HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

Study of Peptide Mimetics of Hepatitis A Virus Conjugated to Keyhole Limpet Hemocyanin and as Multiple Antigen Peptide System

Mimotopes mimic binding properties of natural antigen epitopes. They could be used for vaccine design, drugs development, and diagnostic assays. We have previously identified four bacteriophages displaying hepatitis A virus (HAV) mimotopes from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Three of these HAV mimotopes showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV and the four induced specific anti-HAV antibodies. In the present work, four conjugations were done. In each of them, a linear peptide (46, 53, 54 or 56) containing the amino-acid sequence of the corresponding mimotope was conjugated to keyhole limpet hemocyanin (KLH). Conjugation products were named: 46KLH, 53KLH, 54KLH and 56KLH. A two-arm multiple antigen peptide (MAP) system containing peptide sequence 46, and a second MAP containing two copies of peptide sequence 56 were synthesized and dimerized, to obtain the heterodimeric four-arms MAP (named MAP46-56) containing two copies of peptides 46 and 56. Mice were immunized with peptides conjugated to KLH and MAP46-56 to evaluate the ability of these two forms of mimotope presentation, to elicit antibodies that bind to the original antigen. KLH conjugated peptides rendered the highest levels of anti-peptide antibodies and were the only ones that induced specific anti-HAV antibodies. The results of immunizations showed that for the mimotopes chosen here, conjugation to a carrier protein was the most effective option to induce antibodies that cross-reacted with the natural antigen.     

Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.     

Synthetic peptides induce antibodies in sheep against Taenia ovis

Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protective T. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated form of 45W, were found to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-terminal peptide was found to be highly immunogenic, whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic. Both these immunogens elicited antibodies that cross-reacted with the parent protein; however, only antibodies directed toward the N-terminal peptide were able to bind antigens from the T. ovis oncosphere. Significant protection against challenge infection was not provided by any of the peptide immunogens used.     

Synthetic peptides induce antibodies in sheep against Taenia ovis

Summary Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protectiveT. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated from of 45W, were found to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-terminal peptide was found to be highly immunogenic, whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic. Both these immunogens elicited antibodies that cross-reacted with the parent protein; however, only antibodies directed toward the N-terminal peptide were able to bind antigens from theT. ovis oncosphere. Significant protection against challenge infection was not provided by any of the peptide immunogens used.     

Studies with antibodies against the conserved cysteine region of progesterone receptor

Polyclonal antibodies were generated against two synthetic peptides corresponding to sequences from the DNA-binding domain of steroid receptors. The sequence for peptide 1 (13 amino acids) lies between the two putative metal-binding loops of the conserved cysteine region while the sequence for peptide 2 (12 amino acids) lies within one loop. Peptide antibodies were generated by injecting rabbits with peptide conjugated to bovine serum albumin. By Western blot analysis, antibodies to peptide 2 recognized chick and human progesterone receptor and human glucocorticoid receptor, but peptide 1 antibodies did not. No cross-reactivity with native chick progesterone receptor was detected with either anti-peptide. These findings suggest that the epitopes for peptide 2 antibodies, and possibly for peptide 1 antibodies, are inaccessible to antibody in the native receptor.     

A novel method for producing anti-peptide antibodies. Production of site-specific antibodies to the T cell antigen receptor beta-chain

