幽门螺杆菌UreB抗原表位预测及其有效表位噬菌体展示的筛选

目的 筛选幽门螺杆菌（Hp）尿素酶B亚单位（UreB）分子中的抗原表位，为研制多抗原肽（MAP）疫苗奠定基础。方法 采用生物信息学技术对UreB的T细胞和B细胞表位进行预测和分析。构建ureB基因原核表达系统，Ni-NTA亲和层析法提纯目的重组表达产物rUreB，常规皮内免疫法制备兔抗血清。选择UreB主要T细胞和B细胞联合表位肽并构建其噬菌体展示系统，PEG／NaCl沉淀法提纯重组噬菌体，SDS-PAGE鉴定目的重组PⅢ蛋白（rPⅢ）。分别以商品化抗跏全菌IsG和rUreB抗血清为一抗，采用Western blot对上述表位肽进行鉴定和筛选。结果 与GenBank中相关序列比较，所克隆的ureB基因核苷酸和氨基酸相似性分别为96％～99．5％和96％～100％。rUreB表达量约为细菌总蛋白的52％，提纯后仅见单一蛋白条带。预测的4个主要表位肽UreB230、UreB322、UreB479和UreB527在M13噬菌体中获得成功表达，采用不同一抗的Westernblot均显示相似的阳性结果，但UreB322和UreB527反应强度明显高于UreB230和UreB479。结论 本研究成功地构建ureB基因高效原核表达系统和UreB主要T细胞和B细胞联合表位肽噬菌体展示系统。所采用的生物信息学技术可有效地预测抗原表位。UreB322和UreB527是UreB有效抗原表位，可作为幽门螺杆菌MAP疫苗的候选表位。     

大肠杆菌重组LTKA63突变体和LTB黏膜免疫佐剂活性的研究

目的构建大肠杆菌不耐热肠毒素A亚单位（heat-labile enterotoxin subunit A,LTA）突变体LTKA63及B亚单位（heat-labile enterotoxin subunit B,LTB）基因原核表达系统,确定rLTKA63和rLTB黏膜免疫佐剂活性.方法采用高保真PCR从大肠杆菌44815株基因组DNA中扩增LTA和LTB基因,采用定位突变技术构建LTKA63突变体,T-A克隆后测序.构建LTKA63和LTB原核表达载体,采用SDS-PAGE鉴定表达产物.采用三色流式细胞术确定rLTKA63激活T细胞途径,以GM1-ELISA检测rLTB结合牛GM1的能力.建立幽门螺杆菌SS1株小鼠感染模型,分别以幽门螺杆菌（Hp）重组尿素酶B亚单位（rUreB）和黏附素（rHpaA）为抗原,分别检测rLTKA63和rLTB对rUreB和rHpaA的免疫保护增强作用.结果从大肠杆菌44815株DNA模板中扩增获得预期大小的LTA和LTB基因条带.LTA基因定位突变后获得序列正确的LTKA63突变体.rLTKA63和rLTB表达量分别占细菌总蛋白的30%和20%左右.LTKA63作用后TH1和TH2细胞较阴性对照组分别增加19.9%和42.3%,GM1-ELISA结果证实rLTB能与牛GM1结合.33.3%（4/12）rUreB和41.7%（5/12）rHpaA免疫小鼠胃黏膜标本中分离出幽门螺杆菌,且rUreB和rHpaA特异性S-IgA检测结果均为阴性.rUreB或rHpaA加用rLTKA63或rLTB免疫后,小鼠胃黏膜标本中幽门螺杆菌分离阳性率下降为16.7%～25.0%（P＞0.05）,rUreB和rHpaA特异性S-IgA阳性率可达41.6%～58.3%（P＜0.01）.结论成功地构建了LTKA63和LTB原核表达系统,所表达的rLTKA63和rLTB均具有较强的黏膜免疫佐剂活性.     

