CN103060325A - Antisense nucleic acid and peptide nucleic acid PNA resisting against porcine epidemic diarrhea virus PEDV - Google Patents
Antisense nucleic acid and peptide nucleic acid PNA resisting against porcine epidemic diarrhea virus PEDV Download PDFInfo
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Abstract
The invention relates to a specific antiviral peptide nucleic acid PNA for preventing and controlling porcine epidemic diarrhea virus PEDV. An efficiently specific antisense nucleic acid sequence is designed on account of conserved gene regions of N and S in the PEDV, and a peptide nucleic acid fragment is artificially synthesized by a specific process, wherein the sequence and the conserved target genes are complementary; according to a base complementarity principle, the antisense nucleic acid sequence is specifically combined with the target genes corresponding to the N and the S in the PEDV to induce ribozyme to degrade the target genes so as to block up the transcription and expression of the target genes and inhibit the reproduction and assembly of the PEDV in vivo, so that a purpose of preventing and controlling the PEDV is achieved. The invention also relates to a pharmaceutical composition containing the antisense nucleic acid sequence and a use of the pharmaceutical composition in animal medicines. The specific antiviral peptide nucleic acid PNA can be used for specifically and directly inhibiting the reproduction of the PEDV, and has no toxic side effect, no drug resistance, good antiviral effect and no medicine residue food security problem.
Description
Technical field
The present invention relates to the antisense nucleic acid of a kind of porcine epidemic diarrhea resisting virus PEDV, the anti sense nucleotide sequence of the virus-specific that especially relates to for the conservative region of N among the PEDV and two kinds of major protein genes of S and by the peptide nucleic acid(PNA) PNA of this nucleotide sequence preparation.
Background technology
Porcine epizootic diarrhea (Porcine epidemic diarrhea, PED) be by Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) causing, take diarrhoea, vomiting, dehydration as principal character, is a kind of height contact infectious intestinal disease.Various ages in week, each product boar all can infect this disease, wherein sucking piglets, feeder pig and growing and fattening pigs are had the greatest impact, and sickness rate is up to 100%, and the sow sickness rate is 15%～90%.The PEDV characteristics of incidence is " age in days is little, severe symptoms, age in days is large, mild symptoms ", and morbidity is anxious, and is popular fast.The common meeting of 1 sucking piglets in age in week dehydration behind diarrhoea 3～4d is dead, case fatality rate about 50%.Wean pig, growing and fattening pigs symptom is lighter, and vomiting and apocleisis only occur for the sustainable 4～7d that suffers from diarrhoea, Adult Pig.Sick pig is main contagium, and after PEDV discharged with ight soil, environment, feed, drinking-water and the apparatus etc. that pollute with PEDV infected, and on occurrence regularity, its approach mainly passes through digestive tube.Normally large pig house is first, sick pig watery diarrhea after the morbidity, and ight soil is grass green, and foul smelling brings out full group's diarrhoea subsequently, then involves adjacent pig house and causes the whole audience or a certain area infection morbidity in succession.
PED is one of swine disease that occurs in the world wide, be in the news the first generation of PED of Britain in 1971, after this a plurality of countries such as Belgium, Canada, Germany, Hungary, Korea S and Japan have reported the generation of PED in succession, and PED whenever causes serious financial loss every year.Current PE D incidence is high, and incidence also becomes increasingly complex, the phenomenon of normal appearance and other virus mixed infection, thereby extremely numerous researchists' attention.China is in the generation of reported first PED in 1976, and just constantly there is the generation of PED in China in the eighties in 20th century, and diseased region spreads all over national each province, has reported that this disease all has generation in 26 provinces, cities and autonomous regions of China.PED occurs at home throughout the year, and morbidity season is multiple with autumn, winter, spring.In recent years, this sick epidemic regions has the trend that enlarges gradually.At present, PED has become one of important dysentery of China's pig industry.
