CN103450351B - Long-acting polypeptide for confronting Newcastle disease virus infection and modifier thereof - Google Patents
Long-acting polypeptide for confronting Newcastle disease virus infection and modifier thereof Download PDFInfo
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Abstract
The invention discloses a polypeptide for confronting the Newcastle disease virus infection and a modifier thereof. The amino acid sequence of the polypeptide is the sequence 1 in the sequence table; the modifier is prepared by modifying the end of the amino group of the polypeptide with cholesterol, the modification is that the first amino acid in the sequence 1 in the sequence table is connected to the cholesterol. The polypeptide modifier provided by the invention can high-effectively prevent viruses from invading the host cells, has a prominent effect on inhibiting virus activity (the cellular level is 12 nM, the chick embryo level is 0.1 mg, and the chicken living body level is 0.5 mg), besides, only if the polypeptide or polypeptide modifier is intramuscularly injected in a time range of two days early to two day later of the Newcastle disease virus infection, the high pathogenicity virus infection which can invade into the hemato encephalic barrier of poultry can be effectively prohibited. The polypeptide for confronting the Newcastle disease virus infection and a modifier thereof provide an important basis for applications of polypeptide preparations and have an important application prospect and an innovative meaning in the field of animal medicine.
Description
Technical field
The invention belongs to biological technical field, relate to polypeptide and the modifier thereof of the infection of a kind of anti-new castle disease virus.
Background technology
The current livestock industry output value accounts for 32% of the National Agricultural gross output value, plays more and more important effect in national economy, and wherein the control of livestock and poultry transmissible disease is in animal and veterinary scientific research Zhong Ju top priority.Avian pneumo-encephalitis virus is serious threat aviculture " the first killer ", the multi-pathogenesis respiratory infectious disease that the pathogenic agent multiple infection being master by Avian pneumo-encephalitis virus causes, the principal element causing poultry farming financial loss at present, the financial loss that China is only caused by newcastle disease is often every year more than 2,000,000,000 yuan according to Ministry of Agriculture's working year report in 2010! The associated loss caused thus is then difficult to estimate.The host range of newcastle disease virus infection also has the trend constantly expanded in recent years, similar to respiratory infectious disease bird flu, this virus variation and rate of evolution are very fast, not only can infect more than 200 kind of aquatic bird, and across kind of an infection Mammals, Human health effects could be can not be ignored! Develop antagonism this virus infection high-efficiency preparation, be now extremely urgent wait solve science proposition.
This seminar proves, at Avian pneumo-encephalitis virus (Newcastle disease virus, NDV) in phagocytic process, heptad repeat (heptad repeat) HR1 and the HR2 of fusion glycoprotein is folded to form six dimeric structure (6-Helix) mutually, the bilayer lipid membrane of virus envelope and host cell is furthered, causes that irreversible film fusion process occurs; The HR2 polypeptide of external synthesis can pass through its part of competition binding HR1 specifically, with the formation of blocking virus self 6-Helix structure, suppresses the film fusion process of fusion glycoprotein mediation thus.Galdiero etc. adopt transgenation strategy to modify being derived from the amino of human Aids virus HR2 Region Polypeptides or a C-terminal 2-4 Methionin or L-glutamic acid, obtain the more obvious novel polypeptide of antiviral invasion activity; The b of HR2 spiral hydrophilic surface, c, f and g amino acids is replaced into Methionin or L-glutamic acid by Nishikawa etc., and result shows that salt bridge effect can strengthen the spiralization trend of polypeptide.
Protein/polypeptide etc. being combined (as chemical crosslink technique) with biodegradable polymer soluble compound by certain chemical means and becoming mixture, the new premium properties of polypeptide drug (as reduced the clearance rate of biotech drug) can being given.Usually macromolecular compound is selected to have dextran, white protein, polyoxyethylene glycol (PEG) etc.Wherein, Pegylation to be connected to by peg molecule on protein molecular to extend its transformation period, reaches corresponding biological effect, due to the attraction of water molecules by less administration number of times, its hydrokinetics size raises more, can reduce the removing transformation period of medicine.
Summary of the invention
The object of this invention is to provide the polypeptide that a kind of anti-new castle disease virus infects.
The aminoacid sequence of polypeptide provided by the present invention is sequence 1 in sequence table.
Wherein, sequence 1 is made up of 33 amino acid.
Another object of the present invention is to provide the peptide modified thing that a kind of anti-new castle disease virus infects.
Peptide modified thing provided by the present invention is the product carrying out modifying gained by cholesterol to described polypeptide; The aminoacid sequence of described polypeptide is sequence 1 in sequence table.
In an embodiment of the present invention, first amino acid that described modification is specially sequence 1 in sequence table connects described cholesterol, what adopted is the conventional modifying method that cholesteryl chloroformate and polypeptide first amino react, and namely the chlorion (CL) of cholesteryl chloroformate is substituted and then connects with polypeptide amino.
Described polypeptide (sequence 1) or described peptide modified thing also belong to protection scope of the present invention being prepared as follows the application in arbitrary product:
1) anti-new castle disease virus product;
2) treat and/or prevent by the product of newcastle disease virus infection associated diseases.
