论文总字数：32653字

摘 要

醛糖还原酶(AKR1B1)作为醛酮还原酶超家族(AKRs)成员之一，生理功能多样，催化底物广泛,可通过介导细胞内多种信号传导途径,参与乳腺癌、肝癌、结肠癌等多种肿瘤的发生发展过程。多项研究表明AKR1B1在多种肿瘤中出现表达量异常，提示AKR1B1是潜在的肿瘤标志物。本实验室前期研究通过免疫组化确证了AKR1B1在肝癌（HCC）中的表达模式，约55%的HCC组织中AKR1B1表达阳性；此外Western Blot结果显示，肝癌细胞系的培养上清中存在分泌的AKR1B1。

为进一步探究AKR1B1能否作为HCC诊断标志物以及其在HCC发生及发展中的作用，我们拟建立针对AKR1B1蛋白的双抗体夹心ELISA快速检测方法，探究AKR1B1作为人肝细胞癌血清标志物的潜力。实验室前期工作已经获得稳定分泌抗AKR1B1单克隆抗体的杂交瘤细胞株，并纯化获得了抗AKR1B1的鼠源单克隆抗体。在此基础上制备了抗AKR1B1的兔源多克隆抗体，并建立了以多抗作为捕获抗体、单抗作为检测抗体的双抗体夹心ELISA法。

1.抗AKR1B1蛋白多克隆抗体的制备

利用重组质粒pGEX-4T-1(His)₆C-AR表达纯化ARK1B1蛋白作为免疫原免疫成年雌性新西兰大白兔。用间接ELISA法测多克隆抗体滴度，达到实验要求后心脏采血收集血清，进行Protein G亲和层析纯化。进一步用Western blot鉴定多克隆抗体特异性,结果显示我们成功获得了抗AKR1B1蛋白的多克隆抗体。用间接ELISA法测多克隆抗体效价为1:12800。

2.检测AKR1B1蛋白的双抗体夹心ELISA方法的建立

我们以制备的多克隆抗体作为包被抗体、单克隆抗体作为检测抗体,成功建立了检测AKR1B1的双抗体夹心ELISA方法。实验优化条件为：包被抗体最佳稀释度为1:2000,检测抗体最佳稀释度为1:1000，最佳封闭液浓度为5%的脱脂奶粉，HRP酶标抗体工作浓度为1：5000。

关键词：AKR1B1，多克隆抗体，单克隆抗体，双抗体夹心ELISA,

ABSTRACT

Aldose reductase (AKR1B1) belong to the aldehyde ketone reductase superfamily member (AKRs), aldose reductase (AKR1B1) catalytic substrate, physiological function is diversiform, can be mediated intracellular signal transduction pathway, involved in a wide variety of tumor development, such as breast cancer, liver cancer, colon cancer, etc. Abnormal AKR1B1 expressed in a wide variety of tumor. Research found prior to the lab AKR1B1 expression in hepatocellular carcinoma (HCC) tissue abnormalities, we proposed to establish rapid detection of serum AKR1B1 method, explore AKR1B1 as the potential of human hepatocellular carcinoma (HCC) serum markers. The early stage of the laboratory work has been in preparation for AKR1B1 monoclonal antibody, to achieve the aim to increase the detection sensitivity. Based on this foundation, further examined the expression of liver cancer patients serum AKR1B1, investigates the possibility as a marker of liver cancer.

1. Preparation of polyclonal antibody against AKR1B1 protein

    The ARK1B1 protein was purified by recombinant plasmid pGEX-4T-1(His)6C-AR in the early stage of the laboratory, and monoclonal antibodies were prepared by hybridoma technique. The ARK1B1 protein was used as an immunogen to immunize adult female New Zealand white rabbits. The polyclonal antibody titer was measured by indirect ELISA, and the blood was collected by cardiac blood collection after the experimental requirements were met. Purification by Protein G affinity chromatography and further identification by Western blot showed that the polyclonal antibody against AKR1B1 protein was successfully obtained. The indirect ELISA assay polyclonal antibody titer was 1:12800.

2. Establishment of double antibody sandwich ELISA method for detection of AKR1B1

The prepared polyclonal antibody was used as a coating antibody and a monoclonal antibody was used as a detection antibody, and a double antibody sandwich ELISA method for detecting AKR1B1 was established. The optimal conditions for the experiment were as follows: the optimal dilution of the coated antibody was 1:2000, the optimal dilution of the detection antibody was 1:1000, the optimal concentration of the blocking solution was 5% skim milk powder, and the working concentration of the HRP enzyme antibody was 1:5000.

KEY WORDS: AKR1B1，polyclonal antibody，monoclonal antibody，ELISA，

目 录

摘要 III

Abstract III

第一章 文献综述………………………………………………………………………………7

1 AKR1B1结构特点及生理功能………………………………………………………7

1.1 AKR超家族成员组成……………………………………………………………7

1.2 AKR1B1基因及其分子结构………………………………………………7 1.2.1 AKR1B1基因……………………………………………………………7

1.2.2 AKR1B1的结构特点……………………………………………………8 2 AKR1B1的生理功能…………………………………………………………………8

3 AKR1B1与肝细胞癌…………………………………………………………………8

4 肿瘤标志物及检测方法……………………………………………………………9

4.1 肿瘤标志物定义………………………………………………………………10 4.2 肿瘤标志物检测方法…………………………………………………………10 4.2.1放射免疫分析…………………………………………………………10

4.2.2 化学发光免疫分析法…………………………………………………11

4.2.3 酶联免疫吸附试验……………………………………………………11 4.2.4 生物传感器……………………………………………………………12

5 展望…………………………………………………………………………………12

第二章 抗AKR1B1蛋白多克隆抗体的制备…………………………………………………13

1实验材料………………………………………………………………………………13

1.1 实验动物……………………………………………………………………13

1.2 主要试剂……………………………………………………………………13

1.3 仪器设备……………………………………………………………………13

1.4 主要溶剂配制………………………………………………………………14

2实验方法………………………………………………………………………………15

2.1 动物免疫……………………………………………………………………15

2.2 多克隆抗体的纯化…………………………………………………………16

2.3 western blot鉴定多克隆抗体特异性……………………………………17

2.4间接ELISA检测多克隆抗体效价…………………………………………19

3实验结果……………………………………………………………………………20

3.1 多克隆抗体特异性鉴定……………………………………………………20

3.2 多克隆抗体效价检测………………………………………………………21

4讨论…………………………………………………………………………………21

第三章 抗AKR1B1蛋白双抗体夹心ELISA检测方法建立…………………………………22

1实验方法……………………………………………………………………………22

1.1最佳多抗包被浓度及单抗工作浓度确定…………………………………22

1.2最佳封闭液确定……………………………………………………………22

1.3 HRP酶标抗体工作浓度确定………………………………………………22

2实验结果……………………………………………………………………………23

3讨论…………………………………………………………………………………24

全文总结………………………………………………………………………………………24

参考文献………………………………………………………………………………………25

致谢……………………………………………………………………………………………29

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