Along with the continuous expansion of intensive culture scale, created favourable condition for the propagation of this disease.At present, mainly adopt vaccine inoculation to prevent and control this disease.Vaccine for PED mainly contains following several form at present: tissue inactivation seedling, PED cell deactivation vaccine, the weak malicious seedling of PED, Transgenic Plant Vaccines, lactobacillus vaccine etc.Serosurveys in Europe and other a lot of areas show that the PEDV strain only has a kind of serotype, and there is no sign and show and have different PED serotype.In long-term popular process, virus can morph with the change of envrionment conditions of living in for the environmental difference of adaptable area better.Thereby want prevention and control to live the popular of this virus, need the specific medicine of exploitation.
ICTV (ICTV) the 5th time in 1991 report is classified PED the possible member of coronavirus genus as, and 1995 the 6th time report classified it full member of coronavirus genus as.PEDV belong to shell type virales (Nidovirales) coronaviridae (Coronaviridae) α coronavirus genus (
Alphacoronavirus) the member, have the single strand plus RNA virus of the non-segmented negative of cyst membrane.The gene structure of PEDV and function PEDV genome are the sub-thread positive chain RNAs, genomic nucleic acids has infectivity, genome structure is similar to other coronavirus, 5 ' end has a cap sequence (cap), it is that 27000～33000nt(PEDV Reference strains CV777 Genome Size is 28 033 nt that 3 ' end has a Poly (A) tail, genome total length).Genome 5 ' end non-translational region (5 ' UTR) is positioned at replicative enzyme polyprotein gene upstream, and size is 296 nt; It is the leader sequence (L) of 65～98 nt and take AUG as initiator codon that 5 ' UTR contains length, has simultaneously Kozak sequence (GUUCaugC) and coding 12 amino acid whose open reading frames (ORF).Up to now, except human corona virus HCV 229E, other coronavirus members that reported have the Kozak sequence, but sequence difference to some extent.Genome 3 ' end non-translational region (3 ' UTR) length is 334 nt, end is connected with Poly (A) sequence, 3 ' UTR contains the conserved sequence that is comprised of 8 bases (GGAAGAGC), originate in 73 nt places of poly (A) upstream, all coronavirus members comprise this sequence, but the position is different in genome.The PEDV genome is near in the 3' end 5kb zone 5 main open reading frame (ORF) being arranged, the 4 kinds of structural protein of encoding: S albumen (spike protein), sM albumen (small membrane protein), M albumen (membrane protein) and N albumen (nucleoportein), be positioned at the ORF of sM upstream region of gene, called after ORF3, ORF3 gene length 675 nt, the coding Nonstructural Protein, between per two adjacent genes, gene intervening sequence (interval sequence is arranged, IS), it has 7～18 nt identical with the L sequence 3 ' end of genome and subgenomic mRNA, viral genome copy with translation process in play a significant role.The replicative enzyme polyprotein gene accounts for full genome 2/3, long 20 346 nt, comprise ORFIa(12 354 nt) and ORFIb(8 037 nt) 2 open reading frames, the overlap that 46 nt are arranged between the two, overlapping has slippery sequence (UUUAAAC) and false knot joint structure, and they can make rrna carry out frameshit reading (frameshifting) thereby the correct translation of assurance gene l.The PEDV genome structure as shown in Figure 1.
Albumen
The N gene of PEDV is one maximum among the ORF that has identified.The N gene reaches 1700bp, 441 amino acid polypeptides of encoding.The N gene is in PEDV genome 3 ' end, and the sequence of 11 Nucleotide is arranged between 3 ' end of N gene and poly (A) tail, and this sequence is all more conservative in other coronavirus of having checked order.PEDV genomic 3 ' holds the sequence of these 11 Nucleotide conservative, is the synthetic recognition site of RNA minus strand in the virus replication.Also there is a sequence that is similar to the 7bp of inner conservative gene in 5 ' end of N gene.The N gene of PEDV is cloned the earliest, so also comparatively detailed to the research of N gene.The PEDV N gene that adapts to Growth of Cells contains three inner open reading frame (internal open reading frames, I ORF), be named as respectively I-1 (113 codons), I-2 (63 codons) and I-3 (72 codons), these three I ORF are all definitely conservative.
