人源M2三联体靶抗原BPO-E2融合蛋白的克隆表达与鉴定

目的用基因工程的方法克隆表达抗线粒体抗体二亚型（AMA-M2）的3个靶抗原侧链二氧酸脱氢酶复合体E2（BCOADC-E2）、丙酮酸脱氢酶复合体E2（PDC-E2）、2-氧戊二酸脱氢酶复合体E2（OGDC-E2）及其连接形成的三联体BPO-E2融合蛋白，以用于人原发性胆汁性肝硬化（PBC）的早期发现和临床诊断。方法针对BCOADC-E2、PDC-E2、OGDC-E2的cDNA序列设计引物，从正常人的淋巴细胞中提取RNA，通过反转录PCR方法扩增得到相应的基因片段，经测定序列验证后插入表达载体pET28a（＋），构建重组表达载体pET28a（＋）-BCOADC-E2、pET28a（＋）-PDC-E2、pET28a（＋）-OGDC-E2、pET28a（＋）-BPO-E2，转化大肠杆菌BL21（DE3）后诱导表达蛋白质。表达蛋白经SDS-PAGE、Western-blot等鉴定。结果经核苷酸序列测定和酶切鉴定结果表明，成功地构建了4种重组质粒。IPTG诱导表达后，获得4种融合蛋白。经免疫学鉴定，重组抗原片段具有AMA-M2的免疫原性。结论重组表达的融合蛋白将有利于原发性胆汁性肝硬化（PBC）的实验室诊断。     

原发性胆汁性肝硬化发病机制研究进展

原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)的病因不清,国内外学者从多个角度入手对其发病机制进行了深入研究并提出了多个假设,取得了较大进展．PBC患者的家庭成员有较高的患病风险,且其一级亲属多伴有其他自身免疫性疾病;PBC与HLA Ⅱ型抗原关系较密切,但欧美各国与中国研究结果存在差异,说明PBC的遗传学与流行病学可能与地理分布有关;非HLA遗传因素单核苷酸多态性 (SNPs)在CTLA-4、IL、维生素D及细胞色素 P450等方面的研究都取得了不同进展;PBC易感性还可能与X连锁基因相关;近期利用重组丙酮酸脱氢酶复合物E2亚单位(PDC-E2)特异性二聚体IgA单克隆抗体(mAbs)杆状病毒表达系统对AMA进行的研究阐述了IgA在PBC 患者胆管上皮细胞(BECs)损伤中的作用,并提出了新的假设．对Sp 100的研究发现其对抗原始内皮细胞血管生成中的作用,与PBC的发病存在一定关系．近来不断有新的物质被鉴定与 PBC发病相关．     

原发性胆汁性肝硬化的诊断和治疗进展

原发性胆汁性胆管炎(PBC)是一种慢性进行性自身免疫性肝脏疾病,多见于中年女性,以肝内小胆管破坏及血清抗线粒体抗体(AMA)阳性为特征。B细胞、CD4~+和CD8~+T细胞针对丙酮酸脱氢酶复合体E2(PDC-E2)反应的抗原表位均参与PBC的发病,尤其是B细胞免疫在PBC的发病机制中发挥了重要作用。熊去氧胆酸(UDCA)具有促进胆汁分泌、保护肝细胞的作用,是唯一经美国食品和药物管理局(FDA)批准的治疗PBC的药物,早期、中剂量、长期应用可延缓疾病进展,提高长期生存率,但高达40%的患者对UDCA应答欠佳,且预后较差,需要寻找新的治疗方法。     

