技术领域technical field
本发明涉及胃癌的筛查/检测技术领域，更具体地，涉及一种Metaxin-2作为胃癌标志物的应用。The present invention relates to the technical field of screening/detection of gastric cancer, and more specifically relates to the application of Metaxin-2 as a gastric cancer marker.
背景技术Background technique
胃癌在我国各种恶性肿瘤中居首位，胃癌发病有明显的地域性差别，在我国的西北与东部沿海地区胃癌发病率比南方地区明显为高。好发年龄在50岁以上，男女发病率之比为2：1。胃癌的预后与胃癌的病理分期、部位、组织类型、生物学行为以及治疗措施有关。Gastric cancer ranks first among all kinds of malignant tumors in my country. The incidence of gastric cancer has obvious regional differences. The incidence of gastric cancer in the northwest and eastern coastal areas of my country is significantly higher than that in the south. The age of onset is more than 50 years old, and the ratio of male to female incidence is 2:1. The prognosis of gastric cancer is related to the pathological stage, location, tissue type, biological behavior and treatment measures of gastric cancer.
遗传与分子生物学研究表明，胃癌病人有血缘关系的亲属其胃癌发病率较对照组高4倍。胃癌的癌变是一个多因素、多步骤、多阶段发展过程，涉及癌基因、抑癌基因、凋亡相关基因与转移相关基因等的改变，而基因改变的形式也是多种多样的。Genetic and molecular biology studies have shown that the incidence of gastric cancer in blood relatives of gastric cancer patients is 4 times higher than that of the control group. The carcinogenesis of gastric cancer is a multi-factor, multi-step, multi-stage development process, involving changes in oncogenes, tumor suppressor genes, apoptosis-related genes, and metastasis-related genes, and the forms of gene changes are also diverse.
早期胃癌多数病人无明显症状，少数人有恶心、呕吐或是类似溃疡病的上消化道症状。疼痛与体重减轻是进展期胃癌最常见的临床症状。病人常有较为明确的上消化道症状，如上腹不适、进食后饱胀，随着病情进展上腹疼痛加重，食欲下降、乏力。根据肿瘤的部位不同，也有其特殊表现。贲门胃底癌可有胸骨后疼痛和进行性吞咽困难；幽门附近的胃癌有幽门梗阻表现；肿瘤破坏血管后可有呕血、黑便等消化道出血症状。腹部持续疼痛常提示肿瘤扩展超出胃壁，如锁骨上淋巴结肿大、腹水、黄疸、腹部包块、直肠前凹扪及肿块等。晚期胃癌病人常可出现贫血、消瘦、营养不良甚至恶病质等表现。Most patients with early gastric cancer have no obvious symptoms, and a few people have nausea, vomiting or upper gastrointestinal symptoms similar to ulcer disease. Pain and weight loss are the most common clinical symptoms of advanced gastric cancer. Patients often have relatively clear upper gastrointestinal symptoms, such as upper abdominal discomfort, fullness after eating, and as the disease progresses, upper abdominal pain increases, loss of appetite, and fatigue. Depending on the location of the tumor, there are also special manifestations. Cancer of the cardia and gastric fundus may have retrosternal pain and progressive dysphagia; gastric cancer near the pylorus may have pyloric obstruction; tumors that destroy blood vessels may have symptoms of gastrointestinal bleeding such as hematemesis and melena. Persistent pain in the abdomen often indicates that the tumor has expanded beyond the gastric wall, such as supraclavicular lymphadenopathy, ascites, jaundice, abdominal mass, and palpable mass in the anterior rectal recess. Patients with advanced gastric cancer often have symptoms such as anemia, weight loss, malnutrition and even cachexia.
目前的诊断方法有X线钡餐检查、纤维胃镜检查、腹部超声、螺旋CT与正电子发射成像检查等。这些方法都存在各自的缺陷，如影像学技术难以发现瘤体较小的肿瘤，早期普查的漏检率较高。目前缺乏一种特异性的胃癌生物标志物，特异性的胃癌生物标志物的相关研究具有重要的意义。The current diagnostic methods include X-ray barium meal examination, fiber optic gastroscopy, abdominal ultrasound, spiral CT and positron emission imaging. These methods have their own defects, such as the difficulty of finding small tumors by imaging techniques, and the high missed detection rate in early general screening. At present, there is a lack of a specific gastric cancer biomarker, and the research on specific gastric cancer biomarkers is of great significance.
发明内容Contents of the invention
本发明的目的是为了克服现有技术的不足，提供一种特异性的胃癌生物标志物。The purpose of the present invention is to provide a specific gastric cancer biomarker in order to overcome the deficiencies of the prior art.
本发明的第一个目的是提供Metaxin-2作为胃癌诊断标志物的应用。The first object of the present invention is to provide the application of Metaxin-2 as a diagnostic marker for gastric cancer.
本发明的第二个目的是提供Metaxin-2蛋白片段在Metaxin-2蛋白定量检测中的应用。The second object of the present invention is to provide the application of Metaxin-2 protein fragments in the quantitative detection of Metaxin-2 protein.
本发明的第三个目的是提供Metaxin-2蛋白在制备胃癌筛查/诊断的试剂盒中的应用。The third object of the present invention is to provide the application of Metaxin-2 protein in the preparation of a kit for gastric cancer screening/diagnosis.
本发明的第四个目的是提供Metaxin-2蛋白在制备胃癌筛查/诊断的试剂中的应用。The fourth object of the present invention is to provide the application of Metaxin-2 protein in the preparation of reagents for gastric cancer screening/diagnosis.
本发明的第五个目的是提供Metaxin-2蛋白片段在制备胃癌筛查/诊断的试剂盒中的应用。The fifth object of the present invention is to provide the application of Metaxin-2 protein fragments in the preparation of gastric cancer screening/diagnosis kits.
本发明的第六个目的是提供Metaxin-2蛋白抗体在制备胃癌筛查/诊断的试剂盒中的应用。The sixth object of the present invention is to provide the application of the Metaxin-2 protein antibody in the preparation of a gastric cancer screening/diagnosis kit.
本发明的第七个目的是提供一种用于胃癌筛查/诊断的试剂盒。The seventh object of the present invention is to provide a kit for screening/diagnosing gastric cancer.
为了实现上述目的，本发明是通过以下技术方案予以实现的：In order to achieve the above object, the present invention is achieved through the following technical solutions:
Metaxin-2 （HGNC登录号为HGNC: 7506；Entrez Gene登录号为10651；Ensembl登录号为ENSG00000128654；OMIM登录号为608555；UniProtKB登录号为 O75431） 是一种线粒体外膜蛋白复合物蛋白。Metaxin-2蛋白在人和小鼠中非常保守。Metaxin-2可以和线粒体膜蛋白Metaxin-1相互作用进行物质转运。Metaxin-2 (HGNC accession number is HGNC: 7506; Entrez Gene accession number is 10651; Ensembl accession number is ENSG00000128654; OMIM accession number is 608555; UniProtKB accession number is O75431) is a mitochondrial outer membrane protein complex protein. Metaxin-2 protein is very conserved in human and mouse. Metaxin-2 can interact with mitochondrial membrane protein Metaxin-1 for material transport.
