筛选日本血吸虫童虫早期诊断抗原的研究

目的筛选日本血吸虫童虫早期诊断抗原及其表位.方法制备日本血吸虫可溶性童虫抗原(SSA),经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot后分别与日本血吸虫感染后10、21、42天兔血清进行酶联免疫印迹试验(EITB),筛选出能被早期感染血清所识别的抗原组分.用日本血吸虫感染后21天血清从噬菌体12随机肽库中筛选、富集、纯化、扩增与早期感染兔血清有高亲和力的噬菌体克隆,经动物实验初步评估其对日本血吸虫感染的早期诊断效果.结果SDS-PAGE显示SSA的总条带数多于可溶性成虫抗原(SAWA),但大多为共同抗原.EITB结果显示SSA共出现12条早期反应抗原带,其中97 kDa和47 kDa抗原分子只能被感染后10天兔血清特异性识别;SAWA仅出现7条反应带,未显示只能被感染后10天兔血清识别的期特异性抗原分子.挑选出3个与感染后21天兔血清具有高亲和力的单克隆噬菌体,混合后分别用于检测感染日本血吸虫10、21 d鼠血清中IgG特异性抗体,阳性检出率分别为48.8%和64.4%.结论SSA中的97 kDa和44 kDa抗原分子及3个噬菌体随机12肽抗原模拟表位可能具有日本血吸虫感染早期诊断价值.     

胃癌MG7-Ag模拟表位减毒鼠伤寒沙门菌活菌疫苗研制及免疫效果

目的以减毒鼠伤寒沙门菌为疫苗载体,构建基于胃癌MG7-Ag模拟表位的口服活菌疫苗,观察Balb/c小鼠口服免疫后诱导的体液和细胞免疫反应。方法构建MG7-Ag模拟表位和HBcAg融合基因的原核表达载体,将其转入减毒鼠伤寒沙门菌得到模拟表位的口服活菌疫苗。用构建的口服鼠伤寒沙门菌疫苗免疫Balb/c小鼠,以PBS和携带pYA3341空载体的减毒鼠伤寒沙门菌作为对照。初次口服接种6周后应用ELISA法检测小鼠血清中抗MG7-Ag抗体的滴度。初次免疫后8周时,取免疫鼠脾细胞,以51Cr释放法检测小鼠脾淋巴细胞对靶细胞的杀伤效果,用表达MG7-Ag的小鼠艾氏腹水瘤细胞进行肿瘤攻击实验,观察疫苗对小鼠的保护作用。结果口服疫苗免疫小鼠后疫苗免疫组小鼠血清抗MG7-Ag抗体较PBS和空载体对照组显著增高,三组分别为0.954±0.040,0.653±0.018和0.692±0.012(P<0.01)。各组小鼠脾淋巴细胞体外杀伤实验未见显著差异,三组分别为234.7±27.7,183.4±26.0和195.7±8.0(P>0.05)。小鼠艾氏腹水瘤的攻击实验显示:疫苗免疫组中5只小鼠有1只未见肿瘤形成,而对照组小鼠则全部成瘤,且免疫组的成瘤重量为(0.05±0.01)g,明显小于两对照组的(0.10±0.04)和(0.09±0.04)g(P<0.01)。结论以胃癌MG7-Ag模拟表位为基础的口服减毒鼠伤寒沙门菌疫苗具有免疫原性,可诱导小鼠产生抗肿瘤免疫,并具有一定的保护作用。     

人白细胞介素-36α蛋白的二级结构及其B细胞抗原表位的预测

目的预测并分析人白细胞介素-36α蛋白的二级结构及其潜在的B细胞抗原表位。方法以人IL-36α蛋白的氨基酸序列为基础,采用生物信息软件和互联网服务器预测人IL-36α蛋白的二级结构及其亲水性、可及性和柔韧性,根据预测结果综合分析IL-36α蛋白可能的B细胞抗原表位。结果IL-36α蛋白潜在的B细胞抗原表位位于N端8～12、33～38、111～116和143～149氨基酸区段。结论预测了IL-36α蛋白的二级结构和B细胞抗原表位,为进一步研究IL-36α的生物学活性部位及利用合成肽制备抗IL-36α的多克隆抗体提供了理论依据。     

