卡氏肺孢子虫MSG抗原片段细胞表位的预测

[目的]预测卡氏肺孢子虫主要表面抗原(MSG)片段的B细胞和T细胞抗原表位. [方法]运用Sig-nalp3.0、EMBOSS等网络服务器推测卡氏肺孢子虫MSG抗原片段的B细胞和T细胞抗原表位,分析预测结果. [结果]MSG抗原片段存在3个潜在的B细胞抗原表位及6个潜在的T细胞抗原表位.B细胞抗原表位在氨基酸序列的位置为69-79、96-101、133-140aa 3个区域内或其附近;T细胞抗原表位在氨基酸残基的位置为7-22、28-43、20-35、40-55、53-68、86-101aa. [结论]MSG抗原表位的预测为有目的的克隆基因片段,研究高效的表位疫苗奠定基础.     

肿瘤抗原SCCAg B细胞抗原表位及HLA I类限制性CTL表位的生物信息学分析

目的预测鳞状细胞癌抗原(squamous cell carcinoma antigen,SCCAg)的B细胞抗原表位及HLA I类限制性细胞毒性T细胞表位。方法获得SCCAg的三维结构后,采用生物信息学方法预测SCCAg蛋白存在的B细胞表位;而后,采用Net CTL、Prot Param等软件方法预测SCCAg中可能存在的HLA I类限制性CTL表位。结果 SCCAg蛋白中包含10个线性B细胞表位和5个构象B细胞表位;结合与HLA I类分子结合力、蛋白酶体切割效率和TAP转运效率,经Net CTL预测发现,针对不同的HLA I类分子,肿瘤抗原SCCAg蛋白中存在多个HLA I类分子的限制性CTL表位。结论综合运用多种方法预测了肿瘤抗原SCCAg蛋白中存在的B细胞抗原表位和HLA I限制性的CTL表位,为下一步针对SCCAg蛋白开展靶向免疫治疗提供了基础。     

登革病毒E蛋白抗原表位研究进展

提要登革病毒是虫媒病毒B组的一个成员,其RNA编码3种结构蛋白和7种非结构蛋白。研究发现,病毒抗原中既存在有中和性抗原表位,又有免疫增强抗原表位。本文阐述了登革病毒主要抗原E蛋白的表位研究进展。     

SARS冠状病毒S基因序列及细胞抗原表位预测

目的 比较SARS冠状病毒各分离株S基因序列及氨基酸序列之间的差异，预测SARS-CoV的S蛋白的抗原表位。方法 利用Lasergene软件包中的EditSeq从21株SARS冠状病毒分离株截取S基因序列并翻译成氨基酸序列，然后用ClustalX软件对截取序列进行比较分析，确定基准株，最后用Protean软件对基准株进行抗原表位预测。结果 在21株分离株的S基因中有8株核苷酸序列、13株氨基酸序列没有发生点突变；预测出78个可能性抗原表位。结论 SARS-CoV的S蛋白相当保守，只发生个别点突变，可能性抗原表位多，且在其中的14个区域中可能性最显著。     

肠道病毒71型衣壳蛋白的抗原表位及其单克隆抗体

肠道病毒71(EV71)是手足口病的主要致病原之一,可引发大部分重症病例和死亡病例,其衣壳蛋白VP1～VP4作为免疫原包含了能够诱导体液免疫(产生抗体)和细胞免疫的抗原表位.迄今为止已有多个免疫显性的线性或构象性B细胞抗原表位被确定,与之相关的单克隆抗体在EV71感染的早期诊断和单克隆抗体治疗方面也已显示一定程度的应用前景,同时也有少数T细胞抗原表位被确定.本文对上述研究情况进行综述.     

间日疟原虫传播阻断疫苗新型候选抗原Pvs48 T.B细胞表位的预测与分析

目的 预测与分析间日疟原虫传播阻断疫苗新型候选抗原Pvs48的T.B细胞表位.方法 运用SYFPEITHTI在线预测Pvs48的T细胞抗原表位,然后再利用表位数据库(IEDB)从抗原性、表面易接近性、柔韧性、亲水性、β转角5个方面在线预测Pvs48的B细胞线性抗原表位.结果 成功预测了Pvs48中的6个B细胞表位肽、7...     

