共查询到20条相似文献,搜索用时 15 毫秒
1.
Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD 总被引:4,自引:0,他引:4
In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment. 相似文献
2.
Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus. 总被引:1,自引:0,他引:1
S Delagrave J Catalan C Sweet G Drabik A Henry A Rees T P Monath F Guirakhoo 《Protein engineering》1999,12(4):357-362
HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection. 相似文献
3.
Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment 总被引:1,自引:0,他引:1
Asano R Kudo T Nishimura Y Makabe K Hayashi H Suzuki M Tsumoto K Kumagai I 《Journal of biochemistry》2002,132(6):903-909
Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody). 相似文献
4.
The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies. 相似文献
5.
Catherine Fallecker Nicolas Tarbouriech Mohammed Habib Marie-Anne Petit Emmanuel Drouet 《FEBS letters》2013
The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics. 相似文献
6.
Yang K Basu A Wang M Chintala R Hsieh MC Liu S Hua J Zhang Z Zhou J Li M Phyu H Petti G Mendez M Janjua H Peng P Longley C Borowski V Mehlig M Filpula D 《Protein engineering》2003,16(10):761-770
The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies. 相似文献
7.
Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described. 相似文献
8.
The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small. 相似文献
9.
The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay. 相似文献
10.
mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA. 相似文献
11.
S Kjaer T Wind P Ravn M ?stergaard B F Clark A Nissim 《European journal of biochemistry》2001,268(12):3407-3415
To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained. 相似文献
12.
Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus. 相似文献
13.
Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization. 相似文献
14.
L Sánchez M Ayala F Freyre I Pedroso H Bell V Falcón J V Gavilondo 《Journal of biotechnology》1999,72(1-2):13-20
A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant. 相似文献
15.
Inhibition of the hepatitis C virus NS3 protease activity by Fv fragment of antibody 8D4 总被引:1,自引:0,他引:1
Kasai N Tsumoto K Niwa S Misawa S Ueno T Hayashi H Kumagai I 《Biochemical and biophysical research communications》2001,281(2):416-424
An antibody variable domain fragment (Fv) is a candidate for a specific inhibitor of the hepatitis C virus (HCV) NS3 protease. Here we report the functional characterization of the Fv of antibody 8D4, which is specific for the active site of the HCV NS3 protease domain. The variable fragments of 8D4 in the forms of Fv and scFv (VH-(G(4)S)(3)-VL) were expressed as insoluble fractions in the periplasm of Escherichia coli, and were subsequently solubilized, purified under denaturing conditions, and refolded. The Fv had an inhibition profile almost identical to that of the parent IgG, with an IC(50) of 71.3 nM, whereas the scFv had a greatly decreased affinity to NS3 and was the same as the isolated VH fragment. To date, this is the first report of an antibody Fv fragment specific for the HCV NS3 protease domain, aimed at designing potent protease inhibitors and antiviral drugs. 相似文献
16.
Ming Ni Bing Yu Yu Huang Zhenjie Tang Ping Lei Xin Shen Wei Xin Huifen Zhu Guanxin Shen 《Journal of biosciences》2008,33(5):691-697
We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïe phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer. 相似文献
17.
J L Atwell K A Breheney L J Lawrence A J McCoy A A Kortt P J Hudson 《Protein engineering》1999,12(7):597-604
Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent. 相似文献
18.
Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. 总被引:4,自引:0,他引:4
A M Wu G J Tan M A Sherman P Clarke T Olafsen S J Forman A A Raubitschek 《Protein engineering》2001,14(12):1025-1033
A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a 'diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization. 相似文献
19.
为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 人p5 3四聚功能域融合基因 .经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列一致 .表达产物经SDS PAGE和Western印迹实验证实为约 35kD的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv ,为进一步临床应用奠定基础 相似文献
20.
Martsev SP Chumanevich AA Vlasov AP Dubnovitsky AP Tsybovsky YI Deyev SM Cozzi A Arosio P Kravchuk ZI 《Biochemistry》2000,39(27):8047-8057