CN103884694A - LKB1(Liver Kinase B1) protein detection methods and kit - Google Patents

Abstract

Translated fromChinese

本发明公开了一种LKB1蛋白检测方法，包括以下步骤：制备LKB1单克隆抗体；用量子点标记LKB1单克隆抗体，得到量子点LKB1单克隆抗体；用量子点LKB1单克隆抗体与标本反应；将反应后的标本置于显微镜下观察。本发明还提供了含有量子点LKB1单克隆抗体的检测试剂盒。本发明的检测方法以量子点LKB1单克隆抗体作为探针，与LKB1蛋白结合的特异性增强，毒性减小使用更安全，着色标本可长期保存，进行重复观察；操作简单、灵敏度高。

The invention discloses a method for detecting LKB1 protein, comprising the following steps: preparing LKB1 monoclonal antibody; marking the LKB1 monoclonal antibody with quantum dots to obtain the quantum dot LKB1 monoclonal antibody; reacting the quantum dot LKB1 monoclonal antibody with a sample; The reacted samples were observed under a microscope. The invention also provides a detection kit containing the quantum dot LKB1 monoclonal antibody. The detection method of the present invention uses the quantum dot LKB1 monoclonal antibody as a probe, the specificity of binding to the LKB1 protein is enhanced, the toxicity is reduced and the use is safer, and the colored specimen can be stored for a long time for repeated observation; the operation is simple and the sensitivity is high.

Description

Translated fromChinese

一种LKB1蛋白检测方法及试剂盒A kind of LKB1 protein detection method and kit

技术领域technical field

本发明涉及生物技术领域，具体涉及一种LKB1蛋白检测方法及试剂盒。The invention relates to the field of biotechnology, in particular to a method and kit for detecting LKB1 protein.

背景技术Background technique

人LKB1（Liver Kinase B1）基因或称STK11（Serine-Threonine kinase 11）基因，定位于人染色体19p13.3位置，含10个外显子，编码的LKB1蛋白由433个氨基酸组成，分子量约为50kDa，包括激酶区域（44-309）、N端调节域和C端调节域。N端调节域含一个核定位序列，使LKB1蛋白定位于细胞核内。LKB1基因编码一种丝-苏氨酸蛋白激酶，参与多种生物学过程，包括细胞周期阻滞、p53介导的凋亡、细胞极性诱导等，介导TGF-β、G蛋白偶联等信号通路，在细胞生长、分化调控中发挥着重要作用，几乎在人体多种组织中均有表达，以上皮、睾丸生精小管、肝脏、小肠和骨骼肌最多。LKB1基因是肿瘤易患综合症，即PJ综合征（Peutz-Jeghers syndrome，PJS）的致病基因，在高达90%的PJS病人中可检测到LKB1基因的胚系突变。LKB1基因的功能异常还与全身多种散发性肿瘤的发生密切相关，如在30-40%的非小细胞肺癌、20%的宫颈癌、19%鳞状细胞癌及13%乳腺侵润性导管癌中均有LKB1基因的失活。另外，LKB1基因在恶性肿瘤发生中起着重要作用，已有研究证实LKB1能够使细胞周期停滞在G1期，抑制细胞生长。LKB1基因是一个较为普遍的抑癌基因。LKB1基因表达情况对肿瘤发生和预后影响都是一个重要的中间指标，对揭示LKB1基因抑制细胞恶性增殖的分子背景有重要的意义，因此，LKB1基因表达情况的检测，即LKB1蛋白水平检测或LKB1mRNA的水平检测是亟待解决的问题。The human LKB1 (Liver Kinase B1) gene or STK11 (Serine-Threonine kinase 11) gene is located at the position of human chromosome 19p13.3 and contains 10 exons. The encoded LKB1 protein consists of 433 amino acids with a molecular weight of about 50kDa , including the kinase domain (44-309), the N-terminal regulatory domain, and the C-terminal regulatory domain. The N-terminal regulatory domain contains a nuclear localization sequence that localizes the LKB1 protein to the nucleus. The LKB1 gene encodes a serine-threonine protein kinase that participates in various biological processes, including cell cycle arrest, p53-mediated apoptosis, cell polarity induction, etc., and mediates TGF-β, G protein coupling, etc. The signaling pathway plays an important role in the regulation of cell growth and differentiation, and is expressed in almost all kinds of tissues in the human body, most of which are epithelium, testicular seminiferous tubules, liver, small intestine and skeletal muscle. The LKB1 gene is the causative gene of the tumor susceptibility syndrome, PJ syndrome (Peutz-Jeghers syndrome, PJS), and germline mutations of the LKB1 gene can be detected in up to 90% of PJS patients. The abnormal function of LKB1 gene is also closely related to the occurrence of various sporadic tumors in the whole body, such as 30-40% of non-small cell lung cancer, 20% of cervical cancer, 19% of squamous cell carcinoma and 13% of breast invasive duct Inactivation of the LKB1 gene is found in all cancers. In addition, the LKB1 gene plays an important role in the occurrence of malignant tumors. Studies have confirmed that LKB1 can arrest the cell cycle in the G1 phase and inhibit cell growth. LKB1 gene is a common tumor suppressor gene. The expression of LKB1 gene is an important intermediate indicator for tumor occurrence and prognosis, and it is of great significance to reveal the molecular background of LKB1 gene inhibiting cell malignant proliferation. Therefore, the detection of LKB1 gene expression is the detection of LKB1 protein level or LKB1mRNA Level detection is an urgent problem to be solved.

