Mapping of Taenia solium TSOL18 antigenic epitopes by phage display library

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. TSOL18 has been identified as a host-protective oncosphere antigen. To obtain mouse monoclonal antibodies (mAbs) against TSOL18 and to map its antigenic epitopes are potentials to develop a vaccine for the prevention of T. solium infection. In this study, mAbs were produced by the hybridoma technique using purified glycosylated TSOL18 produced in Pichia pastoris as the immunogen. mAb was used to define the B-cell epitopes of TSOL18 with phage-displayed random dodecapeptide library (Ph.D.-12), and some of the positive phage clones were sequenced and analyzed. The predominant mimotopes were ETTKLQRFQAML (L1) found in 83%, followed by DHTXF in 15% (L2: DHTLFAASHNHR, DHTLFSTGHSHG, and DHTFMQRYHTHQ). Comparison of the peptide sequences with native TSOL18 protein sequence using Clustal W software showed that they did not completely match, suggesting that the ETTKLQRFQAML and DHTXF sequences should be conformational epitopes. The sera of mice immunized with the selected phage clones obviously recognized the TSOL18 protein. Meanwhile, sera collected from TSOL18-vaccinated pigs reacted to both epitopes in enzyme-linked-immunosorbent serologic assay test. Our work demonstrated that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAbs and a mimotope of TSOL18, which could provide an alternative approach for the diagnosis and development of a vaccine for T. solium.     

Klebsiella pneumoniae MrkD adhesin-mediated immunity to respiratory infection and mapping the antigenic epitope by phage display library

In this study, the K. pneumoniae MrkD adhesin was identified an immunodominant antigen which correlated with protection against infection by K. pneumoniae. The mouse monoclonal antibodies (mAbs) against MrkD adhesin were produced by the hybridoma technique using recombinant MrkD-GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 36 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Sixteen phage clones were sequenced and their amino acids were deduced. One mimotope (QKTLAKSTYMSA) showed good match with MrkD adhesion at 148–159 aa and the serum of mice induced by the phage clone clearly recognized MrkD adhesion.     

噬菌体表达短肽模拟乙脑病毒糖蛋白的研究

为研究日本脑炎病毒 (JEV )E蛋白模拟肽 ,将抗JEVE蛋白的mAb 2H4淘筛噬菌体 15肽库。经夹心ELISA、竞争ELISA鉴定后 ,随机挑取 10个阳性克隆 ,测序并与JEVE蛋白同源比较。将阳性噬菌体免疫小鼠 ,检测血清中特异性抗体。ELISA结果显示筛选到的噬菌体能特异地与mAb 2H4结合 ,并且这种结合可被JEV天然抗原所竞争抑制。 10个阳性克隆的氨基酸序列相同 :—RQDPQWPYANSTIAR— ,同源分析得到的序列STXAR可能为mAb 2H4识别的模拟表位。阳性噬菌体表达的 15肽能够刺激小鼠产生特异性抗体。该噬菌体表达短肽模拟JEVE蛋白的部分抗原性。     

Immune response to an 18-kilodalton outer membrane antigen identifies lipoprotein 20 as a Helicobacter pylori vaccine candidate

Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.     

Peptide mimotopes of Neisseria meningitidis group B capsular polysaccharide

The antigenic similarity between Neisseria meningitidis group B (NMGB) capsular polysaccharide (PS) and human polysialic acid (PSA) has hampered the development of a NMGB PS-based vaccine. But the possibility of a safe vaccine based on NMGB PS has been demonstrated by the existence of the NMGB PS-associated nonautoreactive epitope, which is distinct from those present on human PSA. To obtain peptide mimotopes of NMGB PS, we used HmenB3, a protective and nonautoreactive monoclonal antibody, to screen a phage library with 12 amino acids. We obtained 23 phage clones that bound to HmenB3 but not in the presence of E. coli K1 PS [which is alpha(2-8)-linked PSA like NMGB PS]. The clones contained 3 mimotopes and differed from previously described NMGB PS mimotopes. Immunization with a synthetic peptide of one mimotope elicited anti-NMGB antibodies in BALB/c mice. These mimotopes may be useful in the development of group B meningococcal vaccines.     