Another object of the present invention is to provide a kind of product of anti-new castle disease virus, or treats and/or prevents by the product of newcastle disease virus infection associated diseases.
The activeconstituents of product provided by the present invention is described polypeptide or described peptide modified thing.
In the present invention, described Avian pneumo-encephalitis virus is newcastle disease virus, is specially F48E9 strain, La Sota strain, K strain or R strain.
The preparation method of described modifier also belongs to protection scope of the present invention.
The preparation method of modifier provided by the present invention comprises the following steps: Preparation of amino acid sequence is the polypeptide of sequence 1 in sequence table, connects cholesterol, obtain described modifier by first of sequence 1 amino acid.
The encoding gene of described polypeptide (sequence 1), or recombinant vectors, reconstitution cell, recombinant bacterium or the recombinant virus containing described encoding gene also belongs to protection scope of the present invention.
The present invention carries out genetic modification and modification on the basis to togavirus fusion glycoprotein heptad repeat region domain structure and functional study, obtain the polypeptide shown in sequence 1 and modifier thereof in sequence table, this polypeptide cholesterol modifier can suppress poisoning intrusion host cell efficiently, in antiviral activity, (cell levels is 12nM unit to successful, chicken embryo level is 0.1mg unit, chicken live body level is 0.5mg unit), and intramuscular injection in 2 days before and after newcastle disease virus infection, all effectively can resist this highly pathogenic virus infection of invading bird hemato encephalic barrier.Polypeptide drug possess dosage little, not easily produce resistance, and outstanding advantage such as toxic side effect low grade, and modify the antiviral time lengthening of rear polypeptide, the application for polypeptide class preparation provides important evidence, has important application prospect and innovative significance in field of animal medicine.
Accompanying drawing explanation
Fig. 1 is heptad repeat region (heptad repeat, HR) each site (a-g site) relative tertiary location schematic diagram.A left side is side elevational view, and the right side is vertical view.Wherein, a site is the most important critical area that alpha-helix is formed, and mostly is leucine (Leu, L) and Isoleucine (Ile, I); D, e site is the secondary critical area that alpha-helix is formed, Yi Shu protein binding district; B, c, f, g site mostly is polare Aminosaeren, is water-soluble region.
Fig. 2 is by polypeptide each under 12nM final concentration is on the cytopathogenic impact of NDV virus infection.Wherein, A represents polypeptide shown in sequence 1, and B represents the cholesterol modifier of polypeptide, and C represents the polypeptide of contrast shown in sequence 2.In A-C, 1 all represents that virus and polypeptide add cell jointly; 2 all represent that virus adds cell after mixing 1 hour with polypeptide 37 DEG C; 3 all represent and first add virus, 37 DEG C foster 45 minutes after, then add polypeptide; 4 all represent that first adding polypeptide mixes 30 minutes in 4 DEG C, and then infect virus.
Fig. 3 is in the experiment of chicken embryo, inoculation NDV virus strain F48E9(200 TCID
50, namely virus stock solution used 1ml carries out 10
6100 μ L virus liquids are got after dilution) and 0.1mg polypeptide (0.25mM, 100 μ l), chicken embryo pathology situation analysis caused by Different periods virus infection.Wherein, wherein, A represents polypeptide shown in sequence 1; B represents the cholesterol modifier of polypeptide; C represents the polypeptide of contrast shown in sequence 2.
Fig. 4 is in chicken experiment in vivo, inoculation NDV virus strain F48E9 and 0.5mg polypeptide (1.25mM, 100 μ l), and viral different extent of dilution infects caused chicken body pathology situation analysis.Wherein, A represents the cholesterol modifier of polypeptide; B represents polypeptide shown in sequence 1; C represents the polypeptide of contrast shown in sequence 2.
Fig. 5 is in chicken experiment in vivo, inoculation NDV virus strain F48E9(200 TCID
50) and 0.5mg polypeptide (1.25mM, 100 μ l), Different periods (my god) virus infection is on the impact of chicken body survival rate.Wherein, A represents the front cholesterol modifier adding polypeptide for 1 day of virus infection; B represents the front cholesterol modifier adding polypeptide for 2 days of virus infection; C represents the cholesterol modifier that virus infection adds polypeptide simultaneously; The cholesterol modifier of polypeptide within 1 day, is added after D represents virus infection; The cholesterol modifier of polypeptide within 2 days, is added after E represents virus infection; F represents virus infection and adds the polypeptide of contrast shown in sequence 2 simultaneously.
Fig. 6 is cholesteryl chloroformate structure, and molecular formula is C
28h
45clO
2, molecular weight is 449.11.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In the present invention, Avian pneumo-encephalitis virus used is newcastle disease virus (NDV) strain F48E9, standard weak malicious La Sota strain and isolated strain K and R, and all purchased from China Veterinary Drugs Supervisory Inst., catalog number is strain full name.
9-10 day instar chicken embryo: Beijing Cimmeria company, catalog number is " φ SPF chicken embryo ".