目前，LKB1蛋白的检测方法主要为免疫组织化学法，一种现有技术利用兔源多克隆抗体作为一抗，然后用标记了化学显色试剂或者荧光基团的二抗对一抗进行标记，一抗与LKB1蛋白结合后再用化学显色试剂的颜色或者荧光基团的荧光信号来判断LKB1蛋白的丰度。这种间接的检测方法步骤繁琐耗时长，并且由于多克隆抗体的特异性不强导致非特异性信号的产生，对检测结果的准确性有很大影响。还有一种现有技术是利用DAB显色的原理，对LKB1鼠源单克隆抗体进行显色标记，然后LKB1抗原免疫组化染色，再用光学显微镜观察，出现棕黄色或棕褐色的为阳性，综合考虑阳性细胞着色深浅和阳性细胞占所观察同类细胞的比例两项指标，进行半定量判断结果。这种方法中使用的DAB染色剂有毒性，为疑似致癌物质，不安全，另外，显色剂显色反应受温度影响大，且显色时间受到限制，不能长期保存，实验人员无法将组织切片保存进行重复观察。At present, the detection method of LKB1 protein is mainly immunohistochemical method. One existing technology uses rabbit-derived polyclonal antibody as the primary antibody, and then labels the primary antibody with a secondary antibody labeled with a chemical chromogenic reagent or a fluorescent group. After the primary antibody is combined with the LKB1 protein, the abundance of the LKB1 protein is judged by the color of the chemical chromogenic reagent or the fluorescence signal of the fluorophore. This indirect detection method is cumbersome and time-consuming, and non-specific signals are generated due to the low specificity of polyclonal antibodies, which has a great impact on the accuracy of detection results. Another existing technology is to use the principle of DAB color development to color-label the LKB1 mouse monoclonal antibody, then immunohistochemically stain the LKB1 antigen, and then observe it with an optical microscope. If it appears brown or brown, it is positive. The results were semi-quantitatively judged by comprehensively considering the two indicators of the coloring depth of positive cells and the proportion of positive cells in the observed cells of the same type. The DAB staining agent used in this method is toxic, suspected carcinogen, and unsafe. In addition, the color reaction of the chromogenic agent is greatly affected by temperature, and the color development time is limited, so it cannot be stored for a long time, and the experimenter cannot slice the tissue Save for repeat observations.

量子点具有宽的激发光谱和窄的发射光谱（光谱宽在20-40nm之间），通过改变量子点的尺寸或者改变量子点内核的组分可以在很大范围内调节量子点的发射光谱。量子点与有机荧光团相比有几个突出的光学特点：窄的发射光谱能减少光谱重叠，从而能同时区别多个荧光团；宽的激发光谱能在单一波长下激发多种颜色的光谱。此外量子点具有光稳定性和抗降解性，对细胞的毒性小。这些独特的光学性质使量子点能在体内跟踪多个细胞和制备体外多个分子生物传感器，而且由于量子点的可调谐的发光波长，使量子点能促进组织深层的细胞成像。Quantum dots have a wide excitation spectrum and a narrow emission spectrum (the spectral width is between 20-40nm), and the emission spectrum of quantum dots can be adjusted in a wide range by changing the size of quantum dots or changing the composition of the quantum dot core. Compared with organic fluorophores, quantum dots have several outstanding optical features: a narrow emission spectrum can reduce spectral overlap, so that multiple fluorophores can be distinguished simultaneously; a wide excitation spectrum can excite multiple color spectra at a single wavelength. In addition, quantum dots have photostability and degradation resistance, and have little toxicity to cells. These unique optical properties enable quantum dots to track multiple cells in vivo and prepare multiple molecular biosensors in vitro, and due to the tunable emission wavelength of quantum dots, quantum dots can promote cell imaging in deep tissues.

发明内容Contents of the invention

本发明旨在解决上述现有技术中存在的问题，提出一种LKB1蛋白检测方法，包括以下步骤：The present invention aims to solve the problems existing in the above-mentioned prior art, and proposes a method for detecting LKB1 protein, comprising the following steps:

用量子点标记LKB1单克隆抗体，得到量子点LKB1单克隆抗体；Label LKB1 monoclonal antibody with quantum dots to obtain quantum dot LKB1 monoclonal antibody;

用量子点LKB1单克隆抗体与标本反应；Use the quantum dot LKB1 monoclonal antibody to react with the specimen;

将反应后的标本置于显微镜下观察，The reacted specimens were observed under a microscope,

其中，所述的标本为细胞标本或组织标本，所述的细胞标本为组织印片、细胞爬片或细胞涂片，所述的组织标本为石蜡切片或冰冻切片。Wherein, the sample is a cell sample or a tissue sample, the cell sample is a tissue print, a cell slide or a cell smear, and the tissue sample is a paraffin section or a frozen section.

优选地，所述的用量子点标记LKB1单克隆抗体包括以下步骤：Preferably, said labeling LKB1 monoclonal antibody with quantum dots comprises the following steps:

将量子点表面用GHS进行修饰，得到GSH-量子点；Modify the surface of quantum dots with GHS to obtain GSH-quantum dots;

在GSH-量子点中加入SMCC，形成SMCC-GHS-量子点；Add SMCC to GSH-quantum dots to form SMCC-GHS-quantum dots;

将SMCC-GHS-量子点与LKB1单克隆抗体反应，得到量子点LKB1单克隆抗体。SMCC-GHS-quantum dots were reacted with LKB1 monoclonal antibody to obtain quantum dot LKB1 monoclonal antibody.

优选地，所述的将量子点表面用GHS进行修饰为：Preferably, the surface of the quantum dot is modified with GHS as follows:

将量子点加入到GHS中进行反应，得到第一反应液；adding the quantum dots to the GHS for reaction to obtain the first reaction solution;

将第一反应液离心，收集沉淀；centrifuging the first reaction solution to collect the precipitate;

将所得沉淀用potassium t-butoxide溶液溶解，得到第二液体；Gained precipitation is dissolved with potassium t-butoxide solution, obtains the second liquid;

将第二液体透析，去除第二液体中的GSH与potassium t-butoxide，得到GSH-量子点。Dialyzing the second liquid to remove GSH and potassium t-butoxide in the second liquid to obtain GSH-quantum dots.

