CN103242547B - A kind of film boiomacromolecule crosslinking agent and preparation method and application - Google Patents
A kind of film boiomacromolecule crosslinking agent and preparation method and application Download PDFInfo
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Abstract
本发明涉及一种膜用生物高分子交联剂及其制备方法与应用，属于生物技术领域。该膜用生物高分子交联剂包括如下按质量百分比计的组分多聚赖氨酸0.001～0.025％，精氨酸0.05～0.2％，色氨酸0.01～1％，酪氨酸0.01～1％，Triton‑X‑1000.0002～0.005％(V/V)，余量为PH＝8‑10的水。将上述制备百分比的组分混匀得到膜用生物高分子交联剂。本发明提供的膜用生物高分子交联剂能使核酸和蛋白直接交联到核酸/蛋白转印膜上，省去了固定核酸或蛋白对仪器的依赖，提高了核酸/蛋白的检测灵敏度。The invention relates to a biopolymer crosslinking agent for membranes, a preparation method and application thereof, and belongs to the field of biotechnology. The biopolymer cross-linking agent for membranes comprises the following components in terms of mass percentage: 0.001-0.025% polylysine, 0.05-0.2% arginine, 0.01-1% tryptophan, and 0.01-1% tyrosine %, Triton‑X‑1000.0002～0.005% (V/V), the balance is water with PH=8‑10. The above-mentioned components of the preparation percentage are mixed to obtain a biopolymer crosslinking agent for membranes. The biopolymer cross-linking agent for membranes provided by the invention can directly cross-link nucleic acids and proteins to nucleic acid/protein transfer membranes, eliminating the need for instruments to fix nucleic acids or proteins, and improving the detection sensitivity of nucleic acids/proteins.
Description
技术领域technical field
本发明属于生物技术领域，涉及一种膜用生物高分子交联剂及其制备方法与其在核酸/蛋白领域的应用。The invention belongs to the field of biotechnology, and relates to a biopolymer crosslinking agent for membranes, a preparation method thereof and its application in the nucleic acid/protein field.
背景技术Background technique
Blotting技术是核酸和蛋白检测的一大常用基本技术。核酸是以Northernblotting为代表，蛋白以Western blotting为代表。这两大技术是分子生物学中最经典最常用的基本技术。它们共同的一点是要将生物分子固定在商品膜上。核酸的固定方法有高温干燥和紫外交联两种方法，蛋白则主要用干燥方法。固定对于提高生物分子在膜上的结合能力以提高检测灵敏度极为重要。紫外交联和高温干燥必须特定的仪器来实现，而且交联的剂量和干燥的强度会对检测的灵敏度有很大影响(Nucleic Acids Research，2004，32(22)：e175；Nucleic Acids Res.2007；35(8)：e60；Nat Protoc.2008；3(6)：1077-84)。另外，紫外照射对核酸蛋白分子有破坏作用，并且对人体也有伤害作用。蛋白通过干燥固定在膜特别是硝酸纤维膜上并不牢固，会在后面的漂流过程中损失，影响检测的灵敏度。Blotting technology is a common basic technology for nucleic acid and protein detection. Nucleic acid is represented by Northern blotting, and protein is represented by Western blotting. These two techniques are the most classical and commonly used basic techniques in molecular biology. What they all have in common is the immobilization of biomolecules on commercial membranes. Nucleic acid can be immobilized by high-temperature drying and ultraviolet crosslinking, while protein is mainly dried. Immobilization is extremely important to increase the binding capacity of biomolecules on the membrane to improve detection sensitivity. Ultraviolet cross-linking and high-temperature drying must be realized by specific instruments, and the dosage of cross-linking and the intensity of drying will have a great impact on the sensitivity of detection (Nucleic Acids Research, 2004, 32(22): e175; Nucleic Acids Res.2007 ; 35(8):e60; Nat Protoc. 2008; 3(6):1077-84). In addition, ultraviolet radiation has a damaging effect on nucleic acid and protein molecules, and it is also harmful to the human body. The protein is not firmly fixed on the membrane, especially the nitrocellulose membrane by drying, and will be lost in the subsequent drifting process, which will affect the sensitivity of the detection.
发明内容Contents of the invention
本发明的首要目的在于克服现有技术的缺点与不足，提供一种膜用生物高分子交联剂。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a biopolymer cross-linking agent for membranes.
