日本血吸虫雌虫抗原模拟表位的筛选及免疫保护性

目的筛选日本血吸虫雌虫抗原的模拟表位，并探讨其抗日本血吸虫的免疫保护效果。方法用日本血吸虫雌虫免疫兔血清IgG作配体对噬菌体随机12肽库进行3轮亲和筛选，随机挑取18个噬菌体克隆用Dot-ELISA检测其特异性，并对其中的4个阳性克隆进行测序。分别在0、2、4周用混合噬菌体克隆免疫小鼠3次，第6周每鼠经腹部感染40条日本血吸虫尾蚴，42d后剖杀冲虫，计数虫数和每克肝卵数。结果经3轮筛选，特异性噬菌体富集了200多倍，随机挑取的18个克隆经Dot-ELISA鉴定有17个能与雌虫抗原免疫兔血清呈阳性反应。DNA自动测序的4个序列与GenBank的已知序列均无同源性。与对照组相比，混合噬菌体克隆免疫小鼠的减虫率为26．57％，减卵率为65．34％。结论采用噬菌体随机肽库技术可获得模拟日本血吸虫雌虫特异性抗原表位的短肽分子，这些短肽分子能诱导一定程度的保护性免疫。     

噬菌体表达短肽模拟旋毛虫抗原表位及其抗血吸虫保护性免疫研究

目的　从噬菌体随机肽库中筛选出模拟旋毛虫特异性抗原表位的短肽分子 ,探讨其抗血吸虫的交叉免疫保护效果。　方法　以纯化的旋毛虫感染鼠血清IgG为配基 ,亲合筛选法富集特异性噬菌体 ,随机挑取噬菌体克隆用ELISA检测其特异性 ;混合噬菌体克隆经皮下免疫小鼠 3次 ,攻击感染后第 4 5天剖杀小鼠 ,观察减虫和减卵效果。　结果　经 3轮筛选 ,特异性噬菌体得到了有效的富集 ,第三轮洗脱噬菌体的产量约为第一轮的 15 0倍。随机挑取 2 4个噬菌体克隆经ELISA测定 ,有 2 1个克隆能与旋毛虫感染鼠血清IgG特异性反应。与对照组相比 ,混合噬菌体克隆免疫小鼠的减虫率与减卵率分别为 4 2 8%与 6 6 3% (P <0 0 0 1)。　结论　利用噬菌体随机肽库技术可获得模拟旋毛虫特异性抗原表位的短肽分子 ,这些短肽分子能诱导明显的抗血吸虫的保护性免疫。     

噬菌体表达短肽模拟血吸虫致弱尾蚴特异性抗原表位的研究

目的　从噬菌体随机肽库中筛选模拟血吸虫致弱尾蚴特异性抗原表位的噬菌体克隆 ,并探讨其免疫保护效果。方法　用紫外线致弱尾蚴免疫兔血清IgG筛选以融合蛋白形式在丝状噬菌体外壳蛋白Ⅲ表达的随机 7肽库 ,三轮免疫筛选后获得的噬菌体克隆经皮下免疫小鼠 3次 ,攻击感染 45d后剖杀 ,观察减虫和减卵效果。结果　经三轮筛选 ,特异性结合的噬菌体富集增加了 10 0多倍 ,随机挑取 2 4个噬菌体克隆经ELISA测定 ,有 2 2个克隆能与致弱尾蚴免疫兔血清IgG特异性反应。混合噬菌体克隆免疫小鼠试验的减虫率与减卵率分别为 33.5 7%和 5 6 .0 7% (P均 <0 .0 0 1)。结论　采用噬菌体随机肽库技术可获得模拟致弱尾蚴特异性抗原表位的短肽分子 ,这些短肽分子能诱导一定程度的保护性免疫     