本发明研究发现，胃癌患者Metaxin-2基因或蛋白的表达水平与健康人或者癌旁组织相比，表达水平低，因此可用于胃癌诊断。The research of the present invention finds that the expression level of Metaxin-2 gene or protein in patients with gastric cancer is lower than that in healthy people or adjacent tissues, so it can be used for the diagnosis of gastric cancer.
其中，Metaxin-2基因的核苷酸序列如SEQ ID NO.1所示，Metaxin-2蛋白的氨基酸序列如SEQ ID NO.2所示。Wherein, the nucleotide sequence of the Metaxin-2 gene is shown in SEQ ID NO.1, and the amino acid sequence of the Metaxin-2 protein is shown in SEQ ID NO.2.
因此，如下应用均应在本发明的保护范围之内：Therefore, the following applications all should be within the protection scope of the present invention:
Metaxin-2作为胃癌诊断标志物的应用。Application of Metaxin-2 as a diagnostic marker for gastric cancer.
Metaxin-2蛋白片段在Metaxin-2蛋白定量检测中的应用，所述Metaxin-2蛋白片段的氨基酸序列如SEQ ID NO.3～20任一所示。The application of the Metaxin-2 protein fragment in the quantitative detection of the Metaxin-2 protein, wherein the amino acid sequence of the Metaxin-2 protein fragment is shown in any one of SEQ ID NO.3-20.
Metaxin-2蛋白在制备胃癌筛查/诊断的试剂盒中的应用。Application of Metaxin-2 protein in preparation of gastric cancer screening/diagnosis kit.
Metaxin-2蛋白在制备胃癌筛查/诊断的试剂中的应用。Application of Metaxin-2 protein in preparation of reagents for gastric cancer screening/diagnosis.
Metaxin-2蛋白片段在制备胃癌筛查/诊断的试剂盒中的应用，所述Metaxin-2蛋白片段的氨基酸序列如SEQ ID NO.3～20任一所示。The application of the Metaxin-2 protein fragment in the preparation of a gastric cancer screening/diagnosis kit, the amino acid sequence of the Metaxin-2 protein fragment is shown in any one of SEQ ID NO.3-20.
Metaxin-2蛋白抗体在制备胃癌筛查/诊断的试剂盒中的应用。Application of Metaxin-2 protein antibody in preparation of gastric cancer screening/diagnosis kit.
一种用于胃癌筛查/诊断的试剂盒，所述试剂盒含有可以检测Metaxin-2基因或蛋白质的表达水平的试剂，也属于本发明的保护范围。A kit for screening/diagnosing gastric cancer, said kit containing reagents capable of detecting the expression level of Metaxin-2 gene or protein, also belongs to the protection scope of the present invention.
优选地，所述试剂包括SEQ ID NO. 3～20所示肽段中的一条或几条。Preferably, the reagent includes one or more of the peptides shown in SEQ ID NO. 3-20.
优选地，所述试剂包括Metaxin-2蛋白抗体。Preferably, the reagent includes Metaxin-2 protein antibody.
优选地，所述抗体为单克隆抗体或多克隆抗体。Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
所述试剂盒的使用方法，包括以下步骤：The using method of described kit comprises the following steps:
S1. 获得个体的疑似癌症组织；S1. Obtaining suspected cancer tissue from an individual;
S2. 测定其中的Metaxin-2的表达水平；S2. Determining the expression level of Metaxin-2 wherein;
S3. 获得正常个体的同一组织或同一个体的正常组织；S3. Obtain the same tissue from a normal individual or normal tissue from the same individual;
S4. 测定其中的Metaxin-2的表达水平作为对照表达水平；S4. measure the expression level of Metaxin-2 wherein as control expression level;
S5 将S1中获得的Metaxin-2的表达水平与S4中获得的Metaxin-2的对照表达水平进行比较，当S1中获得的Metaxin-2的表达水平与S4中获得的Metaxin-2的对照表达水平相比降低时，确定所述个体患有胃癌。S5 compares the expression level of Metaxin-2 obtained in S1 with the control expression level of Metaxin-2 obtained in S4, when the expression level of Metaxin-2 obtained in S1 is compared with the control expression level of Metaxin-2 obtained in S4 When the ratio is lower, the individual is determined to have gastric cancer.
或者，所述试剂盒的使用方法，包括以下步骤：Alternatively, the method for using the kit comprises the following steps:
S1. 获得个体的血清；S1. Obtaining serum from the individual;
S2. 测定其中的Metaxin-2的表达水平；S2. Determining the expression level of Metaxin-2 wherein;
S3. 获得正常个体的血清；S3. Obtain the serum of a normal individual;
S4. 测定其中的Metaxin-2的表达水平作为对照表达水平；S4. measure the expression level of Metaxin-2 wherein as control expression level;
S5. 将S2中获得的Metaxin-2的表达水平与S4中获得的Metaxin-2的对照表达水平进行比较，当S2中获得的Metaxin-2的表达水平与S4中获得的Metaxin-2的对照表达水平相比降低时，确定所述个体患有胃癌。S5. The expression level of Metaxin-2 obtained in S2 is compared with the control expression level of Metaxin-2 obtained in S4, when the expression level of Metaxin-2 obtained in S2 is compared with the control expression of Metaxin-2 obtained in S4 When the level is lower than that, the individual is determined to have gastric cancer.
另外，本发明给出了具有参考价值的Metaxin-2的检测方法。In addition, the present invention provides a method for detecting Metaxin-2 with reference value.
采用肽段如SEQ ID NO.3～20任一所示Metaxin-2蛋白的片段（表1），所述肽段应当与样品中的蛋白或其经过酶解（如胰蛋白酶酶解）后得到的片段的序列完全一致。所述片段宜经过重同位素（例如13C，14N）标记，以便将其与未经标记的样品相区分，从而进行Metaxin-2蛋白定量检测（例如iTRAQ技术）。此外，所述片段的最佳Transition应有较好的信噪比。Use peptides such as the fragments of Metaxin-2 protein shown in any one of SEQ ID NO.3~20 (Table 1), and the peptides should be obtained from the protein in the sample or after enzymatic hydrolysis (such as trypsin hydrolysis) The sequences of the fragments are completely identical. The fragments should be labeled with heavy isotopes (such as 13C, 14N) to distinguish them from unlabeled samples for quantitative detection of Metaxin-2 protein (such as iTRAQ technology). In addition, the optimal Transition of the segment should have a better signal-to-noise ratio.
