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SSA/Ro-52kD抗原优势表位分析

探讨自身抗原ＳＳＡ／Ｒｏ－５２ｋＤ的抗原优势表位,为疾病机制的研究提供依据,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ＳＳＡ／Ｒｏ－５２ｋＤ多肽片段的ｃＤＮＡ,定向插入表达载体ＰＧＥＸ－５Ｘ－１,并且导入大肠杆菌中表达重组融合蛋白,用ＧＳＴ亲和层析柱进行纯化,经免疫印迹法与病人阳性血清进行反应,结果表明片段Ｒｏ５２３具有较强的抗原性,这提示ＳＳＡ／Ｒｏ－５２ｋＤ的抗原优势表位     

干燥综合征特异性α-胞衬蛋白的抗原表位筛选及鉴定

目的：筛选和鉴定干燥综合征特异性α-胞衬蛋白（α-Fodrin）的抗原表位.方法：采用蛋白质计算机分析软件预测α-胞衬蛋白的抗原决定簇,设计8条重叠蛋白片段序列,应用GST融合表达系统原核表达该系列融合蛋白.通过Western blot实验检测融合蛋白片段的抗原性,并对抗原性片段以外的序列片段进行融合表达和检测,最终确定抗体所识别的部位.结果：8条重叠融合蛋白片段均有抗原性,最小片段以外序列的表达片段检测不具有抗原性.结论：α-胞衬蛋白的第1～59位氨基酸是其抗原表位区域,为进一步研究α-胞衬蛋白在干燥综合征中的作用机制和应用奠定了基础.     

人Fas抗原表位预测

采用可及性和可塑性方案,结合抗原性方案、二级结构方案及综合位点预测方案对人Ｆａｓ抗原的Ｂ细胞表位进行预测,结果显示：人Ｆａｓ抗原Ｂ细胞表位可能位于Ｎ－末端残基５１－７８,１０２－１１０,１４５－１５７等区域内或附近。由于残基１０２、１２０位有２个潜在的Ｎ－糖基化位点（Ａｓｎ－Ｘ－Ｓｅｒ／Ｔｈｒ）,对表位的形成有一定影响,人Ｆａｓ抗原的Ｂ细胞表位可能位于５１－７８,１００－１５７区域内。     

人CD80—Linker—SEA重组毒素的设计及生物学特性预测

目的：构建人重组ＣＤ８０－Ｌｉｎｋｅｒ－ＳＥＡ毒素的真核表达载体并预测Ｌｉｎｄｅｒ的合理性和可行性。方法：应用核酸和蛋白质序列分析软件ＧｏｌｇＫｅｙ对融合基因及Ｌｉｎｋｅｒ部位翻译后在二级结构水平上的一些生物学特性,诸如柔性、抗原性、亲水性及表位等加以预测。结果：ＣＤ８０－Ｌｉｎｋｅｒ－ＳＥＡ融合基因的氨基酸序列经软件分析,并分别与ＣＤ８０与ＳＥＡ单独的分析结果相比较,发现在二级结构的水平未出现新的抗原性,同时在Ｌｉｎｋｅｒ部位具有很低的抗原性,亲水性没有改变,Ｌｉｎｋｅｒ部位呈中性,ＣＤ８０和ＳＥＡ具有原来各自的表位特征,无新的表位出现。结论：本研究通过计算机软件对ＣＤ８０－Ｌｉｎｋｅｒ－ＳＥＡ融合蛋白的预测,并与ＣＤ８０、ＳＥＡ各加以对比分析和比较,以利于在重组过程中有一个合理的设计,有可能最大程度的保留Ｃ     

当前流行的乙型流感病毒血凝素蛋白抗原性及其基因特性

通过抗原性分析弄清了近年来连续２次乙型流感病毒在我国人群中造成流感流行，是由于乙型流感病毒株发生抗原性变异所造成。通过毒粒基因分析发现，我国人群中流行的Ｂ／Ｐａｎａｍａ／４５／９０类毒株，其ＨＡ１区氨基酸序列比国际代表性毒株Ｂ／Ｐａｎａｍａ／４５／９０病毒在１６５位点上多一个氨基酸，乙型流感病毒流行株可能具有地区性差异。     

