CN102827254A

CN102827254A - Cell penetrating peptide hPP10 and use thereof

Info

Publication number: CN102827254A
Application number: CN2012102670507A
Authority: CN
Inventors: 柳长柏; 马节兰; 蔡三金; 吴江锋; 张洁; 贺晓敏; 杨英桂
Original assignee: China Three Gorges University CTGU
Current assignee: Shenzhen Zhenzhen Biomedical Technology Co.,Ltd.
Priority date: 2012-07-30
Filing date: 2012-07-30
Publication date: 2012-12-19
Anticipated expiration: 2032-07-30
Also published as: CN102827254B

Abstract

The invention relates to the field of biomedicine, and in particular relates to novel human cell penetrating peptide hPP10 and a use of using the human cell penetrating peptide as an intracellular drug delivery carrier. The hPP10 deviates from a section of polypeptide sequence of lysine-specific demethylase 4A of human cell nuclear proteins, has a function of penetrating a cell membrane and can carry the proteins and nucleic acid (deoxyribonucleic acid DNA plasmid) to enter a plurality of cells in a transmembrane mode; and therefore, the cell penetrating peptide hPP10 is a transmembrane delivery carrier of bioactive molecules such as protein and nucleic acid which has good development prospect.

Description

Cell-penetrating peptide hPP10 and uses thereof

Technical field

The present invention relates to biomedical sector, specifically, relate to a kind of novel human-derived sexual cell and wear film peptide and uses thereof.

Background technology

Along with the completion of the Human Genome Project and the rise of proteomics, it is found that increasing biomolecules all might become medicine such as protein, polypeptide, nucleic acid etc.But different with conventional medicament is that the stable in vivo molecule low, that need in born of the same parents, bring into play function of these treatment molecules is difficult to get into cell, thereby can limit its applying as medicine.So it is the problem of needing solution badly that the carrier system of these therapeutic biomacromolecules entering target cells, economy, safety can be effectively carried in exploitation.

In recent years, non-virus drugs transport agent is placed high hopes with advantages such as its security, hypotoxicity, low immunoreations.Lead-in mode possibly exist just like electroporation, liposome transfection and organic polymer nano particle etc. in the toxic action, born of the same parents of potential safety hazard such as pair cell and discharge difficulty, is difficult to assembling and operation in the biomacromolecule cell relatively commonly used so far, is difficult to be applied to such-and-such shortcomings such as individuality.Therefore seek novel, the non-virus drugs delivery system of ideal and caused scholars' extensive interest (Figure 21).

Between in the past more than 20 year, the fundamental research that nucleic acid, polypeptide, albumen etc. is had technology in bioactive macromolecule transmembrane transport to the cell of therapeutic action has obtained breakthrough.Chinese scholars has been found a proteinoid structural domain in succession in to some virus infection The Characteristic Study; As: HIV-1Tat (48～60), VP22 (267～300); And drosophila protein ANTP (43～58) etc. has bioactive macromolecules such as mediation heterologous protein, nucleic acid oligomer, metallo-chelate and directly strides across cytolemma and get into the function in endochylema and the nuclear; This type be rich in positively charged ion, have the small peptide of wearing the film function is referred to as cytolemma penetrating peptide (cell-penetrating peptides; CPP), wear film peptide or protein transduction domain (protein transduction domains, PTDs).Green in 1988 and Frankel etc. find TAT first---human immunodeficiency virus's (HIV) NlmR can get into cytoplasm/nucleus by permeates cell membranes/nuclear membrane; Discovering subsequently, the peptide section that this albumen 48-60 amino acids residue (YGRKKRRQRRR) forms can be brought into play it fully and wear the film function.Found later the CPP of multiple source again in succession from virus or other biological; Like I herpes simplex virus type (herpes simplex virus type 1; HSV-1) proteic VP22, fruit bat homology feeler albumen (drosophila hemeoprotein antennapedia transcription protein; ANTP), hepatitis B virus (Hepatitis B virus, pro-S antigen HBV) etc.Can the CPP that have been found that roughly be divided into two types according to its source is different: one type for deriving from viral small peptide etc. like above mentioned TAT, VP22, ANTP and pro-S antigen; Another kind of is according to the small peptide of the characteristics synthetic of natural CPP such as poly l-arginine, MPG, PEP-1, MAP, transportan and various based on unlike signal sequence synthetic peptide section etc.CPP can mediate a series of bioactive moleculess such as DNA, siRNA, polypeptide, protein even nano particle etc. and get into cell as the medicine carrying polypeptide in external or body, performance biological effect separately.These CPP are because of itself having low toxic side effect, do not disturb entrained biomacromolecule activity etc., and are widely used in external or/and in cell, transport bioactive molecules in the body, and are especially noticeable especially aspect the applied research of antineoplaston.CPP research all has very big meaning in fundamental biological knowledge and applied research, as vehicle in a kind of effective born of the same parents, (Figure 22) has a wide range of applications.

Yet derive from the CPP of virus or other kind bioprotein structural domains, in clinical application, possibly still have certain potential safety hazard, such as possible cytotoxicity and immunogenicity problem.People have carried out big quantity research to TAT as CPP, and abdominal injection Tat-beta-galactosidase enzymes can get into the various organs and tissues of mouse, even can see through hemato encephalic barrier entering cerebral tissue.But, fail to be used for clinical study because TAT comes from the HIV viral protein and probably has safe suffering always.Other reports, uses the serious lung's pathologic reaction of the upper respiratory tract spraying initiation that TAT carries medicine.It is thus clear that, need to the difference treatment, develop safer, novel cell-penetrating peptide--the humanized wears film peptide (hCPP) and is very important.

2002, Beck-Sickinger AG group found that in a creative way first humanized wears the film peptide--derive from the residue of the 9-32 of people's calcitonin (hCT).The humanized who reports in succession subsequently wears the film peptide and comes from hCLOCK albumen (a kind of and biorhythm adjusting proteins associated in addition; 2004), Hph-1 (a kind of people source transcription factor; 2006), Bag-1 albumen (a kind of can with the albumen of the interactional activation GR of Bcl-2; 2006), p14ARF albumen (a kind of human tumor suppressor gene albumen; 2008), the CPP of rhLF (2009), people Cytc77-101 and Cytc86-101 (2010) and TCTP albumen (derive from the people and translate 10 amino-acid residues of control oncoprotein N-terminal, 2011) etc.Compare with the CPP of other species dietary protein origins, humanized CPP causes that the immunoreactive possibility of human body is little, and the potential unsafe factor is few relatively, and the pharmaceutical carrier as human disease treatment has absolute predominance, and more wide development, application prospect are arranged.

