CN104548073A - Application of Prx I recombinant protein in preparing medicine for resisting bacterial septicaemia - Google Patents
Application of Prx I recombinant protein in preparing medicine for resisting bacterial septicaemia Download PDFInfo
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- CN104548073A CN104548073A CN201410766259.7A CN201410766259A CN104548073A CN 104548073 A CN104548073 A CN 104548073A CN 201410766259 A CN201410766259 A CN 201410766259A CN 104548073 A CN104548073 A CN 104548073A
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses application of peroxiredoxin Prx I recombinant protein in preparing a medicine for resisting bacterial septicaemia. The recombinant protein is constructed by connecting transfusion peptide protein at the N or C terminal of the amino acid sequence of peroxiredoxin Prx I; and the amino acid sequence of the transfusion protein is as shown in 5'-AAVLLPVLLAAP-3'. The invention provides the peroxiredoxin Prx I recombinant protein for the first time, which has the classical biochemical function of decomposing H2O2 and further has a new immunological function of resisting acute bacterial septicaemia; through recombination and transformation of protein molecules, the efficiency that active protein enters cells is significantly improved; by improving utilization efficiency, the peroxiredoxin Prx I recombinant protein has higher bioactivity, so that production cost is reduced; and the peroxiredoxin Prx I recombinant protein has good application prospects.
Description
(1) technical field
The present invention relates to a kind of recombiant protein and application, particularly a kind of Peroxiredoxin Prx I recombiant protein is preparing the application in bacteria resistance septicemia medicine.
(2) background technology
Peroxiredoxin (Peroxiredoxins, Prx, EC 1.11.1.15) is a class peroxiredoxins albumen, is extensively present in the cells such as animal and plant.In the vertebrates comprising mammal, there are six kinds of Prxs albumen (Prx I ~ VI), they have conservative aminoacid sequence, functional activity site and similar catalyst mechanism, be positioned in the organelles such as nucleus, Cytoplasm, mitochondrion, peroxisome, lysosome and Golgi body respectively, form antioxidant network system important in cell.In Prxs protein family, Prx I is the high and the most widely distributed member of content.Think that the major function of Prxs protein family passes through decomposing H at present
2o
2and play antioxidation.Prxs decomposing H
2o
2molecular mechanism be realized by the redox of cysteine (Cys) residue in its aminoacid sequence, namely conservative in Prxs albumen reproducibility Cys-SH is by H
2o
2oxidation, generates oxidation state Cys-SOH, thus by H
2o
2be reduced into H
2o; And oxidation state Cys-SOH residue can react with the reproducibility Cys-SH in another Prx albumen, form intermolecular or intramolecular disulfide bond, form homotype or heterodimer; Then this dimer can further under the Trx restoring system of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH composition or the effect of reduced glutathion GSH, again monomer reduction state albumen is reduced into, circulate in this way, make H
2o
2constantly be decomposed.Due to H
2o
2one of important biochemical products of biologic artifact physiological metabolism, the H of high concentration
2o
2accumulate and will destroy the oxidation-reduction balance of body, oxidative damage is caused to lipid, nucleic acid, saccharide and protein equimolecular in cell, thus toxic action is produced to cell, bring out the generation of body aging and numerous metabolic disease.Therefore, Prxs albumen may play a significant role in maintenance body oxidation-reduction balance.
Except enzymatic decomposition H
2o
2biochemical function outside, for Prxs, whether there is other biological function and it be unclear that.The present invention with Prx I for object of study, Late Cambrian Prxs albumen has the immunology New function of bacterial-infection resisting, and be that one is distributed in intracellular activated protein for Prx I, need to play a role in born of the same parents, and the Prx I albumen of in-vitro recombination expression is not because effectively entering cell and this application difficult problem that is difficult to play a role, design and a kind of there is the peptide sequence (be called for short feed back peptide) feeding back and enter cell ability, by by this feedback peptide and Prx I molecular recombination, construct a kind of novel Prx I recombiant protein with feedback cell function, the antibacterial immunity function of Prx I albumen is significantly improved.