本发明的再一目的在于提供一种上述膜用生物高分子交联剂的制备方法。Another object of the present invention is to provide a method for preparing the biopolymer crosslinking agent for membranes.
本发明的另一目的在于提供上述膜用生物高分子交联剂的应用。Another object of the present invention is to provide the application of the above biopolymer crosslinking agent for membranes.
本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:
一种膜用生物高分子交联剂，包括如下按质量百分比计的组分：A biopolymer crosslinking agent for membranes, comprising the following components by mass percentage:
余量为碱性水(PH＝8-10)。The balance is alkaline water (PH=8-10).
优选的，一种膜用生物高分子交联剂，包括如下按质量百分比计的组分：Preferably, a biopolymer crosslinking agent for membranes comprises the following components by mass percentage:
余量为碱性水(PH＝8-10)。The balance is alkaline water (PH=8-10).
上述膜用生物高分子交联剂的制备方法包括如下步骤：取所述质量百分比的多聚赖氨酸、精氨酸、色氨酸、酪氨酸、Triton-X-100和水，混匀得到膜用生物高分子交联剂。The preparation method of the biopolymer cross-linking agent for the above film comprises the steps of: taking the polylysine, arginine, tryptophan, tyrosine, Triton-X-100 and water in the mass percentage, mixing A biopolymer cross-linking agent for membranes was obtained.
上述膜用生物高分子交联剂在核酸/蛋白领域中的应用。The application of the biopolymer cross-linking agent for membranes in the nucleic acid/protein field.
所述的膜用生物高分子交联剂在核酸/蛋白领域中的应用，包括如下步骤：The application of the membrane biopolymer cross-linking agent in the field of nucleic acid/protein comprises the following steps:
(1)将核酸/蛋白转印膜用上述膜用生物高分子交联剂溶液20～40℃浸泡处理5～30秒。(1) Soak the nucleic acid/protein transfer membrane in the biopolymer cross-linking agent solution for the membrane at 20-40° C. for 5-30 seconds.
(2)步骤(1)处理过的核酸/蛋白转印膜干燥后用于blotting技术(印迹技术)。(2) The nucleic acid/protein transfer membrane treated in step (1) is dried and used for blotting technique (imprinting technique).
步骤(1)中所述的浸泡处理优选为室温浸泡处理10秒。The soaking treatment described in step (1) is preferably soaking treatment at room temperature for 10 seconds.
步骤(1)中所述的核酸/蛋白转印膜优选为PVDF膜、硝酸纤维素膜或尼龙膜。The nucleic acid/protein transfer membrane described in step (1) is preferably PVDF membrane, nitrocellulose membrane or nylon membrane.
步骤(2)中所述的blotting技术(印迹技术)指Southern Blotting(DNA印迹技术)、Northern blotting(RNA印迹技术)、Western blotting(蛋白质印迹技术)和/或Dotblotting(斑点印迹技术)。The blotting technique (imprinting technique) described in step (2) refers to Southern Blotting (Southern Blotting technique), Northern blotting (Northern Blotting technique), Western blotting (Western Blotting technique) and/or Dotblotting (Dotblotting technique).
本发明基本原理是充分利用所述交联剂的强电荷效应等其它分子间作用力，与待固定核酸或蛋白相互作用，从而将核酸或蛋白固定在膜上。The basic principle of the present invention is to make full use of other intermolecular forces such as the strong charge effect of the cross-linking agent to interact with the nucleic acid or protein to be immobilized, thereby immobilizing the nucleic acid or protein on the membrane.
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
(1)本发明提供的膜用生物高分子交联剂能使核酸和蛋白直接交联到核酸/蛋白转印膜上，省去了固定核酸或蛋白对仪器的依赖。(1) The biopolymer cross-linking agent for membranes provided by the present invention can directly cross-link nucleic acids and proteins to nucleic acid/protein transfer membranes, eliminating the need for instruments to fix nucleic acids or proteins.
(2)将本发明提供的膜用生物高分子交联剂应用到blotting技术提高了核酸/蛋白的检测灵敏度，特别是对于特定的膜如硝酸纤维膜，因为硝酸纤维膜没有电荷也没有供有效紫外交联的化学基团如氨基，硝酸纤维膜是公认的检测灵敏度相对很低的膜。(2) Applying the membrane biopolymer cross-linking agent provided by the invention to the blotting technology improves the detection sensitivity of nucleic acid/protein, especially for specific membranes such as nitrocellulose membranes, because nitrocellulose membranes have no charge and do not provide effective UV cross-linked chemical groups such as amino, nitrocellulose membranes are recognized as relatively low detection sensitivity membranes.
