共查询到20条相似文献,搜索用时 15 毫秒
1.
Xuelian Bai Moonseon Jang Nam Ju Lee Thi Thu Ha Nguyen Mooyoung Jung Jeong Yeon Hwang Hyunbo Shim 《International journal of molecular sciences》2022,23(11)
Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries. 相似文献
2.
Karin N. Westlund Marena A. Montera Aleyah E. Goins Sascha R. A. Alles Nikita Suri Sabrina L. McIlwrath Robyn Bartel Ravi V. Durvasula Adinarayana Kunamneni 《International journal of molecular sciences》2021,22(24)
Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain. 相似文献
3.
Hirose M; Hayano T; Shirai H; Nakamura H; Kikuchi M 《Protein engineering, design & selection : PEDS》1998,11(3):243-248
We have isolated anti-glutathione antibodies from a human synthetic phage
antibody scFv library (Nissim,A., Hoogenboom,H.R., Tomlinson,I.M.,
Flynn,G., Midgley,C., Lane,D. and Winter,G., 1994, EMBO J., 13, 692-698).
Glutathione (GSH) conjugates with carrier proteins, such as bovine serum
albumin (BSA), keyhole limpet hemocyanin (KLH) and human lysozyme (LZM),
were used as antigens. After four cycles of panning and affinity
chromatography, clones that recognized GSH- conjugated proteins, but not
BSA, KLH or LZM, were isolated. The isolated phage antibodies and the
soluble scFv fragments were characterized by immunoblotting, and the
nucleotide sequences of the VH segments of selected clones were determined.
The binding of several isolates to GSH-BSA was competitively inhibited by
GSH in an ELISA. These observations have demonstrated that antibodies
against GSH, a tripeptide, can be isolated from the library. We constructed
the tertiary models of several scFv fragments and discussed the mechanism
of antigen binding sites.
相似文献
4.
Brockmann EC Akter S Savukoski T Huovinen T Lehmusvuori A Leivo J Saavalainen O Azhayev A Lövgren T Hellman J Lamminmäki U 《Protein engineering, design & selection : PEDS》2011,24(9):691-700
Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range. 相似文献
5.
Isolation of anti-T cell receptor scFv mutants by yeast surface display 总被引:13,自引:0,他引:13
Kieke MC; Cho BK; Boder ET; Kranz DM; Wittrup KD 《Protein engineering, design & selection : PEDS》1997,10(11):1303-1310
Yeast surface display and sorting by flow cytometry have been used to
isolate mutants of an scFv that is specific for the Vbeta8 region of the T
cell receptor. Selection was based on equilibrium binding by two
fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to
the c-myc epitope tag present at the carboxy-terminus of the scFv. The
mutants that were selected in this screen included a scFv with threefold
increased affinity for the Vbeta8 and scFv clones that were bound with
reduced affinities by the anti-c-myc antibody. The latter finding indicates
that the yeast display system may be used to map conformational epitopes,
which cannot be revealed by standard peptide screens. Equilibrium antigen
binding constants were estimated within the surface display format,
allowing screening of isolated mutants without necessitating subcloning and
soluble expression. Only a relatively small library of yeast cells (3 x
10[5]) displaying randomly mutagenized scFv was screened to identify these
mutants, indicating that this system will provide a powerful tool for
engineering the binding properties of eucaryotic secreted and cell surface
proteins.
相似文献
6.
Pharaoh Fellow Mwale Chi-Hsin Lee Peng-Nien Huang Sung-Nien Tseng Shin-Ru Shih Hsin-Yuan Huang Sy-Jye Leu Yun-Ju Huang Liao-Chun Chiang Yan-Chiao Mao Wei-Chu Wang Yi-Yuan Yang 《International journal of molecular sciences》2021,22(8)
Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children. 相似文献
7.
Nadya Panagides Lucia F. Zacchi Mitchell J. De Souza Rodrigo A. V. Morales Alexander Karnowski Mark T. Liddament Catherine M. Owczarek Stephen M. Mahler Con Panousis Martina L. Jones Christian Fercher 《International journal of molecular sciences》2022,23(15)
Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins. 相似文献
8.
Directed evolution for the development of conformation-specific affinity reagents using yeast display 总被引:1,自引:0,他引:1
Weaver-Feldhaus JM Miller KD Feldhaus MJ Siegel RW 《Protein engineering, design & selection : PEDS》2005,18(11):527-536
Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform. 相似文献
9.