将一定量的经过重同位素标记的片段（表1所示）加入经酶（如胰蛋白酶）消化的患者血清样品中，用定量蛋白质组学的技术检测患者血清中所述片段的水平，从而确定血清中Metaxin-2水平；将所测得的水平与根据正常人的水平确定的临界值比较，如果所测得的水平高于临界值，则表示胃癌几率大。Add a certain amount of heavy isotope-labeled fragments (shown in Table 1) to patient serum samples digested with enzymes (such as trypsin), and use quantitative proteomics techniques to detect the levels of the fragments in patient serum to determine Metaxin-2 level in serum; compare the measured level with the critical value determined according to the level of normal people, if the measured level is higher than the critical value, it means that the probability of gastric cancer is high.
表1.Table 1.
所述临界值可以由本领域技术人员根据常规手段来确定。例如，本领域技术人员可以根据测得的正常人（组）的血清蛋白质含量数据来绘制受试者工作特征曲线（ROC曲线），随后确定临界值（cut-off）。The critical value can be determined by those skilled in the art according to conventional means. For example, those skilled in the art can draw a receiver operating characteristic curve (ROC curve) according to the measured serum protein content data of normal people (group), and then determine the cut-off value.
将定量蛋白质组学技术可以和以合成肽段为基础的绝对定量技术（absolutequantification analysis, AQUA）相结合，这样就可以直接对多个样品中的Metaxin-2直接进行绝对含量的检测。将所测得的水平与正常对照血清的水平比较，如果所测得的水平高于正常对照血清的水平，则表示该对象患有胃癌。Quantitative proteomics technology can be combined with synthetic peptide-based absolute quantification analysis (AQUA), so that the absolute content of Metaxin-2 in multiple samples can be directly detected. The measured level is compared with the normal control serum level, and if the detected level is higher than the normal control serum level, it indicates that the subject has gastric cancer.
利用与人Metaxin-2蛋白特异性结合的抗体进行检测。此处的术语“抗体”具有最广义地含义，其具体地包括:单克隆抗体（包括全长的单克隆抗体）、多克隆抗体、多特异性抗体（例如双特异性抗体）。术语“抗体”还包括完整分子的片段，例如能够结合响应抗原的Fab和F（ab' ）2。Fab和F（ab' ）2片段缺少完整抗体的Fe片段，能够更迅速地从循环系统中清除，并且与完整抗体相比具有更低的非特异性组织结合性。本发明中有用的Fab和F（ab'）2以及其他抗体片段也可用于检测和定量测定人Metaxin-2，只要它们表现出所需的特异性结合的活性。这些片段通常可用木瓜酶（产生Fab片段）或胃蛋白酶（产生F（ab' ）2片段）通过蛋白酶水解切割产生。Detection is performed using an antibody that specifically binds to human Metaxin-2 protein. The term "antibody" here has the broadest meaning, which specifically includes: monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies). The term "antibody" also includes fragments of intact molecules, such as Fab and F(ab')2, which are capable of binding a responsive antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the circulation, and have lower nonspecific tissue binding than intact antibodies. Fab and F(ab')2 and other antibody fragments useful in the present invention can also be used to detect and quantify human Metaxin-2, provided they exhibit the desired specific binding activity. These fragments are typically produced by proteolytic cleavage with papain (to yield Fab fragments) or pepsin (to yield F(ab')2 fragments).
其中，用于本发明的多克隆抗体和单克隆抗体可用常规方法制得。通常，首先用蛋白来免疫合适的动物，较佳的是小鼠、大鼠、家兔或山羊。由于可获得的血清体积多，能获得标记的抗家兔和抗山羊抗体，因此对于制备多克隆抗血清来说，家兔和山羊是较佳的。免疫通常这样进行:将蛋白以盐水（较佳的以佐剂如弗氏完全佐剂）混合或乳化，然后肠胃外（通常是皮下或肌内）注射该混合物或乳剂。2-6周后用盐水（较佳的是用弗氏不完全佐剂）配的蛋白质注射一次或多次以强化免疫。将免疫后的动物血液抽取到玻璃或塑料容器中，25℃培育该血液I小时，然后4℃培育2-18小时，获得多克隆抗血清。单克隆抗体可用Kohler和Milstein的标准方法[Nature （1975） 256:495-96]或其改进方法制得。通常，如上所述对小鼠或大鼠免疫。然而，并非是对动物取血然后抽提血清，而是取出脾脏（以及任选地取出几个大的淋巴结），将其分散成单细胞。如果需要，可将细胞悬液（在除去非特异性粘附的细胞后）加入包被了蛋白质抗原的板或孔中，对脾细胞进行筛选。表达抗原特异性的膜结合免疫球蛋白的B细胞结合到板上，不象悬液其它物质那样被洗去。然后对所得B细胞或所有解离的脾细胞进行诱导，使其与骨髓瘤细胞融合形成杂交瘤，培养在选择性培养基（如次黄嘌呤、氨基蝶呤、胸苷培养基，“HAT”）中。通过有限稀释接种所得杂交瘤，并测定特异性结合免疫抗原（且不结合无关抗原）的抗体的产生。然后，体外（例如在组织培养瓶或中空纤维反应器中）或体内（如小鼠腹水中）培养所选的分泌单克隆抗体的杂交瘤。Among them, polyclonal antibodies and monoclonal antibodies used in the present invention can be produced by conventional methods. Typically, a suitable animal, preferably a mouse, rat, rabbit or goat, is first immunized with the protein. Rabbit and goat are preferred for the preparation of polyclonal antisera due to the large volume of serum available and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is usually carried out by mixing or emulsifying the protein with saline (preferably with an adjuvant such as Freund's complete adjuvant), and then injecting the mixture or emulsion parenterally (usually subcutaneously or intramuscularly). After 2-6 weeks, one or more injections of protein in saline (preferably in Freund's incomplete adjuvant) are used to boost the immunization. The blood of the immunized animal is drawn into a glass or plastic container, and the blood is incubated at 25° C. for 1 hour, and then incubated at 4° C. for 2-18 hours to obtain polyclonal antiserum. Monoclonal antibodies can be prepared by the standard method of Kohler and Milstein [Nature (1975) 256:495-96] or a modification thereof. Typically, mice or rats are immunized as described above. However, instead of bleeding the animal and extracting the serum, the spleen (and optionally several large lymph nodes) is removed and dispersed into single cells. If desired, splenocytes can be selected by adding the cell suspension (after removal of non-specifically adhered cells) to plates or wells coated with protein antigens. B cells expressing antigen-specific membrane-bound immunoglobulins bind to the plate and are not washed away like the rest of the suspension. The resulting B cells or all dissociated splenocytes are then induced to fuse with myeloma cells to form hybridomas and cultured in selective medium (e.g. hypoxanthine, aminopterin, thymidine medium, "HAT" )middle. The resulting hybridomas are inoculated by limiting dilution and assayed for the production of antibodies that specifically bind to the immunizing antigen (and not to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured in vitro (eg, in tissue culture flasks or hollow fiber reactors) or in vivo (eg, in mouse ascites).
