基于幽门螺杆菌UreB优势抗原表位多抗原肽疫苗制备及其免疫保护作用

目的 构建串联式幽门螺杆菌UreB抗原优势表位UreB322和UreB527原核重组表达系统,制备UreB322和UreB527表位与多肽载体Poly-Asp-Lys的多抗原肽(MAP)疫苗并确定其免疫原性和免疫保护性.方法 采用连接引物PCR构建含肠激酶(EK)位点的串联式UreB322和UreB527表位基因及其原核表达系统.表达的目的重组融合蛋白8×[rEK-UreB322-EK-UreB527-EK]经EK水解后用Sephadex G-25柱分离rUreB322-EK和rUreB527-EK片段.采用碳二亚胺法将rUreB322-EK、rUreB527-EK与Poly-Asp-Lys载体分子交联,制备出多抗原肽疫苗MAP-rUreB322/B527.采用ELISA和Western blot分别检测各重组表位肽及MAP-rUreB322/527的抗原性和免疫反应性.采用幽门螺杆菌SS1株感染BALB/c小鼠模型检测MAP-rUreB322/527免疫保护效果.结果 获得了8次重复串联的UreB322和UreB527编码基因及其原核表达系统,目的融合蛋白8×[rEKUreB322-EK-rUreB527-EK]表达量可达细菌总蛋白的48％.肠激酶可将目的融合蛋白完全水解为rUreB322-EK和rUreB527-EK片段.rUreB322-EK和rUreB527-EK与Poly-Asp-Lys交联率高达92.5％.幽门螺杆菌全菌抗体及rUreB-IgG均能识别rUreB322-EK、rUreB527-EK和MAP-rUmB322/527并与之结合.MAP-rUreB322/527免疫小鼠血清抗体水平明显高于rUreB(P＜0.05).50或100μgMAP-rUreB322/527对幽门螺杆菌SS1株感染小鼠的保护率(83.3％和91.7％)明显高于等量rUreB(41.7％和50.0％)(P＜0.05).结论 本研究成功地构建了幽门螺杆菌UreB抗原优势表位UreB322和UreB527重复串联式编码基因及其原核表达系统,基于UreB优势抗原表位的多抗原肽疫苗MAPrUreB322/527能显著提高免疫保护效果.     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

目的 分析并确定幽门螺杆菌黏附素A(HpaA)分子中有效T细胞和B细胞联合(T/B)抗原表位.方法 重组表达HpaA(rHpaA)并免疫家兔制备抗血清.采用生物信息学技术预测和分析HpaA分子中T细胞和B细胞表位,PCB扩增T/B联合表位肽片段并构建其噬菌体展示系统.采用PEG/NaCl沉淀法提纯展示了T/B联合表位的重组噬菌体PⅢ蛋白(rPⅢ).分别以商品化幽门螺杆菌全菌IgG抗体和rHpaA抗血清为一抗,采用Western blot和ELISA对rPⅢ蛋白中展示的T/B联合表位进行筛选和鉴定.采用MTT检测rHpaA免疫小鼠脾细胞在不同重组噬菌体蛋白刺激下的增殖情况.结果 HpaA分子中共有5个T/B联合表位:HpaA10、HpaA37、HpaA79、HpaA116和HpaA143.所有T/B联合表位均成功地展示于M13噬菌体PⅢ蛋白表面.Western blot、ELISA和淋巴细胞增殖试验结果 均显示,HpaA116是优势抗原表位,HpaA37和HpaA79为有效抗原表位,HpaA10和HpaA143为无效抗原表位.结论 本研究成功地构建了幽门螺杆菌HpaA的T/B联合表位肽噬菌体展示系统.HpaA37和HpaA79,尤其是HpaA116是HpaA有效T/B联合抗原表位.     

丙型肝炎病毒NS5ab区的B细胞模拟表位的确认

目的：用噬菌体展示随机肽库研究丙型肝炎病毒(HCV)NS5ab区的B细胞表位。方法：在原核系统中表达了HCV NS5ab重组蛋白，亲和层析纯化血清中抗HCV NS5ab的特异多抗，用此多抗来筛选噬菌体递呈随机12肽库，获得HCV NS5ab区的B细胞表位。结果：成功地表达了HCV NS5ab重组蛋白，用此蛋白检验了130份HCV阳性血清，阳性率为36％。在筛选的噬菌体展示随机肽库后，挑取13个阳性克隆测序，有9个序列不同，最终通过噬菌体表位表达的方法确认了3个表位。结论：筛选噬菌体展示随机肽库研究HCV的B细胞模拟表位是可行的。     