重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及黏膜佐剂活性研究

目的　对大肠杆菌不耐热肠毒素B亚单位(LTB)进行基因克隆、重组表达、蛋白纯化和黏膜佐剂活性分析。方法　应用PCR技术从产肠毒素大肠杆菌基因组中扩增出不耐热肠毒素B基因,将此构建于原核表达载体pET 11C,并在大肠杆菌BL21(DE3)中表达,然后采用D(+) immobilizedgalactose亲和层析柱对重组蛋白进行纯化。以纯化后重组LTB为佐剂,与幽门螺杆菌重组尿素酶B亚单位蛋白(rUreB)共同口服和滴鼻免疫BALB c小鼠,分析重组LTB的黏膜佐剂活性。结果　PCR扩增出390 bp LTB基因,与GenBank公布的核苷酸序列的相似性为99.5%。重组LTB蛋白的表达率为6%,以胞周质分泌形式表达,经亲和层析后蛋白纯度大于95%。该重组蛋白保持了与神经节苷脂GM1结合的生物学活性和良好的免疫原性及免疫反应性。动物试验表明,重组LTB与rUreB抗原共同口服和滴鼻免疫小鼠后的特异性血清IgG及鼻腔、气管、胃黏膜和小肠黏膜sIgA水平显著高于单独rUreB抗原免疫组(P<0.05)。结论　所获得的重组LTB具有较好的黏膜佐剂活性,为其在黏膜疫苗中的应用奠定基础。     

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.     

乙型肝炎病毒多表位抗原DNA疫苗的免疫原性

目的 :探讨HBV复合多表位DNA疫苗诱导体液及细胞免疫的可行性。方法 :将HBV多表位抗原基因BPT克隆到真核表达载体pcDNA3.1中 ,构建重组真核表达载体pcDNA3.1/BPT。将其通过肌肉注射免疫BALB/c小鼠 ,用间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验 ,检测特异性体液免疫和细胞免疫的水平 ,并观察其对免疫小鼠的毒副作用。结果 :用重组质粒pcDNA3.1/BPT免疫BALB/c小鼠后 ,在效靶比为 10 0∶1时 ,可诱导显著地特异性CTL应答 (P <0 .0 5 )。ELISA检测免疫小鼠血清特异性IgG的水平明显升高 (P <0 .0 5 )。在BPT基因原核表达蛋白的刺激下 ,免疫小鼠脾淋巴细胞增殖显著 (P <0 .0 5 )。RT PCR分析表明 ,IL 12mRNA的水平亦明显升高。结论 :HBV多表位基因疫苗可诱发特异性免疫应答 ,为预防、治疗性HBV疫苗的研制提供了一定的实验依据     

HCV多表位抗原融合肽的表达与抗原性的研究

目的观察HCV复合多表位抗原融合肽的抗原性,探讨利用这种抗原融合肽制备的抗体在丙型肝炎抗原检测方面的潜在可行性。方法将人工合成的HCV复合多表位抗原基因B2克隆到表达载体pThioHis A中,并在大肠杆菌中表达融合蛋白(Trx-B2),纯化该蛋白后免疫小鼠及家兔,分析表达产物的免疫特异性及抗原性。结果融合蛋白(Trx-B2)能在原核表达系统中高效表达,并可诱发小鼠及家兔产生高滴度的特异性HCV抗体。结论偶联了融合蛋白的HCV复合多表位抗原具有良好的抗原性,其免疫实验动物后产生的抗体有望用于HCV的抗原检测。     

HCV复合多表位抗原基因的克隆表达及其免疫学特性分析

目的构建丙型肝病炎病毒（HCV）截短C基因和多表位基因重组原核表达质粒，表达纯化融合蛋白，分析其免疫原性和抗原性。方法PCR方法扩增核心区羧基端部分缺失的基因片段Ct；合成HCVE2区模拟表位与NS3～NS57个表位基因Em；分别将Ct、Em克隆人原核表达质粒pQE30，筛选阳性重组质粒pQE30-CtEm，转化E．coli M15，IPTG诱导融合蛋白表达，薄层扫描分析表达蛋白；可溶性分析后用Ni^2＋-NTA凝胶亲和层析柱纯化、透析并浓缩融合蛋白；Westernblot分析纯化蛋白的特异性和抗原性；纯蛋白免疫小鼠后分析其免疫原性。结果成功构建了HCV复合多表位抗原基因的原核表达质粒pQE30-CtEm，目的基因可高效表达，表达产物主要以包涵体形式存在，Ni^2＋-NTA纯化可获得目的蛋白，纯化蛋白具有良好的抗原性和免疫原性。结论HCV复合多表位抗原基因融合蛋白可高效表达并得到纯化，该融合蛋白可作为HCV诊断抗原，也为丙型肝炎新型疫苗的研究提供了靶抗原。     