4 week age chicken: Beijing Cimmeria company, catalog number is " φ SPF chicken ".
Term involved in the present invention:
Cytopathy (cytopathic effect, CPE): be cytopathic effect, refers to that viruses into tissues culturing cell infects the cytopathy of rear generation, utilizes this kind of pathology effect to carry out Viral Quantification.Virus infection forms the common synplasm of cytopathy (namely multiple cell aggregation forms the maxicell of multinuclear together) and plaque (cell detachment formation plaque) two kinds.
Pathology fusion rate: form the ratio that plasmodial sick cell accounts for all cells.Form giant cell after synplasm and multiple cell membrane fusion, a lot of nucleus in the inside is crowded together.
IC
50: in the antiviral inhibition test of albumen, commonly use IC
50, its ratio (i.e. pathology fusion rate) accounting for all cells for sick cell (as synplasm) for 50% time albumen corresponding to concentration, i.e. 50%inhibition constraction, IC
50.
The preparation of embodiment 1, polypeptide and modifier thereof
According to Avian pneumo-encephalitis virus (NDV) F48E9 strain fusion glycoprotein HR2 region, (aminoacid sequence is as shown in sequence in sequence table 3,467-497 position corresponding to the aminoacid sequence of GenBank No.:AF079172) higher structure (each site of HR (a-g site) relative tertiary location schematic diagram as shown in Figure 1), amino acid mutation and restructuring are carried out to it, namely first keeps a site to be leucine or Isoleucine; Hydrophilic site b, c, f, g intersection is sported Charged acids (as Methionin K and L-glutamic acid E), simultaneously by the N-terminal of new improvement on synthesis with two continuous print Methionins (KK), C-terminal carries out modifying to maintain charge balance with two continuous print L-glutamic acid (EE); Again 484 amino acids of protein binding site d are sported Val by Ser, through software prediction (http://www.ch.embnet.org/software/helix form.html), this sudden change can be urged alpha-helix and be formed trend.The polypeptide shown in sequence 1 in sequence table is obtained after said mutation with restructuring.Contrast polypeptide shown in sequence 2 and aforementioned polypeptides structural similitude, specifically, in view of the action site that HR1 is HR2 simulated albumin, according to characteristics such as amino acid polarity or hydrophobicitys to polypeptide d, same sense mutation is carried out in e site, make forecasting software (http://www.ch.embnet.org/software/COILS form.html) mutually according to HR again to select with HR1 target position in conjunction with the minimum sequence of possibility, namely possibility is less than 30%, to guarantee that the antiviral activity of designed polypeptide (unmodified polypeptide) is not due to introducing caused by a large amount of charged amino acids EE or KK, and get rid of non-specific reason simultaneously.In addition, on the basis of polypeptide shown in sequence 1, then adopt cholesterol (cholesteryl chloroformate) to modify in its N-terminal (first amino acid of sequence 1), namely obtain the cholesterol modifier of polypeptide shown in sequence 1.
In brief, 1) resin swelling: resin wang resin first uses methylene dichloride DCM (15ml/g) to act on and the 30min that vibrates, make resin have good swelling behavior, solid is a space net structure, reactant molecule can move at resin internal freedom.2) amino acid connects: fall solvent by husky core suction filtration; add Fmoc-Lys (the Boc)-OH(N-fluorenylmethyloxycarbonyl-N '-tertbutyloxycarbonyl-1B of 3 times of molar excess) protected amino acid; add the DMAP(4-Dimethylamino pyridine of 10 times of molar excess again); DCC(dicyclohexylcarbodiimide); finally add DMF(dimethyl formamide) dissolve, vibration 30min.Close with acetic anhydride.3) joint detection: adopt ninhydrin method (Kaiser) to detect joint efficiency, get tens grainy resins, wash three times, add triketohydrindene hydrate, potassium cyanide with ethanol, each one of phenol solution, 105 DEG C-110 DEG C heating 5min, deepening blue is positive reaction.4) wash: DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, DMF (10ml/g) washes twice.5) peptide modified: with volume ratio for 2: 1, mixing DMF and dimethyl sulfoxide (DMSO) DMSO lysate, be added in the chloro-formic ester of triplication, ultrasonic to dissolving completely (time can not be long), adds pyridine and regulate pH=8-9, with above-mentioned 4) mix.6) detect: add a small amount of DCM and do not have resin to liquid, be heated to 40 degrees Celsius, nitrogen bubble reaction 120min, triketohydrindene hydrate detects and should be negative, and namely reacts completely.7) wash resin: DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice, drains 10min.8) cutting resin; Will by volumn concentration 94%(v/v) trifluoroacetic acid TFA, 2.5%(v/v) water, 2.5%(v/v) 1,2-dithioglycol EDT and 1%(v/v) thioanisole TIS form mixing solutions as carbonium ion capture reagent, to reduce the side reaction that the attack of positive ion to partial amino-acid side chain causes, clipping time is 120min.9) cholesterol modified polypeptide purifying: conclude required purity 80% and the accuracy of molecular weight with HPLC and MS.Namely select HPLC to carry out the purity check of modified polypeptide, adopt Kromasil 100-5C18, the pillar of 4.6mm × 250mm, 5micron; Moving phase: Buffer A is 0.1%TFA/H
2o(volumn concentration is the TFA aqueous solution of 0.1%); Buffer B to be 0.1%TFA/CAN(volumn concentration be 0.1% TFA/ acetic acid-cellulose nitrate cellulose solution); Gradient is: 10%-50%(v/v) Buffer B; Time is 16min.Select electron spray(ES) ESI-MS mass spectroscopy to carry out the determination of molecular mass, namely adopt turnover rate to be 0.2ml/min, the reaction times is 1 minute, and Buffer A is 0.1%HCOOH/H
2o(volumn concentration is the first aqueous acid of 0.1%), acetic acid-cellulose nitrate the cellulose solution of Buffer B to be 0.1%HCOOH/CAN(volumn concentration the be formic acid of 0.1%), to survey modified polypeptide molecular mass be 4322.22, wherein polypeptide self-molecules present quality 3909.61, cholesteryl chloroformate molecular formula is C
28h
45clO
2, molecular weight is 449.11, and as shown in Figure 6, result shows that modified polypeptide synthesis and purity thereof all meet requirement of experiment to its structure iron.
