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CN118914559A - Protein composition and application thereof in diagnostic field - Google Patents

Protein composition and application thereof in diagnostic field Download PDF

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CN118914559A
CN118914559A CN202410912508.2A CN202410912508A CN118914559A CN 118914559 A CN118914559 A CN 118914559A CN 202410912508 A CN202410912508 A CN 202410912508A CN 118914559 A CN118914559 A CN 118914559A
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lung cancer
protein composition
ddx53
mmrn2
mknk1
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温颖声
杨安力
季奥
伊格纳西奥·皮诺
胡少晖
李宇欣
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Jiaxing Xinyuan Biotechnology Co ltd
Nanjing Xinyuan Biotechnology Co ltd
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Jiaxing Xinyuan Biotechnology Co ltd
Nanjing Xinyuan Biotechnology Co ltd
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    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

本申请公开了蛋白组合物及其在诊断领域的应用,属于生物医药技术领域。该蛋白组合物包括本申请新筛选的抗原蛋白DDX53、MMRN2,以及抗原蛋白MKNK1、FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种或多种进行组合,将其组合并将其用于检测抗体并进行肺癌早期诊断,其敏感性为68~84%,特异性为90~100%,提高了肺癌早期诊断的敏感性和特异性。

The present application discloses a protein composition and its application in the field of diagnosis, belonging to the field of biomedical technology. The protein composition comprises the antigenic proteins DDX53 and MMRN2 newly screened in the present application, and any one or more of the antigenic proteins MKNK1, FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2 are combined, and the combination is used to detect antibodies and perform early diagnosis of lung cancer, with a sensitivity of 68-84% and a specificity of 90-100%, which improves the sensitivity and specificity of early diagnosis of lung cancer.

Description

蛋白组合物及其在诊断领域的应用Protein composition and its application in diagnosis field

本发明专利申请是针对申请号为202311526662.8的分案申请,原申请的申请日为:2023-11-15,发明创造名称为:蛋白组合物及其在诊断领域的应用。The patent application of this invention is a divisional application with application number 202311526662.8. The application date of the original application is: 2023-11-15. The name of the invention is: protein composition and its application in the field of diagnosis.

技术领域Technical Field

本申请属于生物医药技术领域,具体涉及蛋白组合物及其在诊断领域的应用。The present application belongs to the field of biomedical technology, and specifically relates to a protein composition and its application in the field of diagnosis.

背景技术Background Art

蛋白质是生命的物质基础,是构成细胞的基本有机物,是生命活动的主要承担者。在人体中,蛋白质种类多达上万种。蛋白质具有多种重要的生物学功能,包括结构功能、酶催化作用、激素调节作用、免疫功能、运输作用和储能作用等。鉴于蛋白质的多种生物学功能,自然界存在的或者人工合成的蛋白质可以作为生物标记物用于多种疾病的早期诊断、可以作为抗体进行多种疾病的治疗等。Protein is the material basis of life, the basic organic matter that constitutes cells, and the main bearer of life activities. In the human body, there are tens of thousands of types of proteins. Proteins have many important biological functions, including structural functions, enzyme catalysis, hormone regulation, immune function, transportation and energy storage. In view of the various biological functions of proteins, proteins existing in nature or artificially synthesized can be used as biomarkers for early diagnosis of various diseases, and can be used as antibodies for the treatment of various diseases.

早在上世纪60年代,Robert W.Baldwin就已经证实在癌症发展的早期,人体免疫系统可以产生自身抗体。由于自身抗体的产生是免疫系统对肿瘤相关抗原的体液免疫反应,因此血清中自身抗体的水平远比常规的抗原水平高。并且,由于抗原-抗体反应的高特异性和高敏感性,即使在血清中有大量白蛋白干扰的情况下,自身抗体也较其它标志物更易准确检测到。换言之,肿瘤自身抗体比相应肿瘤抗原更容易被检测到。因此,通过检测自身抗体进行肺癌早期诊断具有更加广阔的前景。As early as the 1960s, Robert W. Baldwin had confirmed that the human immune system can produce autoantibodies in the early stages of cancer development. Since the production of autoantibodies is a humoral immune response of the immune system to tumor-associated antigens, the level of autoantibodies in serum is much higher than the conventional antigen level. In addition, due to the high specificity and sensitivity of the antigen-antibody reaction, autoantibodies are easier to detect accurately than other markers even in the presence of a large amount of albumin interference in the serum. In other words, tumor autoantibodies are easier to detect than the corresponding tumor antigens. Therefore, early diagnosis of lung cancer by detecting autoantibodies has a broader prospect.

肺癌是我国及全球范围内发病率和死亡率最高的恶性肿瘤之一。肺癌的预后与其发展阶段紧密相关,局限性肺癌五年存活率为53%,而在出现转移的情况下五年存活率仅为4%。因此肺癌的早期筛查与早期诊断已成为延长患者生存期、挽救患者生命的关键,肺癌的早期发现和及时防治能显著提高患者的存活率和生活质量。Lung cancer is one of the malignant tumors with the highest morbidity and mortality in my country and around the world. The prognosis of lung cancer is closely related to its development stage. The five-year survival rate of localized lung cancer is 53%, while the five-year survival rate is only 4% when metastasis occurs. Therefore, early screening and early diagnosis of lung cancer have become the key to prolonging the survival of patients and saving their lives. Early detection and timely prevention and treatment of lung cancer can significantly improve the survival rate and quality of life of patients.

在相关技术中,如Chapman等使用酶联免疫法比较了采用6个抗原蛋白(p53、NY-ESO-1、CAGE、GBU4-5、Annexin I和SOX2)和7个抗原蛋白(p53、NY-ESO-1、CAGE、GBU4-5、SOX2、HuD和MAGE A4)来检测肺癌患者和健康对照组血清中相应自身抗体的含量,结果显示采用6个自身抗体联合检测得到的敏感性和特异性分别为39%和89%,而采用7个自身抗体联合检测的敏感性和特异性分别为41%和91%。又如公开号为CN103869086A的中国发明专利记载了一种血清自身抗体检测试剂盒,该试剂盒包括选自p53、Annexin1、CAGE、NY-ESO-1、HuD、Cyclin D、PGP9.5、GBU4-5、MDM2、GAGE7、XAGE1b、SOX2、MAGE A1和MAGE A4等14种抗原蛋白中的五种或以上的组合的抗原蛋白,但是其发现GBU4-5、SOX2、HuD、Cyclin D、NY-ESO-1、Annexin1、MAGE A1和XAGE1b相对应的抗体在中国正常人和肺癌患者中几乎没有区别,不适于在中国人中用作早期或辅助诊断的生物标志物,这样的差距有可能是由于检测人种的不同造成的差异,并具体公开了12种组合及其对应的敏感性和特异性,包括4~7个抗原蛋白的组合,其敏感性为27.5%~62.5%,特异性为74.5%~92.7%。当选取4个抗原蛋白(p53、NY-ESO-1、P62、CAGE)时,其敏感性为27.5%,特异性为87.3%,因此并未采用4个抗原蛋白的技术方案。又如公开号为WO2018187228A1、WO2020069637A1和US20220003768A1的发明专利,记载了本申请人早期的研究成果,包括ETHE1、p53、CTAG1A、C1QTNF1、TEX264、CLDN2、NSG1和HRas 8种抗原蛋白中的3~5种抗原蛋白的组合,进一步利用IgA筛选了28种抗原蛋白与上述8种抗原蛋白组合,并对其中任意2~6种抗原蛋白的组合进行评估,公开了200种组合的结果,其中67种组合物辨别能力为高,67种为中等,其余为低。又如公开号为CN116482365A的中国发明专利记载了用于检测血清抗体的蛋白组合物及其应用,包括XAGE1A、RBPJ、CEACAM5、NSG1、TP53、DDHYLS1、NUDT14、PGK1、KRT19、MAGEC2和SULT2B1共计11个抗原蛋白,将其任意两种或多种组合,当组合蛋白的数量为2时,选用XAGE1A和CEACAM5两种蛋白效果最好(敏感性为45.45%,特异性为100%,平均值为72.73%);当组合蛋白的数量为3时,选用XAGE1A、CEACAM5和RBPJ三种蛋白效果最好(敏感性为59.10%,特异性为94.74%,平均值为76.91%);当选用全部11个蛋白时,其敏感性为86.36%,特异性为84.21%,平均值为85.29%。In related technologies, Chapman et al. used enzyme-linked immunosorbent assay to compare the use of 6 antigen proteins (p53, NY-ESO-1, CAGE, GBU4-5, Annexin I and SOX2) and 7 antigen proteins (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD and MAGE A4) to detect the levels of corresponding autoantibodies in the serum of lung cancer patients and healthy controls. The results showed that the sensitivity and specificity of the combined detection of 6 autoantibodies were 39% and 89%, respectively, while the sensitivity and specificity of the combined detection of 7 autoantibodies were 41% and 91%, respectively. For another example, the Chinese invention patent with publication number CN103869086A records a serum autoantibody detection kit, which includes antigen proteins selected from five or more of 14 antigen proteins, including p53, Annexin1, CAGE, NY-ESO-1, HuD, Cyclin D, PGP9.5, GBU4-5, MDM2, GAGE7, XAGE1b, SOX2, MAGE A1 and MAGE A4. However, it was found that the antibodies corresponding to GBU4-5, SOX2, HuD, Cyclin D, NY-ESO-1, Annexin1, MAGE A1 and XAGE1b were almost indistinguishable between normal Chinese and lung cancer patients, and were not suitable for use as biomarkers for early or auxiliary diagnosis in Chinese. Such a gap may be due to differences caused by different ethnic groups detected. The patent specifically discloses 12 combinations and their corresponding sensitivities and specificities, including a combination of 4 to 7 antigen proteins, with a sensitivity of 27.5% to 62.5% and a specificity of 74.5% to 92.7%. When four antigen proteins (p53, NY-ESO-1, P62, CAGE) were selected, the sensitivity was 27.5% and the specificity was 87.3%, so the technical solution of four antigen proteins was not adopted. For example, the invention patents with publication numbers WO2018187228A1, WO2020069637A1 and US20220003768A1 record the early research results of the applicant, including a combination of 3 to 5 of the eight antigen proteins ETHE1, p53, CTAG1A, C1QTNF1, TEX264, CLDN2, NSG1 and HRas, and further screened 28 antigen proteins and the above eight antigen proteins using IgA, and evaluated the combination of any 2 to 6 antigen proteins, disclosing the results of 200 combinations, of which 67 combinations had high discrimination ability, 67 were medium, and the rest were low. For another example, the Chinese invention patent with publication number CN116482365A records a protein composition for detecting serum antibodies and its application, including 11 antigenic proteins, namely XAGE1A, RBPJ, CEACAM5, NSG1, TP53, DDHYLS1, NUDT14, PGK1, KRT19, MAGEC2 and SULT2B1. Any two or more of them are combined. When the number of combined proteins is 2, the two proteins XAGE1A and CEACAM5 have the best effect (sensitivity is 45.45%, specificity is 100%, and the average value is 72.73%); when the number of combined proteins is 3, the three proteins XAGE1A, CEACAM5 and RBPJ have the best effect (sensitivity is 59.10%, specificity is 94.74%, and the average value is 76.91%); when all 11 proteins are selected, the sensitivity is 86.36%, the specificity is 84.21%, and the average value is 85.29%.

