应用噬菌体肽文库筛选mAb F3特异性结合肽

目的 应用噬菌体展示肽文库筛选可与汉坦病毒囊膜蛋白中和性单抗（mAb） F3特异性结合的配体肽。方法 以F3mAb为筛选配基，对噬菌体展示和随机12肽文加进行3轮生物亲和淘选；用夹心ELISA和竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果 通过3轮生物淘选，能被抗体捕获的噬菌体克隆为21／22，ELISA测定显示，筛选到的噬菌体短肽能与F3mAb特异性结合。序列分析表明，7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT；同源性分析显示，该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性。结论 本研究为基于表位水平的HFRSV特异性分子多肽疫苗的设计提供了重要的依据。     

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。     

用噬菌体多肽库筛选日本血吸虫表膜抗原的模拟表位

为探索可用于诊断的模拟表膜抗原。采用纯化的兔抗表膜抗原IgG作探针 ,免疫筛选噬菌体随机十二肽库 ,经 3轮生物淘洗后 ,随机挑选 30个噬菌体克隆 ,用ELISA检测其与筛选抗体的特异性结合 ,选择两个阳性克隆进行DNA序列测定 ,并用斑点ELISA比较检测正常人和日本血吸虫病患者血清各 10份。结果显示 ,随机挑选的 30个克隆中有 9个噬菌体克隆与筛选的抗体有特异性的结合反应。DNA测序结果显示 ,两个阳性噬菌体克隆 (携带的抗原表位 )所演绎的氨基酸序列与GenBank已知的氨基酸序列无同源性。斑点ELISA结果显示两个抗原表位可被血吸虫病患者血清呈特异性识别     

登革2型病毒E蛋白免疫优势表位的筛选鉴定

目的 用噬菌体展示肽库筛选登革2型病毒(DEN2)E蛋白的抗原表位，并确定该抗原表位性质。方法 以DEN2型特异的E单克隆抗体作为筛选分子，生物淘洗噬菌体随机12肽库，将筛选的噬菌体阳性克隆进行ELISA检测、DNA序列测定及展示肽的氨基酸序列推导，通过噬菌体展示肽序列与DEN2E蛋白的氨基酸一级结构的对比，初步确定E蛋白的抗原表位；用模拟该表位线性序列的合成十肽进行抗体结合试验、噬菌体竞争抑制试验及与DEN感染患者的血清学试验，确定其为免疫优势线性表位。结果 肽库淘洗获得的11个ELISA阳性的噬菌体克隆有相似的结构基序WFKKGSS，其展示肽与DEN2E蛋白390～398 AA序列有3～5个氨基酸相同。对应于DEN2E蛋白390～399AA的合成十肽能与淘洗单抗特异反应，并可抑制噬菌体阳性克隆与该单抗结合。该合成肽与DEN2感染患者血清有较高的免疫反应性。结论 本实验通过噬菌体随机肽库的生物淘洗确定的DEN2E蛋白(E390～398AA)线性序列为免疫优势表位，其对应的合成肽E10可望用于DEN2感染的快速诊断。     

细粒棘球蚴AgB亚单位基因的联合表达和血清学评价

目的为进一步提高AgB抗原在诊断中的敏感性和特异性,将细粒棘球蚴AgB1和AgB2两个亚单位基因在同一载体中进行联合表达,并对联合表达的重组抗原和单基因表达的AgB1、AgB2在血清抗体检测中的差异进行比较研究。方法在表达载体PET32a中构建AgB1＋AgB2（AgBs）联合基因的重组质粒,用ELISA检测重组抗原与病人血清的反应性。结果构建了AgBs重组质粒,经测序证实插入的联合基因片段序列正确。AgBs重组质粒表达的融合蛋白分子量为38kDa,为不溶性蛋白。联合表达抗原AgBs对细粒棘球蚴病（CE）血清的敏感性为84．4％,特异性为80．5％;单基因表达的AgB1和AgB2的敏感性分别为75．6％和57．8％,特异性分别为72．6％和91．2％。结论成功构建了AgB亚单位联合表达基因,联合表达的AgBs抗原对CE血清的诊断价值优于单基因表达的AgB1或AgB2抗原。     

肺癌T7噬菌体文库中MUC1蛋白抗原模拟表位的筛选

目的利用噬菌体表面展示技术,从肺癌T7噬菌体展示文库中筛选MUC1蛋白抗原的模拟表位。方法以MUC1单克隆抗体为靶分子,对肺癌T7噬菌体文库进行淘洗筛选,得到的阳性克隆进行测序并推导其氨基酸序列,对得到的阳性噬菌体克隆进行鉴定。结果经3轮淘选,得到20个阳性克隆。测序获得AAPDFRP、SAPDDRP 2种氨基酸序列,对MUC1单抗的抑制率均在50%以上,与肺癌血清结合特异性较高,此2种蛋白序列可模拟MUC1蛋白抗原表位。结论通过噬菌体展示技术成功筛选到MUC1蛋白的模拟表位序列XAPDXRP(X为任意氨基酸),对肺癌的早期诊断有一定的参考价值。     

