CN103980352B - A kind of for suppressing polypeptide and the application thereof of goose tembusu virus cells infected - Google Patents
A kind of for suppressing polypeptide and the application thereof of goose tembusu virus cells infected Download PDFInfo
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- CN103980352B CN103980352B CN201410208557.4A CN201410208557A CN103980352B CN 103980352 B CN103980352 B CN 103980352B CN 201410208557 A CN201410208557 A CN 201410208557A CN 103980352 B CN103980352 B CN 103980352B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of for suppressing polypeptide and the application thereof of goose tembusu virus cells infected.Does is this polypeptid acid sequence selected from SEQ? ID? aminoacid sequence shown in No.1 or SEQ? ID? the truncated sequence of sequence shown in No.1.Polypeptide of the present invention suppresses the infection of goose tembusu virus by suppressing virus with the combination of target cell specific receptor, and does not have an impact and side effect to host itself, makes this polypeptide suppress to have very high specificity and better application prospect.
Description
Technical field
The invention belongs to biology field, relating to a kind of for suppressing polypeptide and the application thereof of goose tembusu virus cells infected.
Background technology
Goose tembusu virus belongs to flaviviridae, Flavivirus, and this virus causes tembusu virus sick, also known as egg drop syndrome, infectivity ovaritis, is significantly decline with laying rate, food consumption, occurs that encephalitis sample nervous symptoms is the Novel infectious disease of feature.Domestic existing multidigit scholar report this virus to the poultry such as duck, goose, chicken all have virulence (Huang Xinmei, Li Yin, Zhao Dongmin etc. the Isolation and ldentification of novel light yellow viral JS804 strain. Jiangsu's agriculture journal, 2011,27 (2): 354-360; Su Jingliang. the egg drop syndrome that the novel flavivirus BYD of duck causes. animal doctor's guide, 2011,4:27-29; Chen Shilong, old few warbler, Wang Shao etc. a kind of separation of the novel flavivirus causing Egg Production of Laying Hens to decline and preliminary evaluation. Fujian Journal of Agricultural Sciench, 2011,26 (2): 170-174; Li Yufeng, horse is beautiful, in ringing. a kind of flavivirus First Report of Studies be newly separated from duck, journal of animal science and veterinary medicine, 2011,42 (6): 885-891).The sickness rate of this disease can up to 100%, mortality ratio is (Teng Qiaoyang between 5% ~ 10%, Yan Pixi, Zhang Xu etc. a kind of new flavivirus causes egg duck egg drop reduction and death. Chinese zoonosis journal, 2010,18 (6): 1-4. ten thousand spring and, Shi Shaohua, Cheng Longfei, cause kind of (egg) duck to lay eggs the separation of rapid drawdown new virus and preliminary evaluation etc. one. Fujian Journal of Agricultural Sciench, 2010,25 (6): 663-666).Break out also bamboo telegraph to domestic major poultry cultivation area from April, 2010 in China, cause serious financial consequences to China's duck goose aquaculture.
Newly send out epidemic disease as one, the morbidity of goose tembusu virus disease is anxious, and propagate rapidly, there is no effective vaccine at present can use, and medication effect is also undesirable, clinically more difficult control.Although the pertinent literature report of existing duck Tan Busu inactivated vaccine and attenuated vaccine, is still in the laboratory study stage, does not carry out clinical application.In addition, the result for the treatment of of anti-duck tembusu virus yolk antibody and antiviral is unsatisfactory.
Goose tembusu virus belongs to flaviviridae family member, and this genus virus mainly comprises several important human pathogen, comprises dengue virus, west nile virus and encephalitis b virus etc.So far, this genus virus does not have the specific therapy infected, and is difficult to be controlled by vaccine inoculation.
Flavivirus is the single strand plus RNA virus with cyst membrane, and its envelope protein E belongs to II class fusion rotein, the combination of mediate retroviral and acceptor and fusion process.What the employing of togavirus target cell infection was similar enters born of the same parents' mechanism, namely after virus E protein is combined with host cell receptor, start E protein conformational change, be folded to form Trimeric structures, further virus envelope and cytolemma distance, cause two films to merge, viral genome is discharged into infection host in host cell subsequently.
