CN102286085A - Peganum harmala lipid transfer protein and preparation method and use thereof - Google Patents

Abstract

The invention discloses a peganum harmala lipid transfer protein (PHP) and relates to the preparation and use of new protein with antifungal, anticancer and antivirus activities. The method uses seeds of a peganum harmala plant as raw materials and comprises the following steps: preparing a coarse product, performing cationic exchange chromatographic separation and purifying by using a molecular sieve; and gradually separating and collecting a purified component having antibacterial activity, dialyzing, desalting, freeze-drying and thus, obtaining purified PHP. In the invention, a filter paper disc agar diffusion method is adopted, and researches prove the protein has growth inhibiting effect on five plant pathogenic fungi, namely alternaria alternate, penicillium degitatum, rhizopus stuolonifer, magnaporthe grisea and penicillium italicum. An 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method proves that the protein has inhibiting effect on proliferation of human esophageal carcinoma cell Eca109, human cervical cancer cells HeLa, human stomach cancer cells MGC-7, mouse melanoma cells B16 and the like. The protein can inhibit the activity of human immunodeficiency virus 1 (HIV-1) reverse transcriptase. The protein can be used for developing medicines for resisting agricultural pathogenic fungi, tumors and viruses.

Description

A kind of Herba pegani harmalae fat transfer protein and its production and application

Technical field:

The invention belongs to biological technical field, the fat transfer protein PHP that to relate to a kind of molecular weight that separation and purification goes out from seed of peganum harmala be 16kDa, more specifically, the present invention relates to a kind of antimycotic, as to suppress tumor promotion and inhibition HIV-1 reverse transcriptase activity Herba pegani harmalae fat transfer protein that has, and utilize seed of peganum harmala for raw material carries out the crude product preparation, cation-exchange chromatography separates and molecular sieve purification prepares described method of protein and described proteinic application.

Background technology:

Plant-growth regular meeting is subjected to the invasion and attack of various fungies, and in the long-term evolution process, plant materials has formed the natural defense system of a cover and come and the antagonism of external source invader.(antifungal protein AFP) is exactly wherein a class to the plant antifungal protein, and they can suppress fungal growth or kill fungi.Mechanism of action according to the plant antifungal protein, structure and sequence characteristic, it can be divided into substantially following a few class: pathogenesis-related proteins (pathogenesis-related proteins, PRs), alexin (defensins), class cyclophilin albumen (cyclophilin-like proteins), ribosome inactivating protein (ribosome inactivating Protein, RIPs), the fat transfer protein (lipid transfer proteins, LTPs), proteinase inhibitor (protease inhibitor), 2S white protein class (2S albumin proteins) and other albumen.Fat transfer protein (lipid transfer protein, LTP) be the less relatively basic protein of molecule amount, owing to can shift phosphatidylcholine, the pure and mild phosphatidylserine of phosphatidyl-4, and can promote the phosphatide between plastosome and the microbody to exchange and (the Gincel.E that gains the name, etal.1994), in animal, plant, yeast, fungi and some bacteriums, discovery is arranged all.So far (non-specific lipid transfer protein, nsLTP), its fat transfer activity has popularity and uncertainty only to find that in plant non-specific fat transfer protein is arranged.From various plants, separated obtaining nsLTPs now, as finding in many different plant tissues such as corn, paddy rice, barley, millet, onion, cowpea, Chinese cabbage, spinach, sweet tangerine, square-bottomed bamboo basket fore-telling, peach, Arabidopis thaliana.And to resolving from the molecular structure of the nsLTPs in the plants such as paddy rice, corn, peach.Non-special fat transfer protein is soluble proteins in plant, and content is abundant, can reach 4% of soluble protein total amount.It is reported, many organs of plant all have the distribution of LTP, fruit and floral organ as seed, stem, leaf, root, Cruciferae, and find around the epidermic cell of plant organ and the content in peripheral cells, cell walls and the face tissue's stratum corneum high especially (Trends Microbiol, etal.1995).At first the research of the non-characteristic fat of plant transfer protein is mainly concentrated on the transhipment of lipid material between the microbial film.Along with continuous further investigation, find that nsLTP albumen also has important effect at plant disease-resistant insect pest, aspect such as antitumor and antiviral.In recent years, from traditional herbal medicine, continue to excavate new active protein composition,, become an important research direction of researcher in the hope of finding some at agricultural and the protein or the polypeptide that pharmaceutically have using value.

Herba pegani harmalae (Peganum harmala L.) is that zygophyllaceae (Zygophyllaceae) Herba pegani harmalae belongs to (Peganum) per nnial herb.Be born in arid meadow, salinization desert belt more.Be distributed in provinces and regions such as the Inner Mongol, Hebei, Shanxi, Shaanxi, Ningxia, Gansu, Qinghai, Xinjiang; Also there are distribution in Mongolia, the Soviet Union.As the existing use history for a long time of Uygur's tradition medicinal material, its seed or herb can be used for treating illnesss such as cough and asthma, rheumatic arthralgia and nameless gall.Chemical ingredients such as alkaloid has antibiotic, desinsection and antitumor isoreactivity in the Herba pegani harmalae, but simultaneously body is had some neural toxic side effect.The present invention first from seed of peganum harmala separation and purification go out the fat transfer protein PHP that a kind of molecular weight is 16kDa, have antimycotic and anti-tumor activity and suppress the effect of HIV-1 reverse transcriptase activity, do not see existing bibliographical information.The present invention provides foundation for the function of activated protein in the scientific knowledge Herba pegani harmalae, has important theory and using value aspect the screening of plant genetic engineering, the sick preparation of agriculture antimycotic cause of disease and anticancer protein medicine.