优选地，所述的SMCC-GHS-量子点的制备方法为：Preferably, the preparation method of described SMCC-GHS-quantum dots is:

将GSH-量子点与sulfo-SMCC水溶液混合反应，得到第三反应液；Mixing and reacting GSH-quantum dots and sulfo-SMCC aqueous solution to obtain a third reaction solution;

在第三反应液中加入巯基乙醇，得到第四反应液；Add mercaptoethanol to the third reaction solution to obtain the fourth reaction solution;

将第四反应液进行层析，得到第五反应液，再将层析后得到的第五反应液离心，离心所得沉淀重新溶解，得到SMCC-GHS-量子点。The fourth reaction solution is subjected to chromatography to obtain the fifth reaction solution, and then the fifth reaction solution obtained after the chromatography is centrifuged, and the precipitate obtained by centrifugation is redissolved to obtain SMCC-GHS-quantum dots.

优选地，所述的SMCC-GHS-量子点与LKB1单克隆抗体反应为：Preferably, the reaction of the SMCC-GHS-quantum dots with the LKB1 monoclonal antibody is:

LKB1单克隆抗体中加入DTT溶液进行还原，得到还原的LKB1单克隆抗体；Add DTT solution to the LKB1 monoclonal antibody for reduction to obtain reduced LKB1 monoclonal antibody;

将SMCC-GHS-量子点与还原的LKB1单克隆抗体混合反应，得到量子点LKB1单克隆抗体。The SMCC-GHS-quantum dots were mixed with the reduced LKB1 monoclonal antibody to obtain the quantum dot LKB1 monoclonal antibody.

优选地，所述的用量子点LKB1单克隆抗体与标本反应包括以下步骤：Preferably, the reaction of the quantum dot LKB1 monoclonal antibody with the sample comprises the following steps:

将标本用固定液进行固定；Fix the specimen with fixative;

用洗涤液冲洗固定后的标本；Rinse the fixed specimen with washing solution;

过氧化氢处理标本；Treat specimens with hydrogen peroxide;

封闭液处理标本；Blocking solution to process specimens;

将量子点LKB1单克隆抗体与标本进行反应。React the quantum dot LKB1 monoclonal antibody with the sample.

本发明还提供了LKB1蛋白检测试剂盒，包括量子点LKB1单克隆抗体。The invention also provides a LKB1 protein detection kit, which includes a quantum dot LKB1 monoclonal antibody.

优选地，还包括固定液和洗涤液。Preferably, fixative and washing solutions are also included.

优选地，还包括封闭液。Preferably, a blocking solution is also included.

优选地，还包括过氧化氢。Preferably, hydrogen peroxide is also included.

优选地，还包括阳性对照品和阴性对照品，所述的阳性对照品为过表达LKB1蛋白的细胞，所述的阴性对照品为LKB1表达沉默的细胞。Preferably, a positive control substance and a negative control substance are also included, the positive control substance is cells overexpressing LKB1 protein, and the negative control substance is cells expressing silenced LKB1.

优选地，所述的固定液选自戊二醛、多聚甲醛、乙醇、甲醇或丙酮。Preferably, the fixative is selected from glutaraldehyde, paraformaldehyde, ethanol, methanol or acetone.

优选地，所述的洗涤液为PBS溶液。Preferably, the washing solution is PBS solution.

优选地，所述的封闭液为正常山羊血清。Preferably, the blocking solution is normal goat serum.

本发明的LKB1蛋白检测方法利用抗原抗体特异性结合的原理，以量子点LKB1单克隆抗体作为探针与抗原（LKB1蛋白）结合，利用量子点发出的荧光对LKB1蛋白进行定位、定性及定量研究。细胞样品或组织样本被制成标本后，加入量子点LKB1单克隆抗体反应，将反应后的标本置于荧光显微镜下观察。LKB1蛋白与量子点LKB1单克隆抗体特异性的结合后，LKB1蛋白发出荧光，能够发出荧光的细胞为阳性细胞，不能发出荧光的为阴性细胞，还可通过综合考虑阳性细胞荧光强度和阳性细胞占所观察同类细胞的比例两项指标，进行半定量判断结果。The LKB1 protein detection method of the present invention utilizes the principle of antigen-antibody specific binding, uses the quantum dot LKB1 monoclonal antibody as a probe to bind to the antigen (LKB1 protein), and utilizes the fluorescence emitted by the quantum dot to conduct localization, qualitative and quantitative research on the LKB1 protein . After the cell sample or tissue sample is made into a specimen, the quantum dot LKB1 monoclonal antibody is added for reaction, and the reacted specimen is observed under a fluorescent microscope. After the LKB1 protein is specifically combined with the quantum dot LKB1 monoclonal antibody, the LKB1 protein emits fluorescence. The cells that can emit fluorescence are positive cells, and the cells that cannot emit fluorescence are negative cells. The positive cell fluorescence intensity and the proportion of positive cells can also be considered comprehensively. The two indicators of the ratio of the observed cells of the same type are semi-quantitatively judged.

本发明的有益效果在于，本发明的检测方法以量子点LKB 1单克隆抗体作为探针，与LKB 1蛋白结合的特异性增强，毒性减小使用更安全，着色标本可长期保存，进行重复观察；本发明的检测方法的有益效果还在于操作简单、灵敏度高。The beneficial effect of the present invention is that the detection method of the present invention uses the quantum dot LKB 1 monoclonal antibody as a probe, the specificity of binding to the LKB 1 protein is enhanced, the toxicity is reduced and the use is safer, and the colored specimen can be stored for a long time for repeated observation The beneficial effect of the detection method of the present invention also lies in simple operation and high sensitivity.

附图说明Description of drawings

图1是荧光显微镜下乳腺癌细胞BT474涂片图。Figure 1 is a smear of breast cancer cell BT474 under a fluorescence microscope.

图2是荧光显微镜下胃癌细胞BGC823涂片图。Figure 2 is a smear of gastric cancer cell BGC823 under a fluorescence microscope.