Kipriyanov SM; Moldenhauer G; Martin AC; Kupriyanova OA; Little M 《Protein engineering, design & selection : PEDS》1997,10(4):445-453
Recombinant antibody fragments directed against cell surface antigens have
facilitated the development of novel therapeutic agents. As a first step in
the creation of cytotoxic immunoconjugates, we constructed a single-chain
Fv fragment derived from the murine hybridoma OKT3, that recognizes an
epitope on the epsilon-subunit of the human CD3 complex. Two amino acid
residues were identified that are critical for the high level production of
this scFv in Escherichia coli. First, the substitution of glutamic acid
encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to
a more than a 30- fold increase in the production of soluble scFv. Second,
the substitution of cysteine by a serine in the middle of CDR-H3
additionally doubled the yield of soluble antibody fragment without any
adverse effect on its affinity for the CD3 antigen. The double mutant scFv
(Q,S) proved to be very stable in vitro: no loss of activity was observed
after storage for 1 month at 4 degrees C, while the activity of scFv
containing a cysteine residue in CDR-H3 decreased by more than half. The
results of production yield, affinity, stability measurements and analysis
of three-dimensional models of the structure suggest that the sixth amino
acid influences the correct folding of the VH domain, presumably by
affecting a folding intermediate, but has no effect on antigen binding.
相似文献
10.
Redesigned anti-human placental alkaline phosphatase single-chain Fv: soluble expression, characterization and in vivo tumour targeting 总被引:1,自引:0,他引:1
Deonarain MP; Rowlinson-Busza G; George AJ; Epenetos AA 《Protein engineering, design & selection : PEDS》1997,10(1):89-98
Although much progress has been made in the production of recombinant
antibodies and their fusions, there are still problems with solubility and
folding. Useful antibodies produced from cloned hybridomas do not always
result in scFvs behaving favourably. We report here further work on an scFv
(H17E2) against the oncofetal antigen human placental alkaline phosphatase.
The overall expression was greatly improved and the H17E2 scFv was
redesigned by manipulation of the interdomain linker, resulting in much
higher expression levels of the soluble scFv in its active conformation at
0.2-0.5 mg/l of bacterial culture. We show that the new soluble version of
this scFv has similar characteristics to the refolded version in terms of
antigen and tumour cell binding, stability and in vivo pharmacokinetics.
The final tumour uptake behaviour of these scFvs is superior to that of the
parental whole antibody with respect to tumour:organ ratios, but still
requires further development before considering it as a suitable molecule
for clinical use in ovarian or testicular cancer.
相似文献
11.
Chi-Hsin Lee Peng-Nien Huang Pharaoh Fellow Mwale Wei-Chu Wang Sy-Jye Leu Sung-Nien Tseng Shin-Ru Shih Liao-Chun Chiang Yan-Chiao Mao Bor-Yu Tsai Nhlanhla Benedict Dlamini Tien-Cuong Nguyen Chen-Hsin Tsai Yi-Yuan Yang 《International journal of molecular sciences》2022,23(21)
Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously. 相似文献
12.
Patrick J. Krohl Kook Bum Kim Lance Lew Derek VanDyke Seth D. Ludwig Jamie B. Spangler 《American Institute of Chemical Engineers》2020,66(12):e16995
The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed “biofloating” that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows. 相似文献
13.
Antibody affinity maturation using bacterial surface display 总被引:9,自引:0,他引:9
Daugherty PS; Chen G; Olsen MJ; Iverson BL; Georgiou G 《Protein engineering, design & selection : PEDS》1998,11(9):825-832
A quantitative system for screening combinatorial single-chain Fv (scFv)
antibody libraries was developed utilizing surface display on Escherichia
coli and fluorescence-activated cell sorting (FACS). This system was
employed to isolate clones with high-affinity to a fluorescently-labeled
hapten from libraries constructed by randomizing heavy and light-chain
residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of
flow cytometry enabled the detection of rare library members directly in
heterogeneous populations and the optimization of selection conditions
prior to sorting. A heavy-chain mutant having wild-type affinity (KD =
0.91+/-0.22 nM) and an expected representation frequency of less than 1 x
10(6), was selected to homogeneity after three rounds utilizing
increasingly stringent selection conditions. The isolated clone possessed
two distinct point mutations relative to the wild-type DNA sequence, yet
still coded for the wild-type amino acid sequence, suggesting that the
wild-type residues may be optimal at the randomized positions. An affinity
improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant
approximately threefold lower than the wild-type antibody, was isolated
from a smaller light-chain library in a single sorting step. Flow cytometry
was shown to be a simple and rapid method for the determination of the
relative hapten dissociation rate constants of selected clones without
requiring subcloning. The relative rate constants estimated by FACS were
confirmed by producing the scFv antibodies in soluble form and measuring
hapten binding kinetics by surface plasmon resonance (SPR). These results
demonstrate that E.coli surface display, coupled with quantitative
selection and analysis using FACS, has the potential to become a powerful
tool for rapid isolation and characterization of desirable mutants from
large polypeptide libraries.
相似文献
14.
Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting 总被引:1,自引:0,他引:1
The complementary determining regions (CDRs) from the fluorescein- binding
antibody 4-4-20, which yields almost no soluble protein in periplasmic
expression in Escherichia coli, were transplanted to the framework of the
humanized antibody 4D5. The resulting single-chain Fv fragment (scFv)
4D5Flu showed both a dramatic improvement in soluble expression, even at 37
degrees C, and an improved thermodynamic stability. Antigen affinity was
maintained upon this engineering by paying attention to crucial
framework-CDR contacts. This demonstrates that the use of superior
frameworks is a robust strategy to improve the physical properties of scFv
fragments. We also report that the grafted version was selected in phage
display over several competing variants of the same antibody with identical
binding constant but poorer folding or stability properties. The selection
required four panning rounds and a temperature of 37 degrees C and we show
that the underlying reason for this selection is a higher fraction of
phages carrying functional scFv molecules.
