CN109321645A - A kind of active detection method of telomerase catalytic protein subunit hTERT - Google Patents
A kind of active detection method of telomerase catalytic protein subunit hTERT Download PDFInfo
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- CN109321645A CN109321645A CN201811246277.7A CN201811246277A CN109321645A CN 109321645 A CN109321645 A CN 109321645A CN 201811246277 A CN201811246277 A CN 201811246277A CN 109321645 A CN109321645 A CN 109321645A
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- htert
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- catalytic protein
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- 108010017842 Telomerase Proteins 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 230000003197 catalytic effect Effects 0.000 title claims abstract description 24
- 102000002067 Protein Subunits Human genes 0.000 title claims abstract description 17
- 108010001267 Protein Subunits Proteins 0.000 title claims abstract description 17
- 102000007469 Actins Human genes 0.000 claims abstract description 34
- 108010085238 Actins Proteins 0.000 claims abstract description 34
- 210000002966 serum Anatomy 0.000 claims abstract description 9
- 238000010839 reverse transcription Methods 0.000 claims abstract description 8
- 230000003321 amplification Effects 0.000 claims abstract description 6
- 238000001962 electrophoresis Methods 0.000 claims abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 238000012408 PCR amplification Methods 0.000 claims abstract description 4
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 239000002299 complementary DNA Substances 0.000 claims description 21
- 238000011144 upstream manufacturing Methods 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 238000012772 sequence design Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 108091035539 telomere Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 210000003411 telomere Anatomy 0.000 description 10
- 102000055501 telomere Human genes 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000032823 cell division Effects 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
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- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
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- 239000000376 reactant Substances 0.000 description 1
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- 210000000130 stem cell Anatomy 0.000 description 1
- 108010057210 telomerase RNA Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
Abstract
The present invention relates to molecular biology and field, in particular to the active detection method of a kind of telomerase catalytic protein subunit hTERT, compared with prior art, the beneficial effects of the invention are as follows, the total serum IgE that the present invention passes through extraction tissue, and further progress reverse transcription, PCR amplification is carried out using hTERT primer and β-Actin primer, electrophoresis detection is carried out again, it is control with β-Actin primer amplification segment, improve the sensitivity and identification of result, one kind is provided more rapidly for the detection of telomerase catalytic protein active, sensitiveer and more convenient and fast detection method and testing product.
Description
Technical field
The present invention relates to molecular biology and field, in particular to a kind of telomerase catalytic protein subunit hTERT activity
Detection method.
Background technique
Telomerase is responsible for a kind of extended enzyme of telomere in cell, is basic nucleoprotein reverse transcriptase, can be by telomere
DNA adds to eukaryocyte end of chromosome, and the telomere that DNA replication dna loses is filled up, and extends by telomere reparation, can be with
Telomere will not be lost because of cell division, so that fissional number increases.Telomere is right in different plant species cell
In keeping chromosome stability and cell activity to play an important role, Telomerase, which can extend, shortens telomere (its cell of the telomere of shortening
Replication capacity is limited), to enhance the proliferative capacity of cell in vitro.Activity inhibited of the Telomerase in normal human tissue,
It is reactivated in tumour, so as to participate in vicious transformation.Telomere Stability, genome be complete, cell keeping for Telomerase
Long-term activity and the potential proliferative capacity etc. that continues play an important role.The presence of Telomerase is exactly lacking DNA replication dna
It falls into and fills up, i.e., by telomere reparation is extended, telomere can be allowed not to be lost because of cell division, so that cell division
Number increase.But in normal human cell, the activity of Telomerase is only thin in hematopoiesis by quite strict regulation
Born of the same parents, stem cell and reproduction cell among these cells that must constantly divide, can just detect active Telomerase.
After cell differentiation maturation, it is necessary to it is responsible for the demand of various different tissues in body, Each performs its own functions, then, the activity of Telomerase
It will disappearance gradually.For cell, itself whether can continue division go down it is not important, but the cell of differentiation and maturation will
More great mission is born, exactly histoorgan is allowed to operate, continues life.
Telomerase is a kind of enzyme being made of catalytic protein and RNA template, can synthesize the DNA of end of chromosome, is assigned thin
The immortality of born of the same parents' duplication.
Telomerase includes three parts, i.e. human telomerase RNA, telomerase catalytic protein subunit hTERT and Telomerase phase
Albumen is closed, organizes minute at these three, telomerase catalytic protein subunit hTERT is the deciding factor of telomerase activation, detection
Telomerase catalytic protein subunit hTERT activity, is of great significance for the diagnosis of cancer, but there is no maturations at present
The active detection method of telomerase catalytic protein subunit hTERT and product.