附图说明Description of drawings
图1是核酸膜用生物高分子交联剂固定BCIP/NBT显色结果图；其中，A：紫外交联，B：交联剂固定。Figure 1 is the chromogenic result diagram of BCIP/NBT immobilized by biopolymer cross-linking agent for nucleic acid membrane; where, A: UV cross-linking, B: cross-linking agent immobilization.
图2是蛋白膜用生物高分子交联剂固定BCIP/NBT显色结果图；其中，A：风干固定，B：交联剂固定。Figure 2 is a picture of the color development results of BCIP/NBT immobilized by a biopolymer cross-linking agent for the protein film; where, A: air-dried and fixed, B: cross-linked agent immobilized.
图3是内源RNA分子的检测结果图。Fig. 3 is a diagram of detection results of endogenous RNA molecules.
图4是内源蛋白分子的检测结果图。Fig. 4 is a diagram of detection results of endogenous protein molecules.
具体实施方式Detailed ways
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
实施例1膜用生物高分子交联剂的制备The preparation of embodiment 1 film biopolymer cross-linking agent
取10mg精氨酸、10mg色氨酸、10mg酪氨酸、1ul Triton-X-100(商品化为液体)溶于10ml0.01％多聚赖氨酸溶液(商品化可得)混匀得到膜用生物高分子交联剂。Take 10mg of arginine, 10mg of tryptophan, 10mg of tyrosine, 1ul Triton-X-100 (commercialized as a liquid) dissolved in 10ml of 0.01% polylysine solution (commercially available) and mix well to obtain a film with biopolymer crosslinkers.
实施例2RNA分子的检测The detection of embodiment 2 RNA molecules
(1)由GenePharma(吉玛)公司合成RNA分子miR-21；由上海博尚生物技术有限公司合成biotin标记的DNA探针(miR-21的反义序列)。(1) RNA molecule miR-21 was synthesized by GenePharma Company; a biotin-labeled DNA probe (antisense sequence of miR-21) was synthesized by Shanghai Boshang Biotechnology Co., Ltd.
(2)分别取上述合成RNA与DNA探针10pmol按LHCD(液相杂交显色法)(X.Li，M.Ni，andY.Zhang，Methods85(2)，151(2012))进行液相杂交。(2) Take 10 pmol of the above-mentioned synthetic RNA and DNA probe and carry out liquid phase hybridization according to LHCD (liquid phase hybridization chromogenic method) (X.Li, M.Ni, and Y.Zhang, Methods85(2), 151(2012)) .
(3)将尼龙膜用实施例1制备的膜用生物高分子交联剂室温浸泡10秒，风干或者按专业标准方法紫外交联。(3) Soak the nylon membrane prepared in Example 1 with a biopolymer cross-linking agent at room temperature for 10 seconds, air-dry or UV cross-link according to professional standard methods.
(4)取步骤(2)的杂交液1μL点于步骤(3)处理的尼龙膜上，风干。(4) Spot 1 μL of the hybridization solution from step (2) on the nylon membrane treated in step (3), and air-dry.
(5)接下来的步骤封闭和加入ABC(亲合素生物素复合物)信号放大及其后的洗膜均按LHCD(液相杂交显色法)(X.Li，M.Ni，and Y.Zhang，Methods85(2)，151(2012))进行。(5) The next step is to block and add ABC (avidin-biotin complex) signal amplification and subsequent washing of the membrane according to LHCD (liquid phase hybridization color method) (X.Li, M.Ni, and Y . Zhang, Methods 85(2), 151(2012)).
(6)使用碱性磷酸酶显色液BCIP/NBT显色30秒，再用水终止显色。(6) Use alkaline phosphatase chromogenic solution BCIP/NBT to develop color for 30 seconds, and then stop color development with water.
结果如图1所示，1：未加探针，2：未加RNA，3：加无关的RNA(吉玛公司合成通用对照)，4：加与miR-21相同的DNA，5：加miR-21。结果表明，A图的紫外交联对RNA分子的检测效果与B图的交联剂固定效果相当。The results are shown in Figure 1, 1: no probe added, 2: no RNA added, 3: irrelevant RNA added (common control synthesized by Gemma), 4: the same DNA as miR-21 added, 5: miR added -twenty one. The results show that the detection effect of UV cross-linking in Figure A on RNA molecules is equivalent to that of cross-linking agent immobilization in Figure B.