Summary of the invention:

The invention provides a kind of Herba pegani harmalae fat transfer protein PHP, its N end contains the aminoacid sequence ITCPQVTQSLAPCVPYLISG shown in the SEQID NO:1, and by the homology comparison of N terminal amino acid residue, explanation is a kind of new fat transfer protein.Described dietary protein origin is in medicinal plant Herba pegani harmalae (Peganum harmala L.) seed, be to be the homodimer that two subunits are formed under native state, molecular weight is 16kDa, and the molecular weight of single subunit is about 8kDa, and this albumen is that a kind of iso-electric point pI is 8.4 basic protein.The present invention has carried out the separation and purification of Herba pegani harmalae fat transfer protein first, is raw material with the seed of peganum harmala, and by ammonium sulfate precipitation, cation-exchange chromatography and gel-filtration sieve chromatography have obtained a kind of Herba pegani harmalae fat transfer protein PHP.This albumen antimycotic, anti-tumor activity and inhibition HIV-1 reverse transcriptase activity and applied analysis have been carried out.

A kind of Herba pegani harmalae fat transfer protein and its production and application may further comprise the steps:

(1) crude product preparation: with the seed of peganum harmala is material, after its pulverizing, adding pH is the PBS phosphoric acid buffer of the neutrality of 7.0-7.5 to weak basic condition, low temperature stirs under the extracting and spends the night, collect supernatant liquor behind the low-temperature centrifugation, slowly adding ammonium sulfate in supernatant liquor, to transfer to saturation ratio be between the 30%-50%, stand at low temperature 4h, centrifugal collection supernatant liquor is abandoned precipitation.Continuation slowly adds ammonium sulfate in supernatant liquor to transfer to saturation ratio be between the 60%-80%, once more behind the stand at low temperature 4h, and centrifugal collecting precipitation, and be dissolved in the small amounts of phosphoric acid damping fluid, the dialysis desalination, lyophilize gets the albumen crude product;

(2) cation-exchange chromatography separation and purification: adopt Resource S or the cationic exchange coloum suitable with its character, the raw protein that above-mentioned steps (1) is obtained carries out chromatography, be the unconjugated albumen of PBS phosphoric acid buffer elder generation flush away of 0.02-0.05mol/L with proper concn behind the last sample, carry out gradient elution with the phosphate buffered saline buffer that contains 0.1-1mol/L NaCl again, collection has antibiotic and component anti-tumor activity after the detection of active, preserves standby after fully dialysing with phosphate buffered saline buffer;

(3) molecular sieve purification: Superdex 75 type molecular sieve columns on the sample that above-mentioned steps (2) is obtained, with the concentration that contains finite concentration NaCl is the PBS phosphoric acid buffer wash-out of 0.02-0.05mol/L, substep is collected, collecting after the detection of active has antibiotic and component anti-tumor activity, the dialysis desalination, lyophilize promptly obtains the pure product of Herba pegani harmalae fat transfer protein PHP of purifying.

(4) the Herba pegani harmalae fat transfer protein PHP of the purifying that obtains of above-mentioned steps (3) have antimycotic, suppress tumor promotion and suppress the HIV-1 reverse transcriptase activity, at anti-agricultural disease fungal pathogens and having a good application prospect aspect tumour and the antiviral protein drug treatment.

Here the proteic preparation scheme of PHP that it is pointed out that the natural origin that the present invention provides is not the unique scheme that obtains natural PHP.Finding that the Herba pegani harmalae protein crude extract has under the prerequisite that suppresses fungi and tumor growth, come separation and purification Herba pegani harmalae neutral grease transfer protein with various method for purifying proteins, for example ammonium sulfate precipitation, positively charged ion or anionresin, reversed phase chromatography and sieve method are applied to combination, increase and decrease or the replacement of each step of the present invention program, the target protein separation and purification can be obtained the Herba pegani harmalae fat transfer protein PHP of purifying.

The new Herba pegani harmalae fat transfer protein PHP that the present invention obtains has tangible anti-mycotic activity, to the 5 kind of plant pathogenic fungies that can cause that fruit rots and grain goes mouldy, as hand over lattice pink mold (Alternaria alternata), penicillium digitatum (Penicillium degitatum), head mold germ (Rhizopus stuolonifer), Pyricularia oryzae (Magnaporthe grisea) and penicillium italicum (Penicillium italicum) etc. all have the growth-inhibiting effect, wherein to handing over the lattice pink mold that very strong inhibition ability is arranged, described albumen can be used to prepare the biological pesticide preparation of agent of food fruit and vegetables corrosion protection and anti-agricultural disease fungal pathogens.

The new Herba pegani harmalae fat transfer protein PHP that the present invention obtains also has tangible antitumour activity, tumour cells such as human cervical carcinoma HeLa, people's Gastric Cancer MGC, human esophagus cancer Eca-109 and mouse melanin tumour b16 all there is significant inhibition growth effect, but less to normal Vero cyto-inhibition; Simultaneously, PHP also has the ability that suppresses the HIV-1 reverse transcriptase activity, and described albumen can be used to prepare the pharmaceutical preparation of treatment tumour and HIV virus associated diseases.

The invention still further relates to the preparation method's of Herba pegani harmalae fat transfer protein PHP optimization, the preparation method after the optimization may further comprise the steps:

(1) the crude product preparation condition of You Huaing is: be material with the seed of peganum harmala, after its pulverizing, add 0.02mol/L, the PBS phosphoric acid buffer of pH 7.2, supernatant liquor is collected in 4 ℃ of stirred overnight extractings behind the low-temperature centrifugation, slowly adding ammonium sulfate in supernatant liquor, to transfer to saturation ratio be 50%, leave standstill 4h at 4 ℃, centrifugal collection supernatant liquor is abandoned precipitation; Continuation slowly adds ammonium sulfate in supernatant liquor to transfer to saturation ratio be 70%, 4 ℃ leave standstill 4h after, centrifugal collecting precipitation, and being dissolved in the small amounts of phosphoric acid damping fluid, the dialysis desalination, lyophilize gets the albumen crude product.

(2) the cation-exchange chromatography separation and purification condition of You Huaing is: adopt Resource S cationic exchange coloum, resulting albumen crude product in the above-mentioned steps (1) is carried out chromatography, use 0.02mol/L behind the last sample, the unconjugated albumen of PBS phosphoric acid buffer elder generation's flush away of pH 7.2, carry out gradient elution with the phosphate buffered saline buffer that contains 0.1-1mol/L NaCl again, collection has antibiotic and component anti-tumor activity after the detection of active, preserves standby after fully dialysing with phosphate buffered saline buffer.

