CN1388131A - Serial medicine carriers, tyrosine hydrozylase fusion protein as new carrier and its prepn - Google Patents

Abstract

The serial medicine carriers are named polypeptide mediated penetrating vector and belongs to membrane penetrating peptide family. They have the functions of penetrating cell membrane and cytoblast membrane, and may carry medicine molecule to penetrate physiological barriers, such as cell membrane, blood brain barrier, placenta barrier, etc. The new carrier-tyrosinie hydrozylase fusion protein is one new kind of protein with the features of both new carrier and tyrosinie hydrozylase, and it may reach human brain substantia nigra pathologic change part via blood brain barrier to treat parkinsonism.

Description

Series new drug carrier, novel carriers-tyrosine hydroxylase fusion rotein and preparation thereof

Technical field:

The present invention relates to the chemistry of peptides field, more specifically say, relate to a kind of novel carriers and preparation thereof and purposes, and novel carriers-tyrosine hydroxylase (tyrosine hydroxylase, TH) preparation of fusion rotein and purposes.

Background technology:

Up to now, most drug is because the selective permeation effect of Human Physiology barrier, particularly cytolemma, hemato encephalic barrier, placental barrier, blood-testis barrier etc., make medicine can not directly arrive diseased region, significantly reduced medicine in the intravital drug effect of machine, limit medicine to treatment of diseases, influenced curative effect.

Parkinson's disease (Parkinson ' s disease, PD) be a kind of central nervous system property carried out disease of being more common in the elderly, black substance Dopamine HCL (the dopamine that its main pathological characters is a human brain, DA) serotonergic neuron degeneration necrosis, make to be transported to striatal DA by black substance one striatum path and significantly to reduce, thus occur trembling, clinical symptom such as few moving, myotony.The precursor substance levodopa (L-DOPA) of oral supplementation DA is a therapy measure the most at present, but generally behind the 3-5 that takes medicine curative effect just significantly descend and serious toxic side effect occurs.Discover: (membrane penetrating peptide MPP) can permeates cell membranes, Human Physiology barrier such as nuclear membrane, it is combined with medicine improve the amount that medicine arrives diseased region, brings into play higher drug effect for the cytolemma penetrating peptide.The membrane property of wearing of MPP is applied in pharmacological agent disease aspect, has important clinical value.

Purpose of the present invention is to provide a kind of novel and effective film to penetrate carrier, has the protein polypeptide that film penetrates function, and preparation PMPV-TH fusion rotein in the hope of in clinical treatment, improves the amount that medicine arrives diseased region, brings into play therapeutic action better.

Summary of the invention:

The invention provides and be called as the new drug carrier of " polypeptide mediation penetrate carrier (Peptide-mediatedpenetrating vector; PMPV) ", can carry exogenous tyrosine hydroxylase (TH) and arrive the human brain diseased region, under the TH effect, people's substantia nigra generates Dopamine HCL to be increased, and reaches the purpose of treatment disease.

New drug carrier PMPV provided by the invention---belong to MPP family, comprise a series of polypeptide and albumen, function with direct permeates cell membranes, nuclear membrane, can the direct permeates cell membranes of bound drug, hemato encephalic barrier, placental barrier, blood-testis barrier performance drug effect, have the characteristic of MPP: 1, non-energy dependence; 2, non-acceptor dependency; 3, non-classical endocytosis and efflux effect; 4, can be with other more macromolecular polypeptide, protein, nucleic acid, plasmid combines and preserve biology and wear film activity etc.Its structural formula is PMPV or its pharmacy acceptable salt, ester or the precursor compound of A-B-C (1) expression, and wherein A is not for existing or being nitrogen-protecting group group, and C is arbitrary amino acid sequence, nucleotide sequence or other molecule, and B is the group that 13 Methionins constitute.

PMPV-TH fusion rotein provided by the invention is meant that comprising the amino acid coding of taking out from 2 or a plurality of independent albumen connects; make these two (or more) frames in same frame; form fusion rotein by expressing such polynucleotide; the PMPV-TH fusion rotein or its pharmacy acceptable salt, ester or the prodrug that comprise structural formula A-B-G-C (2) expression; wherein A is not for existing or nitrogen-protecting group group; C is not for existing or the carboxylic acid protective group; B is for having the active group of PMPV, and G is for having the active group of TH.

The sequence of assert in the scope of the invention comprises those analogue sequences that are characterized as amino acid residue sequence or type change, and whether wherein said change or not the essential property and the biologic activity of above-mentioned PMPV and/or fusion rotein.

