CN104498494B - Detect plasmid and its construction method and the application of ribosome inactivating protein activity - Google Patents
Detect plasmid and its construction method and the application of ribosome inactivating protein activity Download PDFInfo
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- CN104498494B CN104498494B CN201510005119.2A CN201510005119A CN104498494B CN 104498494 B CN104498494 B CN 104498494B CN 201510005119 A CN201510005119 A CN 201510005119A CN 104498494 B CN104498494 B CN 104498494B
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- ribosome inactivating
- inactivating protein
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Abstract
The invention provides a kind of plasmid for detecting ribosome inactivating protein activity and its construction method and application, belong to gene engineering technology field.The plasmid includes the genetic fragment F28RNA with ribosome inactivating protein recognition site, and its nucleotides sequence is classified as SEQ ID No.1.The plasmid is connected by gene cloning, digestion with restriction enzyme, and the genetic engineering means such as conversion are built.The plasmid can in vitro and it is intracellular detection ribosome inactivating protein activity.
Description
Technical field
The present invention relates to gene engineering technology field, a kind of matter for being used to detect ribosome inactivating protein activity is particularly belonged to
Construction method and the application of grain and the plasmid.
Background technology
Ribosome inactivating protein (ribosome-inactivating protein, RIP) is widely present in plant, can
Ribosomal function is specifically destroyed, suppresses the biosynthesis of protein, in plant disease and pest resistance or rugged environment
Play an important role.At present in Curcurbitaceae, Euphorbiaceae is found that about 50 in 14 families such as grass family and Caryophyllales
A variety of ribosome inactivating proteins.According to the primary structure of ribosome inactivating protein, RIP is mainly segmented into following two:I types,
Be made up of skin chain more than one, with N- glycosidase activities, in active site region have highly conserved avtive spot and
Secondary structure, such as trichosanthin (Trichosanthin, TCS), pokeweed antiviral protein (pokeweed
Antiviral protein, PAP), saporin, luffin, barley translation inhibitor etc..II types, A chains have RNA N-
Glycosidase activity, B chains can be with some carbohydrates of specific bond, with activity of lectin, such as ricin.RIP activity egg
The medicine and agricultural chemicals of antiviral, antibacterial and anticancer are may be used as in vain, with boundless potential commercial application value.Early in
1888, people just extracted II type ribosome inactivating protein ricin from castor seeds, but are due to its violent toxicity,
Limit its application (Sharon N etc., Glycobiology.2004,14 (11) in terms of tumour is suppressed:53R-62R)；Mongolian oak
II types ribosome inactivating protein (ML-I) in parasitism, just has begun to as cancer therapy drug be used before a century,
But be due to its toxicity, be not widely popularized, only using the ML-I of low concentration as treatment of cancer ancillary drug (Bar-Sela
G, Harefuah.2006145 (1):42-46).
Although ribosome inactivating protein has potential application value, the premise of its large-scale industrial production is exploitation
Go out one to be economically separated purifying process to reclaim, separate and purify required ribosome inactivating protein.And design and develop one
Purifying process needs the content and activity that an accurate laboratory facilities take quantitative analysis RIP activity egg.Conventional at present
The method of detection ribosome inactivating protein is mainly immunoassay, depurination analytic approach, rabbit reticulocyte lysate etc..Exempt from
Epidemic disease analytic approach can apply to determine various antigens, haptens or antibody, with very high selectivity and very low detection limit, should
Method requirement prepares corresponding ribosome inactivating protein antibody first, is then detected using the antibody.Yang Yunyun etc. is established
The how anti-toxin of ricin (WA)-monoclonal antibody double sandwich-ELISA detection technique, by many anti-high accumulation abilities to antigen and monoclonal antibody
High selectivity combines, with improve detection method sensitivity and specificity (Yang Yunyun etc., analytical chemistry, 2007,35 (3),
439-442).Immunoassay is applied to the detection of known function and the albumen of property, for some unknown RIP activities
Albumen can not then be detected.Depurination analytic approach is also usually utilized to the new ribosome inactivating protein of analysis detection, detects by acidity
Aniline catalysis is broken the ribosome rRNA fragment produced in ribosome inactivating protein apurinic sites, but ribosome inactivating protein is de-
The sensitivity of purine analytic approach depends on the velocity constant that ribosome inactivating protein is catalyzed the reaction of ribosomes depurination, and ribosomes loses
Living protein depurination analytic approach be likely to occur higher false positive and false negative result (S.Ranil Wickramasinghe etc.,
University Of Qingdao's journal (engineering technology version), 2006,21 (1), 10-17).Rabbit reticulocyte lysate cell free translation system
Can be for detection ribosome inactivating protein activity.Ribosome inactivating protein all has N- glycosidase activities, can cut ribose
The N-C glycosidic bonds of rRNA specific site adenylates in body large subunit carry out depurination, make ribosomes 60S subunits irreversible
Inactivation, influence the combination of itself and elongation factor (EF-2) so as to suppress protein synthesis (Endo Y etc., J Biol Chem,
1987,262(17):8128-8130).This method is during Activity determination, it is necessary to measure radial pattern amino acid in translation process
In infiltration capacity come determine ribosome inactivating protein suppress protein translation activity.This method is improved afterwards, is used
Luciferase Assay System, by detecting that the amount of luciferase detects the activity of ribosome inactivating protein.Although this method is avoided
It is radioisotopic to use, make detection process easy and safety, but this method needs the rabbit net bought or prepared to knit red
Cell, it is expensive and do not allow easy to maintain.