(3) the molecular sieve purification condition of You Huaing is: molecular sieve is Superdex 75 type posts, with the molecular sieve column on the sample after the ion exchange treatment that obtains in the above-mentioned steps (2), with the 0.02mol/L that contains 0.15mol/L NaCl, the PBS phosphoric acid buffer wash-out of pH 7.2, substep is collected, collection has antibiotic and component anti-tumor activity after the detection of active, the dialysis desalination, and lyophilize promptly obtains the pure product of Herba pegani harmalae fat transfer protein PHP of purifying.

The present invention obtains crude protein by the ammonium sulfate precipitation of different saturation, in conjunction with cation-exchange chromatography and gel permeation chromatography, obtain a kind of Herba pegani harmalae fat transfer protein PHP, through SDS-PAGE electrophoresis and high performance liquid chromatography (HPLC) chromatographic analysis, this albumen is the homodimer that two subunits are formed under native state, molecular weight is 16kDa, and the molecular weight of single subunit is about 8kDa.Isoelectric focusing electrophoresis is analyzed, and it is that a kind of iso-electric point pI is 8.4 basic protein.Adopt the EDMAN edman degradation Edman, on Applied Biosystems Procise-PROCISE, carry out the mensuration that N terminal amino acid residue is formed, the N terminal amino acid sequence is: ITCPQVTQSLAPCVPYLISG (different bright-Su-half Guang-dried meat-glutamine-figured silk fabrics-Su-glutamine-Si-bright-third-dried meat-half Guang figured silk fabrics-dried meat-junket-bright-different is bright-Si-glycine, Ile-Thr-Cys-Pro-Gln-Val-Thr-Gln-Ser-Leu-Ala-Pro-Cys-Val-Pro-Tyr-Leu-Ile-Ser-Gly).20 amino acid residue sequences that the present invention is obtained, amino acid residue sequence with Blast and ncbi database login carries out the homology comparison, the fat transfer protein of finding it and other plant has homology, wherein the N terminal sequence homology with sweet orange LTP is up to 75%, next is that root of Beijing euphorbia LTP is 70%, rape LTP is 70%, and European Chinese chestnut LTP is 60%, illustrates that it is a kind of new fat transfer protein.

Described albumen PHP of the present invention has tangible anti-mycotic activity, and particularly to handing over the lattice pink mold that very strong restraining effect is arranged, the half-inhibition concentration (IC of lattice pink mold 48h is handed in described albumen effect ₅₀) be 24 μ g/mL.This albumen suppresses the activity stabilized of fungi below 80 ℃ in the pH4-10 scope.

Described albumen PHP of the present invention all has high restraining effect external to kinds of tumor cells (people source and mouse source), but very low to normal cytotoxicity.Experiment confirm, use mtt assay to measure PHP common cancer cells such as human cervical carcinoma cell HeLa, gastric carcinoma cells MGC, esophageal cancer cell Eca-109 and mouse melanoma cell B16 are had significant inhibition growth effect, wherein remarkable to the effect of Eca-109 cell inhibiting, effect during 24h to the half-inhibition concentration (IC of esophageal cancer cell ₅₀) only be 11.2 μ g/mL, and to the IC of normal cell such as Vero cell ₅₀Then be 800 μ g/mL.Observe demonstration by fluorescence colour, behind this albumen effect cancer cells 24h, cell number reduces, karyopyknosis, and dyeing is deepened, and has apoptotic body to form.Detect by LDH is active, find to increase progressively trend, have time-effect relationship along with the prolongation apoptosis rate in PHP treatment time and necrosis rate all present.Illustrate that PHP can be by cancer cell specific induction of apoptosis and to its efficient inhibition.After detecting PHP effect cancer cells, intracellular important apoptosis enzyme caspase-9, caspase-3,7 and the activity of pan-caspase, find when using enzyme inhibitors z-DEVD-fmk (caspase-3,7 inhibitor), when z-LEHD-fmk (caspase-9 inhibitor) and z-VAD-fmk (pan-caspase inhibitor), cell survival rate all has rising.The Caspase enzyme suppresses to have experimental results show that this albumen mainly is by the mitochondrial apoptotic pathway cell death inducing.

Described albumen PHP of the present invention has the active effect that suppresses the HIV-1 ThermoScript II, detect the restraining effect of the PHP of different concns with HIV-1RT test kit (Roche company) to the HIV-1 ThermoScript II, found that inhibiting rate is respectively 9.77%, 16.09%, 32.31%, 49.60%, 63.71% and 69.10% when protein concentration is 2.5,5,10,20,40,60 μ g/mL.Its half-inhibition concentration IC ₅₀Value is 18.5 μ g/mL.The result shows that PHP shows strong restraining effect to the activity of HIV-1 ThermoScript II.

These experiments show that PHP is at anti-food engineering, agricultural disease fungal pathogens biological pesticide preparation and having a good application prospect aspect the protein drug treatment of tumour and HIV virus associated diseases.Preparation, character and activity etc. about PHP do not see that bibliographical information is arranged.

Description of drawings:

Fig. 1 is the SDS-PAGE electrophoresis evaluation figure of PHP separation and purification and with high performance liquid chromatography (HPLC method) purity of protein is identified figure.Accompanying drawing 1A figure is SDS-PAGE electrophoresis detection molecular weight subunit figure, and M is protein standard molecular weight Marker among the figure, and a is that the single subunit band of PHP is 8kDa.B figure is that the employing model is a C18 ZORBAX ODS reversed-phase liquid chromatography post (Agilent) in the accompanying drawing 1, by high performance liquid chromatography (HPLC) method PHP albumen being carried out purity identifies, elutriant A liquid is 100% acetonitrile, B liquid is redistilled water, gradient elution by 5% A liquid through the A of 20min to 95% liquid, flow velocity 1mL/min.The result shows that the PHP sample purity is higher than 95%.

Fig. 2 is PHP apparent molecular weight mensuration figure.The performance liquid chromatographic column that with model is TSK-G4000PWxl detects Herba pegani harmalae PHP molecular weight of albumen, protein standard substance molecular weight: lactoferrin (71kDa), oralbumin (44kDa), carbonic anhydrase (29kDa), beta-lactoglobulin (18kDa), N,O-Diacetylmuramidase (14kDa).The molecular weight standard product use (●) to represent with (◆) expression, PHP.From the standard molecular weight detected result of Fig. 2 as can be known, the apparent molecular weight of PHP is 16kDa.

