CN110804554A - Cell lysis solution and application thereof - Google Patents

Cell lysis solution and application thereof Download PDF

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CN110804554A
CN110804554A CN201911103403.8A CN201911103403A CN110804554A CN 110804554 A CN110804554 A CN 110804554A CN 201911103403 A CN201911103403 A CN 201911103403A CN 110804554 A CN110804554 A CN 110804554A
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inhibitor
cell lysate
distilled water
activity
sodium
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CN110804554B (en
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王淑一
吴鹏飞
李允章
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Nanchang Adicon Clinical Laboratories Ltd
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    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
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Abstract

The invention discloses a cell lysis solution and application thereof. The cell lysate consists of nonionic detergent, protease inhibitor, phosphatase inhibitor, RNase inhibitor and distilled water. The volume ratio of the nonionic detergent to the protease inhibitor to the phosphatase inhibitor to the RNase inhibitor to the distilled water is as follows: 2:1:2:1:14. The cell lysate can effectively release DNA of cells and simultaneously reserve the protease activity of telomeres, can be applied to the detection of the telomerase activity, and provides an accurate and effective sample for the detection of the telomerase activity.

Description

Cell lysis solution and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cell lysate for detecting the activity of cell telomerase in the field of biological and enzyme detection and application thereof.
Background
Telomeres are specific DNA-protein structures at the ends of chromosomes in eukaryotes. Human telomeres consist of 6 base repeats (TTAGGG) and binding proteins, which have important biological functions, such as chromosome stabilization and prevention of chromosome end fusion; protecting a chromosome structural gene; regulating normal cell growth. In normal somatic cells, because the 5' end of linear DNA replication is deleted, the telomere can be gradually shortened along with the continuous proliferation of the cells, when the telomere of the cells is shortened to a certain degree, the cells stop dividing and are in a static state, and then the cells can enter an aging stage. Telomerase is a ribonucleoprotein, which consists of RNA and protein, wherein the RNA is used as a reverse transcription template, the protein is used as reverse transcriptase, and the telomere end is used as a primer to synthesize a telomere DNA repetitive sequence. Human normal somatic cells are generally negative for telomerase activity, whereas 90% of malignant cells are positive. Research results show that telomeres and telomerase are closely related to normal cell growth regulation and malignant tumor formation.
The traditional telomerase research method is based on probe extension to measure telomerase activity. Because a large amount of samples are needed and the detection sensitivity is limited, the method is quickly replaced by a telomere repeat amplification method (TRAP), the basic principle is that the reverse transcriptase activity of a telomere protein part is utilized, an RNA part is used as a reverse transcription template, one sequence which is artificially designed is used as a lead primer, the other sequence is used as a subsequent primer for carrying out RT-PCR, and the size of the enzyme activity is judged through product analysis. The subsequent developed telomerase activity detection method is also based on TRAP method, such as TRAP-ELISA, qPCR-TRAP, etc. Based on these methods for detecting telomerase activity, there is a need for an effective cell lysate that releases cellular nucleic acids while retaining protein activity.
Therefore, based on the needs, the invention develops the cell lysate, the lysate can simultaneously obtain nucleic acid and protein, the activity of protease is ensured, and the components are safe and green.
Disclosure of Invention
The invention aims to provide a cell lysate for detecting telomerase activity. The cell lysate can simultaneously obtain nucleic acid and protein, ensures the activity of protease, has safe and green components, can be used for detecting the activity of telomerase, and has the advantages of simplicity, rapidness and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the cell lysate consists of a nonionic detergent, a protease inhibitor, a phosphatase inhibitor, an RNase inhibitor and distilled water, wherein the volume ratio of the nonionic detergent to the protease inhibitor to the phosphatase inhibitor to the RNase inhibitor to the distilled water is 2:1:2:1: 14.
Further, the nonionic detergent comprises 150mM sodium chloride, 1% Nonidet P-40(NP-40), Tris-HCL having a 50mMPH value of 7.4, and distilled water.
Further, the protease inhibitor comprises 0.2. mu.M Aprotinin, 10. mu.M Leupeptin, 1. mu.M Leupeptin A and distilled water.