幽门螺杆菌尿素酶B亚单位H-2d限制性TH表位的预测与鉴定

目的 鉴定幽门螺杆菌（Hp）尿素酶B亚单位（ureB）的H-2^d限制性TH表位，为研究场表位疫苗奠定实验基础。方法 用RANKPEP软件预测尿素酶B亚单位可能的H-2^d。限制性TH细胞表位，合成表位肽，采用淋巴细胞增殖试验、流式细胞分析及CD4^＋T淋巴细胞增殖试验进行表位鉴定。结果 经软件预测获得7条可能的H-2^d限制性TH细胞表位，多肽合成经分析各表位肽纯度均〉85％，其中3条表位肽U546-561,U229-244,U237-251能够刺激rUreB致敏的BALB／c（H-2^d）小鼠的CD4^＋T淋巴细胞增殖（SI〉2）。结论U546,561,U229-244,U237-251是UreB的H-2^d限制性TH细胞表位，可进一步用于Hp表位疫苗的研究。     

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.     

人类肝素酶B细胞表位的预测和免疫原性鉴定

目的 预测并鉴定肝素酶(heparanase)蛋白B细胞表位免疫原性.方法 以肝素酶蛋白的氨基酸序列为基础,采用DNAStar分析软件以及Bcepred在线二级结构分析工具分析其蛋白二级结构并预测B细胞表位.根据预测结果 ,采用8分支多抗原肽结构合成针对该表位的抗原肽,将后者与通用型T辅助表位人IL-1β线性短肽(VQGEESNDK,氨基酸163～171)联合免疫日本白毛黑眼兔,检测免疫血清效价,鉴定其特异性和免疫原性.结果 软件预测显示,肝素酶蛋白大亚基的第1～15位(MAP1)、第279～293位(MAP2)及175～189位(MAP3)氨基酸序列最可能为其优势B细胞表位.间接酶联免疫吸附试验、免疫印迹及免疫组化分析,证实MAP1、MAP2及MAP3均能诱导机体产生高滴度抗体,但仅MAP1、MAP2抗体具有高特异性,MAP2抗体与肝癌组织的结合力最强.结论 肝素酶大亚基的第1～15位、第279～293位氨基酸为其优势B细胞表位,其中第279～293位氨基酸的免疫原性最强,这为肝素酶多肽抗体及B细胞优势短肽疫苗研制提供了理论依据.     

问号钩端螺旋体属特异性外膜蛋白OmpL1和LipL21抗原表位预测及鉴定

目的 筛选问号钩端螺旋体(简称钩体)属特异性外膜蛋白OmpL1和LipL21有效T和B细胞联合抗原表位,为研制多抗原肽(multiple antigenic peptide,MAP)疫苗提供基础.方法 采用生物信息学方法预测OmpL1和LipL21分子中T和B细胞联合抗原表位.采用PCR扩增候选联合抗原表位片段并分别构建其噬菌体展示系统.分别以rOmpL1或rLipL21、黄疸出血群赖株、钩体患者抗血清为一抗,采用Western blot检测各抗血清与目的表位的免疫反应性及其强度.结果 通过抗原表位预测,选择了高分值的4个OmpLl和2个LipL21联合表位.经扩增获得了预期的各抗原表位片段,各目的表位序列均准确插入噬菌体PⅢ蛋白N端并有效表达.各抗血清均能识别上述6个联合表位.其中LipL21的97～112和176-184表位对任一抗血清均显示相似强度的杂交条带.综合4个OmpL1表位对3种抗血清的不同Western blot结果及其实际意义,杂交信号从强到弱依次为173～191、87～98、297～320和59～78表位.结论 所研究的6个联合表位均分别为LipL21和OmpL1的有效抗原表位,其中LipL21的97～112、176～184和OmpL1的87～98、173～191表位可应用于钩体MAP疫苗研制.     

