塑料毛细管空气热循环PCR法获取人脾Ro,La多肽基因片段

利用塑料毛细管通过空气热循环PCR获取了人脾Ro和La多肽基因片段。该法高效省时，节省试剂，操作安全简便。     

Ro52蛋白的分子生物学研究进展

Ro52蛋白作为靶抗原,存在于多种自身免疫性疾病,如干燥综合征、系统性红斑狼疮、皮肌炎等患者的血清中。Ro52蛋白是TRIM蛋白家族成员(TRIM21),其分子内含有RING-finger、B-box、卷曲螺旋结构域,分别具有不同的生物学功能。Ro52蛋白在免疫防御中的作用日渐为研究者所关注,如Ro52与IgG分子的作用、Ro52作为一种E3泛素连接酶与其他信号分子的作用等。其自身泛素化和广泛的泛素化作用已经在Ro52与干扰素的作用中得到了体现。迄今,Ro52在自身免疫性疾病中的具体致病机制还不清楚。我们尝试通过介绍Ro52的分子生物学特点及其与相关活性分子的相互作用的最新研究进展,初步探索其可能的致病机制。     

核糖核蛋白SSA/Ro的结构及其特性

从组织细胞中提取的核糖核蛋白抗原SSA/Ro被广泛应用于临床中各种自身免疫性疾病检测,特别在检测SS、SLE、NLE等病人的血清中阳性率较高。因而在临床诊断上尚有十分重要的参考价值。本综述了SSA／Ro抗原的结构、生物特性方面的研究情况,并对其今后的研究进行了展望。     

猪脾转移因子对La Sota株鸡新城疫弱毒疫苗的免疫增强作用

【目的】本文旨在研究猪脾转移因子（TF）对新城疫（ND）病毒弱毒疫苗La Sota株的免疫增强作用及机理,为兽医临床防控提供理论依据。【方法】以4种不同剂量La Sota疫苗株分别单独、与TF联合免疫SPF鸡,14 d后以ND病毒（NDV）参考强毒F₄₈E₉株(10^4.7 ELD₅₀)进行攻毒,采用蛋白质芯片技术测定鸡外周血IL-6、IL-10、IL-16和IL-21浓度,并以血凝抑制（HI）试验和荧光定量RT-PCR方法分别检测鸡ND HI抗体效价和F₄₈E₉株的病毒血症水平。【结果】攻毒保护试验表明,单独免疫10^5.17、10^4.17、10^3.17和10^2.17 EID₅₀剂量时疫苗攻毒保护率分别为100%、55%、0%和0%,半数保护量(PD₅₀)为12 023 EID₅₀;对应剂量联合免疫时疫苗攻...     

纯化,测定植物中细胞分裂素的免疫亲和层析法和酶联免疫法

近年来微量的植物激素的定量分析方法有很大发展,Weiler将放射免疫技术引入细胞分裂素的测定,许多研究者对植物激素的免疫测定做了改进。细胞分裂素的酶联免疫吸附分析(ELISA)具有灵敏度高、特异性强、不需要昂贵设备、无放射性污染等优点,日益为各实验室采用。如何简化植物样品的纯化步骤,提高检测的选择性和灵敏度仍然是细胞分裂素免疫测定的限制因素。本     

猪脾和马脾铁蛋白理化特性的比较

Ｈ＾＋,ＯＨ＾－均能参与猪脾和马脾铁蛋白铁核组成,迫使它们分别释放铁核中对酸碱不稳定的铁组份。在可见光谱中,猪脾和马脾铁蛋释放铁的动力学过程可分为一级快速反应和零级慢速反应,但猪脾铁蛋白释放铁一级反应速度明显大于马脾铁蛋白释放铁的一级反应的速率,推测这些现象均与各自蛋白的蛋白壳自身调节能力有着密切联系。     

人整合素α4(CD49d)β7的纯化、检测及有关应用的讨论

使用亲和层析的方法,对人整合素α4β7进行纯化,通过ELISA检测尤其是对于免疫小鼠应答的监测,相应改变免疫策略,从讨论中获取正确的指导思路和适当的试验方法。在人整合素β7和抗体的研制方面取得了显著的进展。     