There is very big dependency quantity of wearing arginine residues in film ability and the peptide sequence and position that the film peptide is worn in discoveries such as Futaki S.We are in being engaged in cell-penetrating peptide research; Through to the retrieval of primary structure in the albumen database, to analyze a segment length in the Methionin specificity demethylation transferring enzyme 4A that finds the humanized be that the primary structure of 20 amino acid whose small peptides is rich in l-arginine and the Methionin that belongs to basic aminoacids; Very strong positive charge is distributing; This constructional feature with most of known CPP is very similar, analyzes its secondary structure then, finds that it can form classical alpha-helix conformation.Infer that this section small peptide possibly be to have the novel humanized who independently wears the film function to wear the film peptide.We have synthesized this section small peptide and called after hPP10 then; Observed its membrane efficiency of wearing to culturing cell; Cytotoxicity and wear film mechanism; Also observed, assessed it simultaneously and in cell, sent the effect of green fluorescent protein (GFP) and DNA, for hPP10 provides scientific basis as a kind of exploitation of novel human-derived property medicament transport carrier.

Summary of the invention

The object of the present invention is to provide a kind of novel human-derived sexual cell to wear film peptide hPP10 and with its purposes as medicament transport carrier in the cell.

On the one hand, the invention provides a kind of novel human-derived sexual cell and wear film peptide hPP10.HPP10 comes from human cell's nucleoprotein, one section peptide sequence of Methionin specificity demethyl enzyme 4A (lysine-specific demethylase 4A), and the peptide sequence of hPP10 is following:

Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg (Methionin-Isoleucine-proline-leucine-proline(Pro)-l-arginine-phenylalanine(Phe)-Methionin-leucine-Methionin-halfcystine-Isoleucine-Phe-Cys-Methionin-Methionin-l-arginine-l-arginine-Methionin-l-arginine).On the other hand, the invention provides with said novel human-derived sexual cell wear film peptide hPP10 as medicament transport carrier in the cell purposes.We find that first this peptide section has the function of permeates cell membranes; And portability albumen, nucleic acid (DNA plasmid) etc. stride film and get in the various kinds of cell (comprising adherent culture cell, suspended culture cell and primary cultured cell etc.), be bioactive moleculess such as a kind of albumen that has a DEVELOPMENT PROSPECT, nucleic acid stride the film transport agent.

The invention has the advantages that compare with the CPP of other biological dietary protein origin, humanized CPP-hPP10 causes that the immunoreactive possibility of human body is little, the potential unsafe factor is few relatively.Therefore, the drug molecule carrier as clinical application has more wide application prospect.

Description of drawings

Fig. 1 is the secondary wheel-like structure synoptic diagram that the novel human-derived property of the present invention is worn film peptide hPP10.

Fig. 2 is that the novel human-derived property of the present invention is worn spiral and the folded folding analysis synoptic diagram that exists in the secondary structure of film peptide hPP10.

Fig. 3 is that the novel human-derived property of the present invention is worn the prokaryotic expression of film peptide hPP10 and the electrophoresis photo of purification result.Wherein

M: molecular weight of albumen sign

1:pET15b-hPP10 plasmid transform bacteria IPTG induces preceding lysate

2:pET15b-hPP10 plasmid transform bacteria IPTG induces the back lysate

3: the hPP10-6xHis behind the purifying

Fig. 4 is that fluorescently-labeled hPP10 (hPP10-FITC) wears film and gets into behind the different tumour cells fluorescence distribution situation in the born of the same parents.

Fig. 5 is that hPP10-FITC wears film and gets into behind former generation human peripheral lymphocyte fluorescence distribution situation in the born of the same parents.

Fig. 6 different concns hPP10-FITC wears membrane efficiency.Fluoroscope is seen down in the A:ECV-304 born of the same parents; Fluorescent quantitation in the B:ECV-304 born of the same parents.

Fig. 7 representes that incubation time goes into born of the same parents' influence to hPP10-FITC.

Fluorescent quantitation after the A:hPP10-FITC short period of time hatches;

Fluorescent quantitation after the B:TAT-FITC short period of time hatches;

Fluorescent quantitation after C:hPP10-FITC is hatched for a long time;

Fluorescent quantitation after D:TAT-FITC is hatched for a long time.

Fig. 8 representes that serum wears the influence of membrane efficiency to hPP10-FITC.

Fig. 9 representes that different cells wear the influence of membrane efficiency to hPP10-FITC through the DMSO pre-treatment.Fluorescence distribution situation in the born of the same parents behind the various dissimilar cells of A:DMSO pre-treatment; Fluorescent quantitation in the born of the same parents behind the different culturing cells of B:DMSO pre-treatment.

Figure 10 representes that former generation lymphocyte wears the influence of membrane efficiency to hPP10-FITC through the 5%DMSO pre-treatment.Fluorescence distribution situation in the born of the same parents behind the former foster mouse spleen lymphocyte of being commissioned to train of A:DMSO pre-treatment; Fluorescence distribution situation in the born of the same parents behind the former foster human peripheral lymphocyte of being commissioned to train of B:DMSO pre-treatment.

Figure 11 representes the effect of hPP10 mediation plasmid pDsRed transfection.

Figure 12 representes that the pcDNA3.1-Gluc construction of recombinant plasmid extracts and the double digestion of BamH I/Xbal I is identified.

Swimming lane 1:pcDNA3.1-Gluc; The double digestion of the Nco I/BamH I of swimming lane 2:pcDNA3.1-Gluc.

Figure 13 representes the effect of hPP10 mediation plasmid pcDNA3.1-Gluc transfection.

Figure 14 representes the double digestion evaluation of pET15b-hPP10-GFP construction of recombinant plasmid and Nde I/BamH I.

Swimming lane 1:pET15b-hPP10-GFP; The double digestion of the Nde I/BamH I of swimming lane 2:pET15b-hPP10-GFP.

Figure 15 representes that SDS-PAGE analyzing IP TG induces pET15b-GFP, pET15b-hPP10-GFP expression and the purifying in the Rosetta bacterium.

Swimming lane

3 and 4 is respectively: the GFP of purifying and hPP10-GFP fusion rotein;

Swimming lane

2 and 5 is respectively: GFP and hPP10-GFP plasmid transform bacteria are induced through IPTG;

Swimming lane

1 and 6 is respectively: GFP and hPP10-GFP plasmid transform bacteria are induced without IPTG.

Figure 16 representes that the hPP10-GFP fusion rotein wears the distribution situation that film gets into the L929 cell.

Figure 17 representes that the hPP10-GFP fusion rotein wears the distribution situation that film gets into former generation human peripheral lymphocyte.