日本血吸虫抗原模拟表位免疫学活性的初步研究

目的　研究从噬菌体 12肽库中筛选得到的日本血吸虫抗原模拟表位的免疫学活性。　方法　以日本血吸虫急感患者血清免疫球蛋白 (Ig)作为靶分子 ,筛选噬菌体 12肽库。经过 3轮吸附 -洗脱 -扩增的淘筛过程 ,在筛选得到的阳性噬菌斑中随机挑取 42个扩增 ,用ELISA检测不同寄生虫病患者和正常人血清 ,以分析其敏感性和特异性。并进一步免疫昆明株小鼠 ,经日本血吸虫尾蚴攻击感染后剖检 ,计算减虫率和减卵率 ,以分析其在小鼠体内诱导的免疫保护性。　结果　在挑取的 42个阳性克隆中 ,有 9个与日本血吸虫急感患者血清有不同程度的结合 (6.2 5 %～ 10 0 % )。除其中 1个灵敏性较低的克隆与其它寄生虫病患者血清有一定的交叉反应外 ,其余均未发现。而经其免疫后的小鼠也分别获得了 2 9.3 %减虫率和 3 5 .9%减卵率。　结论　从噬菌体 12肽库中筛选到的日本血吸虫抗原模拟表位具有较高的特异性和敏感性 ,并能够在小鼠体内诱导一定的免疫保护力。     

日本血吸虫抗原模拟表位免疫学活性的初步研究

目的研究从噬菌体12肽库中筛选得到的日本血吸虫抗原模拟表位的免疫学活性。方法以日本血吸虫急感患者血清免疫球蛋白(Ig)作为靶分子，筛选噬菌体12肽库。经过3轮吸附-洗脱-扩增的淘筛过程．在筛选得到的阳性噬菌斑中随机挑取42个扩增，用ELISA检测不同寄生虫病患者和正常人血清，以分析其敏感性和特异性。并进一步免疫昆明株小鼠，经日本血吸虫尾蚴攻击感染后剖检，计算减虫率和减卵率，以分析其在小鼠体内诱导的免疫保护性。结果在挑取的42个阳性克隆中，有9个与日本血吸虫急感患者血清有不同程度的结合(6．25％～100％)。除其中1个灵敏性较低的克隆与其它寄生虫病患者血清有一定的交叉反应外，其余均未发现。而经其免疫后的小鼠也分别获得了29．3％减虫率和35．9％减卵率。结论从噬菌体12肽库中筛选到的日本血吸虫抗原模拟表位具有较高的特异性和敏感性，并能够在小鼠体内诱导一定的免疫保护力。     

日本血吸虫模拟短肽诱导小鼠的免疫保护性研究

目的　研究日本血吸虫模拟短肽对小鼠的免疫保护效果,预测其在抗血吸虫感染中的作用。　方法　用粗提纯大鼠血清IgG为配基对噬菌体肽库进行3轮免疫学筛选。随机挑取噬菌体克隆,检测其特异性并进行序列分析;用阳性克隆免疫小鼠,以40条日本血吸虫尾蚴攻击感染,感染后42d剖杀取虫,计算减虫率和减卵率;用ELISA测定免疫鼠抗体反应。　结果　经过3轮筛选,特异性噬菌体得到有效富集;自动测序仪测序获得2个模拟肽分子;将其用于免疫小鼠后,各免疫组与对照组相比,2个模拟肽混合免疫组的减虫率为34.9%P<0.05,肝内减卵率为67.6%(P<0.001)。 2个模拟肽分子分别免疫小鼠 ,各组的减虫率为31.0%(P<0.05)和14.5%(P>0.05)及肝内减卵率为61.2%(P<0.001)和35.7%(P<0.05)。ELISA检测其免疫组鼠血清特异性IgG抗体滴度均大于1∶6 400以上。　结论　利用噬菌体表面呈现技术获得的2个模拟肽分子均可诱导部分抗日本血吸虫感染的免疫保护力     

用日本血吸虫感染新模型兔血清筛选噬菌体随机肽库的初步研究

目的在建立一种有成虫寄生但无虫卵肉芽肿形成的日本血吸虫感染小鼠及家兔新模型,并观察其具有较高的抗攻击感染保护力的基础上,用新模型兔血清筛选噬菌体随机12肽库,并初步鉴定阳性噬菌体克隆诱导小鼠抗日本血吸虫感染的免疫保护作用.方法用大肠杆菌抗原吸收后的新模型兔血清,经抗体捕获法筛选噬菌体12肽库,经3轮亲和筛选、富集后获得阳性多克隆.用常规ELISA法检测阳性多克隆噬菌体的抗原性.用滴度为1×1014pfu的阳性多克隆噬菌体按0-2-4周方案皮下多点注射免疫小鼠,末次免疫4周后,用日本血吸虫尾蚴攻击感染,同时设立常规感染兔血清筛获的阳性克隆免疫组、原始肽库免疫组、攻击感染对照组.结果①新模型兔血清筛获的多克隆噬菌体(滴度为1×1014pfu)与新模型兔血清(稀释度为1:400)反应为强阳性,与常规感染兔血清(1:400)反应为弱阳性,与正常兔血清(1:400)反应为阴性.②用新模型兔血清筛获的多克隆噬菌体、常规感染兔血清筛获的多克隆噬菌体及原始肽库分别免疫小鼠后诱导抗攻击感染的减虫率及每克肝脏减卵率分别为27.2%和38.8%、17.8%和35.0%、4.5%和6.0%.结论与用日本血吸虫常规感染模型兔血清筛获的多克隆噬菌体比较,经新模型兔血清筛获的含有模拟日本血吸虫抗原表位的多克隆噬菌体能诱导小鼠更高的抗攻击感染减虫率(P＜0.05)及相似的减卵率(P＞0.05);与原噬菌体随机肽库相比较,能诱导小鼠较高的抗攻击感染减虫率(P＜0.01)及减卵率(P＜0.05).     