尿激酶型纤溶酶原激活剂单克隆抗体的生产及其在抗原纯化中的应用

从ＣＨＯ工程细胞培养上清初步纯化的ｕＰＡ免疫ＢＡＬＢ／ｃ小鼠,通过杂交瘤技术制备单克隆抗体细胞株,取其中一株３８－１－７株作高密度大量培养,细胞密度达１３．２×１０６／ｍＬ时,抗体滴度为１∶６１．４４×１０４。用自制的ｕＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱纯化抗体。抗体滴度提高２４３倍。纯化后的抗体与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ珠交联,制成ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析抗体柱,亲和力常数：１．２８×１０９（ｍｏｌ／Ｌ）－１,交联率：８３．５％。直接从培养上清纯化ｕＰＡ,纯度为９６．３％,回收率：８１．６％±１９,纯化倍数：５０倍左右,比活１．１１±０．２９×１０５。试验结果表明该法效果好,方法简单、操作方便、值得进一步研究和应用。     

一种分离人血浆脂蛋白(a)的简便方法

短时间超速离心(4h)结合抗人 apo(a)免疫亲和层析分离纯化了人血浆 Lp (a),所得制品经聚丙烯酰胺凝胶电泳和 double-decker 火箭免疫电泳等鉴定为纯品.与国外常规分离 Lp (a)的方法相比,该法具有简便,经济,提纯周期短和 Lp (a)纯度高等优点,得率提高一倍以上.     

Isolation of melatonin by immunoaffinity chromatography

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.     

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

Activation of the T‐cell receptor (TCR) and that of the B‐cell receptor (BCR) elicits tyrosine‐phosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large‐scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine‐phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine‐phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine‐phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.     

Ubiquitination of Ro52 autoantigen

Anti-Ro/SSA antibodies are antinuclear antibodies most commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52/SSA (52 kDa), whose function is still unknown. In this study, the ubiquitination of Ro52 was investigated. We found that Ro52 was strongly conjugated by a single molecule of ubiquitin in cells. Although the biological relevance of this mono-ubiquitination was not defined, the function of Ro52 might be modified by the mono-ubiquitination. We also found that Ro52 was conjugated with poly-ubiquitin chain in cells (poly-ubiquitination), suggesting that Ro52 may be downregulated by the ubiquitin-proteasome pathway in vivo. Interestingly, sera from patients with Sj?gren's syndrome showed heterogeneity in their reactivity to poly-ubiquitinated Ro52, probably because of their differing antigenic determinants. This heterogeneity of the reactivity might be associated with the varying clinical features found in patients with Sj?gren's syndrome.     

免疫亲和层析在纯化可提取性核抗原SSB/La中的应用

生化提纯高纯度有生物活性的蛋白质一直是一项艰巨的工作.主要综述了免疫亲和层析的纯化特点,以及用此方法在分离纯化具有临床诊断价值的可提取性核抗原SSB La中的有效应用.     

大连蛇岛蝮蛇类凝血酶在大肠杆菌中的表达与纯化

将编码大连蛇岛蝮蛇类凝血酶 (Gloshedobin)的基因克隆于表达载体pET-32a( + )中 ,以融合蛋白形式在大肠杆菌中获得表达。在 2 5℃下经 1mmol/LIPTG诱导 6h ,SDS-PAGE和蛋白质印迹分析表明 ,部分融合蛋白以可溶形式存在于大肠杆菌的细胞质中。针对金属螯合亲和层析分离某些含His-标签重组蛋白质时专一性不高 ,并且存在配基泄漏的缺陷 ,设计合成了以抗重组类凝血酶的鸡卵黄免疫球蛋白为配基的免疫亲和层析柱。通过疏水色谱OctylSepharoseFF ,IgY免疫亲和层析以及强阴离子交换色谱SourceQ等三步柱色谱分离纯化获得比活力为 454.7U/mg的重组蛇毒类凝血酶 ,活力回收率为 34.8%。蛋白质印迹分析和纤维蛋白原凝结活性分析表明 ,该表达产物具有相应的免疫活性和酶活性