Figure 18 representes that MTT detects the influence that hPP10-FITC handles back pair cell vigor.

Figure 19 representes under the differing temps content of fluorescence small peptide in born of the same parents after the DMSO pre-treatment.

Figure 20 representes that different endocytosis suppressor factor wear membrane efficiency influence to hPP10.

A: heparin is handled behind the various dissimilar cells fluorescence intensity in the born of the same parents;

B: fluorescence intensity in the born of the same parents behind the different culturing cells of chloroquine pre-treatment;

C: CHLORPROMAZINE HCL is handled fluorescence intensity in the born of the same parents behind the different culturing cells.

Figure 21 is a delivery strategies synoptic diagram in the novel therapeutic molecular born of the same parents.

Figure 22 is the principle of work synoptic diagram that cell-penetrating peptide (CPP) and portability thereof are gone into born of the same parents' bio-pharmaceutical.

Embodiment

To combine embodiment that scheme of the present invention is done more detailed explanation below.

Embodiment 1, CPP primary structure, secondary structure analysis, prediction, the novel human-derived property CPP of evaluation:

Secondary structure analysis has adopted the on-line analysis program of emboss, and (its routine analyzer sees also webpage: the wheel-like structure of http://emboss.bioinformatics.nl/cgi-bin/emboss/pepwheel on-line analysis polypeptide; The spiral of http://emboss.bioinformatics.nl/cgi-bin/emboss/ on-line analysis secondary structure, folded folding etc.).The wheel-like structure synoptic diagram of hPP10, spiral, folded folded structure synoptic diagram are distinguished as depicted in figs. 1 and 2.

The hPP10 of chemosynthesis green fluorescence mark:

FITC-

Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg（hPP10-FITC）

With redgreen fluorescent mark hPP10:

Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg(hPP10)

Quantitative, the freezing preservation of purifying is subsequent use.

2.hPP10 route of synthesis have two kind 1) chemosynthesis; 2) gene engineering expression.

HPP10 comes from nature albumen, can synthesize at laboratory level or industrial level.

2.1 chemical synthesis process: this kind method is to select carboxy resin (Fmoc-Tyr (tBu)-Wang resin), adopts solid phase Fmoc method synthetic.Concrete synthesis step is following:

(1) Methionin of with the Fmoc group alpha-amino group being protected is attached on the insoluble carrier Wang resin (Wang resin) through a support arm;

(2) with TFA (trifluoroacetic acid) solution washing Methionin-support arm-resin, make the alpha-amino group deprotection;

(3) second Isoleucine of protecting with suitable condensing agent DCC activatory alpha-amino group in advance being formed common ester through coupled reaction and Methionin is connected up;

(4) with TFA (trifluoroacetic acid) solution washing Isoleucine-support arm-resin, make the alpha-amino group deprotection;

(5) the 3rd proline(Pro) of protecting with suitable condensing agent DCC activatory alpha-amino group in advance being formed common ester through coupled reaction and Isoleucine is connected up.Repeat above-mentioned deprotection, coupling couplet, go up last amino acid---l-arginine, slough the Fmoc blocking group, the synthetic completion up to coupling.

(6) cutting reagent being joined peptide---in the resin, cut down peptide chain from resin, also the side chain protected group on each amino acid is cut down from peptide chain simultaneously, add ether, the deposition polypeptide obtains the polypeptide bullion.Utilization HPLC/MS carries out the evaluation and the purifying of polypeptide, finally obtains required polypeptide.

2.2 gene engineering expression method: adopt prokaryotic expression method, concrete operations are following:

(1) two strand cDNA of design coding hPP10; Both sides have NdeI and XhoI restriction enzyme site; Transfer to Shanghai and give birth to the synthesizing single-stranded oligonucleotide chain of worker company, again two single stranded DNA equivalent are added in the aqueous solution, through 95 ℃ 5 minutes; Naturally cool to room temperature and make it accomplish annealing, form complementary double chain DNA fragment (hPP10);

(2) utilize two kinds of restriction enzymes of NdeI/XhoI to carry out double digestion, 37 ℃ of incubations two hours are with expression plasmid pET15b (available from Novagen) linearizing;

(3) carry out agarose gel electrophoresis, under the long wave ultraviolet light irradiation of ultraviolet transilluminator, cut glue and reclaim the band that contains linearization plasmid pET15b.Instantaneous centrifugal, gel is concentrated to the pipe end, reclaim operation instructions in the test kit and accomplish and cut glue and reclaim according to cutting glue;

(4) will reclaim, the film peptide cDNA fragment (hPP10) of wearing after the linearization plasmid dna fragmentation of purifying and the annealing carries out agarose gel electrophoresis respectively, confirms the connection ratio, places the connection that is incubated overnight of 16 ℃ of water baths;

(5) use CaCl ₂Legal system is equipped with the DH5a competent cell; With the heat-shocked method with above-mentioned connection product pET15b-hPP10 transformed competence colibacillus cell; After 37 ℃ of 0.1g/L penbritin agar plates spent the night screening, picking list colony inoculation was in containing penbritin LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night;

(6) the amplification transform bacteria is collected in centrifugation, uses the alkaline lysis method of extracting recombinant plasmid, screens the positive colony pET15b-hPP10 that successfully makes up, and enzyme is cut and sequence verification;

(7) will verify that correct reorganization protokaryon plasmid pET15b-hPP10 transforms Rosetta competence bacterium.Spend the night for 37 ℃ through 0.1g/L penbritin agar plate;

(8) picking mono-clonal colony inoculation is in containing penbritin LB substratum, and 37 ℃ of shaking culture are spent the night;

(9) with the aseptic centrifuge tube dress of 15ml 3.8ml Amp (+) LB liquid nutrient medium, the bacteria suspension (1:20) of inoculation 0.2ml logarithmic phase.37 ℃, 250rpm continues to cultivate 3h;

(10) in the logarithmic phase bacterial cultures, add 40 μ l 0.1M IPTG to final concentration 1.0mM, 37 ℃, 250rpm continues to cultivate;

(11) the 6h 200 μ l bacteria suspensions of taking a sample after adding IPTG;

(12) centrifugal collecting precipitation, resuspended with equal-volume 1 * Sample Buffer, boiling water bath 5min.All article on the thalline whole protein of preparation;

(13) SDS-PAGE detects the Recombinant Protein Expression situation, like Fig. 3.Expression and the purifying confirmed.

