To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
Amyloid beta-Peptides ; genetics ; immunology ; Antibody Specificity ; immunology ; Humans ; Peptide Fragments ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

OBJECTIVETo investigate the antigenicity of the peptide vaccine HDS from Streptococcus mutans glucosyltransferase and its ability to induce protective immune responses in an experimental rat model of dental caries.

METHODSArtificial antigen HDS-KLH, peptide HDS, glycosyltransferase were injected to immunize rats. Measurement of the specific anti-HDS, GTF IgG or IgA concentration in saliva and serum were undertaken by ELISA among the experimental groups. Gnotobiotic rat model was developed when challenged S. mutans and a caries promoting diet. The jaws of the rats were selected and dyed. The Keyes caries score for each jaw were counted.

RESULTSThe level of serum and salivary specific anti-HDS IgG and IgA in the group immunized by HDS-KLH was significantly higher than that in control group (P < 0.05). The Keyes caries score of GTF, HDS and HDS-KLH immunized group were significantly lower than that of control group, especially lower in smooth tooth surface.

CONCLUSIONArtificial antigen HDS-KLH could induce immune response. As a peptide vaccine, HDS-KLH could reduce the caries incidence in experimental rat model.

Animals ; Dental Caries ; prevention & control ; Glucosyltransferases ; genetics ; immunology ; Male ; Peptides ; immunology ; Rats ; Rats, Sprague-Dawley ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; enzymology ; genetics ; immunology

Animals ; Antigens ; chemistry ; genetics ; immunology ; therapeutic use ; Drug Therapy ; Humans ; Peptides ; chemistry ; genetics ; immunology ; therapeutic use ; Vaccines ; chemistry ; genetics ; immunology ; therapeutic use

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product.

METHODSThe coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein.

RESULTSA gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein.

CONCLUSIONThe constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.

Animals ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Mice ; Mice, Inbred C57BL ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; immunology ; tau Proteins ; biosynthesis ; genetics ; immunology

To develop polyclonal antibodies against predicted B cell epitopes in HIV-1 accessory protein Vpr, the prepared consensus Vpr amino acid sequence was used to predict potential B cell epitopes by online softwares (ABCpred and Bcepred), the synthesized polypeptides of B-cell epitopes were subsequently conjugated with keyhole limpet hemocyanin (KLH) and then used to immunize rabbits. The antibody titers were determined by ELISA, and antibody specifity was analyzed by Western-Blotting and immunoprecipitation, respectively. Amino acid residues 3-19 (N) and 82-95 (C) of Vpr were predicted as the potential B cell epitopes. After inoculation of the conjugation of synthesized peptide to KLH, the antibody titers in rabbit sera against N and C peptides reached more than 1:100000 by ELISA. Western-Blotting analysis showed that the polyclonal antibodies reacted with both wild Vpr and fusion protein of GFP with Vpr, no matter Vpr was derived from HIV-1 B subtype or CRF07_BC recombinant form; Immunoprecipitation analysis showed similar reactions to Western-Blotting results. Two B cell epitopes of Vpr were successfully predicted by Bio-informatics methods and polyclonal antibodies against those peptides could be successfully prepared.
Animals ; Antibodies, Viral ; blood ; immunology ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; HIV Infections ; blood ; immunology ; virology ; HIV-1 ; genetics ; immunology ; Humans ; Peptides ; genetics ; immunology ; Rabbits ; vpr Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
AC133 Antigen ; Animals ; Antibodies ; genetics ; Antigens, CD ; immunology ; Antigens, CD34 ; immunology ; Base Sequence ; Cadherins ; immunology ; Female ; Glycoproteins ; immunology ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Library ; Peptides ; immunology

Amino Acid Sequence ; Amyloid beta-Peptides ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Epitopes ; genetics ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Molecular Sequence Data ; Peptide Library ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

AIMBy using of Escherichia coli DH5alpha to express GST-Ccd1 fusion protein and which was purified by affinity chromatographic separation. The purified target protein was used to immunize rabbits to prepare polyclonal antibody.

METHODSThe previously constructed recombinant prokaryotic expression vector pGEX-5X-1-Ccd1 was transformed into Escherichia coli DH5alpha and was induced to expression by IPTG. The recombinant target protein was expressed with soluble state in Escherichia coli expression system which was separated and purified by chromatographic column stuffed with glutathione Sepharose 4B. The prepared antigen was used to immunize rabbits to get anti-Ccd1 specific rabbit original polyclonal antibody.

RESULTSELISA data demonstrated that the antibody titer of the serum was up to 1:40 000. Immunohistochemistry analysis indicated that the "home-made" antibody had a specific interaction with Ccd1 protein and which could be used for extended experimental research.

CONCLUSIONThe anti-Ccd1 polyclonal antibody we had prepared had a high quality of potency and specificity. The antibody was demonstrated by experiments that which could totally fulfill the requirement of immunoblotting and immunohistochemistry study of Ccd1. The antibody provided an useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of Ccd1.

Antibodies ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigen, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. To obtain the specific peptide binding with PSCA for targeted immunotherapy, PSCA gene was obtained by RT-PCR from human prostate cancer cell line DU145 and the transcated PSCA (tPSCA) gene was cloned into vector pQE30 for soluble expression in E. coli. The identity of recombinant tPSCA was confirmed through ELISA and western blot by use of anti-PSCA monoclonal antibody. Then the 12-peptide phage display library was screened with the purified tPSCA protein for its specific binding peptide through 3 rounds panning. For identifying the peptide's specificity, the peptide was coupled with EGFP (enhanced green fluorecent protein) by recombinant DNA technology and the recombinant coupled protein was termed 11-EGFP. The binding specificity with tPSCA of 11-EGFP was further confirmed by ELISA and competitive inhibition experiment. Flow cytometry demonstrated its binding specificity with cell line DU145. In conclusion, a 12-amino-acid peptide which could bind with PSCA specifically was found and it may be a potential tool for targeted immunotherapy of prostate carcinoma.
Antibodies, Monoclonal ; immunology ; Antigens, Neoplasm ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; Flow Cytometry ; GPI-Linked Proteins ; Humans ; Immunotherapy ; Male ; Membrane Glycoproteins ; genetics ; immunology ; Neoplasm Proteins ; genetics ; immunology ; Peptide Library ; Peptides ; immunology ; Prostatic Neoplasms ; therapy