实施例3蛋白分子的检测The detection of embodiment 3 protein molecules
(1)由吉尔生化有限公司合成FLAG TAG肽段。(1) The FLAG TAG peptide was synthesized by Gill Biochemical Co., Ltd.
(2)将硝酸纤维膜用实施例1制备的膜用生物高分子交联剂室温浸泡10秒，风干。(2) Soak the nitrocellulose membrane with the biopolymer cross-linking agent prepared in Example 1 at room temperature for 10 seconds, and air-dry.
(3)取步骤(1)的蛋白50ng点于步骤(2)处理的膜上，风干或者不用步骤(2)处理直接点样风干。(3) Spot 50ng of the protein from step (1) on the membrane treated in step (2), and air-dry or directly spot and air-dry without step (2) treatment.
(4)用质量体积比10％BSA(牛血清白蛋白)室温下将硝酸纤维素膜封闭1小时。(4) Block the nitrocellulose membrane with 10% BSA (bovine serum albumin) at a mass volume ratio for 1 hour at room temperature.
(5)用biotin标记兔抗FLAG TAG抗体(购自上海博耀生物试剂有限公司)室温杂交1小时。(5) Hybridize with biotin-labeled rabbit anti-FLAG TAG antibody (purchased from Shanghai Boyao Biological Reagent Co., Ltd.) for 1 hour at room temperature.
(6)TBST(按《分子克隆》配制)洗3次，每次10分钟。(6) TBST (prepared according to "Molecular Cloning") washed 3 times, 10 minutes each time.
(7)用ABC(亲合素生物素复合物)信号放大及其后的洗膜均按LHCD(液相杂交显色法)(X.Li，M.Ni，and Y.Zhang，Methods85(2)，151(2012))进行。(7) Amplify the signal with ABC (avidin-biotin complex) and then wash the membrane according to LHCD (liquid phase hybridization color method) (X.Li, M.Ni, and Y.Zhang, Methods85 (2 ), 151(2012)).
(8)使用碱性磷酸酶显色液BCIP/NBT显色1min，再用水终止显色。(8) Use the alkaline phosphatase chromogenic solution BCIP/NBT to develop the color for 1 min, and then stop the color development with water.
结果如图2所示，1：未加FLAG TAG蛋白，2：未加兔抗FLAG TAG抗体，3：加无关的蛋白(按文献Acta Biochim Biophys Sin(2008)：855-863表达的肽段MacGAP-N)，4：FLAG TAG蛋白和兔抗FLAG TAG抗体均加，5：4的重复点样。结果表明，A图的风干处理对蛋白分子的检测信号强度明显低于B图的交联剂固定效果。The results are shown in Figure 2, 1: No FLAG TAG protein, 2: No rabbit anti-FLAG TAG antibody, 3: Add irrelevant protein (according to the literature Acta Biochim Biophys Sin (2008): 855-863 expressed peptide MacGAP -N), 4: FLAG TAG protein and rabbit anti-FLAG TAG antibody were added, 5: 4 repeated spotting. The results show that the air-drying treatment in Figure A has significantly lower detection signal intensity on protein molecules than the cross-linking agent immobilization effect in Figure B.
实施例4内源RNA分子的检测The detection of embodiment 4 endogenous RNA molecules
(1)用CO2气体法人道处死或断颈处死大鼠，用Trizol试剂提取大鼠肝脏RNA(提取方法按照INVITROGEN公司Trizol试剂盒说明书操作)。(1) Rats were killed humanely or by neck dislocation with CO 2 gas, and RNA from rat liver was extracted with Trizol reagent (the extraction method was operated according to the instructions of the Trizol kit from INVITROGEN Company).
(2)所提取的RNA用biotin标记的DNA探针(miR-122的反义序列)按LHCD(液相杂交显色法)(X.Li，M.Ni，and Y.Zhang，Methods85(2)，151(2012))进行液相杂交。(2) The extracted RNA is labeled with biotin DNA probe (antisense sequence of miR-122) according to LHCD (liquid phase hybridization color method) (X.Li, M.Ni, and Y.Zhang, Methods85 (2 ), 151(2012)) for liquid phase hybridization.
