CN103571863A - Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome - Google Patents

Abstract

The invention discloses a novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome. Correlation sequences of pigs on NCBI are utilized to design the protein coding gene of DRACO which is provided with activity and capable of entering cells, then the DRACO protein is expressed and purified in a prokaryotic expression system, the antivirus activity of DRACO protein to HP-PRRSV (Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus) is researched at the Marc-145 cellular level by qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction), Western-Blot, cellular supernatant TCID50 (Tissue Culture Infectious Dose 50) detection, IFA (Indirect Immunofluorescence Assay) and other methods, and the antiviral mechanism of DRACO in two processes of absorbing viruses and entering cells are further explained. The DRACO produced by the method disclosed by the invention has good antiviral effect to HP-PRRSV, and is expected to be a novel drug for preventing and treating porcine reproductive and respiratory syndrome.

Description

A kind of newtype drug protein D RACO and the application in control pig blue-ear disease thereof

Technical field

The present invention relates to biology field.More specifically, relate to a kind of newtype drug protein D RACO and the application in control pig blue-ear disease thereof.

Background technology

Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) claim again pig blue-ear disease, to be caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome viruse, PRRSV).This disease, early than the outburst of the 1987 Nian U.S., spread to Europe subsequently, and China was separated to this virus in 1996.At present, according to genome sequence and antigenic specificity, PRRSV is divided into two kinds of genotype, a kind ofly take the Europe class that Lelystad Virus (LV) strain is representative, another kind ofly take the american type that ATCC VR-2332 strain is representative.In China, PRRSV mainly be take american type as main, but it is reported and be also separated to Europe class strain.2006, porcine hyperthermia has been broken out in China, pig industry is caused to serious financial loss, afterwards this strain is defined as to high pathotype strain (Highly pathogenic porcine reproductive and respiratory syndrome viruse, HP-PRRSV).PRRS mainly causes particularly piglet respiratory symptom of pregnant sow miscarriage, stillborn foetus, mummy tire, weak son and each age group pig, and characteristic pathology is interstitial pneumonia, and mortality ratio is high, is a kind of global Important Infectious Diseases of height contact.

PRRSV belongs to the many viraleses of Buddhist nun (Nidovirales), Arteriviridae (Arteriviridae), Arterivirus (Arterivirus) member, and electric Microscopic observation virus particle is spherical in shape or oval, has cyst membrane.Viral genome is a sub-thread positive chain RNA, total length is about 15Kb, contain 5 ' and 3 ' non-coding region, centre is 10 open reading frame (open reading frame, ORF), wherein ORF2-7 translates respectively viral glycoprotein (glycoprotein GP) GP2a, GP2b, GP3, GP4, GP5, GP5a, M and N albumen.Wherein the most important thing is GP5 and N albumen, they are not only the chief component of virus particle, and produce important effect in packing, maturation, immune evasion and the antibody induction of virus particle.

The prevention and control of PRRSV are the difficult problems in current China and even the world.PRRSV is difficult to prevention and control and is mainly manifested in following several respects: (1) has a liking for scavenger cell and immunosuppressive disease, the pulmonary alveolar macrophage of PRRSV main infection pig (Porcine alveolar macrophages, PAMs), PAMs is immunocyte, destroy PAMs, thereby destruction body immune system, thereby cause immunosuppression; (2) antigenic variability, PRRSV variation is at present very fast, and the use of attenuated vaccine is a reason impelling virus variation, and vaccine does not have cross-protection; (3) antibody dependent strengthens, and the infection of PRRSV can stimulate body to produce antibody, but the antibody of low liter not only can not neutralize virus, on the contrary viral propagation is had to promoter action; (4) viral persistent infection, PRRSV can detect viremia after infecting for a long time in pig body; (5) polyinfection, polyinfection clinically, particularly PCV-II, haemophilus parasuis etc. at present makes PRRSV prevention and control extremely difficult with the polyinfection of PRRSV.

At present PRRSV prevention and control mainly contain inactivated vaccine and attenuated vaccine, and the more of use is attenuated vaccine clinically.Wherein inactivated vaccine has following shortcoming: (1) needs heavy dose of inoculation or application concentrated antigen, and duration of immunity is short, often needs strengthening inoculation; (2) can not cause local immunity, so that a little less than the effect of cellular immunization; (3) producing complete immunizing power needs 2-3 week, is unfavorable for urgent preventive vaccination and reduces vaccine expense; (4) exist deactivation not thoroughly and loose malicious possibility.And attenuated vaccine exists that virulence is returned by force, the danger of restructuring and latent infection.Therefore vaccine exposes increasing problem in anti-PRRSV processed.

Summary of the invention

The problem that the present invention exists in order to overcome the vaccine of existing prevention and control PRRSV virus, a kind of medicine that PRRSV virus is had to good antiviral effect is found in research, can prevent and treat better pig blue-ear disease.