具体实施方式Detailed ways

为了使本领域的技术人员更好的理解本申请的技术方案，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整的描述。本发明的LKB1蛋白检测方法利用抗原抗体特异性结合的原理，以量子点LKB1单克隆抗体作为探针与抗原（LKB1蛋白）结合，利用量子点发出的荧光对LKB1蛋白进行定位、定性及定量研究。In order to enable those skilled in the art to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. The LKB1 protein detection method of the present invention utilizes the principle of antigen-antibody specific binding, uses the quantum dot LKB1 monoclonal antibody as a probe to bind to the antigen (LKB1 protein), and utilizes the fluorescence emitted by the quantum dot to conduct localization, qualitative and quantitative research on the LKB1 protein .

本发明的LKB 1蛋白检测方法采用以下技术方案实现：LKB1 protein detection method of the present invention adopts following technical scheme to realize:

用量子点标记LKB1单克隆抗体，得到量子点LKB1单克隆抗体；Label LKB1 monoclonal antibody with quantum dots to obtain quantum dot LKB1 monoclonal antibody;

用量子点LKB1单克隆抗体与标本反应；Use the quantum dot LKB1 monoclonal antibody to react with the specimen;

将反应后的标本置于显微镜下观察，The reacted specimens were observed under a microscope,

其中，所述的标本为细胞标本或组织标本，所述的细胞标本为组织印片、细胞爬片或细胞涂片，所述的组织标本为石蜡切片或冰冻切片。Wherein, the sample is a cell sample or a tissue sample, the cell sample is a tissue print, a cell slide or a cell smear, and the tissue sample is a paraffin section or a frozen section.

LKB1单克隆抗体可选用鼠源LKB1单克隆抗体，本实施例中选用的是abcam公司的鼠抗人LKB1单克隆抗体ab15095，也可选用其他LKB1单克隆抗体。The LKB1 monoclonal antibody can be selected from the mouse LKB1 monoclonal antibody. In this example, the mouse anti-human LKB1 monoclonal antibody ab15095 from abcam company is used, and other LKB1 monoclonal antibodies can also be used.

量子点可选用CdSe、CdTe、CdSe/ZnS、CdTe/ZnS、CdTe/CdS/ZnS、CdSe/CdS/ZnS、CdSe/CdZnS、CdSe:Mn²⁺、CdTe：Mn²⁺、CdSe:Zn²⁺或CdTe:Zn²⁺，不限于以上。本实施例中使用的量子点是CdSe/CdZnS。Quantum dots can be selected from CdSe, CdTe, CdSe/ZnS, CdTe/ZnS, CdTe/CdS/ZnS, CdSe/CdS/ZnS, CdSe/CdZnS, CdSe:Mn²⁺ , CdTe:Mn²⁺ , CdSe:Zn²⁺ or CdTe:Zn²⁺ , not limited to the above. The quantum dots used in this example are CdSe/CdZnS.

本实施例中，量子点LKB1单克隆抗体采用以下方法制备：In this example, the quantum dot LKB1 monoclonal antibody was prepared by the following method:

将量子点表面用GHS进行修饰，得到GSH-量子点；Modify the surface of quantum dots with GHS to obtain GSH-quantum dots;

在GSH-量子点中加入SMCC，形成SMCC-GHS-量子点；Add SMCC to GSH-quantum dots to form SMCC-GHS-quantum dots;

将SMCC-GHS-量子点与LKB1单克隆抗体反应，得到量子点LKB1单克隆抗体。SMCC-GHS-quantum dots were reacted with LKB1 monoclonal antibody to obtain quantum dot LKB1 monoclonal antibody.

本实施例中采用SMCC（Succinimidyl 4-（N-maleimidomethyl）cyclohexane-1-carboxylate，琥珀酰亚胺-4-环已烷-1-碳酸酯）作为螯合剂，连接LKB1单克隆抗体与GHS-量子点，SMCC分子一端的NHS酯基团与LKB1单克隆抗体的伯氨反应形成稳定的酰胺键，SMCC的另一端（马来酰亚胺基团一端）可与GHS-量子点的巯基发生特异性交联。In this example, SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, succinimidyl-4-cyclohexane-1-carbonate) was used as a chelating agent to connect the LKB1 monoclonal antibody to GHS-Quantum The NHS ester group at one end of the SMCC molecule reacts with the primary amino group of the LKB1 monoclonal antibody to form a stable amide bond, and the other end of the SMCC (maleimide group end) can specifically interact with the sulfhydryl group of the GHS-quantum dot. couplet.

LKB1单克隆抗体与量子点的连接方法还可以通过EDC HCl和NHS共同作用等其他方式实现，并不限于以上。The connection method between LKB1 monoclonal antibody and quantum dots can also be realized by other methods such as the joint action of EDC HCl and NHS, and is not limited to the above.

另外，GHS-量子点的制备方法优选为：In addition, the preparation method of GHS-quantum dots is preferably:

将量子点加入到GHS中进行反应，得到第一反应液；adding the quantum dots to the GHS for reaction to obtain the first reaction solution;

将第一反应液离心，收集沉淀；centrifuging the first reaction solution to collect the precipitate;

将所得沉淀用potassium t-butoxide溶液溶解，得到第二液体；Gained precipitation is dissolved with potassium t-butoxide solution, obtains the second liquid;

将第二液体透析，去除第二液体中的GSH与potassium t-butoxide，得到GSH-量子点。Dialyzing the second liquid to remove GSH and potassium t-butoxide in the second liquid to obtain GSH-quantum dots.

在SMCC-GHS-量子点制备中，为了提高SMCC-GHS-量子点的回收率，在进行SMCC-GHS-量子点纯化前在反应液中加入巯基乙醇，再对反应液进行层析，本实施例中采用的是柱层析的方法，再将层析后得到的反应液离心，离心所得沉淀重新溶解，得到SMCC-GHS-量子点。In the preparation of SMCC-GHS-quantum dots, in order to improve the recovery rate of SMCC-GHS-quantum dots, mercaptoethanol is added to the reaction solution before SMCC-GHS-quantum dots are purified, and then the reaction solution is chromatographed. This implementation In the example, the method of column chromatography is used, and the reaction solution obtained after the chromatography is centrifuged, and the precipitate obtained by centrifugation is redissolved to obtain SMCC-GHS-quantum dots.