应用噬菌体表面展示技术筛选流感病毒H3N2抗原模拟表位

目的 筛选特异性流感病毒H3N2抗原模拟表位,为开展新的流感病毒疫苗研究探索新的途径.方法 应用噬菌体表面展示技术,以抗-H3N2的单克隆抗体作为固相筛选分子,对人工合成的噬菌体随机肽库进行5轮"吸附-洗脱-扩增"的筛选过程,在第5轮筛选后,随机挑取48个克隆,经噬菌体酶联免疫吸附法(ELISA)鉴定并进行交叉反应实验以及竞争抑制性实验,最后对所选克隆进行DNA序列分析,以确定H3N2抗原的模拟表位.结果 经噬菌体富集后,从随机筛选的48个克隆中得到21个阳性克隆,经ELISA鉴定及交叉反应实验、竞争抑制性实验后,确定氨基酸序列XTXPYXX为H3N2的模拟表位.结论 用噬菌体7肽库筛选得到H3N2的模拟表位,为开展用流感病毒模拟表位探索新的防治方法研究奠定了基础.     

应用噬菌体展示技术筛选、鉴定抗汉滩病毒单克隆抗体的模拟表位

目的 :应用噬菌体 12肽文库筛选抗汉滩病毒单抗 (mAb)F3、B11的模拟配体肽 ,并对其免疫学特性进行初步分析。方法 :以纯化的mAb为筛选配基 ,进行噬菌体肽库的生物亲和淘选。用ELISA法鉴定筛选克隆的结合活性 ,对阳性克隆进行序列测定和分析。用动物免疫试验初步分析噬菌体颗粒展示的抗原肽的免疫特性。结果 :通过 3～ 4轮生物淘选 ,ELISA显示筛选到的多数噬菌体克隆均可与mAb特异性结合 ;与mAbF3结合的阳性克隆的氨基酸序列高度一致 ,均为 MHGP TKNQMWHT ,与HTNV/SEOVM蛋白G2区第 75 0～ 75 9位氨基酸具有较高的同源性 ;而mAbB11特异性的结合肽在氨基酸水平上表现出一定的多态性 ,其序列的基序尚未能确定 ;动物免疫试验表明 ,噬菌体肽抗原具有良好的免疫反应性和免疫原性 ,是天然病毒抗原较好的免疫原模拟物。结论 :获得了具有良好结合活性的模拟表位肽 ,为基于表位的HFRSV多肽疫苗及DNA疫苗的研制奠定了基础。     

Selection of phage-displayed peptides recognised by monoclonal antibodies directed against the lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76-12G12 mAb, which is specific for LPS of all strains of Brucella, either A- or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66-4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66-2C8 and A15-6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

Selection of Phage-displayed Peptides Recognised by Monoclonal Antibodies Directed against the Lipopolysaccharide of Brucella

Panning and screening of various phage display libraries with monoclonal antibodies (mAbs) directed against the O-chain of the lipopolysaccharide (LPS) of Brucella sp. allowed the identification of peptidic mimotopes of some O-chain epitopes. Four mAbs were tested. The A76–12G12 mAb, which is specific for LPS of all strains of Brucella, either A-or M-dominant, did not yield any peptidic mimotope, despite a specific yield enrichment during the rounds of panning. The B66–4F9 mAb, that recognises an epitope common to both Brucella sp. and Yersinia enterocilitca O:9 strains, allowed the selection of only one phage clone that was shown to be an antigenic but not immunogenic mimotope. The B66–2C8 and A15–6B3 mAbs, respectively, specific for the LPS of A-dominant and M-dominant Brucella sp., yielded several sequences, which allowed the determination of consensus sequences. These consensus will be of high interest for the construction of second generation libraries. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed for some peptides. These data suggest that a subset of the selected peptides are immunogenic mimotopes of the LPS epitopes.     