由此可见,抗原蛋白的选择,包括抗原蛋白种类和抗原蛋白数量,显著影响了肺癌早期诊断的敏感性和特异性。是否有其余更加适用的抗原蛋白?新的抗原蛋白组合是否具有临床意义?新的抗原蛋白组合是否能够提高肺癌早期诊断的敏感性和特异性?随着我国肿瘤发病率的逐年攀升,对高敏感性的、高通量的、可推广使用的自身抗体的检测方法成为业界急需解决的一个问题。It can be seen that the selection of antigen proteins, including the type and quantity of antigen proteins, significantly affects the sensitivity and specificity of early diagnosis of lung cancer. Are there other more suitable antigen proteins? Does the new antigen protein combination have clinical significance? Can the new antigen protein combination improve the sensitivity and specificity of early diagnosis of lung cancer? With the increasing incidence of tumors in my country, the detection method of autoantibodies with high sensitivity, high throughput and scalable application has become an urgent problem that the industry needs to solve.

发明内容Summary of the invention

1.要解决的问题1. Problem to be solved

本申请针对现有技术中基于肺癌自身抗体的肺癌早期诊断方法的敏感性和特异性需要进一步提高的问题,在前期研究的基础上,进一步筛选了肺癌相关抗原,并将筛选的肺癌相关抗原进行组合,并将其用于检测肺癌抗体,进一步用于肺癌的早期诊断。This application aims to address the problem that the sensitivity and specificity of the prior art early diagnosis method for lung cancer based on lung cancer autoantibodies need to be further improved. Based on previous research, lung cancer-related antigens are further screened, and the screened lung cancer-related antigens are combined and used to detect lung cancer antibodies, which are further used for the early diagnosis of lung cancer.

2.技术方案2. Technical solution

为了解决上述问题,本申请所采用的技术方案如下:In order to solve the above problems, the technical solutions adopted in this application are as follows:

本申请提供了蛋白组合物在制备用于肺癌早期诊断产品中的应用,该产品包含蛋白组合物,该蛋白组合物包括抗原蛋白DDX53和MMRN2,还包括抗原蛋白MKNK1、FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种或多种,该诊断产品利用蛋白组合物检测血清中肺癌自身IgG抗体,从而用于肺癌早期诊断。The present application provides an application of a protein composition in the preparation of a product for early diagnosis of lung cancer. The product comprises a protein composition, which includes antigenic proteins DDX53 and MMRN2, and also includes any one or more of antigenic proteins MKNK1, FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2. The diagnostic product utilizes the protein composition to detect lung cancer autologous IgG antibodies in serum, thereby being used for early diagnosis of lung cancer.

进一步地,上述蛋白组合物包括抗原蛋白DDX53和MMRN2,还包括抗原蛋白MKNK1、FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes the antigenic proteins DDX53 and MMRN2, and also includes any one of the antigenic proteins MKNK1, FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和MKNK1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and MKNK1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和FGF12。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and FGF12.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和PIP4K2C。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和NME3。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和CTAG1A。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and CTAG1A.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和HYLS1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and HYLS1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和TIA1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和SSBP4。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and SSBP4.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和ENO2。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和MKNK1,还包括抗原蛋白FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2 and MKNK1, and also includes any one or more of antigenic proteins FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2和MKNK1,还包括抗原蛋白FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2 and MKNK1, and also includes any one of antigenic proteins FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和FGF12。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and FGF12.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和CTAG1A。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and CTAG1A.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和HYLS1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and HYLS1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和SSBP4。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and SSBP4.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和TIA1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和PIP4K2C。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和NME3。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和ENO2。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和FGF12,还包括抗原蛋白PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and FGF12, and also includes any one or more of antigenic proteins PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1和FGF12,还包括抗原蛋白PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1 and FGF12, and also includes any one of antigenic proteins PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和HYLS1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and HYLS1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和CTAG1A。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and CTAG1A.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和TIA1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和SSBP4。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and SSBP4.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和NME3。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和PIP4K2C。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和ENO2。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和HYLS1,还包括抗原蛋白PIP4K2C、NME3、CTAG1A、TIA1、SSBP4和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and HYLS1, and also includes any one or more of antigenic proteins PIP4K2C, NME3, CTAG1A, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12和HYLS1,还包括抗原蛋白PIP4K2C、NME3、CTAG1A、TIA1、SSBP4和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12 and HYLS1, and also includes any one of antigenic proteins PIP4K2C, NME3, CTAG1A, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和CTAG1A。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and CTAG1A.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和SSBP4。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and SSBP4.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和TIA1。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和NME3。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和PIP4K2C。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和ENO2。Furthermore, the above protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和CTAG1A,还包括抗原蛋白PIP4K2C、NME3、TIA1、SSBP4和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and CTAG1A, and also includes any one or more of antigenic proteins PIP4K2C, NME3, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1和CTAG1A,还包括抗原蛋白PIP4K2C、NME3、TIA1、SSBP4和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1 and CTAG1A, and also includes any one of antigenic proteins PIP4K2C, NME3, TIA1, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和SSBP4。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and SSBP4.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和TIA1。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和NME3。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和PIP4K2C。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和SSBP4,还包括抗原蛋白PIP4K2C、NME3、TIA1和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and SSBP4, and also includes any one or more of antigenic proteins PIP4K2C, NME3, TIA1 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A和SSBP4,还包括抗原蛋白PIP4K2C、NME3、TIA1和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A and SSBP4, and also includes any one of antigenic proteins PIP4K2C, NME3, TIA1 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和TIA1。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and TIA1.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和NME3。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和PIP4K2C。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和TIA1,还包括抗原蛋白PIP4K2C、NME3和ENO2中的任意一种或多种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and TIA1, and also includes any one or more of antigenic proteins PIP4K2C, NME3 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4和TIA1,还包括抗原蛋白PIP4K2C、NME3和ENO2中的任意一种。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4 and TIA1, and also includes any one of the antigenic proteins PIP4K2C, NME3 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1和NME3。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1 and NME3.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1和PIP4K2C。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1和ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1和NME3,还包括抗原蛋白PIP4K2C和/或ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1 and NME3, and also includes antigenic proteins PIP4K2C and/or ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1、NME3和PIP4K2C。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1, NME3 and PIP4K2C.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1、NME3和ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1, NME3 and ENO2.

进一步地,上述蛋白组合物包括抗原蛋白DDX53、MMRN2、MKNK1、FGF12、HYLS1、CTAG1A、SSBP4、TIA1、NME3、PIP4K2C和ENO2。Furthermore, the above-mentioned protein composition includes antigenic proteins DDX53, MMRN2, MKNK1, FGF12, HYLS1, CTAG1A, SSBP4, TIA1, NME3, PIP4K2C and ENO2.