Fig. 3 is PHP anti-mycotic activity figure.(A): rhizopus Rhizopus stuolonifer; (B): penicillium italicum Penicilliumitalicum; (C): hand over lattice pink mold Alternaria alternata; (D): Pyricularia oryzae Magnaporthe grisea; (E): penicillium digitatum Penicillium degitatum.The PHP sample is dissolved among the pH7.2PBS, carries out the mensuration of plant pathogenic fungi bacteriostatic activity with the filter paper agar diffusion method, with the PBS of sterilization in contrast.Put in 28 ℃ the humidity incubator and cultivate 24h.(a)：60μg?PHP；(b)：30μg?PHP；(c)：PBS。Show that this albumen all has the growth-inhibiting effect to 5 kind of plant pathogenic fungies.

Fig. 4 is thermostability and the optimal pH range detection figure that PHP suppresses fungi.The protein sample PHP of purifying is dissolved in the 0.02mol/L that contains 0.15mol/L NaCl, among the PBS of pH7.2 to 1mg/mL, respectively get 2mL solution 4 ℃ of standing over night immediately after 4 ℃ of differing tempss, 20 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ are handled 30min, adopt the filter paper agar diffusion method to measure variant Temperature Treatment after PHP fungi is handed over the restraining effect of lattice pink mold growth.In addition the protein sample PHP of purifying is dissolved in respectively and returns to pH7.2 after handling 4h in the damping fluid of pH 2,4,6,8,10,12, adopt the filter paper agar diffusion method to measure the restraining effect that the PHP after different pH values are handled hands over lattice pink mold cell to grow to fungi then.Show this albumen below 80 ℃, suppress the activity stabilized of fungi in the pH4-10 scope.

Fig. 5 is the MTT detection figure that PHP suppresses cancer cell multiplication.The PHP (3.125,6.25,12.5,25,50 μ g/mL) that gets different concns detects it respectively to ECa-109 (human esophagus cancer cell), MGC (gastric carcinoma cells), HeLa (human cervical carcinoma cell), the inhibited proliferation of B16 (mouse melanoma cell) and Vero (African green monkey kidney cell).The MTT experimental result shows PHP to MGC, and HeLa, B16 and ECa-109 cell all have the growth of inhibition effect, and be wherein stronger to the ECa-109 cyto-inhibition, the IC of its effect 24h ₅₀Value is about 11.2 μ g/mL.But its to normal cell Vero restraining effect a little less than.

Fig. 6 is that PHP induces the apoptotic Fluirescence observation figure of ECa-109.Left hand view A, C be not for adding the negative control cell aspect graph of PHP, and right part of flg B, D add behind the PHP effect 24h of 11.2 μ g/mL microscopic examination figure as a result.Upper strata figure is inverted microscope observation of cell metamorphosis figure, and lower map is for fluorescent dye liquid Hoechst33258 colour developing back observation of cell apoptosis figure as a result under fluorescent microscope.Magnification is 100 times.The PHP treatment group is compared with control group, and cell number reduces, karyopyknosis, and dyeing is deepened, and has apoptotic body to form.

Fig. 7 is the LDH detection figure that PHP handles the ECa-109 cell.ECa-109 cell inoculation 24 well culture plates in vegetative period of taking the logarithm, after if blank group, negative control group, experimental group (dosing hole, release aperture) fully are adherent, add after 11.2 μ g/mL PHP act on 0h, 12h, 24h, 36h, 48h respectively, detect lactate dehydrogenase activity in each hole with the LDH detection kit, measure the OD value in the 450nm place, return to zero with the blank group.Calculate apoptosis rate and necrosis rate.

Apoptosis rate %=LDHp/ (LDHp+LDHi+LDHe) * 100% necrosis rate %=LDHe/ (LDHp+LDHi+LDHe) * 100%

As seen from the figure, along with the prolongation in albumen PHP treatment time, apoptosis rate and necrosis rate all present and increase progressively trend, and apoptosis rate is greater than necrosis rate.Illustrate that protein induced ECa-109 apoptosis of PHP and necrosis have time-effect relationship.After 24 hours, apoptosis and downright bad cell are respectively 38% and 9%, show after necrosis is also handled the ECa-109 cell by PHP to cause.Along with the increase of PHP concentration, apoptosis and downright bad cell also get more and more, but the apoptotic cells number is more much more than the cell number of necrosis, shows that it may be apoptosis rather than necrosis that PHP induces the major cause of ECa-109 necrocytosis.Fig. 8 is the action diagram of PHP to the caspase enzyme inhibitors.The ECa-109 cell is adding and is not adding z-DEVD-fmk (caspase-3,7 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor), after hatching 1h in the substratum of z-AEVD-fmk (caspase-10 inhibitor) and z-VAD-fmk (pan-caspase inhibitor), add and detect each hole light absorption value with mtt assay after PHP handles 24h, relatively calculate cell survival rate with negative control.Experimental result is the mean value of three independent experiment data.As seen from the figure, to caspase-9,3,7 and the influence of general caspase extremely significantly (P＜0.05), cell survival rate is respectively: 75.76%, 76.06% and 82.93%.

Fig. 9 is the detection that the PHP of different concns suppresses the HIV-1 reverse transcriptase activity.Use non-radioactive reverse transcriptase ELISA test kit (Roche company) to detect the restraining effect of the PHP of different concns to HIV-1RT, with concentration be the protein dissolution of 2.5,5,10,20,40,60 μ g/mL in DMSO, each concentration is established 3 multiple holes.Take out in the test kit and react bar, every hole drips 20 μ L HIV-1 RT solution, 20 μ L are dissolved in the albumen of DMSO, the reaction buffer that 20 μ L contain template and Nucleotide.Positive control hole and not protein-contg negative control hole are set.Put 37 ℃ of incubation 1h.Wash 5 times, every hole adds the anti-DIG-POD antibody of 200 μ L working fluid.37 ℃ of incubation 1h wash 5 times.Every hole adds the ABTS substrate reactions liquid of 200 μ L.Room temperature reaction 15-30min, microplate reader is measured OD value (405nm/490nm).And relatively draw inhibiting rate and IC with negative control group ₅₀Value.