The content of the invention
It is an object of the invention to for traditional detection ribosome inactivating protein method sensitivity is low, step is numerous and diverse, into
There is provided a kind of plasmid and its construction method and application for being used to detect ribosome inactivating protein activity for the problems such as this is higher.Use
The plasmids detection ribosome inactivating protein has the advantages that sensitivity height, high specificity, step be simple, cost is relatively low.
The genetic fragment F28RNA with ribosome inactivating protein recognition site that the present invention is provided, its nucleotides sequence is classified as
SEQ ID No.1。
A kind of plasmid for being used to detect ribosome inactivating protein activity that the present invention is provided, contains above-described gene piece
Section F28RNA.
A kind of construction method for being used to detect the plasmid of ribosome inactivating protein activity that the present invention is provided, including following step
Suddenly：
1) be template according to the people 28S ribosomal RNA sequences reported in GenBank, in avtive spot CGAGAG upstreams and
Downstream separately designs primer, introduces Xho I and Not I restriction enzyme sites at 5 ' and 3 ' ends respectively, and gene piece is obtained by PCR amplifications
Section F28RNA；
The primer is：F28F:CTCGAGIt is at AGCTCGCTTGATCTTGATTTTCA (SEQ ID No.2), line
Xho I restriction enzyme sites, F28R:CGCCGGCGIt is Not I enzymes at TCTACGAATGGTTTAGCGCCA (SEQ ID No.3), line
Enzyme site；
2) the above-mentioned genetic fragment F28RNA comprising Xho I and Not I restriction enzyme sites is passed through into restriction enzyme Xho I
With Not I digestion, the same psiCHECK digested through Xho I and Not I is connected toTMOn -2；
3) conversion of above-mentioned connection product is entered after bacillus coli DH 5 alpha, screening positive clone, largely expanded；Using
Alkaline lysis largely extracts psiCHECKTM- 2-F28RNA plasmids.
The plasmid that the present invention is provided can be used for the activity of vitro detection ribosome inactivating protein, and detection method is as follows：Will
psiCHECKTMThe μ g of -2-F28RNA plasmids 1 are dissolved in 10 μ L buffer solutions (pH6-8), add 0.1-1 μ g ribosome inactivating proteins, 37 DEG C
It is incubated 30-60 minutes, enters row agarose gel electrophoresis detection.According to plasmid state by supercoil be changed into amount when incising ring come
Characterize ribosome inactivating protein activity.
The plasmid that the present invention is provided also can be used for the activity of intracellular detection ribosome inactivating protein, and detection method is as follows：
1) by psiCHECKTMThe μ g of -2-F28RNA plasmids 4 are dissolved in 400 μ L serum free mediums, add 8 μ L transfection reagents,
Incubation at room temperature 15-20 hours；
2) above-mentioned mix reagent is added in the cell culture well to be transfected in cell culture medium, 37 DEG C of culture about 24h；
3) cell of transfection is digested with pancreatin, equivalent is dispensed into hole, ribosome inactivating protein is added into sample well, it is right
According to equivalent PBS is added in hole, as a control group, 37 DEG C are cultivated about 12-24 hours；
4) cell is collected, cell pyrolysis liquid cell lysis, centrifuging and taking supernatant is used；
5) 10-20 μ L cell pyrolysis liquids are taken, luciferase substrate is added, firefly luciferase is determined respectively and sea pansy is glimmering
Light element enzymatic activity.The activity of ribosome inactivating protein is characterized according to the reduction of renilla luciferase activity.