Summary of the invention
In view of the deficiency of the prior art, one aspect of the present invention provides a kind of sensitive, easy telomerase catalytic
On the other hand the active detection method of protein subunit hTERT provides a kind test side granzyme catalytic protein and grinds unit mRNA
Active product.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of active detection method of telomerase catalytic protein subunit hTERT, comprising the following steps:
Step 1, primer synthesis, draws according to the upstream telomerase catalytic protein subunit hTERT sequence design hTERT first
Object and hTERT downstream primer are related to β-Actin upstream primer and β-Actin then according to known β-Actin sequence information
Downstream primer；
Step 2, Total RNAs extraction, take 200-300g tissue be added 1.2-2.0mL TRIZO, place 9-15min, then plus
Enter 0.4-0.8mL chloroform, prevent 5-10min after mixing well, 4 DEG C of 10000-12000r/min are centrifuged 20-30min, in absorption
Clearly, 1-3mL isopropanol is added, places 12-15min after mixing, 4 DEG C of 10000-12000r/min are centrifuged 20-30min, then plus
Enter 75% ethyl alcohol of 2-4mL 0-10 DEG C, 4 DEG C of 8000-10000r/min are centrifuged 12-15min after mixing, go supernatant and drying, use water
Dissolution, obtains total serum IgE；
Step 3, hTERT cDNA amplification, the total serum IgE reverse transcription that step 2 is obtained at the first chain of cDNA, then with
The first chain of cDNA is template, and hTERT upstream primer, hTERT downstream primer, β-Actin upstream primer and the downstream β-Actin is added
Primer is expanded using PCR method, obtains hTERT cDNA amplified production and β-Actin amplified production；
Step 4, hTERT cDNA detection, hTERT cDNA amplified production and β-the Actin amplification that step 3 is obtained produce
Object is detected using polyacrylamide gel electrophoresis, and hTERT cDNA amplified production fragment length should be 356bp, β-Actin amplification
Product sheet segment length should be 500bp, judge testing result with this.
Preferably, hTERT upstream primer sequence is SEQ ID NO:1 in the step 1, in the step 1 under hTERT
Trip primer sequence is SEQ ID NO:2.
Preferably, β-Actin upstream primer sequence is SEQ ID NO:3, β-in the step 1 in the step 1
Actin downstream primer sequence is SEQ ID NO:4.
A kind of detection Telomerase catalytic protein grinds the active product of unit mRNA, the product include total RNA extraction reagent,
RNA Reverse Transcription, PCR amplification reagent and electrophoresis detection reagent.
Compared with prior art, the invention has the advantages that the present invention passes through the total serum IgE for extracting tissue, and further
Reverse transcription is carried out, PCR amplification is carried out using hTERT primer and β-Actin primer, then carry out electrophoresis detection, with β-Actin primer
Amplified fragments are control, improve the sensitivity and identification of result, provide one kind for the detection of telomerase catalytic protein active
More rapidly, sensitiveer and more convenient and fast detection method and testing product.
Detailed description of the invention
Fig. 1 is pcr amplified fragment of embodiment of the present invention polyacrylamide gel electrophoresis figure.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, but the present invention can be defined by the claims and
The multitude of different ways of covering is implemented.
Embodiment 1
1 Preparatory work of experiment
Cancer patient's cancerous issue sample, TRIZO reactant, Taq DNA polymerase (Gibco BRL company), reverse transcription examination
Agent box etc..
2 primers synthesis, according under telomerase catalytic protein subunit hTERT sequence design hTERT upstream primer and hTERT
It swims primer and is related to β-Actin upstream primer and β-Actin downstream primer then according to known β-Actin sequence information；
HTERT upstream primer sequence is that SEQ ID NO:1, hTERT downstream primer sequence is SEQ ID NO:2；β-Actin upstream primer
Sequence is that SEQ ID NO:3, β-Actin downstream primer sequence are SEQ ID NO:4.
3 Total RNAs extractions take 300g tissue that 2.0mL TRIZO is added, place 9-15min, 0.8mL chloroform is then added, fills
Dividing after mixing prevents 10min, and 4 DEG C of 10000-12000r/min are centrifuged 30min, draw supernatant, 3mL isopropanol is added, after mixing
12min is placed, 4 DEG C of 10000-12000r/min are centrifuged 20min, 10 DEG C of 75% ethyl alcohol of 4mL are then added, 4 DEG C after mixing
8000-10000r/min is centrifuged 12min, goes supernatant and drying, is dissolved with water, obtains total serum IgE；
4hTERT cDNA amplification, the total serum IgE reverse transcription that step 2 is obtained is at the first chain of cDNA, then with cDNA first
Chain is template, and hTERT upstream primer, hTERT downstream primer, β-Actin upstream primer and β-Actin downstream primer is added, adopts
It is expanded with PCR method, obtains hTERT cDNA amplified production and β-Actin amplified production；
5hTERT cDNA detection, the hTERT cDNA amplified production and β-Actin amplified production that step 3 is obtained use
Polyacrylamide gel electrophoresis detection, hTERT cDNA amplified production fragment length should be 356bp, β-Actin amplified production piece
Segment length should be 500bp, and electrophoresis result is as shown in Figure 1, clearly can judge that telomerase catalytic albumen is sub- single from electrophoresis result
Position hTERT activity.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
It simply modifies or converts made by present specification, be applied directly or indirectly in other relevant technical fields,
Similarly it is included within the scope of the present invention.
