CN109456392A - It is a kind of inhibit Porcine epidemic diarrhea virus infection polypeptide and application - Google Patents
It is a kind of inhibit Porcine epidemic diarrhea virus infection polypeptide and application Download PDFInfo
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- CN109456392A CN109456392A CN201811362139.5A CN201811362139A CN109456392A CN 109456392 A CN109456392 A CN 109456392A CN 201811362139 A CN201811362139 A CN 201811362139A CN 109456392 A CN109456392 A CN 109456392A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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Abstract
The invention discloses a kind of polypeptide of inhibition Porcine epidemic diarrhea virus infection and application, the amino acid sequences of the polypeptide are as follows: NATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDL.The present invention devises the derived peptides based on HR2 by amino acid alignment and is synthesized according to Bioinformatics Prediction PEDV HR2.The present invention inhibits PEDV infection host's intestinal epithelial cell IPEC-J2 process using the HR2 derived peptides of synthesis；It is identified by quantitative fluorescent PCR, synthesis polypeptide has the function of inhibiting virus infection host cell, can be applied to anti-PEDV infection.Polypeptide abundant information provided by the invention PEDV fusion core function, for follow-up study PEDV film syncretizing mechanism provides reference；Polypeptide provided by the invention can be applied to preparation PEDV antiviral vaccine and drug.
Description
Technical field
The present invention relates to virology, bioinformatics and cell biologies, popular more particularly to a kind of inhibition pig
Property diarrhea virus (porcine epidemic diarrhea virus, PEDV) infect polypeptide and its application.
Background technique
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is a kind of high degree in contact chitling road transmission
Disease, using morbid pig vomiting, watery diarrhea, dehydration as main feature.PED can impact the pig of each age level, especially
There is high lethal to the suckling pig within 7 ages in days, case fatality rate is up to 100%.PED has caused China's pig breeding industry huge
Economic loss becomes and restricts one of the infectious disease that domestic pig breeding industry develops in a healthy way.PED cause of disease Porcine epidemic diarrhea virus (PED
Virus, PEDV) it is a kind of single strand plus RNA virus with cyst membrane, belong to shell type virales (Nidovirales) coronal disease
Malicious section's (Coronaviridae) α-type coronavirus genus (Alphacoronavirus) has characteristic coronavirus cyst membrane
" fibre is prominent " structure.Wherein, PEDV Spike Glycoprotein (spike glycoprotein, S) repeats 1st area containing conservative septivalency
(heptad repeat1, HR1) and septivalency repeat 2nd area (heptad repeat 2, HR2).HR1 and HR2 are ultimately formed in fusion
The heart (fusion core) completes virus envelope and with host cell target membrane merges this critical process, play it is important cause a disease with
Immunologic function.Although having the antiviral research for other coronavirus HR2, it is based on the antiviral sense of PEDV HR2
Dye still needs to be illustrated comprehensively.
Summary of the invention
For the deficiency of existing research, the object of the present invention is to provide a kind of the more of inhibition Porcine epidemic diarrhea virus infection
Peptide and application.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of polypeptide inhibiting Porcine epidemic diarrhea virus infection, amino acid sequence are as follows: NATYLNLTGEIADLEQRS
ESLRNTTEELQSLIYNINNTLVDL。
Using the polypeptide sequence as core, any optimization to the polypeptide sequence, modification, including being cut to polypeptide fragment
It is short.
A kind of polypeptide is preparing the application in pig epidemic diarrhea antiviral vaccine and drug.
The present invention devises spreading out based on HR2 according to Bioinformatics Prediction PEDV HR2, by amino acid alignment
Raw polypeptide is simultaneously synthesized.The present invention infects host's intestinal epithelial cell IPEC-J2 to PEDV using the HR2 derived peptides of synthesis
Process is inhibited；It is identified by quantitative fluorescent PCR, synthesis polypeptide has the function of inhibiting virus infection host cell, can answer
It is infected for anti-PEDV.
The invention has the advantages that:
1, the technologies such as present invention comprehensive utilization virology, bioinformatics and cell biology, provide a kind of inhibition
The polypeptide of PEDV infection can inhibit PEDV infection host cell using synthesis polypeptide of the present invention；
2, polypeptide abundant information provided by the invention PEDV fusion core function merges for follow-up study PEDV film
Mechanism provides reference；
3, polypeptide provided by the invention can be applied to preparation PEDV antiviral vaccine and drug.
Detailed description of the invention
Fig. 1 is PEDV HR1 and HR2 prediction case.
Fig. 2 is PEDV HR1 and HR2 amino acid sequence schematic diagram.
In figure, PEDV amino acid sequence is upper, and HCoV-NL63 amino acid sequence is under；Amino acid sequence the 982nd to
1121, the 1278th to the 1317th respectively represent the HR1 and HR2 (narrow frame) of prediction, the 1005th to the 1061st,
1273 to the 1314th respectively represent HR1 the and HR2 derived peptides (wide frame) of design.
Fig. 3 is PEDV HR1 and HR2 derived peptides signature analysis situation.