Embodiment 2, polypeptide and the detection of modifier action target spot thereof and the detection of secondary structural stability thereof
Polypeptide involved by the present embodiment and modifier thereof are that embodiment 1 prepares gained, total following four:
1) polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2) the cholesterol modifier of polypeptide: the N end of the polypeptide shown in sequence 1 carries out the product that cholesterol modifies gained.
3) polypeptide is contrasted: the polypeptide of aminoacid sequence as shown in sequence in sequence table 2.
4) wild HR2 polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 3.
The outer binding tests of aleuroplast is adopted to carry out the detection of polypeptide and the research of modifier action target spot and secondary structural stability thereof.In view of HR1 is the action site of HR2, directly adopt the experiment of Jasco J-715 circular dichroism spectrum to conduct a research, service temperature is 25 DEG C, and light path is 0.1cm, and recording wavelength scope is 195-235nm.Concrete operations are as follows: by above-mentioned four peptide species respectively with HR1 polypeptide (aminoacid sequence Aa119-172 position, AAVALNQAQ ENARNIWRLKESIKKTNEAVLELKDGLATTAIALDKVQKFINDDI) etc. mole mixing, make the final concentration of two peptide species summations in every individual system be 10 μMs; Arrange each polypeptide independent system separately in five peptide species (above-mentioned four peptide species and HR1 polypeptide) in contrast, the final concentration of every peptide species in respective system is separately 10 μMs simultaneously.With above-mentioned five independent systems and four mixed systems for research object, study the above-mentioned action target spot of four polypeptide and the thermostability of secondary structure thereof.
The result of study of the action target spot of above-mentioned four polypeptide is shown, all there are two troughs at 208nm and 222nm in all polypeptide, for the α-helixstructure of standard, compare with HR1 polypeptide mixed system with wild HR2 polypeptide, there is not considerable change in the spiralization trend of contrast polypeptide and HR1 polypeptide mixture, and polypeptide (or cholesterol modifier of polypeptide) is more obvious with the spiralization trend of HR1 polypeptide mixture.This result shows, the target position of polypeptide shown in sequence 1 (or cholesterol modifier of polypeptide) is identical with wild HR2 polypeptide, is still HR1.
The result of study of the thermostability of above-mentioned four polypeptide secondary structures is shown, under 222nm, compare with HR1 polypeptide mixed system with contrast polypeptide, polypeptide (or cholesterol modifier of polypeptide) is higher with the thermal denaturation temperature of HR1 polypeptide mixture, concrete, the thermal denaturation temperature of contrast polypeptide and HR1 polypeptide mixed system is about 50 DEG C, and this is close with each monomer thermal denaturation temperature; The cholesterol modifier of polypeptide and the thermal denaturation temperature of HR1 polypeptide mixture are greater than 100 DEG C (when 100 DEG C, secondary structure is not still destroyed); The thermal denaturation temperature of polypeptide and HR1 polypeptide mixture is 85 DEG C.The thermal denaturation temperature of wild HR2 polypeptide and HR1 mixture is 85 DEG C.This result shows, the complex body that polypeptide (or cholesterol modifier of polypeptide) and HR1 polypeptide are formed is very stable.
Embodiment 3, cell-based assay polypeptide and modifier thereof are to the anti-infection activity of Avian pneumo-encephalitis virus
Polypeptide involved by the present embodiment and modifier thereof are that embodiment 1 prepares gained, total following four:
1) polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2) the cholesterol modifier of polypeptide: the N end of the polypeptide shown in sequence 1 carries out the product that cholesterol modifies gained.
3) polypeptide is contrasted: the polypeptide of aminoacid sequence as shown in sequence in sequence table 2.
4) wild HR2 polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 3.