抗原表位预测的免疫信息学方法研究进展

表位是抗原分子中由特殊的化学基团组成的结构，根据与抗原受体结合的不同可分为B细胞表位和T细胞表位。B细胞表位是抗原中可被B细胞抗原受体或抗体特异性识别并结合的线性片段或空间构象性结构。B细胞表位只有很少的一部分是线性表位，而绝大部分（约90％）是构象性表位。定位构象性表位最直接的方法是分析抗原蛋白-单克隆抗体的结晶复合物，但这个实验耗时很长，而且实验所需的单克隆抗体以及纯抗原蛋白．单克隆抗体的结晶复合物制备都有很大的困难。因此通过生物信息学方法准确预测B细胞表位，不仅有助于基础免疫学研究，而且也有助于疫苗和抗体的研究与开发。     

SARS-CoV相关重组B细胞线性多表位肽的表达及鉴定

目的 利用已筛选到的线性B细胞表位组合得到重组多表位肽,并验证其用作表位肽疫苗的可行性。方法 用搭桥PCR和人工合成DNA方法获得多表位肽编码基因片段,构建原核表达载体表达多表位肽。ELISA法测其与SARS病人血清的交叉反应性及其用于免疫动物获得的多克隆抗血清的效价。Western blot检测抗原抗体结合特异性。中和试验检测中和SARS-CoV效果。结果 成功获得PEP1和PEP2编码基因片段并表达重组多表位肽rPEP1和rPEP2。纯化的rPEP1和rPEP2与病人血清的交叉反应率分别为88.7%和97.2%;与免疫动物血清具有结合特异性,结合效价分别为1×106和5×105;中和SARS-CoV的效率分别为70%和80%。结论 成功获得交叉反应性好、免疫原性强的重组多表位肽rPEP1和rPEP2,为制备多表位肽疫苗提供了实验依据。     

T细胞表位预测研究进展

T细胞表位是指抗原经过抗原提呈细胞加工后，由MHC分子提呈给T细胞受体的短肽。随着分子生物医学和分子免疫学技术的发展，尤其是计算机在生物学中的广泛应用，对T细胞表位可以进行初步预测。本文就T细胞表位预测的分子基础、CTL抗原表位和Th抗原表位预测研究进展作一简要综述。     

Naturally occurring hepatitis B surface gene variants in chronic hepatitis B virus infection: correlation with viral serotypes and clinical stages of liver disease

Virus variants escaping from host immunity may be implicated in the pathogenesis of hepatitis B virus (HBV) infection. In this cross-sectional study, the association was evaluated of the frequency of amino acid variation within the immunogenic epitopes of surface gene with different disease stages of chronic HBV infection. The surface gene of HBV encompassing the a determinant (amino acids 124-148) and the putative HLA class I restricted cytotoxic T lymphocyte (CTL) epitope (amino acids 28- 51) were amplified and directly sequenced in 33 asymptomatic carriers (Group I), 31 patients with chronic hepatitis (Group II), 22 with cirrhosis (Group III), and 36 with hepatocellular carcinoma (Group IV). The amino acid sequences were compared subsequently with the consensus sequences of HBV serotype adw or adr. The frequency of amino acid variation per site per sequence (FEQ) was analyzed by generalized estimating equation with Poisson model after stratification by clinical and virological features. The FEQ was 1.21% overall, and was highest in Group IV patients and in patients above 50 years of age. In contrast, nine Group IV patients aged below 50 years who were infected with serotype adw had an inversely higher FEQ than those above 50; the age effect among hepatocellular carcinoma patients was significantly different from that among non-cancerous patients (P = 0.04). Variation of amino acid clustered within a determinant and CTL epitope for serotype adw but was distributed at random for serotype adr. Mutation hotspots differed between serotypes adw and adr. The FEQ of HBV surface protein is correlated positively with advancing age and severity of liver disease, and certain variants may contribute to the persistence of HBV infection.     

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.     

类风湿性关节炎患者单个核细胞对合成肽反应性研究

目的：寻找与类风湿性关节性相关的Ｔ细胞表位。方法：用４种合成的短肽；ＤＷ１５肽，ＨＳＰ肽，ＧＲＰ肽，ＣⅡ肽，体外刺激从类风关患者外周血，关节液，滑膜中得到的单个核细胞。结果：关节内（关节液＋滑膜）ＭＮＣ对ＧＲＰ肽的反应与正常对照差异显著。关节内ＭＮＣ对ＰＨＡ刺激的反应显著低于患者及正常人外周血ＭＮＣ。结论：ＧＲＰ肽是部分类风关患者关节内Ｔ细胞的Ｔ细胞表位。     