用抗体测定血清中抗原可用免疫荧光技术，利用荧光标记的抗体并结合光学显微术、流式细胞计量术或荧光计量术检测而实现。试验方法例如包括使生物样品（如生物液体如血清）在能够鉴别可检测标记抗体存在下进行培育，然后用本领域熟知的众多方法中的任一种方法检测抗体。Antigens in serum can be determined with antibodies using immunofluorescence techniques, using fluorescently labeled antibodies combined with optical microscopy, flow cytometry or fluorometric detection. Assay methods include, for example, incubating a biological sample (eg, a biological fluid such as serum) in the presence of a detectably labeled antibody capable of identifying it, and then detecting the antibody by any of a number of methods well known in the art.
生物样品可以用固相支持物或载体（如硝酸纤维素）、或能固定细胞、细胞颗粒或可溶蛋白的其他固相支持物或载体来处理。然后用合适的缓冲液洗涤支持物或载体，再根据本发明上述那样用可检测的标记的抗体处理。再用缓冲液第二次洗涤固相支持物或载体，除去未结合的抗体。然后可用常规方法检测所述固相支持物或载体上所结合的标记的量。熟知的支持物或载体包括:玻璃、聚苯乙烯、聚丙烯、聚乙烯、葡聚糖、尼龙淀粉酶、天然的和改性的纤维素、聚丙烯酰胺、辉长岩和磁铁石。支持物或载体的结构可以是球形（如在玻珠内）、圆柱形（如试管的内表面或棒的外表面）。另外，表面可以是平坦的，例如板、测试条带等。较佳的支持物或载体包括聚苯乙烯珠。本领域技术人员可知道用于结合抗体或抗原的其他许多合适的载体，或者能够通过常规实验来确定这些载体。Biological samples can be treated with solid supports or carriers such as nitrocellulose, or other solid supports or carriers capable of immobilizing cells, cell particles, or soluble proteins. The support or carrier is then washed with a suitable buffer and treated with a detectably labeled antibody as described above according to the invention. The solid phase support or carrier is then washed a second time with buffer to remove unbound antibody. The amount of label bound to the solid support or carrier can then be detected by conventional methods. Well-known supports or carriers include: glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The structure of the support or carrier can be spherical (such as in a glass bead), cylindrical (such as the inner surface of a test tube or the outer surface of a rod). Additionally, the surface can be flat, such as a plate, test strip, etc. Preferred supports or carriers include polystyrene beads. Many other suitable carriers for binding antibodies or antigens are known to those skilled in the art, or can be ascertained by routine experimentation.
在本发明中，可将抗体与酶相连，然后用于酶连免疫分析。然后，当该酶与合适的底物接触时，酶会与底物反应，从而产生可被检测（例如通过分光光度分析、荧光分析、或肉眼观察）的化学物。可用于可检测地标记抗体的酶包括（但不局限于）:苹果酸脱氢酶、葡萄球菌核酸酶、S-5-类固醇异构酶、酵母乙醇脱氢酶、α-甘油磷酸脱氢酶、磷酸丙糖异构酶、辣根过氧化物酶、碱性磷酸酶、天冬酰胺酶、葡萄糖氧化酶、β—半乳糖苷酶、核糖核酸酶、脲酶、过氧化氢酶、葡萄糖-6-磷酸脱氢酶、葡糖淀粉酶和乙酰胆碱酯酶。采用酶的生色团底物，利用比色方法就可以完成检测。检测还可以通过将酶与底物反应的程度与类似制备的标准物进行肉眼比较来实现。In the present invention, antibodies can be linked to enzymes and then used in ELISA. Then, when the enzyme is contacted with an appropriate substrate, the enzyme reacts with the substrate to produce a chemical that can be detected (eg, by spectrophotometric analysis, fluorescent analysis, or visual inspection). Enzymes that can be used to detectably label antibodies include (but are not limited to): malate dehydrogenase, staphylococcal nuclease, S-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase , triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6 - Phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection is accomplished using a colorimetric method using a chromophore substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzyme reaction with a substrate to similarly prepared standards.
检测也可用其他各种免疫试验实现。例如，通过放射性标记抗体,就可以通过采用放射免疫分析（RIA）来检测。放射性同位素可用闪烁计数器等设备或通过放射性自显影来进行检测。Detection can also be accomplished with various other immunoassays. For example, by radiolabeling antibodies, it can be detected by using radioimmunoassay (RIA). Radioactive isotopes can be detected using equipment such as scintillation counters or by autoradiography.
还可以用荧光化合物来标记本发明的抗体。当荧光标记的抗体被暴露于适当波长的光时，就能因荧光而检测出它的存在。最常用的荧光标记化合物有异硫氰酸荧光素、罗丹明、藻红蛋白、藻青蛋白、别藻蓝蛋白、邻苯二醛和荧光胺。抗体还可用发出荧光的金属（如152E或其他镧系金属）进行可检测地标记。这些金属可通过这些金属的螯合基团（如二乙三胺五乙酸（ETPA））与抗体连接。还可以将抗体偶联化学发光化合物，对抗体进行可检测标记。然后，检测在化学反应过程中发光的存在来检测带有化学发光标记的抗体的存在。特别有用的化学发光标记化合物的例子是鲁米诺、异鲁米诺、热吖啶鎗酯、咪唑、吖啶鎗盐和草酸酯。同样，还可以用生物发光化合物来标记本发明抗体。生物发光是在生物系统中发现的一种化学发光，其中催化性蛋白提高了化学发光反应的效率。生物发光蛋白的存在可通过检测发光来确定。用于标记目的的重要的生物发光化合物是萤光素、萤光素酶和水母发光蛋白。Antibodies of the invention may also be labeled with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. The most commonly used fluorescently labeled compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Antibodies can also be detectably labeled with fluorescent metals such as 152E or other lanthanide metals. These metals can be attached to antibodies via chelating groups for these metals, such as diethylenetriaminepentaacetic acid (ETPA). The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of chemiluminescently labeled antibodies is then detected by detecting the presence of luminescence during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, pyracridinium esters, imidazoles, acridinium salts and oxalates. Likewise, antibodies of the invention may also be labeled with bioluminescent compounds. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins can be determined by detecting luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.
