TH线性肽对人肝素酶B细胞表位多抗原肽的免疫增强作用

目的 探讨提高人肝素酶B细胞表位肽应答效应的免疫策略.方法 预测人肝素酶蛋白B细胞表位,采用8分支多抗原肽(MAP)结构合成多肽,利用ELISA鉴定合成的MAP与肝素酶全蛋白抗体的结合反应.以MAP联合或不联合通用型TH表位线性肽免疫C57BL/6小鼠,动态检测免疫血清效价.结果 软件预测得到3个肝素酶蛋白B细胞表位,即大亚基的第1～15位(MAP1)、第279～293位(MAP2)及175～189位(MAP3)氨基酸序列.ELISA显示,MAP2多肽与全蛋白抗体的结合力明显强于MAP1及MAP3,MAP与TH线性肽联合免疫产生的抗体滴度明显高于单纯MAP免疫组.结论 生物信息学预测得到的3个表位肽均为肝素酶蛋白的B细胞优势表位,T辅助表位线性肽能显著增强MAP的免疫效应.     

TIMP-1B细胞表位的预测和鉴定

目的: 预测并鉴定金属蛋白酶组织抑制物-1(TIMP-1)蛋白B细胞表位。方法: 采用DNAStar和BcePred分析软件联合预测TIMP-1的B细胞表位,以此合成8分支多抗原肽结构的表位肽(MAP),并与通用型T辅助表位肽(VQGEESNDK,氨基酸163~171)联合免疫家兔,检测免疫血清效价,用Western blotting和间接酶联免疫吸附测定等方法鉴定其特异性和抗体亲和力。结果: 软件预测显示,TIMP-1的第27~41 位(MAP1)、第57~71 位(MAP2)、第95~109 位(MAP3)和第193~207 位(MAP4)氨基酸序列最可能为其优势B细胞表位。抗体滴度动态检测表明,MAP2、MAP3和MAP4均能诱导产生特异性抗体,其中MAP2和MAP4诱导的抗体水平最高;免疫印迹证实MAP2、MAP3和MAP4诱导产生特异性的TIMP-1抗体;间接ELISA证实MAP4与商品化TIMP-1抗体具有最高的亲和力。结论: TIMP-1的第27~41位和第193~207位氨基酸为其优势B细胞表位,其中第193~207位氨基酸的免疫原性最强,这为TIMP-1多肽抗体和B细胞优势短肽疫苗研制提供了理论依据。     

鼠TRAM蛋白B细胞表位预测及其免疫原性检测

目的 预测TRAM蛋白的二级结构和B细胞表位,为抗鼠TRAM单克隆抗体制备奠定基础。方法以TRAM蛋白的氨基酸序列为基础,采用Goldkeu计算机分析软件以及网络nnpredict二级结构分析软件对TRAM蛋白二级结构及B细胞表位预测。合成针对该表位的多肽,以此多肽为免疫原免疫兔,对其免疫原性进行检测。结果用多参数预测TRAM蛋白的二级结构和B细胞表位,综合评判表明：TRAM分子的第216～229位氨基酸满足亲水性、可及性和可塑性,在二级结构上位于蛋白伸展结构或无规则卷曲结构内,最可能为其优势B细胞表位。此多肽能诱导机体产生较高的抗体滴度,多克隆抗体具有高的特异性。结论TRAM分子的第216～229位氨基酸为其优势B细胞表位,这为制作B细胞优势短肽单克隆抗体提供了理论依据。     