The present invention also comprises a composition, comprise the strand of separated coding PMPV or fusion rotein or double-stranded nucleotide sequence, dna molecular preferably also comprises the carrier of the dna sequence dna that contains coding PMPV or fusion rotein, when wherein this carrier is present in the cell, can express PMPV or fusion rotein.The present invention also comprises the composition of the cell that contains this carrier, and wherein said carrier contains the dna sequence dna of coding PMPV or fusion rotein.

When the present invention is mainly used in the pharmacological agent disease, new drug carrier mediation medicine enters diseased region performance drug effect by the Human Physiology barrier, use the PMPV-TH fusion rotein when particularly treating Parkinson's disease, make TH arrive diseased region by hemato encephalic barrier smoothly, the treatment Parkinson's disease.

Main points of the present invention are fusion rotein and the preparation thereof of this pharmaceutical carrier PMPV and PMPV-TH, thereby set up a kind of recruit's combined protein with dual-use function of membrane penetration effect and other biological effectiveness, set up the membrane biological-activated albumen system of wearing.

Embodiment 1 preparation PMPV genophore:

1). preparation PMPV gene:

I.e. 13 Methionin gene series connection preparations---entrust Shanghai to give birth to the preparation of worker company limited.

2) the .PMPV gene combines with carrier and forms the PMPV genophore:

Carrier TransVectortm is provided by Q.BIO gene company.

Get carrier TransVectortm and each 1ug of PMPV gene, digest with II type restriction enzyme EcoRI and PstI respectively, use plasmid extraction kit (Wizard PCR Preps DNA PurificationSystem) to reclaim again.Carrier and PMPV gene add the T of 5U with 1: 3 mixed ₄Ligase enzyme, 16 ℃ were reacted 16 hours.Reaction product CaC ^l ₂The heat-shocked method transforms the BL21 bacterium.Transformed bacteria is coated and is carried out the white bacterium colony screening of indigo plant on the LB fixed flat planar substratum that contains penbritin and X-gal.Picking contains the white colony of recon, called after BL21PMPV.

Plasmid DNA extracting in a small amount: single bacterium colony inserts and contains in the 3mlLB nutrient solution of penbritin (100ug/m1) the 37C overnight incubation.Bacterium liquid is moved in the 1.5mlEppendorf pipe, and the centrifugal 2min of 5000rpm abandons supernatant.Add 100ul solution I (50mM glucose, 10mMEDTA, 25mMTris-HCL, pH8.0) suspension thalline, add 200ul solution II (0.2N NaOH1m%SDS), gentle mixing is put on ice, add 150ul solution III (3MNaAC, pH5.0), abundant mixing, the centrifugal 10min of 12000rpm, collect supernatant, with equal-volume phenol/chloroform: the Virahol extracting, add 2 times of volume dehydrated alcohol deposit D NA then, precipitation is washed once with 70% ethanol, after draining, be dissolved among the 20ulTE (1mMEDTA, 10mMTris-HCL, pH8.0), handle 30min with 2000ug/mlRNase ,-20 ℃ of preservations.Embodiment 2 preparation TH genes:

Get the total RNAlug of people's tire, add each reacted constituent successively by cDNA synthesis system operational guidance.Reaction cumulative volume 20ul, behind 42 ℃ of reaction 15min, 99 ℃ of deactivation 5min are as pcr template.The cumulative volume of PCR reaction system is 50ul, contains reverse transcription product 10ul, and all the other each composition dosages still carry out with reference to above-mentioned operational guidance.The first step: 95 ℃ of 12min start Golden Tap enzyme; Second step: 94 ℃ of sex change 50s; The 3rd step: 54 ℃ of 1min anneal; The 4th step: extend 72 ℃ of 1.5min; The 5th step: circulate 35 times.The pcr amplification band is checked in 2% agar-agar gel electrophoresis.Embodiment 3 preparation PMPV-TH antigen-4 fusion protein gene carriers (Fig. 1):

1), get PMPV carrier and each 1ug of TH gene, digest with II type restriction enzyme EcoRI and PstI respectively, use plasmid extraction kit (Wizard PCR Preps DNA PurificationSystem) to reclaim again.PMPV carrier and TH gene add the T of 5U with 1: 3 mixed ₄Ligase enzyme, 16 ℃ were reacted 16 hours.Reaction product CaCl ₂The heat-shocked method transforms the BL21 bacterium.Transformed bacteria is coated and is carried out the white bacterium colony screening of indigo plant on the LB fixed flat planar substratum that contains penbritin and X-gal.Picking contains the white colony of recon, called after BL21PMPV-TH.