丙型肝炎病毒包膜糖蛋白高变区1多抗原肽设计及？…

目的 应用多抗原肽（ＭＡＰ）研究丙型肝炎病毒包膜糖蛋白高变区１（ＨＶＲ１）的抗原性。方法 根据已经获得的ＨＣＶ－ＢＪ株Ｅ２／ＮＳ１区氨基酸序列,参照国内外得所报道的ＨＶ ＨＶＲ１序列及抗原性参数,设计并合成含ＨＣＶ ＨＶＲ１３９０－４１１ａａ序列２２个氨基酸的线性表位多肽（以ＬＰ表示）及ＭＡＰ（对称８分枝）,分别以ＬＰ和ＭＡＰ免疫Ｂａｌｂ／Ｃ小鼠及家兔,比较其免疫原性。结果 ＭＡＰ免疫原性明显强于     

当前流行的乙型流感病毒血凝素蛋白的抗原性及其基因特性

通过抗原性分析弄清了近年来连续２次乙型流感病毒在我国人群中造成流感流行，是由于乙型流感病毒株发生抗原性变异所造成。通过毒粒基因分析发现，我国人群中流行的Ｂ／Ｐａｎａｍａ／４５／９０类毒株，其ＨＡＩ区氨基酸序列比国际代表性毒株Ｂ／Ｐａｎａｍａ／４５／９０病毒在１６５位点上多一个氨基酸（天冬酰胺），乙型流感病毒流行株可能具有地区性差异。     

肠毒素性大肠杆菌CS3菌毛呈现载体的构建

目的 构建大肠杆菌ＣＳ３菌毛呈现载体,实现外源表位在细菌表面的呈现。方法 通过对ＣＳ３亚基蛋白二级结构、抗原表位、亲水性及柔韧性的预测分析,确定外源表位的插入位点,重叠延伸ＰＣＲ方法进行定点突变,将口蹄疫病毒ＶＰＩ插入到ＣＳ３中以验证表现呈现能力;用重组菌腹腔注射免疫小鼠以探讨其抗原性。结果 在大肠杆菌ＣＳ３的１３６位氨基酸残在后突变插入ＢａｍＨⅠ酶切点构建成呈现载体,全细胞ＥＬＩＳＡ、电镜和免疫     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.     

SS-B/La antigen purification, and ELISA detection of anti-SS-B/La antibodies in sera from patients with inflammatory connective tissue diseases

SS-B/La antigen was purified by immunoadsorbent columns with immunoglobulin from a patient with primary Sjögren's syndrome. Monitoring of the purification was facilitated by the fused rocket-immunoelectrophoresis technique. Technical ELISA variables for the detection of serum antibodies against the SS-B/La antigen were evaluated, and a recommended procedure is described. Prospective investigation of anti-SS-B/La antibodies in 103 blood donors and 131 patients with chronic inflammatory connective tissue diseases, including 43 patients with primary Sjögren's syndrome was performed. Anti-SS-B/La antibody concentrations were above normal in 65% of the patients with primary Sjögren's syndrome (verified by the strictly objective Copenhagen criteria for keratoconjunctivitis sicca and xerostomia) and 9% of patients with other chronic connective tissue diseases. The predictive value for primary Sjögren's syndrome among patients with increased levels of the anti-SS-B/La antibodies attending a rheumatology clinic was 78%.     

Purification and Characterization of a Nuclear SS-B Antigen

A nuelear SS-B antigen was isolated from a saline extract of acetone powder of rabbit thymus by precipitation with ammonium sulphate, affinity chromatography with Blue Sepharose CL-6B, and preparative agarose gel electrophoresis. The mol. wt of the antigen was 68,000. Its electrophoretic mobilily was similar to that of pro-albumin, and the iso-electric point was around pH 4.0. The main amino acids of the antigen were gluiamie acid, leucine, lysine and alanine. Both histidine and tyrosine were also found. The purified antigen precipitated with anti-SS-H sera but not with any other reference antisera. It resembled La and Ha antigens in susceptibility to proteolytic and nucleolytic enzymes and to heat. The purified SS-B antigen, however, had a higher molecular weight than did the Ha and La antigens. The molecule eould not be split inlo subunits with mercaptoethanol or acid. Counter-electrophoresis showed antibodies to the SS-B antigen in sera from patients wilh rheumatic diseases, including rheumatoid arthritis, systemic lupus erythematosus and Sjögren's sjndrome, but not in any of the control sera.     