人源M2二联体靶抗原BO-E2融合蛋白的克隆表达与鉴定

目的:表达二联体BO-E2重组融合蛋白,以用于人原发性胆汁性肝硬化(PBC)的早期发现和临床诊断.方法:通过逆转录聚合酶链式反应(RT- PCR)方法扩增得到抗线粒体抗体二亚型(AMA-M2)的二个靶抗原侧链:二氧酸脱氢酶复合体E2(BCOADC-E2)、2-氧戊二酸脱氢酶复合体E2(OGDC-E2)相应的基因片段,经测定序列验证后插入表达载体pET28a( ),构建重组表达载体pET28a( )-BCOADC-E2,pET28a( )-OGDC-E2,pET28a( )-BO-E2,转化大肠杆菌BL21(DE3)后诱导表达蛋白质.表达蛋白经SDS-PAGE、westem blot等鉴定.结果:经核苷酸序列测定和酶切鉴定结果表明,成功地构建了3种重组质粒.异丙基-B-D-硫代半乳糖苷(IPTG)诱导表达后,获得3种融合蛋白.经免疫学鉴定,重组抗原片段具有AMA-M2的免疫原性.结论:重组表达的BO-E2融合蛋白将有利于PBC的实验室诊断.     

原发性胆汁性胆管炎免疫发病机制

原发性胆汁性胆管炎（PBC）是一种以慢性进行性胆汁淤积为主要特征的自身免疫性肝病,针对自身抗原——丙酮酸脱氢酶复合体E2亚单位（PDC-E2）免疫耐受性的丧失是PBC发病的根源。肝内胆管上皮细胞特有的免疫生物学特性使其成为PBC发病的积极参与者。近年来,PBC的检出率逐年提高,但临床上仍未改变熊去氧胆酸单一用药的格局,...     

乙肝病毒PreS2基因在大肠埃希菌中的表达

目的在大肠埃希菌(Escherichiacoli)中表达乙肝病毒前S2(HBVPreS2)蛋白,并对其进行鉴定和纯化。方法利用DNA重组技术,将全长的HBVPreS2基因克隆至pMAL-C2x[融合表达载体含麦芽糖结合蛋白(maltosebindingprotein,MBP)标签蛋白]质粒中,构建pMAL-C2/S2表达载体,转化至E.coli,经IPTG诱导表达,SDS-PAGE分析目的蛋白的表达形式,依据其不同的表达形式采取适当的纯化方案,纯化产物经Westernblot和ELISA法检测反应原性,经Xa因子切割去除MBP标签蛋白。结果成功构建了表达载体pMAL-C2/S2,目的蛋白为PreS2-MBP,以包涵体和可溶性2种形式表达。纯化产物能与anti-HepatitisBvirusPreS2Antigen发生特异性反应。融合蛋白经Xa因子切割去除了MBP标签蛋白。结论在E.coli中成功表达了HBVPreS2蛋白,为进一步研制新型HBV预防及免疫治疗制剂打下了基础。     

PBC患者CD8~+效应T细胞的细微表型与功能特征

【据Hepatoatology 2011年11月报道】题:在人类原发性胆汁性肝硬化患者中,CD8+效应T细胞的细微表型与功能特征（作者Tsuda M等）原发性胆汁性肝硬化（PBC）患者对丙酮酸脱氢酶E2（PDC-E2）产生了多种特异反应,包括自身反应性CD4⁺、CD8⁺T细胞和自身抗体等。美国加利福尼亚大学戴维斯分校的研究者检测了132例研究对象（包括76例PBC患者和56例对照）的CD8⁺T细胞表型。研究发现PBC患者外周血单核细胞中一种效应记忆T细胞（TEM）出现频率较高,其细胞表型的特征是表达CD45RO^highCD57⁺CD8^high,同时也表达肠道归巢整合素、α4β7。这些CD8^high TEM细胞在TCR被激活后膜联蛋白V表达降低。CD45RO^highCD57⁺CD8^highT细胞较其他CD8^high T细胞表达更高水平的粒酶A、粒酶B、穿孔素、CCR5     

