CN106905419B - Micromolecule polypeptide Prdx5 truncate and its carrier and application - Google Patents
Micromolecule polypeptide Prdx5 truncate and its carrier and application Download PDFInfo
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- CN106905419B CN106905419B CN201710093364.2A CN201710093364A CN106905419B CN 106905419 B CN106905419 B CN 106905419B CN 201710093364 A CN201710093364 A CN 201710093364A CN 106905419 B CN106905419 B CN 106905419B
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- prdx5
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- micromolecule polypeptide
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- 101100191327 Mus musculus Prdx6 gene Proteins 0.000 title claims abstract description 35
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of micromolecule polypeptide Prdx5 truncate and its carrier and applications, micromolecule polypeptide Prdx5 truncate, and amino acid sequence is as shown in SEQ ID NO.1.The present invention develops new small molecule polypeptide Prdx5 truncate of the targeting on Nrf2, and the polypeptide molecular weight is small, and penetration performance is good；It can be expressed in prokaryotic expression system, production cost is low；With good stability, can tolerance acid-base degree and temperature in a certain range variation, will have a wide range of applications in the preparation of antitumor drugs.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to micromolecule polypeptide Prdx5 truncate and its carrier and answer
With.
Background technique
Anti-tumor drug is by one of extensive concern and the hot issue of research for a long time.At present to tumour or cancer
Treatment usually uses the composite treatment of operative treatment combination radiation and chemotherapy.Radioactive ray and chemotherapeutic lead to Apoptosis, from
And killing tumor cell.The radiation and chemotherapy drug of high dose can destroy or eliminate cancer cell, but also damage is normal thin simultaneously
Born of the same parents decline patient immune function, cause a series of toxic side effects to human body.
Polypeptide drugs are the brand-new fields of great development prospect in current biomedicine field, efficient with it, safe, special
The features such as property is strong is gradually available for the prevention and treatment of cancer.The function of gene will finally pass through its expression product --- protein come
It realizes, the activities or function of organism all be unable to do without the material base of protein.It was found that and identifying that there is critical function
Protein can bring conclusive influence for the exploitation of new drug.
It occurrence and development based on tumour and treats in the Mechanism Study of prognosis, other than traditional former cancer antioncogene, mesh
Preceding about the effect of active oxygen (reactive oxidative species, ROS) in oncobiology is a hot spot
Research field.ROS is constantly generated and is removed in various types of cells, plays its normal and pathology function.Nrf2 exists
It maintains to play an important role on the ROS Relative steady-state of cell, ROS can be repaired by the oxidation of the 151st cysteine of Keap1
Decorations change the conformation of Keap1 to discharge Nrf2, so that Nrf2 is detached from ubiquitin degradation state, and then enters core and be incorporated into downstream
The Antioxidant responsive element of the promoter region of multiple genes adjusts the transcriptions tables such as anti-oxidant target gene NQO1, GSTs and HMOX1
It reaches, reduces the level of ROS.
It is many experimental studies have found that, in tumor tissues (including lung cancer, liver cancer, breast cancer, oophoroma etc.) Nrf2 and
Prdx5 gene high expression, while finding that its height expression causes existing anti-tumor drug invalid again.
Summary of the invention
Goal of the invention: the deficiencies in the prior art are directed to, the object of the present invention is to provide a kind of micromolecule polypeptides
Prdx5 truncate has anti-tumor function, meets biological medicine use demand.It is a further object of the present invention to provide in one kind
State the expression vector of micromolecule polypeptide Prdx5 truncate.Further object of the present invention is to provide above-mentioned micromolecule polypeptide Prdx5 and cuts
The application of short body.
Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
Micromolecule polypeptide Prdx5 truncate, amino acid sequence is as shown in SEQ ID NO.1.
The encoding gene of the micromolecule polypeptide Prdx5 truncate, DNA sequence dna is as shown in SEQ ID NO.2.
The carrier of encoding gene containing the micromolecule polypeptide Prdx5 truncate.
The micromolecule polypeptide Prdx5 truncate application in preparation of anti-tumor drugs.
The utility model has the advantages that compared with prior art, the present invention develops new small molecule of the targeting on Nrf2
Polypeptide Prdx5 truncate, the polypeptide molecular weight is small, and penetration performance is good；It can be expressed in prokaryotic expression system, production cost is low；
With good stability, can tolerance acid-base degree and temperature in a certain range variation.