猪血管内皮细胞上人天然抗体识别的抗原表位的研究

为了测定猪血管内皮细胞膜表面可能存在的碳水化合物抗原表位,5个人工合成的糖结合物(还原胺化法)和猪胃粘膜糖肽片断被用于中和人天然抗体.然后再用这类中和之后的人血清与猪血管内皮细胞行补体依赖的细胞毒反应(MTT法),并与牛血清白蛋白对比.有2个糖结合物能部分和人天然素体,它们是:(1)去唾液酸分枝双糖-牛血清白蛋白;(2)Le~a-Le~x钥孔虫戚血蓝素.这些糖结合物均含有Gal(β1,3)GlcNAc或Gal(β1,4)GlcNAc.猪胃粘膜糖肽片断具有较强地抑制人血清细胞毒性的作用,除含有Gal(β1,3)GlcNAc、Gal(β1,4)GlcNAc之外,它的主要结构为GlcNAc(χ1,4)Gal.用钥孔虫戚血蓝素作载体能加强糖结合物中和人天然抗体的作用.提示天然抗体认别成簇状的糖表位结构.     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori.     

幽门螺杆菌黏附素A有效T和B联合抗原表位噬菌体展示和抗原性测定

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori.     

嵌合的βhCG-CTP免疫原性的研究

通过分子改进提高βｈＣＧ－ＣＴＰ避孕疫苗的免疫原性。方法根据分子设计及免疫识别原理,利用蛋白质固相合成技术人工合成一小分子表位嵌合多肽,内含βｈＣＧ－ＣＴＰ上的两个Ｂ细胞表位以及一个源自麻疹病毒融合蛋白Ｔ细胞表位,以紫多肽免疫家兔,以ＷＨＯ的βｈＣＧ－ＣＴＰ疫苗标准品为对照,用ＥＬＩＳＡ测定产生的抗体滴度,比较两者的免疫原性。结果该嵌合多肽免疫家兔后产生的抗体滴度显著高于ＷＨＯβｈＣＧ－ＣＴＰ疫苗     

Identification of a nephritogenic immunodominant B and T cell epitope in experimental autoimmune glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG) can be induced in Wistar Kyoto (WKY) rats by immunization with the non‐collagenous domain (NC1) of the alpha 3 chain of type IV collagen, α3(IV)NC1. In patients with Goodpasture's disease, the major B cell epitope is located at the N‐terminus of α3(IV)NC1. In order to investigate whether B and T cell responses in EAG are directed towards immunodominant peptides within the same region of rat α3(IV)NC1, we immunized WKY rats with recombinant rat α3(IV)NC1 (positive control) and five 15‐mer overlapping synthetic peptides from the N‐terminus of rat α3(IV)NC1: pCol(17–31), pCol(24–38), pCol(31–45), pCol(38–52) and pCol(45–59). Positive control animals immunized with α3(IV)NC1 produced an antibody response directed towards α3(IV)NC1 and pCol(24–38). Splenic T cells from these animals proliferated in response to α3(IV)NC1 and pCol(24–38). No significant antibody or T cell responses were observed to the other peptides examined. Animals immunized with pCol(24–38) developed linear deposits of immunoglobulin G on the glomerular basement membrane, albuminuria and focal necrotizing glomerulonephritis with crescent formation by week 6 after immunization. Circulating antibodies from these animals recognized pCol(24–38) and α3(IV)NC1, and their T cells proliferated in response to pCol(24–38) and α3(IV)NC1. Animals immunized with the other peptides developed no significant immune response to α3(IV)NC1 and no disease. In conclusion, these results demonstrate that a 15‐mer peptide from the N‐terminus of α3(IV)NC1 [pCol(24–38)] is recognized by B and T cells from rats immunized with recombinant α3(IV)NC1, and that the same peptide is capable of inducing crescentic glomerulonephritis. Identification of this immunodominant peptide will be of value in designing new therapeutic strategies for inducing mucosal tolerance in EAG, which may be applicable to patients with glomerulonephritis.     

嗜肺军团菌MompS抗原二级结构分析及表位预测

目的分析嗜肺军团菌MompS抗原的二级结构并预测其B细胞表位,初步判断其活性多肽所在区域。方法联合运用多种方法对MompS的二级结构和表面特性,如亲水性、理化性质、可及性、免疫原性和可塑性等方面进行分析,预测其抗原表位。结果 MompS存在多个潜在抗原表位位点,可能的蛋白质抗原表位分布于三个区域：M1（353-546）、M2（1-215）、M3（216-399）。体外实验证明,所预测抗原表位区域基本能与免疫血清发生抗原特异性反应。结论应用DNA Star和胞外区在线分析软件能够成功预测MompS抗原的B细胞表位,M3区域可用于后续的活性肽表位筛选区域。