旋毛虫Ts87抗原单克隆抗体的制备及其识别肽段的研究

将原核系统成功表达的Ts87重组蛋白纯化后免疫动物,利用淋巴细胞杂交瘤技术制备抗Ts87抗原的单克隆抗体.通过ElISA、免疫印迹、免疫组化等方法筛选出能分泌抗Ts87御抗原的抗体的阳性克隆,在1000个融合的杂交瘤细胞中,有3株融合细胞分泌的单克隆抗体(2A2,5A3,6G12)鉴定结果为阳性.这3株融合细胞分泌的抗体均能识别Ts87重组蛋白、旋毛虫成虫和幼虫的Ts87抗原以及旋毛虫幼虫组织切片.获得的抗Ts87抗原的单克隆抗体在将来的研究中可作为旋毛虫病的诊断试剂.为了进一步鉴定抗体识别的相应抗原表位和模拟抗原表位,选择了其中一株单克隆抗体5A3筛选噬菌体十二肽库.噬菌体M7展示的肽段是抗体识别Ts87抗原的线性表位,其他的噬菌体克隆展示的肽段是Ts87抗原的模拟表位.该技术为旋毛虫病多表位疫苗的构建奠定了基础.     

幽门螺杆菌GroEL蛋白重组表达及表达产物小鼠免疫保护作用

目的 检测幽门螺杆菌临床菌株groEL基因序列差异,确定重组表达的GroEL蛋白(r-GroEL)抗原性、免疫反应性以及对幽门螺杆菌感染小鼠的免疫保护性.方法 采用PCR及测序法了解幽门螺杆菌临床菌株groEL基因序列.构建幽门螺杆菌groEL基因pET42a-E.coli BL21DE3原核表达系统,采用SDS-PAGE及凝胶图像分析系统确定rGroEL表达量,Ni-NTA亲和层析法提纯rGroEL.采用ELISA和Western blot检测rGroEL的抗原性和免疫反应性,采用幽门螺杆菌全菌为包被抗原的ELISA确定GroEL膜定位.采用幽门螺杆菌小鼠感染模型了解rGroEL免疫保护作用.结果 35株幽门螺杆菌临床菌株groEL基因序列相似性高达99.4％～ 100％.rGroEL表达量可达细菌总蛋白的56％,SDS-PAGE后提纯的rGroEL在凝胶中显示为单一的条带.rGroEL可诱导家兔和小鼠产生高效价IgG抗体,同时也能被幽门螺杆菌全菌抗体(Hp-IgG)或rGroEL-IgG识别并与之结合.GroEL是幽门螺杆菌表面蛋白抗原.50、100或200 μg rGroEL免疫可分别使50.0％ (6/12)、75.0％ (9/12)和91.7％ (11/12)小鼠免于幽门 螺杆菌SS1株的感染,200 μg rGroEL免疫保护率(91.7％)显著高于50μg rGroEL(50.0％)( P＜0.05).结论 幽门螺杆菌GroEL蛋白是序列保守、具有良好免疫原性和免疫保护性的表面抗原,可作为基因工程疫苗候选抗原.     