Detailed description of the invention
Fig. 1 is interaction and the Immunoprecipitation studies figure of Nrf2 and Prdx5 full-length proteins；
Fig. 2 is the vector encoded genes plasmid figure of micromolecule polypeptide Prdx5 truncate；
Fig. 3 is the structural domain figure of Prdx5 and Nrf2 interaction；
Fig. 4 is after interference micromolecule polypeptide Prdx5 truncate is expressed on transcellular influence result figure.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.The experiment of actual conditions is not specified in embodiment
Method, usually according to normal condition, such as (third edition, J. Pehanorm Brooker etc. write Molecular Cloning:A Laboratory guide, Huang Peitang etc.
Translate, Science Press, 2002) described in condition, or carry out according to the normal condition proposed by manufacturer.
Embodiment 1
Material: Protein A/G beads, cell pyrolysis liquid, Nrf2 antibody, Prdx5 antibody, IgG.
Method: the 0.01M PBS that cell gives pre-cooling is washed 2 times；After 1mLPBS is added, cell scraper scraping cells on ice are moved
Into 1.5mL centrifuge tube, cell is collected by centrifugation in 1000g × 5min；Configured 500 μ L cell pyrolysis liquid is added and is placed in rotation instrument
Supernatant is transferred to new EP and managed by the thorough lytic cell of upper 4 DEG C of rotation 30min, 4 DEG C of centrifugation 10min of 12000g.Presettling adds
The Protein A/G beads for entering 20 μ L mixing, rotates 1 hour on DNA mixed instrument, and 900g is centrifuged 5min (4 DEG C), by supernatant
Liquid is transferred to new centrifuge tube, abandons pearl.It takes 40-60 μ L supernatant as Input, 2 × SDS of equivalent loading is added
Buffer, boiling water boiling 5-10min freeze after centrifugation.Rest part is bisected into two equal parts, is separately added into equivalent (1 μ g) Normal
IgG or corresponding antibodies rotate 2 hours (4 DEG C) on DNA mixed instrument.30 μ L mixing is respectively added in each centrifuge tube
Protein A/G beads is continued to rotate 2 hours.Then 900g is centrifuged 5min (4 DEG C), abandons supernatant.1mL washing buffer is added
Wash pearl.900g is centrifuged 5min (4 DEG C), washs 3 times altogether.Finally 40-60 μ L 2 × SDS loading buffer is added
In washed pearl, boiling water boiling 5-10min freezes after centrifugation.Western blot detection is carried out together with Input sample.
As a result there is the interaction of Nrf2 and Prdx5 full-length proteins in tissue IP and cell IP as shown in Figure 1:.
Embodiment 2
The present invention provides a kind of stable micromolecule polypeptide that can inhibit Nrf2 high expression tumour cell proliferation.The small molecule
Polypeptide is encoded by the gene of designed, designed, is inserted into expression plasmid after aggregated enzyme chain reaction (PCR) synthesis.Expression vector is transferred to
E. coli bl21 is cultivated in LB culture medium, inducing expression at 37 DEG C.Broken bacterium takes supernatant, takes out using the DNA of OMEGA company
Extraction reagent kit obtains the target DNA of purifying, is named as Prdx5；Its nucleotide sequence is as shown in SEQ ID NO.1, corresponding volume
The amino acid sequence of code albumen is as shown in SEQ ID NO.2.Specific building process is as follows:
1) building of expression vector: design primer is connected through PCR method, passes through restriction enzyme Hind III and Kpn I
After double digestion, it is inserted into p3XFLAG-Myc-CMV plasmid, as shown in Figure 2.The expression vector of building is identified correct through digestion.
Material: E.coli BL21/DE3 engineered strain is that this room freezes；T4 DNA ligase is the production of Gibco BRL company
Product；Restriction enzyme is Biolab Products；Taq archaeal dna polymerase is Promega Products；Plasmid extraction kit
Purchased from the vast Tyke biotech company in Beijing；Ni ion affinity chromatography medium is purchased from this yuan of Zhenyang company；Fetal calf serum
(FCS), DMEM culture medium, 1640 culture mediums are Hyclone Products.
Method: the 1. building of expression vector
The PCR primer of genetic fragment is synthesized by Shanghai Sani company, and primer sequence is as follows:
Upstream primer: 5 '-CCAAGCTTACCATGTCCAAGACACACCTGCC-3 '；
Downstream primer: 5 '-GGGGTACCGAAAGCTGTGAGATGATATTGGGTGCC-3 '.