Inhibiting rate (%)=1-(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) * 100%

Found that inhibiting rate is respectively 9.77%, 16.09%, 32.31%, 49.60%, 63.71% and 69.10% when 2.5,5,10,20,40 and 60 μ g/mL, its half-inhibition concentration IC ₅₀Value is 18.5 μ g/mL.The result shows that PHP shows stronger restraining effect to the HIV-1RT activity.

Adopt the mode of specific embodiment to explain the present invention below, but the present invention is not limited to embodiment.

Embodiment:

Embodiment 1: the preparation of Herba pegani harmalae fat transfer protein PHP

(1) crude product preparation: with the seed of peganum harmala is material, it is ground into powder, add the 0.02mol/L PBS phosphoric acid buffer of pH7.2,4 ℃ of stirred overnight extractings, collect supernatant liquor behind the low-temperature centrifugation, slowly adding ammonium sulfate in supernatant liquor, to transfer to saturation ratio be 50%, leaves standstill 4h at 4 ℃, centrifugal collection supernatant liquor is abandoned precipitation.Continuation slowly adds ammonium sulfate in supernatant liquor to transfer to saturation ratio be 70%, 4 ℃ leave standstill 4h after, centrifugal collecting precipitation, and being dissolved in the small amounts of phosphoric acid damping fluid, the dialysis desalination, lyophilize gets the albumen crude product;

(2) cation-exchange chromatography separation and purification: adopt Resource S cationic exchange coloum, the raw protein that the first step is obtained carries out chromatography, the unconjugated albumen of 0.02mol/LPBS phosphoric acid buffer elder generation's flush away with pH 7.2, carry out gradient elution with the phosphate buffered saline buffer that contains 0.1-1mol/L NaCl then, collection has antibiotic and component anti-tumor activity after the detection of active, preserves standby after fully dialysing with phosphate buffered saline buffer;

(3) molecular sieve purification

Molecular sieve is Superdex 75 gel permeation chromatography posts, the ion-exchange sample that previous step is obtained carries out chromatography again, 0.02mol/LPBS phosphoric acid buffer wash-out with the pH 7.2 that contains 0.15mol/L NaCl, flow velocity is 0.5ml/min, substep is collected, collection has antibiotic and component anti-tumor activity after the detection of active, the dialysis desalination, and lyophilize promptly obtains the Herba pegani harmalae fat transfer protein of purifying.

Embodiment 2: the part physico-chemical property research of Herba pegani harmalae fat transfer protein

(1) purity and molecular weight detection

The PHP albumen that obtains among the embodiment 1 is measured with Tricine-SDS-PAGE electrophoresis (gel strength 12%), and experimental result is shown in A figure in the accompanying drawing 1, and the proteic molecular weight subunit of PHP is about 8kDa.Adopting model is that C18ZORBAX ODS reversed-phase liquid chromatography post (Agilent) carries out high performance liquid chromatography (HPLC) evaluation albumen PHP purity, and the sample purity result shows that the PHP sample purity is higher than 95% (B figure in the accompanying drawing 1).The performance liquid chromatographic column (HPLC) that with model is TSK-G4000 PWxl detects Herba pegani harmalae PHP molecular weight of albumen.Adopt 25 ℃ of column temperatures, the detection wavelength is 280nm, and moving phase is 0.05mol/LPBS (pH7.2), and solvent is 0.05mol/L PBS (pH7.2), and sample size is 20 μ L, and flow velocity is 0.8mL/min.Take off the row standard substance respectively and make the typical curve equation: lactoferrin 71kDa, oralbumin 44kDa, carbonic anhydrase 29kDa, beta lactoglobulin 18kDa, N,O-Diacetylmuramidase 14kDa.Analytical procedure adopts normalization method.From the standard molecular weight detected result of accompanying drawing 2 as can be known, PHP albumen apparent molecular weight is 16kDa.It under the native state homodimer that two subunits are formed.

(2) isoelectrofocusing (IEF-PAGE) detects:

The PHP sample of purifying is added on the adhesive tape, uses the disk electrophoresis instrument, with about 200V constant voltage electrophoresis 1h, about 400V electrophoresis 2h, stop electrophoresis more earlier.Take out adhesive tape, be soaked in fixing about 0.5h in 10% trichoroacetic acid(TCA), again adhesive tape is cleaned with distilled water, dye decolouring.Measurement standard albumen and sample protein focus on the distance of band apart from the adhesive tape lower end, and do typical curve.The iso-electric point pI that finds protein sample from typical curve is 8.4.

(3) thermostability experiment:

The protein sample PHP of purifying is dissolved among the 0.02mol/L PBS of the pH 7.2 that contains 0.15mol/L NaCl to 1mg/mL, respectively get 2mL solution 4 ℃ of standing over night immediately after 4 ℃ of differing tempss, 20 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ are handled 30min, adopt the filter paper agar diffusion method to measure treatment of different temperature after PHP fungi is handed over the restraining effect of lattice pink mold growth.Experimental result shows that PHP is activity stabilized below 80 ℃ shown in A figure in the accompanying drawing 4.

(4) ph stability experiment:

The protein sample PHP of purifying is dissolved in respectively returns to pH7.2 after handling 4h in the damping fluid of pH 2,4,6,8,10,12, adopt the filter paper agar diffusion method to measure the restraining effect that the PHP after different pH values are handled hands over lattice pink mold cell to grow to fungi then.Experimental result shows that PHP is active more stable in the pH4-10 scope shown in B figure in the accompanying drawing 4.