N albumen is phosphorprotein, and relative molecular weight is 57kD.The N protein content is the abundantest in the virus infected cell.This albumen iso-electric point (pI) height lacks glycosylation and RNA binding site.This albumen is 12%～19% with the consistence of other coronavirus MHV, IBV, HCV OC43 and BCV, and the consistence of same FIPV, CCV, PRCV, TGEV and HCV229E is 32%～37%.The N albumen of PEDV and HCV229E is the most approaching.N albumen can with the virus polymerase effect, perhaps also might same Cytokine, thus change transcribing of host cell.
Albumen
The S of PEDV (spike) albumen is by the S genes encoding.Setting up password of this gene is not for starting the synthetic of protein usually, is the polypeptide of 1383 Aa of 151 kD but translate molecular weight, and this polypeptide contains 29 potential glycosylation sites, lacks the proteolysis site.Bioinformatics Prediction shows that the most of glycosyls of PEDV S gene site all has biological significance.Sequence conservation to the S gene the analysis showed that PEDV S gene has respectively 60. 6% (S2 district) and 37. 0%(S1 with the sequence of HCV229E) identical; With TGEV, FIPV compares with CCV, and the S sequence then has respectively 59. 6%-60. 0% (S1) and 35. 5%-, 35. 9%(S2 districts) identical.PEDV is identical with HCV229E flanking sequence KWPWWVWL, and this sequence is compared from KWPWYVWL only has an amino acid different, and the latter has shown conservative property in all S protein genes of measuring so far.S albumen plays an important role in immune-mediated, and S albumen also has other biological action, such as the identification target cell, promotes the fusion of virus and cytolemma.
Antisense nucleic acid (antisense nucleic acid) be one section with the natural existence of certain section sequence complementation of target gene (mRNA or DNA) or the nucleotide sequence of synthetic, antisense nucleic acid is combined by the specific and viral target gene of base pairing mode and is formed hybrid molecule, thereby copying, transcribing the expression of regulating target gene with translation skill, or induce RNase H identification and cutting mRNA, and then make its afunction.
Antisense nucleic acid comprises sense-rna (antisense RNA) and antisense DNA (antisense DNA), has synthetic convenient, the characteristics such as sequences Design is simple, easily modification, selectivity is high, avidity is strong.Antisense nucleic acid is as a kind of new antiviral, antitumor drug, started the revolution of an area of pharmacology, namely new drug receptor mRNA by novel receptor combination (Watson-Crick hybridization), cause new drug receptor in conjunction with afterreaction: the degraded of the target RNA that (1) RNase H mediates; (2) suppress processing after the copying and transcribe and transcribe of DNA and translation etc.Can say that antisense oligonucleotide (ODNs) therapy has higher specificity than traditional class of medications.From twentieth century end of the seventies till now, in this time in 30 years, antisense nucleic acid medicament has been walked out the laboratory, has entered practical clinical.After particularly first antisense nucleic acid medicament Fomivirsen went on the market by the FDA approval, people particularly paid close attention to antisense therapy.
The antisense nucleic acid action principle is based on basepairing rule, can participate in regulation and control to related gene expression by carrying out mode that base pairing is combined with target RNA.Its mode of action may have: 1. sense-rna is combined the combination that forms complementary double-stranded blocking-up rrna and virus mRNA with virus mRNA, thereby has suppressed the process that virus mRNA is translated into protein.2. antisense DNA can form a kind of three chain nucleic acid (triple helix nucleic acid) with target gene, and transcripton, enhanser and promoter region that it is transcribed by acting on controlling gene transcribe to gene.3. the combination of antisense nucleic acid and virus mRNA can stop that mRNA is to cytoplasmic transportation.4. antisense nucleic acid is combined rear so that mRNA is more easily degraded by nuclease identification with virus mRNA, thereby greatly shortens the transformation period of mRNA.Above-mentioned four kinds of action pathway all can show as inhibition or the regulation and control to viral gene expression, and this regulation and control are high degree of specificity.