SLE患者血清中病原体相关噬菌体7肽的检测

目的用噬菌体7肽库筛选系统性红斑狼疮（SLE）患者血清特异性抗体,测序分析其实际意义。方法先用30例正常人混合血清与噬菌体7肽库反应,未与正常人白清结合的7肽再与30例SLE患者混合血清结合,获得SLE血清特异性结合的噬菌体克隆。用患者混合血清进行Dot—ELISA实验鉴定获得的噬菌体克隆,进一步用SLE患者及正常人血清各12例筛选阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况,并对最终鉴定的噬菌体克隆进行测序与比对分析。结果混合的阳性噬菌体克隆与SLE患者个体血清反应阳性率明显高于其与正常人血清的反应率;序列分析显示阳性噬菌体克隆的抗原表位与杆菌、球菌、弧菌等微生物有同源性,与裂殖酵母属、链球菌属、立克次（氏）体属等有100％同源性,与人类抗原表位无关。结论SLE患者血清中存在与病原体抗原表位结合的抗体成分,提示SLE可能与病原体感染有关。     

重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用

目的：制备人自身抗原细胞色素P4502D6（CYP2D6）257-351位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗LKM-1抗体的敏感性和特异性。方法：以肝脏的cDNA混合文库为模板作PCR,将PCR产物与真核表达载体pEGH共同转化酿酒酵母Y258,碱裂解法进行质粒制备,PCR扩增鉴定。表达载体构建成功后,在半乳糖的诱导下表达产生重组融合蛋白,经GST亲和层析法进行纯化后,免疫印迹法鉴定抗原性,ELISA检测抗LKM-1抗体阳性血清及部分其他结缔组织病患者血清中的抗LKM-1抗体。结果：重组融合蛋白在宿主菌中获得表达,免疫印迹法鉴定表明其能与标准抗LKM-1抗体阳性血清反应,而与正常血清、其他抗血清无反应。在26份抗LKM-1抗体阳性血清中,6份抗HCV抗体阳性血清用重组多肽ELISA检测有5份呈阳性,其余20份血清用重组多肽ELISA检测均呈阳性；20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论：重组的257-351位氨基酸片段是CYP2D6抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗LKM-1抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。     

噬菌体表面呈现抗人红细胞血型A抗原单链抗体的研究

目的 构建表达抗人红细胞血型Ａ抗原５０Ａ杂交瘤细胞的单链抗体（ＳｃＦｖ）。方法 应用重组噬菌体抗体技术,从５０Ａ杂交瘤细胞中分离、构建单链抗体基因,并将其克隆入噬粒ｐＣＡＴＮＡＢ５Ｅ中,转化Ｅ．ｃｏｌｉ ＸＬ－Ｂｌｕｅ,辅助噬菌体援救构建５０Ａ噬菌体单链抗体库;采用完整红细胞亲和富集法淘选阳性重组噬菌体,鉴定重组噬菌体并进行序列测定分析;免疫印迹试验检测重组单链抗体的特异性抗原活性。结果 用Ｍ１３     

应用噬菌体肽文库筛选McAb F3特异性结合肽

目的：应用噬菌体展示肽文库筛选可与抗汉坦病毒囊膜蛋白单抗F3特异性结合的配体肽。方法：采用protein-A亲和层析纯化的F3单抗为筛选配基，对噬菌体展示的随机12肽文库进行生物亲和淘洗，夹心ELISA、竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果：通过3轮生物淘洗，能被抗体捕获的噬菌体克隆为91．7％（11／12）；ELISA测定显示筛选到的噬菌体短肽能与F3单抗特异性结合；序列分析表明7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT，同源性分析显示该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性，结论：获得了具有良好结合活性的模拟表位肽，为基于表位水平的HFRSV（肾病综合征出血热病毒）特异性分子多肽疫苗的设计提供了重要依据。     

应用噬菌体随机肽库技术筛选SARS-CoV S蛋白模拟表位

目的筛选SARS病毒(Severe acutere spiratorysyndromeas sociated coronavirus,SARSCoV)结构蛋白S(Spike)特异性的模拟表位,为抗SARS疫苗研究提供基础。方法以抗SARS病毒S蛋白的单克隆抗体为固相筛选分子,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,随机挑选30个克隆,经噬菌体酶联免疫吸附(ELISA)法和交叉反应实验鉴定阳性克隆,再进行DNA序列分析和竞争抑制结合实验,以确定SARS病毒S蛋白的模拟表位。结果经噬菌体富集后,从随机挑选的30个克隆中得到了29个编码8种12肽的阳性克隆,8条肽与S蛋白的320～350氨基酸序列有较高同源性,确定YF、W、E和K氨基酸残基为模拟表位的骨架结构。结论用噬菌体12肽库成功筛选到了SARS病毒S蛋白的模拟表位,为基于S蛋白的肽疫苗研制提供了基础。     

Mimotopes of the Vi Antigen of Salmonella enterica Serovar Typhi Identified from Phage Display Peptide Library

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.     