In figure, septivalency, which repeats amino acid residue position, to be indicated with a, b, c, d, e, f and g, wherein the amino of the position a and d
Sour residue is hydrophobic amino acid or the amino acid containing very bulky side chain.
Fig. 4 is that HR1 and HR2 synthesis derived peptides inhibit PEDV infection host cell situation.
In figure, NS indicates that p>0.05, * * * indicate p<0.001.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Embodiment 1.PEDV HR2 prediction
Pass through online software LearnCoil-VMF (http://night-ingale.lcs.mit.edu/cgi-bin/
Vmf) HR1 and HR2 of PEDV CH/hubei/2016 strain S protein are predicted.The identification of LearnCoil-VMF software is typical
HR feature, i.e., about each own 3~4 septivalencys repeat in HR1 and HR2, and each repetition is from leucine or isoleucine
Start.As a result as shown in Figure 1, the HR1 and HR2 of the prediction of PEDV S protein are located at 982-1121 amino acids and the
1278-1317 amino acids.
The design of embodiment 2.PEDV HR2 derived peptides
Human corona virus NL63 (HCoV-NL63) HR1 the and HR2 ammonia parsed with reference to fusion core crystal structure
Base acid sequence (Fig. 2, wide frame), is designed PEDV HR1 and HR2 derived peptides, finally chooses 1005-1061 respectively
As HR1 and HR2 derived peptides sequence, amino acid sequence is respectively SAIGNITS for amino acid and 1273-1314 amino acids
AFESVKEAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQ (SEQID NO.1) and NATYLNLTGEIAD
LEQRSESLRNTTEELQSLIYNINNTLVDL(SEQ ID NO.2).Through analyzing, amino acid composition meets coronavirus seven
The septivalency of valence repeated characteristic, i.e., one group proper alignment repeats amino acid residue, and resi-dues can use a, b, c, d, e, f and g table
Show, wherein the amino acid residue of the position a and d is hydrophobic amino acid or the amino acid (Fig. 3) containing very bulky side chain.By HR1 and
HR2 derived peptides transfer to Shanghai gill biochemistry Co., Ltd to synthesize, and purity is all larger than 95%.
Embodiment 3.PEDV HR2 derived peptides inhibit PEDV to infect host cell
3.1PEDV HR2 derived peptides inhibit PEDV proliferation
PEDV host's intestinal epithelial cell IPEC-J2 is pressed 2 × 105A/mL, 500 holes μ L/ are laid with 24 orifice plates, in 37 DEG C
CO2It is cultivated for 24 hours in incubator.PEDV CH/hubei/ is diluted respectively using plasma-free DMEM medium (Gibco company, the U.S.)
2016 strains and HR1, HR2 synthesize derived peptides, and virus infection plural number (MOI) is 1, synthesize the final concentration of 0.1- of derived peptides
1000nM, to be control containing only the DMEM culture medium of virus and dimethyl sulfoxide (DMSO).By the virus and final concentration of MOI=1
After the synthesis derived peptides mixing of 0.1-1000nM, 37 DEG C of placement 1h.Using PBS board-washing 3 times, 500 μ L mixing is added in every hole
Three repeating holes are arranged in liquid, every group of processing cell.After 37 DEG C of incubation 1h, PBS board-washing 3 times, wash away not with IPEC-J2 cell knot
The virus and synthesis derived peptides of conjunction, are added 500 μ L cell maintenance mediums (penicillin containing 100U/mL, 100 μ g/mL streptomysins, matter
Measure the DMEM culture medium of score 0.3% tryptone phosphate broth and 0.03% yeast extract powder of mass fraction), 37 DEG C of cultures
12h.It discards supernatant, collects cell, the content of intracellular infection virus is detected by fluorescence quantifying PCR method.The experiment is independent
In triplicate.
3.2 fluorescence quantitative PCR detection PEDV HR2 derived peptides inhibit PEDV to infect host cell
The total serum IgE that reagent TRIzol (Invitrogen company, the U.S.) extracts PEDV infection cell is extracted using RNA, is used
PrimeScriptTMReverse transcription reagent box (TaKaRa company) generates complementary DNA (cDNA) and is used as quantitative fluorescent PCR template.Using
Relative fluorescence quantitative PCR is measured the viral level in infection cell, is indicated with PEDV nucleocapsid protein (N) mRNA content
The content of virus, with glyceraldehyde 3-phosphate dehydro-genase (GAPDH) for internal reference, using 2-ΔΔCtMethod analyzes data.Primer
Tables 1 and 2 is seen with reaction system, and response procedures carry Fast using ABI fluorescence quantitative PCR instrument and (U.S. Applied is sequenced
Biosystems company).
1 relative fluorescence quantification PCR primer sequence of table
2 relative fluorescence quantitative PCR reaction system of table
Experimental data indicates that statistical analysis is non-using Origin software with cell mean and standard error of mean (SEM)
It matches double tail Student t and examines progress.P>0.05 is no difference of science of statistics, and p<0.05 is to have statistical difference, and p<0.01 is
There is significant statistical difference, p < 0.001 is to have highly significant statistical difference.