Antisense nucleic acid is to identify the gene of practicing shooting by the principle of base complementrity pairing, analyze from theory, the researchist is take zooblast as example, the nearly tens pairs of bases of its karyomit(e), if the number of 4 bases (A, G, C and T) is roughly the same, and in whole gene stochastic distribution, so according to Principle of Statistics, little greater than the possibility that antisense nucleic acid and the non-target gene of 17 bases are hybridized, so it is unique that the antisense nucleic acid molecule that length surpasses 17 bases and the combination of target gene can be described as, thereby make antisense nucleic acid have specificity highly.
Studies show that, can produce 200-300 bar mRNA at the gene of a copy of cell interior, translate 100,000 and have bioactive protein molecule.The several action sites of conventional medicament Main Function on certain structural domain of the protein molecular that biological function is arranged, in fact the structure of albumen is very complicated, and in vivo, the space structure of activated protein is again Protean, dynamic and the whole function of controlling target molecule with the limited several action sites of conventional medicament is difficult to the effect that reaches desirable, thereby is not difficult to find out the limitation of conventional medicament.Can translate tens to a hundreds of albumen by mRNA, antisense nucleic acid is directly regulated and control target gene in the mRNA level, this step is equivalent to the conventional medicament effect has been amplified tens of to hundreds of times, and the regulation and control of visible antisense nucleic acid are extremely economical rationality.
Toxicologic study shows that antisense nucleic acid has very low toxicity in vivo, although its retention time in vivo has and long weak point is arranged, and the elimination that finally all will be degraded, this has been avoided such as the danger of exogenous origin gene integrator in the transgenosis therapy to the host chromosome.Compare with conventional medicament, antisense nucleic acid medicament has high specificity, and efficient height and less toxic side effect and other advantages are suppressing tumor growth and the antiviral aspect such as copy has shown good using value.At present existing a plurality of medicines enter US and European market, also have in addition more than 30 kind of antisense nucleic acid medicament carrying out having entered I, II and the experiment of III phase after the research of preclinical phase or the exploitation.
Owing to have a large amount of exonucleases in the animal body, if antisense nucleic acid without chemically modified, is degraded soon, lose activity.At present the chemically modified of antisense nucleic acid there are a lot of methods, common are thio-modification antisense nucleic acid and 2 '-methoxyl group and modify antisense nucleic acid etc.And the research of thio-modification medicine is the most comprehensive at present, and it can effectively resist the degraded of nuclease, can promote the activity of Nuclease R ase H simultaneously, and present this modifying method successfully is used for clinical antisense nucleic acid medicament.But these are the modifying method of first-generation antisense nucleic acid also, and along with development and the progress of technology, new modification approach and method are developed, so that the research of antisense nucleic acid has entered second and third generation, wherein the modification of peptide nucleic acid(PNA) is the most noticeable.
Peptide nucleic acid(PNA) (peptidenucleic acids, PNAs), be a kind of brand-new DNA analogue take neutral amido linkage as skeleton, the structural unit of its skeleton is the N(2-amino-ethyl)-glycine, base portion is connected on the amino N of main framing by the methylene radical carbonyl.Although PNA structurally relative oligonucleotide has had significant change, the base complementrity pair principle is still followed in the combination between PNA and the complementary nucleic acid, even has higher affinity than natural nucleotide.But PNA sequence-specific ground targeting is in DNA or RNA, is second and third product of antisense nucleic acid in generation.PNA forms stable PNA-DNA or PNA-RNA structure with corresponding DNA or RNA, and this structure is highly stable, the impact that changed by environmental factors, and such as ion, pH value etc.In recent years, the scientific research personnel further optimizes the PNA that develops at first, done significant improvement (connecting the type of chirality and achiral group, base etc. such as skeleton structure, at N-(2-amino-ethyl) glycine) in structure aspects, improve its biological stability and availability, target binding characteristic and pharmacokinetic properties, and PNA is applied to the aspects such as diagnosis and treatment.