2), plasmid DNA extracting in a small amount: single bacterium colony inserts and contains in the 3mlLB nutrient solution of penbritin (100ug/ml) 37 ℃ of overnight incubation.Bacterium liquid is moved in the 1.5mlEppendorf pipe, and the centrifugal 2min of 5000rpm abandons supernatant.Add 100ul solution I (50mM glucose, 10mMEDTA, 25mMTris-HCL, pH8.0) suspension thalline, add 200ul solution 2 (0.2N NaOH1m%SDS), gentle mixing is put on ice, add 150ul solution 3 (3MNaAC, pH5.0), abundant mixing, the centrifugal 10min of 12000rpm, collect supernatant, with equal-volume phenol/chloroform: the Virahol extracting, add 2 times of volume dehydrated alcohol deposit D NA then, precipitation is washed once with 70% ethanol, after draining, be dissolved among the 20ulTE (1mMEDTA, 10mMTris-HCL, pH8.0), handle 30min with 2000ug/mlRNase ,-20 ℃ of preservations.

3) evaluation of recombinant chou:

(1) the restriction endonuclease map analysis of recombinant plasmid

Get the BL21 bacterium colony at LB culture medium culturing 16 hours, reference literature J Sambrook, Fritsch EF, Maniatis T, et al.Molecular cloning (M) .New York:Cold Spring HarborLaboratory Press, 1989.16,34,304,672.1 method extract plasmid, with plasmid called after BL21PMPV-TH, digest with II type restriction enzyme EcoRI, PstI, EcoRI+PstI respectively, 2% agarose gel electrophoresis inspection, enzyme analysis is cut the result.

(2) the sequential analysis BL21PMPV-TH of recombinant plasmid checks order with full-automatic foranalysis of nucleic acids instrument, and the result is analyzed with the PCGENE analysis software and with the GENEBANK sequence.Embodiment 4 preparation PMPV-TH fusion roteins:

1, the expression of PMPV-TH and separation and purification:

1) expression of PMPV-TH:

The expression strain BL21PMPV-TH that transforms is inoculated in the LB nutrient solution, and 38 ℃ of shaking culture begin to be warming up to 42 ℃ after 3-4 hour, continue to cultivate 6 hours, finish to cultivate centrifugal receipts bacterium.

2) collection of inclusion body and cracking:

4 ℃ 3000 rev/mins centrifugal 30 minutes, abandon supernatant, claim weight in wet base, add lysate (50mol/L tri methylol amino methane/hydrochloric acid 1mol/L EDTA pH8.0,1mg/ml N,O-Diacetylmuramidase) by the concentration of 10g bacterium/100ml, 0 ℃ of effect 30 minutes.Ice-bath ultrasonic 30-45 second then, intermittently 2 minutes, ultrasonic 3 times altogether.Ultrasonic finishing, 4 ℃ 8000 rev/mins, abandon supernatant, collecting precipitation is an inclusion body.With the 8mol/L urea cracking inclusion body of purifying, 8mol/L urea is prepared with 50mol/L tri methylol amino methane/hydrochloric acid PH7.0 solution.With containing the resuspended inclusion body of 0.5% mercaptoethanol 8mol/L urea, the room temperature magnetic agitation is spent the night, 8000 rev/mins centrifugal 20 minutes, supernatant is the inclusion body lysate.

3) separation of PMPV-TH and purifying:

A, Ulltrogel ACA44 gel filtration chromatography:

(be dissolved in the 8mol/L urea of 50mol/L tri methylol amino methane/hydrochloric acid with balanced solution, pH7.0) balance Ulltrogel ACA44 gel-filtration column (1.6cm*80cm), with this gel-filtration column on the above-mentioned inclusion body lysate, each sample 3ml that goes up, last sample finishes and uses the level pad wash-out, use the 15%DSD-PAGE analysis again and respectively separate component, reach slightly pure.

B, CM Sepharose FF ion-exchange chromatography:

(be dissolved in the 8mol/L urea of 50mol/L tri methylol amino methane/hydrochloric acid with balanced solution, pH7.0) balance CM Sepharose FF post is directly gone up sample with the protein peak that gel-filtration obtains, after last sample finishes, behind the balanced solution wash-out, then with being dissolved in different concentrations of sodium chloride eluant solution in the balance liquid, collecting elution peak SDS-PAGE and analyze each distribution.

The renaturation of c, PMPV-TH:

Adopt the dilution process renaturation.Target protein renaturation buffer (10mol/L tri methylol amino methane/hydrochloric acid with purifying, pH7.0) doing 10 times dilution makes the protein content after the dilution remain on 50ug/ml, place 4 ℃ of refrigerator overnight, next day, place dialysis tubing that same damping fluid is fully dialysed to remove denaturing agent.

