自身抗原52 kD-Ro/SSA的序列分析及抗原性预测

目的：分析自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ的序列结构，预测其抗原位点。方法：将自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ的氨基酸序列输入“蛋白质结构预测”软件包，分析蛋白质分子生、表面可及性、柔性，并模拟其二级结构，预测其主要抗原位点。结果：自身抗原５２ｋＤ－Ｒｏ／ＳＳＡ是多抗原位点，其氨基酸序列中１２０￣１３０、３００￣３２０、３６０￣３８０位间抗原性较强。结论：确定５２ｋＤ－Ｒｏ／ＳＳＡ的抗原优势表位，为研     

自身抗原SSB/La抗原优势表位分析

探讨自身抗原Ｌａ的抗原优势表位，为疾病机制的研究提供依据。方法：根据计算机软件进行的蛋白质序列结构分析，采用ＰＣＲ法克隆自身抗原Ｌａ多肽片段的ｃＤＮＡ，定向插入表达载体ＰＧＥＸ－２Ｔ，并且导入大肠杆菌中表达重组事蛋白，用ＧＳＴ亲人事层析柱进行纯化，经免疫印迹法与患者阳性血清进行反应，结果：ＳＳＢ／Ｌａ的抗原优势表位主要存在于１－２８、８０－１０７，１０８１８８，２８３－３３８位。不同病人在不同病程     

自身抗原Ro 52 kd多肽分子cDNA表达克隆的构建及其融合蛋白在大肠杆菌中的表达

采用ＰＣＲ法克隆自身抗原Ｒｏ５２ｋｄ多肽分子的ｃＤＮＡ，定向插入表达载体ＰＧＥＸ－４Ｔ－１，并导入大肠杆菌中表达重组融合蛋白，经免疫印迹法表明，重组融合蛋白具有Ｒｏ５２ｋｄ的抗原性。这为今后对Ｒｏ５２ｋｄ抗原表位的精确定位，分析特定抗原表位与疾病的相关性及制备重组抗原用于临床检测奠定了基础。     

自身抗原Ro52kd多肽分子cDNA表达克隆的构建及其融合蛋白在大 …

采用ＰＣＲ法克隆自身抗原Ｒｏ５２ｋｄ多肽分子的ＣＤＮＡ，定向插入表达载体ＰＧＥＸ－４Ｔ－１，并导入大肠杆菌中组融合蛋白，经免疫印迹法表明，重组融合蛋白具有Ｒｏ５２ｋｄ的抗原性，这为今后对Ｒｏ５２ｋｄ抗原表位的精确定位地分析特定抗原表位与疾病的相关性及制备重组抗原用于临床检测奠定了基础。     

表达人自身抗原SSA/Ro60重组体的构建及鉴定

目的：构建表达人自身抗原ＳＳＡ／Ｒｏ６０的重组体,为研究自身免疫病中的抗原体作用提供物质基础。方法：采用逆转录－ＰＣＲ技术克隆自身抗原ＳＳＡ／Ｒｏ６０ ｃＤＮＡ,定向插入表达载体ＰＧＥＸ－２Ｔ,并经酶切电泳,ＤＮＡ测序鉴定分析。结果：经鉴定,证明成功构建了表达人自身抗原ＳＳＡ／Ｒｏ６０的重组体。结论：ＳＳＡ／Ｒｏ６０表达型重组体可用于自身抗原ＳＳＡ／Ｒｏ６０的大量生产并用于诊断。     

SS—A／Ro抗原分子生物学特性及其临床意义

ＳＳ－Ａ／Ｒｏ抗原为细胞内ＲＮＡ－蛋白质复合体的一种，依赖细胞功能状态的不同分别存在于细胞核和细胞浆内。该抗原主要由连接四种ＲＮＡ（ｈＹ１，ｈＹ３，ｈＹ４，ｈＹ５）的５２ＫＤ和６０ｋＤ蛋白多肽组成。ＳＳ－Ａ／Ｒｏ抗原自身抗体存在于多种结缔组织疾病中，尤其是系统性红斑狼疮（ＳＬＥ）及干燥综合症（ＳＳ）。ＳＳ－Ａ／Ｒｏ抗体可作为新生儿红斑狼疮（ＮＬＥ）的血清学标志，在病理过程中可能起一定作用。ＳＳ－Ａ／Ｒｏ抗原分子克隆的成功不仅为研究该抗原的结构特征提供了新的手段而且提供了大量用于检测ＳＳ－Ａ／Ｒｏ抗体的纯化抗原。     