One, the preparation of infection model
1, the cell toxicant propagation of chick embryo fibroblast (CEF)
The acquisition of primary chick embryo fibroblast (CEF) cell: aseptically, after 9-10 day instar chicken embryo is taken out, careful will end to end, bone and fatty tissue are rejected, remaining part PBS is cut into small pieces after washing 3 times as far as possible, add 0.25% trysinization 30 minutes, PBS adds 70 milliliters of DMEM nutrient solutions containing 10% serum after washing 3 times, Beijing Mai Chen company, catalog number is CM15019), flush out inoblast energetically, nutrient solution containing cell crosses three layers of gauze, with 5ml/ bottle or 200 μ L/24 orifice plate packing, 37 DEG C of cell cultures after 24 hours (fraction of coverage reaches more than 80%) next step experiment can be carried out.
According to the virus stock solution used of amount inoculation newcastle disease virus (NDV) the strain F48E9 of cell bottle nutrient solution 1/10 volume, reach 75%(until primary chick embryo fibroblast pathology (forming large synplasm) and be generally 48 hours) time, adopt multigelation method results virus, put into-20 DEG C after frozen 2 hours by cell bottle and place room temperature to partial melting, now yawing cell bottle makes attached cell take off wall, again put into-20 DEG C frozen, so 3 times repeatedly, make virus discharge from cell, cell toxicant put-20 DEG C or-70 DEG C frozen.
2, the cytotoxic toxicity test of step 1 gained
Adopt cytopathy (synplasm) to test the cytotoxic virulence of detecting step 1 gained, concrete operations are as follows:
Cell toxicant step 1 obtained carries out 10 doubling dilutions and obtains 10
1-10
12different dilution cell toxicant suspension; CEF cell is cultivated to individual layer with 96 orifice plates, cell toxicant suspension is added in each hole, 50 μ L/ holes, totally ten two extent of dilution, each extent of dilution establishes eight holes, arrange simultaneously and normally do not add cytotoxic cell controls, in the CO2gas incubator of 37 DEG C, place observation of cell pathology (CPE) after 30 hours and calculate TCID
50(forming cytopathic virus titer).Experiment repetition 3 times, result gets 3 mean values repeated.
TCID
50method of calculation: record occur the rate value in the hole of CPE phenomenon higher than and closest to 50% viral dilution, and lower than and closest to 50% viral dilution, and calculate than distance, thus obtain TCID
50result.Wherein, than apart from=(higher than and closest to 50% the rate value-50% in hole of appearance CPE phenomenon)/(higher than and closest to 50% appearance CPE phenomenon hole rate value-lower than and closest to 50% the rate value in hole of appearance CPE phenomenon); L TCID
50=than apart from × (lower than and closest to 50% appearance CPE phenomenon hole viral dilution logarithm-higher than and closest to 50% the logarithm of viral dilution in hole of appearance CPE phenomenon)+higher than and the logarithm of viral dilution in hole of appearance CPE phenomenon closest to 50%.
Result shows the viral TCID of NDV
50tire is 2 × 10
9, by virus stock solution used 1ml according to 1:10
6volume ratio dilute after, inoculate 48 porocyte plates, with 50 μ L/ holes, separately add 50 μ L/ hole nutrient solutions, cultivate after 30 hours, these cell plate are by appearance 100 TCID
50the plaque of unit, this is the Virus Standard reference quantity of later step.
Two, Inhibition test
By cell toxicant stoste frozen in above-mentioned steps 1 dilution 1:10
6, prepare for inoculating 48 porocyte plates, 50 μ l/ holes, separately add 50 μ L/ hole nutrient solutions (catalog number is CM15019 for DMEM, Beijing Mai Chen company), namely inoculum size is 100 TCID
50.By above-mentioned ready viral dilution liquid according to above-mentioned inoculum size and different final concentration (virus+polypeptide+cell system concentration) (5,10,50,250,1000,5000,20000,40000nM) above-mentioned four peptide species solution in any one (polypeptide, or polypeptide cholesterol modifier, or contrast polypeptide, or wild HR2 polypeptide; Solvent is aseptic ultrapure water, 50 μ l/ holes), add the monolayer of the CEF cell obtained in step one 1 in 48 porocyte plates simultaneously.37 DEG C, CO
2after incubator continues to cultivate 30h, with 0.5%(0.5g/100ml) Giemsa stain dyeing 3-5min, microscopy after washing, every orifice plate is got five visuals field and is calculated IC
50.Experiment in triplicate.Result shows, the cholesterol modifier of polypeptide and polypeptide effectively can suppress virus infection in nM level concentration, obviously be better than wild HR2 polypeptide (μM level, refer to reference: Structure and function study of paramyxovirus fusion protein heptad repeat peptides, Archives of Biochemistry and Biophysics, 2005,436 (2), 316-322).In addition, the polypeptide of unmodified and the cholesterol modifier of polypeptide, with the IC of wild HR2 polypeptide
50be respectively 10.5 ± 1.4nM, 8.8 ± 1.0nM, 10 ± 3.2 μMs, show that the cholesterol modifier wilder HR2 polypeptide antiviral effect of unmodified polypeptide and polypeptide is stronger.