(3) summary of the invention
Order of the present invention is to provide the New function that a kind of Peroxiredoxin Prx I recombiant protein has bacteria resistance septicemia; By Prx I albumen is carried out molecular structure alteration, at it, amino or carboxyl terminal has been recombinated and has a kind ofly been had the peptide sequence (feedback peptide) feeding back cell ability, and the antisepsis function of Prx I albumen is significantly improved.
The technical solution used in the present invention is:
The invention provides a kind of Peroxiredoxin Prx I recombiant protein and preparing the application in bacteria resistance septicemia medicine, described recombiant protein be Peroxiredoxin Prx I aminoacid sequence N end or C end connect feed back peptide protein build form; The aminoacid sequence of described feedback peptide is 5 '-AAVLLPVLLAAP-3 ', and the nucleotides sequence of this signal peptide aminoacid sequence of encoding is classified as GCAGCCGTTCTTCTCCCTGTTCTTCTT GCCGCACCC.
Bacterial septicemia of the present invention is that Aeromonas hydrophila (Aeromonas hydrophil, A.h) infects the acute sepsis caused.
Peroxiredoxin Prx I albumen of the present invention is coded by the Prx I gene of vertebrates Fugu ocellatus, utilize escherichia coli in-vitro recombination expression to form, the gene order (gb DQ003333.1) of coding Prx I albumen is for shown in SEQ ID NO.1.
Prx I recombiant protein of the present invention is used in particular for Aeromonas hydrophila (Aeromonas hydrophil, the preparation of the anti-infectious immunity medicine of the acute sepsis A.h) caused, it significantly to the resistance of acute bacterial septicemia, can increase substantially survival rate by Enhancement test animal.
The Prx I recombiant protein carrying feedback peptide sequence of the present invention, successfully solves it as activated protein in a kind of born of the same parents, after vivoexpression is produced, how to enter this application difficult problem of cells play function to efficient lossless wound.Distinguish the feedback cell ability of cultured cell and in-vivo tissue horizontal detection Prx I recombiant protein in vitro, utilize laboratory animal (Brachydanio rerio) the acute sepsis model of infected by Aeromonas hydrophila simultaneously, Prx I albumen (not connecting the normal Prx I albumen feeding back peptide and the restructuring Prx I albumen the being connected feedback peptide) difference to acute bacterial septicemia resistivity relatively before and after transformation, thus prove that the function of the Prx I albumen through transformation is significantly improved.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: (1) the present invention provides Peroxiredoxin Prx I first except having decomposing H
2o
2classical biochemical function outside, also there is the immunologic function of the bacillary acute sepsis of new opposing; (2) by carrying out modified recombinant to this protein molecular, it is made to carry a kind of peptide sequence with feedback cell function, successfully establish a kind of undamaged approach giving cytoactive albumen, this approach significantly can enter the efficiency of cell by enhanced activity albumen, give it by improving utilization ratio and there is higher biologic activity, thus reduction production cost, have a good application prospect.
(4) accompanying drawing explanation
Fig. 1 is the cDNA amplification of Prx I recombiant protein; M:DNA Marker; The cDNA fragment of 1:Prx I-NS; The cDNA fragment of 2:Prx I-CS; The cDNA fragment of 3:Prx I.
Fig. 2 is the abduction delivering result of Prx I recombiant protein; M: albumen Marker; 1,3,5: transformed Prx I-NS, Prx I-CS and Prx I recombinant expression plasmid but without the matched group of induction; The abduction delivering result of 2:Prx I-NS recombiant protein; The abduction delivering result of 4:Prx I-CS recombiant protein; 6: the abduction delivering result of normal Prx I albumen.
Fig. 3 is the feedback cell function qualification result (200 ×) of Prx I recombiant protein; The first row: the cell image under ordinary optical microscope; Second row: the cell image under fluorescence microscope, represents the result sending fluorescence signal after FITC, FITC-Prx I, FITC-Prx I-NS and FITC-Prx I-CS recombiant protein feeds back cell respectively.