The object of the present invention is to provide a kind of encoding gene that has activity and can enter the newtype drug protein D RACO of cell.

Another object of the present invention is to provide a kind of newtype drug protein D RACO that has activity and can enter cell.

Another object of the present invention is to provide the application of above-mentioned protein D RACO in preparing viral diseases medicine.

Preferably, described virus be transcribe with reproduction process in can produce the virus of long dsRNA helical region.

More preferably, described virus is PRRSV virus.

Above-mentioned DRACO gene order is shown in SEQ ID NO.1.DRACO aminoacid sequence is that shown in SEQ ID NO.2, DRACO albumen is comprised of 3 parts: transduction label, dsRNA detection architecture territory and apoptosis-inducing structural domain.

Transduction label be PTD and be present in albumen n end, aminoacid sequence is SEQ ID NO.3.DsRNA detection architecture domain amino acid sequence is the 14th to the 194th of DRACO Argine Monohydrochloride sequence, as SEQ ID NO.6, apoptosis-inducing structural domain aminoacid sequence is the 195th to the 291st of DRACO Argine Monohydrochloride sequence, as SEQ ID NO.7.

, be by the multiple clone site of the carrier pET-28A that sets out, to insert DRACO gene order SEQ ID NO.1 structure to form.

A bacterial strain, the coding gene sequence that contains above-mentioned DRACO.

DRACO Antiviral Mechanism is: most of virus transcribe with reproduction process in all can produce long dsRNA helical region, and not infected cell can not produce, therefore can be detected by DRACO by the cell of virus infection, active cell apoptotic signal path, thereby make the apoptosis of virus infection, and normal cell is unaffected.

PRRSV virus transcribe with reproduction process in can produce longer dsRNA helical region, therefore the present invention studies the preventive and therapeutic effect of DRACO to PRRSV virus, and at cell levels proof DRACO, PRRSV has been had to good antiviral effect.

Above-mentioned purpose of the present invention is achieved by the following technical programs:

First according to the genes involved sequences Design DRACO protein coding gene of the upper pig of NCBI, then utilize escherichia coli prokaryotic expression system to express and be purified into DRACO albumen, and then on cell levels, study the antiviral effect of DRACO to HP-PRRSV.Specific experiment design is as follows:

According to NCBI(http: the pig protein kinase R(Protein Kinase R that //www.ncbi.nlm.nih.gov/) searches acquisition, PKR) (being dsRNA detection architecture territory) (sequence number is AB104654.1) Gene Partial sequence (as shown in SEQ ID NO.4) and caspase-3 incitant 1(apoptotic protease activating factor 1, Apaf-1, sequence number is AF013263.1) Gene Partial sequence (as shown in SEQ ID NO.5), N end adds transduction peptide sequence PTD, design obtains DRACO protein coding gene, sequence is shown in SEQ ID NO.1.

The DRACO sequence that design is obtained send Sangon Biotech (Shanghai) Co., Ltd. (http://sangon.com/) synthetic; The synthetic DRACO protein coding gene obtaining is cloned into prokaryotic expression carrier pET-28A, builds and obtain prokaryotic expression carrier pET-DRACO, then at expression strain BL21(DE3) in IPTG abduction delivering; Finally by crossing purifying, obtain DRACO albumen.

DRACO cell toxicity test: AlamarBlue(is purchased from Invitrogen company) as viable cell metabolism indicator, under the reaction of plastosome enzymatic reduction, can produce measurable fluorescence meta-bolites, by measuring its fluorescence intensity, can monitor cytoactive.Use multi-functional microplate reader to read respectively 540nm exciting light and 590nm utilizing emitted light fluorescent value, make DRACO cytotoxicity figure.

DRACO antivirus test: cultivating Marc-145 cell to cell degree of converging with the DMEM nutrient solution containing 10% foetal calf serum is 60-70%, discards nutrient solution, and PBS washes 3 times, adds respectively the PFU=0.7 * TCID of plaque forming unit containing MOI=0.1 He1( ₅₀, infection multiplicity MOI=virus number/cell count) and the DMEM nutrient solution of 2% foetal calf serum of HP-PRRSV virus, 37 ℃ are continued to cultivate 5h, and PBS washes 3 times, adds the DMEM nutrient solution of 2% foetal calf serum of the DRACO of different concns, cultivates 36h for 37 ℃, collects supernatant and is TCID ₅₀detect; Collecting cell is done qRT-PCR(N gene quantification primer and is seen SEQ ID NO.8 and SEQ ID NO.9) and Western-Blot detection.