应用噬菌体随机肽库技术筛选幽门螺杆菌中性粒细胞激活蛋白的抗原表位

目的 筛选和鉴定幽门螺杆菌(Helicobacter pylori,Hp)中性粒细胞激活蛋白(neutrophil-activating protein,NAP)的有效抗原表位,为Hp疫苗的研制提供基础.方法 以抗NAP的单克隆抗体作为固相筛选分子,经3轮吸附-洗脱-扩增免疫,筛选噬菌体随机7肽库,随机挑选噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、交叉反应试验及竞争抑制试验鉴定阳性克隆,测定阳性克隆所携带DNA序列并进行计算机辅助分析.以制备的阳性噬菌体克隆短肽液免疫小鼠,免疫血清与NAP经Western blot分析,以验证NAP的模拟表位.结果 经3轮免疫筛选后挑选到45个阳性克隆,经ELISA鉴定有12个阳性克隆,测序结果显示5种表位,其中P17噬菌体展示肽FAHLATQ与NAP氨基酸序列(137～143)高度同源,位于NAP高抗原区域(118～140),免疫血清可识别NAP.结论 用噬菌体随机7肽库成功筛选到了NAP的模拟表位,为基于NAP的诊断和疫苗的研制提供了基础.     

Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.     

Characterization of murine coronavirus neutralization epitopes with phage-displayed peptides

Phage-displayed peptide libraries were used to map immunologically relevant epitopes on the surface (S) glycoprotein of a neurotropic murine coronavirus (MHV-A59). Three in vitro virus-neutralizing and in vivo protective mAbs against either continuous or discontinuous epitopes on the S glycoprotein were used to screen 12 different peptide libraries expressed on the pVIII major coat protein of the fd filamentous bacteriophage. Consensus sequences that matched short sequences within the S glycoprotein were identified. The sequence of a tight-binding, mAb-selected peptide suggested the location of a discontinuous epitope within the N-terminal S1 subunit. Several tightly binding phage were amplified and used directly as immunogens in BALB/c and C57BL/6 mice. Partial protection of C57BL/6 mice against a lethal acute virus infection was achieved with a phage preparation that displayed a linear epitope. Protection correlated with the presence of sufficient levels of specific antiviral antibodies recognizing the same immunodominant domain and 13-mer peptide, located within the C-terminal S2 subunit, as the selecting mAb. Thus, the direct use of phage-displayed peptides to evaluate protective antiviral immune responses complements their use to characterize antibody-binding epitopes. This is the first evaluation of protective immunization induced by mAb-selected phage-displayed peptides.     

Evaluation of neutralizing efficacy of monoclonal antibodies specific for 2009 pandemic H1N1 influenza A virus in vitro and in vivo

Pandemic influenza A virus (H1N1) 2009 poses a serious public-health challenge worldwide. To characterize the neutralizing epitopes of this virus, we generated a panel of eight monoclonal antibodies (mAbs) against the HA of the A/California/07/2009 virus. The antibodies were specific for the 2009 pdm H1N1 HA, as the antibodies displayed HA-specific ELISA, hemagglutination inhibition (HAI) and neutralization activity. One mAb (mAb12) showed significantly higher HAI and neutralizing titers than the other mAbs. We mapped the antigenic epitopes of the HA by characterizing escape mutants of a 2009 H1N1 vaccine strain (NYMC X-179A). The amino acid changes suggested that these eight mAbs recognized HA antigenic epitopes located in the Sa, Sb, Ca1 and Ca2 sites. Passive immunization with mAbs showed that mAb12 displayed more efficient neutralizing activity in vivo than the other mAbs. mAb12 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge in mice. In addition, a single injection of 10 mg/kg mAb12 outperformed a 5-day course of treatment with oseltamivir (10 mg/kg/day by gavage) with respect to both prophylaxis and treatment of lethal viral infection. Taken together, our results showed that mouse-origin mAbs displayed neutralizing effectiveness in vitro and in vivo. One mAb in particular (mAb12) recognized an epitope within the Sb site and demonstrated outstanding neutralizing effectiveness.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定