Fig. 4 is the body histiocytic shows fluorescent microscopy images of interior feedback (200 ×) of Prx I recombiant protein; The result of fluorescence signal is sent after representing FITC-Prx I-NS recombiant protein, cell such as different tissues such as FITC-Prx I and FITC feedback liver,spleen,kidney, intestinal etc. respectively.
Fig. 5 is the immunologic function qualification result that Prx I recombiant protein antisepsis infects.N is the number of experiment fish.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
CDNA clone and the structure of embodiment 1, Prx I and recombinant molecule thereof
RNA is extracted from Fugu ocellatus spleen according to the explanation of Trizol test kit (TaKaRa), 3'-Full RACE Core Set test kit (TaKaRa) is utilized to be cDNA by RNA reverse transcription, using this cDNA as template, by the primers F 1 and R1 amplification feeding back peptide nucleotide sequence containing coding, there is the Prx I (Prx I-NS) that N end feeds back peptide; By primers F 2 with containing the primer R2 amplification of coding feedback peptide nucleotide sequence, there is the Prx I (Prx I-CS) that C end feeds back peptide; The normal Prx I of peptide is fed back by primers F 0 and R0 amplification nothing.
F1：5'-CCATGGCAGCCGTTCTTCTCCCTGTTCTTCTTGCCGCACCCGCTGCAGGCAAAGCTC-3'
R1：5'-CTCGAGGTGCTTGGAGAAGAACTC-3'
F2：5'-CCATGGGCCACCACCACCACCACCACGCTGCAGGCAAAGCTC-3'
R2：5'-CTCGAGTT AGGGTGCGGCAAGAAGAACAGGGAGAAGAACGGCTGCGTGCTTG GAGAAGAACTC-3'
F0：5'-CCATGGCTGCAGGCAAAGCTC-3'
R0：5'-CTCGAGGTGCTTGGAGAAGAACTC-3'
PCR amplification system composition is in table 1; PCR condition is: 94 DEG C of denaturation 4min, 94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 30s, and 72 DEG C extend 10min, totally 35 circulations.PCR primer, through 1.2% agarose gel electrophoresis and order-checking qualification, obtains and expects the cDNA (Fig. 1) of Prx I-NS, Prx I-CS that size conforms to sequence and Prx I.
PCR amplification system prepared by table 1.Prx I and recombinant molecule cDNA thereof
The preparation of embodiment 2, Prx I and recombiant protein thereof
NcoI and XhoI restriction enzyme site is added respectively in the PCR primer described in embodiment 1, then respectively PCR primer is recombinated in carrier T, build Prx I, Prx I-NS and Prx I-CS plasmid (pT/Prx I, pT/Prx I-NS and pT/Prx I-CS); With NcoI and XhoI enzyme action pT/Prx I, pT/Prx I-NS and pT/Prx I-CS plasmid respectively, object product through 1.2% agarose gel electrophoresis qualification and check order errorless after, recombinate in pET28 (a) expression plasmid again, obtain three kinds of corresponding prokaryotic expression plasmids.With these three kinds of prokaryotic expression plasmids transformation of E. coli expression strain Rosetta (BL21) respectively, transformed bacteria is inoculated in 5ml containing Amp (ampicillin, final concentration 50 μ g/ml) LB fluid medium (Shanghai raw work) in, put 37 DEG C of shaking tables, 250rpm cultivates 5h, again by culture fluid with the volume ratio of 1:100, add in the LB fluid medium containing Amp (final concentration 50 μ g/ml) and spread cultivation, by above-mentioned condition by antibacterial culturing to OD
600between 0.6 ~ 1.0 time, add IPTG (isopropylthiogalactoside, final concentration 0.1mM), proceed to 20 DEG C of low temperature shaking tables, 100rpm overnight incubation.Next day, collect and transform bacterial strain, after ultrasonication, collect albumen supernatant, adopt SDS-PAGE to identify the expression of Prx I, Prx I-NS and Prx I-CS recombiant protein, and by Ni-NTA protein purification post (the raw work in Shanghai) separation and purification.Result shows, and said method success induction of the expression of solubility Prx I, Prx I-NS and Prx I-CS recombiant protein, and obtains corresponding purifying protein (Fig. 2).