Goose tembusu virus disease belongs to emerging infectious disease, and correlative study still belongs to blank, and current goose tembusu virus cells infected mechanism is not yet clear and definite, does not also have the peptide inhibitor that can suppress virus infection specifically.Therefore, the peptide inhibitor that qualification goose tembusu virus infects enters born of the same parents' mechanism by what contribute to understanding goose tembusu virus, and is significant to the prevention and control of tembusu virus disease and the design of antiviral.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, providing a kind of for suppressing the peptide inhibitor of goose tembusu virus cells infected.
Object of the present invention realizes by following technical scheme:
A kind of for suppressing the polypeptide of goose tembusu virus cells infected, its aminoacid sequence is selected from the truncated sequence of sequence shown in the aminoacid sequence shown in SEQIDNo.1 or SEQIDNo.1, and shown in described SEQIDNo.1, the truncated sequence of sequence at least comprises sequence shown in SEQIDNO.2; Preferably sequence as shown in SEQIDNO.2.
To encode the gene of polypeptide of the present invention.
Gene nucleotide series of the present invention is preferably as shown in SEQIDNO.3.
Recombinant expression vector containing gene of the present invention.
The application in the anti-goose tembusu virus medicine of preparation of polypeptide of the present invention, gene or recombinant expression vector.
A pharmaceutical composition for anti-goose tembusu virus, comprises polypeptide of the present invention.
Prepare the method for polypeptide of the present invention, directly synthesize by artificial or obtained by vivoexpression.Such as by the microbial expression peptide of recombinant nucleic acid molecules of peptide needed for the coding under controlling at suitable transcripting promoter, and from peptide needed for affiliated microorganism-collecting.
Beneficial effect:
To achieve these goals, the present invention with goose tembusu virus envelope protein E for design basis, with improvement on synthesis competition binding goose tembusu virus specific receptors, the combination of interference goose tembusu virus and target cell specific receptor, thus suppress goose tembusu virus target cell infection.Result shows, the polypeptide shown in SEQIDNo.1 has the effect that interference goose tembusu virus is combined with target cell specific receptor, and presents the effect suppressing goose tembusu virus cells infected.Those skilled in the art can design truncate based on SEQIDNo.1 or analogue, in order to disturb the combination of goose tembusu virus and target cell specific receptor according to aforesaid method.
Polypeptide provided by the invention, by suppressing virus to suppress the infection of goose tembusu virus with the combination of target cell specific receptor, and not having an impact and side effect to host itself, making this polypeptide have very high specificity and better application prospect.
Accompanying drawing explanation
Fig. 1 shows flavivirus envelope protein E structural representation
Fig. 2 shows polypeptide design principle, the position view of the E protein of display polypeptide simulation.
Fig. 3 is that the polypeptide of synthesis has dose-dependent effect to suppressing the infection of goose tembusu virus.Final concentration is the infection that the polypeptide of 50 μ g/ml effectively can suppress goose tembusu virus, and suppression efficiency is 60%.But contrast bovine serum albumin group does not have this inhibition.
Fig. 4 shows polypeptide of the present invention can be incorporated into target cell membrane surface, disturbs light yellow virus to be combined with target cell specific receptor, thus suppresses light yellow virus infection target cell, and this combination has dose-dependent effect.But contrast bovine serum albumin group does not detect the combination of albumen and target cell.
Embodiment
The acquisition of embodiment 1 polypeptide
According to goose tembusu virus envelope protein E the 198th to the 280th amino acids sequence, design polypeptide, adopt the method for synthetic to obtain, aminoacid sequence is as shown in SEQIDNO.1.
Embodiment 2 polypeptide suppresses goose tembusu virus cells infected
1, by normal cell cultural method cultivate BHK ?21 cell to 80% saturated levels.
2, (concentration is 200TCID to the peptide inhibitor (final concentration is respectively 5,10,50 μ g/ml) embodiment 1 synthesized with goose tembusu virus
50) 4 DEG C hatch 1 hour; Control group adopts BSA (final concentration is respectively 5,10,50 μ g/ml) goose tembusu virus, and (concentration is 200TCID
50) 4 DEG C hatch 1 hour.
3, mixtures incubated in step 2 is inoculated respectively BHK ?21 cell 2 hours, remove nutrient solution, wash 3 times with PBS, add normal cell nutrient solution, cell cultures 72 hours, fluorescent quantitation RT ?PCR detect viral nucleic acid content.