在SMCC-GHS-量子点与LKB1单克隆抗体反应制备量子点LKB1单克隆抗体的步骤中，为了提高SMCC-GHS-量子点与LKB1单克隆抗体的作用，SMCC-GHS-量子点与LKB1单克隆抗体混合反应之前，在LKB1单克隆抗体中加入DTT溶液进行还原，得到还原的LKB1单克隆抗体。In the step of preparing quantum dot LKB1 monoclonal antibody by reacting SMCC-GHS-quantum dot with LKB1 monoclonal antibody, in order to improve the effect of SMCC-GHS-quantum dot and LKB1 monoclonal antibody, SMCC-GHS-quantum dot and LKB1 monoclonal antibody Before the antibody mixing reaction, add DTT solution to the LKB1 monoclonal antibody for reduction to obtain the reduced LKB1 monoclonal antibody.

另外，所述的用量子点LKB1单克隆抗体与标本反应包括以下步骤：In addition, the reaction of the quantum dot LKB1 monoclonal antibody with the sample includes the following steps:

将标本用固定液进行固定；Fix the specimen with fixative;

用洗涤液冲洗固定后的标本；Rinse the fixed specimen with washing solution;

过氧化氢处理标本；Treat specimens with hydrogen peroxide;

封闭液处理标本；Blocking solution to process specimens;

将量子点LKB1单克隆抗体与标本进行反应。React the quantum dot LKB1 monoclonal antibody with the sample.

为了使细胞内的物质尽量保持原有的形态、结构、位置，在标本上加入了固定液，本实施例中固定液优选为戊二醛、多聚甲醛、乙醇、甲醛或丙酮，更优选为多聚甲醛或丙酮，最优选为丙酮。洗涤液优选为PBS溶液。为了减少量子点LKB1单克隆抗体与除LKB1蛋白外的其他蛋白的非特异性结合，在量子点LKB1单克隆抗体处理标本之前加入封闭液，所述的封闭液优选为血清，更优选为正常山羊血清或牛血清蛋白（BSA），最优选为正常山羊血清。In order to maintain the original shape, structure, and position of the substances in the cells as much as possible, a fixative is added to the specimen. In this embodiment, the fixative is preferably glutaraldehyde, paraformaldehyde, ethanol, formaldehyde or acetone, more preferably Paraformaldehyde or acetone, most preferably acetone. The washing solution is preferably PBS solution. In order to reduce the non-specific binding of the quantum dot LKB1 monoclonal antibody to other proteins except the LKB1 protein, a blocking solution is added before the quantum dot LKB1 monoclonal antibody treats the specimen, and the blocking solution is preferably serum, more preferably normal goat serum or bovine serum albumin (BSA), most preferably normal goat serum.

本发明还提供了LKB1蛋白检测试剂盒，包括量子点LKB1单克隆抗体，还包括固定液、洗涤液、过氧化氢、封闭液、阳性对照品和阴性对照品。The invention also provides a LKB1 protein detection kit, which includes a quantum dot LKB1 monoclonal antibody, and also includes a fixing solution, a washing solution, hydrogen peroxide, a blocking solution, a positive control substance and a negative control substance.

所述的阳性对照品为过表达LKB1蛋白的细菌，所述的阴性对照品为LKB1表达沉默的细菌。所述的固定液选自戊二醛、多聚甲醛、乙醇、甲醇或丙酮。所述的洗涤液为PBS溶液。所述的封闭液为正常山羊血清。The positive control substance is a bacterium overexpressing LKB1 protein, and the negative control substance is a silencing bacterium expressing LKB1. The fixative is selected from glutaraldehyde, paraformaldehyde, ethanol, methanol or acetone. The washing solution is PBS solution. The blocking solution is normal goat serum.

以下为实施例。The following are examples.

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

实施例1Example 1

LKB1蛋白检测步骤：LKB1 protein detection steps:

（1）CdSe/CdZnS量子点表面GSH修饰(1) GSH modification on the surface of CdSe/CdZnS quantum dots

在1ml的CdSe/CdZnS量子点溶液中缓慢加入1ml浓度为200mg/ml的GSH溶液，加热至60℃进行反应。GSH包被CdSe/CdZnS量子点形成GSH-量子点，GSH-量子点沉淀，离心收集沉淀。用1ml浓度为100mg/ml的potassiumt-butoxide溶解沉淀，水浴超声5min。水浴超声后的水溶液用0.2μm滤膜过滤，然后在10mM PBS溶液中透析除去多余的GSH和potassium t-butoxide。Slowly add 1 ml of GSH solution with a concentration of 200 mg/ml into 1 ml of CdSe/CdZnS quantum dot solution, and heat to 60° C. for reaction. GSH is coated with CdSe/CdZnS quantum dots to form GSH-quantum dots, and the GSH-quantum dots are precipitated, and the precipitates are collected by centrifugation. Dissolve the precipitate with 1ml of potassiumt-butoxide with a concentration of 100mg/ml, and ultrasonicate for 5min in a water bath. The aqueous solution after sonication in a water bath was filtered with a 0.2 μm filter membrane, and then dialyzed in 10 mM PBS solution to remove excess GSH and potassium t-butoxide.

（2）SMCC螯合的量子点合成(2) Synthesis of SMCC chelated quantum dots

在上述1ml GSH-量子点PBS溶液中加入100μl浓度为1mM的sulfo-SMCC水溶液，反应30min。然后加入1mM巯基乙醇。再将反应混合物过NapTM-5柱子，用PBS溶液洗脱。洗脱液15000g离心5min去除聚合的量子点。Add 100 μl of 1mM sulfo-SMCC aqueous solution to the above 1ml GSH-quantum dot PBS solution, and react for 30min. Then 1 mM mercaptoethanol was added. Then the reaction mixture was passed through NapTM-5 column and eluted with PBS solution. The eluate was centrifuged at 15000 g for 5 min to remove aggregated quantum dots.

