结肠癌肿瘤自身抗体谱筛选及其应用

 目的 本研究拟寻找鉴定结肠癌自身抗体谱，研究这些自身抗体作为结肠癌诊断候选血清标志物的可能性，同时鉴定这些自身抗体的抗原为研究结肠癌发生、发展相关的基因提供线索。方法 用结肠癌组织建立了库容量达5×105pfu的cDNA表达文库，用结肠癌患者血清进行了文库血清学分析（SEREX），筛选获得了阳性抗原克隆，进一步分析了其中4个克隆与30例结肠癌和30例正常人血清的反应情况。结果 获得的33个阳性克隆中，31个剪切成功，2个克隆与已知EST序列明显无同源性，另外29个克隆与已知基因高度同源。Uracil DNA glycosylase等4个抗原克隆与结肠癌患者和正常人血清反应阳性率分别为76%（23%）、80%（6%）、77%（0）、73%（66%）。结论 本研究发现的29个结肠癌抗原可能参与了结肠癌的发生发展，可能作为结肠癌的治疗潜在分子靶点和结肠癌诊断新的候选血清学标志物。Uracil DNA glycosylase等3个克隆与结肠癌患者血清的反应阳性率明显高于正常人的血清，其相关自身抗体可作为结肠癌诊断的血清标志物。     

应用SEREX技术筛选肿瘤抗原的研究进展

SEREX (serologicalidentificationofantigenbyrecombi nantcDNAexpressionlibraries)技术 ,即重组克隆表达抗原的血清学鉴定技术 ,是由德国血清学家Sahin等[1] 建立的从分子水平鉴定肿瘤抗原的方法。其理论基础是肿瘤能刺激机体产生免疫反应 ,人体B淋巴细胞可以识别自身肿瘤抗原 ,产生高价抗体 ,引起强烈的IgG反应。SEREX技术不需要特异CTL和瘤细胞的体外建株 ,而是应用cDNA表达文库使抗原浓度成百倍的增高 ,解决了既往肿瘤抗原抗体反应中的关键问题 ,新发现了大量肿瘤抗原。1　SEREX技术SEREX技术的基本步骤[1,2 ] :①患者…     

SEREX方法在筛选和鉴定骨肉瘤抗原中的应用

引言肿瘤抗原及其编码基因的理论研究和实验技术使肿瘤抗原的筛选和肿瘤免疫的研究进入一个新阶段[1]。肿瘤抗原的筛选与鉴定是临床特异性免疫治疗的前提和关键,随着对肿瘤生物学和肿瘤免疫学的深入认识,设计了基于体液免疫的重组cDNA文库的血清学分析(serological analysis of recombi-nant cDNAexpressionlibraries,SEREX)方法,简便易行[2]。本文对SEREX方法的原理、技术步骤、及其在骨肉瘤肿瘤基因筛选的应用及研究现状作一简要介绍。1SEREX方法原理重组cDNA表达文库的血清学分析最早由Pfreundsch小组建立[2]。SEREX方法将分子克隆技术和利用患者自体血清对肿瘤细胞的自体分型技术融为一体,不仅可检测抗体反应,而且能在抗原与患者自体血清反应的基础上,直接从分子水平确定具有免疫原性的肿瘤蛋白质(抗原)。此方法的流程主要是以新鲜瘤组织或细胞样本构建cDNA文库并连接入噬菌体表达载体,以重组的噬菌体转染E.coli大肠杆菌,将细菌表达的重组蛋白转移至NC膜上,与稀释后的病人自体血清共孵育,用酶联特异性抗人IgG二抗识别与高滴度血清抗体反应的克隆,阳...     

应用SEREX技术筛选和鉴定非小细胞肺癌肿瘤相关抗原

背景与目的 目前用于非小细胞肺癌诊断的标志物为数不多,为寻找更多的早期诊断和靶向治疗相关的标志物,本研究应用SEREX技术筛选与鉴定非小细胞肺癌肿瘤相关抗原.方法 使用非小细胞肺癌患者血清对人肺鳞癌和腺癌噬菌体展示文库进行生物淘选和SEREX筛选.PCR扩增阳性克隆插入片段后测序,结果 与GeneBank数据库中已知基因进行同源性比较,分析其生物学特性.结果用非小细胞肺癌血清对噬菌体展示文库进行筛选,共获得31个阳性克隆,测序后发现其代表15个基因,其中12个与已知cDNA序列同源,与肺癌或其它肿瘤的发生、发展关系密切;另外3个未发现有同源基因,可能是新基因.结论 应用SEREX技术发现15个肺癌相关抗原和3个肺癌相关抗原的候选基因,为进一步的深入研究打下良好的基础.     

