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.

Construction of Large Human Single-chain Antibody Phage Display Library

A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half nest PCR, and assembly by two way fusion PCR. In this study, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other researches.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

A Co-expression System Based on Phage and Phagemid to Select Cognate Antibody-antigen Pairs in vivo

A modified selectively infective phage (SIP) is developed to facilitate the selection of interacting antibody antigen pairs from a large single chain antibody (scFv) library in vivo. The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5 E. The antigen fused to the C terminal of N1 N2 domain and the scFv to the N terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively. The phages produced by co transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm. In a model system, the scFv fragment of the anti hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 10 5 fold excess of a non interacting control pairs, which demonstrates this system to be very sensitive and facile to screen a large single chain antibody library.

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.

Identification of a gene engineering antibody against cystic echinococcosis in liver

OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.

Construction and Expression of a Single Chain Antibody Mimicing Human Ovarian Cancer Antigen CA125

One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody W J02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02, could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.

Davana： A New Host Plant for Ralstonia solanacearum from India

Ralstonia solanacearum infecting Davana （Artemisia pallens Wall.） from commercial nurseries in India was isolated on modified semi selective media （SMSA）. Here, we report a new host for Ralstonia solanacearum i.e. davana. It has huge demand in medicinal and aromatic industries. Isolate was confirmed as race-l, biovar-3 by morphological, physiological, biochemical and pathogenicity studies. Two sets of primers （OLI 1 ＆ Y2 and Y 1 ＆ Y2） were used in this study. Further, the identity of the isolate was confirmed by serological diagnostic kit obtained from International Potato Research Center, Lima, Peru and single chain variable fragment antibody specific to Ralstonia solanacearum used to confirm the casual organism.

Engineering a cell-penetrating hyperstable antibody scFv(Ras)-An extraordinary approach to cancer therapeutics

In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment.Additionally,it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system.As an alternative,this study modifies the hyperstable single-chain fragment variable(scFv)antibody to eliminate cancer using its linear shape.The scFv(F8)antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site.Furthermore,a cell-penetrating peptide(CPP)was attached to the scFv(Ras)antibody model to allow entrance to the cell,creating CPP-scFv(Ras).Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blotting,and the binding assay were performed to prove its effectiveness.As a result,CPP-scFv(Ras)was successfully engineered and bound to the antigen,HRas(G12V).