进一步地,上述抗原蛋白DDX53是包括具有如SEQ ID NO:1所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:1有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:1所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MSHWAPEWKRAEANPRDLGASWDVRGSRGSGWSGPFGHQGPRAAGSREPPLCFKIKNNMVGVVIGYSGSKIKDLQHSTNTKIQIINGESEAKVRIFGNREMKAKAKAAIETLIRKQESYNSESSVDNAASQTPIGRNLGRNDIVGEAEPLSNWDRIRAAVVECEKRKWADLPPVKKNFYIESKATSCMSEMQVINWRKENFNITCDDLKSGEKRLIPKPTCRFKDAFQQYPDLLKSIIRVGILKPTPIQSQAWPIILQGIDLIVVAQTGTGKTLSYLMPGFIHLDSQPISREQRNGPGMLVLTPTRELALHVEAECSKYSYKGLKSICIYGGRNRNGQIEDISKGVDIIIATPGRLNDLQMNNSVNLRSITYLVIDEADKMLDMEFEPQIRKILLDVRPRQTVMTSATWPDTVRQLALSYLKDPMIVYVGNLNLVAVNTVKQNIIVTTEKEKRALTQEFVENMSPNDKVIMFVSQKHIADDLSSDFNIQGISAESLHGNSEQSDQERAVEDFKSGNIKILITTDIVSRGLDLNDVTHVYNYDFPRNIDVYVHRVGYIGRTGKTGTSVTLITQRDSKMAGELIKILDRANQSVPEDLVVMAEQYKLNQQKRHRETRSREPGQRRKEFYFLS(SEQ ID NO:1)。Furthermore, the antigen protein DDX53 comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide having at least 80% homology with the sequence SEQ ID NO: 1 and having the same or substantially the same function, or a polypeptide having SEQ ID NO: A polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in NO:1, MSHWAPEWKRAEANPRDLGASWDVRGSRGSGWSGPFGHQGPRAAGSREPPLCFKIKNNMVGVVIGYSGSKIKDLQHSTNTKIQIINGESEAKVRIFGNREMKAKAKAAIETLIRKQESYNSESSVDNAASQTPIGRNLGRNDIVGEAEPLSNWDRIRAAVVECEKRKWADLPPVKKNFYIESKATSCMSEMQVINWRKENFNITCDDLKSGEKRLIPKPTCRFKDAFQQYPDLLKSIIRVGILKPTPIQSQAWPIILQGIDLIVVAQTGTGKTLSYLMPGFIHLDSQPISRE QRNGPGMLVLTPTRELALHVEAECSKYSYKGLKSICIYGGRNRNGQIEDISKGVDIIIATPGRLNDLQMNNSVNLRSITYLVIDEADKMLDMEFEPQIRKILLDVRPRQTVMTSATWPDTVRQLALSYLKDPMIVYVGNLNLVAVNTVKQNIIVTTEKEKRALTQEFVENMSPNDKVIMFVSQKHIADDLSSDFNIQGISAESLHGNSEQSDQ ERAVEDFKSGNIKILITTDIVSRGLDLNDVTHVYNYDFPRNIDVYVHRVGYIGRTGKTGTSVTLITQRDSKMAGELIKILDRANQSVPEDLVVMAEQYKLNQQKRHRETRSREPGQRRKEFYFLS (SEQ ID NO: 1).

进一步地,上述抗原蛋白MMRN2是包括具有如SEQ ID NO:2所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:2有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:2所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MILSLLFSLGGPLGWGLLGAWAQASSTSLSDLQSSRTPGVWKAEAEDTGKDPVGRNWCPYPMSKLVTLLALCKTEKFLIHSQQPCPQGAPDCQKVKVMYRMAHKPVYQVKQKVLTSLAWRCCPGYTGPNCEHHDSMAIPEPADPGDSHQEPQDGPVSFKPGHLAAVINEVEVQQEQQEHLLGDLQNDVHRVADSLPGLWKALPGNLTAAVMEANQTGHEFPDRSLEQVLLPHVDTFLQVHFSPIWRSFNQSLHSLTQAIRNLSLDVEANRQAISRVQDSAVARADFQELGAKFEAKVQENTQRVGQLRQDVEDRLHAQHFTLHRSISELQADVDTKLKRLHKAQEAPGTNGSLVLATPGAGARPEPDSLQARLGQLQRNLSELHMTTARREEELQYTLEDMRATLTRHVDEIKELYSESDETFDQISKVERQVEELQVNHTALRELRVILMEKSLIMEENKEEVERQLLELNLTLQHLQGGHADLIKYVKDCNCQKLYLDLDVIREGQRDATRALEETQVSLDERRQLDGSSLQALQNAVDAVSLAVDAHKAEGERARAATSRLRSQVQALDDEVGALKAAAAEARHEVRQLHSAFAALLEDALRHEAVLAALFGEEVLEEMSEQTPGPLPLSYEQIRVALQDAASGLQEQALGWDELAARVTALEQASEPPRPAEHLEPSHDAGREEAATTALAGLARELQSLSNDVKNVGRCCEAEAGAGAASLNASLDGLHNALFATQRSLEQHQRLFHSLFGNFQGLMEANVSLDLGKLQTMLSRKGKKQQKDLEAPRKRDKKEAEPLVDIRVTGPVPGALGAALWEAGSPVAFYASFSEGTAALQTVKFNTTYINIGSSYFPEHGYFRAPERGVYLFAVSVEFGPGPGTGQLVFGGHHRTPVCTTGQGSGSTATVFAMAELQKGERVWFELTQGSITKRSLSGTAFGGFLMFKT(SEQ IDNO:2)。Further, the above-mentioned antigen protein MMRN2 is a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:2, or a polypeptide having at least 80% homology with the sequence SEQ ID NO:2 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:2, MILSLLFSLGGPLGWGLLGAWAQASSTSLSDLQSSRTPGVWKAEAEDTGKDPVGRNWCPYPMSKLVTLLALCKTEKFLIHSQQPCPQGAPDCQKVKVMYRMAHKPVYQVKQKVLTSLAWRCCPGYTGPNCEHHDSMAIPEPADPGDSHQEPQDGPVSFKPGHLAAVINEVEVQQEQQEHLLGDLQNDVHRVADSLPGLWKA LPGNLTAAVMEANQTGHEFPDRSLEQVLLPHVDTFLQVHFSPIWRSFNQSLHSLTQAIRNLSLDVEANRQAISRVQDSAVARADFQELGAKFEAKVQENTQRVGQLRQDVEDRLHAQHFTLHRSISELQADVDTKLKRLHKAQEAPGTNGSLVLATPGAGARPEPDSLQARLGQLQRNLSELHMTTARREEELQYTLEDMRATLTRHV DEIKELYSESDETFDQISKVERQVEELQVNHTALRELRVILME KSLIMEENKEEVERQLLELNLTLQHLQGGHADLIKYVKDCNCQKLYLDLDVIREGQRDATRALEETQVSLDERRQLDGSSLQALQNAVDAVSLAVDAHKAEGERARAATSRLRSQVQALDDEVGALKAAAAEARHEVRQLHSAFAALLEDALRHEAVLAALFGEEVLEEMSEQTPGPLPLSYEQIRVALQDAASGLQEQALGWDELAARVTALEQASEPPRPA EHLEPSHDAGREEAATTALAGLARELQ SLSNDVKNVGRCCEAEAGAGAASLNASLDGLHNALFATQRSLEQHQRLFHSLFGNFQGLMEANVSLDLGKLQTMLSRKGKKQQKDLEAPRKRDKKEAEPLVDIRVTGPVPGALGAALWEAGSPVAFYASFSEGTAALQTVKFNTTYINIGSSYFPEHGYFRAPERGVYLFAVSVSVEFGPGPGTGQLVFGGHHRTPVCTTGQGSGSTATVFAMAEL QKGERVWFELTQGSITKRSLSGTAFGGFLMFKT(SEQ IDNO:2).

进一步地,上述抗原蛋白MKNK1是包括具有如SEQ ID NO:3所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:3有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:3所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MVSSQKLEKPIEMGSSEPLPIADGDRRRKKKRRGRATDSLPGKFEDMYKLTSELLGEGAYAKVQGAVSLQNGKEYAVKIIEKQAGHSRSRVFREVETLYQCQGNKNILELIEFFEDDTRFYLVFEKLQGGSILAHIQKQKHFNEREASRVVRDVAAALDFLHTKDKVSLCHLGWSAMAPSGLTAAPTSLGSSDPPTSASQVAGTTGIAHRDLKPENILCESPEKVSPVKICDFDLGSGMKLNNSCTPITTPELTTPCGSAEYMAPEVVEVFTDQATFYDKRCD LWSLGVVLYIMLSGYPPFVGHCGADCGWDRGEVCRVCQNKLFESIQEGKYEFPDKDWAHISSEAKDLISKLLVRDAKQRLSAAQVLQHPWVQGQAPEKGLPTPQVLQRNSSTMDLTLFAAEAIALNRQLSQHEENELAEEPEALADGLCSMKLSPPCKSRLARRRALAQAGRGEDRSPPTAL(SEQ ID NO:3)。Further, the antigen protein MKNK1 comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:3, or a polypeptide having at least 80% homology with the sequence SEQ ID NO:3 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:3, MVSSQKLEKPIEMGSSEPLPIADGDRRRKKKRRGRATDSLPGKFEDMYKLTSELLGEGAYAKVQGAVSLQNGKEYAVKIIEKQAGHSRSRVFREVETLYQCQGNKNILELIEFFEDDTRFYLVFEKLQGGSILAHIQKQKHFNEREASRVVRDVAAALDFLHTKDKVSLCHLGWSAMAPSGLTAAPTSLGSSDPPTSASQVAGTTGIAHRDLKPENILCESPEKVSPVKICDFDLGSGMKLNNSCTPITTPELTTPCGSAEYMAPEVVEVFTDQATFYDKRCD LWSLGVVLYIMLSGYPPFVGHCGADCGWDRGEVCRVCQNKLFESIQEGKYEFPDKDWAHISSEAKDLISKLLVRDAKQRLSAAQVLQHPWVQGQAPEKGLPTPQVLQRNSSTMDLTLFAAEAIALNRQLSQHEENELAEEPEALADGLCSMKLSPPCKSRLARRRALAQAGRGEDRSPTAL (SEQ ID NO: 3).