西尼罗病毒多表位基因的融合表达及其免疫保护作用研究

目的研究西尼罗病毒（WNV）多表位基因的融合表达及其免疫保护作用。方法利用生物信息软件Biosun分析西尼罗全基因序列，确定表位基因片段并选择合适片段连接构成多表位基因。采用重叠PCR方法扩增该基因，构建多表位基因的原核重组表达载体pET-43a-M，进行融合表达和纯化。将表达蛋白加弗氏佐剂免疫小鼠并进行攻毒试验，观察其保护作用。结果分子克隆和构建了原核重组表达质粒pET-43a-M，表达和纯化了融合蛋白Nus-M，该蛋白免疫后的小鼠能够部分抵御西尼罗病毒的攻击。结论构建的西尼罗病毒多表位基因能够在原核细胞内表达，表达产物有较弱的免疫保护反应，为该病毒多表位抗原的串联及多表位疫苗研究提供了试验资料，但需要进一步改进。     

Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children

Because 21 immunized children (13%) among the 162 confirmed Japanese encephalitis (JE) cases during 1986–1991 occurred in Taiwan, we collected 320 serum samples from Taiwan children aged 15–31 and 27–44 months immediately before the 1st dose (n = 41) and 1–3 months after the 2nd dose (n = 78, 27 pairs), and immediately before (n = 58) and 1–3 months after the 3rd dose (n = 143,44 pairs) to determine neutralization antibody (Nt Ab) against the Nakayama (N) and Beijing-1 (B) strains and two Taiwan wild type JE viruses (JEV): CC-27 and CH-1392. Our Nt results showed that (1) B vaccine stimulated a better homologous Ab response than N vaccine for Nt Ab seropositivity rate (NASR), produced a higher level of Nt titer after the primary immunization [2 doses = 100% vs. 91%, geometric mean titer (GMT) = 115 vs. 22], had a greater booster effect (3 doses: 100% vs. 95%; GMT = 320 vs. 33), and showed a better capability to neutralize two local Taiwan JEV strains, particularly only after 3 doses (ave. NASR for B vs. N = 90% vs. 10%; and GMT for B vs. N = 154 vs. 1); (2) the two wild type JEV strains had different plaque morphology and antigenic variation and the CC-27 strain was not neutralized as well as the CH-1392 strain after 3 doses of vaccine (BBB or NNN or NNB); and (3) 30% of the children had lost JEV Nt Ab one year after the 2nd dose of N vaccine and natural infection with JE virus did occur among those children after immunization. In conclusion, (1) three doses of mouse-brain vaccine are the minimum requirement to protect children against the local Taiwan JEV; (2) the best strain for a JE vaccine depends on level of Nt Ab it induced, the molecular epidemiology and antigenic variation of the JEV in each local area; and (3) future vaccine must produce better B- and T-cell memory. © 1994 Wiley-Liss, Inc.     

Mumps virus strains isolated in Croatia in 1998 and 2005: Genotyping and putative antigenic relatedness to vaccine strains

Two mumps virus strains 9218/Zg98 and Du/CRO05 were isolated in two locations in Croatia in 1998 and 2005, respectively. Genetic characterization of these temporally distinct mumps virus isolates was carried out in order to determine their genotype and putative antigenic relatedness to mumps virus vaccine strains. Sequence analysis of the small hydrophobic (SH) gene revealed that isolate 9218/Zg98 shows less than 95% of similarity to any reference strain, thus representing a potential reference strain for a new genotype. Isolate Du/CRO05 clearly belongs to genotype G with the 97% of homology to the reference strain Glouc1/UK96. When compared to each other, the two Croatian strains have extremely low level of homology of only 89% indicating no relatedness between them. Putative antigenic properties of the HN protein of these two isolates were compared to different vaccine strains. The results reveal a higher level of homology of antigenic determinants to non-A genotype vaccine strains than to A genotype vaccine strain.     

人嗜铬颗粒蛋白A抗原结构的研究

人嗜铬颗粒蛋白A(CgA)是存在于许多神经内分泌细胞和内分泌细胞的分泌颗粒内的酸性蛋白质.我们通过CgA多抗、单抗和人CgA合成多肽间的结合反应研究人CgA的线性抗原位点,结果表明合成多肽CgA1-20、47-67、107-158、254-297、331-375、395-439和CgA25-46、163-210、231-253、298-314呈现无抗原性或弱抗原性.而合成多肽CgA68-106、222-230、315-330和376-394则呈现强抗原性.两株人CgA鼠源单抗B4E11和A11识别的氨基酸残基分别为CgA68-70(GAK)、CgA81-90(GFEDELSEVL).我们的研究将有助于选择针对CgA片段特异性的抗体,而这些抗体则可应用于CgA分子的结构和功能的研究,或提供对CgA阳性的内分泌和神经内分泌肿瘤进行体内外诊断的新方法.     