4种血清型登革病毒NS1蛋白序列及B细胞抗原表位特异性分析

目的比较4种血清型登革病毒NS1蛋白型特异性抗原表位基因序列及氨基酸序列之间的差异,为利用基因差异进行血清学分型及疫苗研究提供新的线索。方法利用DNAstar数据包中的Editseq程序,从20株登革病毒分离株的全基因组序列中将NS1基因型特异性抗原表位序列截取出来,再用ClustalX软件进行多序列比对,进行同源性分析,找出型内最为保守的抗原表位序列。并将比对结果在120株登革病毒序列中进一步验证。结果 NS1蛋白36～45和71～85位氨基酸为型特异性抗原表位,高度保守,型内完全相同,型间不同。结论 NS1蛋白36～45位氨基酸可以作为登革病毒血清分型和研制亚单位疫苗的靶标。     

麻疹病毒血凝素蛋白抗原表位的预测与分析

目的 探讨麻疹病毒(MeV)流行株血凝素蛋白(H)抗原表位上氨基酸(aa)变异对病毒抗原性的可能影响。方法 利用生物信息学软件预测MeV H蛋白上B细胞线性表位,设计并合成来源于疫苗株和流行株表位以及同一区域非表位上的多肽对。间接ELISA法检测合成多肽的免疫原性,并制备多肽免疫血清。采用交叉ELISA法分析两条多肽间的抗原性差异,计算抗原比。结果 合成的多肽均能与MeV免疫血清结合,其中设计在表位区的多肽对CW23/CW22(273～282 aa)结合能力最强,而非表位区多肽对CW150/CW151(418～427 aa)结合能力最弱。多肽对中来源不同两条多肽间抗原性差异较大,其中CW23(疫苗株来源)与CW22(流行株来源)间抗原比为16,CW123(疫苗株来源)与CW124(流行株来源)(236～246 aa)间的抗原比为2.877±0.583。非表位多肽对中,CW125与CW126(356～364 aa)间抗原比为1.631±0.481,而CW150与CW151间抗原比为10.367±1.617。结论 麻疹流行株上仍存在保守的抗原表位,但预测的抗原表位及非表位区上的部分aa变异导致疫苗株与流行株间抗原性存在差异。     

汉坦病毒G2糖蛋白多表位抗原基因的设计与构建

目的构建汉坦病毒SEO型代表株L99G2蛋白的多表位抗原基因(mea)。方法通过生物信息学软件对L99株G2蛋白氨基酸序列进行综合分析及预测,优选B细胞表位,引入GPG间隔序列串联表位,设计mea,然后应用重叠PCR法构建mea,并将其克隆到原核表达质粒pET32a(+)。结果优选出5个B细胞表位,设计并成功构建mea,PCR定向克隆获得pET32a-mea重组表达质粒。结论首次构建了汉坦病毒G2糖蛋白mea及其原核表达系统E.coliBL21/pET32a-mea,为其表达及免疫学应用奠定基础。     

小麦过敏原Tri a Bd 27k蛋白抗原表位预测及三维结构模拟

目的 通过生物信息学方法了解小麦过敏原Tri a Bd 27k的结构特征,为小麦变态反应性疾病的诊断和预防提供依据.方法 在Uniprot数据库中获得Tri a Bd 27k蛋白序列,通过DNAStar预测B细胞抗原表位,NetMHCⅡ和Syfpeithi预测T细胞抗原表位;使用Swiss-Model软件预测其三维结构并用Ramachandran进行稳定性评估.在Uniport 数据库搜索Tria Bd 27k的同源蛋白,应用Clustalx 1.83及Mega 3.1构建同源进化树.结果 小麦过敏原Tri a Bd 27k的B/T细胞共同抗原表位优势区域为34-42、102-117,Ramachandran软件预测结果显示小麦Tri a Bd 27k蛋白的空间构象稳定.结论 通过对小麦过敏原Tria Bd 27k进行生物信息学分析获得了该过敏原优势区域及三维结构模型,为进一步了解和掌握小麦过敏原Tri a Bd 27k的结构功能打下理论基础.     