In addition, respectively in following four kinds of situations, virus (100 TCID
50) add the monolayer of the CEF cell obtained in the step one 1 of cultivating in 48 orifice plates with 12nM polypeptide: 1) virus and polypeptide add jointly; 2) add after virus mixes 1 hour with polypeptide 37 DEG C; 3) first add virus, 37 DEG C foster 45 minutes after, then add polypeptide; 4) first add polypeptide in 4 DEG C of mixing 30 minutes, and then infect virus.The results detailed in Fig. 2, as can be seen from the figure, when peptide concentration is 12nM, the antiviral activity of the cholesterol modifier of polypeptide all has superiority in the virus infection different treatment stage compared with the polypeptide of unmodified, and it is relative significantly to add (A4 with B4 in Fig. 2 compares) effect before virus infection, but under all experiment conditions two peptide species antiviral activities still at an order of magnitude.
In embodiment 4, chicken embryo, experiment detection polypeptide and modifier thereof are to the anti-infection activity of Avian pneumo-encephalitis virus
Polypeptide involved by the present embodiment and modifier thereof are that embodiment 1 prepares gained, total following four:
1) polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2) the cholesterol modifier of polypeptide: the N end of the polypeptide shown in sequence 1 carries out the product that cholesterol modifies gained.
3) polypeptide is contrasted: the polypeptide of aminoacid sequence as shown in sequence in sequence table 2.
4) wild HR2 polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 3.
One, the preparation of infection model
1, the propagation of embryo toxicity
The Avian pneumo-encephalitis virus F48E9 that 9-10 day instar chicken embryo is bought through allantoic cavity every embryonic breeding kind 10 μ l, allantoic fluid (about 65 hours) is collected after being cultured to chicken embryo death, be embryo toxicity, carry out cytopathy (synplasm) experiment to measure its TCID according to method described in embodiment 3 step one 2
50, frozen in-70 DEG C subsequently.The visible bleeding of virus infection chicken embryo.
2, the toxicity test of the embryo toxicity of step 1 gained
By the embryo toxicity of 9-10 day instar chicken embryo (5) through allantoic cavity inoculation step 1 gained, every egg inoculation 50 TCID
50unit, namely virus stock solution used 1ml is according to 1:10
6volume ratio dilute after, get 25 μ L, after chicken embryo death, observe chicken embryo pathology and record the death time.
Result shows, and chicken embryo presents systemic bleeding venereal disease and becomes (hemorrhage lesion degree into +++), and the death time is 65 hours, and all chicken embryonic knob fruits are consistent.Virus causes that chicken embryo pathology is characterized by hemorrhage (NDV), and naked eyes and observable also distinguish normal and infection state.The hemorrhage lesion degree of chicken embryo divides following grade :-(not occurring hemorrhage embryo) ,+(slightly), ++ (medium), +++ (seriously).
Two, Inhibition test
Divide 13 groups at random by 9-10 day instar chicken embryo, often organize 5.By 13 groups of chicken embryos all through the embryo toxicity stoste of allantoic cavity inoculation step 1 gained, every egg inoculation 50 TCID
50unit), and simultaneously through allantoic cavity inoculation different concns polypeptide (or polypeptide cholesterol modifier, or contrast polypeptide, or wild-type HR2 polypeptide) (0.1mg/ only, 0.5mg/ only or 1.0mg/ only), the each dosage of every peptide species all inoculates 1 group, and another 1 group only adds virus.Observe and add up the chicken embryo survival time, after chicken embryo death, observing pathology simultaneously.Experiment in triplicate.Result shows, every egg inoculation 0.1mg(0.25mM, 100 μ l) the polypeptide of unmodified, or the cholesterol modifier of polypeptide, or wild-type HR2 polypeptide all can suppress the pathology of NDV infected chicken embryo (namely chicken embryo is not hemorrhage, hemorrhage lesion degree be-) completely, chicken embryo can be survived more than 140 hours; The polypeptide of the unmodified of every egg inoculation 0.5mg, or the cholesterol modifier of polypeptide, or wild-type HR2 polypeptide all can suppress the pathology of NDV infected chicken embryo (namely chicken embryo is not hemorrhage, hemorrhage lesion degree be-) completely, and chicken embryo can be survived more than 140 hours; The polypeptide (all polypeptide) of every egg inoculation 1mg all affects chicken embryo self and grows, thus increases the weight of NDV(poison by force) murder by poisoning to chicken embryo, similar with chicken embryo lesion degree in infection model (hemorrhage lesion degree is +++), all survived to 65 hours.Under same experiment condition (0.1mg/ only, 0.5mg/ only or 1.0mg/ only), chicken embryo lesion degree all similar (hemorrhage lesion degree is +++) in contrast polypeptide and infection model, also survived to 65 hours.
