HBV前S_1蛋白检测研究初报

<正> 乙型肝炎病毒前S基因可以为前S_1和前S_2肽段编码,后者含有HBV白蛋白受体且被证明和病毒的活动性复制有关。最近证明前S_1和前S_2蛋白对于诱导HBV的免疫应答有重要意义。对前S_1蛋白的研究最近方有进展,国外已证明了其和HBV DNA具有十分密切的关系,并且可能和病毒对肝细胞的吸附直接有关。为了探索前S_1蛋白的临床意义,静安区中心医院临床免疫研究室用抗前S_1单克隆抗体MQ19/7(西德Gott-     

抗聚合人血清白蛋白的抗独特型抗体

一种针对兔抗聚合人血清白蛋白抗体(anti-PHSA,Ab_1)的抗独特型抗体(anti-anti-PHSA,Ab_2),已通过免疫同种动物制成。该Ab_2具有以下特性:(1)它既可与兔anti-PHSA反应,也可与鼠的单克隆的anti-PHSA反应。(2)它与鼠的单克隆的anti-PHSA的反应,可受到PHSA和乙型肝炎病毒(HBV)上的聚合人血清白蛋白受体(PHSA-R)的抑制。(3)它可在异种动物体内诱导产生具有anti-PHSA活性的抗-抗独特型抗体(anti-anti-anti-PHSA,Ab_3),而且在收获的Ab_2血清中也查见了Ab_3。本研究不仅表明了针对PHSA的免疫应答中独特型网络调节的客观存在,而且在其抗独特型抗体(anti-Id)中,的确存在具有PHSA内映像的成分。     

抗HBV前S_2蛋白单克隆抗体的制备及其相应抗原的免疫组化定位研究

本文报告用聚合人血清白蛋白(PHSA)亲和层析柱纯化具有前S_2抗原活性的HBsAg颗粒作为免疫原,通过常规融合、筛选和克隆化,获得3株分泌抗前S_2蛋白单克隆抗体杂交瘤细胞系(E8、C9和F3)。3种单克隆抗体对肝组织的免疫组化试验表明,前S_2抗原在肝炎肝硬化和肝癌周围组织中均有不同程度的表达,其染色表型,E8呈局灶胞浆型,C9为弥漫胞浆型,F3为膜型。     

HBV-前S_2抗原和抗体在乙型肝炎中的临床意义

本文用酶免疫法检测了HBV-前S_2抗原和抗体在乙型肝炎患者血清中的变动。结果表明,在急性HBV感染时,病程一月内前S_2抗原的阳性效为86.9％,抗前S_2抗体的阳性率为27.3％;在病程1～6个月间,前S_2抗原的阳性率降至34.7％,抗前S_2抗体的阳性率升至60％。前S_2抗原在HBeAg和HBV-DNA阳性血清中的检出率(84.2％)明显高于在HBeAg和HBV-DNA阴性血清中的检出率(20.0％),而抗前S_2抗体则相反。在慢性活动性肝炎时,前S_2抗原的检出率为80％,在慢性迁延性肝炎时的检出率为82.6％,抗前S_2抗体在慢性乙型肝炎病人血清中的检出率均很低。本文结果提示前S_2抗原的表达与HBV病毒的复制相关,而抗前S_2抗体的出现可作为预示HBV感染恢复的指标。     

多聚人血清白蛋白受体检测及其临床意义

多聚人血清白蛋白受体(polymeried human serum albumin receptor简称PHSA-R)具有种属特异性，存在于人类乙型肝炎病毒(HBV)颗粒(又称Dane颗粒)表面，也存在于肝细胞膜上。PHSA-R具有HBV DNA长链上的前-S2基因编码。PHSA-R与多聚人血清白蛋白(PHSA)的结合是通过氢键，疏水相互作用等非特异性结合。在HBV侵入宿主肝细胞，并在肝细胞复制过程中PHSA起着桥梁作用。     