2, the determination of activity of PMPV-TH:

1) uses 6-OHDA and prepare inclined to one side side PD mouse model

Get 182 of male Sprague-Duwley rats, body weight 230-250g, laboratory animal is fixed in animal on the stereotactic apparatus behind 0.25% vetanarcol (1M/kg) intraperitoneal injection of anesthesia.Calvarium portion skin is cut in aseptic technique down, exposes the bregma point, gets two target spots (a point: preceding cranium tail side 5.0mm, center line right side 1.9mm, endocranium veutro 7.4mm; B point: preceding cranium tail side 5.3mm, center line right side 2.6mm, endocranium veutro 6.5mm).With 10ug 6-OHDA stereotactic injection in two target spots of right substantia nigra a, b (every some injection volume 5ug), injection speed 0.3ul/min, let the acupuncture needle remain at a certain point 15 minutes annotate to finish the back.

2) behavior detects

After 3 weeks of modelling, with apomorphine (Apomotphine, APO) by the 0.25mg/kg abdominal injection bring out rat (do not damage side) to the left the rotation, show the Modelling success, earlier rat is placed homemade commentaries on classics basin, adapt to after 5 minutes, injection APO, record internal rotation number of times half an hour, selecting 210 animals that change more than/30 minutes is successful PD model, is used for next step experiment.

3) import and measure

After the PD mouse model is eventually stablized three months, get 40 on the above model of 7 commentariess on classics/min, randomly assigne divides three groups, 20 of treatment groups, and each 10 of two control groups, treatment group blood injection PMPV-TH albumen, control group is only to TH.Detect once before the administration, detected once in per three days after the administration.

Experimental result shows, the rotation times that the 3rd, 6,9,12 and 15 day APO brings out behind the treatment group transplantation treatment reduces 46.5%, 41.5%, 33.4%, 30.3% and 26.3% respectively, decreased average 35.6% before than treatment; During to the 18th day, rotation times has returned to the preceding level of treatment.Through DUNCAN check, before the rotation times of different time is transplanted in the treatment group 15 days significant difference (p＜0.01) is arranged, and the per-cent of rotation times has no significant change before and after two control group administrations, through the DUNCAN check, p＞0.05 (Fig. 2).The invention effect:

Utilize means such as genetically engineered, polypeptide synthesize, preparation has the new drug carrier of wearing film activity and wears film activity and the double activity peptide of other biologic activity, and guide the bio-carrier medicine of preparing armour-piercing nuclear bomb function of new generation with pharmaceutical use, be applied to treatment of diseases, have important medical research and clinical use value.

Description of drawings:

Fig. 1-PMPV-TH gene recombined vector makes up

Bring out the rotation times experiment before and after Fig. 2-rat treatment group and the control group administration

The contrast of TH-tyrosine hydroxylase

The PMPV-carrier proteins

PMPV-TH-carrier proteins-tyrosine hydroxylase fusion rotein

Claims (7)

1, peptide species mediation penetrates carrier PMPV, and its structural formula is PMPV or its pharmacy acceptable salt, ester or the precursor compound of A-B-C (1) expression, wherein

A is not for existing or nitrogen-protecting group group;

B is the group that 13 Methionins constitute;

C is arbitrary amino acid sequence, nucleotide sequence or other molecule.

2, a kind of fusion rotein PMPV-TH, it contains the amino acid coding of taking out from 2 or how independent albumen and connects, make these 2 or more the mutiread frame in same frame, form fusion rotein by expressing such polynucleotide, comprise that structural formula is PMPV-TH fusion rotein or its pharmacy acceptable salt, ester or the precursor compound of A-B-G-C (2) expression, wherein

A is not for existing or nitrogen-protecting group group;

C is not for existing or the carboxylic acid protective group;

B is for having the active group of PMPV;

C is the TH active group.

3, a composition comprises strand or double-strandednucleic acid sequence, the preferably dna molecular of separated coding PMPV or fusion rotein.

4, composition as claimed in claim 3, it comprises the carrier of the dna sequence dna that contains coding PMPV or fusion rotein.

5, composition as claimed in claim 4 when wherein said carrier is present in the cell, can be expressed PMPV or fusion rotein.

6, as claim 4 or 5 described compositions, wherein said carrier contains the dna sequence dna of coding PMPV or fusion rotein.

7, the application of PMPV-TH fusion rotein in the treatment Parkinson's disease.

Images