The antiviral indirect immunofluorescence assay of DRACO (Indirect Immunofluorescent Assay, IFA): the same antivirus test method, collecting cell, 4% paraformaldehyde is 10min fixedly, PBS washes 10min, the 10%Triton-100 15min that bores a hole, PBS washes 10min, then with the 1%BSA sealing 30min of PBS dilution, add the primary antibodie of anti-PRRSV virus N albumen, room temperature 1h, add again the anti-effect of anti-mouse two 1h, Hoechst dyes core 5min, and PBS washes 10min, then at fluorescence microscopy Microscopic observation DRACO antiviral effect.

DRACO Antiviral Mechanism research: (1) thus whether DRACO adsorbs by affect HP-PRRSV the research that Marc-145 cell reaches the object of anti-HP-PRRSV: first, when Marc-145 cell is when in 6 orifice plates, degree of converging is 70%, PBS washes 3 times, then with MOI=0.1, meet malicious HP-PRRSV, and add the DRACO of concentration gradient, hatch 2h for 4 ℃, after having hatched, with PBS, wash 3 times, the Virus eliminating medicine that is not adsorbed onto cell surface is washed off, then at 5%CO ₂, cultivate 24h in 37 ℃ of incubators, N gene is done to qRT-PCR to collecting cell and Western-Blot detects;

(2) whether DRACO enters by suppressing HP-PRRSV the research that Marc-145 cell reaches the object of anti-HP-PRRSV: first, 6 orifice plates at Marc-145 cell degree of converging 70% are washed 3 times with PBS, then with MOI=0.1, meet malicious HP-PRRSV, hatch 2h for 4 ℃, after having hatched, with PBS, wash 3 times, the Virus eliminating medicine that is not adsorbed onto cell surface is washed off, then add the nutritive medium of DRACO of concentration gradient at 5%CO ₂, cultivate 6h in 37 ℃ of incubators, PBS washes 3 times, renews the fresh nutritive medium containing 2% foetal calf serum and continues to cultivate 24h, collecting cell carries out Western-Blot detection;

(3), when PRRSV enters after cell 4h and 6h, whether PG-1 also has the research of antivirus action.First, at 6 orifice plates of Marc-145 cell degree of converging 70%, with PBS, wash 3 times, then with MOI=0.1, meet malicious HP-PRRSV, hatch 2h for 4 ℃, PBS washes 3 times, the Virus eliminating medicine that is not adsorbed onto cell surface is washed off to 5%CO ₂, cultivate 4h and 6h in 37 ℃ of incubators, PBS washes 3 times, by concentration, be respectively 40,60, the DMEM nutrient solution of 2% foetal calf serum of 80mg/L DRACO adds 37 ℃, cell to cultivate 6h, PBS washes 3 times, renew the fresh nutritive medium containing 2% foetal calf serum and continue to cultivate 24h, discard nutritive medium, PBS washes 3 times, and collecting cell carries out Western-Blot detection.

Statistical analysis.At least 3 independent repetitions of all tests above, result adopts mean value and standard error to represent, uses student ' s t testanalyze.All statistical study all adopt with p<0.05as the inspecting standard with remarkable significant difference, analysis software is SPSS 16.0 and GraphPad Prism 5.

Compared with prior art, the present invention has following beneficial effect:

The present invention designs the medicine DRACO albumen that obtains a kind of new control pig blue-ear disease first, and final design synthetic DRACO have activity and can enter cells play effect.

Pass through TCID ₅₀, the several different methods such as qRT-PCR, IFA and Western-Blot proved that DRACO has good antivirus action to HP-PRRSV, for developing the newtype drug of anti-pig blue-ear disease, lay a good foundation.

The present invention is studied the mechanism of the anti-HP-PRRSV of DRACO, comprises virus absorption onto cell and the research that enters two processes of cell.Result shows that DRACO can reduce to significance N gene and N protein expression level, and the rising along with DRACO concentration, inhibition is more obvious, and DRACO antivirus action mainly occurs in PRRSV and enters in cell processes, when PRRSV enters after cell 4h and 6h, DRACO antiviral effect is not obvious.

Accompanying drawing explanation

Fig. 1 is DRACO prokaryotic expression SDS-PAGE figure.

Fig. 2 is purifying figure after DRACO expresses.1:100mM imidazole buffer wash-out; 2:300mM imidazole buffer is wash-out for the first time; 3:300mM imidazole buffer is wash-out for the second time.

Fig. 3 is DRACO cell toxicity test result statistical graph.

Fig. 4 is in DRACO antivirus test, the virus titer figure in the Marc-145 cell conditioned medium that the HP-PRRSV after different concns DRACO processes infects respectively.

Fig. 5 is in DRACO antivirus test, and after 80mg/L DRACO effect 36h, N gene is with respect to the expression level figure of internal reference GAPDH.

Fig. 6 is in DRACO antivirus test, after different concns DRACO processes 36h, and N protein expression level figure.

Fig. 7 is that the antiviral indirect immunofluorescence assay of DRACO detects DRACO antiviral effect figure; Green is anti-PRRSV N albumen color, and blueness is Hoechst transfect cell core color.