Identification of hepatitis A virus mimotopes by phage display, antigenicity and immunogenicity

A phage-displayed peptide approach was used to identify ligands mimicking antigenic determinants of hepatitis A virus (HAV) for the first time. Bacteriophages displaying HAV mimotopes were isolated from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Selected phage-peptides were screened for reactivity with sera from HAV infected patients and healthy controls. Four cloned peptides with different sequences were identified as mimotopes of HAV; three of them showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV. One clone was recognised by 92% of the positive sera. The phagotopes competed effectively with HAV for absorption of anti-HAV-specific antibodies in human sera, as determined by ELISA. The four phage clones induced neutralising anti-HAV antibodies in immunised mice. These results demonstrate the potential of this method to elucidate the disease related epitopes of HAV and to use these mimotopes in diagnostic applications or in the development of a mimotope-based hepatitis A vaccine without the necessity of manipulation of the virus.     

GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies

Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS(M)) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.     

人泌尿系上皮肿瘤疫苗的初步研究

目的通过筛选噬菌体随机12肽库获得MUC1糖链抗原模拟表位。方法利用纯化获得的M a695抗体筛选噬菌体随机12肽库,通过夹心ELISA分析噬菌体克隆,测定阳性克隆DNA序列并进行同源性及氨基酸分析,竞争性抑制实验鉴定噬菌体克隆。结果经3轮筛选,获得了14个阳性克隆,DNA序列分析并推导出氨基酸序列:KHYDPFHHRMPQ,QADTARSVALAG,VPSKPDLHVRSI,MTPIHYWNHNRV。鉴定结果表明4个噬菌体展示肽克隆抑制率均在50%以上。结论所得序列KHYDPFH-HRMPQ,QADTARSVALAG,VPSKPDLHVRSI,及MTPIHYWNHNRV模拟了MUC1抗原表位。     

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.     

从噬菌体环肽库中筛选TNF-α表位模拟肽的研究

目的：用具有中和活性的抗TNF—α单抗(mAb)J1D9筛选噬菌体环七肽库，从中获得可模拟TNF—α表位的短肽。方法：筛选TNF—α表位模拟肽，并用夹心ELISA法鉴定阳性克隆。结果：对环七肽库进行3轮筛选后，随机挑选20个噬菌体克隆，经ELISA鉴定出9个阳性克隆。DNA序列分析后，推导的氨基酸序列共3种：c—RRPAQSG—c、c—NKHNRKI—c和c—RGMSRKI—c。结论：筛选的环七肽克隆展示肽具有TNF—α的抗原性，为TNF—α表位模拟肽。     

大肠杆菌脂多糖2630模拟位的研究

目的 :利用纯化的抗大肠杆菌L2 6 30多抗筛选噬菌体环七肽库 ,以获得可模拟脂多糖 (LPS)表位的短肽克隆。方法 :以亲和层析纯化的抗大肠杆菌L2 6 30多克隆抗体为靶分子 ,筛选噬菌体随机环七肽库 ,用双夹心ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆的抗原性。结果 :对噬菌体环肽库进行 3轮筛选后 ,随机挑选 2 0个克隆 ,经鉴定其中 12个可与抗L2 6 30抗体结合 ,即为阳性克隆 ;其中有 5个阳性噬菌体克隆表现出与抗鼠伤寒沙门氏菌LPS多抗结合的活性 ,提示这 5个噬菌体展示的短肽具有模拟大肠杆菌LPS及鼠伤寒沙门氏菌LPS共同表位的性质。经DNA序列分析显示 ,其中 8个克隆的氨基酸序列具有X DGLL XX或X EDGLL X保守序列 ,其余 4个克隆的序列均不相同。结论 :筛选获得的噬菌体环七肽克隆具有模拟大肠杆菌LPS表位的活性 ,为大肠杆菌L2 6 30多表位模拟短肽。其中 5个阳性噬菌体克隆短肽具有模拟大肠杆菌LPS及鼠伤寒沙门氏菌LPS共同表位的活性     

Mimotopes of the Vi antigen of Salmonella enterica serovar typhi identified from phage display peptide library

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.     

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.