3.hPP10 have and wear film usefulness very by force:

3.1hPP10-FITC have and significantly wear the film characteristic and in born of the same parents, be evenly distributed;

3.1.1hPP10-FITC can wear that film gets into the cell such as tumour of vitro culture and in cell uniform distribution, have and significantly wear the film characteristic;

Four kinds of culturing cell: the ECV-304, HeLa, HepG2, the PC3 that take the logarithm vegetative period are according to 1 * 10 ⁵The density of individual cells/well is inoculated in the conventional cultivation of 12 orifice plates, establishes experimental group and control group;

During to logarithmic phase (80% density), the RPMI-1640 nutrient solution that changes serum-free into continues to cultivate 1 hour;

Add final concentration 10 μ M hPP10-FITC, the conventional cultivation 1 hour;

Discard nutrient solution, PBS washing 3 times;

Fluorescent microscope is observed fluorescence and the interior location of born of the same parents situation thereof in the culturing cell down.

The result sees Fig. 4, and fluorescence microscope is found, without redgreen fluorescence in the various cells of hPP10-FITC processing.Behind the hPP10-FITC incubation, can in born of the same parents, see obvious green fluorescence, and these four kinds of different clones all there are fluorescence in the obvious born of the same parents, prompting hPP10 wears film and gets into cell and do not have obvious cell to have a liking for the tropism.

3.1.2hPP10-FITC human peripheral lymphocyte is worn the film characteristic

Freshly take, isolating healthy human peripheral blood lymphocyte, according to 1 * 10 ⁵The density of individual cells/well is inoculated in 12 orifice plates, establishes experimental group and control group;

The conventional cultivation after 2 hours in incubator, the RPMI-1640 nutrient solution that changes serum-free into continues to cultivate 1 hour;

Discard Incubating Solution, PBS washing 3 times;

The result sees Fig. 5, and fluorescence microscope is found, loses green fluorescence in the human peripheral lymphocyte without the hPP10-FITC processing.Behind the hPP10-FITC incubation, can see green fluorescence in the tangible born of the same parents, prompting hPP10 can efficiently wear the human peripheral lymphocyte that film gets into prepared fresh.

Has concentration dependent 3.2hPP10-FITC wear film entering cell

The ECV-304 cell is after the RPMI-1640 through serum-free handles 1h, and after the hPP10-FITC (2.5 μ M, 5.0 μ M, 7.5 μ M, 10.0 μ M) of adding concentration gradient was hatched 1h, PBS cleaned 3 times, observes fluorescence situation in the culturing cell.And adopt fluorescence microplate reader to detect fluorescence intensity numerical value in the cell; Concrete grammar is following: cell is after hatching end through different concns hPP10-FITC, and PBS cleans 3 times, adds 0.1M NaOH by 300 μ l/ holes; Room temperature lysing cell 10min, the centrifugal 3min of 1000rpm.Get cell pyrolysis liquid supernatant 50 μ l in 96 orifice plates in fluorescence microplate reader, exciting light/absorb light is that 490nm/520nm measures down the fluorescence intensity value.The experiment repetition is averaged for 3 times.In experiment, that adopts classics wears film peptide TAT-FITC as positive control.

Fig. 6 A shows that fluorescence intensity raises with hPP10-FITC concentration and strengthens in the born of the same parents, and under the visible same concentrations, hPP10-FITC fluorescence is stronger than TAT-FITC in the born of the same parents.Fig. 6 B is visible, and along with the rising of hPP10-FITC concentration, fluorescence intensity strengthens thereupon in the born of the same parents, and prompting hPP10-FITC wears film entering cell and has concentration dependent.When hPP10-FITC concentration was 5 μ M, fluorescence intensity had all selected for use the hPP10-FITC of 5 μ M to experimentize as final concentration in subsequent experimental than higher.

3.3hPP10 the time length significantly is longer than TAT in the born of the same parents

COS7, ECV-304, PC3, HeLa are after the RPMI-1640 through serum-free cultivates processing 1h, and the adding final concentration is after the hPP10-FITC of 5 μ M is hatched 0.5h, 1.0h, 2.0h, 4.0h, 10h, 20h and 30h respectively.PBS washing three times, lysing cell detects 490nm/520nm according to preceding method with fluorescence microplate reader and detects cell pyrolysis liquid supernatant fluorescent value.Experiment all repeats 3 times and averages.

The different culture cell is along with the prolongation of hPP10-FITC incubation time, and its fluorescent value all strengthens to some extent, is the highest (Fig. 7 A) to its fluorescent value of 4h.And TAT-FITC is when hatching 1h, and fluorescence intensity be a maximum in the born of the same parents, and along with the further prolongation of incubation time, fluorescent value weakens (Fig. 7 B) gradually.In order to confirm that further hPP10-FITC holds time in born of the same parents, incubation time is extended to 30h.Find that hPP10-FITC can be maintained until at least 30 hours (7C) in ECV-304, PC3 and three kinds of cells of HeLa; And TAT-FITC fluorescence in cell sharply descends as time passes, in the time of 10 hours, can not detect fluorescence basically and have (7D).This research shows that novel human-derived property wears film peptide hPP10 and in born of the same parents, hold time and can reach 30 hours at least, and TAT only can keep short several hours time, significantly is shorter than hPP10.

3.4 reducing hPP10-FITC, serum wears membrane efficiency

Culturing cell HepG2, ECV-304 and B16 are respectively after cultivating 1h through the RPMI-1640 that serum and serum-free are arranged; The adding final concentration is after the hPP10-FITC of 5 μ M continues to hatch 1h; PBS cleans 3 times; Lysing cell is got its supernatant and is detected 490nm/520nm wavelength fluorescence light absorption value with fluorescence microplate reader.The experiment repetition is averaged for 3 times.

The result sees Fig. 8, and the existence of serum can influence cell to wearing the endocytosis picked-up of film peptide generally speaking.Detect to find that through fluorescent quantitation the existence of serum is worn film to hPP10-FITC and gone into born of the same parents very big influence is arranged.Serum-free group hPP10 wears membrane efficiency apparently higher than serologic group (P＜0.05) is arranged.The result shows that the existence of serum influences the membrane efficiency of wearing of hPP10.