Peptide antigens used to generate site-specific antibodies to proteins are of interest in the development of vaccines. The need to conjugate them to a carrier protein for optimal immunogenicity results in a number of problems including a possible immune response to the carrier. Here we describe a new method of synthesizing an immunogenic peptide antigen, referred to as multiple antigenic peptide (MAP), which may render the need for a carrier protein obsolete. A 14-residue sequence derived from the human T cell antigen receptor beta-chain constant region was selected, and the peptide was synthesized directly onto a branching lysine core with 8 copies of the 14-residue peptide linked to the core by the COOH-terminal amino acid. The molecular weight of this structure was 13,422 of which only 7% represents the lysine residues of the core. The octameric MAP was highly immunogenic in mice and rabbits, allowing production of polyclonal and monoclonal antibodies. The majority of these antibodies reacted with the peptide in its monomeric form as well as its octameric form. Moreover, the antibodies reacted with the intact beta-chain protein. The antigenic determinants of the peptide that were recognized by the antibodies included continuous determinants and conformational determinants. The NH2-terminal residues of the octameric MAP appeared to be most immunogenic. There were no antibodies to the central lysine core. This method of direct synthesis of a polymeric peptide provides accurate knowledge of the conformation and quantity of the peptide prior to immunization, which is usually not the case when peptides are conjugated to carriers. The method is versatile because the possibility exists to synthesize MAP with 16 or 32 peptide arms or to synthesize polymers containing two different peptides.     

C-terminal region of human T cell lymphotropic virus type I (HTLVI) p19 core protein is immunogenic in humans and contains an HTLVI-specific epitope

To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis

To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue (312)R for biological activity against mosquito larvae.     

Rat and Mouse Monoclonal Antibodies to Human Myelin Basic Protein

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.     

Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase

Antibodies were produced against 5 peptides corresponding to segments of the (Ca2+-Mg2+)-ATPase of fast-twitch rabbit skeletal muscle sarcoplasmic reticulum (SR) including the N- and C-terminal regions. With the exception of antibodies directed against the peptide corresponding to residues 567-582 all antibodies bound strongly to the ATPase in intact SR vesicles, indicating that the epitopes were located on the cytoplasmic face of the SR. When the vesicles were disrupted, by solubilisation in SDS, binding of these antibodies was unchanged, further supporting the idea that these epitopes were located on the cytoplasmic face of SR. This is the first demonstration of the location of the N- and C-terminal regions of SR (Ca2+-Mg2+)-ATPase. These observations are discussed in the light of current structural models of the ATPase.     

Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria

To identify vaccine relevant T cell epitopes on the circumsporozoite (CS) protein of Plasmodium falciparum, the lymphocyte proliferative responses to 10 CS protein derived peptides were studied in 28 adult Kenyans, and correlated with resistance to malaria. Eight peptides, six of which were not overlapping, induced proliferation of lymphocytes from one to five volunteers, suggesting either genetic restriction of response to each of the T epitopes, or dominance of some T sites on the immunizing sporozoites. The 28 volunteers were radically cured of malaria and during the next 126 days 25 of the 28 were reinfected. Resistance to malaria was not correlated with antibodies to malaria Ag, but was significantly correlated with lymphocyte responses to CS protein residues 361-380 and 371-390. Among the 25 volunteers who became re-infected with malaria, lymphocytes from only two responded to a peptide including residues 361-380 of the P. falciparum CS protein, and only one to peptide 371-390. In contrast, lymphocytes from all three volunteers who did not become infected responded to peptide 361-380 (p = 0.003), and lymphocytes from two of the three responded to peptide 371-390 (p = 0.023). The significant correlation between proliferation to peptides 361-380 and 371-390 and resistance to malaria suggests that at least one epitope within these overlapping peptides is involved in a protective cellular immune response. The data support inclusion of these residues in future CS protein vaccines.     

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.     

Synthetic peptide vaccines against foot-and-mouth disease. II. Comparison of the response of guinea-pigs, rabbits and mice to various formulations

The immune response of guinea-pigs to vaccination with either of two aphthovirus-specific synthetic peptides was investigated. One peptide copied the sequence of amino acids 141 to 160 from the VP1 of aphthovirus strains O1BFS 1860 and O1 Kaufbeuren (O peptide), the other copied the equivalent sequence from aphthovirus strain A24 Cruzeiro (A peptide). The immune response was enhanced when the peptides were conjugated to a carried protein, but the choice of carrier protein and cross-linking agent was not critical in obtaining enhancement. The response was greatest when the peptide or peptide-carrier conjugate was adjuvanted in Freund's complete adjuvant (FCA). The O peptide was poorly immunogenic and did not confer protection against challenge with infectious virus, whereas the A peptide had good immunogenicity and did confer protection. This reflected the relative immunogenicity of the parent viruses. Rabbits, three strains of guinea-pigs and seven strains of mice were vaccinated with the O peptide conjugated to keyhole limpet haemocyanin (KLH). Considerable variation was observed between the responses of each strain and it is proposed that this relates to their repertoire of immune response genes.     