HBV-前S_2抗原单克隆抗体制备、特性和应用

用HBsAg制备了一株分泌乙型肝炎病毒前S_2抗原单克隆抗体(MAb)的杂交瘤细胞株(1E_2),小鼠腹水中MAb效价达10~7。1E_2MAb有很强的抑制多聚人血清白蛋白(pHSA)和HBsAg中前S_2抗原结合的能力;与重组痘菌病毒表达的中分子蛋白(含S和前S_2抗原)而不与小分子蛋白(含S抗原)反应;与人工合成的前S_2肽段(N端12O-146氨基酸序列)特异性结合。应用1E_2MAb的酶免疫法检测乙肝病毒感染者血清中前S_2抗原,表明前S_2抗原滴度和HBsAg滴度有较好的相关性,在EBeAg阳性血清中更为明显;HBeAg阳性血清前S_2抗原的阳性率和几何平均滴度明显高于HBeAg阴性/HBsAg阳性血清。     

HBV上PHSA受体与循环中PHSA结合情况研究

乙型肝炎病毒(HBV)是环状部分双股的DNA病毒,它在许多特性上不同于其它已知的人类病毒。目前尽管在分子水平上进行了比较深入的研究,但关于HBV的嗜肝机理至今没有得到令人满意的解释。1979年Imai等证实了HBV上有一种与人血清中老化白蛋白分子(PHSA)结合的受体,Thung等于1982年证实肝细胞表面也有PHSA受体。因此不少学者提出HBV上的PHSA受体通过PHSA的桥介导作用以达到嗜肝的目的。为了研究PHSA在HBV嗜肝过程中的桥介导作用,作者应用ELISA法着重检测了血循环中PHSA与HBV的结合情况,现简要报告如下。     

乙型肝炎病毒前S2蛋白与病毒复制标志的关系

乙型肝炎病毒(HBV)感染时,血清前S_2蛋白(Pre-S_2)与其病情转归及病毒复制的关系,近年来已成为HBV研究的新课题。为进一步了解Pre-S_2与HBV复制指标的关系,我们采用酶联免疫吸附试验(ELISA)检测126例乙肝患者活动期与恢复期血清Pre-S_2抗原,同时与血清乙肝标志物、HBV-DNA、多聚白蛋白受体(PHSAr)和谷丙转氨酶(ALT)进行比较,结果报告如下。     

乙型肝炎患者前S_2抗体检测的意义

<正> 乙肝病毒(HBV)前S_2蛋白(Pre-S_2)与HBV的感染和复制有密切关系,而抗前S_2抗体(Pre-S_2Ab)则认为与HBV的清除和疾病恢复有关。我们采用ELISA抑制法进行了检测,并探讨其有关意义。     

乙肝表面抗原（HBsAg）在哺乳动物细胞中表达研究的回顾与展望

哺乳动物细胞表达的乙肝表面抗原(HBsAg),可分泌至胞外,正确糖基化,已成为理想的表达系统,其中提高表达水平后选择合适的表达体系是当前这一领域的研究热点。可采用强启动子及扩增HEsAg基因拷贝数的方法提高整合性载体系统的HBsAg基因的表达水平;非整合性基因载体(如BPV)也广泛应用于HBsAg基因的表达中;利用热休克蛋白启动子hsp70及昆虫细胞多角体蛋白启动子表达HBsAg是最新的研究进展;牛痘病毒载体是制备HEsAg的一个独特的研究方向。由于pre—S_2编码的55氨基酸顺序台聚人血清白蛋白(PHSA)受体,对病毒的消除起关键作用;另外还含抗原决定簇,与S-抗原的抗原决定簇同时免疫效果更好,因此制备含PHSA受体与HBsAg的多肽是今后乙肝疫苗制备的发展方向。除重组乙肝疫苗外,第三代的乙肝疫苗——合成肽疫苗,以及第四代的乙肝疫苗——抗独特型抗体疫苗,其研究工作正在迅速展开。     

用RIA法研究血循环中PHSA和AAA的存在情况

作者采用敏感的RIA法,分别检测179例人血清中PHSA和85例血清中的AAA。结果表明,血循环中的确存在PHSA,阳性检出率为11.7％,HBsAg阳性组和阴性组有显著性差异。AAA在血清中也有不同程度检出。作者根据检测情况,对弄清血循环中是否存在PHSA的意义,以及与Thung等提出的在HBV上的PHSA受体与血循环中PHSA结合再嗜肝的假说之关联等进行了讨论,并指出了用McAb在RIA检测方法中应注意的问题。     