3.5DMSO promoting hPP10 to wear film, pre-treatment gets into culturing cell

Take the logarithm adherent culture cell HSC-T6 in vegetative period, Caski, ECV-304, suspended culture cell THP1 and the former foster fibrocyte of being commissioned to train are according to 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in 12 orifice plates and carries out the routine cultivation.Every kind of cell all sets up experimental group and control group separately;

During to logarithmic phase (80% density), be changed to the RPMI-1640 nutrient solution of serum-free, cultivated again 1 hour;

Every hole adding final concentration is 5% DMSO, continues to cultivate 1 hour.After the adding final concentration is the hPP10-FITC or TAT-FITC of 5 μ M, hatched 1 hour;

Discard nutrient solution, PBS washing 3 times;

Fluorescent microscope is observed HSC-T6, Caski, ECV-304, suspended culture cell THP1 and former being commissioned to train down and is supported fluorescence and the interior location of born of the same parents situation thereof in the fibrocyte born of the same parents; Or add 300 μ l 0.1M NaOH by every hole; Room temperature cracking ECV-304; Caski or B16 cell 10 minutes, centrifugal 3 minutes of 1000rpm.Get cell pyrolysis liquid supernatant 50 μ l to 96 orifice plates in fluorescence microplate reader, measure down the fluorescence light absorption value in 490nm/520nm.The experiment repetition is averaged for 3 times.

Inventor's early-stage Study finds that DMSO can obviously strengthen TAT and wear membrane efficiency, at this, has also observed DMSO and has worn the film influence to what novel human-derived property was worn film peptide film hPP10.Find that through fluorescence microscope DMSO wears film to hPP10-FITC and has obvious reinforced effects.With wear membrane efficiency through TAT-FITC after the DMSO pre-treatment and compare, hPP10-FITC wears membrane efficiency and TAT quite or slightly is better than TAT-FITC (Fig. 9 A) behind the DMSO pretreatment cell.After the DMSO pre-treatment; HPP10-FITC shows in born of the same parents and does the density green fluorescence; And be uniformly distributed in endochylema and karyon, even to common transfection or the transduction very suspension cell or the primary cell of difficulty, hPP10-FITC also has very ideal and wears the film effect.Fluorescent quantitation detects to be found, after the 5%DMSO pre-treatment, without DMSO pretreatment cell strong (Fig. 9 B), fluorescence intensity obviously is better than TAT-FITC to fluorescence intensity in the born of the same parents of hPP10-FITC in the born of the same parents of hPP10-FITC and TAT-FITC.Prompting 5%DMSO pretreatment cell has obviously deposited into the membrane efficiency of wearing of hPP10 and TAT, and what DMSO can significantly strengthen CPP wears membrane efficiency and acellular specificity (Fig. 9).

3.6DMSO pre-treatment promotes hPP10 to wear the lymphocyte that film gets into prepared fresh

Freshly take, prepare human peripheral lymphocyte, or mice spleen lymph cell, according to 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in 12 orifice plates;

Discard nutrient solution, PBS washing 3 times;

Fluorescent microscope is observed fluorescence intensity and the interior fluorescence of born of the same parents location situation in the culturing cell down.

The result finds that after the DMSO pre-treatment, hPP10-FITC also can efficiently wear human peripheral or the mouse spleen lymphocyte that film gets into prepared fresh, and is uniformly distributed in endochylema and karyon, and Figure 10 A is hPP10-FITC density and distribution in the mouse spleen lymphocyte; Figure 10 B is hPP10-FITC density and distribution in the human peripheral lymphocyte.Prompting hPP10 not only can wear film and get into the adherent culture cell, in the suspended culture cell, can also wear human peripheral lymphocyte or mouse spleen lymphocyte that film gets into prepared fresh, has shown extremely application prospects.

Embodiment 2, hPP10 mediate plasmid DNA transfection

The method of hPP10 mediation plasmid DNA transfection may further comprise the steps: (1) the culturing cell ECV-304 in vegetative period that takes the logarithm, and with 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in the conventional cultivation of 12 orifice plates, and the corresponding positive and negative hole are set simultaneously;

(2) put 37 ℃ of 5%CO ₂In the incubator, cultivate 20 ~ 24h, when cell density reaches 70%-80%, change nutrient solution the RPMI-1640 nutrient solution of serum-free into, continue to cultivate 1 hour;

(3) meanwhile prepare the transfection sample:

A. getting final concentration is that 10 μ M hPP10 adding contains in the centrifuge tube of 50 μ l Opti-MEM, at room temperature hatches 5min gently behind the mixing; B. the plasmid of getting 0.8 μ g adds and contains in the another one centrifuge tube of 50 μ l Opti-MEM, at room temperature hatches 5min gently behind the mixing.B is joined among a lentamente, and room temperature leaves standstill 30min behind the mixing;

(4) above-mentioned hPP10+ DNA mixing solutions is joined in the corresponding culturing cell jog mixing equably.With commercially available liposome Lipofectamine 2000 as the transfection experiment positive control;

(5) put cell and in CO2gas incubator, continue incubation 4-6h after, change into and contain the calf serum nutrient solution, continue to cultivate 24-48h;

(6) observe the transfection effect of hPP10 mediation, with liposome Lipofectamine 2000 transfection reagents and positive control peptide TAT as the transfection positive control.

2.1hPP10 mediation red fluorescent protein expression plasmid pDsRed transfection

Be replaced with in 4-6 hour after the transfection after normal cultured liquid continues to cultivate 48h, fluorescent microscope is observed the effect of ECV-304 cell through hPP10 mediation transfection plasmid pDsRed (available from Clontech company) down.Visible by Figure 11, hPP10 can mediate transfection plasmid pDsRed, has red fluorescent protein to express in some culturing cells.

2.2 the effect of the structure of plasmid pcDNA3.1-Gluc and its transfection of hPP10 mediation

2.2.1 eukaryon recombinant plasmid pcDNA3.1-Gluc makes up and enzyme is cut evaluation

(1) utilize two kinds of enzymes of BamHI/XbalI to carry out double digestion eukaryon expression plasmid pcDNA3.1 (+) (available from invitrogen company) and plasmid pGLuc-Basic Vector (eukaryotic expression secretor type luciferase carrier; Available from NEB company); 37 ℃, incubation 2h;

(2) carry out agarose gel electrophoresis respectively; Cut cDNA fragment that glue reclaims linearizing expression plasmid pcDNA3.1 (+) and secretor type luciferase (Gaussia Luciferase) respectively to centrifuge tube; Centrifugal, reclaim test kit operation instructions recovery dna fragmentation according to cutting glue;

(3) fragment that is recovered to is carried out agarose gel electrophoresis, confirm to connect and to use the DNA ratio, join the link reaction system, place the connection of spending the night of 16 ℃ of water baths;

(4) get the DH5a competent cell; With the heat-shocked method to connect product pcDNA3.1-Gluc transformed competence colibacillus cell; After 37 ℃ of 0.1g/L penbritin agar plates spent the night screening, the single colony inoculation of picking was in containing penbritin LB liquid nutrient medium, and 37 ℃ of overnight shakings are cultivated;

(5) use the alkaline lysis method of extracting recombinant plasmid; Screen the positive colony that successfully makes up; Restriction enzyme is identified with BamHI/XbalI double digestion recombinant plasmid; Obtain enzyme and cut product, the pcDNA3.1he theoretical value is the insertion sheet segment DNA band (Figure 12) of 590bp, and electrophoresis result shows consistent with the expection size; And confirm to insert fragment through order-checking and do not have sudden change.Show and successfully made up eukaryon recombinant plasmid pcDNA3.1-Gluc.