日本血吸虫保护性抗原模拟表位的筛选及其免疫保护作用的研究

目的在前期已建立日本血吸虫感染兔新模型的基础上,用该血清从噬菌体随机12肽库中筛选阳性单克隆噬菌体,并鉴定其诱导小鼠的免疫保护效果。方法通过给感染日本血吸虫家兔注射酚氧化酶抑制剂,抑制其体内日本血吸虫雌虫产卵,建立无虫卵肉芽肿的日本血吸虫感染动物新模型,用其血清筛选出阳性多克隆噬菌体,经与可溶性虫卵抗原(SEA)免疫兔血清充分吸附后,从中随机挑取14个单克隆分别纯化、扩增,用常规ELISA法检测阳性单克隆噬菌体的抗原性;挑选出与新模型兔血清呈强阳性反应,但与SEA免疫兔血清呈阴性或弱阳性反应的单克隆噬菌体,按1×1015pfu/次剂量,0-2-4周方案,皮下多点注射免疫小鼠。末次免疫4周后,每鼠经腹部皮肤攻击感染(40±1)条日本血吸虫尾蚴,感染42d后观察减虫率及减卵率。结果所选14个单克隆噬菌体均能与新模型兔血清和常规感染模型兔血清反应,尤其是第8号和第13号2个克隆与新模型兔血清呈强阳性反应,但与SEA免疫兔血清呈阴性反应;8号、13号单克隆噬菌体及原始噬菌体肽库免疫小鼠后诱导抗攻击感染的减虫率及每克肝脏减卵率分别为35.81%和63.32%、32.09%和52.02%、14.90%和30.64%。结论阳性多克隆噬菌体与SEA免疫兔血清免疫吸附后能去除大部分表达SEA抗原模拟表位的克隆,所选14个单克隆噬菌体均表达     

随机肽库筛选弓形虫特异性抗原表位诱导保护性免疫的研究

目的　从噬菌体随机肽库中筛选出模拟弓形虫特异性抗原表位的短肽分子 ,并探讨其对弓形虫的保护性效果。方法　以纯化的弓形虫免疫兔血清IgG为配基 ,亲和筛选法富集特异性噬菌体 ,随机挑取噬菌体克隆用夹心ELISA法和Dot-ELISA法检测其特异性 ,混合 4个阳性克隆免疫小鼠 ,1月后攻击感染 ,观察小鼠发病时间和死亡时间。结果　经 3轮筛选 ,特异性噬菌体得到了有效富集 ,第 3轮洗脱噬菌体的产量为第 1轮的 16 7倍 ,随机挑取 2 7个噬菌体经Dot -Bloting和夹心ELISA法检测 ,有 2 4个能与弓形虫免疫兔血清及单克隆抗体特异反应。用致死量弓形虫攻击感染经阳性克隆免疫的小鼠 ,存活时间明显长于对照组。结论　免疫筛选噬菌体随机多肽库可获得具有保护性的弓形虫抗原表位 ,为弓形虫疫苗研制提供了新途径。     

用噬菌体十二肽库筛选日本血吸虫病诊断抗原

目的　从噬菌体十二肽库中筛选出特异、灵敏的日本血吸虫病诊断抗原。　方法　以日本血吸虫急感患者血清免疫球蛋白(Ig)作为靶分子,免疫筛选噬菌体随机十二肽库。经过3轮吸附洗脱扩增的淘筛过程,随机挑取10个蓝色噬菌斑扩增,用ELISA检测其免疫活性,并通过对并殖吸虫病及旋毛虫病患者血清的检测分析其诊断日本血吸虫病的价值。　结果　6个克隆可以与日本血吸虫急感患者血清免疫球蛋白(Ig)特异结合。其中A492值最高的克隆(SjA1)具有较好的特异性和灵敏性。　结论　从噬菌体随机十二肽库中筛选到的克隆对日本血吸虫病具有较高的诊断价值。     

Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice. METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced. Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P= 0.001), 14.5% (P= 0.074) worm reduction and 61.2% (P= 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA. CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.     