Fig. 8 is that in DRACO Antiviral Mechanism-virus absorption onto cell process, N gene is with respect to the expression level figure of internal reference GAPDH.

Fig. 9 is in DRACO Antiviral Mechanism-virus absorption onto cell process, and N albumen Western-Blot detects figure.

Figure 10 is in DRACO Antiviral Mechanism-cell entry cell processes, and N albumen Western-Blot detects figure.

After Figure 11 is DRACO Antiviral Mechanism-cell entry cell 4h, N albumen Western-Blot detects figure.

After Figure 12 is DRACO Antiviral Mechanism-cell entry cell 6h, N albumen Western-Blot detects figure.

Embodiment

Below in conjunction with Figure of description and specific embodiment, further explain the present invention, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts test method, reagent and equipment are the art ordinary method, reagent and equipment.

embodiment 1 DRACO gene design is synthesized and expression analysis

1, DRACO gene design is synthetic: according to NCBI(http: the pig protein kinase R(Protein Kinase R that //www.ncbi.nlm.nih.gov/) searches acquisition, PKR) (sequence number is AB104654.1) Gene Partial sequence (as shown in SEQ ID NO.4) and caspase-3 incitant 1(apoptotic protease activating factor 1, Apaf-1, sequence number is AF013263.1) Gene Partial sequence (as shown in SEQ ID NO.5), N end adds transduction peptide sequence PTD, PTD aminoacid sequence is shown in SEQ ID NO.3, design obtains DRACO protein coding gene, sequence is shown in SEQ ID NO.1.The DRACO Argine Monohydrochloride sequence of this genes encoding is SEQ ID NO.2.The DRACO sequence that design is obtained send Sangon Biotech (Shanghai) Co., Ltd. (http://sangon.com/) synthetic.

2, build DRACO gene recombination prokaryotic expression bacterial strain: the above-mentioned synthetic DRACO gene clone obtaining, to prokaryotic expression carrier pET-28A, is built and obtains prokaryotic expression carrier pET-DRACO, and its insertion restriction enzyme site is kpn Iwith hind III; Then prokaryotic expression carrier pET-DRACO is transformed to bacterial strain BL21(DE3) competent cell, Kan ⁺resistance screening, evaluation, order-checking, positive recombinant strains glycerine is stored in-80 ° of C refrigerators.

3, the abduction delivering of recombinant strains: draw the recombination bacillus coli 50 μ L of above-mentioned glycerine conservation, add 5mL Kan is housed ⁺the bacteria culture bottle of the LB liquid nutrient medium of resistance, 37 ° of C shaking culture are spent the night, and draw respectively the bacterium liquid 50 μ L of above-mentioned recovery, add two 200mlLKan is housed ⁺the bacteria culture bottle of the LB liquid nutrient medium of resistance, 37 ° of C shaking tables are cultivated 4h, to OD ₆₀₀reach at 0.6 ~ 1.0 o'clock, one bottle add IPTG to final concentration be 0.8mmol/L(5 μ l) abduction delivering, another bottle does not add, and carries out mark, continue to cultivate after 3h, the centrifugal 2min of 8000rpm collects thalline.

4, DRACO soluble analysis: with 500 μ lLPBS suspension thalline, it is suitable cursory to select, and is placed in frozen water, cleans 3 times, chooses suitable probe (probe is placed under liquid level, prevents foam), regulates working hour 10min; Regulate broken 5S, stop 5S; Select power 30%(150 W), after completing, fragmentation in 4 ° of centrifugal 15 ~ 20min of C 12000rpm, collects supernatant liquor, and precipitation suspends with 300 μ lLPBS, in-80 ° of C, preserves.

5, DRACO protein SDS-PAGE is identified: get the precipitation 40 μ L of supernatant liquor and suspension, add 5 * loading buffer of 10 μ L, boil 10min in boiling water, carry out SDS-PAGE analysis, analyze the solubility of DRACO expressing protein.See accompanying drawing 1, in figure, 1 for not adding IPTG abduction delivering, and 2 is IPTG abduction delivering, and M is for dying in advance protein molecular Marker.

the purifying of embodiment 2 DRACO albumen

Utilize His-tag to cross column purification DRACO albumen, and BCA protein quantification test kit (purchased from Thermo Scientific company, article No.: 23227) measure DRACO protein concentration.

1. be ready to protein sample, phosphate buffered saline buffer, Binding buffer, Washing buffer, Elution buffer, 20% ethanol, distilled water install Ni-NTA chromatography column on iron stand.

2. with syringe, add the ddH2O of 4 times of column volumes (5mL) to clean Ni-NTA chromatography column, balance chromatography column, Binding buffer with 4 times of volumes of cylinder, balance pillar, Binding buffer has played adhesive attraction, and object is for ensuing, the albumen filtering containing His label sticks to Ni-NTA resin, the good albumen of filtration by containing His label, by identical method, is added to the chromatography column that Ni-NTA resin is housed.