Sequence table
<120>the active detection method of a kind of telomerase catalytic protein subunit hTERT
<141> 2018-10-07
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 1
ctgcactggc tgatgagtgt 20
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<211> 20
<212> DNA
<213>artificial sequence ()
<400> 2
ctgaacagtg ccttcaccct 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 3
gtggggcgcc ccaggcacca 20
<210> 4
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 4
ctccttaatg tcacgcacga tttc 24
Claims (4)
1. a kind of active detection method of telomerase catalytic protein subunit hTERT, which comprises the following steps:
Step 1, primer synthesis, first according to telomerase catalytic protein subunit hTERT sequence design hTERT upstream primer and
HTERT downstream primer is related to β-Actin upstream primer and the downstream β-Actin then according to known β-Actin sequence information
Primer；
Step 2, Total RNAs extraction take 200-300g tissue that 1.2-2.0mL TRIZO is added, place 9-15min, be then added
0.4-0.8mL chloroform prevents 5-10min, 4 DEG C of 10000-12000r/min to be centrifuged 20-30min, draw supernatant after mixing well,
1-3mL isopropanol is added, 12-15min is placed after mixing, 4 DEG C of 10000-12000r/min are centrifuged 20-30min, 2- is then added
75% ethyl alcohol of 4mL 0-10 DEG C, 4 DEG C of 8000-10000r/min are centrifuged 12-15min after mixing, go supernatant and drying, use is water-soluble
Solution, obtains total serum IgE；
Step 3, hTERT cDNA amplification, the total serum IgE reverse transcription that step 2 is obtained is at the first chain of cDNA, then with cDNA the
One chain is template, and hTERT upstream primer, hTERT downstream primer, β-Actin upstream primer and β-Actin downstream primer is added,
It is expanded using PCR method, obtains hTERT cDNA amplified production and β-Actin amplified production；
Step 4, hTERT cDNA detection, hTERT cDNA amplified production and β-Actin amplified production that step 3 obtains are adopted
It is detected with polyacrylamide gel electrophoresis, hTERT cDNA amplified production fragment length should be 356bp, β-Actin amplified production
Fragment length should be 500bp, judge testing result with this.
2. a kind of active detection method of telomerase catalytic protein subunit hTERT according to claim 1, feature exist
In hTERT upstream primer sequence is SEQ ID NO:1 in the step 1, and hTERT downstream primer sequence is in the step 1
SEQ ID NO:2。
3. a kind of active detection method of telomerase catalytic protein subunit hTERT according to claim 1, feature exist
In β-Actin upstream primer sequence is SEQ ID NO:3, β-Actin downstream primer sequence in the step 1 in the step 1
It is classified as SEQ ID NO:4.
4. a kind of detection Telomerase catalytic protein grinds the active product of unit mRNA, which is characterized in that the product includes total serum IgE
Extract reagent, RNA Reverse Transcription, PCR amplification reagent and electrophoresis detection reagent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811246277.7A CN109321645A (en) | 2018-10-23 | 2018-10-23 | A kind of active detection method of telomerase catalytic protein subunit hTERT |
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| CN201811246277.7A CN109321645A (en) | 2018-10-23 | 2018-10-23 | A kind of active detection method of telomerase catalytic protein subunit hTERT |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108789A2 (en) * | 1999-12-16 | 2001-06-20 | F. Hoffmann-La Roche Ag | Quantitation of hTERT mRNA Expression |
| CA2534871A1 (en) * | 2006-02-15 | 2007-08-15 | The Ohio State University Research Foundation | Three-gene test to differentiate malignant from benign thyroid nodules |
| CN104059882A (en) * | 2013-03-19 | 2014-09-24 | 翁炳焕 | Fluorescence in situ hybridization hTERT transfected external quality assessment cell line and preparation method thereof |
| CN107974505A (en) * | 2017-12-14 | 2018-05-01 | 中源协和基因科技有限公司 | HTERT genes are directly detecting the purposes in blood plasma in cfDNA contents using qPCR as reference gene |
-
2018
- 2018-10-23 CN CN201811246277.7A patent/CN109321645A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108789A2 (en) * | 1999-12-16 | 2001-06-20 | F. Hoffmann-La Roche Ag | Quantitation of hTERT mRNA Expression |
| CA2534871A1 (en) * | 2006-02-15 | 2007-08-15 | The Ohio State University Research Foundation | Three-gene test to differentiate malignant from benign thyroid nodules |
| CN104059882A (en) * | 2013-03-19 | 2014-09-24 | 翁炳焕 | Fluorescence in situ hybridization hTERT transfected external quality assessment cell line and preparation method thereof |
| CN107974505A (en) * | 2017-12-14 | 2018-05-01 | 中源协和基因科技有限公司 | HTERT genes are directly detecting the purposes in blood plasma in cfDNA contents using qPCR as reference gene |
Non-Patent Citations (1)
| Title |
|---|
| 邱广斌等: "反转录聚合酶链式反应法检测端粒酶催化蛋白亚单位mRNA", 《中国实验诊断学》 * |
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