Chitosan is alkaline polysaccharide unique in the natural polysaccharide, has superior functional property and the unique molecular structures such as source abundant, nontoxic, easy chemical modified, biocompatibility and recyclability.Chitosan is used for Novel Drug Delivery Systems as Biodegradable material, can greatly improve curative effect of medication by the change route of administration, have the release of control, increase targeting, minimizing stimulation and reduction toxic side effect and raising hydrophobic drug and pass through the effect characteristics such as cytolemma, increase medicine stability.
Porcine epizootic diarrhea is a kind of height contact infectious intestinal disease of the pig that caused by Porcine epidemic diarrhea virus (PEDV), and morbidity is anxious, and is popular fast, drops to principal character with watery diarrhea, vomiting, dehydration and appetite.Although developed inactivated vaccine and the attenuated vaccine of anti-PEDV, because very large variation had appearred in epidemic characteristic and the incidence of PED in recent years, brought many difficulties to preventing and controlling, pig industry is caused huge financial loss.The new tool that exploitation prevention and treatment PEDV infect seems particularly urgent, and the present invention takes the lead in peptide nucleic acid(PNA) technology and antisense technology are combined, and applies to prevention and treatment Porcine epidemic diarrhea virus.
Summary of the invention
The technical problem to be solved in the present invention is, for PEDV(N and
S) Antisensedigonucleotsequence sequence of virus-specific of conservative region design of two kinds of major protein genes, adopt that special process is synthetic, peptide is modified and chitosan packing antisense oligonucleotide, make it possess high-caliber bioavailability, stablize the curative effect of physicochemical property and highly effective and safe.
For solving the problems of the technologies described above, the present invention has adopted following technical scheme to be: this antisense nucleic acid is selected from one or more groups the combination in the following nucleotide sequence:
N-1: 5’-ttctaaggtacttgcaaataacg -3’；
N-2: 5’-ctcctacttcacgtgcaaattca -3’；
N-3: 5’-ctcttacgagattacatacaatt -3’；
S-1: 5’-gtgaaaaccagggtgtcaattca -3’；
S-2: 5’-tcgtaattgcctatttaacaaag -3’；
S-3: 5’-ttcttaaagtggatacttacaac-3’。
Anti sense nucleotide sequence is as shown in the table
Main raw and reagent:
Virus stain
The PEDV strain: CCV-10 strain, malicious valency are 10
5.2TCID
50/mL, provided by the gloomy Disease Diagnosis of Veterinary Technical Research Center of nanmu laboratory.
Cell strain
The Vero cell, the gloomy Disease Diagnosis of Veterinary Technical Research Center of nanmu laboratory provides.
Synthesizing of antisense nucleic acid
Adopt the synthesis technique of specific peptide nucleic acid(PNA) to carry out the synthetic of antisense nucleic acid.
The modification of antisense nucleic acid
In order to obtain good practical application effect, further PNA is carried out chemically modified with chitosan.
The formulation of antisense nucleic acid
1, adopt the controlled release pharmaceutical technology that the peptide nucleic acid(PNA) of modified mistake is prepared into the molten controlled release micro pill preparation of colon;
2, adopt the lyophilize pharmaceutical technology that the peptide nucleic acid(PNA) of modified mistake is prepared into freeze-drying preparation for injection;
3, adopt after the freeze-drying again granulating process that the peptide nucleic acid(PNA) of modified mistake is prepared into water-soluble granular formulation for oral use.
The medicament stability analysis
High temperature: 105 ℃ of flowing steam high temperature, sterilization in 20 minutes does not affect its biological activity.
Extreme temperature: depositing for 50 ℃ did not affect its biological activity in 6 months.
Room temperature: depositing did not affect its biological activity in 24 months.
Low temperature: depositing for-20 ℃ did not affect its biological activity in 48 months.