3. use the Washing buffer of 4 times of column volume, by identical method, be injected into the target protein that the chromatography column that Ni-NTA resin is housed is removed some weak bindings and non-specific binding, Elution buffer with 4 times of column volumes, with identical method inject Ni-NTA chromatography column from nickel post under wash-out with the target protein of His label, with 1.5mlLEP, manage packing, every pipe 1mL ,-80 ° of C preserve.

4. use the phosphate buffered saline buffer containing 300mM imidazoles of 4 times of column volumes, inject Ni-NTA chromatography column, remove all foreign proteins, clean chromatography column, with the ddH2O of 4 times of column volume, clean the chromatography column that Ni-NTA resin is housed, preserve chromatography column, with 20% ethanol of 4 times of column volume, clean after Ni-NTA chromatography column, 4 ° of C preserve.The DRACO albumen that purifying obtains carries out SDS-PAGE analysis, and result is as Fig. 2.

embodiment 3 DRACO cell toxicity tests

AlamarBlue(is purchased from Invitrogen company) as viable cell metabolism indicator, under the reaction of plastosome enzymatic reduction, can produce measurable fluorescence meta-bolites, by measuring its fluorescence intensity, can monitor cytoactive.With the DMEM nutrient solution containing 10% foetal calf serum, cultivate Marc-145 cell to 60-70%, discard nutrient solution, add the nutritive medium effect 36h containing DRACO doubling dilution, set PBS control group, then add 10%(V/V) ratio AlamarBlue continuation cultivation 3h, use multi-functional microplate reader to read respectively 540nm exciting light and 590nm utilizing emitted light fluorescent value, make DRACO cytotoxicity figure.See accompanying drawing 3, using PBS cellular control unit activity as 100%, the fluorescent value of the cell that the DRACO of doubling dilution processes is the relative cytoactive of DRACO under different concns than upper PBS control group fluorescent value, as shown in Figure 3, when DRACO concentration is 80mg/L, it does not have toxicity to Marc-145 cell, cytoactive 100%, and this concentration is the peak concentration of later tests.

embodiment 4 DRACO antivirus tests

1. while cultivating Marc-145 cell to cell degree of converging 70% in 6 orifice plates containing the DMEM substratum of 10% foetal calf serum, discard nutrient solution, PBS washes 3 times, meets malicious HP-PRRSV respectively with MOI=0.1 and 1, and in the DMEM of 2% foetal calf serum nutrient solution, 37 ℃ are continued to cultivate 5h.

2.PBS washes 3 times, and the DMEM nutrient solution that is 2% foetal calf serum of 80mg/L DRACO by concentration adds 37 ℃, cell to cultivate 36h, and establishes PBS control group, and PBS washes 3 times, and every hole adds 400 μ L Trizol, carries RNA, is qRT-PCR and detects.The results are shown in Figure 5, result shows on transcriptional level, and DRACO can reduce N gene expression dose in significance ground.

3. the samely with MOI=0.1, meet malicious HP-PRRSV, in the DMEM of 2% foetal calf serum nutrient solution, 37 ℃ are continued to cultivate 5h, PBS washes 3 times, by concentration, be respectively 40,60, the DMEM nutrient solution of 2% foetal calf serum of 80mg/L DRACO adds 37 ℃, cell to cultivate 36h, and establish PBS control group, collect 1mL supernatant and be TCID ₅₀detect, the results are shown in Figure 4, result shows that DRACO can reduce to significance the viral yield in cell conditioned medium, suppresses virus and is discharged in cell conditioned medium.Discard in addition remaining culture liq, PBS washes 3 times, 0.25% trysinization, and lysing cell, surveys protein concentration, and Western-Blot detects, and the results are shown in Figure 6, and result shows in protein translation level, and DRACO can significantly reduce N protein expression level.

4. while cultivating Marc-145 cell to cell degree of converging 70% in 12 orifice plates containing the DMEM substratum of 10% foetal calf serum, discard nutrient solution, PBS washes 3 times, with MOI=0.1, meet malicious HP-PRRSV, in the DMEM of 2% foetal calf serum nutrient solution, 37 ℃ are continued to cultivate 5h, PBS washes 3 times, by concentration, be 40 respectively, 60, the DMEM nutrient solution of 2% foetal calf serum of 80mg/L DRACO adds 37 ℃, cell to cultivate 36h, and establish PBS control group, PBS washes 3 times, 4% paraformaldehyde is 10min fixedly, PBS washes 10min, the 10%Triton-100 15min that bores a hole, PBS washes 10min, then with PBS dilution 1%BSA sealing 30min, anti-PRRSV N albumen primary antibodie room temperature 1h, the anti-effect of anti-mouse two 1h, Hoechst dyes core 5min, PBS washes 10min, IFA detects.The results are shown in Figure 7, as shown in Figure 7, DRACO concentration is 40,60, during 80mg/L, PRRSV virus N albumen fluorescent value is starkly lower than PBS control group, show that PRRSV virus N albumen is not almost at cells, illustrate that DRACO antiviral effect is obvious, and when DRACO concentration is 80mg/L, antiviral effect is the most obvious.