尤文肉瘤EWS-FLI1融合蛋白B淋巴细胞表位的预测及鉴定

目的:预测及鉴定尤文肉瘤EWS-FLI1融合蛋白的B淋巴细胞表位。方法:采用综合法预测EWS-FLI1蛋白的二级结构及B淋巴细胞表位,运用标准Fmoc方案合成预测的表位肽,HPLC和MS进行表位肽的纯度分析及分子量鉴定,ELISA法检测表位肽的抗原性,并测定表位肽特异性免疫血清效价,Western blot鉴定免疫血清与EWS-FLI1蛋白亲合力。结果:通过综合法预测得到3个高分值的B淋巴细胞表位,HPLC分析合成的表位肽纯度>85%,MS鉴定表位肽的分子量无误,ELISA法检测证实3个B淋巴细胞表位肽均可获得强的抗原抗体反应,其中表位肽P2的抗原性最强,在1∶40时A450=2.46,达到最高,1∶10 240稀释后抗原抗体反应仍呈阳性;用这3个B淋巴细胞表位肽免疫新西兰兔也能获得理想的抗体效价,其中表位肽P2获得的抗体效价最高,1∶512 000稀释时A450=1.11;Western blot鉴定表位肽P1、P2免疫血清能够结合EWS-FLI1蛋白。结论:尤文肉瘤EWS-FLI1蛋白的B淋巴细胞表位肽P1、P2具有潜在的抗原性和免疫原性。     

SARS病毒基因组所编码的E蛋白的二级结构和B细胞表位预测

目的：预测SARS病毒E蛋白的B细胞表位和二级结构。方法：以SARS病毒基因组序列为基础，采用Garnier-Robson方法、Chou-Fasman方法和Karplus-Schuhz方法预测E蛋白质的二级结构；用Kyte-Doohttle方案预测蛋白质的亲水性；用Emini方案预测蛋白质的表面可能性；用Jameson-Wolf方案预测氨基酸的抗原性指数。综合评判，预测SAPS病毒E蛋白的B细胞表位。结果：在SARS病毒E蛋白N-端的第1～6、13～19、39～43、47～64区段和第73～76区段有β-折叠中心；第6～12区段和第67～69区段可能形成转角或无规则卷曲，是柔性区域。E蛋白N端第2～13区段和第61～74区段为B细胞优势表位区域。结论：用多参数预测SARS病毒E蛋白的二级结构和B细胞表位，为实验方法探索SARS冠状病毒E蛋白的B细胞表位提供理论依据。     

E血清型沙眼衣原体主要外膜蛋白B细胞表位的预测

基于E血清型沙眼衣原体(Chlamydia trachomatis,CT)主要外膜蛋白(Major outer membrane protein, MOMP)氨基酸序列,采用Hopp-Woods的亲水性方案、Emini表面可及性方案、Jameson-Wolf 抗原指数方案和Janin可及性方案等,辅以对MOMP蛋白的二级结构中的柔性区域及跨膜区域的分析,预测CT MOMP蛋白的B细胞表位.推测最有可能的B细胞表位位于MOMP蛋白N端第73～81区段、217～225区段、第377～386区段、第261～270区段和第161～175区段内或它们的附近.用多参数预测CT MOMP蛋白的B细胞表位,为进一步研究蛋白特性及表位疫苗研制奠定了基础.     

杨絮蛋白的组分分析及其T、B细胞表位预测

杨絮是引起过敏性疾病的重要气传变应原之一。为了分析杨絮的蛋白组分并预测其T、B细胞表位,本研究采用有机溶媒法提取杨絮总蛋白,二喹啉甲酸(bicinchoninic acid,BCA)试剂盒检测蛋白浓度。胰酶酶解总蛋白,经高效液相色谱法(high performance liquid chromatography,HPLC)分级、液相色谱-质谱进行组分分析以及质谱质控鉴定;生物信息学方法在线预测不同组分中的T细胞和线性B细胞表位。结果显示,杨絮蛋白总浓度为5.53μg/μL,经酶解及液相色谱-质谱进行组分分析共获得22 023个肽段,质谱质控显示所获肽段的质量偏移(mass error)10 ppm,肽段长度主要分布在8～20个氨基酸残基之间;生物信息学在线预测结果表明,所得肽段含16个高亲和力T细胞表位、2 300个中等亲和力的T细胞表位以及10个线性B细胞表位。该研究结果表明,杨絮的蛋白组分含有具有潜在诱发Ⅰ型过敏性疾病的T和B细胞表位。     