Embodiment 3, Prx I and recombiant protein thereof feed back the qualification of cell function
1, FITC fluorescent labeling Prx I and recombiant protein Prx I-NS thereof and Prx I-CS
Crosslinked fluid is prepared: get 7.56g NaHCO
3, 1.06g Na
2cO
3with 7.36g NaCl, adjust pH to 9.5, add water and be settled to 1L after being dissolved in water, 121 DEG C of sterilizings 30 minutes are for subsequent use.The preparation of FITC labelling crosslinked fluid is standby: be dissolved in above-mentioned crosslinked fluid by fluorescein FITC powder, final concentration is 1mg/ml.Prepared by protein-crosslinking liquid: the Prx I-NS, the Prx I-CS that obtain with purification in above-mentioned crosslinked fluid dialysis embodiment 2 and Prx I recombiant protein, trapped fluid is the protein-crosslinking liquid of these three kinds of albumen.
With the ratio of F/P (FITC/Protein)=15 μ g/100 μ g, FITC labelling crosslinked fluid is added in Prx I-NS, Prx I-CS and Prx I protein-crosslinking liquid respectively, 4 DEG C of lucifuge mix homogeneously.4 DEG C, the centrifugal 10min of 10000g, abandons precipitation, and supernatant is albumen (FITC-Prx I-NS, FITC-Prx I-CS and the FITC-Prx I) solution of FITC labelling.Utilize PBS (pH 7.2) dialysis three kinds of labelled proteins, collect trapped fluid, measure light absorption value A
280and A
495, utilize formula: protein concentration (mg/mL)=[A
280– 0.31 × A
495]/1.4, calculate the concentration of FITC labelled protein.
2, the external feedback cell function qualification of FITC labelled protein
The Hela cell that IMDM culture fluid (U.S. CORNING, containing 10%FBS) is cultivated is accessed 96 well culture plates, 5000, every hole cell, 37 DEG C, 5%CO
2cultivate 24h.Next day, discard culture fluid, change the IMDM culture fluid without FBS, in cell culture well, add 10 μ L concentration be respectively the FITC solution of 10 μMs and 10 μ L concentration is the labelled protein (FITC-Prx I-NS, FITC-Prx I-CS and FITC-Prx I) of 10 μMs, often organize sample and 3 repetitions are set, 37 DEG C, 5%CO
2hatch 0.5h.Then clean cell with PBS, repeat twice, then in every porocyte, add 150 μ l PBS, observe under putting inverted fluorescence microscope (ZEISS Axiovert40) and take pictures.Result shows, add the cell of FITC and FITC-Prx I albumen, have no obvious fluorescence signal, and add the cell being connected to the Prx I recombiant protein (FITC-Prx I-NS and FITC-Prx I-CS) feeding back peptide, then present obvious fluorescence signal, show that feeding back Toplink effectively guides Prx I albumen permeates cell membranes and enter (Fig. 3) in cell.
3, the external feedback histiocyte Function Identification of FITC labelled protein
Get 15 tail Brachydanio rerio, be divided into 3 groups at random, 5 tails/group, lumbar injection 10 μ l concentration to be FITC solution, the 10 μ l concentration of 10 μMs the be FITC-Prx-I of 10 μMs and 10 μ l concentration are 10 μMs of FITC-Prx-I-NS respectively, after raising 2h in 28 DEG C of recirculated waters, get liver, spleen, kidney and intestinal tissue respectively, put frozen section embedding medium (Tissue-Tek O.C.T) embedding,-80 DEG C are fixedly spent the night, freezing microtome (LeiCa 1950) is cut into slices, slice thickness 4.5 μm, observes and takes pictures under inverted fluorescence microscope (ZEISS Axiovert40).Result shows, the tissue in the individual source of FITC and FITC-Prx-I injection has no obvious fluorescence signal, and individual the organizing of source of FITC-Prx-I-NS injection all shows strong fluorescence signal, show that feeding back peptide effectively can not only guide the outer cultured cells of Prx I albumen penetrator, can also guide Prx I albumen penetrator inner tissue's cell (Fig. 4) effectively.