人类肿瘤抗原的鉴定及应用进展

人类肿瘤抗原的鉴定是肿瘤免疫研究中极其重要的一环,T细胞识别的肿瘤抗原以各种分子生物学和免疫学方法被鉴定,包括CTL筛选法、血清学鉴定重组cDNA表达文库（SER-EX）法、组合肽库技术及多种系统性基因分析法.通过对肿瘤抗原的鉴定,可更好地了解抗原的本质、抗肿瘤免疫反应过程和免疫逃逸机制,对促进和发展肿瘤免疫治疗,发现肿瘤标记物具有重要意义.     

人类肿瘤抗原的鉴定及应用进展

人类肿瘤抗原的鉴定是肿瘤免疫研究中极其重要的一环,T细胞识别的肿瘤抗原以各种分子生物学和免疫学方法被鉴定,包括CTL筛选法、血清学鉴定重组cDNA表达文库(SER-EX)法、组合肽库技术及多种系统性基因分析法.通过对肿瘤抗原的鉴定,可更好地了解抗原的本质、抗肿瘤免疫反应过程和免疫逃逸机制,对促进和发展肿瘤免疫治疗,发现肿瘤标记物具有重要意义.     

肿瘤相关抗原与自身抗体检测对食管癌早期发现和高危人群预警的意义

目的：探讨检测食管癌相关抗原与自身抗体对食管癌早期发现和高危人群预警的意义.方法：应用间接酶联免疫反应法和肿瘤相关抗原微阵列（包含8个重组的癌抗原蛋白C-myc、p53、cyclin B1、p16、p62、Koc、Imp1和Survivin）检测河南食管癌高发区无症状居民血清自身抗体.根据自身抗体变化对部分居民进一步作食管内镜检查和黏膜活检及免疫组织化学检测,了解血清自身抗体阳性和阴性居民食管上皮组织病理学变化特征及组织中相应肿瘤相关抗原变化的关系.结果：391例无症状居民中,79例（20%）至少呈现1种自身抗体阳性.对64例自身抗体阳性组和67例自身抗体阴性组居民进行内镜检查和黏膜活检组织病理学检查,发现2例早期食管癌,且均在抗体阳性组.血清自身抗体阳性组食管癌前病变发生率明显高于自身抗体阴性组,P＜0.01.阳性百分率随食管上皮病变加重呈现线性增高趋势.食管上皮组织中,8种肿瘤相关抗原表达与血清中相应自身抗体有明显相关性,P＜0.05.结论：血清肿瘤相关自身抗体检测有助于提高食管癌高发区无症状居民食管癌前癌变和早期食管癌的检出率.血清自身抗体与食管上皮组织中相应肿瘤抗原的表达变化明显相关.血清肿瘤相关自身抗体检测结合黏膜活检可能是食管癌早期发现和高危人群预警的重要手段.     

应用蛋白印迹-免疫沉淀-质谱法筛选卵巢癌肿瘤抗原

背景与目的肿瘤抗原的筛选对卵巢癌的早期诊断有重要意义。目前,噬菌体抗体库技术(phageantibodylibrary)、核糖体展示技术(ribosomedisplay)和血清学筛选重组cDNA表达文库(serologicalanalysisofrecombinantcDNAexpressionlibraries,SEREX)技术等作为常用的肿瘤抗原筛选方法已得到广泛应用。本研究采用蛋白印迹鄄免疫沉淀鄄质谱方法筛选和鉴定卵巢癌肿瘤抗原,以期建立一种新的筛选肿瘤抗原的方法及技术路线。方法应用蛋白印迹(Westernblot)法筛选出差异明显的卵巢癌患者血清,用该血清与SKOV3细胞裂解物反应,免疫沉淀富集抗原,与正常血清的免疫沉淀样品比较,获得差异蛋白条带,然后进行质谱鉴定、生物信息学分析。结果采用蛋白印迹筛选到标记为7号的卵巢癌患者血清,与其他血清比较有明显的差异,经免疫沉淀、聚丙烯酰胺凝胶电泳与正常血清免疫沉淀样品比较,发现在接近66.2ku的位置处有一差异蛋白条带,经质谱鉴定、生物信息学分析为热休克蛋白(heatshockprotein,HSP)70及细胞角蛋白(cytokeratin)9。结论采用蛋白印迹鄄免疫沉淀鄄质谱法进行卵巢癌肿瘤抗原的筛选和鉴定是一种行之有效的方法。     