PCR reaction system are as follows: H222 μ L of O, 1 μ L of upstream primer, 1 μ L, PCMV-N-FLAG-PRDX5 plasmid 1 of downstream primer
25 μ L of μ L, Taq archaeal dna polymerase.
PCR reaction condition are as follows: 94 DEG C of denaturation 4min, 55 DEG C of annealing, 72 DEG C of primer extends.
Two restriction enzyme sites of Kpn I and Hind III are devised at genetic fragment both ends, double digestion is connected into
P3XFLAG-Myc-CMV plasmid.
Linked system are as follows: H210 μ L, T4 ligase buffer1 μ L, T4 ligase of O, 21 μ L of μ L, Fragment.
Control group separately is set, fragment is not added, with H2O supplies volume.It is connected overnight in PCR machine.
JM109 competence bacteria is converted with connection product.Step of converting are as follows: 1) 100 μ L competence bacterias are placed in ice water, are added
DNA0.5 μ L stands 30 minutes；2) 42 DEG C water-bath 2 minutes；3) ice bath 5 minutes；4) the not antibiotic LB culture medium of addition, 37
At DEG C, 150RPM shake culture 50 minutes.5) transformed bacteria is inoculated in LB agar plate with ampicillin, sets 37 DEG C of incubators
Overnight incubation.6) positive colony 4 are selected, is inoculated in LB liquid medium respectively, sets 37 DEG C of shaking table shake cultures.
2. the identification of carrier: 1) extracting positive colony plasmid DNA with kit.2) Hind III and Kpn I double digestion are used
It identifies Plasmid DNA, serves extra large Sani biotech company sequencing.Double digestion system are as follows: H214 μ L, Green buffer of O, 2 μ
2 μ L, Hind III of L, DNA, 1 μ L, Kpn I 1 μ L, 37 DEG C water-bath 4 hours.2) inducing expression in Escherichia coli: conversion is big
Enterobacteria BL21/DE3, picking single colonie are inoculated in the LB culture medium of the ampicillin containing 100mg/L, 37 DEG C of shake cultures
12h.3) it purifies: collecting transformed bacteria, solution I 250uL is added, centrifuge 13000R is centrifuged 10min；Solution is added
II 250uL is overturned 20 times repeatedly, is centrifuged 10min in centrifuge 13000R after wire drawing shape；Solution III 350uL is added,
Centrifuge 13000R is centrifuged 10min；DNA washing buffer 750uL is added, centrifuge 13000R is centrifuged 1min；Finally
The target DNA of purifying is obtained using the DNA extraction agent box of OMEGA company.
3. truncating IP: the expression vector of Prdx5 truncate being transferred in lung carcinoma cell, cellular immunity co-precipitation, tool are carried out
Body step is the same as embodiment 1.
As a result as shown in Figure 3: the interaction between Prdx5 truncate and Nrf2 albumen.Wherein in addition to Prdx5 overall length egg
White (wt) can be with outside Nrf2 protein-interacting, and Prdx5 truncated mutant (2) can also interact with Nrf2 albumen.
Embodiment 3
Material: H1299 cell, serum free medium, PBS, 6 orifice plates, marker, ruler, pipette tips.
Method: 1. first being compared in 6 orifice plates behind with ruler with marker, uniform to draw horizontal line, per every about 0.5~
1cm together, crosses via hole.Every hole is at least across 5 lines.About 5 × 10 are added in hole5A cell, particular number because of cell not
It is same and different, it grasps as that can be paved with overnight.Compare ruler, as far as possible the horizontal line scratch perpendicular to behind, pipette tips with pipette tips within second day
It is vertical, it cannot tilt.It is washed cell 3 times with PBS, serum free medium is added in the cell under place to go stroke.It is put into 37 degree of 5%CO2
Incubator, culture.It sampled, takes pictures by 0,6,12,24 hour.
2. the cell for transfecting Prdx5 truncate and the cell for transfecting unloaded label are added separately in six orifice plates, method
Ibid.
As a result as shown in figure 4, wild type H1299 cell (left side) and the unloaded H1299 cell of transfection (in) showed
Transfer case to be significantly stronger than transfection Prdx5 truncate H1299 cell (right side), illustrate Prdx5 truncation physical efficiency inhibit swell
The transfer of oncocyte.