利用重组OGDC—E2检测M2抗体及其临床意义

目的重组表达人OGDC-E2融合蛋白，并用于检测自身抗体。方法重组表达的OGDC—E2，分别采用免疫印迹法（IBT）法和酶联免疫吸附试验（ELISA）方法检测PBC患者的M2抗体。50例确诊的PBC患者为PBC组，以其他肝病患者70例、自身免疫病患者70例和健康体检者70例作对照。结果50例确诊的PBC患者中41例OGDC-E2抗体阳性。9例阴性，阳性率为82．0％。而疾病对照组和健康体检者血清中M2抗体检测均为阴性。结论用重组人2-氧戊二酸脱氢酶复合物E2亚单位检测M2抗体，有较好的敏感性及一定的特异性。有助于临床对PBC的诊断。     

采用重组侧链二氧酸脱氢酶复合物E2亚单位检测M2抗体及其临床意义

目的 探讨用重组表达的侧链二氧酸脱氢酶复合物E2亚单位（BCOADC—E2）检测M2抗体，以早期诊断原发性胆汁性肝硬化（PBC）。方法 重组表达BCOADC—E2，采用酶联免疫吸附试验（ELISA）和免疫印迹法（IBT）来检测PBC患者的M2抗体，并以健康体检者、自身免疫病患者、其他非胆汁性肝硬化患者为对照组。结果 60例肝病患者血清中检测出抗BCOADC—E2抗体阳性33例，阴性者为27例，阳性率为55％，而健康体检者和疾病对照组血清中M2抗体检测均为阴性。结论 采用重组表达的人侧链二氧酸脱氢酶复合物E2亚单位检测M2抗体，有一定的特异性，对于早期诊断PBC有一定的辅助参考作用。     

蓝氏贾第鞭毛虫沉默信息调节因子2(Sir2)基因的克隆、表达与纯化

目的获取蓝氏贾第鞭毛虫(Giardia lamblia,Gl)沉默信息调节因子2(Sir2)基因序列及其重组蛋白。方法以蓝氏贾第鞭毛虫中国C2克隆株基因组DNA为模板,PCR扩增GlSir2基因编码序列,将所得片段连接至pMD-19T质粒。转化大肠埃希菌(E.coli)JM109,挑取阳性克隆进行测序。将GlSir2基因插入原核表达载体pET28b,构建表达载体pET28b-GlSir2,转化E.coli BL21(DE3),用异丙基硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的表达情况。收集重组表达产物,用8 mol/L尿素溶解包涵体并收集上清,镍离子亲和层析进行纯化。将纯化产物透析复性,蛋白质印迹(Western blotting)进行免疫学鉴定。结果获得了蓝氏贾第鞭毛虫中国C2克隆株GlSir2基因编码序列,该序列包含一个1 680 bp的开放阅读框架,可编码559个氨基酸,预测其蛋白的相对分子质量(Mr)约为62 800。构建表达载体pET28b-GlSir2,经IPTG诱导,重组蛋白以包涵体形式表达。溶解包涵体并经纯化后的目的蛋白纯度达80%以...     

Antibody to two forms of dihydrolipoamide acetyltransferase (PDC-E2) in primary biliary cirrhosis

ABSTRACT— Dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), is the major autoantigen in primary biliary cirrhosis. By immunoblotting with sera from patients with primary biliary cirrhosis, we observed a double band, of molecular weight 70 and 74 kD for PDC-E2, when a preparation of bovine heart mitochondria was not boiled prior to electrophoresis. This double band could also be detected using antisera raised in rats or rabbits against intact PDC or PDC-E2, but not in antisera raised against a synthetic decamer representing the lipoic acid binding sequence of PDC-E2; the latter reacted only with the 74 kD component. Antibody eluted from either the 70 or 74 kD component reacted with both 70 and 74 kD components. By ELISA, sera from patients with primary biliary cirrhosis reacted more strongly with a non-boiled than a boiled PDC-E2, whereas immune animal sera reacted equally with both preparations. Thus, according to whether preparations of PDC are boiled or not, two conformationally alternative forms of the PDC-E2 protein can be revealed by immunoblotting. The two forms in non-boiled preparations migrate at molecular weights corresponding to 70 and 74 kD.     