Antisense nucleic acid is prepared into peptide nucleic acid(PNA) PNA, is used for the specificity antivirus peptide nucleic acid(PNA) PNA of prevention and control Porcine epidemic diarrhea virus (PEDV), for PEDV(
NAnd S) gene is guarded the efficient specific anti sense nucleotide sequence of partial design, and adopts special process artificial synthesis peptide nucleic acid fragment, this sequence and the complementation of above-mentioned conservative property target gene, according to the base complementrity principle, anti sense nucleotide sequence specifically with PEDV(
NThe target gene of correspondence and S) combines, induce ribozyme that target gene is degraded, thereby block transcribing and expressing of target gene, suppress PEDV and in body, copy and assemble, to reach the purpose of prevention and control porcine epizootic diarrhea, have no side effect, have no drug resistance, the energy specificity directly suppresses Porcine epidemic diarrhea virus PEDV and copies, and antiviral effect is good, the food-safety problems such as no drug residue.
Peptide nucleic acid(PNA) PNA and urocanic acid chitosan UAC make UAC-PNA nano molecular micro-capsule; Passable, adopt chitosan that peptide nucleic acid(PNA) PNA is carried out chemically modified.
Adopt the controlled release pharmaceutical technology that modified peptide nucleic acid(PNA) PNA is later made the molten controlled release micro pill preparation of colon.
Adopt the lyophilize pharmaceutical technology that the peptide nucleic acid(PNA) of modified mistake is prepared into freeze-drying preparation for injection.
Adopt after the freeze-drying again granulating process that the peptide nucleic acid(PNA) of modified mistake is prepared into water-soluble granular formulation for oral use.
Also can contain pharmaceutically acceptable carrier or vehicle in the molten controlled release micro pill preparation of above-mentioned colon, freeze-drying preparation for injection and the water-soluble granular formulation for oral use.
Antisense nucleic acid can be used for preparing the purposes on the animal pharmaceuticals.
Description of drawings
The present invention is described in detail below in conjunction with the drawings and specific embodiments:
Fig. 1 is PEDV genome structure synoptic diagram.
Embodiment
Embodiment 1: for the peptide nucleic acid(PNA) extracorporeal antivirus effect effect analysis of PEDV
Go out the genome of PEDV from the GenBank database retrieval, particularly report in recent years the sequence of popular PEDV strain in China, carry out sequential analysis with biological software, consider the conservative property of sequence, G+C% content, then the base distribution characteristics select suitable zone design antisense nucleic acid, and last determined N and S target site for two kinds of key proteins of virus is as follows:
N albumen
N-1: 5’-ttctaaggtacttgcaaataacg -3’
N-2: 5’-ctcctacttcacgtgcaaattca -3’
N-3: 5’-ctcttacgagattacatacaatt -3’
S albumen
S-1: 5’-gtgaaaaccagggtgtcaattca -3’
S-2: 5’-tcgtaattgcctatttaacaaag -3’
S-3: 5’-ttcttaaagtggatacttacaac-3’
1.1 analyze their antiviral effects with different concns
Get the good Vero cell of growth conditions, after trysinization, be plated in the 24 porocyte plates 0.5 mL/ hole (2.0 * 10
5Individual cell), 5%CO
2Suck nutrient solution after cultivating 24 h under 37 ℃ of conditions, every hole adds 300 μ l medicine to be screened, makes diluent with perfect medium, peptide concentration (0.05 μ M, 0.1 μ M, 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M), and each medicine gradient is established 4 parallel holes.It is 0.1 ratio with the above-mentioned cell of PEDV CCV-10 virus strain infection that dosing is processed behind the 6h in MOI, 48h after infecting, and cleer and peaceful cell collections in, utilization TaqMan probe Real-time PCR standard measure detects the viral copy number of PEDV in each treatment group.Establish simultaneously the virus infection positive controls, blank group, cell negative control group.
The primer that the present invention provides with reference to Shirato K. etc. adopts real-time PCR detection by quantitative PEDV.
Primer PEDV-P1: 5 '-ACGGCGACTACTCAGC-3 '
Primer PEDV-P2: 5 '-GGGCATAAAGGGATAAT-3 '
Probe PEDV-probe:FAM-5 '-CCGCAAACGGGTGC-3 '
Calculate the inhibiting rate that different peptide nucleic acid(PNA)s produces the PEDV virus replication with following formula.