肌幼虫可溶性抗原免疫小鼠抗旋毛虫感染作用

目的观察不同剂量旋毛虫肌幼虫可溶性抗原（muscle larvae soluble antigen，MLSAg）滴鼻免疫小鼠诱导的抗旋毛虫感染作用，确定MLSAg滴鼻免疫适宜剂量。方法C57BL／6J小鼠50只，随机分为5组，分别用5，10，20，30ugMLSAg溶于20ul生理盐水滴鼻免疫小鼠2次，间隔2周；对照组用0．9％NaCl20ul滴鼻。末次免疫后7d，用旋毛虫肌幼虫150条／只灌胃攻击全部小鼠，观察健康状况，记录体重。攻击后28d，检测血清和肠液IgG、IgA。计数舌肌、咬肌、膈肌及后肢肌肉的虫荷。结果攻击后对照组小鼠体重逐渐减轻，而4个免疫组小鼠体重仍呈增高趋势。5，10，20和30ug组小鼠血清IgG水平分别为1．255，1．281，1．394和1．441；血清IgA水平分别为1．108，1．282，1．262和1．326，均高于对照组（1．158，1．035）。肠液IgA水平分别为1．974，2．026，2．057和2．015明显高于对照组（1．785）；10，20和30ug组小鼠肌肉虫荷均明显低于对照组及5ug组（P〈0．05）。结论不同剂量MLSAg滴鼻免疫小鼠均可诱导抗旋毛虫感染，滴鼻免疫剂量以20ug为佳。     

Identification and characterization of protective epitope of Trichinella spiralis paramyosin

Trichinella spiralis paramyosin (Ts-Pmy) is a protective antigen that induces partial immunity against T. spiralis infection in mice. To identify protective epitope of Ts-Pmy, a monoclonal antibody (mAb) 7E2 against the recombinant protein was generated, which partially protected against T. spiralis infection following passive transfer. The mAb was used to screen a random phage-displayed peptide library. Ten positive clones were identified, most of which matched amino acids 88-107 or 108-127 of Ts-Pmy. Expression of overlapping fragments of Ts-Pmy in E. coli confirmed that region 88-107 was specifically recognized by 7E2. A peptide based on this epitope region (YX1) was synthesized and shown to compete with native Ts-Pmy for binding to 7E2. Mice immunized with KLH-conjugated YX1 were protected against T. spiralis larval challenge. The identification of a protective epitope within Ts-Pmy highlights the possibility of developing a subunit vaccine against T. spiralis infection.     

醋与酱油对旋毛虫肌幼虫杀伤作用

目的 观察醋、酱油对旋毛虫幼虫的杀伤作用.方法 将旋毛虫肌幼虫在体外经醋、酱油处理不同时间后观察其活力;48只小鼠随机分为3组,分别经口感染300条经醋、酱油及生理盐水处理后的肌幼虫,感染后42 d收集肌幼虫,测定生殖力指数(RCI).结果 肌幼虫经醋处理3,6,12,24 h的死亡率分别为0.99%,2.94%,24.53%及100%;经酱油处理24,48,72,96,120,144,168,192,216,240 h的死亡率分别为0.98%,2.86%,3.06%,4.81%,5.50%,9.35%,10.42%,11.43%,12.50%及13.59%;幼虫死亡率随醋或酱油处理时间的延长而升高(P<0.05);用醋处理3 h幼虫的RCI(4.07)与正常幼虫的RCI(178.10)差异有统计学意义(P<0.05),处理6 h肌幼虫的RCI为0;用酱油处理12,24,36,48h肌幼虫的RCl分别为13.78,10.94,1.00及0.00;肌幼虫的RCI均随醋与酱油处理时间的延长而下降(P<0.05).结论 醋对旋毛虫肌幼虫有较强的杀伤作用,而酱油对旋毛虫肌幼虫的杀伤作用较弱.     