Further, the phosphatase inhibitor comprises 1mM sodium orthovanadate, 1mM sodium fluoride, 4mM sodium pyrophosphate, 1.15mM sodium cuprate and distilled water.
Further, the RNase inhibitor comprises guanidinium isothiocyanate in an amount of 20U/ml.
The invention also provides an application of the cell lysate, wherein the cell lysate is used for detecting the activity of telomerase.
The invention has the following advantages:
(1) the cell lysate of the present invention can simultaneously obtain nucleic acid and protein by processing one sample.
(2) The cell lysate of the present invention does not affect the protease activity in the sample.
(3) The cell lysate avoids the use of toxic substances such as phenol, chloroform and the like, does not influence the health of operators, and is green and environment-friendly.
(4) The cell lysate of the present invention can be used for detecting the enzymatic activity of telomeres.
Drawings
FIG. 1 QPCR response curves for 3 samples taken and 75 degrees enzyme inactivation treated samples from example 3.
Figure 2 internal reference Actin response curves for 3 samples extracted in example 3 and samples treated for 75 degree enzyme inactivation.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
Example 1: preparation of cell lysate
The preparation method comprises the following steps:
(1) preparing 100ml of 10X non-ionic detergent stock solution: 150mM sodium chloride, 10% Nonidet P-40(NP-40),500mM Tris-HCl (pH 7.4);
(2) preparing 1ml of 100X protease inhibitor stock solution of 20 mu M Aprotinin,1000 mu M Leupeptin and 100 mu M Pepstatin A
(3) A10 Xphosphatase inhibitor stock solution (100 ml) was prepared containing 10mM sodium orthovanadate, 10mM sodium fluoride, 40mM sodium pyrophosphate, and 11.5mM sodium cuprate.
(4) Preparing mixed liquor as required before use, and preparing 1ml of mixed liquor as required: taking 100ul of 10X non-ionic detergent stock solution; 10ul of 100X protease inhibitor stock solution; 100ul of 100X protease inhibitor stock solution; rnase inhibitor 20U, i.e. 20 ul; finally 770ul of distilled water was added.
Example 2: extraction of nucleic acids and proteins from whole blood using cell lysates
1.1 ml of fresh peripheral blood was taken.
Removing erythrocytes by treatment with erythrocyte lysate, centrifuging at 1500rpm for 5min to collect cells, washing with PBS, and quantifying to 5 × 105Cells were harvested by centrifugation at 1500rpm for 5 min.
2. Cell lysis
The cells were collected in a 1.5mL centrifuge tube, 200ul of the fresh cell lysate prepared in example 1 was added, and lysed on ice for 20min, during which time the tube was shaken vigorously every 5min for 30s each time.
3.12000rpm, 4 ℃ for 25min, and taking the supernatant, wherein the supernatant contains the nucleic acid and the protein required by the experiment.
Example 3
The supernatants of 3 whole blood samples extracted in example 2 were subjected to telomerase treatment as follows:
1. a part of the telomerase-containing supernatant was taken and treated at 75 ℃ for 20min to serve as a negative control (i.e., inactivation of protease activity).
2. Preparing a telomerase treatment reaction solution:
3. telomerase treatment reaction procedure:
Figure BDA0002269300770000042
the purpose of the above reaction is: the telomerase in the sample supernatant plays a role of reverse transcriptase, because the telomerase is a ribonucleoprotein and consists of RNA and protein, the RNA is used as a reverse transcription template, the protein is used as the reverse transcriptase, and the telomere terminal is used as a primer to synthesize a telomere DNA repetitive sequence.
EXAMPLE 4 QPCR reaction
0.3ul of ACX primer was added to 49.7ul of the system of example 3 and a total volume of 50ul was used for QPCR reaction, FIG. 1 is a QPCR reaction curve for 3 samples extracted in example 3 and a 75 degree enzyme inactivation treated sample; figure 2 is an internal reference Actin response curve for 3 samples extracted in example 3 and samples treated for 75 degree enzyme inactivation.
QPCR reaction procedure:
Figure BDA0002269300770000051
the purpose is as follows: the reaction product of example 3 corresponds to cDNA, and the QPCR reaction can be carried out using cDNA as a template.
The results of fig. 1 and 2 are summarized below:
sample(s) Telomerase Activity Ct Internal reference Actin Ct
Sample
1 33.91 22.26
Sample 2 33.12 24.32
Sample 3 33.75 24.25
75 degree treatment of sample 1 38.12 25.32
Description of the drawings: after 75 degrees of treatment, the protein in the extracted sample loses the enzyme activity, the reverse transcription reaction cannot be carried out, and the RNA template cannot be converted into cDNA, so that the QPCR reaction cannot be carried out or the reaction capacity is very low; such as: the Ct of the sample is more than 38, and the telomere activity is judged to be inactivated after the sample is treated at 75 ℃ and compared with an untreated sample; meanwhile, the lysate prepared by the method is used for obtaining RNA and protein simultaneously after cell lysis, and the enzyme activity of the protein is reserved.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (6)