(3)将尼龙膜用实施例1制备的膜用生物高分子交联剂室温浸泡10秒，风干。(3) Soak the nylon membrane prepared in Example 1 with the biopolymer cross-linking agent at room temperature for 10 seconds, and air-dry.
(4)取步骤(2)的杂交液1μL点于步骤(2)处理的尼龙膜上，风干。(4) Take 1 μL of the hybridization solution from step (2) and spot on the nylon membrane treated in step (2), and air dry.
(5)接下来的步骤封闭和加入ABC(亲合素生物素复合物)信号放大及其后的洗膜均按LHCD(液相杂交显色法)(X.Li，M.Ni，and Y.Zhang，Methods85(2)，151(2012))进行。(5) The next step is to block and add ABC (avidin-biotin complex) signal amplification and subsequent washing of the membrane according to LHCD (liquid phase hybridization color method) (X.Li, M.Ni, and Y . Zhang, Methods 85(2), 151(2012)).
(6)使用碱性磷酸酶显色液BCIP/NBT显色1min，再用水终止显色。(6) Use the alkaline phosphatase chromogenic solution BCIP/NBT to develop the color for 1 min, and then stop the color development with water.
结果如图3所示，1：未加探针，2：未加RNA，3：加无关的RNA，4：加与miR-122相同的DNA分子，5：大鼠肝脏RNA。所示结果表明，本发明所制生物高分子交联剂能用于膜上检测内源性RNA分子。The results are shown in Figure 3, 1: no probe, 2: no RNA, 3: irrelevant RNA, 4: the same DNA molecule as miR-122, 5: rat liver RNA. The results shown show that the biopolymer cross-linking agent prepared by the present invention can be used to detect endogenous RNA molecules on the membrane.
实施例5内源蛋白分子的检测The detection of embodiment 5 endogenous protein molecule
(1)用CO2气体法人道处死大鼠，用SIGMA公司蛋白提取试剂RIPA提取大鼠脑总蛋白。(1) Rats were killed humanely with CO 2 gas, and the total protein of rat brain was extracted with protein extraction reagent RIPA of SIGMA Company.
(2)将硝酸纤维素膜用实施例1制备的膜用生物高分子交联剂室温浸泡10秒，风干。(2) Soak the nitrocellulose membrane with the biopolymer cross-linking agent prepared in Example 1 at room temperature for 10 seconds, and air-dry.
(3)取步骤(1)的蛋白10μg划线状点于步骤(2)处理的硝酸纤维素膜上，风干。(3) Take 10 μg of the protein from step (1) as a streak and spot on the nitrocellulose membrane treated in step (2), and air-dry.
(4)用质量体积比10％BSA(牛血清白蛋白)室温下将硝酸纤维素膜封闭1小时。(4) Block the nitrocellulose membrane with 10% BSA (bovine serum albumin) at a mass volume ratio for 1 hour at room temperature.
(5)用兔抗大鼠GS(谷氨酰胺合成酶)一抗(购自SIGMA公司)室温杂交1小时。(5) Hybridize with rabbit anti-rat GS (glutamine synthetase) primary antibody (purchased from SIGMA) for 1 hour at room temperature.
(6)TBST(按《分子克隆》配制)洗3次，每次10分钟。(6) TBST (prepared according to "Molecular Cloning") washed 3 times, 10 minutes each time.
(7)用羊抗兔二抗(HRP标记)(购自ABCAM公司)室温杂交1小时。(7) Hybridize with goat anti-rabbit secondary antibody (HRP-labeled) (purchased from ABCAM Company) at room temperature for 1 hour.
(8)TBST洗3次，每次10分钟。(8) Wash 3 times with TBST, 10 minutes each time.
(9)ECL显色5分钟，X光片暴光5秒。(9) ECL color development for 5 minutes, X-ray film exposure for 5 seconds.
结果如图4所示，1：未加一抗，2：未加二抗，3：一抗二抗都加。所示结果表明，本发明所制生物高分子交联剂能用于膜上检测内源性蛋白分子。The results are shown in Figure 4, 1: no primary antibody was added, 2: no secondary antibody was added, 3: both primary and secondary antibodies were added. The results shown show that the biopolymer cross-linking agent prepared by the present invention can be used to detect endogenous protein molecules on the membrane.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
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