In summary, can in vitro and cell provided by the present invention for the plasmid of detection ribosome inactivating protein activity
The activity of interior detection ribosome inactivating protein, compared with prior art with it is convenient, fast, sensitive the characteristics of, it is to avoid it is conventional
Prepared in method, rabbit granulophilocyte, the limitation of ribosomes, extraction RNA instrument and technical conditions, the time of detection is by original
Shorten within several days about 120 minutes, greatly reduce the expense of detection, be to study ribosome inactivating protein extensively
There is provided favourable condition, solve that current ribosome inactivating protein detection technique medium sensitivity is low, step is numerous and diverse, cost is higher
The problems such as, with larger application potential.
Brief description of the drawings
Fig. 1：Genetic fragment F28RNA agarose gel electrophoresis figure
Fig. 2：psiCHECKTM- 2-F28RNA plasmid agarose gel electrophoresis qualification figures
Fig. 3：Agarose gel electrophoresis detects buckwheat ribosome inactivating protein to psiCHECKTM- 2-F28RNA plasmids are cut
Cut
Fig. 4：psiCHECKTM- 2-F28RNA plasmids detect buckwheat ribosome inactivating protein activity in the cell
Embodiment
Embodiment 1：psiCHECKTMThe structure of -2-F28RNA plasmids
It is template according to the people's 28S ribosomes sequence reported in GenBank, in avtive spot CGAGAG upstream and downstreams point
Primer, F28F are not designed:CTCGAGAGCTCGCTTGATCTTGATTTTCA,F28R:CGCCGGCGTCTACGAATGGTTTAGCGCCA, introduces Xho I and Not I restriction enzyme sites at 5 ' and 3 ' ends respectively；Using
Trizol methods extract HCT116 cell RNAs；Using RNA as template, F28F and F28R are that primer obtains genetic fragment by PCR amplifications
F28RNA (Fig. 1)；By the genetic fragment F28RNA and psiCHECK of acquisitionTM- 2 plasmids pass through restriction enzyme Xho I and Not
I digests, the separation of 1% agarose gel electrophoresis, reclaims genetic fragment F28RNA and psiCHECKTM- 2 carriers；Utilize DNA ligase
By the genetic fragment F28RNA and psiCHECK of recoveryTM- 2 carriers are attached, and connection product conversion is entered into bacillus coli DH 5
After α, screening positive clone (Fig. 2), largely expanded；PsiCHECK is largely extracted using alkaline lysisTM- 2-F28RNA matter
Grain.
Embodiment 2：The detection of vitro detection ribosome inactivating protein activity
By psiCHECKTMThe μ g of -2-F28RNA plasmids 1 are dissolved in 10 μ L PBSs (pH7), add the buckwheat of different content
Wheat ribosome inactivating protein, 37 DEG C are incubated 30 minutes, enter row agarose gel electrophoresis detection.Changed with plasmid state by supercoil
Ribosome inactivating protein is characterized to incise amount during ring active (see Fig. 3).