小鼠感染旋毛虫后血清中IL-2、IL-8含量变化的实验研究

目的通过检测白细胞介素-2(IL-2)、白细胞介素-8(IL-8)的含量,探讨小鼠感染旋毛虫后细胞免疫功能的变化. 方法将80只小鼠随机分为两组,实验组每只小鼠喂食感染旋毛幼虫200条,对照组小鼠不做处理,分别于小鼠感染旋毛虫后第1、2、3、4周随机取实验组和对照组小鼠各10只,常规眼眶静脉取血分离血清,用放射免疫法测定其IL-2、IL-8的值. 结果实验组IL-2的含量在感染后第1周低于对照组(P<0.05),在第2、3、4周均低于相应对照组(P<0.01);实验组IL-2含量随感染时间的延长而呈逐渐下降趋势;实验组IL-8的含量在感染各期均低于对照组(P<0.01),而实验组在感染各期之间IL-8含量差异均无显著性(P>0.05). 结论实验组IL-2、IL-8的含量在感染各期均低于对照组,表明宿主的特异性和非特异性细胞免疫功能均降低,且特异性细胞免疫功能随感染时间的延长而呈不断下降趋势,提示旋毛虫在感染宿主后破坏和逃避了宿主的免疫系统和免疫功能,给其在宿主中的生存和发育等创造了有利条件,对宿主造成进一步的损害.     

卵泡刺激素受体(FSHR)胞外区B细胞表位的优化筛选

目的:采用生物信息学技术,对前期研究获得的卵泡刺激素受体(FSHR)的B细胞表位肽段进一步筛选、优化,以获得理想的FSHR靶标抗原。方法:①利用Insight II分子模拟软件寻找FSHR与FSH相互作用的区域。②应用DNA STAR软件中的Protein模块对优化后抗原进行综合分析。③肽结合实验对优化后抗原进行免疫原性检测。结果:≥5个氨基酸片段的柔性区域位于FSHR第30-44、52-65、112-121、174-182、190-210、223-245区段。当FSHR胞外区第37位缬氨酸被赖氨酸或谷氨酸替代时,具有较好的亲水性及抗原性。而肽结合实验证实肽32-44具有更好的免疫原性。结论:FSHR胞外区肽段32-44有可能成为避孕疫苗的理想抗原。     

Molecular modeling of the human sperm associated antigen 11 B (SPAG11B) proteins

Abstract

Antimicrobial proteins and peptides are ubiquitous in nature with diverse structural and biological properties. Among them, the human beta-defensins are known to contribute to the innate immune response. Besides the defensins, a number of defensin-like proteins and peptides are expressed in many organ systems including the male reproductive system. Some of the protein isoforms encoded by the sperm associated antigen 11B (SPAG11) gene in humans are beta-defensin-like and exhibit structure dependent and salt tolerant antimicrobial activity, besides contributing to sperm maturation. Though some of the functional roles of these proteins are reported, the structural and molecular features that contribute to their antimicrobial activity is not yet reported. In this study, using in silico tools, we report the three dimensional structure of the human SPAG11B proteins and their C-terminal peptides. web-based hydropathy, amphipathicity, and topology (WHAT) analyses and grand average of hydropathy (GRAVY) indices show that these proteins and peptides are amphipathic and highly hydrophilic. Self-optimized prediction method with alignment (SOPMA) analyses and circular dichroism data suggest that the secondary structure of these proteins and peptides primarily contain beta-sheet and random coil structure and alpha-helix to a lesser extent. Ramachandran plots show that majority of the amino acids in these proteins and peptides fall in the permissible regions, thus indicating stable structures. The secondary structure of SPAG11B isoforms and their peptides were not perturbed with increasing NaCl concentration (0-300?mM) and at different pH (3, 7, and 10), thus reinforcing our previously reported observation that their antimicrobial activity is salt tolerant. To the best of our knowledge, for the first time, results of our study provide vital information on the structural features of SPAG11B protein isoforms and their contribution to antimicrobial activity.     

Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine

Biodefense vaccine are destined to be stockpiled for periods of time and deployed in the event of a public health emergency. In this report, we compared the potency of liquid and lyophilized (thermostabilized) formulations of a candidate ricin toxin subunit vaccine, RiVax, adsorbed to aluminum salts adjuvant, over a 12-month period. The liquid and lyophilized formulations were stored at stressed (40 °C) and unstressed (4 °C) conditions and evaluated at 3, 6 and 12-month time points for potency in a mouse model of lethal dose ricin challenge. At the same time points, the vaccine formulations were interrogated in vitro by competition ELISA for conformational integrity using a panel of three monoclonal antibodies (mAbs), PB10, WECB2, and SyH7, directed against known immunodominant toxin-neutralizing epitopes on RiVax. We found that the liquid vaccine under stress conditions declined precipitously within the first three months, as evidenced by a reduction in in vivo potency and concomitant loss of mAb recognition in vitro. In contrast, the lyophilized RiVax vaccine retained in vivo potency and conformational integrity for up to one year at 4 °C and 40 °C. We discuss the utility of monitoring the integrity of one or more toxin-neutralizing epitopes on RiVax as a possible supplement to animal studies to assess vaccine potency.     

乙型肝炎表面抗原和e抗原阳性产妇所生新生儿乙型肝炎母婴传播阻断效果的影响因素

目的探讨乙型肝炎(乙肝)表面抗原(HBsAg)和e抗原(HBeAg)阳性产妇所生新生儿在出生后乙肝疫苗(HepB)和乙肝免疫球蛋白(HBIG)联合免疫以及完成HepB全程免疫后乙肝病毒(HBV)突破性感染的影响因素。方法2016年6月-2017年5月在南昌市2个县(区)选择HBsAg和HBeAg阳性产妇所生新生儿,在联合免疫和HepB全程免疫完成后1-2个月检测血清HBsAg和乙肝表面抗体(HBsAb),分析儿童母婴传播阻断失败率(HBsAg阳性率)。结果本研究共纳入278名婴儿,母婴传播阻断失败率为2.52%(7/278),HBsAb阳性率为96.8%(269/278)。产妇HBsAg阳性时间在2年以上是阻断失败的危险因素,而分娩方式、喂养方式、母亲和婴儿HBIG的使用情况和婴儿性别等与HBV阻断失败率无相关性。结论HepB和HBIG联合免疫对HBsAg和HBeAg阳性产妇所生新生儿具有较好的乙肝母婴传播阻断效果,建议加强育龄妇女HBsAg和HBeAg筛查。     

Investigation of memory B cell responses to hepatitis B surface antigen in health care workers considered as non-responders to vaccination

Up to 20% of health care workers are considered as non-responders to hepatitis B vaccination (anti-HBs < 10 mUI/ml in serum). We have explored memory B cells differentiated in vitro into anti-HBs antibody-secreting cells (anti-HBs-SCs) by ELISPOT assay. Anti-HBs-SCs were detected in vaccinated responders (n = 11) and non-responders (n = 10) but IgG anti-HBs-SCs were significantly lower in the non-responder group (p < 0.001). Low amounts of HBs antibodies were also quantified by ELISA in non-responders’ sera. These results indicate that a suboptimal B cell response exists in non-responders to HBV vaccination. This B cell response may mediate a protection against clinically significant breakthrough hepatitis B infection.     

Silencing an immunodominant epitope of hepatitis B surface antigen reveals an alternative repertoire of CD8 T cell epitopes of this viral antigen

Immunodominance hierarchies operating in immune responses to viral antigens limit the diversity of the elicited T cell responses. The L^d/S_28–39-restricted CD8 T cell response to the hepatitis B surface antigen (HBsAg or S) prevents copriming of D^d- and K^b-restricted CD8 T cell responses. We exchanged L to V at position S₃₉ of HBsAg to construct mutant S_L39V. Comparable levels of wild-type S and mutant S_L39V were produced by transiently transfected cells, and mice immunized with the pCI/S and pCI/S_L39V DNA vaccines showed comparable serum antibody responses to HBsAg. The pCI/S but not pCI/S_L39V DNA vaccination induced L^d/S_28–39-specific CD8 T cell responses. However, the pCI/S_L39V DNA vaccine efficiently primed CD8 T cell responses to the subdominant D^d- and K^b-restricted epitopes, confirming the immunosuppressive phenotype of the L^d/S_28–39-specific CD8 T cell response. A single point mutation within the HBsAg can hence completely silence a ‘dominant’ CD8 T cell response thereby facilitating priming of a multispecific repertoire of suppressed, ‘subdominant’ epitopes. The data have practical implications for understanding HBV-specific CD8 T cell responses and for the design of novel vaccination strategies.