NY-ESO-1基因在人食管癌中的表达及其基因克隆

背景与目的：NY-ESO-1是从食管癌cDNA文库中筛选到的肿瘤抗原,但是该基因在食管癌组织中的表达情况尚未见报道。本文研究NY-ESO-1基因在食管癌中的表达率,以便于确定食管癌患者是否可以使用以NY-ESO-1抗原为基础的疫苗治疗。方法：采用RT-PCR方法检测了NY-ESO-1基因在30例食管癌组织和相应癌旁正常组织中的表达;采用分子克隆的方法从食管癌组织中克隆了NY-ESO-1cDNA。结果：NY-ESO-1基因在食管癌组织中的表达率为50%（15/30）,在30例相应癌旁正常组织中未见表达;克隆的NY-ESO-1cDNA序列与GenBank报道一致。结论：NY-ESO-1基因在食管癌中高频率表达,NY-ESO-1抗原可以被具有HLA-1类分子限制性CTL识别。     

人源性食管鳞癌cDNA文库的构建及鉴定

目的:构建人源性食管鳞癌cDNA噬菌体表达文库。方法:从人食管鳞癌组织中提取总RNA并分离纯化mRNA,用RT-PCR合成cDNA单链,LD-PCR扩增双链cDNA,除去<500bp的小片段,与载体λTriplEx2噬菌体左右臂连接,经体外包装即构成全长cD-NA噬菌体表达文库。转化E.coliXL1-Blue感受态细胞,测定文库的滴度,确定文库的容量大小,用蓝白筛选测定文库的重组率,PCR鉴定cDNA插入片段的大小。结果:构建的人源性食管鳞癌cDNA噬菌体表达文库滴度为1.64×106pfu/mL,重组率为98.6%,cDNA插入片段的大小为0.7～2.5kb,平均长约1.5kb。结论:成功构建1个人源性食管鳞癌cDNA噬菌体表达文库,适合于大批量筛选cDNA克隆的食管鳞癌肿瘤抗原基因。     

Isolation and characterization of human lung cancer antigens by serological screening with autologous antibodies

Serological analysis of a recombinant cDNA expression library (SEREX) derived from two lung adenocarcinoma cancer cell lines using autologous sera led to the isolation of 41 positive cDNA clones comprising 28 different antigens. They coded for a variety of nuclear and cytoplasmic proteins. Among the antigens, nucleoporin 107 (NUP107) was isolated most frequently (5 of 41 clones). The second most frequently isolated antigen was coded for by C21orf58 (4 of 41 clones). During serological analysis of selected antigens based on their reactivity to sera from normal individuals and lung cancer patients, none of the antigens showed a cancer-restricted recognition pattern. However, five genes including NUP107 showed higher expression when we examined the changes in gene expression in five different adenocarcinoma cell lines, including those used in SEREX, compared with their levels in normal lung tissues by cDNA microarray analysis. On the other hand, the expression levels of five genes including C21orf58 were down regulated in all adenocarcinoma cell lines. This SEREX study combining comprehensive gene expression assays has added to the growing list of lung cancer antigens, which may aid the development of diagnostic and immunotherapeutic reagents for patients with lung cancer.     

Serological identification of tumor antigens of esophageal squamous cell carcinoma

Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma.     