Antigenic structure of phytohemagglutininstimulated mouse lymphocytes studied with the help of specific antiserum

Experiments with antiserum against intact and phytohemagglutinin (PHA)-transformed lymphocytes showed that a new antigenic determinant (or determinats), not present on the surface of intact lymphocytes or those stimulated briefly (2 h) by PHA, appears on the surface of mouse lymphocytes subjected to prolonged (68 h) PHA stimulation. Meanwhile on the surface of lymphocytes stimulated for a long time by PHA there is a decrease in the number (or density) of antigenic determinants present on the surface of intact lymphocytes.Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 836–838, July, 1976.     

HLA-DR-positive cells in the human amniochorion

Human amniochorion has been studied by indirect immunofluorescence using a monoclonal antibody to a monomorphic determinant of HLA-DR antigens. Positive staining was noted only on significant numbers of large cells with irregular cytoplasmic processes in the mesenchymal connective tissue layer of the membranes. These cells resemble the dendritic-type, strongly HLA-DR-positive cells described by Steinman [3] and may be particularly relevant in immunobiological events at this important materno-foetal interface.     

Molecular epidemiology of chronic hepatitis B virus infection in Greece

Virological data on chronic hepatitis B virus (HBV) infection in Greece are limited. HBV genotypes, surface antigen (HBsAg) subtypes, and HBsAg “a” determinant mutations among patients infected chronically with HBV, were investigated. Serum samples from 135 HBsAg positive patients were tested. Serologic (HBsAg, anti‐HBs, HBeAg, and anti‐HBe), virologic (HBV‐DNA quantitation) and biochemical markers (serum alanine aminotransferase/ALT and aspartate aminotransferase/AST) were analyzed. HBV genotypes and HBsAg subtypes were determined by partial sequencing of the S gene. Genotyping was performed by using the National Center for Biotechnology Information online Genotyping tool and phylogenetic analysis. Nucleotide sequences were aligned pair wise with ClustalW and phylogenetic trees were constructed by the neighbor‐joining method. Sequences were also used to predict HBV HBsAg subtypes. In six patients (4%), simultaneous presence of HBsAg and anti‐HBs was determined, whereas 47 patients (35%) were HBeAg positive, 84 (62.5%) were anti‐HBe positive, and four patients (3%) were characterized by the simultaneous presence of HBeAg and anti‐HBe. Mean ALT was 238 IU/L (standard deviation = 576.84), and HBV‐DNA levels ranged from 1.02 × 10⁵ to 2.2 × 10⁷ IU/ml. Genotype D was predominant (98%), with viral groups D/ayw2 (73%) and D/ayw3 (27%). Group A/adw accounted for 1% of cases. Genotypes B and C were found exclusively in the Chinese immigrants (1%). Single or multiple point mutations were found in 35 cases (26%). Some of the most common mutations occurred at amino acid positions 129, 133, 134, 144, 145, including the “vaccine escape” mutation G145R. Mutations analysis revealed that amino acid substitutions did not affect detection by commercial immunoassays. J. Med. Virol. 83:245–252, 2011. © 2010 Wiley‐Liss, Inc.     

A filter paper sandwich method using small volumes of reagents for the detection of antigens electrophoretically transferred onto nitrocellulose

A method is described which allows antigens electrophoretically 'blotted' onto nitrocellulose to be probed using small volumes (less than 0.3 ml) of antibody solutions. Reagent volumes required for this method are more than 10-fold lower than the minimum volumes attainable using either custom made or commercial incubation trays. The procedure is simple and economical and should be applicable to any situation where it is desirable to either conserve or reduce the amount of screening reagent used.