1. A cell lysate, which consists of nonionic detergent, protease inhibitor, phosphatase inhibitor, RNase inhibitor and distilled water, wherein the volume ratio of the nonionic detergent, the protease inhibitor, the phosphatase inhibitor, the RNase inhibitor and the distilled water is as follows: 2:1:2:1:14.
2. The cell lysate of claim 1, wherein the non-ionic detergent comprises 150mM sodium chloride, 1% Nonidet P-40(NP-40),50 mM Tris-HCL at PH7.4, and distilled water.
3. Cell lysate according to claim 1, characterised in that the protease inhibitor comprises 0.2 μmaprotin, 10 μ M Leupeptin,1 μ M Pepstatin a and distilled water.
4. Cell lysate according to claim 1, characterised in that the phosphatase inhibitor consists of 1mM sodium orthovanadate, 1mM sodium fluoride, 4mM sodium pyrophosphate, 1.15mM sodium cuprate and distilled water.
5. Cell lysate according to claim 1, characterised in that the rnase inhibitor comprises guanidinium isothiocyanate, in an amount of 20U/ml.
6. Use of a cell lysate according to any one of claims 1-5, in the detection of telomerase activity.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102220418A (en) * 2011-04-22 2011-10-19 天津医科大学 Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit
CN102876792A (en) * 2012-09-27 2013-01-16 浙江今复康生物科技有限公司 AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and detection method
CN104797246A (en) * 2012-09-25 2015-07-22 新加坡科技研究局 Telomerase inhibitors for use in therapy
CN105582525A (en) * 2014-10-24 2016-05-18 佛山市龙杰生物科技有限公司 Application of CIRP (cold-inducible RNA binding protein) in preparing telomerase binding agent or telomerase activity regulating agent
CN105648051A (en) * 2015-11-25 2016-06-08 厦门多维生物医药科技有限公司 Method for quantifying activity of cell telomerase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102220418A (en) * 2011-04-22 2011-10-19 天津医科大学 Dual temperature rapid cycling fluorescence quota PCR method for detecting telomerase activity and kit
CN104797246A (en) * 2012-09-25 2015-07-22 新加坡科技研究局 Telomerase inhibitors for use in therapy
CN102876792A (en) * 2012-09-27 2013-01-16 浙江今复康生物科技有限公司 AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and detection method
CN105582525A (en) * 2014-10-24 2016-05-18 佛山市龙杰生物科技有限公司 Application of CIRP (cold-inducible RNA binding protein) in preparing telomerase binding agent or telomerase activity regulating agent
CN105648051A (en) * 2015-11-25 2016-06-08 厦门多维生物医药科技有限公司 Method for quantifying activity of cell telomerase

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