本发明的抗体也可用于免疫测定试验，如夹心试验。在典型的免疫测定试验中，将一定量的未标记抗体（或抗体片段）结合于固相支持物或载体上，加入一定量的带可检测标记的可溶抗体，从而可以检测和/或定量分析在固相抗体、抗原和标记抗体之间形成的三元复合物。典型的且较佳的免疫测量试验包括“正向”试验，在该试验中与固相结合的抗体先与待测试样品接触，通过形成二元固相抗体-抗原复合物从而将抗原从样品中抽提出。在培育合适的时间后，洗涤固相支持物或载体以去除液体样品残余物（包括未反应的抗原，如果有的话），然后再与含有未知量的标记抗体的溶液接触。在第二次培育使标记抗体通过未标记抗体与结合于固相支持物或载体上的抗原复合之后，第二次洗涤固相支持物或载体，除去未反应的标记抗体。Antibodies of the invention may also be used in immunoassays, such as sandwich assays. In a typical immunoassay, a certain amount of unlabeled antibody (or antibody fragment) is bound to a solid support or carrier, and a certain amount of detectably labeled soluble antibody is added so that it can be detected and/or quantified Analysis of ternary complexes formed between solid-phase antibody, antigen, and labeled antibody. Typical and preferred immunoassays include "forward" assays, in which antibodies bound to a solid phase are first contacted with the sample to be tested and the antigen is removed from the sample by the formation of a binary solid phase antibody-antigen complex. Extracted. After an appropriate incubation time, the solid support or carrier is washed to remove liquid sample residues (including unreacted antigen, if any) before being contacted with a solution containing an unknown amount of labeled antibody. After the second incubation to complex the labeled antibody with the antigen bound to the solid support or carrier by the unlabeled antibody, the solid support or carrier is washed a second time to remove unreacted labeled antibody.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明提供了一种新的特异性的胃癌生物标志物Metaxin-2，并给出了具有参考价值的Metaxin-2的检测方法。该胃癌生物标志物Metaxin-2用于胃癌的胃癌筛查/诊断效果好，准确性高，具有很好的应用前景，值得大范围推广。The invention provides a new specific gastric cancer biomarker Metaxin-2, and provides a reference value detection method for Metaxin-2. The gastric cancer biomarker Metaxin-2 is used for gastric cancer screening/diagnosis of gastric cancer with good effect and high accuracy, has a good application prospect, and is worthy of widespread promotion.
附图说明Description of drawings
图1为对Metaxin-2的免疫印迹图谱进行数据分析得出的蛋白质表达量相对分布图。Fig. 1 is a relative distribution diagram of protein expression obtained by data analysis of Metaxin-2 western blot pattern.
图2为对Metaxin-2免疫组织化学实验结果进行数据分析得出的蛋白质表达量相对分布图。Fig. 2 is a graph showing the relative distribution of protein expression obtained by data analysis of Metaxin-2 immunohistochemical experiment results.
图3为对Metaxin-2血清中蛋白质含量实验结果进行数据分析相对分布图。Fig. 3 is a relative distribution diagram of data analysis on the experimental results of protein content in Metaxin-2 serum.
具体实施方式Detailed ways
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The present invention will be further elaborated below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆:实验室手册（New York:Cold Spring Harbor Laboratory Press，1989）中所述的条件，或按照制造厂商所建议的条件。The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.
在本发明的下述实施例中，尿素、3-［3-（胆酰胺丙基）二甲氨基］丙磺酸内盐（CHAPS）、十二烷基磺酸钠（SDS）、二硫苏糖醇（DTT）、三（羟甲基）氨基乙烷（Tris）、碘代乙酰胺（IAA）购自Bio-Rad公司；β-巯基乙醇等化学试剂购自Sigma公司；胰蛋白酶（Trypsin,sequencing grade）购自 Promega 公司；iTRAQ（isobaric tags for relative andabsolute quantitation）试剂购自AB SCIEX；SCX 柱和 SAX柱以及pH缓冲液试剂盒购自waters公司。In the following examples of the present invention, urea, 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS), sodium dodecylsulfonate (SDS), dithiothreonate Sugar alcohol (DTT), tris(hydroxymethyl)aminoethane (Tris), and iodoacetamide (IAA) were purchased from Bio-Rad; chemical reagents such as β-mercaptoethanol were purchased from Sigma; trypsin (Trypsin, sequencing grade) were purchased from Promega; iTRAQ (isobaric tags for relative and absolute quantitation) reagents were purchased from AB SCIEX; SCX columns, SAX columns and pH buffer kits were purchased from waters.
在本发明的下述实施例中，使用的溶液配方如下:In the following examples of the present invention, the solution formula used is as follows:
裂解液：8mol/L尿素、4% CHAPS、40mmol/L Tris和65mmol/L DTT。Lysis solution: 8mol/L urea, 4% CHAPS, 40mmol/L Tris and 65mmol/L DTT.
TBST：Tris 2.42g/L，氯化钠8g/L，Tween-20 1ml/L，用HCl调节pH到7.6。TBST: Tris 2.42g/L, sodium chloride 8g/L, Tween-20 1ml/L, adjust the pH to 7.6 with HCl.
实施例1 iTRAQ差异蛋白质组学质谱定量分析Metaxin-2蛋白的表达含量Example 1 Quantitative analysis of Metaxin-2 protein expression by iTRAQ differential proteomics mass spectrometry
1，样本概况1. Sample overview
8例胃癌患者的癌组织及癌旁组织，该8例患者，均由3名病理科医生确诊，患有胃癌，8例患者的病理资料如表2所示。The cancer tissues and paracancerous tissues of 8 gastric cancer patients were all diagnosed by 3 pathologists as suffering from gastric cancer. The pathological data of the 8 patients are shown in Table 2.
表2：Table 2:
2，样品制备2. Sample Preparation
用酶解样品制备法（enzymatic sample preparation, ESP）制备的胃癌的癌组织与癌旁组织蛋白质样品。Gastric cancer tissue and paracancerous tissue protein samples prepared by enzymatic sample preparation (ESP).
以溶液内酶解样品制备法制备上述8例患者的胃癌的癌组织及癌旁组织蛋白质样品（所用癌组织及癌旁组织样品均为取自同一胃癌患者的成对样品），具体过程如下：The protein samples of gastric cancer tissues and paracancerous tissues of the above 8 patients were prepared by in-solution enzymatic sample preparation (the cancer tissues and paracancerous tissue samples used were paired samples from the same gastric cancer patient), and the specific process was as follows:
手术切除的新鲜组织块迅速置于冰上，快速切成几个肉眼可见、无坏死区域的小块。用预冷的PBS溶液洗涤组织小块数次后，在液氮中快速研磨成细胞沉淀，然后将细胞沉淀分别溶于裂解液中，然后于冰浴条件下，使用超声细胞破碎仪（Soniprep 150，英国，MSE公司）间歇超声破碎2min后，于15000g/min、4℃离心1h，取上清，以改良的Bradford法（见Bio-Rad公司产品说明书）进行总蛋白质定量。The surgically excised fresh tissue block is quickly placed on ice and quickly cut into several small pieces that are visible to the naked eye without areas of necrosis. After washing the small pieces of tissue with pre-cooled PBS solution several times, they were quickly ground into cell pellets in liquid nitrogen, and then the cell pellets were dissolved in the lysate respectively. , UK, MSE Company) after intermittent sonication for 2min, centrifuge at 15000g/min, 4°C for 1h, take the supernatant, and quantify the total protein by the improved Bradford method (see Bio-Rad product manual).