embodiment 5 DRACO Antiviral Mechanism researchs: HP-PRRSV adsorbs inhibition test

1. at 6 orifice plates of Marc-145 cell degree of converging 70%, with PBS, wash 3 times, then with MOI=0.1, meet malicious HP-PRRSV, and add respectively that concentration is 40,60,80mg/L DRACO, hatch 2h for 4 ℃.

2.PBS washes 3 times, the Virus eliminating medicine that is not adsorbed onto cell surface is washed off, then at 5%CO ₂, continue to cultivate 24h in 37 ℃ of incubators, discard nutrient solution, PBS washes 3 times, collecting cell, every hole adds 400 μ L Trizol, carries RNA, N gene is done to qRT-PCR and detect, and sees Fig. 8; Lysing cell, surveys protein concentration, and Western-Blot detects, see Fig. 9, result shows that DRACO can reduce to significance N gene and N protein expression level, and along with the rising of DRACO concentration, inhibition is more obvious, illustrates that DRACO has antiviral effect in HP-PRRSV adherent cell process.

embodiment 6 DRACO Antiviral Mechanism researchs: HP-PRRSV enters inhibition test

1, at 6 orifice plates of Marc-145 cell degree of converging 70%, with PBS, wash 3 times, then with MOI=0.1, meet malicious HP-PRRSV, hatch 2h for 4 ℃.

2, PBS washes 3 times, and the Virus eliminating medicine that is not adsorbed onto cell surface is washed off, then adds respectively that concentration is 40,60,80mg/L DRACO, at 5%CO ₂, cultivate 6h in 37 ℃ of incubators.

3, PBS washes 3 times, renew the fresh nutritive medium containing 2% foetal calf serum and continue to cultivate 24h, discard nutritive medium, PBS washes 3 times, and collecting cell carries out Western-Blot detection, see Figure 10, result shows to enter in cell processes at HP-PRRSV, and DRACO has good antiviral effect, when even concentration is 40mg/L, all can't detect N protein expression level, illustrate that DRACO antivirus action mainly occurs in PRRSV and enters in cell processes.

4,, when PRRSV enters after cell 4h and 6h, whether PG-1 also has antivirus action.First, at 6 orifice plates of Marc-145 cell degree of converging 70%, with PBS, wash 3 times, then with MOI=0.1, meet malicious HP-PRRSV, hatch 2h for 4 ℃, PBS washes 3 times, the Virus eliminating medicine that is not adsorbed onto cell surface is washed off to 5%CO ₂, in 37 ℃ of incubators, cultivate 4h and 6h, PBS washes 3 times, by concentration, be 40 respectively, 60, the DMEM nutrient solution of 2% foetal calf serum of 80mg/L DRACO adds 37 ℃, cell to cultivate 6h, PBS washes 3 times, renew the fresh nutritive medium containing 2% foetal calf serum and continue to cultivate 24h, discard nutritive medium, PBS washes 3 times, collecting cell Western-Blot detects, see accompanying drawing 11(4h) and 12(6h), result shows, when HP-PRRSV processes with different concns DRACO after entering cell 4h and 6h at 37 ℃ again, DRACO antiviral effect is not obvious, only have when DRACO concentration is 80mg/L, just there is antiviral effect, explanation is after cell entry cell, DRACO antiviral effect is not obvious.

SEQUENCE LISTING

<110> Zhongshan University

<120> newtype drug protein D RACO and the application in control pig blue-ear disease thereof