自身核抗原U1SnRNP70kD多肽分子中U1RNA结合功能区的cDNA克隆及其在大肠杆菌中的表达

本研究采用逆转录－ＰＣＲ技术克隆自身核抗原Ｕ１ＳｎＲＮＰ７０ｋＤ多肽分子中Ｕ１ＲＮＡ结合功能区（Ｕ１ＲＮＡｂｉｎｄｉｎｇｄｏｍａｉｎ，Ｕ１ＢＤ）的ｃＤＮＡ，经ＤＮＡ测序证实以后定向插入原核表达载体ＰＧＥＸ－２Ｔ，进而导入大肠杆菌中表达重组蛋白。免疫印迹法研究表明：９６％（４８／５０）的抗Ｕ１ＳｎＲＮＰ７０ｋＤ抗体阳性血清能够识别该重组蛋白，证实Ｕ１ＢＤ重组蛋白具有Ｕ１ＳｎＲＮＰ７０ｋＤ抗原性，而且Ｕ１ＢＤ是Ｕ１ＳｎＲＮＰ７０ｋＤ多肽上主要抗原表位区域，能够被大多数抗Ｕ１ＳｎＲＮＰ７０ｋＤ抗体阳性血清识别。这为今后对Ｕ１ＢＤ抗原表位精确定位，分析特定抗原表位与疾病的相关性以及制备重组抗原用于临床检测等研究奠定基础。     

自身核抗原U1RNP 70 kD多肽全长基因克隆及鉴定

应用逆转录ＰＣＲ方法从Ｈｅｌａ细胞中成功地克隆了编码自身核抗原Ｕ１ＲＮＰ７０ｋＤ多肽的全长基因，将此基因重组入原核表达载体系统ＰＧＥＸ－２Ｔ中，以限制性酶切及核苷酸测序鉴定扩增的ＤＮＡ片段，经计算机分析，基因全长１．３ｋｂ，编码４３７个氨基酸，并进行了基因同源性比较。结果表明，克隆得到的基因与Ｇｅｎｂａｎｋ报道的完全一致，蛋白质一级结构分析表明编码的７０ｋＤ多肽在羧基片段有３个可能的抗原性区域，该多肽的抗原表位可能主要存在于羧基半段上，为重组蛋白的表达及抗原表位的定位研究奠定基础。     

自身核抗原U1SnRNP 70kD多肽分子中U1RNA结合功能区的cDNA克隆…

本研究采用逆转录－ＰＣＲ技术克隆自身核抗原Ｕ１ＳｎＲＮＰ ７０ｋＤ多肽分子中Ｕ１ＲＮＡ结合功能区的ｃＤＮＡ，经ＤＮＡ测序证实以后定向插入原核表达载体ＰＧＥＸ－２Ｔ，进而导入大肠杆菌中表达重组蛋白。     

大肠癌相关抗原CA-Hb3的提取、亲和层析纯化及其生物学特性

方法：应用ＴｒｉｔｏｎＸ１００和正丁醇分别提取了大肠癌组织粗提抗原和大肠癌细胞株ＨＲＴ－１８的ＣＢＥ（正丁醇提取物），二者均有抗原活性。抗原经抗大肠癌单抗Ｈｂ３－亲和层析进一步纯化，获得纯大肠癌相关抗原ＣＡ－Ｈｂ３。结果：经鉴定，ＣＡ－Ｈｂ３为糖蛋白，其抗原活性与糖链有关。其分子量随变性程度而异，变性完全（１００℃巯基乙醇作用５ｍｉｎ）时为５０ｋＤ，而变性不完全（１００℃３ｍｉｎ）约２７ｋＤ，故推测该抗原是由两个亚基（约２７ｋＤ）组成的同源二聚体。结论：在ＳＤＳ－ＰＡＧＥ鉴定蛋白质分子量时，使用不同变性时间处理样品可研究其亚基组成     

The evolution of red blood cell and lymphocyte Ro/SSA.