3，Metaxin-2检测3. Metaxin-2 detection
以iTRAQ（isobaric tags for relative and absolute quantitation）差异蛋白质组学质谱定量，具体过程如下：Quantification by iTRAQ (isobaric tags for relative and absolute quantitation) differential proteomics mass spectrometry, the specific process is as follows:
取上一步获得的8对胃癌组织及癌旁组织蛋白质样品各200ug，运用溶液内酶解技术将其酶解成肽段，然后置换成iTRAQ反应试剂，其中，采用肽段如SEQ ID NO.3～20任一所示。各取100ug分别与iTRAQ试剂混合，室温静置 1h。加入 8uL 羟胺，室温静置 15min，终止反应。合并样品，震荡使充分混合，除去样品中的盐等杂质。Take 8 pairs of gastric cancer tissue and paracancerous tissue protein samples obtained in the previous step, 200ug each, use in-solution enzymatic hydrolysis technology to enzymatically hydrolyze them into peptides, and then replace them with iTRAQ reaction reagents, in which peptides such as SEQ ID NO.3 are used Any of ~20 is shown. Take 100ug of each and mix with iTRAQ reagent respectively, and let it stand at room temperature for 1h. Add 8uL hydroxylamine and let stand at room temperature for 15min to terminate the reaction. Combine the samples and oscillate to mix thoroughly to remove impurities such as salt in the samples.
溶液内酶解技术得到的肽段混合物首先用SCX/SAX柱进行分析，然后用完全自动化的多维液相色谱串联质谱仪LTQ™Q-Exactive™ （购自Thermo Fisher公司）进行分析，得到的原始数据采用Maxquant软件（德国马普研究所）进行数据库搜索，并对鉴定到的蛋白质进行定量分析，定量结果如表3所示。The peptide mixture obtained by in-solution enzymatic hydrolysis technology was first analyzed by SCX/SAX column, and then analyzed by fully automated multidimensional liquid chromatography tandem mass spectrometer LTQ™Q-Exactive™ (purchased from Thermo Fisher). The data was searched using Maxquant software (Map Planck Institute, Germany), and the identified proteins were quantitatively analyzed. The quantitative results are shown in Table 3.
4，结果4. Results
根据表3的结果，Metaxin-2在胃癌组织中的表达含量与癌旁组织相比，在胃癌癌旁组织中高表达2.7倍以上，胃癌组织中表达明显下调。According to the results in Table 3, the expression level of Metaxin-2 in gastric cancer tissues was more than 2.7 times higher in gastric cancer tissues than in adjacent tissues, and the expression in gastric cancer tissues was significantly down-regulated.
表3：胃癌组织及癌旁组织中Metaxin-2的定量结果。Table 3: Quantitative results of Metaxin-2 in gastric cancer tissues and adjacent tissues.
结果发现Metaxin-2 在胃癌癌组织中表达下调。因此，以Metaxin-2 作为一个蛋白质分子标记物对它的表达量进行检测可以用于检测胃癌，即Metaxin-2 可以用作检测胃癌的蛋白质分子。It was found that the expression of Metaxin-2 was down-regulated in gastric cancer tissues. Therefore, using Metaxin-2 as a protein molecular marker to detect its expression level can be used to detect gastric cancer, that is, Metaxin-2 can be used as a protein molecule to detect gastric cancer.
实施例2 免疫印迹定量分析Metaxin-2蛋白的表达含量Example 2 Quantitative analysis of the expression content of Metaxin-2 protein by western blot
1，样本概况1. Sample overview
取6对胃癌组织及相应的癌旁组织的蛋白质样品，其病理资料如表4所示。The protein samples of 6 pairs of gastric cancer tissues and corresponding paracancerous tissues were taken, and their pathological data are shown in Table 4.
表4：6例胃癌标本的病理资料。Table 4: Pathological data of 6 gastric cancer specimens.
2，样品制备2. Sample Preparation
同时实施例2中的2，样品制备。Meanwhile, 2 in Example 2, sample preparation.
3，Metaxin-2检测3. Metaxin-2 detection
使用购买的抗Metaxin-2 抗体对上述6对胃癌组织及相应的癌旁组织的蛋白质样品进行免疫印迹分析，过程如下：Use the purchased anti-Metaxin-2 antibody to perform Western blot analysis on the protein samples of the above 6 pairs of gastric cancer tissues and corresponding paracancerous tissues, the process is as follows:
每个样品取20ug蛋白质样品用12% SDS-PAGE分离，转移至PVDF膜（购自GE Healthcare公司）上；Take 20ug protein samples from each sample and use 12% SDS-PAGE to separate and transfer to PVDF membrane (purchased from GE Healthcare);
一抗使用兔抗人Metaxin-2 多克隆抗体（购自Abcam公司，1: 1000稀释），4℃孵育过夜，用TBST洗涤三次，每次5分钟；Rabbit anti-human Metaxin-2 polyclonal antibody (purchased from Abcam, diluted 1:1000) was used as primary antibody, incubated overnight at 4°C, washed three times with TBST, 5 minutes each time;
二抗为抗兔抗体（购自Santa Cruz公司，I: 10000稀释），室温孵育I小时，再用TBST洗涤三次，每次10分钟；The secondary antibody was an anti-rabbit antibody (purchased from Santa Cruz Company, diluted at 1: 10000), incubated at room temperature for 1 hour, and then washed three times with TBST, each time for 10 minutes;
加入ECL plus试剂（购自GE Healthcare公司）反应5分钟后，以X-光片曝光检测。After adding ECL plus reagent (purchased from GE Healthcare) and reacting for 5 minutes, it was detected by X-ray film exposure.
对免疫印迹图谱采用Gel-Pro Analyzer凝胶定量分析软件（Media Cybernetic公司）进行数据分析，得出蛋白质表达量的相对分布图。Gel-Pro Analyzer gel quantitative analysis software (Media Cybernetic Company) was used for data analysis of the western blot pattern, and the relative distribution map of protein expression was obtained.
4，结果4. Results
结果如图1所示。图1结果显示，在胃癌癌旁组织中的表达量明显高于相应的癌组织，其平均比值为2.67，P值（配对t检验）为0.00066。根据图1的结果，Metaxin-2 在胃癌癌旁组织存在高表达，该结果与质谱结果一致。The result is shown in Figure 1. The results in Figure 1 show that the expression level in gastric cancer paracancerous tissues is significantly higher than that in corresponding cancer tissues, with an average ratio of 2.67 and a P value (paired t test) of 0.00066. According to the results in Figure 1, Metaxin-2 is highly expressed in gastric cancer paracancerous tissues, which is consistent with the results of mass spectrometry.