（3）SMCC-GSH-量子点与LKB1单克隆抗体螯合(3) Chelation of SMCC-GSH-quantum dots with LKB1 monoclonal antibody

1mg LKB1单克隆抗体溶于1ml水中，加入20μl浓度为1M的DTT对LKB1单克隆抗体进行还原；在10mM PBS溶液中进行透析；还原后的LKB1单克隆抗体加入到上述制备的SMCC-GSH-量子点中孵育1h，让LKB1单克隆抗体与量子点结合；然后在10mMPBS溶液中进行透析；0.2μm滤膜过滤去除聚合的量子点。1mg of LKB1 monoclonal antibody was dissolved in 1ml of water, and 20μl of 1M DTT was added to reduce the LKB1 monoclonal antibody; dialyzed in 10mM PBS solution; the reduced LKB1 monoclonal antibody was added to the SMCC-GSH-quantum prepared above Incubate the dots for 1 h to allow the LKB1 monoclonal antibody to bind to the quantum dots; then dialyze in a 10 mMPBS solution; filter through a 0.2 μm filter membrane to remove aggregated quantum dots.

（4）利用量子点-LKB1单克隆抗体进行冰冻切片的免疫组化检测(4) Immunohistochemical detection of frozen sections using quantum dot-LKB1 monoclonal antibody

a.冰冻切片4～8μm，室温放置30min后，浸入4℃丙酮中固定10分钟；a. Frozen sections with a thickness of 4-8 μm were placed at room temperature for 30 minutes, then immersed in acetone at 4°C for 10 minutes;

b.PBS冲洗，5min×3次；b. Rinse with PBS, 5min×3 times;

c.用3%过氧化氢孵育5～10分钟消除内源性过氧化物酶活性；c. Incubate with 3% hydrogen peroxide for 5-10 minutes to eliminate endogenous peroxidase activity;

d.PBS冲洗，5min×3次；d. Rinse with PBS, 5min×3 times;

e.5-10%正常山羊血清封闭，室温孵育10分钟。倾去血清，勿洗，滴加适当比例稀释的QDs-LKB1抗体，37℃孵育1~2小时或4℃过夜；e. Block with 5-10% normal goat serum and incubate at room temperature for 10 minutes. Pour away the serum, do not wash, add the QDs-LKB1 antibody diluted in an appropriate proportion dropwise, and incubate at 37°C for 1-2 hours or overnight at 4°C;

f.PBS冲洗，2分钟×3次；f. Rinse with PBS, 2 minutes x 3 times;

g.复染，脱水透明、封片；g. Counterstaining, dehydration, transparency, and mounting;

h.在荧光显微镜下观察。h. Observation under a fluorescent microscope.

其中，CdSe/CdZnS量子点合成过程如下：在25ml三颈烧瓶中混合1gTOPO，3g HDA，66mg cadmium,4-pentanedionate，250mg月桂酸（stearic acid），冲入氩气，加热至320℃，恒温搅拌30min，快速加入0.2ml 100mg/ml TBP溶液，冷却至300℃恒温，CdSe量子点开始合成，检测其荧光发射波谱，当发射波谱达到635nm时，继续冷却至250℃，恒温，于三颈烧瓶中逐滴加入0.25mlCd-Zn-Se储存溶液，伴随剧烈搅拌，同时检测发射波谱，当红移15nm后继续冷却至100℃，搅拌5h，冷却至50℃时，加入过量的甲醇使量子点产物沉淀，后离心收集沉淀，将所得沉淀用20ml四氢呋喃溶解备用。Among them, the synthesis process of CdSe/CdZnS quantum dots is as follows: mix 1gTOPO, 3g HDA, 66mg cadmium, 4-pentanedionate, 250mg lauric acid (stearic acid) in a 25ml three-necked flask, pour into argon, heat to 320°C, and stir at constant temperature 30min, quickly add 0.2ml 100mg/ml TBP solution, cool to 300°C constant temperature, CdSe quantum dots start to synthesize, detect its fluorescence emission spectrum, when the emission spectrum reaches 635nm, continue cooling to 250°C, constant temperature, in a three-necked flask Add 0.25ml of Cd-Zn-Se storage solution dropwise, with vigorous stirring, and detect the emission spectrum at the same time, when the red shift is 15nm, continue to cool to 100°C, stir for 5h, and when cooled to 50°C, add excess methanol to precipitate the quantum dot product, Afterwards, the precipitate was collected by centrifugation, and the resulting precipitate was dissolved with 20 ml of tetrahydrofuran for subsequent use.

应用例1Application example 1

将乳腺癌细胞BT474制成细胞涂片，用实施例1中的LKB 1蛋白检测方法对乳腺癌细胞涂片进行观察。图1为荧光显微镜下的观察到的乳腺癌细胞BT474。The breast cancer cell BT474 was made into a cell smear, and the breast cancer cell smear was observed with the LKB 1 protein detection method in Example 1. Fig. 1 is the observed breast cancer cell BT474 under the fluorescence microscope.

应用例2Application example 2

将胃癌细胞BGC823制成细胞涂片，用实施例1中的LKB 1蛋白检测方法对胃癌细胞涂片进行观察。图2为荧光显微镜下的观察到的胃癌细胞BGC 823。Gastric cancer cell BGC823 was made into a cell smear, and the gastric cancer cell smear was observed by the LKB 1 protein detection method in Example 1. Figure 2 shows gastric cancer cell BGC 823 observed under a fluorescence microscope.

应用例3Application example 3

将胃癌组织标本与正常胃粘膜标本按照实施例1中的方法在显微镜下观察，综合每张切片中阳性细胞荧光强度和阳性细胞所占观察同类细胞的百分比两个指标，进行半定量检测。Gastric cancer tissue specimens and normal gastric mucosal specimens were observed under a microscope according to the method in Example 1, and semi-quantitative detection was performed based on two indicators, the fluorescence intensity of positive cells in each section and the percentage of positive cells in the same type of observed cells.