猴B病毒囊膜蛋白gD的B细胞表位预测及鉴定

目的鉴定猴B病毒囊膜蛋白gD的抗原表位。方法利用生物信息学分析猴B病毒囊膜蛋白gD的二级结构、亲水性、表面可及性、抗原性指数以及柔韧性,得到可能的表位肽段;再利用合成肽与B病毒阳性血清进行ELlSA验证,评价表位肽段的特异性和敏感性。结果在异D蛋白上预测得到7个表位肽段;ELISA测定结果显示,4个肽段（序列分别为^46 LPPLEQKTD^54、^106 RGAPEATRSDA^126、^291291PELAPEERGTSRFPGD＾３０６和＾３６１AVYLVRRRGR＾３７０可与B病毒阳性血清发生免疫学反应,敏感性在40％～70％之间。结论B病毒gD蛋白上至少存在4个线性化表位。     

幽门螺杆菌多表位重组原核表达工程菌的构建及表达特性研究

目的 构建含有幽门螺杆菌尿素酶（UreI、UreB）及粘附素（HpaA）的多表位原核表达质粒pET28a(+)/ureI-ureB-hpaA〔pET28a(+)/IBA〕及相应的原核表达工程菌,研究其表达特性。 方法 通过生物信息学方法从ureI 、ureB和 hpaA基因中筛选T细胞和B细胞优势表位基因序列,人工合成并构建原核重组表达质粒pET28a(+)/IBA。经限制性内切酶（Nde Ⅰ,Xho Ⅰ）酶切鉴定及DNA测序鉴定后导入大肠杆菌BL21 (DE3) 。该工程菌经IPTG诱导后,用SDS-PAGE检测重组蛋白（rIBA）的表达情况,以抗幽门螺杆菌悉尼株（SS1株）特异卵黄抗体采用Western blot检测rIBA的抗原性。 结果 构建的多表位原核表达质粒pET28a(+)/IBA双酶切和测序鉴定与设计序列100%一致。工程菌经 IPTG 诱导后SDS-PAGE电泳显示在相对分子质量40×10 ³左右有一条明显蛋白条带,Western blot显示有相应位置特异反应条带。 结论 成功构建幽门螺杆菌多表位重组原核表达质粒pET28a(+)/IBA及相应原核表达工程菌,该工程菌表达的rIBA具有抗原性,表明该重组蛋白与幽门螺杆菌有高度相关性。     

Monoclonal Antibodies to Human Renin: Properties and Applications

A series of 11 different monoclonal antibodies generated against human kidney renin have been characterised. Their binding affinity, inhibition of renin activity, epitope distribution, crossreactivity with related enzymes and finally in vivo pharmacological effects were analysed. All antibodies were found to be specific for primate renin recognising 6 independent antigenic structures on the renin molecule. They expressed different effects on renin activity namely (1) no inhibition, (2) only partial, or (3) complete inhibition. Partially inhibiting antibodies demonstrated specific degrees of inhibition (30, 60 or 80%). One antibody, R-36-16, demonstrated an IC 50 of 1.3 × 10^?11 M/L and, when injected into marmosets, induced complete inhibition of plasma renin activity and reduction of blood pressure. Using a selected pair of antibodies a radioimmunoassay has been established providing a fast and highly reproducible determination of human and marmoset immunoreactive renin, detecting both active and inactive renin down to concentrations of 10 pg/ml (1.25 × 10^?17 moles of renin per 50 μl sample).     

肺吸虫模拟抗原诊断价值及序列分析

目的　探讨噬菌体展示肺吸虫模拟抗原的诊断价值并分析与天然抗原的同源性。方法　建立肺吸虫噬菌体模拟抗原的ELISA ,对肺吸虫病人、日本血吸虫病人、旋毛虫病人及健康者血清进行检测 ,分析其敏感性和特异性 ;提纯噬菌体模拟肽的DNA模板进行序列测定 ,以Blast软件分析其序列同源性。结果　肺吸虫模拟抗原的 6个阳性克隆中P5、P6、P8、P13、P16的阳性、阴性预测值及诊断效率均为 10 0 % ,P7的阳性、阴性预测值及诊断效率分别为 10 0 %、78.9%、86.7%。上述 6个克隆有 2个克隆的DNA序列完全相同 ,即 6个克隆包含了 5种不同的抗原表位 ;Blast分析表明 5个表位与已知的肺吸虫抗原表位无DNA序列的同源性。结论　肺吸虫模拟抗原对肺吸虫病具有一定的诊断价值 ,这 5种表位可能从空间结构上模拟了天然抗原表位。     

