Anti-La antibody production by MRL-1pr/1pr mice. Analysis of fine specificity

In evaluating the origin of autoantibodies, patterns of self-Ag recognition have been interpreted to reflect the relative role of Ag in stimulating a response. Few studies, however, have assessed whether human autoantibodies display patterns of autoantigen recognition similar to those of SLE-prone mice. In previous studies, anti-La antibodies from humans have been shown to bind multiple epitopes on recombinant human La Ag, including immunoreactivity with a large fragment, termed La C, representing the middle portion of the La sequence. We report herein for the first time that MRL-1pr mice also spontaneously produce antibodies to recombinant human La protein and resemble human autoantibodies in their reactivity with La C. To further investigate the fine specificity of this response, we tested for antibody binding to six synthetic La peptides representing sequences within La C. Whereas two of the synthetic La peptides reacted with MRL-1pr sera containing anti-La binding, low reactivity was observed with a large panel of human anti-La sera. Our results therefore show that patterns of La antigen recognition displayed by MRL-1pr antibodies differ from those of human autoantibodies, possibly reflecting differences between mouse and man in the induction of these responses.     

Characteristics and epitope mapping of a cloned human autoantigen La

The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sj?gren's syndrome. To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line. This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein. The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence. We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence. The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities. Only the sera containing anti-La antibodies reacted with the cloned La. By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein. No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies. Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sj?gren's syndrome.     

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

The epitope study on the SARS-CoV nucleocapsid protein

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this prot     

Mapping of multiple B cell epitopes on the 70-kilodalton autoantigen of the U1 ribonucleoprotein complex.

High titer IgG autoantibodies to the 70-kDa polypeptide component (p70) of the U1 ribonucleoprotein (RNP) complex occur in the sera of patients with mixed connective tissue disease, SLE, and related rheumatic diseases. To gain insight into the pathogenesis and diversity of this antibody response we have used recombinant DNA technology to map the linear B cell epitopes on p70. A full length 1.7-kb cDNA clone encoding p70 was isolated from a human placental library and restriction fragments or polymerase chain reaction-generated fragments of the gene subcloned into the bacterial expression vector pGEX. Purified fusion proteins representing specific regions of p70 were immunoblotted with a panel of 70 anti-(U1)RNP+ sera containing anti-p70 antibodies. Six epitopes, four major (A, B, C, and F) and two minor (D and E) were mapped and were located throughout the molecule. The anti-(U1)RNP sera displayed heterogeneity in their pattern of reactivity to the six epitopes although reactivity to epitope C was more frequently associated with SLE rather than mixed connective tissue disease. The identification of multiple B cell epitopes on p70 is consistent with the concept that this self Ag drives the autoantibody response.     

Human recombinant anti-La (SS-B) autoantibodies demonstrate the accumulation of phosphoserine-366-containing la isoforms in nucleoplasmic speckles

Using the recombinant La (SS-B) protein or a phosphorylated peptide derived thereof 27 La-specific human recombinant autoantibodies were selected from anti-La-positive systemic lupus erythematosus and systemic sclerosis patient-derived combinatorial phage display antibody libraries. Binding of these anti-La antibodies to various isoforms of the La protein present in normal and apoptotic cell extracts was analysed by Western blotting. Twenty-four of the selected antibodies recognize most, if not all isoforms of La, whereas three are exclusively reactive with the protein phosphorylated at serine-366. Sequence analysis of the selected antibodies showed a restricted spectrum of diversity in their VH germline gene usage. Remarkably, the recombinant antibodies recognizing exclusively the phosphoserine-366-containing isoform of La displayed a spleckled nucleoplasmic staining pattern in immunofluorescence analysis of HeLa and HEp-2 cells. This pattern differed markedly from those obtained with anti-La antibodies recognizing all isoforms of the La protein. Colocalization experiments with marker antibodies for spliceosomal UsnRNPs and RNA polymerase III subunits revealed that the anti-phosphorylated La antibodies stain the same nucleoplasmic speckles as anti-UsnRNP antibodies. In contrast to anti-UsnRNP antibodies the anti-phosphorylated La antibodies did not stain the Cajal bodies. In addition, no colocalization of phosphorylated La with RNA polymerase III was observed. Potential functional implications of the accumulation of phosphorylated La in nucleoplasmic speckles are discussed.     

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316-335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128-285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316-335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316-335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.     

Mapping of epitopes on the La(SS-B) autoantigen of primary Sj?gren's syndrome: identification of a cross-reactive epitope.

Autoepitopes on the ribonucleoprotein La(SS-B) were identified by using recombinant La(SS-B) polypeptides and sera from 166 patients with the antinuclear autoantibody anti-La(SS-B). The La(SS-B) polypeptides were encoded by polymerase chain reaction-derived overlapping or nonoverlapping fragments of the La(SS-B) gene, which encodes a protein of 408 amino acids (aa). Of the 166 sera tested, 99% reacted with a fusion protein comprising the first 107 N-terminal aa (LaA); 91% reacted with a fusion protein comprising aa 111 to 242 (LaC), and 91% reacted with a fusion protein comprising aa 346 to 408 (LaL2/3) at the C terminus of La(SS-B). The order of immunodominance as assessed by the number of sera reacting with each epitope and the strength of the reactivity was LaA (aa 1 to 107) greater than LaC (aa) 111 to 242) much greater than LaL2/3 (aa 346 to 408). Cross-reactivity was observed between antibodies eluted from LaC (aa 111 to 242) and LaL2/3 (aa 346 to 408), but there was no significant primary sequence homology between the two regions. The LaC region contained at least two epitopes, one encompassing a putative RNA-binding motif (aa 112 to 187) which was recognized by 83% of patient sera. Serial serum samples from three patients showed that the antibody response to La(SS-B) was initially directed to the N terminus (LaA, aa 1 to 107), but over a period of time all three major epitopes, including that encompassing the putative RNA-binding motif, were recognized. This result suggests that the primary immune response to La(SS-B) is restricted to an immunodominant epitope. As the specificity of the autoantibody response broadens, it includes the RNA-binding motif, which may have important implications for the expression of disease.     