表1荧光强度评价Table 1 Fluorescence intensity evaluation

表2阳性细胞比例评价Table 2 Positive cell ratio evaluation

标本评分=荧光强度值评分×阳性细胞比例评分Specimen score = fluorescence intensity value score × positive cell ratio score

根据标本评分确定LKB1蛋白的水平：Determine the level of LKB1 protein according to the specimen score:

0-1分为阴性；0-1 is divided into negative;

2-3分为弱阳性；2-3 is divided into weak positive;

4分以上为强阳性。A score of 4 or more is strongly positive.

40例胃癌组织标本（胃癌组）与40例正常胃粘膜标本（正常组）LKB1蛋白的水平比较结果见表3，正常胃粘膜标本LKB1蛋白水平全部为强阳性，胃癌组织标本LKB1蛋白水平没有强阳性，37.5%为弱阳性，62.5%为阴性，根据SPSS 13.0统计软件的数据处理结果，胃癌组与正常组的LKB 1蛋白水平差异显著（P<0.05）。The comparison results of LKB1 protein levels in 40 gastric cancer tissue samples (gastric cancer group) and 40 normal gastric mucosa samples (normal group) are shown in Table 3. The LKB1 protein levels of normal gastric mucosa samples were all strongly positive, while the LKB1 protein levels of gastric cancer tissue samples were not strongly positive. Positive, 37.5% were weakly positive, and 62.5% were negative. According to the data processing results of SPSS 13.0 statistical software, there was a significant difference in the level of LKB 1 protein between the gastric cancer group and the normal group (P<0.05).

表3各组LKB 1蛋白的水平比较Table 3 Comparison of the levels of LKB 1 protein in each group

实施例2Example 2

按照以下配方制作LKB1蛋白检测试剂盒：Make the LKB1 protein detection kit according to the following formula:

量子点LKB1单克隆抗体：按照实施例1步骤（1）-（3）合成量子点LKB1单克隆抗体；Quantum dot LKB1 monoclonal antibody: synthesize quantum dot LKB1 monoclonal antibody according to steps (1)-(3) of Example 1;

固定液：丙酮；Fixative: acetone;

洗涤液：PBS溶液；Washing solution: PBS solution;

封闭液：5-10%正常山羊血清；Blocking solution: 5-10% normal goat serum;

过氧化氢：浓度为3%-5%。Hydrogen peroxide: the concentration is 3%-5%.

本实施例中制备的LKB 1蛋白检测试剂盒的使用方法：The method of use of the LKB 1 protein detection kit prepared in this embodiment:

将标本用固定液进行固定；Fix the specimen with fixative;

用洗涤液冲洗固定后的标本；Rinse the fixed specimen with washing solution;

过氧化氢处理标本；Treat specimens with hydrogen peroxide;

封闭液处理标本；Blocking solution to process specimens;

将量子点LKB1单克隆抗体与标本进行反应。React the quantum dot LKB1 monoclonal antibody with the sample.

实施例3Example 3

按照以下配方制作LKB1蛋白检测试剂盒：Make the LKB1 protein detection kit according to the following formula:

量子点LKB1单克隆抗体：按照实施例1步骤（1）-（3）合成量子点LKB1单克隆抗体；Quantum dot LKB1 monoclonal antibody: synthesize quantum dot LKB1 monoclonal antibody according to steps (1)-(3) of Example 1;

固定液：丙酮；Fixative: acetone;

洗涤液：PBS溶液；Washing solution: PBS solution;

封闭液：5-10%正常山羊血清；Blocking solution: 5-10% normal goat serum;

过氧化氢：浓度为3%-5%；Hydrogen peroxide: the concentration is 3%-5%;

阴性对照：构建靶向LKB 1基因的siRNA表达质粒，将所述质粒转染到HEK293T细胞；Negative control: constructing an siRNA expression plasmid targeting the LKB 1 gene, and transfecting the plasmid into HEK293T cells;

阳性对照：构建LKB1基因表达质粒，将所述质粒转染到HEK293T细胞。Positive control: LKB1 gene expression plasmid was constructed, and the plasmid was transfected into HEK293T cells.

本实施例中制备的LKB1蛋白检测试剂盒的使用方法：The method of using the LKB1 protein detection kit prepared in this example:

将标本用固定液进行固定；Fix the specimen with fixative;

用洗涤液冲洗固定后的标本；Rinse the fixed specimen with washing solution;

过氧化氢处理标本；Treat specimens with hydrogen peroxide;

封闭液处理标本；Blocking solution to process specimens;

将量子点LKB1单克隆抗体与标本进行反应。React the quantum dot LKB1 monoclonal antibody with the sample.

阴性对照和阳性对照使用时将阴性对照细胞和阳性对照细胞制作成细胞涂片或细胞爬片，用上述方法进行处理，在荧光显微镜下观察，阴性对照和阳性对照作为判断荧光强度的基准。When negative control and positive control are used, negative control cells and positive control cells are made into cell smears or cell slides, processed by the above method, observed under a fluorescent microscope, and negative control and positive control are used as benchmarks for judging the fluorescence intensity.

虽然本发明参照当前的较佳实施方式进行了描述，但本领域的技术人员应能理解，上述较佳实施方式仅用来说明本发明，并非用来限定本发明的保护范围，任何在本发明的精神和原则范围之内，所做的任何修饰、等效替换、改进等，均应包含在本发明的权利保护范围之内。Although the present invention has been described with reference to the current preferred embodiments, those skilled in the art should understand that the above-mentioned preferred embodiments are only used to illustrate the present invention, and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and scope of principles shall be included in the protection scope of the present invention.