Embodiment 3：The detection of intracellular detection ribosome inactivating protein activity
By psiCHECKTMThe μ g of -2-F28RNA plasmids 4 are dissolved in 400 μ L serum free mediums, add 8 μ L transfection reagents
(TurboFect Transfection Reagent), is incubated at room temperature 15 minutes；Above-mentioned mix reagent is added to cell culture
In the cell culture well to be transfected in base, Cell abundance will reach more than 95%, and 37 DEG C are continued to cultivate about 24 hours；By transfection
Cell is digested with pancreatin, and equivalent is dispensed into 3 holes, wherein No. 1 hole, No. 2 holes are separately added into 1 μ g and 5 μ g buckwheats ribosomes lose
Living protein, No. 3 holes add equivalent PBS as blank control group, and 37 DEG C are cultivated about 12 hours；Cell is collected, is split with cell
Solve liquid cell lysis, centrifuging and taking supernatant；20 μ L cell pyrolysis liquids are taken, measurement bottom of the tube is added, adds 100 μ L firefly luciferins
Enzyme detection reagent, is put into instrument after 30 seconds after mixing and determines immediately, and record luminous value is firefly luciferase RLU；Complete
Into after above-mentioned firefly luciferase determination step, 100 μ L renilla luciferase detection reagents are added, are put into after mixing after 30 seconds
Determined immediately in instrument, record luminous value is firefly luciferase RLU；In the case of using renilla luciferase as internal reference,
The RLU values that obtained RLU values divided by renilla luciferase measure are obtained are determined with firefly luciferase.According to obtained ratio
The activation degree for carrying out the different sample room purpose reporter genes of comparison is computed.As a result as shown in figure 4, adding 1 μ g and 5 μ g buckwheat cores
After sugared body inactivating protein, the activity of firefly luciferase reduces by 23% and 57% respectively.
Claims (2)
1. a kind of construction method for being used to detect the plasmid of ribosome inactivating protein activity, it is characterised in that comprise the following steps：
1) it is template according to the people 28S ribosomal RNA sequences reported in GenBank, in avtive spot CGAGAG upstream with
Trip separately designs primer, introduces Xho I and Not I restriction enzyme sites at 5 ' and 3 ' ends respectively, and genetic fragment is obtained by PCR amplifications
F28RNA；
The primer is：F28F:CTCGAGAGCTCGCTTGATCTTGATTTTCA,F28R:
CGCCGGCGTCTACGAATGGTTTAGCGCCA；
2) by the above-mentioned genetic fragment F28RNA comprising Xho I and Not I restriction enzyme sites by restriction enzyme Xho I and
Not I digest, and are connected to the same psiCHECK digested through Xho I and Not ITMOn -2；
3) conversion of above-mentioned connection product is entered after bacillus coli DH 5 alpha, screening positive clone, largely expanded；Using alkali cracking
Solution largely extracts psiCHECKTM- 2-F28RNA plasmids.
2. application of the plasmid that method as claimed in claim 1 is built in detection ribosome inactivating protein activity.
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| CN106434848B (en) * | 2016-06-21 | 2019-08-09 | 成都医学院 | A kind of active method of measurement ribosome inactivating protein |
| CN114813900A (en) * | 2021-01-29 | 2022-07-29 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting active II type ribosome inactivating protein |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263107A (en) * | 1999-02-11 | 2000-08-16 | 中国科学院福建物质结构研究所 | Ribosome deactivated protein-pumpkin protein |
| CN1763087A (en) * | 2005-11-08 | 2006-04-26 | 中国人民解放军军事医学科学院野战输血研究所 | Sponge gourd seed ribosome deactivated protein and encoding gene and application |
| CN101070342A (en) * | 2007-05-22 | 2007-11-14 | 山西康宝生物制品股份有限公司 | Balsm-pear-seed ribosome inactivated protein and its coding gene and use |
| CN101738480A (en) * | 2009-09-18 | 2010-06-16 | 中国计量科学研究院 | Nanoparticle probes for detecting ribosome inactivating protein, manufacturing method thereof and use thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1263107A (en) * | 1999-02-11 | 2000-08-16 | 中国科学院福建物质结构研究所 | Ribosome deactivated protein-pumpkin protein |
| CN1763087A (en) * | 2005-11-08 | 2006-04-26 | 中国人民解放军军事医学科学院野战输血研究所 | Sponge gourd seed ribosome deactivated protein and encoding gene and application |
| CN101070342A (en) * | 2007-05-22 | 2007-11-14 | 山西康宝生物制品股份有限公司 | Balsm-pear-seed ribosome inactivated protein and its coding gene and use |
| CN101738480A (en) * | 2009-09-18 | 2010-06-16 | 中国计量科学研究院 | Nanoparticle probes for detecting ribosome inactivating protein, manufacturing method thereof and use thereof |
Non-Patent Citations (2)
| Title |
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| M11167;登录号;《NCBI Genbank》;19930803;序列 * |
| 用质粒作底物测定核糖体失活蛋白酶活性的新方法;王洪涛等;《生物化学与生物物理进展》;20071231;第34卷(第9期);991-993 * |
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