15 m o l/L N aC、l1 mm o l/L G SSH和3 mm o l/L G SH的50 mm o l/L T ris-HC l(pH 8.0)缓冲液作为A n ti-CTLA 4 scFv的复性液,4℃下透析复性48 h,可获得较高复性得率和结合活性。