(5) N terminal amino acid order-checking experiment:

Adopt the EDMAN edman degradation Edman, on Applied Biosystems Procise-PROCISE, carry out the mensuration that N terminal amino acid residue is formed.The N terminal amino acid sequence is: ITCPQVTQSLAPCVPYLISG (different bright-Su-half Guang-dried meat-glutamine-figured silk fabrics-Su-glutamine-Si-bright-third-dried meat-half Guang-figured silk fabrics-dried meat-junket-bright-different is bright-Si-glycine, Ile-Thr-Cys-Pro-Gln-Val--Thr-Gln-Ser-Leu-Ala-Pro-Cys-Val-Pro-Tyr-Leu-Ile-Ser-Gly).20 amino acid residue sequences that the present invention is obtained, amino acid residue sequence with Blast and ncbi database login carries out the homology comparison, the fat transfer protein of finding it and other plant has homology, wherein the N terminal sequence homology with sweet orange LTP is up to 75%, next is that root of Beijing euphorbia LTP is 70%, rape LTP is 70%, and European Chinese chestnut LTP is 60%, illustrates that it is a kind of new fat transfer protein.

Embodiment 3: Herba pegani harmalae fat transfer protein PHP is to the restraining effect of fungi

Can cause the 5 kind of plant pathogenic fungies that fruit rots and grain goes mouldy, as rhizopus Rhizopus stuolonifer, penicillium italicum Penicillium italicum, hand over lattice pink mold Alternaria alternata, Pyricularia oryzae Magnaporthe grisea and penicillium digitatum Penicillium degitatum are inoculated on the potato culture respectively, cultivate the 48h activation, obtain the activatory novel bacterial for 28 ℃.Get 1mL sterile distilled water flushing media surface, fungal spore is come off and make bacteria suspension.Contain the preparation of bacterium flat board: will be autoclaving and be cooled to 50 ℃-60 ℃ substratum 20mL and be poured in the plate that diameter is 10cm, to be cooled, draw 200 μ L bacteria suspensions and drip to dull and stereotyped central authorities, make and contain the bacterium flat board.When treating the about 2-3cm of disc diameter that mycelia outwards spreads, at the filter paper of placing the bacterium of going out of 6mm diameter from the about 0.5cm in mycelia edge place, after cultivating 24h, on filter paper, drip testing protein respectively, put in 28 ℃ the humidity incubator and cultivate 72h, observe the bacteriostatic action of the PHP of record different concns also, in contrast with the PBS of sterilization to plant pathogenic fungi.Experimental result sees shown in the accompanying drawing 3 that PHP all has the growth-inhibiting effect to five kinds of plant pathogenic fungis that can cause that fruit rots and grain goes mouldy for examination.

Respectively with 1 * 10 ⁴Five kinds of individual/mL supply the examination fungal spore to be inoculated in 96 well culture plates, Yu Kongzhong adds the PHP albumen that final concentration is respectively 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL respectively, control group only adds PBS, respectively establish 3 multiple holes, after placing 28 ℃ humidity incubator to cultivate 24h, under the 595nm wavelength, survey the OD value, calculate inhibiting rate and half-inhibition concentration (IC ₅₀).

Table 1PHP albumen is to the inhibition situation of five kinds of fungies

Bacterial classification	IC 50Value (μ g/mL)	Active
			Penicillium italicum (Penicillium italicum)	300	++
Penicillium digitatum (Penicillium degitatum)	600	+
			Head mold germ (Rhizopus stuolonifer)	135	++
Hand over lattice pink molds (Alternaria alternata)	24	+++
			Pyricularia oryzae (Magnaporthe grisea)	195	++

Embodiment 4: Herba pegani harmalae fat transfer protein PHP is to the vitro inhibition effect of cancer cells

This example adopts mtt assay to detect the vitro inhibition effect of PHP to cancer cells.The ECa-109 that takes the logarithm vegetative period (human esophagus cancer cell), MGC (gastric carcinoma cells), HeLa (human cervical carcinoma cell), B16 (mouse melanoma cell) and Vero (African green monkey kidney cell), be resuspended in the DMEM substratum that contains 10%FBS, through trysinization with 5 * 10 ⁴The final concentration of individual/mL is inoculated sub 96 well culture plates, and every hole 100 μ L add PBS solution edge sealing with each hole around 96 orifice plates.Place 37 ℃, 5%CO ₂The saturated humidity incubator in cultivate 24h and make cell attachment.Sop up substratum, add the solution (filtration sterilization in advance) that is respectively 3.125,6.25,12.5,25 and 50 μ g/mL with the PHP concentration of substratum preparation, every hole 100 μ L, the every hole of blank group adds 200 μ L perfect mediums.Behind the effect 48h, remove supernatant, every hole adds MTT and the 80 μ L substratum that 20 μ L concentration are 0.5mg/mL and continues to hatch 4h, as seen black-and-blue first a ceremonial jade-ladle, used in libation particle, the careful suction goes supernatant, every hole to add 150 μ L dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on the shaking table, crystallisate is fully dissolved.Under the 570nm wavelength, survey the OD value, and calculate inhibitory rate of cell growth and half-inhibition concentration IC ₅₀Value.With the protein concentration is transverse axis, and inhibiting rate is the longitudinal axis, curve plotting.By 4 multiple holes of each concentration, each experiment repeats 3 times, and averaging is net result.Inhibitory rate of cell growth (%)=1-(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) * 100%.The measurement result of various cells is seen Fig. 5 and table 2.

Table 2PHP albumen is to the half-inhibition concentration (IC of five kinds of cancer cells ₅₀) mensuration

Cell strain	The cell source	IC 50(μg/mL)
			Vero	African green monkey kidney cell	800
MGC	Gastric carcinoma cells	50
			HeLa	Human cervical carcinoma cell	43.8
B16	The mouse melanoma cell	23.5
			ECa-109	Human esophagus cancer cell	11.2

PHP is to MGC as can be seen by showing, and HeLa, B16 and ECa-109 cell all have the growth of inhibition effect, and be wherein stronger to the ECa-109 cyto-inhibition, its IC during effect 24h ₅₀Value is about 11.2 μ g/mL.But its to normal cell Vero restraining effect a little less than.