Autoepitopes reactive with anti-SS-B/La

Sera from 120 patients with suspected autoimmune rheumatic disease and antinuclear antibodies of anti-SS-B/La specificity were examined by Western blotting for reactivity with the SS-B/La polypeptide of HeLa cells and recombinant SS-B/La derived from a 1.4 kilobase (kb) cDNA encoding approximately 90% of the SS-B/La molecule. All sera reacted with the HeLa cell and the recombinant SS-B/La. One hundred and fourteen (95%) reacted with a set of three Staph. aureus V8 protease-resistant peptides of Mr 30,000, 29,00 and 28,000 from a methionine-rich region of HeLa cell SS-B/La designated the X domain, and 98 (82%) reacted with another set of two protease-resistant peptides of Mr 24,000 and 23,000 from a phosphorylated region of HeLa cell La designated the Y domain. One reacted weakly with the Y domain only. All sera that reacted with X and Y reacted more strongly with X, suggesting that X was the major epitope. Antibodies affinity purified from the X domain reacted strongly with the X peptides but not with the Y peptides and conversely, antibodies affinity purified from the Y domain reacted with the Y peptides but not with the X peptides. Both antibodies reacted with a fusion protein comprising 102 amino acids at the carboxyl terminus of the SS-B/La molecule. This protein contained no methionine, demonstrating that methionines were not involved in the antibody-binding site. Over 80% of patients whose only criteria for selection was the presence of anti-SS-B/La had the clinical, histologic, serologic and phenotypic features of Sj?gren's syndrome whilst the remaining 20% had at least two of the features.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of human hybridoma monoclonal anti-Sm and anti-U1RNP antibodies spontaneously produced from normal human lymphocytes.

A panel of human:human hybridomas secreting monoclonal anti-Sm/RNP antibodies was established by the fusion of normal human tonsillar lymphocytes to the lymphoblastoid B cell line GM4672. The specificity of these antibodies was studied by direct binding and competitive inhibition in enzyme linked immunosorbent assays (ELISAs), and by immunoblotting. The stable subclones of these hybridoma monoclonal anti-Sm/RNP antibodies could be classified into four groups according to ELISA. Group I bound Sm/RNP only, group II bound Sm-RNP, Ro/SS-A and La/SS-B, group III bound Sm/RNP and ssDNA, and group IV bound Sm/RNP, Ro/SS-A, La/SS-B and ssDNA. When antibodies from each of the groups were tested by immunoblotting, the following pattern of reactivity emerged. Group II and IV antibodies reacted with U1RNP-A, Sm-B'/B, Sm-D and Sm-E proteins, as well as the Ro/SS-A and La/SS-B proteins. In contrast, group I and III antibodies did not bind to any individual protein components of Sm/RNP,Ro/SS-A or La/SS-B antigens, but recognized their conformational epitopes. These results, therefore, directly demonstrate for the first time that normal-derived B cells have the genetic potential, revealed here by somatic cell hybridization, to produce anti-Sm/RNP antibody responses which are ordinarily only associated with systemic lupus erythematosus (SLE) and related connective tissue diseases.     

Molecular analysis of the RNA and protein components recognized by anti-La(SS-B) autoantibodies.