用噬菌体随机肽库鉴定日本血吸虫22.6kDa抗原分子的表位

目的　筛选和鉴定日本血吸虫 (中国大陆株 ) 2 2 .6kDa抗原 (Sj2 2 .6)的表位。　 方法　用纯化的抗Sj2 2 .6的多克隆抗体IgG对噬菌体十二肽库进行 5轮免疫学筛选 ,挑取克隆 ;采用Westernblotting免疫识别 ,将获得的阳性克隆免疫小鼠 ,并采用dot ELISA筛选能刺激小鼠产生较高滴度抗Sj2 2 .6抗体的阳性克隆 ;测定核苷酸序列 ,分析其表位与Sj2 2 .6抗原的同源性。　结果　经 5轮免疫学筛选后挑取的 1 4个克隆 ,用Westernblotting方法均能被抗Sj2 2 .6抗体识别。经动物免疫初步实验筛选 ,共获得 6个免疫原性较强的阳性克隆 ,测序结果获得 4种不同表位 ,其中 1种表位与Sj2 2 .6抗原具有较高的同源性 ,其余 3种表位与其无一级结构的同源性。　结论　获得的 4种日本血吸虫中国大陆株抗原表位 ,1种可能为结构表位 ,3种为模拟表位     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV~37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0&#177;1.6) %to (39.0&#177;2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pⅢ in order to get some information on mimotopes.METHODS: Through affinity enrichment and ELISA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times ofenrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6)%to (39.0±2.7)%.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

噬菌体表达的短肽模拟蚯蚓与日本血吸虫共同表位研究

目的采用噬菌体呈现技术,获得蚯蚓与血吸虫的共同表位。方法依次以日本血吸虫病患者血清(SjIg)、蚯蚓免疫兔血清(LtIg)和LtIg、SjIg作为靶分子,以噬菌体12肽库的第三轮扩增肽库为源肽库,进行2轮吸附-洗脱-扩增免疫筛选。每轮随机挑取蓝色噬菌斑各21个,用ELISA方法检测其抗原性,并对其反应性较好的阳性克隆进行测序。结果获得4个阳性克隆可与SjIg、LtIg较好的结合。得到3个阳性克隆的氨基酸序列,它们有蚯蚓与血吸虫共同的线性表位。结论结果说明,从12肽库筛选蚯蚓与寄生蠕虫抗原共同表位是可行的,同时也为获得寄生蠕虫共同抗原提供了一条新的途径。     

Protective immunity induced by phage displayed mitochondrial related peptides of Schistosoma japonicum

New antigens and strategies are necessary for vaccine development against schistosomiasis japonica. Using a pool of 43 high titred anti-SWA sera from individuals residing in an Schistosoma japonicum endemic area of China, we have cloned a S. japonicum gene by cDNA library screening. The recombinant Sj338 protein has 44-46% identity to a mitochondrial precursor receptor protein of humans and rats. Immunization of mice with the recombinant Sj338 conferred 27-32% (p<0.01) reduction in worm burdens following cercarial challenge. In an effort to identify protective epitopes in Sj338 and increase the level of protection, we screened a random 12-mer peptide library constructed in M13 using a polyspecific anti-Sj338 rabbit serum. After five rounds of biopanning, we identified 30 reactive clones consisting of 11 distinct peptide sequences. These clones shared limited primary sequence homology with the recombinant Sj338 protein. Anti-sera raised against these phage clones recognized recombinant Sj338 and SWAP by Western blot. In murine vaccination experiments using whole recombinant phage without adjuvant, four of these clones demonstrated worm reductions of 11.6-25.1% (p=ns - 0.05) compared to M13 vaccinated animals. Animals vaccinated with all four of these phage demonstrated 34.2% (p<0.01) worm reduction compared to controls vaccinated with M13 clone. These data suggest that mimotope peptides are potential vaccine candidates for S. japonicum.