Serological analysis of human renal cell carcinoma

Serological analysis of cDNA expression libraries (SEREX) has proven to be a useful technique in the quest to elucidate the repertoire of immunogenic gene products in human cancer. We have applied the SEREX method to human renal cell carcinoma (RCC) in order to identify associated immunogenic gene products. cDNA expression libraries were prepared from a RCC tumor, a RCC cell line and human testis. The 3 libraries were screened with sera from 35 RCC patients and 15 healthy controls. Approximately 4.5 x 10(6) phage plaques were screened resulting in 234 positive clones, which corresponded to 74 different gene products. The seroreactivity toward 49 of these antigens was assessed. Seroreactivity to 21 (43%) of the antigens was similar in RCC patients and healthy controls, 9 antigens (18%) elicited antibodies more frequently and 19 antigens (39%) solely in RCC patients. In the reverse setting, reactivity of RCC patients' sera was tested against a panel of 44 previously identified "tumor-associated" antigens via the SADA (serum antibody detection array) method; 6 antigens reacted with RCC patients' and healthy donors' sera, 8 were reactive only with RCC patients' sera. From the 27 antigens identified by SEREX and SADA, which did not react with sera from healthy controls, 10 antigens reacted with a significant proportion of RCC patients' sera and 77% of RCC patients' sera reacted at least with one of these antigens. Sera from patients with non-malignant renal diseases or an autoimmune disease did not react with these 10 antigens.     

Serological identification of immunogenic antigens in acute monocytic leukemia

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent leukemia antigen. Here, we defined the acute monocytic leukemia-associated antigen (LAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on acute monocytic leukemia (FAB M5), followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other leukemia sera with positive clones were performed. Thirty-five distinct novel antigens reactive with autologous IgG were identified by SEREX analysis on an acute monocytic leukemia patient and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Twenty of the 35 antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining 15 were recognized exclusively by sera from allogeneic leukemia patients but not by normal donor sera, suggested that the immune response to these 15 antigens are leukemia related. The 15 immunogenic antigens detected by immune responses in the autologous host facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL) and are potential candidates for diagnosis and immunotherapy in acute myeloid leukemia (AML).     

Characterization of human colon cancer antigens recognized by autologous antibodies

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1–NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer. Int. J. Cancer76:652–658, 1998.© 1998 Wiley-Liss, Inc.     

Serological identification and bioinformatics analysis of immunogenic antigens in osteosarcoma

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent osteosarcoma antigen. Here, we defined the osteosarcoma-associated antigen (OSAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on osteosarcoma, followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other bone tumor sera with positive clones were performed. Seven distinct novel antigens reactive with IgG antibodies were identified by SEREX analysis on osteosarcoma patients and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Five of the seven antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining two were recognized exclusively by sera from allogeneic osteosarcoma patients but not by normal donor sera, suggested that the immune response to the two antigens are osteosarcoma related. Crude lysate ELISA methodology indicated that the optical density value of OSAA-3 and OSAA-5 were significantly higher in OS patients than in healthy donors. The two immunogenic antigens detected by immune responses might facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL). In summary, the antigens identified in this study may be potential candidates for diagnosis and targets for immunotherapy in osteosarcoma (OS).     

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.     

Analysis of the antibody repertoire of astrocytoma patients against antigens expressed by gliomas

The molecular characterization of antigens preferentially or exclusively expressed by astrocytomas and recognized by the autologous immune system are a prerequisite for the development of specific vaccines. To identify such antigens, we screened 5 cDNA expression libraries derived from astrocytomas and other gliomas for reactivity with high-titered IgG antibodies in the sera of astrocytoma patients using SEREX, the serologic identification of antigens by recombinant cDNA expression cloning. Autologous and allogeneic SEREX analysis of >5 x 10(6) clones with the sera of 18 astrocytoma patients revealed 10 antigens: the differentiation antigen glial fibrillary acidic protein (GFAP), Bax-inhibitor 1 (which was overexpressed in all glioma samples tested), 3 other molecules involved in the regulation of gene expression and proliferation (the nm23-H2-encoded nucleoside diphosphate kinase B, the Ran binding protein-2 and a DNA binding protein encoded by the son gene), SP40,40 (a complement inhibitory molecule), the chaperonin TCP-1, calnexin and 2 new gene products. No immune responses were detected against the "shared tumor" or "cancer testis antigens" that are regularly expressed in gliomas. Antibody responses in astrocytoma patients against antigens expressed by gliomas were rare and, with the exception of Bax-inhibitor 1 and the product of the son gene, were also found in apparently healthy controls. We conclude that although astrocytomas express a broad spectrum of antigens, they elicit antibody responses only rarely, most likely because of their intrinsic immunosuppressive effects.