Experimental result is shown in Fig. 3: (bovine serum albumin final concentration is respectively 5 to control group, 10, 50 μ g/ml) viral nucleic acid relative content is respectively 97%, 95.9%, 96.2%, (the polypeptide final concentration of embodiment 1 synthesis is respectively 5 to test group, 10, 50 μ g/ml) viral nucleic acid relative content is respectively 80.6%, 45.1%, 36.6%, by with control group comparative analysis, add the polypeptide that embodiment 1 is synthesized BHK ?in 21 cells goose tembusu virus content decline 60%, illustrate and use polypeptide of the present invention that goose tembusu virus can be suppressed to infect, cell is well protected.
The vivoexpression of embodiment 3 peptide inhibitor
Polypeptide coding genes shown in synthetic SEQIDNO.1, adds EcoRI restriction enzyme site (GAATTC) at 5 ' end, and 3 ' end adds SalI restriction enzyme site (GTCGAC).By the gene clone of synthesis between protein expression vector pET28 (a) multiple clone site EcoRI and SalI site.The gene order of this polypeptide is as shown in SEQIDNo.3, and the polypeptide of restructuring is with 6 × His label.Transform BL21 (DE3), ordinary method is cultivated and abduction delivering, obtains recombinant polypeptide inhibitor by protein expression and purification system (Ni ?NTASuperflowCartridges) by specification method purifying.SDS ?PAGE electrophoresis detection with expection consistent.Adopt the method for embodiment 2 carry out goose tembusu virus BHK ?the inhibition test copied in 21 cells, the final concentration of recombinant polypeptide is respectively 5,10,50 μ g/ml, corresponding viral nucleic acid relative content is respectively 76.8%, 43.7%, 37.2%, and result shows that it is consistent with the polypeptide result of directly synthesizing.
Embodiment 4 peptide inhibitor suppresses the mechanism analysis of goose tembusu virus cells infected
Relevant for determining that whether the retarding effect observed takies virus receptor with polypeptide competitiveness, by polypeptide and BHK ?4 DEG C, 21 cell hatch 1h, after PBS washing Yong ?the acetone of 20 DEG C of precoolings: ethanolic soln (2: 3) room temperature is fixed, the positive serum prepared with polypeptide immune mouse is primary antibodie, with FITC mark sheep anti-mouse igg be two resist, carry out Immunofluorescence test.Control group is bovine serum albumin and cell incubation.
Experimental result: polypeptide of the present invention can with BHK ?21 Cell bindings, competitive stop virus absorption onto cell thus suppress virus infection (Fig. 4).
Claims (7)
1., for suppressing a polypeptide for goose tembusu virus cells infected, it is characterized in that its aminoacid sequence is as shown in SEQIDNo.1.
2. the gene of coding polypeptide according to claim 1.
3. gene according to claim 2, is characterized in that nucleotide sequence is as shown in SEQIDNO.3.
4. the recombinant expression vector containing gene described in Claims 2 or 3.
5. the application in the medicine of the anti-goose tembusu virus of preparation of the gene described in polypeptide according to claim 1, Claims 2 or 3, recombinant expression vector according to claim 4.
6. a pharmaceutical composition for anti-goose tembusu virus, is characterized in that comprising polypeptide according to claim 1.
7. prepare the method for polypeptide described in claim 1, it is characterized in that by artificial directly synthesis or obtained by vivoexpression.
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| CN105709170B (en) * | 2014-12-18 | 2020-03-27 | 山东省农业科学院家禽研究所 | Traditional Chinese medicine compound for preventing and treating tembusu virus diseases of ducks and geese |
| CN105968180B (en) * | 2016-06-30 | 2019-08-06 | 江苏省农业科学院 | Polypeptide for inhibiting tembusu virus infection cell and its application in the drug that preparation inhibits tembusu virus |
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| 虫媒黄病毒包膜E蛋白的研究进展;邓永强;《军事医学科学院院刊》;20061231;第30卷(第6期);575-579 * |
| 鹅坦布苏病毒囊膜蛋白结构域II 在大肠杆菌中的表达及免疫学鉴定;赵冬敏;《江苏农业学报》;20150630;第31卷(第2期);619-623 * |
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