In view of the polypeptide of above-mentioned unmodified is close with the antiviral activity of wild-type HR2 polypeptide, therefore adopt 200 TCID
50(namely virus stock solution used 1ml is according to 1: 10 for virus strain F48E9 titre
6volume ratio dilute after, get 100 μ L) study the polypeptide of unmodified, the cholesterol modifier 0.1mg(0.25mM of polypeptide further, 100 μ l) antiviral effect, the concrete operation step of testing other parts is the same, and what arrange that a virus inoculation do not inoculate polypeptide adds malicious control group simultaneously.Result shows, under this virus titer, wild-type HR2 polypeptide fails effectively to suppress the pathology of NDV infected chicken embryo (hemorrhage lesion degree is +++), and chicken embryo is survived to 55 hours only, consistent with adding the malicious control group death time.And the polypeptide of unmodified under this concentration and the cholesterol modifier of polypeptide demonstrate efficient anti-virus ability, the polypeptide of the unmodified of every egg inoculation 0.1mg or the cholesterol modifier of polypeptide effectively can suppress the pathology of NDV infected chicken embryo, and (namely chicken embryo is not hemorrhage, hemorrhage lesion degree is-), chicken embryo can be survived more than 140 hours.Adopt identical method, carry out the experiment of other three virus strain of NDV (La Sota, K and R strain), result shows, the polypeptide of the unmodified of egg inoculation 0.1mg or the cholesterol modifier of polypeptide effectively can suppress the pathology of NDV infected chicken embryo (namely chicken embryo is not hemorrhage, hemorrhage lesion degree be-).
For effect of cholesterol modifier antiviral effect in chicken embryo of the polypeptide and polypeptide of evaluating unmodified further, by a NDV virus strain F48E9(200 TCID
50) infect the 36 little different time points (every 6h as a time point) up to 36 hours afterwards before chick embryo allantoic cavity and add polypeptide 0.1mg(0.25mM, 100 μ l), to study its impact on chicken embryo death and lesion degree.The concrete operation step of testing other parts is the same.As shown in Figure 3, the polypeptide of unmodified only 6 littlely plays function (not occurring hemorrhage embryo, A) before virus infection simultaneously in time adding chicken embryo; But the cholesterol modifier of polypeptide all can suppress pathology (namely not occurring hemorrhage embryo, B) before infection 12 little adding up to simultaneously completely; And contrast polypeptide for showing pathology retarding effect (namely occurring hemorrhage embryo, C).Under same experiment condition, wild-type HR2 polypeptide processes infected chicken embryo at virus infection any time, and result is all consistent with contrast polypeptide group death time and symptom, and result is unlisted.
Embodiment 5, chicken experiment in vivo detection polypeptide and modifier thereof are to the anti-infection activity of Avian pneumo-encephalitis virus
Polypeptide involved by the present embodiment and modifier thereof are that embodiment 1 prepares gained, total following four:
1) polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2) the cholesterol modifier of polypeptide: the N end of the polypeptide shown in sequence 1 carries out the product that cholesterol modifies gained.
3) polypeptide is contrasted: the polypeptide of aminoacid sequence as shown in sequence in sequence table 2.
4) wild HR2 polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 3.
One, viral polypeptide is injected simultaneously
Divide 20 groups at random by chicken in 4 week age, often organize 5.By embryo toxicity stoste from the 5 kind different extent of dilution (10 of 20 groups of chickens through collunarium eye droppings inoculation step 1 gained
2, 10
4, 10
6, 10
8, 10
10doubly dilution) under virus strain F48E9100 μ L, and the 0.5mg of intramuscular injection simultaneously polypeptide (concentration is 1.25mM, 100 μ l; Polypeptide relate to unmodified polypeptide, polypeptide cholesterol modifier and contrast polypeptide), every peptide species all inoculates 1 group, and another 5 groups only add corresponding extent of dilution (10
2, 10
4, 10
6, 10
8, 10
10doubly dilution) virus.Observe and add up the chicken body survival time, observing survival time and record dead chicken body pathology simultaneously, wherein chicken body was recorded as survival (death of chicken body is blocked) more than 40 days.
Result shows, in contrast polypeptide group (in Fig. 4 C), even if NDV virus stock solution used 1ml dilutes 10
10doubly, chicken body death (21 days) can still be caused; And 10
6under extent of dilution, 100 μ L(i.e. 200 TCID
50) the chicken body about 10 days of virus infection is dead; And the cholesterol modifier (in Fig. 4 A) of polypeptide, in virus 10
6the chicken body death caused can be infected by blocking virus completely under extent of dilution; In addition, the polypeptide (in Fig. 4 B) of unmodified only protects part chicken body from infection, 10
6under extent of dilution 75% chicken body still can in infect latter 35 days time death, namely chicken body test in, cholesterol modified polypeptide also shows long-effect active.