<130>

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 876

<212> DNA

<213> DRACO encoding gene

<400> 1

ggctatgcgc gcgcagcggc tcgccaggcg cgcgcaggca tggccagtgg tcgttcaccg 60

tgtttctaca tagaagaact taataaatac caacaaaaga acgatgtcat acttaagtat 120

cgtgaactgt gtaagacagg acctgcacat aacttgaggt ttacgtatca agttgtaata 180

gatgagaaag aattccccaa agctgaaggt agatcaaaga aggaagccaa aaatgctgca 240

gtcaaattag cttttgaaat aattaataaa gaacacaagg caaatagttc ttcatcactg 300

ccgacagcaa atcctccaga tagacaactc actgagaatt acataggccg tattaatacg 360

atttcccaaa agaaaaacct atctgtaaat tatgaaccat gtgaaccggg ggaagatggg 420

cctgaaaaat ttcattataa atgcaaaatc ggacggaagg tatatggtag tggtgtagga 480

tccactaaac aggaggcaaa acaattggct gccaaacagg cttatgaaaa gttacagtca 540

gaaaacttta tgaatgctga cccaccatcc tctagttgtc tcatggatgc aaaagctcga 600

aattgtttgc ttcaacatag agaagccctg gaaagggaca tcaaaacatc ctacatcatg 660

gatcacatga ttagtaatgg cgttttaaca ctttcagagg aggaaaaagt gaaaaatgag 720

cctactcaat gccaacgagc agctctgcta attaaaatga tacttaaaaa agataattat 780

gcctacatat cattctacaa tgctctgcta catgaagggt ataaagatct tgctgccctt 840

ctccatggtg gccttcctgt cgtctcttct tcctaa 876

<210> 2

<211> 291

<212> PRT

<213> protein D RACO aminoacid sequence

<400> 2

Gly Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala Gly Met Ala Ser

1 5 10 15

Gly Arg Ser Pro Cys Phe Tyr Ile Glu Glu Leu Asn Lys Tyr Gln Gln

20 25 30

Lys Asn Asp Val Ile Leu Lys Tyr Arg Glu Leu Cys Lys Thr Gly Pro

35 40 45

Ala His Asn Leu Arg Phe Thr Tyr Gln Val Val Ile Asp Glu Lys Glu

50 55 60

Phe Pro Lys Ala Glu Gly Arg Ser Lys Lys Glu Ala Lys Asn Ala Ala

65 70 75 80

Val Lys Leu Ala Phe Glu Ile Ile Asn Lys Glu His Lys Ala Asn Ser

85 90 95

Ser Ser Ser Leu Pro Thr Ala Asn Pro Pro Asp Arg Gln Leu Thr Glu

100 105 110

Asn Tyr Ile Gly Arg Ile Asn Thr Ile Ser Gln Lys Lys Asn Leu Ser

115 120 125

Val Asn Tyr Glu Pro Cys Glu Pro Gly Glu Asp Gly Pro Glu Lys Phe

130 135 140

His Tyr Lys Cys Lys Ile Gly Arg Lys Val Tyr Gly Ser Gly Val Gly

145 150 155 160

Ser Thr Lys Gln Glu Ala Lys Gln Leu Ala Ala Lys Gln Ala Tyr Glu

165 170 175

Lys Leu Gln Ser Glu Asn Phe Met Asn Ala Asp Pro Pro Ser Ser Ser

180 185 190

Cys Leu Met Asp Ala Lys Ala Arg Asn Cys Leu Leu Gln His Arg Glu

195 200 205

Ala Leu Glu Arg Asp Ile Lys Thr Ser Tyr Ile Met Asp His Met Ile

210 215 220

Ser Asn Gly Val Leu Thr Leu Ser Glu Glu Glu Lys Val Lys Asn Glu

225 230 235 240

Pro Thr Gln Cys Gln Arg Ala Ala Leu Leu Ile Lys Met Ile Leu Lys

245 250 255

Lys Asp Asn Tyr Ala Tyr Ile Ser Phe Tyr Asn Ala Leu Leu His Glu

260 265 270

Gly Tyr Lys Asp Leu Ala Ala Leu Leu His Gly Gly Leu Pro Val Val

275 280 285

Ser Ser Ser

290

<210> 3

<211> 13

<212> PRT

<213> transduction label PTD aminoacid sequence

<400> 3

Gly Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala Gly

1 5 10

<210> 4

<211> 543

<212> DNA

<213> pig protein kinase R Gene Partial sequence

<400> 4

atggccagtg gtcgttcacc gtgtttctac atagaagaac ttaataaata ccaacaaaag 60

aacgatgtca tacttaagta tcgtgaactg tgtaagacag gacctgcaca taacttgagg 120

tttacgtatc aagttgtaat agatgagaaa gaattcccca aagctgaagg tagatcaaag 180

aaggaagcca aaaatgctgc agtcaaatta gcttttgaaa taattaataa agaacacaag 240

gcaaatagtt cttcatcact gccgacagca aatcctccag atagacaact cactgagaat 300

tacataggcc gtattaatac gatttcccaa aagaaaaacc tatctgtaaa ttatgaacca 360

tgtgaaccgg gggaagatgg gcctgaaaaa tttcattata aatgcaaaat cggacggaag 420

gtatatggta gtggtgtagg atccactaaa caggaggcaa aacaattggc tgccaaacag 480

gcttatgaaa agttacagtc agaaaacttt atgaatgctg acccaccatc ctctagttgt 540

ctc 543

<210> 5

<211> 291

<212> DNA

<213> caspase-3 incitant 1 partial sequence

<400> 5

atggatgcaa aagctcgaaa ttgtttgctt caacatagag aagccctgga aagggacatc 60

aaaacatcct acatcatgga tcacatgatt agtaatggcg ttttaacact ttcagaggag 120