Recent studies have demonstrated that the Ro/SSA autoantigen is heterogeneous as is the corresponding autoimmune response. In addition the autoimmune responses is highly species specific and preferentially reactive with the human antigen. Quantitative ELISA study shows that red blood cell Ro/SSA evolves much more rapidly than lymphocyte Ro/SSA and Western Blot analysis shows that the quantitative ELISA results are mirrored by changes in the 60 kD Ro/SSA molecules but not the 52 kD and 54 kD Ro/SSA molecules. The 52 kD and 54 kD Ro/SSA molecules seem to be relatively conserved as indicated by the Western immunoblotting experiments. These studies add weight to the concept that the antigenic epitopes of these related proteins are under the control of separate genes which have undergone different rates of evolution.     

Autoantibodies to the Ro/SSA antigen are conformation dependent. I: Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous and the autoanti-Ro/SSA response is correspondingly heterogeneous. There are two isoform families; the 60 kD forms and the 52 kD forms. We studied the antigenic difference between the native and denatured Ro/SSA isoforms and found that the autoanti-Ro/SSA response to the native 60 kD antigen is quite homogeneous. All anti-Ro/SSA sera recognize the native kD antigen regardless of the reactivities to the 60 kD band on the Western blot. Surprisingly, no anti-Ro/SSA sera without anti-La/SSB reacts with the native 52 kD Ro/SSA, although sera with both precipitating anti-Ro/SSA and anti-La/SSB can immunoprecipitate the native 52 kD antigen. Anti-Ro/SSA sera exist which react exclusively with the native 60 kD Ro/SSA protein (10/43, 23%) while no anti-Ro/SSA sera have been found which react exclusively with the denatured 52 kD Ro/SSA antigen. In sera with anti-Ro/SSA precipitins alone, only antibody to the denatured 52 kD Ro/SSA molecule is found! In sera with anti-Ro/SSA and anti-U1 RNP precipitins, no antibody to either native or denatured 52 kD Ro/SSA is found, while in sera with both anti-Ro/SSA and anti-La/SSB precipitins, antibodies to both the native and denatured forms of 52 kD Ro/SSA are present. These data suggest that the anti-Ro/SSA response to the 60 kD molecule is driven by the native 60 kD Ro/SSA molecule while the molecular identification of the antigen drive in the anti-52 kD Ro/SSA response is unknown.     

Sjögren's syndrome: autoantibodies to cellular antigens. Clinical and molecular aspects

Autoantibodies to cellular autoantigens are usually found in sera of patients with systemic autoimmune rheumatic diseases. Patients with Sj?gren's syndrome (SS) frequently present autoantibodies to both organ and non-organ-specific autoantigens. The most commonly detected autoantibodies are those directed against the ribonucleoproteins Ro/SSA and La/SSB. The presence of the antibodies in SS is associated with early disease onset, longer disease duration, parotid gland enlargement, higher frequency of extraglandular manifestations and more intense lymphocytic infiltration of the minor salivary glands. Over the past several years, the structure and function of these autoantigens have been extensively studied. Several centers, using different techniques, have investigated the B cell epitopes on the protein components Ro 60 kD, Ro 52kD, and La 48 kD. Finally, increased evidence of direct involvement of anti-Ro/SSA and anti-La/SSB autoantibodies in the pathogenesis of tissue injury has been contributed by several studies.     

A calreticulin-like protein co-purifies with a '60 kD' component of Ro/SSA, but is not recognized by antibodies in Sjögren's syndrome sera.

In this study, we used human tonsils for the isolation of the 60 kD component of the Ro/SSA autoantigen, following the method described by Wu et al. (J Immunol Methods 1989; 121:219-24). Western blot analyses were carried out using Ro/SSA-reactive human Sjögren's syndrome sera, to follow the autoantigen through the purification procedure. A 60 kD Ro/SSA component was eluted as a broad peak from a Mono Q column. Within this peak, a much more abundant protein, co-migrating with the Ro/SSA component on SDS-PAGE, was also eluted. The more abundant protein was further purified on a Superose 12 column and its N-terminal sequence was shown to be identical to that of human calreticulin. The 60 kD Ro/SSA autoantigen was also further purified on the Superose 12 column and was eluted as an asymmetric peak, with the majority being eluted at a position corresponding to 60 kD, whereas the calreticulin-like protein was eluted from the same column as an apparent dimer of approximately 120 kD. A panel of five Ro/SSA-reactive human sera reacted with the purified Ro/SSA antigen, but not with the calreticulin-like protein. Therefore, it is clear that the calreticulin-like protein is not a Ro autoantigen and is distinct from the 60 kD Ro/SSA antigen. As the calreticulin-like protein is a much more abundant protein than the 60 kD Ro/SSA component, its co-purification with the autoantigen on ion-exchange and its close migration with the autoantigen on SDS-PAGE may explain why peptide sequences for human calreticulin were derived from apparent 60 kD Ro/SSA antigen preparations.     