应用合成肽研究柯萨奇B病毒衣壳蛋白VP1的共同抗原表位

对柯萨奇B4病毒(CVB4)衣壳蛋白VPl保守区的亲水性和二级结构进行分析和预测,选择可能代表优势抗原表位的肽段进行合成(VPl-l肽,RIYF KPKHVK AYV),并截取了该肽段的前12个残基(VPI-2肽)用同样方法进行合成.用VP1-1肽免疫家兔制备出高效价抗体,应用酶联免疫吸附法(ELISA)检测,这些抗体与CVBI-6病毒均有结合反应,VP1-1肽与型特异性CVB1-6抗体均有良好的结合反应,表明VP1-1肽是CVB的共同抗原表位.用VP1-1肽检测心肌炎患者血清CVBIgM抗体,阳性率在40%左右.     

人TLR4的B细胞优势表位设计、合成及其免疫原性检测

目的　鉴定TLR4的优势抗原表位 ,为抗人TLR4单克隆抗体制备奠定基础。方法　分别应用Hoop&Woods亲水性参数、抗原性参数、可及性参数和抗原表位的软件分析对TLR4B细胞表位进行预测 ,最后用抗原性指数的方法进行综合评述。合成针对该表位的多肽 ,以此多肽为免疫原免疫小鼠 ,对其免疫原性进行检测。结果　预测TLR4的B细胞优势表位为 189～ 2 0 2氨基酸序列 :NH2 HKLTLRNNFDSLNV COOH ,此多肽能诱导机体产生较高的抗体滴度 ,多抗具有较高的特异性。结论　预测的TLR4短肽是B细胞的优势表位 ,以此制作抗人TLR4单克隆抗体是可行的。     

旋毛虫抗原分子克隆及其T细胞和B细胞表位预测

目的 克隆旋毛虫肌幼虫Mr21 000抗原蛋白基因(Ts21),预测其T细胞和B细胞表位.方法 旋毛虫(河南地理株)感染小鼠后收集肌幼虫,提取肌幼虫总RNA,应用RT-PCR技术获取目的基因Ts21,构建重组质粒pUC18-Ts21并测序,应用生物信息分析软件预测其T细胞和B细胞表位.结果 成功构建克隆载体pUC18-Ts21.旋毛虫Mr21 000抗原的T细胞表位位于第9～23、135～150位氨基酸位点;B细胞表位位于第31～35、53～56、98～104、124～130、133～157、160～172位氨基酸位点.结论 成功克隆了旋毛虫河南地理株肌幼虫Mr21 000抗原的编码基因,T细胞和B细胞表位预测结果表明该抗原蛋白具有较好的抗原性,可作为旋毛虫病免疫诊断和预防的候选抗原.     

Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies.

The multiple antigen peptide (MAP) system has been proposed as a novel and valuable approach for eliciting antibodies to peptides and developing synthetic vaccines. The MAP system consists of a small immunogenically inert core matrix of lysine residues with alpha- and epsilon-amino groups for anchoring multiple copies of the same or different synthetic peptides. Several MAP systems, each containing eight copies of 6-15 residue-long peptides derived from the terminal and central regions of various proteins were analyzed in this study. The immunogenicity of MAPs was compared to that of the same peptides linked to carrier protein by means of conventional conjugation procedures. The various peptide antisera were tested in ELISA with homologous peptides conjugated to a carrier protein via their C terminal (as in the MAP system) or their N terminal end, or with their parent proteins. The antigenic properties of MAPs were studied with anti-peptide sera obtained by classical methods and with anti-protein sera. The results showed that the MAP system was an efficient antigen in ELISA except when the peptide corresponded to a C terminal epitope. However, the value of MAPs for raising anti-peptide antibodies cross-reactive with the cognate protein appeared much more limited. In the case of one N terminal peptide, the MAP construction was not immunogenic while the conventionally conjugated peptide induced antibodies that reacted strongly with the corresponding protein. In the case of the two C terminal peptides tested, the antibodies raised against MAP constructs reacted well with homologous MAPs but did not cross-react with the whole protein. Only in the case of a peptide from an internal domain of histone H2A did immunization with a MAP generate antibodies that cross-reacted with the protein.     