SEQUENCE LISTING
<110>Nantong University
<120>micromolecule polypeptide Prdx5 and its carrier and application
<130> 100
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 114
<212> PRT
<213> Artificial
<220>
<223>micromolecule polypeptide Prdx5
<400> 1
Ser Lys Thr His Leu Pro Gly Phe Val Glu Gln Ala Glu Ala Leu Lys
1 5 10 15
Ala Lys Gly Val Gln Val Val Ala Cys Leu Ser Val Asn Asp Ala Phe
20 25 30
Val Thr Gly Glu Trp Gly Arg Ala His Lys Ala Glu Gly Lys Val Arg
35 40 45
Leu Leu Ala Asp Pro Thr Gly Ala Phe Gly Lys Glu Thr Asp Leu Leu
50 55 60
Leu Asp Asp Ser Leu Val Ser Ile Phe Gly Asn Arg Arg Leu Lys Arg
65 70 75 80
Phe Ser Met Val Val Gln Asp Gly Ile Val Lys Ala Leu Asn Val Glu
85 90 95
Pro Asp Gly Thr Gly Leu Thr Cys Ser Leu Ala Pro Asn Ile Ile Ser
100 105 110
Gln Leu
<210> 2
<211> 342
<212> DNA
<213> Artificial
<220>
<223>micromolecule polypeptide Prdx5
<400> 2
tccaagacac acctgccagg gtttgtggag caggctgagg ctctgaaggc caagggagtc 60
caggtggtgg cctgtctgag tgttaatgat gcctttgtga ctggcgagtg gggccgagcc 120
cacaaggcgg aaggcaaggt tcggctcctg gctgatccca ctggggcctt tgggaaggag 180
acagacttat tactagatga ttcgctggtg tccatctttg ggaatcgacg tctcaagagg 240
ttctccatgg tggtacagga tggcatagtg aaggccctga atgtggaacc agatggcaca 300
ggcctcacct gcagcctggc acccaatatc atctcacagc tt 342
<210> 3
<211> 31
<212> DNA
<213> Artificial
<220>
<223>primer sequence
<400> 3
ccaagcttac catgtccaag acacacctgc c 31
<210> 4
<211> 35
<212> DNA
<213> Artificial
<220>
<223>primer sequence
<400> 4
ggggtaccga aagctgtgag atgatattgg gtgcc 35
Claims (4)
1. micromolecule polypeptide Prdx5 truncate, amino acid sequence is as shown in SEQ ID NO.1.
2. the encoding gene of micromolecule polypeptide Prdx5 truncate described in claim 1, DNA sequence dna such as SEQ ID NO.2 institute
Show.
3. the carrier of the encoding gene containing micromolecule polypeptide Prdx5 truncate as claimed in claim 2.
4. micromolecule polypeptide Prdx5 truncate application in preparation of anti-tumor drugs described in claim 1.
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| CN106110297B (en) * | 2016-07-05 | 2019-12-17 | 南通大学 | Application of GFI1 truncated body |
| CN110078811B (en) * | 2018-01-25 | 2022-02-11 | 中国医学科学院医药生物技术研究所 | Polypeptide IMB-P1 with anti-tumor activity and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101481412A (en) * | 2009-02-09 | 2009-07-15 | 吉林大学 | Polypeptide with antineoplastic function, encoding gene and use thereof |
| CN102140473A (en) * | 2010-01-29 | 2011-08-03 | 香港中文大学 | Anti-tumor nucleic acid and polypeptide and application thereof |
| CN102357246A (en) * | 2011-11-02 | 2012-02-22 | 江苏省中医药研究院 | EGFR and HER2 combined polypeptide epitope vaccine |
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| CN1300829A (en) * | 1999-12-22 | 2001-06-27 | 上海博德基因开发有限公司 | Polypeptide-antioxidizing enzyme 24 of human peroxidase and polynucleotide for coding this polypeptide |
| FR2919804B1 (en) * | 2007-08-08 | 2010-08-27 | Erytech Pharma | COMPOSITION AND ANTI-TUMOR THERAPEUTIC VACCINE |
| CN103417961B (en) * | 2012-05-23 | 2018-02-09 | 烟台聚杰生物工程有限公司 | PRDX2 and/or PRDX6 new application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481412A (en) * | 2009-02-09 | 2009-07-15 | 吉林大学 | Polypeptide with antineoplastic function, encoding gene and use thereof |
| CN102140473A (en) * | 2010-01-29 | 2011-08-03 | 香港中文大学 | Anti-tumor nucleic acid and polypeptide and application thereof |
| CN102357246A (en) * | 2011-11-02 | 2012-02-22 | 江苏省中医药研究院 | EGFR and HER2 combined polypeptide epitope vaccine |
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