As a result such as Fig. 4 is shown, 0.1-1000nM HR1 synthesizes derived peptides without obvious inhibiting effect, and HR2 is synthesized
Derived peptides can significantly reduce the content of intracellular virus under 0.1nM low concentration, show that HR2 synthesizes derived peptides
Have the function of inhibiting virus infection host cell.
The foregoing is merely preferred embodiments of the present invention, and for those skilled in the art, the present invention can have
Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all
It is included within protection scope of the present invention.
Sequence table
<110>Henan Academy of Agricultural Sciences
<120>a kind of polypeptide for inhibiting Porcine epidemic diarrhea virus infection and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57
<212> PRT
<213>pig epidemic diarrhea (Porcine Epidemic Diarrhea Virus)
<400> 1
Ser Ala Ile Gly Asn Ile Thr Ser Ala Phe Glu Ser Val Lys Glu Ala
1 5 10 15
Ile Ser Gln Thr Ser Lys Gly Leu Asn Thr Val Ala His Ala Leu Thr
20 25 30
Lys Val Gln Glu Val Val Asn Ser Gln Gly Ala Ala Leu Thr Gln Leu
35 40 45
Thr Val Gln Leu Gln His Asn Phe Gln
50 55
<210> 2
<211> 42
<212> PRT
<213>pig epidemic diarrhea (Porcine Epidemic Diarrhea Virus)
<400> 2
Asn Ala Thr Tyr Leu Asn Leu Thr Gly Glu Ile Ala Asp Leu Glu Gln
1 5 10 15
Arg Ser Glu Ser Leu Arg Asn Thr Thr Glu Glu Leu Gln Ser Leu Ile
20 25 30
Tyr Asn Ile Asn Asn Thr Leu Val Asp Leu
35 40
<210> 3
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 3
cgcaaagact gaacccacta ac 22
<210> 4
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 4
ttgcctctgt tgttacttgg agat 24
<210> 5
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 5
ccttccgtgt ccctactgcc aac 23
<210> 6
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 6
gacgcctgct tcaccacctt ct 22
Claims (3)
1. a kind of polypeptide for inhibiting Porcine epidemic diarrhea virus infection, which is characterized in that its amino acid sequence are as follows: NATYLNLTG
EIADLEQRSESLRNTTEELQSLIYNINNTLVDL。
2. the polypeptide according to claim 1 for inhibiting Porcine epidemic diarrhea virus infection, which is characterized in that with the polypeptide
Sequence is core, any optimization to the polypeptide sequence, modification, including the truncation to polypeptide fragment.
3. a kind of polypeptide as claimed in claim 1 or 2 is preparing the application in pig epidemic diarrhea antiviral vaccine and drug.
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| CN201811362139.5A CN109456392A (en) | 2018-11-15 | 2018-11-15 | It is a kind of inhibit Porcine epidemic diarrhea virus infection polypeptide and application |
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| CN201811362139.5A CN109456392A (en) | 2018-11-15 | 2018-11-15 | It is a kind of inhibit Porcine epidemic diarrhea virus infection polypeptide and application |
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| CN109456392A true CN109456392A (en) | 2019-03-12 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117695370A (en) * | 2023-12-19 | 2024-03-15 | 青岛市畜牧工作站(青岛市畜牧兽医研究所) | Application of oligopeptide in preparation of porcine epidemic diarrhea virus inhibition drugs |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063801A2 (en) * | 2003-12-30 | 2005-07-14 | Universiteit Utrecht Holding B.V. | Method for decreasing the contact between hepta-repeat region s of the corona-virus spike proteins and use thereof |
| CN107698665A (en) * | 2017-10-26 | 2018-02-16 | 浙江大学 | A kind of antiviral polypeptide, encoding gene, carrier, Host Strains and application |
| WO2018081318A1 (en) * | 2016-10-25 | 2018-05-03 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Prefusion coronavirus spike proteins and their use |
-
2018
- 2018-11-15 CN CN201811362139.5A patent/CN109456392A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063801A2 (en) * | 2003-12-30 | 2005-07-14 | Universiteit Utrecht Holding B.V. | Method for decreasing the contact between hepta-repeat region s of the corona-virus spike proteins and use thereof |
| WO2018081318A1 (en) * | 2016-10-25 | 2018-05-03 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Prefusion coronavirus spike proteins and their use |
| CN107698665A (en) * | 2017-10-26 | 2018-02-16 | 浙江大学 | A kind of antiviral polypeptide, encoding gene, carrier, Host Strains and application |
Non-Patent Citations (1)
| Title |
|---|
| LI,D.: ""S protein [Porcine epidemic diarrhea virus] ，GenBank: ART84225.1"", 《GENBANK》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117695370A (en) * | 2023-12-19 | 2024-03-15 | 青岛市畜牧工作站(青岛市畜牧兽医研究所) | Application of oligopeptide in preparation of porcine epidemic diarrhea virus inhibition drugs |
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