Epitope mapping of anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation of the Transmembrane 4 Superfamily

CD53 is a pan-leukocyte glycoprotein which is a member of the recently described Transmembrane 4 Superfamily (TM4SF) of membrane proteins that are predicted to span the lipid bilayer four times. The major hydrophilic region of murine CD53 was expressed as a glutathione-S-transferase fusion protein, and the epitopes of four mouse anti-rat CD53 monoclonal antibodies (mAb) (OX-44, 2D1, 6E2 and 7D2) were mapped to this region using mouse/rat chimeric fusion proteins. The epitopes of OX-44,6E2 and 7D2 are restored by the substitution of a single isoleucine residue for threonine at position 154 in the mouse protein. The 2D 1 epitope is non-linear and appears to require the juxtaposition of isoleucine at position 154 with one or more of the amino acids arginine (132), methionine (133) and serine (140). All of these epitopes are shown to be sensitive to reduction, thus indicating the importance of disulfide bonding in the correct folding of the CD53 hydrophilic domain. Moreover, as these four mAb recognize CD53 at the cell surface, the data provide direct molecular evidence for the proposed membrane orientation of the TM4SF.     

Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display

Yersinia pestis plasminogen activator (Pla), a surface virulence factor contributes to the highly invasive nature of the pathogen by binding various tissue matrix components. In this study we characterised the laminin-binding site(s) of Pla via phage display and alanine-scanning mutagenesis. Previously we isolated 18 different heptamer peptide sequences from a phage display library with biopanning on laminin, and have shown that two of them with sequences of WSLLTPA or YPYIPTL completely inhibited laminin binding of a Pla-expressing recombinant Escherichia coli strain. These phages themselves strongly bound laminin in an ELISA assay utilising horseradish peroxidase-labelled anti-M 13 antibody. In the present study, with the application of synthetic peptides, a 55% and a 33% inhibition was demonstrated using WSLLTPA and YPYIPTL, respectively. Peptide pattern alignment showed two homologous regions for WSLLTPA and one for YPYIPTL inside the Pla sequence. Amino acids were changed for alanine in one of the WSLLTPA regions and in the YPYIPTL region in order to prove the role of the LTP/PTL motifs in laminin binding. Of the four constructed mutants, the triple mutant L65A-T66A-L67A in the WSLLTPA region and the double mutant G178A-L179A in the YPYIPTL region decreased the laminin binding capacity of the Pla-expressing recombinant E. coli by about 50%.     

Characterization of Factor VIII Inhibitors

Factor VIII (FVIII) inhibitors develop as either alloantibodies against exogenous FVIII in patients with congenital hemophilia A after FVIII-replacement therapy or as autoantibodies against endogenous FVIII in previously healthy, nonhemophilic individuals. The predominant immunoglobulin G (IgG) subclass of FVIII inhibitors is IgG(4). The main epitopic regions are known to be located, however, in the A2, A3, and C2 domains. The A2 and A3 epitopes have been identified between amino acid residues 484 and 509 and residues 558 and 565, respectively. Both of these regions are close to the binding sites for activated FIX (FIXa). Two regions have been identified in the C2 domain, one in the amino-terminal portion of the domain (residues 2181-2243) and the other in the carboxy-terminal portion of the domain (residues 2248-2312 and residues 2315-2330). In addition, a crystallographic analysis of a complex of the C2 domain and a human monoclonal IgG(4)(K) Fab revealed that this type of antibody is in direct contact with hydrophobic and basic residues of the membrane-binding surface. Inactivated FVIII is rapidly cleared from the circulation in the presence of inhibitors. The inhibitors also bind to essential FVIII ligand proteins, including von Willebrand factor, FIXa, FXa, and thrombin, and to surface membrane phospholipid. Some type 2 inhibitors interfere with binding to activated protein C.