实施例3 胃癌组织芯片免疫组织化学研究定量分析Metaxin-2蛋白的表达含量Example 3 Quantitative Analysis of Metaxin-2 Protein Expression in Gastric Cancer Tissue Microarray Immunohistochemical Study
为了进一步证实Metaxin-2 在胃癌组织和癌旁组织之间的表达差异，使用胃癌组织芯片，进行免疫组织化学研究。In order to further confirm the expression difference of Metaxin-2 between gastric cancer tissues and paracancerous tissues, immunohistochemical studies were performed using gastric cancer tissue microarrays.
1，样本概况1. Sample overview
随机选取69对胃癌的癌组织和对应的癌旁组织样本，选取的样本的具体资料分别如表5所示。69 pairs of gastric cancer tissues and corresponding paracancerous tissue samples were randomly selected, and the specific data of the selected samples are shown in Table 5.
表5：胃癌组织芯片资料。Table 5: Gastric cancer tissue microarray data.
2，样品制备及Metaxin-2检测2. Sample preparation and Metaxin-2 detection
（1）将组织切片置于60℃恒温箱烘烤约3个小时，取出后依次使用二甲苯、二甲苯/乙醇（1：1）、100%乙醇、90%乙醇、80%乙醇、70%乙醇、50%乙醇和水，进行脱蜡水化处理；(1) Bake the tissue slices in a constant temperature oven at 60°C for about 3 hours, then use xylene, xylene/ethanol (1:1), 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol Ethanol, 50% ethanol and water for dewaxing and hydration treatment;
（2）PBS洗3次，每次5分钟；0.3% H2O2 （甲醇稀释）浸泡半小时，PBS洗3次，每次5分钟；(2) Wash 3 times with PBS, 5 minutes each time; soak in 0.3% H2 O2 (diluted with methanol) for half an hour, wash 3 times with PBS, 5 minutes each time;
（3）高压修复抗原，双蒸水洗2次，每次5分钟；PBS洗2次，每次5分钟；10%血清室温封闭20分钟；(3) Antigen retrieval by high pressure, wash twice with double distilled water, 5 minutes each time; wash twice with PBS, 5 minutes each time; block with 10% serum at room temperature for 20 minutes;
（4）加入兔抗人Metaxin-2 多克隆抗体（购自Abcam公司，1：50稀释），4℃过夜，PBST洗3次，每次5分钟；(4) Add rabbit anti-human Metaxin-2 polyclonal antibody (purchased from Abcam, diluted 1:50), overnight at 4°C, wash 3 times with PBST, 5 minutes each time;
（5）加入生物素标记的羊抗兔抗体（来自ABC试剂盒，购自VECTOR公司），室温孵育30分钟，PBST洗3次，每次5分钟；PBS洗2次，每次5分钟；(5) Add biotin-labeled goat anti-rabbit antibody (from ABC kit, purchased from VECTOR Company), incubate at room temperature for 30 minutes, wash 3 times with PBST, 5 minutes each time; wash 2 times with PBS, 5 minutes each time;
（6）加入ABC溶液（来自ABC试剂盒，购自VECTOR公司），室温孵育30分钟，PBS洗3次，每次5分钟；(6) Add ABC solution (from ABC kit, purchased from VECTOR), incubate at room temperature for 30 minutes, wash with PBS 3 times, 5 minutes each time;
（7）DAB溶液（购自上海生工生物工程技术服务有限公司）显色；苏木精（购自VECTOR公司，H3404）染色20秒；(7) DAB solution (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.) for color development; hematoxylin (purchased from VECTOR, H3404) for 20 seconds;
（8）组织切片依次使用：50 %乙醇、70 %乙醇、80 %乙醇、90 %乙醇、100 %乙醇、二甲苯/乙醇（1：1）、二甲苯，进行脱水透明处理。(8) Tissue sections were used in turn: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, xylene/ethanol (1:1), xylene for dehydration and transparent treatment.
（9）然后使用中性树脂封片，于显微镜观察，对组织芯片进行评估。(9) Then use neutral resin to seal the slide, observe under a microscope, and evaluate the tissue chip.
3、结果3. Results
结果显示：组织芯片中共69对有效样本，并根据染色强度和阳性率打分，其中，染色强度打分标准为：阴性为0分、弱阳性为1分、中等阳性为2分、强阳性为3分；阳性率打分标准为：低于5%为0分，5%~30%为1分，31%~60%为2分，60%以上为3分。将染色强度的得分与阳性率的得分相加，获得组织切片的综合得分，得分情况如表6所示。The results show that there are 69 pairs of valid samples in the tissue chip, and they are scored according to the staining intensity and positive rate. The staining intensity scoring standard is: 0 points for negative, 1 point for weak positive, 2 points for moderate positive, and 3 points for strong positive The scoring standard for the positive rate is: less than 5% is 0 point, 5%-30% is 1 point, 31%-60% is 2 points, and more than 60% is 3 points. The score of the staining intensity and the score of the positive rate were added to obtain the comprehensive score of the tissue section, and the scores are shown in Table 6.
表6：胃癌组织芯片综合得分Table 6: Comprehensive score of gastric cancer tissue microarray
根据表6的结果计算得到图2：胃癌癌组织的平均综合得分为2，癌旁组织的平均综合得分为3.29，两者具有极显著差异，P值为8.09E-9。统计发现，在超过83% （57对）的样本对中，Metaxin-2 在胃癌癌旁细胞中高表达。该结果与前面的质谱结果、免疫印迹结果相一致。Figure 2 is calculated according to the results in Table 6: the average comprehensive score of gastric cancer tissue is 2, and the average comprehensive score of paracancerous tissue is 3.29, and there is a very significant difference between them, with a P value of 8.09E-9. Statistics found that in more than 83% (57 pairs) of sample pairs, Metaxin-2 was highly expressed in gastric cancer paracancerous cells. This result is consistent with the previous mass spectrometry results and western blot results.
实施例4 酶联免疫吸附定量分析Metaxin-2蛋白的表达含量Example 4 ELISA Quantitative Analysis of Metaxin-2 Protein Expression Content
为了进一步发现Metaxin-2 在胃癌血清和正常人血清之间的表达差异，进行酶联免疫吸附测定。To further discover the expression difference of Metaxin-2 between gastric cancer serum and normal human serum, an enzyme-linked immunosorbent assay was performed.
1，样本概况1. Sample overview
随机选取35例胃癌患者的血清和30例正常人血清样本，其中，选取的样本的具体资料分别如表7和表8所示。Serum samples from 35 gastric cancer patients and 30 normal persons were randomly selected, and the specific data of the selected samples are shown in Table 7 and Table 8, respectively.
表7：胃癌血清样本信息。Table 7: Gastric cancer serum sample information.