Detection of Infectious bursal disease virus by ELISA using an antipeptide antibody raised against VP3 region

Antigenic determinant analysis was carried out on VP3, one of major immunogenic proteins of Infectious bursal disease virus (IBDV) using computer algorithms. Altogether 17 peptides were synthesized for predicted putative regions and were tested for their reactivity with IBDV-positive polyclonal sera as well as with antisera to other common avian viruses to confirm specificity and to rule out cross reactivity. Of 17 peptides tested, three were selected and synthesized in multiple antigenic peptide (MAP) format. The immunization of rabbits with the three MAPs resulted in high humoral immune response. The purified antipeptide antibodies were screened against native IBDV antigen and the respective titers were determined. Out of the three antisera to MAPs that raised against the MAP3, spanning the amino acids (aa) 974-995 region on the VP3 protein had a very high titer (2048) and reacted specifically with IBDV. Thus, the antiserum to MAP3 detected native virus in enzyme-linked immunosorbent assay (ELISA), revealing the presence of a potential antigenic determinant on the C-terminus of the protein. This study proved that an antipeptide antibody could be used as a safe and specific tool for the diagnosis of IBD in chickens.     

Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine.

Chemically defined synthetic polymers, known as multiple antigen peptide systems (MAPs) represent an effective and novel approach for engineering peptide-based vaccines. Ten different mono and diepitope MAP models, containing different arrangements and stoichiometry of functional B and/or T helper epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize mice. High titers of antibody and protective immunity against sporozoite challenge were elicited by MAPs containing T and B epitopes arranged in tandem and in equimolar amounts. These results indicate that MAPs may serve as a basis for developing subunit vaccines to induce high levels of antibodies against sporozoites.     

Immunogenicity of Well-Characterized Synthetic Plasmodium falciparum Multiple Antigen Peptide Conjugates

Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide (MAP) conjugates containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1, and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein central repeat region were tested in this study. Mice of all four strains produced peptide-specific antibodies; however, the magnitude of the humoral response indicated strong genetic restriction between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluorescence assay. In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid-phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.     

Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1

Based on our hypothesis that epitope vaccine may be a new strategy to induce high levels of neutralization antibodies against HIV-1, we prepared multiple-epitope vaccines using three neutralizing epitopes (GPGRAFY, RILAVERYLKD and ELDKWA) of HIV-1 gp160, and characterized their immunogenicity. Peptide 1 [C-G-(ELDKWA-GPGRAFY)(2)-K] and peptide 2 (CG-GPGRAFY-ELDKWA-G-RILAVERYLKD) were synthesized and conjugated with carrier protein bovine serum albumin (BSA). After vaccination antibody responses to these immunogens were induced and evaluated by ELISA. The C-G-(ELDKWA-GPGRAFY)(2)-K-BSA (BSA: carrier protein) multiple-epitope vaccine induced a strong antibody response to the C-G-(ELDKWA-GPGRAFY)(2)-K peptide (antibody titer: 1:25,600) and C-(ELDKWAG)(4) peptide (antibody titer: 1:12,800), but a weak antibody response to the C-(GPCGRAFY)(4) peptide. The CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K-BSA (BSA: carrier protein) multiple-epitope vaccine also induced strong antibody response to the CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K peptide (antibody titer: 1:25, 600) and C-(ELLDKWAG)(4) peptide (antibody titer: 1:6,400), a very strong response to C-(RIVALVERYLKD-G)(2)-K peptide (dilution: 1:102, 400), and a very weak response to the C-(GPGRAFY)(4) peptide (dilution: 1:400) in mice. Both antisera induced by both multiple-epitope vaccines interacted with the recombinant soluble gp41 (rgp41), but did not bind two control peptides. In comparison with both epitope vaccines, the rgp160 subunit vaccine could induce weak epitope-specific antibody response to these three epitopes on the three epitope peptides and V3, N-domain and C-domain peptides (dilution: 1:400-1:1,600). These results indicate that both multiple-epitope vaccines could induce high levels of antibodies to both neutralizing epitopes RILAVERYLKD and ELDKWA, while the GPGRAFY epitope on both vaccines appeared to have weak immunogenicity. Both multiple-epitope vaccines showed significant potency on inducing high levels of epitope-specific neutralization antibodies in comparison with rgp160 subunit vaccine.     

