Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus(ASFV)is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies(mAbs)and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein(p72)single-chain antibody and fused with an IgG Fc fragment(scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide(221MTGYKH226)of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

ISOLATION OF ENDOTOXIN-SPECIFIC ANTIBODIES BY SELECTION OF AN SINGLE CHAIN PHAGE ANTIBODY LIBRARY

Objective: To isolate murine anti endotoxin single chain phage antibody from a constructed library. Methods: Total RNA was firstly extracted from murine splenic cells and mRNA was reverse-transcribed into cDNA. Then the designed primers were used to amplify the variable region genes of the heavy and light chain (VH, VL) with polymerase chain reaction. The linker was used to assemble the VH and VL into ScFv, and the NotI and SfiI restriction enzymes were used to digest the ScFv in order to ligate into the pCANTAB5E phagemid vector that was already digested with the same restriction enzymes. The ligated vector was then introduced into competent E.coli TG1 cells to construct a single-chain phage antibody library. After rescued with M13KO7 helper phage, recombinant phages displaying ScFv fragments were harvested from the supernatant and selected with endotoxin. The enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected to detect the anti endotoxin antibody with indirect ELISA. Results: The titer of anti endotoxin in murine sera was 1:12,800. The concentration of total RNA was 12.38 μg/ml. 1.9×107 clones were obtained after transformed into TG1. 3×104 colonies were gotten after one round panning. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies. Conclusion: A 1.9×107 murine anti endotoxin single chain phage antibody library was successfully constructed. Two anti endotoxin antibodies were obtained from the library.

Isolation of Goose-origin scFv Antibodies Against Goose Parvovirus from Bacterial Display Antibody Libraries

Goose parvovirus(GPV)can cause a highly contagious and fatal gosling plague(GP)disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment(scFv)antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2(protective antigen of GPV)as the bait by flow cytometry(FCM).After screening,the 15 clones with high mean fluorescence intensity(MFI)were isolated and sequenced.These 15 scFvs were expressed by pET-28a(+)in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast(GEF)in vitro,three of the 15 purified scFvs(named scFv-10,scFv-11 and scFv-50)showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.

Construction and Quality Analysis of Full-length cDNA Library of Phyllostachys heterocycla Germinating Seeds

[Objective] This study aimed to construct the full-length cDNA library for ger- minating seeds of Phyllostachys heterocycla [Method] Germinating seeds of P. hetero- cycla were used as experimental materials to construct the full-length cDNA library by using Oligo-capping method. [Result] The constructed library has a total capacity of 6.5×10^6 recombinant clones, and a low proportion of clones without inserted frag- ments; the size of inserted fragments ranges between 0.3-5.0 kb, with strict classifi- cation and ideal consistency. Furthermore, the proportion of clones harboring long in- serted fragments （1.0-5.0 kb） is as high as 30%, achieving the standard for high- quality full-length cDNA library. [Conclusion] The full-length cDNA library of germinat- ing seeds of P. heterocycla was successfully constructed, which laid important foun- dation for the functional genomics research of bamboo plants.

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain kappa and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and kappa gained by RT-PCR were about 650 bp. Fd and kappa PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 10(6).The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART^(TM)

Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.

Staphylococcus aureusβ-hemolysin-neutralizing single-domain antibody isolated from phage display library of Indian desert camel

Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.

Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Isolation of Human Antibodies Against Hepatitis E Virus From Phage Display Library by Immobilized Metal Affinity Chromatography

Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography （1MAC）. Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus （HEV） from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

Glutathione peroxidase（GPX） plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of＇ this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening, Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immu- nosorbent assay（ELISA） analysis with 4 rounds of scelection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester（GStI-s-DNP-Bu） and S-2,4-dinit,-iphenyl t-hexyl ester（GSH-s-I）NP-He）. Nevertheless, several studies need to be condueted to determine whether scFv-B8 and seFv-tI6 possess GPX activity. 1＇o enhance the speed of the selection, selenocysteine（Sec, the catalytic group of GPX） was incorporated directly into the phages, scFv-B8 and seFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8 and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-seFv with GPX activity.

Antibody Phage Library Screening Efficiency Measured by KD Values

An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each additional panning round, which indicates the increase of the binding affinity between the phage and the target molecules. This result shows that the KD value is a reliable intrinsic parameter and a new method for screening efficiency detection is thus provided.

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

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.

Construction of normal human IgE phage antibody library and the screening of the anti-trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.

应用噬菌体展示随机12肽库筛选诺如病毒抗原模拟表位

目的:利用Ph.D.-12噬菌体展示肽库筛选诺如病毒(NoV)的抗原模拟表位。方法:包被与GⅡ.4、GⅡ.6、GⅡ.17型NoV具有高特异性及中和能力较强的单链可变片段抗体(scFv),用Ph.D.-12噬菌体展示肽库进行3轮生物淘选。ELISA鉴定淘选所得噬菌体与scFv的结合活性及其与NoV P蛋白的竞争作用;阳性克隆测序后进行生物信息学分析,合成多肽鉴定其抗原性。结果:发现1段与GⅡ.6 VP1区同源性较高的氨基酸序列“MG-D-W”,综合分析提示其可能为GⅡ.6 NoV的抗原模拟表位,且合成的包含“MG-D-W”的多肽可竞争抑制P蛋白与人类组织血型抗原(HBGAs)受体的结合。结论:“MG-DW”是与NoV单链抗体高亲和力的肽段,可能模拟了GⅡ.6 NoV与scFv结合的抗原表位。

抗多房棘球蚴亮氨酸氨基肽酶纳米抗体筛选及原核表达纯化

目的筛选抗多房棘球蚴亮氨酸氨基肽酶(Em-LAP)纳米抗体并构建该纳米抗体原核表达系统,表达纯化纳米抗体蛋白,获得高特异性抗多房棘球蚴亮氨酸氨基肽酶的纳米抗体。方法利用噬菌体文库展示技术(phage library display technology)筛选亮氨酸氨基肽酶纳米抗体,应用phage-ELISA法检测纳米抗体与靶蛋白的亲和力。将亲和力高的抗体测序后,串联目的序列并优化密码子,合成后接入表达载体pET30a做酶切鉴定。将重组质粒转入大肠杆菌BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白表达,表达的蛋白经金属螯合亲和层析纯化获得目的蛋白LAPNb。结果筛选出抗Em-LAP纳米抗体,构建了重组pET30a-LAPNb质粒,得到了一个大小约为28KD的LAPNb蛋白。结论本研究成功筛选获得了抗Em-LAP纳米抗体,构建了LAPNb原核表达系统,表达纯化了LAPNb蛋白。

Analysis of BAC_5 mcAb-Related Epitope Using Random Peptide library

Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Hepatitis B immunoglobulin （HBIG） is important in the management of hepatitis B virus （HBV） infection. Aiming to develop recombinant monoclona# antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen （HBsAg）-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the FIp recombinase-mediated integration （FIp-ln） system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting （FACS）, eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression （FSE）, fluorescent strength of binding （FSB） and relative binding ability （RBA） were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

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.