进一步地,上述抗原蛋白FGF12是包括具有如SEQ ID NO:4所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:4有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:4所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MESKEPQLKGIVTRLFSQQGYFLQMHPDGTIDGTKDENSDYTLFNLIPVGLRVVAIQGVKASLYVAMNGE GYLYSSDVFTPECKFKESVFENYYVIYSSTLYRQQESGRAWFLGLNKEGQIMKGNRVKKTKPSSHFVPKPI EVCMYREQSLHEIGEKQGRSRKSSGTPTMNGGKVVNQDST(SEQ ID NO:4)。Further, the above-mentioned antigen protein FGF12 includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:4, or includes a polypeptide having at least 80% homology with the sequence SEQ ID NO:4 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:4, MESKEPQLKGIVTRLFSQQGYFLQMHPDGTIDGTKDENSDYTLFNLIPVGLRVVAIQGVKASLYVAMNGE GYLYSSDVFTPECKFKESVFENYYVIYSSTLYRQQESGRAWFLGLNKEGQIMKGNRVKKTKPSSHFVPKPI EVCMYREQSLHEIGEKQGRSRKSSGTPTMNGGKVVNQDST (SEQ ID NO:4).

进一步地,上述抗原蛋白PIP4K2C是包括具有如SEQ ID NO:5所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:5有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:5所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MASSSVPPATVSAATAGPGPGFGFASKTKKKHFVQQKVKVFRAADPLVGVFLWGVAHSINELSQVPPPVMLLPDDFKASSKIKVNNHLFHRENLPSHFKFKEYCPQVFRNLRDRFGIDDQDYLVSLTRNPPSESEGSDGRFLISYDRTLVIKEVSSEDIADMHSNLSNYHQYIVKCHGNTLLPQFLGMYRVSVDNEDSYMLVMRNMFSHRLPVHRKYDLKGSLVSREASDKEKVKELPTLRDMDFLNKNQKVYIGEEEKKIFLEKLKRDVEFLVQLKIMDYSLLLGIHDIIRGSEPEEEAPVREDESEVDGDCSLTGPPALVGSYGTSPEGIGGYIHSHRPLGPGEFESFIDVYAIRSAEGAPQKEVYFMGLIDILTQYDAKKKAAHAAKTVKHGAGAEISTVHPEQYAKRFLDFITNIFA(SEQ ID NO:5)。Further, the above-mentioned antigen protein PIP4K2C includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:5, or includes a polypeptide having at least 80% homology with the sequence SEQ ID NO:5 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:5, MASSSVPPATVSAATAGPGPGFGFASKTKKKHFVQQKVKVFRAADPLVGVFLWGVAHSINELSQVPPPVMLLPDDFKASSKIKVNNHLFHRENLPSHFKFKEYCPQVFRNLRDRFGIDDQDYLVSLTRNPPSESEGSDGRFLISYDRTLVIKEVSSEDIADMHSNLSNYHQYIVKCHGNTLLPQFLGM YRVSVDNEDSYMLVMRNMFSHRLPVHRKYDLKGSLVSREASDKEKVKELPTLRDMDFLNKNQKVYIGEEEKKIFLEKLKRDVEFLVQLKIMDYSLLLGIHDIIRGSEPEEEAPVREDESEVDGDCSLTGPPALVGSYGTSPEGIGGYIHSHRPLGPGEFESFIDVYAIRSAEGAPQKEVYFMGLIDILTQYDAKKKAAHAAKTVKHGAGAEISTV HPEQYAKRFLDFITNIFA (SEQ ID NO:5).

进一步地,上述抗原蛋白NME3是包括具有如SEQ ID NO:6所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:6有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:6所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MICLVLTIFANLFPAACTGAHERTFLAVKPDGVQRRLVGEIVRRFERKGFKLVALKLVQASEELLREHYAEL RERPFYGRLVKYMASGPVVAMVWQGLDVVRTSRALIGATNPADAPPGTIRGDFCIEVGKNLIHGSDSVES ARREIALWFRADELLCWEDSAGHWLYE(SEQ ID NO:6)。Further, the above-mentioned antigen protein NME3 is a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:6, or a polypeptide having at least 80% homology with the sequence SEQ ID NO:6 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:6, MICLVLTIFANLFPAACTGAHERTFLAVKPDGVQRRLVGEIVRRFERKGFKLVALKLVQASEELLREHYAEL RERPFYGRLVKYMASGPVVAMVWQGLDVVRTSRALIGATNPADAPPGTIRGDFCIEVGKNLIHGSDSVES ARREIALWFRADELLCWEDSAGHWLYE (SEQ ID NO:6).

进一步地,上述抗原蛋白CTAG1A是包括具有如SEQ ID NO:7所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:7有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:7所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGG AASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLT AADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR(SEQ ID NO:7)。Further, the above-mentioned antigen protein CTAG1A includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:7, or includes a polypeptide having at least 80% homology with the sequence SEQ ID NO:7 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:7, MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGG AASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLT AADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO:7).

进一步地,上述抗原蛋白HYLS1是包括具有如SEQ ID NO:8所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:8有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:8所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MDPEERMLAAATAFTHIRAGQGEGDVRREAQSIQYDPYSKASVAPGKRPALPVQLQYPHVESNVPSETVSEASQRLRKPVMKRKVLRRKPDGEVLVTDESIISESESGTENDQDLWDLRQRLMNVQFQEDKESSFDVSQKFNLPHEYQGISQDQLICSLQREGMGSPAYEQDLIVASRPKSFILPKLDQLSRNRGKTDRVARYFEYKRDWDSIRLPGEDHRKELRWGVREQMLCRAEPQSKPQHIYVPNNYLVPTEKKRSALRWGVRCDLANGVIPRKLPFPLSPS(SEQ ID NO:8)。Furthermore, the antigen protein HYLS1 comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 8, or comprises a polypeptide having at least 80% homology with the sequence SEQ ID NO: 8 and having the same or substantially the same function, or comprises a polypeptide having SEQ ID The amino acid sequence shown in NO:8 is replaced, deleted or added with one or more amino acids to form a polypeptide with the same or substantially the same function, MDPEERMLAAATAFTHIRAGQGEGDVRREAQSIQYDPYSKASVAPGKRPALPVQLQYPHVESNVPSETVSEASQRLRKPVMKRKVLRRKPDGEVLVTDESIISESESGTENDQDLWDLRQRLMNVQFQEDKESSFDVSQKFNLPHEYQGISQDQLICSLQREGMGSPAYEQDLIVASRPKSFILPKLDQLSRNRGKTDRVARYFEYKRDWDSIRLPGEDHRKELRWGVREQMLCRAEPQSKPQHIYVPNNYLVPTEKKRSALRWGVRCDLANGVIPRKLPFPLSPS (SEQ ID NO:8).

进一步地,上述抗原蛋白TIA1是包括具有如SEQ ID NO:9所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:9有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:9所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MEDEMPKTLYVGNLSRDVTEALILQLFSQIGPCKNCKMIMDTAGNDPYCFVEFHEHRHAAAALAAMNGRKIMGKEVKVNWATTPSSQKKDTSSSTVVSTQRSQDHFHVFVGDLSPEITTEDIKAAFAPFGRISDARVVKDMATGKSKGYGFVSFFNKWDANAIQQMGGQWLGGRQIRTNWATRKPPAPKSTYECRCIGEEKEMWNFGEKYARF(SEQ ID NO:9)。Further, the above-mentioned antigen protein TIA1 is a polypeptide comprising an amino acid sequence as shown in SEQ ID NO:9, or a polypeptide having at least 80% homology with the sequence SEQ ID NO:9 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:9, MEDEMPKTLYVGNLSRDVTEALILQLFSQIGPCKNCKMIMDTAGNDPYCFVEFHEHRHAAAALAAMNGRKIMGKEVKVNWATTPSSQKKDTSSSTVVSTQRSQDHFHVFVGDLSPEITTEDIKAAFAPFGRISDARVVKDMATGKSKGYGFVSFFNKWDANAIQQMGGQWLGGRQIRTNWATRKPPAPKSTYECRCIGEEKEMWNFGEKYARF (SEQ ID NO:9).