Broader cross-reactivity after conjugation of V3 based multiple antigen peptides to HBsAg

Vaccines against highly variable pathogens should elicite antibodies to a huge number of clinical isolates. For this purpose, new strategies to overcome the variability are needed. We have previously reported a useful method to conjugate multiple antigen peptides (MAPs) to carrier proteins. Also, we have suggested that these conjugates might enhance cross-reactivity in comparison to other synthetic structures. In this work, MAPs were synthesized and their respective conjugates to HBsAg were obtained. Two peptides from the V3 loop of HIV-1 were included in the MAPs as B cell epitopes because of their variability. Groups of mice were immunized and the immunogenicity and the level of cross-reaction to a panel of five heterologous V3 peptides were studied. Our results show that sera from mice immunized with MAPs coupled to HBsAg recognize a higher number of heterologous peptides (P < 0.05). This behavior was related neither to the immunogenicity nor the antigenicity of the synthetic structures. These results have important implications for the choice of better immunogens against variable epitopes.     

Alzheimer''s neurofibrillary tangles contain unique epitopes and epitopes in common with the heat-stable microtubule associated proteins tau and MAP2.

Ten monoclonal antibodies raised against Alzheimer's neurofibrillary tangles (ANTs) were characterized for reactivity with heat-stable microtubule fractions from bovine and human brain. Five of the antibodies showed very little reaction, but the other five reacted strongly with heat-stable microtubule associated proteins (MAPs). The proteins recognized by these antibodies have estimated molecular weights similar to those of known heat-stable MAPs, tau (52-68 kd) and MAP2 (200-250 kd). That the proteins are indeed tau and MAP2 is demonstrated by reaction of electroblotted proteins with antibodies raised in mouse and guinea pig against bovine brain tau and MAP2. One anti-ANT antibody reacts only with tau, two bind strongly to tau and weakly to MAP2, one recognizes both tau and MAP2 equally well, and one primarily stains MAP2. Extraction of ANT with 2% SDS does not remove tau or MAP2 epitopes from ANT, indicating that epitopes shared with heat-stable MAPs are integral components of ANT. The existence of tau epitopes in ANT is also demonstrated by immunoblotting of ANT-enriched fractions with anti-tau antibodies. Most of the material recognized by anti-tau antibodies in ANT-enriched fractions is present in large molecules excluded by 3% polyacrylamide gel upon electrophoresis. Anti-tau antibodies immunostain ANT in immunofluorescence and immunoperoxidase studies. The immunostaining can be blocked by absorption of anti-tau antibodies with purified tau proteins from bovine brain. Not all ANTs in any given tissue section or isolated Alzheimer perikarial preparations, however, are stained by anti-tau antibodies. These results are consistent with previous studies that have demonstrated heterogeneity of ANTs. Whether this heterogeneity is due to biochemical modification of MAPs or absence of MAPs in some ANTs is unknown. The significance of what appear to be shared epitopes recognized by monoclonal antibodies in tau and MAP2, and the implications this may have on the pathogenesis of ANT formation, requires further investigation.