目的 :制备抗禽流感病毒 (AIV)H9亚型血凝素蛋白的单克隆抗体 (mAb)。方法 :以AIVH9亚型油乳剂灭活疫苗作为免疫原 ,免疫 8wk龄BALB/c小鼠。采用淋巴细胞杂交瘤技术制备抗AIVH9亚型血凝素蛋白的mAb ;采用ELISA和血凝抑制试验(HI)检测腹水mAb的效价 ;采用ELISA、HI、免疫荧光染色 (IF)及Westernblot鉴定mAb的特异性。结果 :获得 3株可稳定分泌特异性mAb的杂交瘤细胞株 2A3、2H1和 1C8,其腹水mAb的ELISA效价依次为 1× 10 7、1× 10 5和 5× 10 6,血凝抑制效价为 1× 2 8～ 1× 2 13 ;3株mAb的Ig亚类均为IgG1。以mAb 2H1进行Westernblot的结果显示 ,该mAb能与AIV的Mr 为 75 0 0 0的蛋白条带起反应 ,表明其是针对AIVH9亚型血凝素蛋白的mAb。与 32株AIVH9亚型国内分离株进行血凝抑制试验表明 ,mAb 2H1具有良好的广谱性。结论 :成功地制备了抗AIVH9亚型血凝素蛋白的mAb ,为AIV的抗原性分析、血清学诊断、疫苗质量的监测及流行病学调查等奠定了基础     

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti‐HCV sera

Conformational B‐cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti‐E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15‐mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2–CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti‐E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B‐cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B‐cell responses against those specific E2 epitopes that may correlate with immunity. J. Med. Virol. 82:1655–1665, 2010. 2010 Wiley‐Liss, Inc.     

Epitope mapping of Mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes

Phage display techniques have been widely employed to map the epitope structures which served as the basis for developing molecular vaccines. In the present study, we applied this technique to map the epitopes of Mycoplasma hyopneumoniae, the etiologic agent causing swine enzootic pneumonia, and evaluated directly the immune responses in mice of the selected phage-displayed epitopes (phagotopes). Two phage-displayed random peptide libraries were biopanned with the protein A-purified IgG of the rabbit anti-M. hyopneumoniae hyperimmune serum and the selected phage clones were sequenced and analyzed. Some of the inserts of the selected phagotopes showed a good match with the known proteins of M. hyopneumoniae. Others, which did not match with any known proteins, but shared extensive homology with each other, were clustered and classified as the conformational epitopes of M. hyopneumoniae. To evaluate the potential of using these phagotopes as effective vaccines, several phage clones were chosen to immunize mice. IgA coproantibody, IgA in bronchoalveolar lavage fluid and serum IgG responses were assayed. The serum raised by the phage clones clearly recognized several major mycoplasmal proteins indicating that the phagotope-induced immune responses were antigen-specific. The stronger IgG1 response revealed that the immune responses of the epitope-displaying phage were mainly through Th2 activation. The growth inhibition assay showed that the selected phage clones CS4 and varphi58 are potential vaccine candidates and suggested that the mycoplasmal 97 kDa, 56 kDa, 30 kDa and 23 kDa proteins may play important roles in the immune responses. The present work demonstrates that the whole epitope profile of a microorganism can be obtained through screening the phage displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitope-displaying phages as peptide vaccines.     

丙型肝炎病毒非结构蛋白NS4A抗原模拟表位的筛选和鉴定

目的：筛选丙型肝炎病毒（hepatitis C virus,HCV)非结构蛋白NS4A（HCV NS4A)特异性噬菌体模拟表位，为抗HCV的疫苗研究探索新途径。方法：以抗HCV NS4A的单克隆抗体作为固相筛选分子，对人工合成的噬菌体随机12肽库进行5轮“吸附－洗脱－扩增”的筛选过程，随机挑取45个克隆，经噬菌体酶联免疫吸附法（ELISA）鉴定并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV NS4A抗原的模拟表位。结果：经噬菌体富集后，从随机筛选的45个克隆中得到13个阳性克隆，确定氨基酸序列XXRXXMXPXXXI为HCV NS4A的模拟表位。结论：用噬菌体12肽库成功筛选得到HCV NS4A的模拟表位，为开展用HCV模拟表位探索HCV的防治研究创造了条件。     

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library

Summary. Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and E^rns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289–293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of E^rns (aa 117–119) was identified for two independent E^rns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.     