进一步地,上述抗原蛋白SSBP4是包括具有如SEQ ID NO:10所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:10有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:10所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MYAKGGKGSAVPSDSQAREKLALYVYEYLLHIGAQKSAQTFLSEIRWEKNITLGEPPGFLHSWWCVFWDLYCAAPDRREACEHSGEAKAFQDYSAAAAPSPVMGSMAPGDTMAAGSMAAGFFQGPPGSQPSPHNPNAPMMGPHGQPFMSPRFPGGPRPTLRMPSQPPAGLPGSQPLLPGAMEPSPRAQGHPSMGGPMQRVTPPRGMASVGPQSYGGGMRPPPNSLAGPGLPAMNMGPGVRGPWASPSGNSIPYSSSSPGSYTGPPGGGGPPGTPIMPSPGDSTNSSENMYTIMNPIGQGAGRANFPLGPGPEGPMAAMSAMEPHHVNGSLGSGDMDGLPKSSPGAVAGLSNAPGTPRDDGEMAAAGTFLHPFPSESYSPGMTMSV(SEQ ID NO:10)。Furthermore, the antigen protein SSBP4 comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 10, or a polypeptide having at least 80% homology with the sequence SEQ ID NO: 10 and having the same or substantially the same function, or a polypeptide having SEQ ID The amino acid sequence shown in NO:10 is replaced, deleted or added with one or more amino acids to form a polypeptide with the same or substantially the same function, MYAKGGKGSAVPSDSQAREKLALYVYEYLLHIGAQKSAQTFLSEIRWEKNITLGEPPGFLHSWWCVFWDLYCAAPDRREACEHSGEAKAFQDYSAAAAPSPVMGSMAPGDTMAAGSMAAGFFQGPPGSQPSPHNPNAPMMGPHGQPFMSPRFPGGPRPTLRMPSQPPAGLPGSQPLLPGAMEPSPRAQGHPSMGGPMQRVTPPRGMASVGPQSYGGGMRPPPNSLAGPGLPAMNMGPGVRGPWASPSGNSIPYSSSSPGSYTGPPGGGGPPGTPIMPSPGDSTNSSENMYTIMNPIGQGAGRANFPLGPGPEGPMAAMSAMEPHHVNGSLGSGDMDGLPKSSPGAVAGLSNAPGTPRDDGEMAAAGTFLHPFPSESYSPGMTMSV (SEQ ID NO:10).

进一步地,上述抗原蛋白ENO2是包括具有如SEQ ID NO:11所示的氨基酸序列的多肽,或是包括具有与序列SEQ ID NO:11有至少80%同源性且具有相同或基本相同功能的多肽,或是具有SEQ ID NO:11所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而形成的具有相同或基本相同功能的多肽,MSIEKIWAREILDSRGNPTVEVDLYTAKGLFRAAVPSGASTGIYEALELRDGDKQRYLGKGVLKAVDHINSTIAPALISSGLSVVEQEKLDNLMLELDGTENKSKFGANAILGVSLAVCKAGAAERELPLYRHIAQLAGNSDLILPVPAFNVINGGSHAGNKLAMQEFMILPVGAESFRDAMRLGAEVYHTLKGVIKDKYGKDATNVGDEGGFAPNILENSEALELVKEAIDKAGYTEKIVIGMDVAASEFYRDGKYDLDFKSPTDPSRYITGDQLGALYQDFVRDYPVVSIEDPFDQDDWAAWSKFTANVGIQIVGDDLTVTNPKRIERAVEEKACNCLLLKVNQIGSVTEAIQACKLAQENGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRSERLAKYNQLMRIEEELGDEARFAGHNFRNPSVL(SEQ ID NO:11)。Further, the antigen protein ENO2 is a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 11, or a polypeptide having at least 80% homology with the sequence SEQ ID NO: 11 and having the same or substantially the same function, or a polypeptide having the same or substantially the same function formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 11, MSIEKIWAREILDSRGNPTVEVDLYTAKGLFRAAVPSGASTGIYEALELRDGDKQRYLGKGVLKAVDHINSTIAPALISSGLSVVEQEKLDNLMLELDGTENKSKFGANAILGVSLAVCKAGAAERELPLYRHIAQLAGNSDLILPVPAFNVINGGSHAGNKLAMQEFMILPVGAESFRDAMRLGAEVYHTLKG VIKDKYGKDATNVGDEGGFAPNILENSEALELVKEAIDKAGYTEKIVIGMDVAASEFYRDGKYDLDFKSPTDPSRYITGDQLGALYQDFVRDYPVVSIEDPFDQDDWAAWSKFTANVGIQIVGDDLTVTNPKRIERAVEEKACNCLLLKVNQIGSVTEAIQACKLAQENGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRSERLAKY NQLMRIEEELGDEARFAGHNFRNPSVL (SEQ ID NO: 11).

进一步地,上述至少80%同源性包括具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同源性。Furthermore, the at least 80% homology mentioned above includes 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

进一步地,上述抗原蛋白表达于酵母细胞中,并经过纯化获得。Furthermore, the above antigen protein is expressed in yeast cells and obtained through purification.

本申请还提供了上述抗原蛋白的制备方法,具体包括如下步骤:The present application also provides a method for preparing the above antigen protein, which specifically comprises the following steps:

(1)采用Gateway高通量克隆技术,将抗原蛋白的全长cDNA克隆到酵母表达载体pEGH-A中,构建GST融合蛋白的酵母表达质粒;(1) Using Gateway high-throughput cloning technology, the full-length cDNA of the antigen protein was cloned into the yeast expression vector pEGH-A to construct a yeast expression plasmid for the GST fusion protein;

(2)将酵母表达质粒转化到大肠杆菌细胞中,进行质粒扩增和纯化;对纯化的质粒进行酶切和DNA测序验证,选取正确的质粒转化到酵母细胞中;(2) Transforming the yeast expression plasmid into Escherichia coli cells, amplifying and purifying the plasmid; performing restriction digestion and DNA sequencing verification on the purified plasmid, and selecting the correct plasmid to transform into yeast cells;

(3)挑取转化好的酵母克隆,接种于SC-Ura/Glucose液体培养基中,30℃振荡培养20~24小时;转接至SC-Ura/Raffinose(棉子糖)液体培养基中,30℃振荡培养过夜;第二天测OD600吸光度值大于1.0时,加入半乳糖至终浓度为2%,继续振荡培养过夜;离心收集酵母细胞,用无菌水洗2次;(3) Pick the transformed yeast clones, inoculate them into SC-Ura/Glucose liquid medium, and culture them with shaking at 30°C for 20 to 24 hours; transfer them to SC-Ura/Raffinose liquid medium, and culture them with shaking at 30°C overnight; when the OD600 absorbance value is greater than 1.0 on the second day, add galactose to a final concentration of 2%, and continue to culture them with shaking overnight; collect the yeast cells by centrifugation, and wash them twice with sterile water;

(4)加入裂解液和镍珠,振荡破碎细胞后,离心回收上清;在上清中加入Glutathione Sepharose 4B beads,混匀,4℃摇动1小时;离心收集beads,用洗液I和洗液II各清洗3次;加入洗脱液,短暂震荡将beads重悬,4℃摇动1小时;离心,取上清,即获得抗原蛋白。(4) Add lysis buffer and nickel beads, shake to break the cells, and recover the supernatant by centrifugation; add Glutathione Sepharose 4B beads to the supernatant, mix well, and shake at 4°C for 1 hour; collect the beads by centrifugation, and wash them three times with wash solution I and wash solution II respectively; add elution buffer, shake briefly to resuspend the beads, and shake at 4°C for 1 hour; centrifuge and collect the supernatant to obtain the antigen protein.

进一步地,上述抗原蛋白的制备方法中,裂解液的成分包括:50mM Tris,pH 7.5,100mM NaCl,1mMEGTA,10% Glycerol,0.1% TritonX-100,0.1%beta-mercaptoethanol(BME),1mM PMSF(200mM in isopropanol),50μM LLnL(50mM in DMSO),1μM MG-132(50mMin DMSO),Roche Protease inhibitor tablets(without EDTA)1in 50ml。Furthermore, in the above-mentioned method for preparing the antigen protein, the components of the lysate include: 50mM Tris, pH 7.5, 100mM NaCl, 1mM EGTA, 10% Glycerol, 0.1% TritonX-100, 0.1% beta-mercaptoethanol (BME), 1mM PMSF (200mM in isopropanol), 50μM LLnL (50mM in DMSO), 1μM MG-132 (50mMin DMSO), Roche Protease inhibitor tablets (without EDTA) 1in 50ml.

进一步地,上述抗原蛋白的制备方法中,洗液I成分包括:50mM Tris,pH 7.5,500mM NaCl,1mM EGTA,10% Glycerol,0.1% TritonX-100,0.1%beta-mercaptoethanol(BME),1mM PMSF。Furthermore, in the above-mentioned method for preparing the antigen protein, the components of the washing solution I include: 50mM Tris, pH 7.5, 500mM NaCl, 1mM EGTA, 10% Glycerol, 0.1% TritonX-100, 0.1% beta-mercaptoethanol (BME), and 1mM PMSF.

进一步地,上述抗原蛋白的制备方法中,洗液II成分包括:50mM HEPES pH 7.5,100mM NaCl,1mM EGTA,10% Glycerol,0.1%beta-mercaptoethanol(BME),1mM PMSF。Furthermore, in the above method for preparing the antigen protein, the components of the washing solution II include: 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 10% Glycerol, 0.1% beta-mercaptoethanol (BME), and 1 mM PMSF.