Immunopathology of primary biliary cirrhosis.

A major advance in the study of primary biliary cirrhosis was identification of the major B-cell auto-antigen as the mitochondrial enzyme pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PDC-E2). Subsequent studies revealed that PDC-E2 also contained epitopes recognized by patients' T cells. Furthermore, aberrant expression of MHC class II, intercellular adhesion molecules, lymphocyte co-stimulatory molecules and B-cell epitopes of PDC-E2 was observed on patients' biliary epithelium, supporting the concept that biliary epithelial cells are the target of a focused autoimmune reaction. Changes in distribution of auto-antigen on biliary epithelium and the presence of auto-antibody in patient's serum have both been shown to occur very early in the natural history of primary biliary cirrhosis, suggesting an intimate role for these molecules in immunopathogenetic mechanisms.     

Immunoreactivity of antimitochondrial autoantibodies in Japanese patients with primary biliary cirrhosis

The incidence and prevalence of primary biliary cirrhosis show wide geographic differences. The frequency of this disease in Japan is lower than in Northern Europe. To elucidate the immunoreactivity of serum with enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) and the M2 mitochondrial antigenic complex in Japanese patients, we examined sera from 107 patients with primary biliary cirrhosis from three geographically different regions of Japan. The sera were assayed by immunofluorescence on frozen tissue sections, immunoblotting on bovine heart mitochondria and recombinant E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADCE2), ELISA using recombinant E2 subunit of human pyruvate dehydrogenase complex (PDC-E2) and purified porcine 2-oxoglutarate dehydrogenase complex (OGDC), and enzyme inhibition assay using porcine PDC and OGDC. Of the 107 sera, 95 (88%) reacted by immunofluorescence, 102 (95%) by immunoblotting with at least one of the M2 autoantigens, although only 78 (73%) reacted with PDC-E2; 72 (67%) by ELISA with PDC-E2; and 81 (76%) with PDC by the enzyme inhibition assay. Thus, the frequency of reactivity with PDC-E2 by all assays was lower for Japanese than the reported frequency for Caucasian patients with primary biliary cirrhosis, whereas the frequency of reactivity by immunoblotting and ELISA against 2-OADC enzymes other than PDC was relatively higher. The relative frequency of reactivity of autoantibodies to the M2 autoantigens was similar for the three different regions of Japan. The different autoantibody profiles for Japanese and Caucasian patients with primary biliary cirrhosis point to immunogenetic and environmental determinants of this disease, which should provide new insights into its autoimmune origins.     

Differential expression of MHC class II subregion products on bile duct epithelial cells and hepatocytes in patients with primary biliary cirrhosis

To study the expression of MHC Class II subregion gene products on biliary epithelial cells in primary biliary cirrhosis, frozen sections from liver biopsies of 15 patients with primary biliary cirrhosis were studied immunohistochemically using HLA-D subregion specific monoclonal antibodies L243 (HLA-DR), Leu10 (HLA-DQ) and B7/21 (HLA-DP). Patients with early stages of primary biliary cirrhosis showed expression of HLA-DP, HLA-DR and HLA-DQ subregion gene products on bile duct epithelial cells. In advanced stages of disease, no MHC Class II antigens or only HLA-DR and HLA-DP were expressed on bile duct cells. While normal hepatocytes did not express detectable amounts of MHC Class II antigens, hepatocytes from liver biopsies of four patients with primary biliary cirrhosis showed a distinct staining exclusively with monoclonal antibodies specific for HLA-DR. The expression of MHC Class II antigens on parenchymal cells was independent of a lymphocytic infiltration into the tissue. This study demonstrates that bile ductular cells, but not hepatocytes, express a full set of MHC Class II molecules at least during the early stages of primary biliary cirrhosis. We propose, therefore, that the expression of both HLA-DR and HLA-DQ subregion products on bile duct epithelial cells may be a necessary, although not sufficient, condition for the initiation of an autoimmune process leading to the destruction of intrahepatic bile ducts in primary biliary cirrhosis.     