Two, viral polypeptide different time injection
Divide 12 groups at random by chicken in 4 week age, often organize 5 (namely 2 groups of/time experiments repeat 2 times).12 groups of chickens are inoculated 200 TCID through collunarium eye droppings
50embryo toxicity F48E9(that is 10
6100 μ L virus liquids are got) under extent of dilution, and before infected chicken body 2 days (day-2) to the cholesterol modifier of different time points (day-2, day-1, day0, day1, day2) intramuscular injection 0.5mg polypeptide in 2 days afterwards (day 2), (concentration is 1.25mM, 100 μ l) or contrast polypeptide (inject with virus infection simultaneously, i.e. day0), the impact of research on chicken body survival rate, each time period all inoculates 1 group.Observe and add up the chicken body survival time, observing survival time and record dead chicken body pathology simultaneously, wherein chicken body was recorded as survival (death of chicken body is blocked) more than 40 days.
Result shows, the cholesterol modifier of polypeptide adds (in Fig. 5 A-D) after first 2 days to 1 day at virus infection, and the chicken body of more than 85% can be made to obtain available protecting; Add (in Fig. 5 E) after 2 days in infection, the chicken body of more than 80% can be made to obtain available protecting (namely the survival of chicken body was more than 40 days), and contrast in polypeptide group (in Fig. 5 F), chicken body is only survived 10 days.
The cytotoxicity experiment of embodiment 6, polypeptide and modifier thereof
Polypeptide involved by the present embodiment and modifier thereof are that embodiment 1 prepares gained, total following four:
1) polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 1.
2) the cholesterol modifier of polypeptide: the N end of the polypeptide shown in sequence 1 carries out the product that cholesterol modifies gained.
3) polypeptide is contrasted: the polypeptide of aminoacid sequence as shown in sequence in sequence table 2.
4) wild HR2 polypeptide: the polypeptide of aminoacid sequence as shown in sequence in sequence table 3.
Whether adopt each polypeptide of lactate dehydrogenase (LDH) experimental study toxic to cell, concrete utilization completes purchased from the commercial toxicity detection test kit of Roche company.To being cultured in individual layer CEF cell the one added in above-mentioned four peptide species of following final concentration: 5 μMs, 25 μMs, 50 μMs, 100 μMs, 250 μMs, 500 μMs, 1.0mM, 2.5mM, and mix gently, measure poison exponent according to test kit specification sheets after 24 hours.Experiment in triplicate.Result shows, and above-mentioned four peptide species are in 2.5mM concentration and following to the equal free of toxic effects of cell.
Claims (12)
1. polypeptide, is characterized in that: the aminoacid sequence of described polypeptide is sequence 1 in sequence table.
2. the modifier of polypeptide described in claim 1, is characterized in that: described modifier is the product carrying out modifying gained by cholesterol to described polypeptide.
3. modifier according to claim 2, is characterized in that: described in be modified to sequence 1 in sequence table first amino acid on connect described cholesterol.
4. polypeptide according to claim 1 or the modifier described in Claims 2 or 3 are being prepared as follows the application in arbitrary product:
1) anti-new castle disease virus product;
2) treat and/or prevent by the product of newcastle disease virus infection associated diseases.
5. the product of anti-new castle disease virus, is characterized in that: the activeconstituents of described product is the modifier described in polypeptide according to claim 1 or Claims 2 or 3.
6. treat and/or prevent by the product of newcastle disease virus infection associated diseases, it is characterized in that: the activeconstituents of described product is the modifier described in polypeptide according to claim 1 or Claims 2 or 3.
7. the preparation method of modifier described in Claims 2 or 3, comprises the following steps: Preparation of amino acid sequence is the polypeptide of sequence 1 in sequence table, connects cholesterol, obtain the modifier described in Claims 2 or 3 by first of sequence 1 amino acid.
8. the encoding gene of polypeptide described in claim 1.
9. the recombinant vectors containing encoding gene described in claim 8.
10. the reconstitution cell containing encoding gene described in claim 8.
11. recombinant bacteriums containing encoding gene described in claim 8.
12. recombinant viruses containing encoding gene described in claim 8.
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| CN101805395A (en) * | 2010-03-10 | 2010-08-18 | 中国农业大学 | Polypeptide with inhibiting virus mixed infection activity |
| CN101951958A (en) * | 2007-10-22 | 2011-01-19 | P.安杰莱蒂分子生物学研究所 | Cholesterol derivatives of inhibitors of viral fusion |
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| CN101951958A (en) * | 2007-10-22 | 2011-01-19 | P.安杰莱蒂分子生物学研究所 | Cholesterol derivatives of inhibitors of viral fusion |
| CN101805395A (en) * | 2010-03-10 | 2010-08-18 | 中国农业大学 | Polypeptide with inhibiting virus mixed infection activity |
Non-Patent Citations (2)
| Title |
|---|
| SC29EK, a Peptide Fusion Inhibitor with Enhanced -Helicity, Inhibits Replication of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide;Takeshi Naito et al.;《Antimicrobial Agents and Chemotherapy》;20081229;第53卷(第3期);第1014页左栏第1段和图1、第1015页表1、第1017页右栏第2段和第1016页右栏倒数第1段 * |
| 新城疫病毒F蛋白七肽重复序列（HR）三螺旋HR212蛋白的抗体抑制病毒和细胞融合;李影等;《自然科学进展》;20070130;第17卷(第1期);第122-126页 * |
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