进一步地,上述抗原蛋白的制备方法中,洗脱液成分包括:50mM HEPES pH 8.0,100mM NaCl,30%Glycerol,40mM Glutathione(Reduced form),0.03% TritonX-100。Furthermore, in the above method for preparing the antigen protein, the eluent components include: 50 mM HEPES pH 8.0, 100 mM NaCl, 30% Glycerol, 40 mM Glutathione (Reduced form), and 0.03% TritonX-100.

本申请还提供了一种固相载体,该载体包被有上述任一所述的蛋白组合物。The present application also provides a solid phase carrier, which is coated with any of the above-mentioned protein compositions.

本申请还提供了上述蛋白组合物在制备用于肺癌早期诊断试剂盒中的应用,该试剂盒包含上述任一项的蛋白组合物,用于检测受试者血清中肺癌自身IgG抗体,从而用于肺癌早期诊断。The present application also provides the use of the above protein composition in the preparation of a kit for early diagnosis of lung cancer, wherein the kit comprises any of the above protein compositions and is used to detect lung cancer autologous IgG antibodies in the serum of a subject, thereby being used for early diagnosis of lung cancer.

本申请还提供了一种用于肺癌早期诊断的试剂盒,该试剂盒包含上述任一的蛋白组合物,该试剂盒可以利用蛋白组合物检测受试者血清中肺癌自身IgG抗体,从而用于肺癌早期诊断。The present application also provides a kit for early diagnosis of lung cancer, which comprises any of the above-mentioned protein compositions. The kit can use the protein composition to detect lung cancer autologous IgG antibodies in the serum of a subject, thereby being used for early diagnosis of lung cancer.

进一步地,上述试剂盒所用的信号检测方法包括可见光显色法、化学发光法、荧光发光法。Furthermore, the signal detection methods used in the above-mentioned kit include visible light colorimetry, chemiluminescence, and fluorescence.

进一步地,上述试剂盒还包括显色剂、血清稀释缓冲液、洗液和终止液。Furthermore, the above kit also includes a color developer, a serum dilution buffer, a washing solution and a stop solution.

本申请还提供了上述一种用于肺癌早期诊断的试剂盒在肺癌早期诊断中的应用。The present application also provides the use of the above-mentioned kit for early diagnosis of lung cancer in the early diagnosis of lung cancer.

进一步地,上述应用包括如下步骤:Furthermore, the above application includes the following steps:

S1:获取受试者血清样本;S1: Obtain serum samples from subjects;

S2:使用试剂盒检测受试者血清样本中自身抗体的表达水平;S2: Use the kit to detect the expression level of autoantibodies in the serum samples of the subjects;

S3:将检测的血清样本中自身抗体的表达水平与预设值比较,如大于预设值,则判断患有肺癌。S3: Compare the expression level of the autoantibodies in the detected serum sample with the preset value. If it is greater than the preset value, it is determined that the patient has lung cancer.

本申请还提供了一种用于肺癌早期诊断的设备,该设备包括:The present application also provides a device for early diagnosis of lung cancer, the device comprising:

样本获取模块,用于获取受试者血清样本;A sample acquisition module, used to obtain a serum sample from a subject;

样本检测模块,包括上述任一用于肺癌早期诊断的试剂盒,用于检测受试者血清样本中自身抗体的表达水平;A sample detection module, including any of the above-mentioned kits for early diagnosis of lung cancer, used to detect the expression level of autoantibodies in a serum sample of a subject;

数据处理及结果输出模块:将样本检测模块检测的受试者血清样本中自身抗体的表达水平与预设值比较,大于预设值则输出患有肺癌的结果,其他则输出未患肺癌的结果。Data processing and result output module: compare the expression level of autoantibodies in the serum samples of the subjects detected by the sample detection module with the preset value. If it is greater than the preset value, the result of lung cancer is output; otherwise, the result of not having lung cancer is output.

3.有益效果3. Beneficial effects

本申请与现有技术相比,其有益效果在于:Compared with the prior art, the present application has the following beneficial effects:

本申请提供的蛋白组合物及其在诊断领域的应用,其中抗原蛋白其中抗原蛋白DDX53、MMRN2、MKNK1、FGF12、NME3、HYLS1、TIA1、和ENO2均为本申请新筛选的抗原蛋白,其中DDX53和MMRN2两个抗原蛋白的组合,用于检测抗体并进行肺癌早期诊断,其敏感性为58%,特异性为100%,能以较少的抗原蛋白达到相似或更优的效果。与抗原蛋白MKNK1、FGF12、PIP4K2C、NME3、CTAG1A、HYLS1、TIA1、SSBP4和ENO2中的任意一种或多种组合,其敏感性与特异性的平均值(Discriminative ability)为79%~88%,提高了肺癌早期诊断的敏感性和特异性。The protein composition provided by the present application and its application in the field of diagnosis, wherein the antigen protein wherein the antigen protein DDX53, MMRN2, MKNK1, FGF12, NME3, HYLS1, TIA1, and ENO2 are all newly screened antigen proteins of the present application, wherein the combination of the two antigen proteins DDX53 and MMRN2 is used to detect antibodies and perform early diagnosis of lung cancer, and its sensitivity is 58%, and its specificity is 100%, and it can achieve similar or better effects with less antigen protein. With any one or more combinations of antigen proteins MKNK1, FGF12, PIP4K2C, NME3, CTAG1A, HYLS1, TIA1, SSBP4 and ENO2, its average value of sensitivity and specificity (Discriminative ability) is 79% to 88%, which improves the sensitivity and specificity of early diagnosis of lung cancer.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1是HuProt芯片代表性扫描图像。Figure 1 is a representative scan image of the HuProt chip.

图2是抗原蛋白DDX53在芯片上的反应信号,左侧为肺癌样品,右侧为健康样品。Figure 2 shows the reaction signal of the antigen protein DDX53 on the chip, with lung cancer samples on the left and healthy samples on the right.

图3是抗原蛋白CTAG1A的SDA-PAGE凝胶电泳和考马斯亮蓝染色结果。FIG3 is the results of SDA-PAGE gel electrophoresis and Coomassie brilliant blue staining of the antigen protein CTAG1A.

图4-6是各抗原蛋白在在100例受试者样本的OD值分布。Figure 4-6 shows the OD value distribution of each antigen protein in 100 subject samples.

具体实施方式DETAILED DESCRIPTION

下面结合具体实施例对本申请进一步进行描述。The present application is further described below in conjunction with specific embodiments.

需要说明的是,本说明书中所引用的如“上”、“下”、“左”、“右”、“中间”等用语,亦仅为便于叙述的明了,而非用以限定可实施的范围,其相对关系的改变或调整,在无实质变更技术内容下,当亦视为本申请可实施的范畴。It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

除非另有定义,本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同;本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and/or" used herein includes any and all combinations of one or more of the associated listed items.

实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

如本文所使用,术语“约”用于提供与给定术语、度量或值相关联的灵活性和不精确性。本领域技术人员可以容易地确定具体变量的灵活性程度。As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

如本文所使用,术语“......中的至少一个”旨在与“......中的一个或多个”同义。例如,“A、B和C中的至少一个”明确包括仅A、仅B、仅C还包括它们各自的组合。As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and also includes their respective combinations.

浓度、量和其他数值数据可以在本文中以范围格式呈现。应当理解,这样的范围格式仅是为了方便和简洁而使用,并且应当灵活地解释为不仅包括明确叙述为范围极限的数值,而且还包括涵盖在所述范围内的所有单独的数值或子范围,就如同每个数值和子范围都被明确叙述一样。例如,约1至约4.5的数值范围应当被解释为不仅包括明确叙述的1至约4.5的极限值,而且还包括单独的数字(诸如2、3、4)和子范围(诸如1至3、2至4等)。相同的原理适用于仅叙述一个数值的范围,诸如“小于约4.5”,应当将其解释为包括所有上述的值和范围。此外,无论所描述的范围或特征的广度如何,都应当适用这种解释。Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

实施例1Example 1

本实施例提供肺癌相关抗原的筛选,具体包括如下步骤:This embodiment provides screening of lung cancer-associated antigens, which specifically includes the following steps:

从80名肺癌患者和20名健康个体中收集的100份血清样本分别在HuProt人类蛋白质组芯片上进行了分析。HuProt芯片由CDI Laboratories,Inc.提供。每张HuProt v3.0芯片由20,240种人类全长蛋白质组成,约覆盖人类蛋白质组的80%。肺癌组和健康组在年龄、性别或吸烟史构成上没有任何显著差异。100 serum samples collected from 80 lung cancer patients and 20 healthy individuals were analyzed on the HuProt human proteome chip. The HuProt chip was provided by CDI Laboratories, Inc. Each HuProt v3.0 chip consists of 20,240 human full-length proteins, covering approximately 80% of the human proteome. There were no significant differences in age, gender, or smoking history between the lung cancer group and the healthy group.