Embodiment 5:PHP albumen is observed the apoptosis induction of ECa-109 cell

This example adopt can labeled cell nuclear fluorescent reagent Hoechst33258 observe the influence situation of PHP albumen to the metamorphosis of ECa-109 cell.The cell of taking the logarithm vegetative period is resuspended in the DMED substratum that contains 10%FBS, with 2 * 10 through trysinization ⁵The final concentration of individual/mL is inoculated in 6 well culture plates (placing the cover glass of a sterilization), every hole 2ml, and adding concentration after creep plate is adherent respectively is the PHP effect 24h of 50 μ g/mL, PBS gives a baby a bath on the third day after its birth inferior.With stationary liquid (methyl alcohol: after acetate=3: 1) 1ml fixes 20 minutes, PBS gives a baby a bath on the third day after its birth inferior, add 1ml Hoechst33258 staining fluid (final concentration is 5 μ g/mL), 37 ℃ of lucifuges were hatched 30 minutes, PBS gives a baby a bath on the third day after its birth inferior again, observation of cell apoptosis result under fluorescent microscope, a random observation N visual field (100 times) also take pictures, and the result is as shown in Figure 6.

Embodiment 6:LDH detects apoptosis and downright bad experiment

The cell of taking the logarithm vegetative period is resuspended in the DMED substratum that contains 10%FBS, with 3 * 10 after trysinization ⁴The final concentration of individual/mL is inoculated sub 24 well culture plates, every hole 500 μ L, establishing the adherent back of blank group, negative control group, experimental group (dosing hole, release aperture) fully, to add concentration be after the PHP1 of 11.2 μ g/ml acts on 0h, 12h, 24h, 36h, 48h respectively.Release aperture adds the complete lysing cell of 0.2%Triton fully.Each hole substratum supernatant is moved in the 1.5mL centrifuge tube, 4 ℃, 3000rpm/min, centrifugal 2min.Obtain supernatant e and sedimentation cell p.Each hole attached cell trysinization is resuspended in the DMED substratum that contains 10%FBS, 3000rpm/min, centrifugal 2min obtains sedimentation cell i.Detect cytotoxicity with the LDH detection kit, measure the OD value in the 450nm place, return to zero with the blank group.

Apoptosis rate %=LDHp/ (LDHp+LDHi+LDHe) * 100% necrosis rate %=LDHe/ (LDHp+LDHi+LDHe) * 100%

Experimental result as shown in Figure 7.

The experiment of embodiment 7:Caspase inhibitors of apoptosis

The cell of taking the logarithm vegetative period is resuspended in the DMED substratum that contains 10%FBS, with 5 * 10 through trysinization ⁴The final concentration of individual/mL is inoculated sub 96 well culture plates, and every hole 100 μ L add the PBS edge sealing with each hole around 96 orifice plates.The rightmost side one row are made as the blank group, add the acellular serum DMEM substratum that has.Place 37 ℃, 5%CO ₂The saturated humidity incubator in cultivate 24h and make cell attachment.Sop up substratum, experimental group adds respectively and contains z-DEVD-fmk (caspase-3,7 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor), z-AEVD-fmk (caspase-10 inhibitor) and z-VAD-fmk (pan-caspase inhibitor) have blood serum medium 100 μ L, after hatching 1h, add the blood serum medium that has that contains PHP, make every hole final volume 200 μ L, other establishes Caspase enzyme inhibitors control group, add respectively and contain the Caspase-8 inhibitor, the Caspase-9 inhibitor, the Caspase-10 inhibitor, Caspase-3,7 inhibitor, the pan-Caspase inhibitor blood serum medium 200 μ L are arranged, fat transfer protein control group add contain PLTP blood serum medium 200 μ L are arranged.The rightmost side one row are made as the blank group, add the acellular serum DMEM substratum that has.Place 37 ℃, the every hole of blank group to add 200 μ L perfect mediums, handle 24h, experimental result as shown in Figure 8.The Caspase enzyme suppresses experiment and shows z-DEVD-fmk (caspase-3,7 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor), the equal pair cell death of z-AEVD-fmk (caspase-10 inhibitor) andz-VAD-fmk (pan-caspase inhibitor) has influence in various degree.Wherein to caspase-9,3,7 and the influence of general caspase extremely significantly (P＜0.05), cell survival rate is respectively: 75.76%, 76.06% and 82.93%.

Embodiment 8:HIV-1 ThermoScript II suppresses experiment

The PHP of different concns suppresses the detection of HIV-1 reverse transcriptase activity.Use non-radioactive reverse transcriptase ELISA test kit (Roche company) to detect the restraining effect of the PHP of different concns to HIV-1 RT, with concentration be the protein dissolution of 2.5,5,10,20,40,60 μ g/mL in DMSO, each concentration is established 3 multiple holes.Take out in the test kit and react bar, every hole drips 20 μ L HIV-1 RT solution, 20 μ L are dissolved in the albumen of DMSO, the reaction buffer that 20 μ L contain template and Nucleotide.Positive control hole and not protein-contg negative control hole are set.Put 37 ℃ of incubation 1h.After washing 5 times, every hole adds the anti-DIG-POD antibody of 200 μ L working fluid.37 ℃ of incubation 1h wash 5 times.Every hole adds the ABTS substrate reactions liquid of 200 μ L.Room temperature reaction 15-30min, microplate reader is measured OD value (405nm/490nm).And relatively draw inhibiting rate and IC with negative control group ₅₀Value.

Inhibiting rate (%)=1-(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) * 100%

Found that inhibiting rate is respectively 9.77%, 16.09%, 32.31%, 49.60%, 63.71%, 69.10% when 2.5,5,10,20,40,60 μ g/mL, its half-inhibition concentration IC ₅₀Value is 18.5 μ g/mL.The result shows that PHP shows stronger inhibition to HIV-1 RT activity.

Sequence table

SEQUENCE?LISTING

＜110〉Xinjiang University

＜120〉a kind of Herba pegani harmalae fat transfer protein and its production and application

<130>1

<160>1

<170>PatentIn?version?3.3

<210>1

<211>20

<212>PRT

＜213〉Herba pegani harmalae (Peganum harmala L.)

<220>

＜221〉aminoacid sequence of the N of a kind of Herba pegani harmalae fat transfer protein PHP end

<222>(1)..(20)

<300>

<313>(1)..(20)

<400>1

Ile?Thr?Cys?Pro?Gln?Val?Thr?Gln?Ser?Leu?Ala?Pro?Cys?Val?Pro?Tyr

1 5 10 15

Leu?Ile?Ser?Gly

20

 

Claims (10)

1. Herba pegani harmalae fat transfer protein PHP is characterized in that: the N end contains the aminoacid sequence ITCPQVTQSLAPCVPYLISG shown in the SEQID NO:1.