表8：正常血清样本信息。Table 8: Normal serum sample information.
2，样品制备及Metaxin-2检测2. Sample preparation and Metaxin-2 detection
血清与抗体经过一个小时的孵育，然后洗板，加底物，半个小时避光反应后加终止液完成反应。Serum and antibody were incubated for one hour, then the plate was washed, substrate was added, and stop solution was added after half an hour of dark reaction to complete the reaction.
3，结果3. Results
根据酶联免疫吸附测定的结果得到图3，统计发现，Metaxin-2在胃癌的患者血清中低表达。该结果与前面的质谱结果、免疫印迹结果以及免疫组化结果相一致。Figure 3 was obtained according to the results of the enzyme-linked immunosorbent assay, and it was found that Metaxin-2 was lowly expressed in the serum of patients with gastric cancer. This result is consistent with the previous mass spectrometry results, western blot results and immunohistochemistry results.
综上所述，Metaxin-2 在胃癌的癌组织和癌旁组织中存在明显的差异表达，并且在胃癌患者的血清中表达降低，显然与胃癌的发生发展有着密切的相关性，因此它的表达量可以用于检测胃癌。相应的，特异性Metaxin-2的抗体，包括各种Metaxin-2的单克隆抗体和多克隆抗体，由于其能够用于检测Metaxin-2的表达量，因而可以用于检测胃癌，或者用于制备检测胃癌的制剂或试剂盒。To sum up, Metaxin-2 is significantly differentially expressed in gastric cancer tissues and paracancerous tissues, and its expression is reduced in the serum of gastric cancer patients, which is obviously closely related to the occurrence and development of gastric cancer. Therefore, its expression The amount can be used to detect gastric cancer. Correspondingly, specific Metaxin-2 antibodies, including various Metaxin-2 monoclonal antibodies and polyclonal antibodies, can be used to detect gastric cancer because they can be used to detect the expression level of Metaxin-2, or to prepare A preparation or a kit for detecting gastric cancer.
虽然有关Metaxin-2动态的生物学功能及肿瘤相关机制还有待进一步研究，但是将它作为检测胃癌的标记物却是肯定的。Although the biological function and tumor-related mechanism of Metaxin-2 dynamics need to be further studied, it is certain to use it as a marker for detecting gastric cancer.
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<211> 27<211> 27
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 13<400> 13
Gly Glu Gln Ile Leu Leu Ser Asp Asn Ala Ala Ser Leu Ala Val GlnGly Glu Gln Ile Leu Leu Ser Asp Asn Ala Ala Ser Leu Ala Val Gln
1 5 10 151 5 10 15
Ala Phe Leu Gln Met Cys Asn Leu Pro Ile LysAla Phe Leu Gln Met Cys Asn Leu Pro Ile Lys
20 25 20 25
<210> 14<210> 14
<211> 22<211> 22
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 14<400> 14
Tyr Gly Ser Pro Tyr Pro Trp Pro Leu Asn His Ile Leu Ala Tyr GlnTyr Gly Ser Pro Tyr Pro Trp Pro Leu Asn His Ile Leu Ala Tyr Gln
1 5 10 151 5 10 15
Lys Gln Trp Glu Val LysLys Gln Trp Glu Val Lys
20 20
<210> 15<210> 15
<211> 24<211> 24
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 15<400> 15
Val Pro Phe Ile His Val Gly Asn Gln Val Val Ser Glu Leu Gly ProVal Pro Phe Ile His Val Gly Asn Gln Val Val Ser Glu Leu Gly Pro
1 5 10 151 5 10 15
Ile Val Gln Phe Val Lys Ala LysIle Val Gln Phe Val Lys Ala Lys
20 20
<210> 16<210> 16
<211> 22<211> 22
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 16<400> 16
Val Pro Phe Ile His Val Gly Asn Gln Val Val Ser Glu Leu Gly ProVal Pro Phe Ile His Val Gly Asn Gln Val Val Ser Glu Leu Gly Pro
1 5 10 151 5 10 15
Ile Val Gln Phe Val LysIle Val Gln Phe Val Lys
20 20
<210> 17<210> 17
<211> 20<211> 20
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 17<400> 17
Lys Thr Leu Asp Gln Val Leu Glu Asp Val Asp Gln Cys Cys Gln AlaLys Thr Leu Asp Gln Val Leu Glu Asp Val Asp Gln Cys Cys Gln Ala
1 5 10 151 5 10 15
Leu Ser Gln ArgLeu Ser Gln Arg
20 20
<210> 18<210> 18
<211> 19<211> 19
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 18<400> 18
Thr Leu Asp Gln Val Leu Glu Asp Val Asp Gln Cys Cys Gln Ala LeuThr Leu Asp Gln Val Leu Glu Asp Val Asp Gln Cys Cys Gln Ala Leu
1 5 10 151 5 10 15
Ser Gln ArgSer Gln Arg
<210> 19<210> 19
<211> 17<211> 17
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 19<400> 19
Tyr Gly Ser Pro Tyr Pro Trp Pro Leu Asn His Ile Leu Ala Tyr GlnTyr Gly Ser Pro Tyr Pro Trp Pro Leu Asn His Ile Leu Ala Tyr Gln
1 5 10 151 5 10 15
LysLys
<210> 20<210> 20
<211> 17<211> 17
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 20<400> 20
Gly His Ser Leu Ser Asp Gly Leu Glu Glu Val Gln Lys Ala Glu MetGly His Ser Leu Ser Asp Gly Leu Glu Glu Val Gln Lys Ala Glu Met
1 5 10 151 5 10 15
LysLys
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810187002.4ACN108490179A (en) | 2018-03-07 | 2018-03-07 | Application of Metaxin-2 as gastric cancer marker |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810187002.4ACN108490179A (en) | 2018-03-07 | 2018-03-07 | Application of Metaxin-2 as gastric cancer marker |
| Publication Number | Publication Date |
|---|---|
| CN108490179Atrue CN108490179A (en) | 2018-09-04 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810187002.4APendingCN108490179A (en) | 2018-03-07 | 2018-03-07 | Application of Metaxin-2 as gastric cancer marker |
| Country | Link |
|---|---|
| CN (1) | CN108490179A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5858714A (en)* | 1997-05-29 | 1999-01-12 | Incyte Pharmaceuticals, Inc. | Human metaxin protein |
| CN1852974A (en)* | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5858714A (en)* | 1997-05-29 | 1999-01-12 | Incyte Pharmaceuticals, Inc. | Human metaxin protein |
| CN1852974A (en)* | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| Title |
|---|
| PIERRE—FRANCOIS CARTRON等: "《Metaxin 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery, are essential for Bak activation during TNF alpha triggered apoptois》", 《CELLULAR SIGNALLING》* |
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|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20180904 | |
| RJ01 | Rejection of invention patent application after publication |