The Function Identification of embodiment 4, Prx I recombiant protein bacteria resistance septicemia
By Aeromonas hydrophila (Aeromonas hydrophil, A.h) bacterial strain (being provided by Zhejiang Institute of Fresh Water Aquatic Products) access beef-protein medium, 28 DEG C, after 200rpm cultivates 8h, collect thalline, suspend with PBS.Get 150 tail Brachydanio rerio, be divided into 3 groups at random, 50 tails/group, first group of lumbar injection A.h bacterium (0.5 × 10
6cFU/ tail) in contrast; The mixed liquor (0.5 × 10 of second group of lumbar injection A.h bacterium and restructuring Prx I albumen
6cFU+0.25 μ g/ tail); The mixed liquor (0.5 × 10 of the 3rd group of lumbar injection A.h bacterium and restructuring Prx I-NS albumen
6cFU+0.25 μ g/ tail), raise in 28 DEG C of recirculated water.Within after infecting every 3 hours, observe once, Continuous Observation 24 hours, records the mortality rate of each group, and test repetition 3 times, adds up the relative protection ratio of 24 hours.Relative protection ratio=(1-experimental group mortality rate/matched group mortality rate) × 100%.Result shows, the Brachydanio rerio mortality rate of A.h bacterium infectable infection is 76.7 ± 1.1%, the mortality rate of A.h bacterium associating Prx I recombiant protein injection group is altogether 73.3 ± 4.2%, protective rate is 4.4 ± 4.0%, and the mortality rate of A.h bacterium associating Prx I-NS recombiant protein injection group is altogether 57.7 ± 3.7%, protective rate is 25.2 ± 3.9%, compared with combining Prx I protein injection group with A.h bacterium injection group separately and A.h bacterium, the septicemia mortality rate of A.h bacterium associating Prx I-NS recombiant protein injection group significantly reduces (P<0.05), show that Prx I-NS recombiant protein has played the Septicemia: function of significant anti-Aeromonas hydrophila induction.
The Function Identification of table 2.Prx I recombiant protein bacteria resistance septicemia
In the present invention, the restructuring Prx I albumen through molecular modification obtains the ability feeding back cell, and shows the immunologic function of the bacteria resistance septicemia made new advances.But what more than experiment was enumerated is only some instantiations of the present invention; obviously; the present invention also has many distortion, and all distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (3)
1. Peroxiredoxin Prx I recombiant protein is preparing the application in bacteria resistance septicemia medicine, it is characterized in that described recombiant protein be Peroxiredoxin Prx I aminoacid sequence N end or C end connect feed back peptide protein build form; The aminoacid sequence of described feedback peptide is 5 '-AAVLLPVLLAAP-3 '.
2. apply as claimed in claim 1, it is characterized in that the nucleotides sequence of described Peroxiredoxin Prx I is classified as shown in SEQ ID NO.1.
3. apply as claimed in claim 1, it is characterized in that described bacteria resistance septicemia is the acute sepsis that Aeromonas hydrophila (Aeromonas hydrophil) causes.
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| US6835810B2 (en) * | 2002-05-13 | 2004-12-28 | Geneshuttle Biopharma, Inc. | Fusion protein for use as vector |
| CN101616928A (en) * | 2007-01-29 | 2009-12-30 | 株式会社普罗赛尔制药 | Novel macromolecule transduction domains and recognition methods thereof and purposes |
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|---|---|---|---|---|
| US6835810B2 (en) * | 2002-05-13 | 2004-12-28 | Geneshuttle Biopharma, Inc. | Fusion protein for use as vector |
| CN101616928A (en) * | 2007-01-29 | 2009-12-30 | 株式会社普罗赛尔制药 | Novel macromolecule transduction domains and recognition methods thereof and purposes |
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