2. albumen according to claim 1 is characterized in that: described dietary protein origin is in medicinal plant Herba pegani harmalae (Peganum harmala L.).

3. albumen according to claim 1 is characterized in that: be the homodimer that two subunits are formed under the native state, molecular weight is 16kDa, and the molecular weight of single subunit is about 8kDa, is that a kind of iso-electric point pI is 8.4 basic protein.

4. preparation may further comprise the steps as proteic method as described in arbitrary one of the claim 1～3:

(1) crude product preparation: with the seed of peganum harmala is material, after its pulverizing, adding pH is the PBS phosphoric acid buffer of the neutrality of 7.0-7.5 to weak basic condition, low temperature stirs under the extracting and spends the night, collect supernatant liquor behind the low-temperature centrifugation, slowly adding ammonium sulfate in supernatant liquor, to transfer to saturation ratio be between the 30%-50%, stand at low temperature 4h, centrifugal collection supernatant liquor is abandoned precipitation; Continuation slowly adds ammonium sulfate in supernatant liquor to transfer to saturation ratio be between the 60%-80%, once more behind the stand at low temperature 4h, and centrifugal collecting precipitation, and be dissolved in the small amounts of phosphoric acid damping fluid, the dialysis desalination, lyophilize gets the albumen crude product;

(2) cation-exchange chromatography separation and purification: adopt Resource S or the cationic exchange coloum suitable with its character, the raw protein that above-mentioned steps (1) is obtained carries out chromatography, be the unconjugated albumen of PBS phosphoric acid buffer elder generation flush away of 0.02-0.05mol/L with proper concn behind the last sample, carry out gradient elution with the phosphate buffered saline buffer that contains 0.1-1mol/L NaCl again, collection has antibiotic and component anti-tumor activity after the detection of active, preserves standby after fully dialysing with phosphate buffered saline buffer;

(3) molecular sieve purification: Superdex 75 type molecular sieve columns on the sample that above-mentioned steps (2) is obtained, with the concentration that contains finite concentration NaCl is the PBS phosphoric acid buffer wash-out of 0.02-0.05mol/L, substep is collected, collecting after the detection of active has antibiotic and component anti-tumor activity, the dialysis desalination, lyophilize promptly obtains the pure product of Herba pegani harmalae fat transfer protein PHP of purifying;

(4) the Herba pegani harmalae fat transfer protein PHP of the purifying that obtains of above-mentioned steps (3) is a kind of new fat transfer protein, that this albumen has is antimycotic, suppress tumor promotion and suppress the effect of HIV-1 reverse transcriptase activity, antimycotic field of biological pesticide and having a good application prospect aspect tumour and the antiviral protein drug treatment on the agent of food fruit and vegetables corrosion protection, agricultural.

5. the arbitrary described albumen of claim 1～4 is characterized in that: described albumen has the activity that suppresses fungal growth, the purposes in the biological pesticide of preparation food fruit and vegetables corrosion protection agent and anti-agricultural disease fungal pathogens.

6. purposes according to claim 5 is characterized in that: described fungi is for handing over lattice pink molds (Alternaria alternata), penicillium digitatum (Penicillium degitatum), head mold germ (Rhizopus stuolonifer), Pyricularia oryzae (Magnaporthe grisea) and penicillium italicum 5 kind of plant pathogenic fungies such as (Penicillium italicum).

7. the arbitrary described albumen of claim 1～4 is characterized in that: described albumen has the effect that suppresses tumor cell proliferation and inducing apoptosis of tumour cell, the purposes in preparation medicine for treating tumor thing.

8. purposes according to claim 7 is characterized in that: described tumour is cervical cancer, cancer of the stomach, the esophageal carcinoma and melanoma.

9. the arbitrary described albumen of claim 1～4 is characterized in that: described albumen has the effect that suppresses the HIV-1 reverse transcriptase activity, treats purposes in the medicine of HIV disease at the protein drug of preparation anti HIV-1 virus.

10. method according to claim 4 is characterized in that the preparation condition of the Herba pegani harmalae fat transfer protein optimized is:

(1) the crude product preparation condition of You Huaing is: be material with the seed of peganum harmala, after its pulverizing, add 0.02mol/L, the PBS phosphoric acid buffer of pH 7.2, supernatant liquor is collected in 4 ℃ of stirred overnight extractings behind the low-temperature centrifugation, slowly adding ammonium sulfate in supernatant liquor, to transfer to saturation ratio be 50%, leave standstill 4h at 4 ℃, centrifugal collection supernatant liquor is abandoned precipitation; Continuation slowly adds ammonium sulfate in supernatant liquor to transfer to saturation ratio be 70%, 4 ℃ leave standstill 4h after, centrifugal collecting precipitation, and being dissolved in the small amounts of phosphoric acid damping fluid, the dialysis desalination, lyophilize gets the albumen crude product.

(2) the cation-exchange chromatography separation and purification condition of You Huaing is: adopt Resource S cationic exchange coloum, resulting albumen crude product in the above-mentioned steps (1) is carried out chromatography, use 0.02mol/L behind the last sample, the unconjugated albumen of PBS phosphoric acid buffer elder generation's flush away of pH 7.2, carry out gradient elution with the phosphate buffered saline buffer that contains 0.1-1mol/L NaCl again, collection has antibiotic and component anti-tumor activity after the detection of active, preserves standby after fully dialysing with phosphate buffered saline buffer.

(3) the molecular sieve purification condition of You Huaing is: molecular sieve is Superdex 75 type posts, with the molecular sieve column on the sample after the ion exchange treatment that obtains in the above-mentioned steps (2), with the 0.02mol/L that contains 0.15mol/L NaCl, the PBS phosphoric acid buffer wash-out of pH 7.2, substep is collected, collection has antibiotic and component anti-tumor activity after the detection of active, the dialysis desalination, and lyophilize promptly obtains the pure product of Herba pegani harmalae fat transfer protein PHP of purifying.

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