Microbial mimics are major targets of crossreactivity with human pyruvate dehydrogenase in primary biliary cirrhosis

BACKGROUND/AIMS: Previous studies on patients with primary biliary cirrhosis (PBC) have shown extensive cross-reactivity between the dominant B- and T-cell epitopes of human pyruvate dehydrogenase complex-E2 (PDC-E2), and microbial mimics. Such observations have suggested microbial infection as having a role in the induction of anti-mitochondrial antibodies, through a mechanism of molecular mimicry. However the biological significance of these cross-reactivities is questionable, because PDC-E2 is so highly conserved among various species. METHODS: Interrogating protein databases, ten non-PDC-E2 microbial sequences with high degree of similarity to PDC-E2(212-226) were found in Escherichia coli (6), Helicobacter pylori, Pseudomonas aeruginosa, Cytomegalovirus, and Haemophilus influenzae. We report on a study testing reactivity and competitive cross-reactivity against these respective peptides, and in some cases the parent protein, using sera from 55 patients with PBC, compared to reactivity of 190 pathological and 28 healthy controls. RESULTS: Cross-reactivity to E. coli mimics was commonly seen in PBC, and in a subset of pathological controls except where there was no evidence of urinary tract infection and correlated with anti-mitochondrial reactivity. CONCLUSIONS: E. coli/PDC-E2 cross-reactive immunity characterizes primary biliary cirrhosis; the large number of E. coli immunogenic mimics may account for the dominance of the major PDC-E2 autoepitope.     

Apotopes and innate immune system: Novel players in the primary biliary cirrhosis scenario

Our understanding of primary biliary cirrhosis has been rapidly growing over the past decade and the disease is now regarded as a model for other female-predominant, organ-specific autoimmune conditions. Primary biliary cirrhosis ensues from a multi-lineage loss of tolerance to the E2 component of the pyruvate dehydrogenase complex. One of the major unanswered questions in the pathogenesis of primary biliary cirrhosis is the specificity of small intrahepatic bile ducts attack while PDC-E2 is present in mitochondria of all nucleated cells. Recent findings suggest that the uniqueness of the primary target tissue, biliary epithelium, may be of considerable importance for understanding primary biliary cirrhosis and that the biliary epithelial cell is more than an innocent victim. Rather, it attracts an immune attack by virtue of the unique apoptotic mechanisms and by the way it handles PDC-E2. Moreover, recent evidence suggests that apoptotic bodies of biliary epithelial cell are able to activate the innate immune system in the presence of anti-mitochondrial antibodies. This review article is intended to provide a critical overview of the role of apoptosis in biliary epithelial cells, the activation of the innate immune system, and its biological and clinical significance in primary biliary cirrhosis.     

Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the lambda phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC--dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2)--were isolated, appearing at a frequency of 0.01% in the combinatorial immunoglobulin library. These Fab clones recognize human PDC-E2 with high affinity (Ka = 10(-7)-10(-9) M-1). Using both immunoblotting and ELISA, LC1-LC6 showed little cross-reactivity to any of the other autoantigens commonly recognized by PBC sera or to other antigens commonly recognized by PBC sera or to other antigens such as histone, calf thymus DNA, and bovine serum albumin. The Fab monoclonal antibodies show a typical anti-mitochondrial staining pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients with PBC. Our results demonstrate that a recombinant combinatorial immunoglobulin library can be used to isolate high-affinity Fabs against a specific autoantigen. Such reagents will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.     

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.     

Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

AIM:To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). METHODS:Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore,the inner lipoyl peptide 167-184 was used in an unlip oylated and a lipoylated form as well as cou...