gaaaaagtga aaaatgagcc tactcaatgc caacgagcag ctctgctaat taaaatgata 180

cttaaaaaag ataattatgc ctacatatca ttctacaatg ctctgctaca tgaagggtat 240

aaagatcttg ctgcccttct ccatggtggc cttcctgtcg tctcttcttc c 291

<210> 6

<211> 181

<212> PRT

<213> dsRNA detection architecture domain amino acid sequence

<400> 6

Met Ala Ser Gly Arg Ser Pro Cys Phe Tyr Ile Glu Glu Leu Asn Lys

1 5 10 15

Tyr Gln Gln Lys Asn Asp Val Ile Leu Lys Tyr Arg Glu Leu Cys Lys

20 25 30

Thr Gly Pro Ala His Asn Leu Arg Phe Thr Tyr Gln Val Val Ile Asp

35 40 45

Glu Lys Glu Phe Pro Lys Ala Glu Gly Arg Ser Lys Lys Glu Ala Lys

50 55 60

Asn Ala Ala Val Lys Leu Ala Phe Glu Ile Ile Asn Lys Glu His Lys

65 70 75 80

Ala Asn Ser Ser Ser Ser Leu Pro Thr Ala Asn Pro Pro Asp Arg Gln

85 90 95

Leu Thr Glu Asn Tyr Ile Gly Arg Ile Asn Thr Ile Ser Gln Lys Lys

100 105 110

Asn Leu Ser Val Asn Tyr Glu Pro Cys Glu Pro Gly Glu Asp Gly Pro

115 120 125

Glu Lys Phe His Tyr Lys Cys Lys Ile Gly Arg Lys Val Tyr Gly Ser

130 135 140

Gly Val Gly Ser Thr Lys Gln Glu Ala Lys Gln Leu Ala Ala Lys Gln

145 150 155 160

Ala Tyr Glu Lys Leu Gln Ser Glu Asn Phe Met Asn Ala Asp Pro Pro

165 170 175

Ser Ser Ser Cys Leu

180

<210> 7

<211> 97

<212> PRT

<213> apoptosis-inducing structural domain aminoacid sequence

<400> 7

Met Asp Ala Lys Ala Arg Asn Cys Leu Leu Gln His Arg Glu Ala Leu

1 5 10 15

Glu Arg Asp Ile Lys Thr Ser Tyr Ile Met Asp His Met Ile Ser Asn

20 25 30

Gly Val Leu Thr Leu Ser Glu Glu Glu Lys Val Lys Asn Glu Pro Thr

35 40 45

Gln Cys Gln Arg Ala Ala Leu Leu Ile Lys Met Ile Leu Lys Lys Asp

50 55 60

Asn Tyr Ala Tyr Ile Ser Phe Tyr Asn Ala Leu Leu His Glu Gly Tyr

65 70 75 80

Lys Asp Leu Ala Ala Leu Leu His Gly Gly Leu Pro Val Val Ser Ser

85 90 95

Ser

<210> 8

<211> 20

<212> DNA

<213> N gene quantification primer 1

<400> 8

aaaaccagtc cagaggcaag 20

<210> 9

<211> 20

<212> DNA

<213> N gene quantification primer 2

<400> 9

cggatcagac gcacagtatg 20

Claims (10)

1. an encoding gene of newtype drug protein D RACO, is characterized in that, DRACO gene order is as shown in SEQ ID NO.1.

2. a newtype drug protein D RACO, is characterized in that, the aminoacid sequence of DRACO is as shown in SEQ ID NO.2.

3. newtype drug protein D RACO according to claim 2, it is characterized in that, this albumen is comprised of 3 parts: transduction label, dsRNA detection architecture territory and apoptosis-inducing structural domain, and the label of wherein transduceing is PTD and is present in albumen n end, PTD aminoacid sequence is SEQ ID NO.3.

4. newtype drug protein D RACO according to claim 3, is characterized in that, described dsRNA detection architecture domain amino acid sequence is as shown in SEQ ID NO.6, and apoptosis-inducing structural domain aminoacid sequence is as shown in SEQ ID NO.7.

5. a recombinant prokaryotic expression vector, is characterized in that, by SEQ ID NO.1 gene order described in the multiple clone site insertion claim 1 of the carrier that sets out.

6. a recombined pronucleus expression bacterial strain, is characterized in that containing the coding gene sequence of DRACO described in claim 1.

7. the application of the encoding gene of DRACO in preparing viral diseases medicine described in claim 1.

8. the application of newtype drug protein D RACO in preparing viral diseases medicine described in claim 2.

9. according to application described in claim 7 or 8, it is characterized in that, described virus be transcribe with reproduction process in can produce the virus of long dsRNA helical region.

10. according to application described in claim 7 or 8, it is characterized in that, described virus is PRRSV virus.

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