Peptide nucleic acid(PNA) extracorporeal antivirus effect result
Table 1. peptide nucleic acid(PNA) is to the anti-PEDV effect of external Vero cell
Table 1 result shows that each peptide nucleic acid(PNA) is determined the median effective dose concentration of each antisense peptide nucleic acid to the inhibiting rate that the PEDV virus replication produces by statistical analysis.
1.2 with their antiviral effects of different time point analysis
Virus infection and drug treating method are the same, with reference to the N-1 that back filters out, and N-2, N-3, S-1, S-2 and S-3 median effective dose concentration are analyzed respectively each comfortable different time points antiviral effect.After the Vero cell infection PEDV strain 48,60,72,84,96h, every hole adds 300 μ l medicines to be screened (making diluent with perfect medium), 4 parallel holes of each medicine.Dosing is collected upper cleer and peaceful cell after processing 24h, uses with TaqMan probe Real-time PCR standard measure and detects PEDV virus copy number in each treatment group, and viral inhibiting rate between each medication group of statistical study the results are shown in Table 2.
Table 2. peptide nucleic acid(PNA) is to the anti-PEDV effect of external Vero cell
1.3 drug regimen is processed
Method is the same, and for the N-X that back filters out, whether N-X, S-X and S-X(X refer to 1,2,3 wherein any one numeral) carry out determining suitable separately dosage and time point after concentration and the time gradient analysis.Then, on these experiment basis in front, the medicine of effective antivirus action of filtering out is used in combination, relatively the difference between antiviral effect of each group combination.After the Vero cell infection PEDV CCV-10 strain, add respectively N or S drug regimen, establish simultaneously virus control group and negative control group, detect with the Real-time PCR method, viral inhibiting rate between each medication group of statistical study is determined the combinatorial optimization scheme of medicine, such as table 3.
The peptide nucleic acid(PNA) of table 3. various combination is to the anti-PEDV effect of external Vero cell
1.4 cytotoxicity experiment
(1) make detected object with the Vero cell, 96 orifice plates, every hole adds 5000 cells of 100 microlitres.Drug level (0.02,0.1,0.5,1,5,10 μ m), each gradient arranges 3 repeating holes, and other establishes untreated cell contrast, the contrast of acellular substratum.
(2) after processing finished, the per 100 μ l substratum in every hole added 10 μ l MTT Stock, continue to hatch 4 hours in the 37 oC incubators.Also add again MTT Stock behind the fresh serum free medium of replaceable 100 μ l.
(3) suck substratum, every hole adds 100 μ l MTT solvating agents, keeps in each hole liquid volume consistent.
(4) measure the OD absorbancy and compare calculating at 570nm.Attention: for accurate consideration can in the absorbancy OD value of the mensuration unreduced MTT of 699 nm places itself, then be used OD
570 Deduct OD
699
(5) result judges: cell proliferation or toxicity=100% x (OD
Experiment -OD
Background )/(OD
Contrast -OD
Background ).
The OD experiment is the OD value of accepting to process cell, and the OD contrast is untreated cell control tube OD value, and the OD background is acellular substratum contrast OD value.Cell proliferation or toxicity change list are shown the percentage ratio of untreated control after processing.
Detected result shows, equal nontoxicity.