HuProt芯片首先在室温下与封闭缓冲液(含0.1%(v/v)Tween 20和3% BSA的PBS)中孵育1小时,然后加入1:1,000稀释的血清样品并在室温孵育1小时。用1×TBST轻轻摇动洗涤3次,每次10分钟。将芯片与3毫升1,000倍稀释的Alexa647标记的山羊抗人IgG抗体(JacksonImmuno,美国)室温下避光孵育1小时。随后,将HuProt芯片用1×TBST洗涤3次,然后用双蒸水冲洗3次。将HuProt芯片甩干,用Genepix 4000B芯片扫描仪(MolecularDevices,美国)扫描,其扫描结果如图1所示,并使用GenePix Pro 6.0软件获取芯片上的抗体信号。The HuProt chip was first incubated in blocking buffer (PBS containing 0.1% (v/v) Tween 20 and 3% BSA) at room temperature for 1 hour, and then a 1:1,000 diluted serum sample was added and incubated at room temperature for 1 hour. Wash 3 times with 1×TBST with gentle shaking for 10 minutes each time. The chip was incubated with 3 ml of 1,000-fold diluted Alexa647-labeled goat anti-human IgG antibody (JacksonImmuno, USA) at room temperature in the dark for 1 hour. Subsequently, the HuProt chip was washed 3 times with 1×TBST and then rinsed 3 times with double distilled water. The HuProt chip was dried and scanned with a Genepix 4000B chip scanner (Molecular Devices, USA). The scanning results are shown in Figure 1, and the antibody signal on the chip was acquired using GenePix Pro 6.0 software.

为选择候选抗原蛋白,提取HuProt芯片上给定蛋白质点处的前景信号值(Fij)的中值进行分析。由于每种蛋白质在芯片上有两个重复点,因此取每种蛋白质的Fij的平均值为其信号强度Fp。对100张HuProt芯片上的全部Fp进行分位数归一化,然后进行t检验以鉴定肺癌组和健康组之间有显著差异的抗原蛋白,选择标准为t检验的p值小于0.05,并且肺癌组的平均Fp高于健康组的1.5倍以上。以此方法鉴定出抗原蛋白DDX53、MMRN2、MKNK1、FGF12、NME3、HYLS1、TIA1、和ENO2,其在芯片上的ID如表1所示。抗原蛋白DDX53在芯片上的反应信号如图2所示,左侧为肺癌样品,右侧为健康样品。To select candidate antigen proteins, the median of the foreground signal value (Fij) at a given protein point on the HuProt chip was extracted for analysis. Since each protein has two replicate points on the chip, the average value of Fij for each protein is taken as its signal intensity Fp. All Fp on 100 HuProt chips were quantile normalized, and then a t-test was performed to identify antigen proteins with significant differences between the lung cancer group and the healthy group. The selection criteria were that the p value of the t-test was less than 0.05, and the average Fp of the lung cancer group was more than 1.5 times higher than that of the healthy group. Antigen proteins DDX53, MMRN2, MKNK1, FGF12, NME3, HYLS1, TIA1, and ENO2 were identified in this way, and their IDs on the chip are shown in Table 1. The reaction signal of the antigen protein DDX53 on the chip is shown in Figure 2, with lung cancer samples on the left and healthy samples on the right.

此外,发明人还综合了前期的研究,选择了抗原蛋白PIP4K2C、SSBP4和CTAG1A作为肺癌相关抗原蛋白,其中抗原蛋白PIP4K2C、SSBP4、CTAG1A选自69份肺癌样品和30份对照样品在HuProt芯片上的反应结果,选择标准为对肺癌样品的诊断特异性大于89%,敏感性与特异性的平均值(Discriminative ability)大于53%(详见US20220003768A1)。In addition, the inventors also summarized previous studies and selected antigen proteins PIP4K2C, SSBP4 and CTAG1A as lung cancer-related antigen proteins, among which the antigen proteins PIP4K2C, SSBP4, and CTAG1A were selected from the reaction results of 69 lung cancer samples and 30 control samples on the HuProt chip. The selection criteria were that the diagnostic specificity for lung cancer samples was greater than 89%, and the average value of sensitivity and specificity (Discriminative ability) was greater than 53% (see US20220003768A1 for details).

表1抗原蛋白在芯片上的IDTable 1 Antigen protein ID on chip

Clone IdClone Id 抗原蛋白Antigenic protein JHU16271JHU16271 DDX53DDX53 JHU16011JHU16011 MMRN2MMRN2 JHU00052JHU00052 MKNK1MKNK1 JHU00314JHU00314 FGF12FGF12 JHU00059JHU00059 NME3NME3 JHU01186JHU01186 HYLS1HYLS1 JHU01234JHU01234 TIA1TIA1 JHU00604JHU00604 ENO2ENO2 JHU00151JHU00151 PIP4K2CPIP4K2C JHU06511JHU06511 SSBP4SSBP4 JHU17795JHU17795 CTAG1ACTAG1A

实施例2Example 2

本实施例提供实施例1中肺癌相关抗原蛋白的制备方法,具体包括如下步骤:This example provides a method for preparing the lung cancer-associated antigen protein in Example 1, which specifically comprises the following steps:

(1)采用Gateway高通量克隆技术,将抗原蛋白的全长cDNA克隆到酵母表达载体pEGH-A中,构建GST融合蛋白的酵母表达质粒;(1) Using Gateway high-throughput cloning technology, the full-length cDNA of the antigen protein was cloned into the yeast expression vector pEGH-A to construct a yeast expression plasmid for the GST fusion protein;

(2)将酵母表达质粒转化到大肠杆菌细胞中,进行质粒扩增和纯化;对纯化的质粒进行酶切和DNA测序验证,选取正确的质粒转化到酵母细胞中;(2) Transforming the yeast expression plasmid into Escherichia coli cells, amplifying and purifying the plasmid; performing restriction digestion and DNA sequencing verification on the purified plasmid, and selecting the correct plasmid to transform into yeast cells;

(3)挑取转化好的酵母克隆,接种于5毫升SC-Ura/Glucose液体培养基中,30℃振荡培养20~24小时;转接1毫升至300毫升SC-Ura/Raffinose(棉子糖)液体培养基中,30℃振荡培养过夜;第二天测OD600吸光度值大于1.0时,加入半乳糖至终浓度为2%,继续振荡培养过夜;离心收集酵母细胞,用无菌水洗2次;(3) Pick the transformed yeast clones, inoculate them into 5 ml SC-Ura/Glucose liquid medium, and culture them with shaking at 30°C for 20 to 24 hours; transfer 1 ml into 300 ml SC-Ura/Raffinose liquid medium, and culture them with shaking at 30°C overnight; when the OD600 absorbance value is greater than 1.0 on the second day, add galactose to a final concentration of 2%, and continue to culture them with shaking overnight; collect the yeast cells by centrifugation, and wash them twice with sterile water;

(4)加入裂解液和镍珠,振荡破碎细胞后,离心回收上清;在上清中加入Glutathione Sepharose 4B beads,混匀,4℃摇动1小时;离心收集beads,用洗液I和洗液II各清洗3次;加入洗脱液,短暂震荡将beads重悬,4℃摇动1小时;离心,取上清,分装10微升,其余冻存于-80℃冰箱保存;其中,裂解液的成分包括:50mM Tris,pH 7.5,100mM NaCl,1mMEGTA,10% Glycerol,0.1% TritonX-100,0.1%beta-mercaptoethanol(BME),1mMPMSF(200mM in isopropanol),50μM LLnL(50mM in DMSO),1μM MG-132(50mM in DMSO),Roche Protease inhibitor tablets(without EDTA)1in 50ml;洗液I成分包括:50mMTris,pH 7.5,500mM NaCl,1mM EGTA,10% Glycerol,0.1% TritonX-100,0.1%beta-mercaptoethanol(BME),1mM PMSF;洗液II成分包括:50mM HEPES pH 7.5,100mM NaCl,1mMEGTA,10% Glycerol,0.1%beta-mercaptoethanol(BME),1mM PMSF;洗脱液成分包括:50mM HEPES pH 8.0,100mM NaCl,30% Glycerol,40mM Glutathione(Reduced form),0.03% TritonX-100。(4) Add lysis buffer and nickel beads, shake to break the cells, and then centrifuge to recover the supernatant; add Glutathione Sepharose 4B beads to the supernatant, mix well, and shake at 4°C for 1 hour; collect beads by centrifugation, and wash them three times with wash buffer I and wash buffer II; add elution buffer, briefly shake to resuspend the beads, and shake at 4°C for 1 hour; centrifuge, take the supernatant, divide it into 10 μl, and freeze the rest in a -80°C refrigerator; the components of the lysis buffer include: 50mM Tris, pH 7.5, 100mM NaCl, 1mM EGTA, 10% Glycerol, 0.1% TritonX-100, 0.1% beta-mercaptoethanol (BME), 1mMPMSF (200mM in isopropanol), 50μM LLnL (50mM in DMSO), 1μM MG-132 (50mM in DMSO), Roche Protease inhibitor tablets (without EDTA) 1in 50ml; washing solution I components include: 50mM Tris, pH 7.5, 500mM NaCl, 1mM EGTA, 10% Glycerol, 0.1% TritonX-100, 0.1% beta-mercaptoethanol (BME), 1mM PMSF; washing solution II components include: 50mM HEPES pH 7.5, 100mM NaCl, 1mM EGTA, 10% Glycerol, 0.1% beta-mercaptoethanol (BME), 1mM PMSF; elution solution components include: 50mM HEPES pH 8.0, 100mM NaCl, 30% Glycerol, 40mM Glutathione (Reduced form), 0.03% TritonX-100.