Claims (12)

Translated fromChinese

1.一种LKB1蛋白检测方法，其特征在于，包括以下步骤：1. A method for detecting LKB1 protein, comprising the following steps:用量子点标记LKB1单克隆抗体，得到量子点LKB1单克隆抗体；Label LKB1 monoclonal antibody with quantum dots to obtain quantum dot LKB1 monoclonal antibody;用量子点LKB1单克隆抗体与标本反应；Use the quantum dot LKB1 monoclonal antibody to react with the specimen;将反应后的标本置于显微镜下观察，The reacted specimens were observed under a microscope,其中，所述的标本为细胞标本或组织标本，所述的细胞标本为组织印片、细胞爬片或细胞涂片，所述的组织标本为石蜡切片或冰冻切片。Wherein, the sample is a cell sample or a tissue sample, the cell sample is a tissue print, a cell slide or a cell smear, and the tissue sample is a paraffin section or a frozen section.2.根据权利要求1所述的检测方法，其特征在于，所述的用量子点标记LKB1单克隆抗体包括以下步骤：2. detection method according to claim 1, is characterized in that, described LKB1 monoclonal antibody with quantum dot labeling comprises the following steps:将量子点表面用GHS进行修饰，得到GSH-量子点；Modify the surface of quantum dots with GHS to obtain GSH-quantum dots;在GSH-量子点中加入SMCC，形成SMCC-GHS-量子点；Add SMCC to GSH-quantum dots to form SMCC-GHS-quantum dots;将SMCC-GHS-量子点与LKB1单克隆抗体反应，得到量子点LKB1单克隆抗体。SMCC-GHS-quantum dots were reacted with LKB1 monoclonal antibody to obtain quantum dot LKB1 monoclonal antibody.3.根据权利要求2所述的检测方法，其特征在于，所述的将量子点表面用GHS进行修饰为：3. detection method according to claim 2, is characterized in that, described quantum dot surface is modified as:将量子点加入到GHS中进行反应，得到第一反应液；adding the quantum dots to the GHS for reaction to obtain the first reaction solution;将第一反应液离心，收集沉淀；centrifuging the first reaction solution to collect the precipitate;将所得沉淀用potassium t-butoxide溶液溶解，得到第二液体；Gained precipitation is dissolved with potassium t-butoxide solution, obtains the second liquid;将第二液体透析，去除第二液体中的GSH与potassium t-butoxide，得到GSH-量子点。Dialyzing the second liquid to remove GSH and potassium t-butoxide in the second liquid to obtain GSH-quantum dots.4.根据权利要求2所述的检测方法，其特征在于，所述的SMCC-GHS-量子点的制备方法为：4. detection method according to claim 2, is characterized in that, the preparation method of described SMCC-GHS-quantum dot is:将GSH-量子点与sulfo-SMCC水溶液混合反应，得到第三反应液；Mixing and reacting GSH-quantum dots and sulfo-SMCC aqueous solution to obtain a third reaction solution;在第三反应液中加入巯基乙醇，得到第四反应液；Add mercaptoethanol to the third reaction solution to obtain the fourth reaction solution;将第四反应液进行层析，得到第五反应液，再将层析后得到的第五反应液离心，离心所得沉淀重新溶解，得到SMCC-GHS-量子点。The fourth reaction solution is subjected to chromatography to obtain the fifth reaction solution, and then the fifth reaction solution obtained after the chromatography is centrifuged, and the precipitate obtained by centrifugation is redissolved to obtain SMCC-GHS-quantum dots.5.根据权利要求2所述的检测方法，其特征在于，所述的SMCC-GHS-量子点与LKB1单克隆抗体反应为：5. detection method according to claim 2, is characterized in that, described SMCC-GHS-quantum dot and LKB1 monoclonal antibody reaction are:LKB1单克隆抗体中加入DTT溶液进行还原，得到还原的LKB1单克隆抗体；Add DTT solution to the LKB1 monoclonal antibody for reduction to obtain reduced LKB1 monoclonal antibody;将SMCC-GHS-量子点与还原的LKB1单克隆抗体混合反应，得到量子点LKB1单克隆抗体。The SMCC-GHS-quantum dots were mixed with the reduced LKB1 monoclonal antibody to obtain the quantum dot LKB1 monoclonal antibody.6.根据权利要求1所述的检测方法，其特征在于，所述的用量子点LKB1单克隆抗体与标本反应包括以下步骤：6. detection method according to claim 1, is characterized in that, described reaction with sample with quantum dot LKB1 monoclonal antibody comprises the following steps:将标本用固定液进行固定；Fix the specimen with fixative;用洗涤液冲洗固定后的标本；Rinse the fixed specimen with washing solution;过氧化氢处理标本；Treat specimens with hydrogen peroxide;封闭液处理标本；Blocking solution to process specimens;将量子点LKB1单克隆抗体与标本进行反应。React the quantum dot LKB1 monoclonal antibody with the sample.7.一种LKB1蛋白检测试剂盒，其特征在于，包括量子点LKB1单克隆抗体。7. A LKB1 protein detection kit, characterized in that it comprises a quantum dot LKB1 monoclonal antibody.8.根据权利要求7所述的试剂盒，其特征在于，还包括固定液、洗涤液、封闭液和过氧化氢。8. The kit according to claim 7, further comprising fixative, washing solution, blocking solution and hydrogen peroxide.9.根据权利要求8所述的试剂盒，其特征在于，还包括阳性对照品和阴性对照品，所述的阳性对照品为过表达LKB1蛋白的细胞，所述的阴性对照品为LKB1表达沉默的细胞。9. The kit according to claim 8, further comprising a positive control substance and a negative control substance, wherein the positive control substance is a cell overexpressing the LKB1 protein, and the negative control substance is a silencing of LKB1 expression Cell.10.根据权利要求8或9所述的试剂盒，其特征在于，所述的固定液选自戊二醛、多聚甲醛、乙醇、甲醇或丙酮。10. The kit according to claim 8 or 9, wherein the fixative is selected from glutaraldehyde, paraformaldehyde, ethanol, methanol or acetone.11.根据权利要求8或9所述的试剂盒，其特征在于，所述的洗涤液为PBS溶液。11. The kit according to claim 8 or 9, characterized in that the washing solution is a PBS solution.12.根据权利要求8或9所述的试剂盒，其特征在于，所述的封闭液为正常山羊血清。12. The kit according to claim 8 or 9, wherein the blocking solution is normal goat serum.

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