JZ-1,一株分泌抗HLAI类抗原轻链单克隆抗体的小鼠杂交瘤系

β_2微球蛋白对研究主要组织相容性复体(MHC)Ⅰ类抗原的分子结构及其免疫学功能具有重要作用。但它的分子量小,免疫原性弱,较难获得相应的单克隆抗体。本文系国内首次报道,用PHA刺激的人外周血淋巴细胞免疫小鼠。应用杂交瘤技术及极限稀释技术,从21株抗淋巴细胞克隆中筛选出一株分泌抗HLA Ⅰ类抗原轻链——人β_2M单抗的细胞株,命名为JZ-1。 JZ-1细胞株目前已培养8个月,分泌抗体稳定。它所分泌的抗体能与人β_2M专一性反应;反应滴度甚高,可达10~(-6);能封闭其它试剂对β_2M的反应;它与不同群体淋巴细胞的反应格局符合经典的β_2M分布格局;此外JZ-1单抗是一种补体结合性抗体,它对MLR及PHA淋巴细胞转化反应是有明显的抑制功能。     

正常人肝细胞HBV PreS2相关抗原的研究

经斑点免疫印迹试验和Western Blot,用抗P31和抗PreS120-145研究了HBV包膜蛋白抗原同正常人肝细胞膜抗原的关系。结果表明:肝膜纯化物能与抗P31及抗PreS120-145反应,其中抗PreS120-145只能识别肝膜抗原中的两条蛋白带(123KD,68KD)。提示在正常人肝细胞膜上存在有HBV-PHSAr的相关抗原,这种抗原在参预HBV嗜肝过程和乙型肝炎的免疫病理损伤中可能起重要作用。     

人不同来源T细胞对丝裂原、PMA和A23187的反应性比较

本文比较了胎儿胸腺细胞、正常成人外周血Ｔ细胞及儿童扁桃体Ｔ细胞对丝裂原ＰＷＭ、ＰＨＡ和ＣｏｎＡ、ＰＫＣ激活剂ＰＭＡ和Ｃａ２＋导入剂Ａ２３１８７的反应性。结果表明：胎儿胸腺细胞对ＰＷＭ的反应对ＰＨＡ和ＣｏｎＡ的反应性很弱；正常成人外周血Ｔ细胞对ＰＨＡ的反应性最强，对ＰＷＭ和ＣｏｎＡ的反应性较弱；儿童扁桃体Ｔ细胞对３种丝裂原的反应性均很强；ＰＭＡ单独作用能强烈诱导儿童扁桃体Ｔ细对正常成人外周血Ｔ细胞及胸腺细胞作用较弱；ＰＷＭ和ＰＭＡ对胎儿胸腺细胞及正常成人外周血Ｔ细胞均有明显协同作用，但对儿童扁桃体Ｔ细胞无协同作用；Ａ２３１８７单独作用对３种Ｔ细胞的增殖作用均很弱，Ａ２３１８７对ＰＷＭ诱导的增殖均有部分抑制作用。这些结果对于认识处于不同发育阶段及不同部位的的活化途径和功能有一定意义。     

Binding of mitogenic plant lectins to human lymphocytes. Flow cytometric analysis

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.     

Lymphocyte proliferative responses in haemophiliac patients: relations to clinical and immunological findings

Blood lymphocyte proliferative responses to mitogens were studied in 65 patients with haemophilia (haemophilia A: 54 patients, haemophilia B: 11 patients) in parallel with 39 male control subjects. As a group, patients with haemophilia did not demonstrate abnormal proliferative responses to phytohaemagglutinin (PHA), Concanavalin A (ConA) and pokeweed mitogen (PWM) when compared with healthy controls. When the patients were analysed according to their seropositivity for antibody to human immunodeficiency virus (HIV), those who were positive had significantly decreased PHA, ConA and PWM responses. Haemophiliac patients with T4+/T8+ ratios less than 1 had reduced proliferative responses to PHA, ConA and PWM when compared to patients with ratios greater than 1. No significant difference in mitogen responses were found when the patients were analysed according to the presence or absence of palpable lymphadenopathy. Those patients with haemophilia A who had received more than 5 x 10(4) units of factor VIII during the two years preceding the study showed no significant difference in PHA, ConA and PWM responses when compared to patients receiving less.     

Inhibitory activity of monoclonal antibody F35.25 on the interaction between hepatocytes (HepG2 cells) and preS1-specific ligands.