The aim of this study was to determine whether sera with autoantibodies to the La(SS-B) nuclear antigen react with the same or different sets of cellular or viral ribonucleoproteins (RNPs) and whether patients with anti-La(SS-B) comprised a homogeneous group with respect to phenotypic and serological markers. The 34 anti-La(SS-B) sera studied were detected in the course of screening 2,000 sera referred from patients with suspected or defined multisystem autoimmune disease. Analysis of the molecular components of the small nuclear (sn) RNPs isolated from immune complexes developed in vitro between the IgG fractions of the anti-La(SS-B) sera and cell lines selected for their content of viral and cellular (non-viral) RNA showed that all 34 anti-La(SS-B) sera reacted with the same group of cellular RNAs and with two viral RNAs encoded by Epstein-Barr virus. The La(SS-B) RNPs contained one major 50,000 dalton antigenic polypeptide that resolved into 5-6 heterogeneously charged isospecies on two-dimensional immunoblots. In addition to anti-La(SS-B) reactivity, all 34 sera were shown to contain anti-Ro(SS-A) activity by counterimmunoelectrophoresis (CIEP); however, with three exceptions, the antigenic Ro(SS-A) polypeptide was not detectable by immunoblotting. The homogeneity of this group with anti-La(SS-B) was indicated by the findings that of the 34 cases 31 (88%) had hypergammaglobulinaemia, 33 (97%) had rheumatoid factor and 27 (of 30 tested, 90%) were HLA-B8. Thus all anti-La(SS-B) sera react with the same set of RNAs associated with an antigenic 50,000 dalton nucleoprotein, and the presence of anti-La(SS-B) autoantibodies identified a homogeneous group of patients with the serological and phenotypic features of primary Sjögren''s syndrome.     

Different La/SS-B mRNA Isoforms are Expressed in Salivary Gland Tissue of Patients with Primary Sjögren's Syndrome

Recently we isolated a La/SS-B mRNA isoform from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjögren's Syndrome. In the La/SS-B mRNA isoform the exon 1 was replaced. The alternative exon was termed exon 1′. Genomic analysis showed that the exon 1′ La mRNA was the result of a promoter-switch in combination with alternative splicing. Due to the unusual structure of the exon 1′ La/SS-B mRNA, the function and the behaviour under physiological and pathophysiological conditions in tissue of patients with primary Sjögren's syndrome or Systemic Lupus Erythematosus remained obscure. Therefore assays were established allowing a qualitative and quantitative estimation of expression of the exon 1 and 1′ La mRNA form, includingin situand dot blot hybridization as well as reversed PCR. Both mRNA forms were found to represent finally processed cytoplasmic mRNAs belonging to the abundant class of mRNAs. They were expressed and regulated in parallel. A ratio exon 1 to 1′ between 1:1 and 5:1 was determined. Both mRNA forms were downregulated in quiescent cells and upregulated in activated and proliferating cells including non-keratized stratified squamous epithelial, endothelial, salivary gland as well as infiltrating cells.     

Translocation of the nuclear autoantigen La to cell surface: assembly and disassembly with the extracellular matrix.

La (SS-B) protein is known as one major antigenic target for autoantibodies from patients with certain autoimmune diseases such as Sjogren's syndrome or Lupus Erythematosus. La protein belongs to the so called "extractable nuclear antigens". Here we report that La antigen is not restricted to the nucleus as one might deduce from the exclusive nuclear staining pattern of patient anti-La antibodies but after stimulation of serum-starved cells with 10% fetal calf serum (FCS) appears and stays for at least 45 min at the outer surface of CV-1 cells being available for binding of anti-La antibodies. In addition we found that a minor part of La antigen associates with the extracellular fibronectin network. After addition of 10% FCS to serum starved cells this extracellular autoantigen disassembled from the extracellular matrix and was taken up again by the cells. Incubation of serum starved cells with mercuric chloride, a known potent inducer of autoantibodies, also resulted in a detachment of the extracellular matrix associated La protein. From our studies it becomes likely that La protein itself is the antigen during autoimmunization. Moreover, once developed, anti-La antibodies might be able to bind to cell surface expressed La protein resulting in a damage of these cells leading to the inflammational events known to occur during disease.     

Elevation of the levels of SmB, B' and D proteins but not that of the La (SS-B) antigen during HSV infections

Three Sm proteins, B, B' and D, which are highly immunoreactive in systemic lupus erythematosus (SLE), increase in abundance in Vero cells infected with laboratory strains and clinical isolates of herpes simplex virus (HSV) types 1 and 2. The autoimmune antigen La (SS-B) does not accumulate in identical infections. The significance of the Sm antigen accumulation is discussed in terms of its possible role in HSV infection and in connection with the origin of autoantibodies in SLE.