Genetic knock out of 60kD Ro (or SSA), a common lupus autoantigen, induces lupus

Systemic lupus erythematosus (SLE) could have the most complicated phenotype and the most diverse genetics of any genetically complex human disease. Recent research shows that genetic ablation of the gene encoding 60kD Ro, a common autoantigen for SLE patients, results in an SLE-like illness in Ro-/- mice. These data add to the expanding cooperation between mouse and human SLE genetic studies and are the first in which ablation of an autoantigen induces an autoimmune disease.     

Advances in B-cell epitope analysis of autoantigens in connective tissue diseases

The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.     

Autoantibodies to the Ro/SSA autoantigen are conformation dependent. II: Antibodies to the denatured form of 52 kD Ro/SSA are a cross reacting subset of antibodies to the native 60 kD Ro/SSA molecule.

The immune response to the Ro/SSA particles is conformation dependent. In sera with only anti-Ro/SSA precipitins, the autoantibodies to the 60 kD Ro/SSA are largely to the native 60 kD Ro/SSA while autoantibodies to the 52 kD Ro/SSA particle when present are exclusively to the denatured 52 kD Ro/SSA particle. Antibodies eluted from a recombinant 52 kD Ro/SSA fusion protein reacted in a sandwich ELISA which only measures antibody to native 60 kD Ro/SSA antigens and this reaction is largely inhibited by native homogeneous 60 kD Ro/SSA. In addition, antibody binding to the 52 kD Ro/SSA antigen in Western blot is also strongly inhibited by native 60 kD Ro/SSA. These experiment strongly suggest that reactivity of denatured 52 kD Ro/SSA antigen represents a cross reaction with autoantibodies directed to the native 60 kD Ro/SSA antigen. As a corollary of these experiments, data are presented that suggest the hY-RNAs are not associated with the 52 kD Ro/SSA protein but only with the 60 kD Ro/SSA protein. These data are consistent with the hypothesis that the autoanti-Ro/SSA response is driven by native 60 kD Ro/SSA and the immune response to denatured 52 kD Ro/SSA is largely a cross-reactive subset of the immune response to native 60 kD Ro/SSA.     

Heterogeneity of the Ro/SSA antigen and autoanti-Ro/SSA response: evidence of the four antigenically distinct forms.

Precipitating antibodies to the Ro/SSA antigen occur in the sera of 40% of patients with sytemic lupus erythematosus (SLE) and in 40–70% of the sera of patients with primary Sjögren's syndrome. Previous work has shown that lymphocyte extracts contain two Ro/SSA antigens with protein moieties of 60 kD and 52 kD and that erythrocyte haemolysate contain two analogous but antigenically distinct Ro/SSA molecules of 60 kD and 54 kD. Frequency analysis of the various specificities in 43 sera with precipitating anti-Ro/SSA and studies with affinity-eluted antibodies suggest that the lymphocyte 60 kD and erythrocyte 60 kD Ro/SSA molecules are related as are the lymphocyte 52 kD and erythrocyte 54 kD Ro/SSA proteins. Anti-Ro/SSA sera when accompanied by other precipitins (anti-La/SSB and anti-U1RNP) react preferentially with certain Ro/SSA isoforms. Evidence is also presented for a 45 kD form of Ro/SSA. These data suggest that the antigenic heterogeneity of the Ro/SSA antigen is immunologically relevant and that there are two families of Ro/SSA antigens: one comprising of the two 60-kD proteins in the erythrocyte and lymphocyte and the other the erythrocyte 54 kD and lymphocyte 52 kD Ro/SSA proteins, respectively.     

New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sj?gren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.