(5)对分装的10微升蛋白进行SDA-PAGE凝胶电泳和考马斯亮蓝染色,检测目标蛋白的分子量大小、浓度和纯度。(5) Perform SDA-PAGE gel electrophoresis and Coomassie Brilliant Blue staining on 10 μL of the protein to detect the molecular weight, concentration and purity of the target protein.

以抗原蛋白CTAG1A为例,图3为SDA-PAGE凝胶电泳和考马斯亮蓝染色结果(左侧为分子量标尺),其GST融合蛋白的分子量约为48kD,浓度约为1毫克/毫升,纯度约为95%。利用相同的方法,制备实施例1中的其余蛋白。Taking antigen protein CTAG1A as an example, Figure 3 shows the results of SDA-PAGE gel electrophoresis and Coomassie brilliant blue staining (molecular weight scale on the left), and the molecular weight of its GST fusion protein is about 48 kD, the concentration is about 1 mg/ml, and the purity is about 95%. The remaining proteins in Example 1 were prepared using the same method.

实施例3Example 3

本实施例提供实施例1中筛选的肺癌相关抗原在肺癌自身抗体检测的应用,包括如下步骤:This example provides the application of the lung cancer-associated antigens screened in Example 1 in the detection of lung cancer autoantibodies, including the following steps:

S1:获取受试者血清样本,受试者样本来自中山大学附属肿瘤医院,其中健康样本20例(HC1-HC20),肺癌患者80例(LC1-LC80);S1: Obtain serum samples from subjects, which were from the Affiliated Cancer Hospital of Sun Yat-sen University, including 20 healthy samples (HC1-HC20) and 80 lung cancer patients (LC1-LC80);

S2:使用间接ELISA方法检测自身抗体,包括使用ELISA板包被0.1μg的肺癌相关抗原,封闭后加入100倍稀释的受试者血清样本,使用HRP标记抗人IgG作为二抗,清洗、显色后终止反应,并立即用酶标仪检测。具体包括:S2: Use the indirect ELISA method to detect autoantibodies, including using ELISA plates to coat 0.1 μg of lung cancer-related antigens, adding 100-fold diluted serum samples of subjects after blocking, using HRP-labeled anti-human IgG as the secondary antibody, terminating the reaction after washing and color development, and immediately detecting with an enzyme-labeled instrument. Specifically include:

(1)使用ELISA包被液稀释抗原至1μg/mL并加100μL包被液于酶标板上,37℃,1h,其中包被液为10mM PBS(PH=7.4);(1) Dilute the antigen to 1 μg/mL using ELISA coating solution and add 100 μL of the coating solution to the ELISA plate at 37°C for 1 h. The coating solution is 10 mM PBS (PH = 7.4);

(2)清洗:Elisa清洗液加满酶标板,甩干,重复3次,其中清洗液包括:10mM PBS(PH=7.4),4%Tween-20;(2) Cleaning: Fill the ELISA plate with Elisa cleaning solution, spin dry, and repeat 3 times. The cleaning solution includes: 10 mM PBS (PH = 7.4), 4% Tween-20;

(3)封闭:Elisa封闭液200μl/孔,4℃,过夜,其中封闭液包括:10mM PBS(PH=7.4),5%BSA,4%Tween-20;(3) Blocking: Elisa blocking solution 200 μl/well, 4°C, overnight, wherein the blocking solution includes: 10 mM PBS (PH=7.4), 5% BSA, 4% Tween-20;

(4)清洗:Elisa清洗液加满酶标板,甩干,重复5次;(4) Cleaning: Fill the ELISA plate with Elisa cleaning solution, spin dry, and repeat 5 times;

(5)添加血清样品:每板预留两个空白对照孔作为本底,其余94孔可检测47份样品,每个样品做二个重复孔;每孔加入100μL稀释100倍的血清样本,37℃,30min,其中样本稀释液包括:10mM PBS(PH=7.4),5%BSA;(5) Adding serum samples: two blank control wells are reserved on each plate as background, and the remaining 94 wells can detect 47 samples, with two replicate wells for each sample; 100 μL of 100-fold diluted serum sample is added to each well and incubated at 37° C. for 30 min, wherein the sample diluent includes: 10 mM PBS (PH=7.4), 5% BSA;

(6)清洗:Elisa清洗液加满酶标板,甩干,重复5次;(6) Cleaning: Fill the ELISA plate with Elisa cleaning solution, spin dry, and repeat 5 times;

(7)二抗:用Elisa封闭液稀释HRP标记抗人IgG二抗;每孔加入100μL,37℃,30min;(7) Secondary antibody: dilute HRP-labeled anti-human IgG secondary antibody with Elisa blocking solution; add 100 μL to each well and incubate at 37°C for 30 min;

(8)清洗:Elisa清洗液加满酶标板,甩干,重复5次;(8) Cleaning: Fill the ELISA plate with Elisa cleaning solution, spin dry, and repeat 5 times;

(9)显色:显色液A和显色液B1:1混合,现配先用;每孔加入100μL,37℃,5min;(9) Color development: Mix color development solution A and color development solution B in a ratio of 1:1 and use it immediately after preparation; add 100 μL to each well and incubate at 37°C for 5 min;

(10)终止:终止液50μl/孔,终止后立刻用酶标仪读数。(10) Termination: 50 μl/well of stop solution. Read the result immediately using an ELISA reader.

结果分析:Result analysis:

100例受试者样本的OD值如图4-6所示,其中HC代表正常人样本,LC代表患者样本,从图4中可以看出,11种抗原蛋白检测到的正常人样本和患者样本体内的肺癌自身抗体存在表达水平的差异。The OD values of the samples from 100 subjects are shown in Figures 4-6, where HC represents normal samples and LC represents patient samples. It can be seen from Figure 4 that there are differences in the expression levels of lung cancer autoantibodies in normal samples and patient samples detected by 11 antigen proteins.

以正常人样本中最大值或第二高值为CUT-OFF值,当检测的OD值大于CUT-OFF值时,则判断受试者患有肺癌。将上述抗原蛋白进行组合,其不同组合的敏感性及特异性如表2所示。The maximum value or the second highest value in the normal sample is taken as the CUT-OFF value. When the detected OD value is greater than the CUT-OFF value, the subject is judged to have lung cancer. The above antigen proteins are combined, and the sensitivity and specificity of different combinations are shown in Table 2.

表2Table 2

本申请中,选用4个抗原蛋白(DDX53、MMRN2、FGF12和MKNK1)时,其敏感性为73%,已经高于公开号为CN116482365A的中国发明专利中选用8个蛋白时的敏感性(72.7%),选用本申请的8个抗原蛋白(DDX53、MMRN2、TIA1、HYLS1、FGF12、MKNK1、SSBP4和CTAG1A)时,其敏感性为81%,提高了11.42%。由此可见,本申请中的蛋白组合物具有更高的敏感性和特异性,有利于提高肺癌早期诊断的准确性。In the present application, when four antigen proteins (DDX53, MMRN2, FGF12 and MKNK1) are selected, the sensitivity is 73%, which is higher than the sensitivity (72.7%) when eight proteins are selected in the Chinese invention patent with publication number CN116482365A. When the eight antigen proteins (DDX53, MMRN2, TIA1, HYLS1, FGF12, MKNK1, SSBP4 and CTAG1A) of the present application are selected, the sensitivity is 81%, which is an increase of 11.42%. It can be seen that the protein composition in the present application has higher sensitivity and specificity, which is conducive to improving the accuracy of early diagnosis of lung cancer.

Claims (5)

1. Use of a protein composition in the manufacture of a product for early diagnosis of lung cancer, the product comprising a protein composition comprising the antigen proteins DDX53, MMRN2, and further comprising any one or more of the antigen proteins MKNK1, FGF12, NME3, TIA1, and ENO 2; the diagnosis product utilizes the protein composition to detect lung cancer self-IgG antibodies in serum, thereby being used for early diagnosis of lung cancer.
2. The use according to claim 1, wherein the protein composition further comprises any one, two or three of the antigen proteins PIP4K2C, CTAG1A, HYLS1 and SSBP 4.
3. Use of a protein composition in the preparation of a kit for early diagnosis of lung cancer, wherein the kit comprises the protein composition of any one of claims 1 or 2, and the kit uses the protein composition to detect lung cancer autoantibodies in serum of a subject, thereby being used for early diagnosis of lung cancer.
4. The use according to claim 3, characterized by the steps of:
s1: obtaining a serum sample of a subject;
s2: detecting the expression level of lung cancer autoantibody in a serum sample of a subject by using a kit;
S3: and comparing the expression level of the lung cancer autoantibody in the detected serum sample with a preset value, and if the expression level is larger than the preset value, judging that the lung cancer exists.
5. An apparatus for early diagnosis of lung cancer, the apparatus comprising:
a sample acquisition module for acquiring a serum sample of a subject;
A sample detection module comprising the early diagnosis kit for lung cancer of claim 3 for detecting the expression level of lung cancer autoantibody in a serum sample of a subject;
And the data processing and result output module is used for: comparing the expression level of the lung cancer autoantibody in the serum sample of the subject detected by the sample detection module with a preset value, outputting a lung cancer result if the expression level is larger than the preset value, and outputting a lung cancer-free result if the expression level is not larger than the preset value.
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