相似文献
15.
In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ‐aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine‐3‐carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays—the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1‐{2‐[2′‐(1,1’‐biphenyl‐2‐ylmethylidene)hydrazine]ethyl}piperidine‐3‐carboxylic acid and 1‐(2‐{2′‐[1‐(2‐thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine‐3‐carboxylic acid, were found to be potent GAT1 ligands exhibiting pKi values of 6.186±0.028 and 6.229±0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including membrane‐bound targets such as G protein coupled receptors (GPCRs), ion channels and transporters. As such, this strategy displays high potential in the drug discovery process. 相似文献
16.
Sukyo Jeong Hyun Joo Ahn Kyung Jin Min Jae Won Byun Hyun Mi Pyo Mi Young Park Bok Kyung Ku Jinju Nah Soyoon Ryoo Sung Hwan Wee Sang Jick Kim 《International journal of molecular sciences》2021,22(9)
For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals. 相似文献
17.
Engineered bispecific antibodies that recruit cytotoxic lymphocytes to kill specific tumor cells have been showing promising clinical results. Here, we describe a bispecific single-chain Fv (scFv) immunofusion or BIf to target CD123(+) leukemia, that contains an anti-CD123 scFv fused at the N-terminus of human IgG1 hinge-C(H)2-C(H)3, and an anti-CD3 scFv fused at C-terminus. When expressed from transfected CHO-S cells, CD123xCD3 BIf forms a homodimer that provides a structure of N-terminal tumor-targeting domain that closely resembles natural antibody. The CD123xCD3 dimeric structure also provides binding affinity to CD123(+) tumor cells with a Kd of 10(-10) M, one to two orders of magnitude stronger than traditional bispecific antibody constructs. The location of the anti-CD3 scFv at C-terminus of BIf reduces the binding affinity to CD3(+) T cells by two orders, which could help to prevent non-specific T-cell activation. CD123xCD3 BIf is able to achieve T-cell-mediated target cell killing activities at low pM levels with E/T ratios as low as 2. Overall, the inclusion of human IgG1 constant region in BIf construct increases target cell-binding affinity; potentially increases serum half-life by its larger size and FcRn-mediated salvage system; and includes the abilities to activate the additional antibody-mediated cellular cytotoxicities. 相似文献
18.
目的构建多样性良好的抗白念珠菌人源性单链抗体库,筛选抗白念珠菌特异性的噬菌体抗体。方法从20份人外周血淋巴细胞(包括正常成年人5份、新生儿5份和白念珠菌感染恢复期患者10份)中提取总RNA,反转录为cDNA,PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,以重叠延伸PCR法将VH和VL拼接成scFv基因,克隆入噬菌粒载体pCANTAB-5E中,电转化大肠杆菌XL1-Blue,构建人源抗白念珠菌天然噬菌体抗体库,并从中筛选阳性克隆抗体。结果构建的人源性抗白念珠菌天然噬菌体抗体库库容为2.8×109,多样性良好。共筛选出18个阳性克隆。结论已成功构建了1个多样性良好的抗白念珠菌人源性噬菌体抗体库,为筛选有效治疗白念珠菌和耐药性白念珠菌感染的药物提供了条件。 相似文献
19.
Moreland NJ Susanto P Lim E Tay MY Rajamanonmani R Hanson BJ Vasudevan SG 《International journal of molecular sciences》2012,13(3):2618-2635
Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library (>10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III. 相似文献
20.
目的构建人源抗狂犬病病毒单链抗体(Single-chain fragment variable,scFv)噬菌体抗体库,并进行筛选。方法以接种过狂犬病疫苗的21份高效价的健康人外周血为原料,提取淋巴细胞总RNA,PCR扩增VH、Vκ、Vλ基因,重叠延伸PCR扩增获得scFv基因,克隆入噬菌粒pS100,电转化E.coli TG1,建立scFv初级抗体库,并进行基因序列分析及鉴定。以灭活的狂犬病病毒为抗原,进行3轮scFv噬菌体抗体库的富集筛选,随机挑取单克隆,制备噬菌体抗体颗粒,并进行ELISA分析,阳性克隆进行序列分析,采用快速荧光灶抑制试验(Rapid fluorescent facus inhibition test,RFFIT)检测噬菌体抗体颗粒的中和活性。结果获得了库容为1.7×108的人源抗狂犬病病毒scFv噬菌体抗体库,该抗体库具有较好的序列正确率和多样性,经3轮筛选后,ELISA分析显示,共获得24个序列各不相同的针对狂犬病病毒的阳性克隆,对其中7个克隆的噬菌体抗体颗粒进行RFFIT检测,均具有中和活性。结论已成功构建了人源抗狂犬病病毒scFv噬菌体抗体库,并建立了筛选方法。 相似文献