Epitope analysis of an immunodominant domain on the P1 protein of Haemophilus influenzae type b using synthetic peptides and anti-idiotypic antibodies.

Synthetic peptides, anti-idiotypic antibodies (anti-Id) and human and murine monoclonal antibodies (mAbs) were used to further define a major antigenic domain on the outer membrane P1 protein (OMP) of Haemophilus influenzae type b (Hib). Synthetic peptides were elaborated from the known primary sequences of the P1 protein of prototype Hib strains MinnA (OMP subtype 1H) and 8358 (OMP subtype 6U). By peptide mapping, antibodies are categorized into three groups: A, B and C. A first epitope on the P1 from strain MinnA was identified by the reactivity of one set of murine anti-P1 mAbs with the two overlapping peptides 11H and 13H, corresponding to amino acid residues 384-412 and 400-437, respectively. On the basis of their reactivity with both peptides, these mAbs were designated as group A. Anti-Id obtained from mice immunized with two group A mAbs reacted specifically with all group A mAbs. A second epitope on the same P1 protein was identified by the reactivity of the peptide 13H with another distinct set of murine anti-P1 mAbs assigned to group B. This group of mAbs did not recognize the peptide 11H. Murine anti-Id which were prepared against one group B mAb inhibited the attachment of this mAb to outer membrane preparations, whereas the binding of the other group B mAbs was not affected, suggesting that these mAbs represent a heterologous group of mAbs. The epitope(s) recognized by two human anti-P1 mAbs was (were) distinct from the ones recognized by murine mAbs since no reactivity with the peptides was observed. Similarly, the binding of the two human mAbs to the P1 antigen was not inhibited by anti-Id raised against group A or B mAbs. Interestingly, an epitope on a different P1 protein recovered from strain 8358 was identified by the reactivity of group C murine mAbs with the peptide 13U, which occupies the same position on the P1 protein as 13H but differs from the latter by 10 amino acid residues. Our studies demonstrated the presence of several distinct surface-exposed B-cell epitopes within the antigenic domain which was defined previously on the P1 protein of Hib MinnA. Furthermore, we showed the immunodominance of this region on two different P1 proteins. None of the mAbs, however, had a bacteriolytic or protective activity against Hib strains. We suggest that the surface-exposed immunodominant region on the OMP P1 of Hib do not induce protective antibodies against Hib infection.     

交叉保护性抗LPS多克隆抗体的制备与LPS多表位模拟肽的筛选

目的 获得具交叉保护性的抗细菌脂多糖(LPS)多克隆抗体与模拟LPS多表位的噬菌体展示环肽克隆。方法制备具交叉反应性的兔抗鼠伤寒沙门菌抗血清，鉴定抗血清对大肠杆菌和铜绿假单胞菌攻击小鼠的交叉保护性。以亲和纯化的多克隆抗体为靶，亲和筛选噬菌体随机环七肽库。双夹心ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆。结果兔抗血清能与多种来源的LPS反应，对用大肠杆菌和铜绿假单胞菌攻击的小鼠有显著的保护性。用亲和层析纯化的多克隆抗体为靶分子进行3轮筛选，随机挑选46个克隆，其中20个克隆显示与多抗结合。鼠伤寒沙门菌LPS可抑制阳性噬菌体克隆与多抗结合，所有克隆的IC50(达到50％抑制率的LPS浓度)为125ng/ml。挑选其中10个克隆测序并推导氨基酸序列，其中5个克隆具X-QFYP-X-A保守序列；3个克隆具LFTFAHY序列；2个克隆具YQYYPAA序列，所有序列非极性氨基酸含量平均值为80．0％。结论获得能与多种LPS反应且具交叉保护作用的兔多克隆抗体，筛选得到可模拟鼠伤寒沙门菌LPS多表位噬菌体展示环七肽。