The capacity of a preS1-specific monoclonal antibody (McAb) F35.25 to block the attachment of preS1-specific ligands to human hepatoma HepG2 cells was studied. In order to define more precisely the fine epitope specificity of McAb F35.25, its reaction with synthetic peptides derived from the preS1 sequence (12-53) was investigated. McAb F35.25 was found to recognize better synthetic peptide preS(21-47) from the adw 2 and ayw sequences than the synthetic peptide preS(32-53) adw 2. The shortest sequence recognized by McAb F35.25 among the peptide sequence studied was preS(32-47). The corresponding amino acid sequence (for HBV subtype adw 2) is PAFGANSNNPDWDFNP. As expected, it was found that McAb F35.25 inhibited the attachment of HepG2 cells to HBsAg-cellulose, as well as to preS(21-47)-cellulose, corresponding to two HBV subtypes adw 2 and ayw. Finally, the inhibitory effect of different peptides on the interaction of McAb F35.25 with HBsAg particles containing the preS1 sequence was also studied. The peptide preS(12-47) appeared to be the most effective inhibitor. Therefore, the McAb F35.25 is specific for the sequence preS1(X to 47), where (12 less than or equal to X less than 32). These results indicate that McAb F35.25 is probably virus-neutralizing and represents a reagent of great value to study the interaction between HBV and hepatocytes independently of d/y subtype changes.     

Discrimination of hepatitis B virus (HBV) subtypes using monoclonal antibodies to the PreS1 and PreS2 domains of the viral envelope.

We report the production and characterization of murine anti-PreS2 and anti-PreS1 monoclonal antibodies (mAb) and demonstrate their utility in discriminating hepatitis B virus (HBV) subtypes. On the basis of Western blotting and reciprocal competition binding to HBV virions, at least five distinct epitopes have been identified in the PreS domain: two within the PreS1 region and three within the PreS2 region. All PreS2 mAb bind M protein (gp33 and gp36) but only one group binds strongly to M and L proteins (p39 and gp42). This group determinant was mapped to peptide residues 120-145. The second group bound to an endoglycosidase F-sensitive epitope which is defined by a mannose-rich glycan at ASN 123 in the PreS2 region. The third group was mapped to peptide residues 150-174 and was reactive with the M envelope proteins but not L or S proteins on Western blots. All PreS1 mAb bind L protein but not M protein on Western blots. Using these mAb, HBV subtype assays were developed allowing evaluation of the Paris (1975) HBsAg subtype panel members along with other HBsAg-positive specimens. All Paris subtype members (except ayw2 and ayw3) could be easily distinguished by differential PreS2 mAb reactivity. The Paris subtypes, adw2, adw4, and adr, could be classified as distinct groups by PreS2 and PreS1 mAb binding. Specimens from Hong Kong and the United States classified as adw2 in the S region fell into two groups based on PreS2 mAb binding: one having reactivity similar to Paris adw2 subtype and the other having identical reactivity to Paris ayw1 subtype. Furthermore, some specimens classified as adr in the S region gave similar reactivity to the Paris ayr subtype in the PreS2 and PreS1 regions. One complicating factor in this approach toward subtyping was the discovery that some HBsAg positive sera may contain factors which block PreS epitopes. Grouping of HBV subtypes by PreS1, PreS2, and S mAb reactivity may allow better correlation with groupings based on HBV DNA sequence homology.     

人CD137单抗诱导不同状态T细胞增殖和凋亡的研究

为了研究一种新的T细胞共刺激分子—CD137对不同状态T细胞增殖和凋亡的双重调节作用。采用3 H TdR掺入法测定T细胞增殖 ,用流式细胞仪测定细胞凋亡。结果显示 :(1)CD137单抗可与T细胞表面的CD137抗原结合 ,明显增强PHA刺激T细胞增殖 ,使T细胞增殖指数较PHA单独作用高 2～ 3倍 ,但CD137单抗单独不能刺激T细胞增殖 ;(2 )对于慢性活化的T细胞 ,CD137单抗可协同PHA诱导T细胞凋亡 ,使T细胞凋亡率从PHA单独作用的 19 2 0 %增加到 36 31% ,CD137单抗单独并不能诱导慢性活化T细胞凋亡。CD137单抗一方面可协同PHA刺激静止状态T细胞的增殖 ,另一方面可协同PHA诱导慢性活化T细胞凋亡 ,对T细胞起双重调节作用。