2.2.2hPP10 the effect of mediation plasmid pcDNA3.1-Gluc transfection

(1) the culturing cell ECV-304 in vegetative period that takes the logarithm is with 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in the conventional cultivation of 12 orifice plates, and the corresponding positive and negative hole are set simultaneously;

(3) meanwhile preparing transfection sample: a., to get final concentration be that 10 μ M hPP10 add and to contain in the centrifuge tube of 50 μ l Opti-MEM, at room temperature hatches 5min gently behind the mixing; B. the plasmid of getting 0.8 μ g adds and contains in the another one centrifuge tube of 50 μ lOpti-MEM, at room temperature hatches 5min gently behind the mixing.B is joined among a lentamente, and room temperature leaves standstill 30min behind the mixing;

(4) the hPP10+ plasmid mixture is joined in the corresponding culturing cell hole uniformly the jog mixing.With commercially available liposome Lipofectamine 2000 as the transfection experiment positive control;

(5) at 5%CO ₂In the incubator, 37 ℃ continue incubation 4-6h after, be replaced as and contain calf serum normal cultured liquid, continue to cultivate 24-48h;

(6) get 200 μ l nutrient solution supernatants (containing the secretor type luciferase) and place the 1.5ml centrifuge tube; The centrifugal 3min of 5000rpm, according to secretor type luciferase test kit operation instruction, utilization fluorescence micropore determinator; Detect the culture supernatant uciferase activity; As the transfection positive control, TAT and meaningless peptide NCO are as positive and negative peptide contrast with liposome Lipofectamine 2000 transfection reagents, and each is handled, and the uciferase activity result sees Figure 13 in the sample.

Figure 13 is visible, and hPP10 has successfully mediated secretor type luciferase plasmids pcDNA3.1-Gluc transfection, cell expressing the luciferase protein of higher level, and its activity progressively strengthens along with increasing of hPP10 concentration.

This research successful use hPP10 has mediated the transfection of DNA (pDsRed and pcDNA3.1-Gluc); But its transfection efficiency is compared with commercial reagent Lipofectamin 2000 and is seemed lower; We will continue to optimize the transfection experiment condition condition of hPP10 mediation, in the hope of improving transfection efficiency.The transfection of hPP10 success mediated dna plasmid will provide strong instrument for external and intravital gene transfection, may be used on scientific research and gene therapy clinically in the future.

Embodiment 3, pET15b-hPP10-GFP plasmid construction, Expression of Fusion Protein and purifying are worn the research of film effect with it

3.1pET15b-hPP10-GFP construction of recombinant plasmid and enzyme are cut evaluation

(1) two strand cDNA of design coding hPP10; Both sides have NdeI and XhoI restriction enzyme site, transfer to Shanghai and give birth to the synthesizing single-stranded oligonucleotide chain of worker company, two single stranded DNA equivalent are added in the aqueous solution again; Through 95 ℃; 5 minutes, naturally cool to room temperature and make it accomplish annealing, form complementary double chain DNA fragment (hPP10); Designing a pair of primer simultaneously is template with pEGFP (available from Clontech company), and PCR obtains GFP protein gene fragment, and both sides have XhoI and BamHI restriction enzyme site respectively, and purified pcr product is subsequent use;

(2) utilize two kinds of restriction enzymes of BamHI/NdeI to carry out double digestion, 37 ℃ of incubations two hours are with expression plasmid pET15b (available from Novagen) linearizing;

(3) carry out agarose gel electrophoresis, under the long wave ultraviolet light irradiation of ultraviolet transilluminator, cut glue and reclaim the band that contains linearization plasmid pET15b; Instantaneous centrifugal, gel is concentrated to the pipe end, reclaim operation instructions in the test kit and accomplish and cut glue and reclaim according to cutting glue;

(4) will reclaim, after the linearization plasmid dna fragmentation of purifying and the annealing wear film peptide cDNA fragment (hPP10) and the GFP gene fragment is carried out agarose gel electrophoresis respectively, confirm the connection ratio, place the connection that is incubated overnight of 16 ℃ of water baths;

(6) the amplification transform bacteria is collected in centrifugation, uses the alkaline lysis method of extracting recombinant plasmid, screens the positive colony pET15b-hPP10-GFP that successfully makes up, and enzyme is cut and sequence verification;

Utilize the NdeI/BamHI double digestion to identify that recombinant plasmid pET15b-hPP10-GFP obtains enzyme and cuts the product theoretical value and be 795bp (Figure 14), fragment is consistent with the expection size; Confirm not have sudden change through order-checking.Explain that prokaryotic expression plasmid pET15b-hPP10-GFP makes up successfully.Same procedure makes up and obtains pET15b-GFP and pET15b-TAT-GFP

3.2 Expression of Fusion Protein and purifying

3.2.1 the prokaryotic expression of fusion rotein

(1) successful recombinant plasmid pET15b-GFP and the pET15b-hPP10-GFP that makes up transformed the Rosetta competent cell respectively.Spend the night for 37 ℃ through 0.1g/L penbritin agar plate;

(2) picking mono-clonal colony inoculation is in containing penbritin LB substratum, and 37 ℃ of shaking culture are spent the night;

(3) with the aseptic centrifuge tube dress of 15ml 3.8ml LB (+) ^AmpLiquid nutrient medium, the bacteria suspension (1:20) of inoculation 0.2ml logarithmic phase.37 ℃, 250rpm continues to cultivate 3h;

(4) in the logarithmic phase bacterial cultures, add 40 μ l 0.1M IPTG to final concentration 1.0mM, 37 ℃, 250rpm continues to cultivate;

(5) the 6h 200 μ l bacteria suspensions of taking a sample after adding IPTG;

(6) centrifugal collecting precipitation, resuspended with equal-volume 1 * Sample Buffer, boiling water bath 5min.All article on the thalline whole protein of preparation;

(7) SDS-PAGE detects the Recombinant Protein Expression situation.

3.2.2 the purifying of fusion rotein

3.2.2.1 induce in a large number: with containing 300ml LB (+) in the 1L culturing bottle ^AmpLiquid nutrient medium, and abduction delivering soluble recombining albumen (1.0mM IPTG, 30 ℃, 250rpm, 9h).4 ℃ of 6500rpm * 10min, collecting precipitation is also weighed, and-40 ℃ of preservations are subsequent use.