Anti-alpha-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-alpha-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sj?gren's syndrome (SjS). We looked (in Nijmegen) for anti-alpha-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS 6, rheumatoid arthritis [RA] 12, systemic lupus erythematosus [SLE] 6, and scleroderma 6) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-alpha-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The ELISAs for alpha-fodrin showed six (Nijmegen) and four (Hanover) anti-alpha-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-alpha-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-alpha-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-alpha-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-alpha-fodrin autoantibodies does not add much to the diagnosis of Sj?gren's syndrome.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

CTB与HCV抗原决定簇融合蛋白的抗原性及其在抗HCV ELISA试剂盒中的应用

系统研究了通过霍乱毒素B亚基与HCV的4个抗原决定簇进行基因融合所表达的12种融合蛋白中HCV抗原决定簇的反应原性,探索了以融合蛋白为抗原,进行抗-HCV检测的途径。结果表明,多数融合蛋白中HCV抗原决定簇均能与对应的HCV抗体结合。以融合蛋白95082为抗原研制的抗-HCV ELISA试剂检测122名献血员血清,结果与美国雅培公司抗-HCV ELISA试剂检测的结果完全一致,经药品生物制品检定所检定,其特异性、灵敏度、精密性及稳定性均达到国家卫生部抗-HCV ELISA试剂的暂行检定标准。     

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.     

Simplified approaches for the development of an ELISA to detect circulating autoantibodies to p53 in cancer patients

Background  

The recognition that human tumors stimulate the production of autoantibodies has initiated the use of this immune response as serological markers for the early diagnosis and management of cancer. The enzyme-linked immunosorbent assay (ELISA) is the most common method used in detecting autoantibodies, which involves coating the microtiter plate with the tumor associated antigen (TAA) of interest and allowing serum antibodies to bind. The patient's sample is directly in contact with the coating antigen so the protein used for coating must be pure to avoid non-specific binding. In this study, a simplified method to selectively and specifically immobilize TAAs onto microtiter plates in order to detect circulating autoantibodies in cancer patients without prior purification process was described. Wild type full-length p53 protein was produced in fusion with biotin carboxyl carrier peptide (BCCP) or hexahistidine [(His)6] using pAK400 and pET15b(+) vectors, respectively. The recombinant p53 fusion protein produced was then subjected to react with either a commercial p53 monoclonal antibody (mAb) or sera from lung cancer patients and healthy volunteers in an enzyme-linked immunosorbent assay (ELISA) format.     

A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.     

Immunochemical and biochemical analysis of the proliferating cell nuclear antigen (PCNA) in HeLa cells

PCNA, also known a cyclin, a protein of molecular weight (MW) 35 000, accumulates in the nuclei of dividing and transformed cells and reacts with autoantibodies from certain lupus patients. Using an indirect immunofluorescence technique, we show that lupus sera containing anti-PCNA antibodies reveal a heterogeneous nuclear fluorescence pattern upon reaction with asynchronous HeLa cells, whereas with synchronized cells a sequence of distinct patterns is disclosed on progression through the cell cycle. Cell-free translation of HeLa cell mRNA followed by immunoprecipitation with anti-PCNA sera shows a single protein with the same apparent MW as PCNA labelled in vivo, suggesting that PCNA is not derived from a larger precursor protein and not grossly modified by post-translational events. However, a group of at least nine nuclear polypeptides ranging in MW from about 12 000 to 110 000 are recognized by immunoblotting with anti-PCNA sera, indicating either that additional antigenic sites are produced on denaturation of native proteins or that additional autoantibodies are present in these sera. We also show that PCNA and several of these polypeptides are associated with nuclear structures containing chromatin.     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

伪狂犬病病毒gE基因主要抗原表位区的原核表达及其在疫苗接种和自然感染鉴别诊断中的应用

根据伪狂犬病病毒（PRV）Min-A株gE基因序列,利用PCR方法扩增了PRV-gE基因不含信号肽、胞内区和跨膜区的主要抗原表位区,并克隆到原核表达载体pGEX-6p-1中,获得的重组质粒命名为pGEX-tgE。经SDSPAGE电泳分析证实克隆的部分gE基因获得了表达,融合表达产物大小约为63kD,并在终浓度为0.6mmol/L的IPTG诱导下,3.5h其表达量达到高峰。通过改变诱导条件,有效抑制了包涵体形成,提高了重组蛋白的溶解性。Western blot分析证实表达的重组gE蛋白具有抗原反应活性。将表达产物利用亲和层析法纯化后作为ELISA抗原,通过对其特异性、敏感性及工作条件的优化试验,和对48份PRV阴性血清样品的检测结果的统计学分析,建立了猪伪狂犬病tgE-ELISA鉴别诊断方法。通过对400份送检血清样品的检测结果分析,表明其与PRV全病毒ELISA试验的符合率高达95%以上,与基于抗gE蛋白单抗竞争性ELISA的符合率达94%。此方法可用于gE基因缺失PRV疫苗免疫动物和PRV自然感染动物的鉴别诊断。     

SARS冠状病毒NS融合蛋白的重组表达纯化与免疫学性质分析(英文)

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.