Synthesis,Immunogenicity, and Antigenicity of the Glycoprotein E Fragments of the Tick-Borne Encephalitis Virus

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.     

Targeting peptide termini, a novel immunoaffinity approach to reduce complexity in mass spectrometric protein identification

Mass spectrometry and peptide-centric approaches are powerful techniques for the identification of differentially expressed proteins. Despite enormous improvements in MS technologies, sample preparation and efficient fractionation of target analytes are still major bottlenecks in MS-based protein analysis. The complexity of tryptically digested whole proteomes needs to be considerably reduced before low abundance proteins can be effectively analyzed using MS/MS. Sample preparation strategies that use peptide-specific antibodies are able to reduce the complexity of tryptic digests and lead to a substantial increase in throughput and sensitivity; however, the number of peptide-specific capture reagents is low, and consequently immunoaffinity-based approaches are only capable of detecting small sets of protein-derived peptides. In this proof-of-principle study, special anti-peptide antibodies were used to enrich peptides from a complex mixture. These antibodies recognize short amino acid sequences that are found directly at the termini of the peptides. The recognized epitopes consist of three or four amino acids only and include the terminally charged group of the peptide. Because of its limited length, antibodies recognizing the epitope will enrich not only one peptide but a whole class of peptides that share this terminal epitope. In this study, β-catenin-derived peptides were used to demonstrate that it is possible (i) to effectively generate antibodies that recognize short C-terminal peptide epitopes and (ii) to enrich and identify peptide classes from a complex mixture using these antibodies in an immunoaffinity MS approach. The expected β-catenin peptides and a set of 38 epitope-containing peptides were identified from trypsin-digested cell lysates. This might be a first step in the development of proteomics applications that are based on the use of peptide class-specific antibodies.     

Synthesis, immunogenicity and antigenic characteristics of the glycoprotein E fragments from the tick-borne encephalitis virus]

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.     

Sequences that flank subdominant and cryptic epitopes influence the proteolytic generation of MHC class I-presented peptides

The proteasome has been shown to make the proper C-terminal cleavage for the generation of several immunodominant class I-presented peptides whereas aminopeptidases generate their proper N termini. In this study, we show that these two distinct proteolytic processes are also involved in generating a subdominant OVA peptide KVVRFDKL (K-L). Moreover, proteasome inhibitors did not enhance the presentation of any K-L construct, suggesting that destruction of this peptide by proteasomes, if any, does not limit its presentation. We have further examined in intact cells the influence of residues flanking this epitope on these proteolytic processes. When the N-terminal flanking residues of K-L are fused to an immunodominant OVA peptide SIINFEKL (S-L), the presentation of S-L is reduced as compared with a construct with its natural flanking sequence and was not inhibited (or enhanced) by proteasome inhibitors. Similarly, a reduction in presentation was observed when the C-terminal flanking residues of the subdominant epitope were attached to S-L. A detailed analysis revealed that the Pro at the P1' position of K-L was responsible for this reduction, and presentation of these C-terminally extended constructs was sensitive to proteasome inhibitor. The study suggests that both the N- and C-terminal flanks of the subdominant peptide are suboptimal for Ag presentation. Moreover, three of four C-terminal residues that flank other subdominant or cryptic epitopes in OVA reduced the presentation of S-L. Therefore, the residues that flank the C termini of several subdominant and cryptic epitopes are often suboptimal for cleavage and may contribute to the phenomenon of immunodominance.