3.2.2.2 imidazoles purifying

(1) ultrasonic degradation: average 100ml bacterium liquid is with 15ml Lysis/Binding Buffer (300mM NaCl, 50mMNaH ₂PO ₄, the 10mM imidazoles, pH 8.0) resuspended, ice bath.Ultrasonic power 300 ~ 600W, ultrasonic 2sec, 2sec works 90 times at interval.So circulation is 12 ~ 20 times, and extremely even broken bacterium is in aaerosol solution;

(2) 12000rpm * 20min, shifts supernatant to the 15ml centrifuge tube by 4 ℃;

(3) combine: with Lysis/Binding Buffer balance Ni-NTA in the 15ml centrifuge tube of 3 ~ 5 times of volumes, centrifugal collection Ni-NTA uses with to precipitate isopyknic Lysis/Binding Buffer resuspended promptly Balanced 50%Ni-NTA again.Get 1 ~ 4ml 50%Ni-NTA and mix with the cracking supernatant, 37 ℃ are rotated combination 1 ~ 2h in the molecular hybridization stove;

(4) washing removes foreign protein: with at least 1/2 bacteria liquid long-pending Wash Buffer (300mMNaCl, 50mMNaH ₂PO ₄, 20 ~ 30mM imidazoles, pH 8.0) the wash-out foreign protein.Collect preceding 3 ~ 5ml prick post liquid, detection is done in sampling fully;

(5) wash-out target protein: shared 5 ~ 10ml Elution Buffer (300mM NaCl, 50mM NaH ₂PO ₄, the 250mM imidazoles, pH 8.0), add 0.5ml at every turn, collect with the 1.5ml centrifuge tube;

(6) preserve: in 96 orifice plates, respectively get 5 μ l samples and mix, relatively shade with 195 μ l Xylene Brilliant Cyanine G G-250 solution.Retain the darkest number pipe of color, add the aseptic glycerine of 100 ~ 200 μ l respectively, in-40 ℃ of preservations, and detection is done in sampling fully behind the mixing.

GFP and hPP10-GFP fusion rotein relative molecular mass are respectively 27KD and 30KD.Positive colony is analyzed expressed fusion protein through SDS-PAGE after 1.0mM IPTG induces 6 hours, a strongly expressed band, accord with expectation molecular mass size (Figure 15) near 35KD and 27KD, occur.The proteic purifying of gene engineering expression is to utilize 6 * His label, obtains effective purifying through the Ni-NTA affinity chromatography, and the purity of fusion rotein is greater than 80%.Figure 15 shows GFP and the hPP10-GFP albumen that has obtained purifying.

3.3hPP10-GFP fusion rotein is worn the research of film effect

3.3.1hPP10-GFP fusion rotein is striden film and is got into the intracellular capability study of adherent culture

(1) the culturing cell L929 in vegetative period that takes the logarithm is according to 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in the conventional cultivation of 12 orifice plates.Every kind of cell all sets up experimental group and control group separately;

When (2) treating that cell grows to 80% fusion, be changed to the nutrient solution of serum-free, continue to cultivate 1 hour;

(3) every hole adding final concentration is 5%DMSO, continues conventional the cultivation 1 hour;

(4) experimental group adding final concentration is the fusion rotein hPP10-GFP of 5 μ M, and the albumen TAT-GFP that positive controls adds same concentrations establishes negative control simultaneously.37 ℃ of incubators were hatched 1 hour;

(5) discard with the supernatant nutrient solution PBS washing three times;

(6) place under the fluorescent microscope and to observe the hPP10-GFP fusion rotein and wear location situation in film and the born of the same parents.

After adding hPP10-GFP fusion rotein is hatched 1h in the L929 cell, observe clear bright green fluorescence in the cell under the fluorescent microscope, fluorescence distribution even (Figure 16) in the born of the same parents; HPP10-GFP fluorescence obviously strengthens in the born of the same parents behind 5%DMSO pre-treatment L929 cell.Show that hPP10 portability macromole (green fluorescent protein) efficiently wears film and get into cell, the 5%DMSO pre-treatment can significantly strengthen hPP10 and carry green fluorescent protein and stride film and get into cell.

3.3.2hPP10-GFP what fusion rotein strode that film gets into human peripheral lymphocyte wears the film capability study

(1) freshly takes, separates the preparation human peripheral lymphocyte, according to 1 * 10 ⁵The inoculum density of individual cells/well is inoculated in 12 orifice plates, establishes experimental group and control group;

(2) 12 orifice plates are left standstill 2 hours in incubator after, the RPMI-1640 nutrient solution that changes serum-free into continue to be cultivated 1 hour;

(4) experimental group adding final concentration is the fusion rotein hPP10-GFP of 5 μ M, and negative control group adds the GFP albumen of same concentrations, and 37 ℃ of incubators were hatched 1 hour;

(5) discard with nutrient solution PBS washing three times;

The fresh human peripheral lymphocyte of taking, separate preparation adds the hPP10-GFP fusion rotein after the 5%DMSO pre-treatment, observe tangible green fluorescence under the fluorescent microscope, fluorescence distribution even (Figure 17) in the born of the same parents; And it is fainter without hPP10-GFP fluorescence in the DMSO pretreatment cell born of the same parents.

This research prompting hPP10 not only self can wear film and get into cell, also can carry high molecular weight protein (GFP) through the fusion rotein form and stride film entering adherent culture cell and human peripheral lymphocyte.This result of study to cell in is sent high molecular weight protein medicine by hPP10 as carrier for exploitation in the future and is provided the foundation.

Embodiment 4, hPP10 pair cell effect of vigor are very little

(1) the culturing cell ECV-304 and HepG2 in vegetative period that takes the logarithm is with 1 * 10 ⁴The inoculum density of individual cells/well is inoculated in the conventional cultivation of 96 orifice plates, every hole 100 μ l, and every kind of cell is established 3 multiple holes, 37 ℃ of cultivations;

(2) to logarithmic phase, nutrient solution changes the RPMI-1640 nutrient solution of serum-free into, continues to cultivate 1 hour;

(3) change the RPMI-1640 nutrient solution that the hPP10 concentration gradient is 10 μ M, 20 μ M, 30 μ M, 40 μ M and 50 μ M serum-frees respectively into, continue to cultivate 24 hours;

(4) incubation time finishes the back and adds the PBS washing by every hole 100 μ l, 2min * 3 time;

(5) every hole adds normal cultured liquid and 20 μ l MTT (mother liquid concentration 5mg/ml, the i.e. 0.5%MTT) solution that 80 μ l contain serum, cultivates 4h in 37 ℃ of continuation, stops cultivating the back suction and removes nutrient solution.Add DMSO 99.8MIN. with 200 μ l/ holes, vibration 10min fully dissolves the back to crystallisate and uses that the detection wavelength is the light absorption value A of 570nm on the long ELIASA of all-wave, gets 3 MVs in holes again for every group.Measure OD490.Repeat 3 times, calculate cell survival rate.

(6) calculating of cells survival rate is following:

Cells survival rate=(experimental port OD value-control wells OD value-blank well OD value)/(control wells OD value-blank well OD value) * 100%.

For handling cell, clear and definite hPP10 whether can influence its vigor, the situation that influences of pair cell vigor behind this experiment employing mtt assay mensuration different concns hPP10 processing 24h.ECV-304 and HepG2 cell are handled back MTT analytical data demonstration through different concns hPP10, and cell still remained on more than 75% the effect of vigor of pair cell very little (Figure 18) after concentration was higher than the hPP10 long time treatment cell of 20 μ M.HPP10-FITC does not influence cell viability in prompting effective concentration (less than the 5 μ M) scope.

Embodiment 5, hPP10 wear film mechanism

5.1 slightly suppressing hPP10-FITC, low temperature wears film

Use the different culture cell; Like ECV-304, Caski and HeLa after handling 1h through the RPMI-1640 of serum-free; The hPP10-FITC that adds final concentration and be 5 μ M is hatched 1h respectively under 4 ℃ and 37 ℃ of two conditions after; PBS cleans 3 times, gets the fluorescence light absorption value that its supernatant detects the 490nm/520nm wavelength according to the preceding method lysing cell.The experiment repetition is averaged for 3 times.

It is generally acknowledged that the cell endocytic approach is that energy relies on, the energy metabolism of cell is almost stagnated during low temperature, also is that low temperature can be blocked the cell endocytic approach.If it is to realize through endocytic pathway that hPP10 wears the film mode, variation of temperature will inevitably influence fluorescence content in the born of the same parents so.This experiment is selected for use and is explored temperature 4 ℃ and 37 ℃ of two conditions and whether influence it and wear the film mode.Figure 19 shows, it is very little that low temperature is worn the film influence to hPP10, with great majority report results are consistent so far.Prompting hPP10 small peptide is originally worn film maybe be irrelevant with endocytosis mechanism.

5.2 significantly reducing hPP10, heparin wears membrane efficiency

Observe three kinds of endocytosis suppressor factor to hPP10-FITC wear film influence, heparin sodium (PBS dissolving, final concentration 10 μ M), chloroquine (PBS dissolving, final concentration 10 μ M), CHLORPROMAZINE HCL (PBS dissolving, final concentration 30 μ M) are made into mother liquor, the sterilization of 0.22 μ m membrane filtration.Four kinds of different culture cell COS7, ECV-304, PC3 and HeLa are after the RPMI-1640 with serum-free cultivates 1h; Add three kinds of endocytosis suppressor factor of final concentration in the cell respectively, continue to hatch 30min, the adding final concentration is that the hPP10-FITC of 5 μ M is hatched 1h; Remove substratum; PBS cleans 3 times, places fluorescence microplate reader according to preceding method, detects the fluorescence light absorption value of 490nm/520nm.The experiment repetition is averaged for 3 times.

Experiment is hatched with different endocytosis suppressor factor through culturing cell, investigates the endocytosis suppressor factor is gone into born of the same parents' situation to hPP10 influence.Wherein heparin is the competitive inhibitor of surface of cell membrane S-PG; Chloroquine is for suppressing inclusion body acidifying endocytosis regulator; The endocytosis suppressor factor that CHLORPROMAZINE HCL relies on for cage modle albumen.Find out that from Figure 20 A after adding heparin, cell reduces the picked-up of hPP10 significantly.The prompting heparin can significantly suppress hPP10 and absorbed the entering cell, and the S-PG of surface of cell membrane is worn film entering cell to hPP10 and brought into play important effect.Figure 20 B and 20C show that chloroquine and CHLORPROMAZINE HCL are all not obvious to the influence that hPP10 wears film entering cell.Prompting hPP10 wear film get in the process of cell need with negative charge interaction of molecules such as surface of cell membrane S-PG, it is machine-processed not rely on the protein mediated endocytosis of cage modle and inclusion body acidifying.

Conclusion: we are carrying out on the basis of systems analysis existing CPP structure, and through Protein Data Bank is retrieved, methods such as prediction and secondary structure analysis are found a brand-new Humanized cell film penetrating peptide (hCPP), called after hPP10.The fusion rotein (hPP10-GFP) of synthetic hPP10 and prokaryotic expression itself and green fluorescent protein has carried out a series of experiment in vitro researchs.Find that hPP10 has the very strong film ability of wearing; And portability macromole GFP; DNA is striden film and is got into adherent culture cells such as comprising tumour cell, primary cell, multiple culturing cell such as suspension cell and human peripheral lymphocyte; And not influencing the function of entrained bioactive molecules, pair cell has no side effect basically.Show that hPP10 wears the film peptide as a kind of new Humanized cell, can be used for external and the interior bioactive molecules (like protein, polypeptide, medicines such as gene) that in cell, transports of body.The exploitation of hPP10 will provide transportation means in the another novel born of the same parents for scientific research (protein transduction of cultured cell in vitro and gene transfection), clinical treatment (in cell, sending albumen or genomic medicine).

Claims

1. a novel human-derived sexual cell is worn film peptide hPP10, it is characterized in that hPP10 comes from human cell's nucleoprotein, and one section peptide sequence of Methionin specificity demethyl enzyme 4A has the cytolemma penetrativity.

2. Humanized cell as claimed in claim 1 is worn film peptide hPP10, it is characterized in that the said peptide sequence of wearing the film peptide is following:

Lys-Ile-Pro-Leu-Pro-Arg-Phe-Lys-Leu-Lys-Cys-Ile-Phe-Cys-Lys-Lys-Arg-Arg-Lys-Arg。

3. claim 1 or 2 said novel human-derived sexual cells are worn the purposes of film peptide hPP10 as the medicament transport carrier.

4. purposes as claimed in claim 3 is characterized in that said novel human-derived sexual cell is worn film peptide hPP10 portability albumen or nucleic acid is striden film entering various kinds of cell.

5. purposes as claimed in claim 4 is characterized in that said nucleic acid is the DNA plasmid.

6. purposes as claimed in claim 4 is characterized in that said novel human-derived sexual cell wears the cell that film peptide hPP10 portability high molecular weight protein (GFP) gets into and comprise adherent culture cell, suspended culture cell and primary cultured cell.