I Individual Applicant
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Street :
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＜110〉LastName: Korea Spro
＜110〉FirstName: strong precious
<110> MiddleInitial :
<110> Suffix :
Individual Applicant
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Street: Room 526, biological husbantry High Technology Center, No. 4 Nanjing, Tong Wei road, Xuanwu District
City: Nanjing
State: Jiangsu Province
Country: China
PostalCode : 210014
PhoneNumber :
FaxNumber :
EmailAddress :
＜110〉LastName: Korea Spro
＜110〉FirstName: strong precious
<110> MiddleInitial :
<110> Suffix :
Application Project
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＜120〉Title: antisense nucleic acid and the peptide nucleic acid(PNA) PNA of porcine epidemic diarrhea resisting virus PEDV
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
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＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
ttctaaggta cttgcaaata acg 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : N-1
SequenceDescription :
Sequence
--------
＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
ctcctacttc acgtgcaaat tca 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : N-2
SequenceDescription :
Sequence
--------
＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
ctcttacgag attacataca att 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : N-3
SequenceDescription :
Sequence
--------
＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
gtgaaaacca gggtgtcaat tca 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : S-1
SequenceDescription :
Sequence
--------
＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
tcgtaattgc ctatttaaca aag 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : S-2
SequenceDescription :
Sequence
--------
＜213〉OrganismName: artificial sequence
<400> PreSequenceString :
ttcttaaagt ggatacttac aac 23
<212> Type : DNA/RNA
<211> Length : 23
SequenceName : S-3
SequenceDescription :
Claims (9)
1. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV, this antisense nucleic acid are selected from one or more groups the combination in the following nucleotide sequence:
N-1: 5’-ttctaaggtacttgcaaataacg -3’；
N-2: 5’-ctcctacttcacgtgcaaattca -3’；
N-3: 5’-ctcttacgagattacatacaatt -3’；
S-1: 5’-gtgaaaaccagggtgtcaattca -3’；
S-2: 5’-tcgtaattgcctatttaacaaag -3’；
S-3: 5’-ttcttaaagtggatacttacaac-3’。
2. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 1 is characterized in that described antisense nucleic acid is prepared into peptide nucleic acid(PNA) PNA.
3. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 2 is characterized in that described peptide nucleic acid(PNA) PNA and urocanic acid chitosan UAC make UAC-PNA nano molecular micro-capsule.
4. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 2 is characterized in that, adopts chitosan that peptide nucleic acid(PNA) PNA is carried out chemically modified.
5. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 4 is characterized in that, adopts the controlled release pharmaceutical technology that modified peptide nucleic acid(PNA) PNA is later made the molten controlled release micro pill preparation of colon.
6. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 4 is characterized in that, adopts the lyophilize pharmaceutical technology that the peptide nucleic acid(PNA) of modified mistake is prepared into freeze-drying preparation for injection.
7. the antisense nucleic acid of porcine epidemic diarrhea resisting as claimed in claim 4 virus PEDV is characterized in that, adopts after the freeze-drying granulating process that the peptide nucleic acid(PNA) of modified mistake is prepared into water-soluble granular formulation for oral use again.
8. such as the antisense nucleic acid of each described porcine epidemic diarrhea resisting virus PEDV of claim 5 ~ 6, it is characterized in that, also contain pharmaceutically acceptable carrier or vehicle in the preparation.
9. the antisense nucleic acid of porcine epidemic diarrhea resisting virus PEDV as claimed in claim 1 is in the purposes for preparing on the animal pharmaceuticals.
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| CN112868929A (en) * | 2021-01-13 | 2021-06-01 | 江西正邦动物保健品有限公司 | Pig additive containing saccharicterpenin and preparation method and application thereof |
| CN114836418A (en) * | 2021-02-02 | 2022-08-02 | 中国农业大学 | CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus |
| CN115957349A (en) * | 2022-10-08 | 2023-04-14 | 华中农业大学 | Application of preparation for activating PJA1 gene expression of pig in preparation of pig epidemic diarrhea virus infection resisting medicine |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434650A (en) * | 2016-07-18 | 2017-02-22 | 张则斌 | Anti-chicken newcastle disease virus peptide nucleic acid and application thereof |
| CN106434650B (en) * | 2016-07-18 | 2017-11-17 | 张则斌 | A kind of peptide nucleic acid of anti-newcastle disease virus and application |
| CN112868929A (en) * | 2021-01-13 | 2021-06-01 | 江西正邦动物保健品有限公司 | Pig additive containing saccharicterpenin and preparation method and application thereof |
| CN114836418A (en) * | 2021-02-02 | 2022-08-02 | 中国农业大学 | CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus |
| CN114836418B (en) * | 2021-02-02 | 2024-07-12 | 中国农业大学 | CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus |
| CN115957349A (en) * | 2022-10-08 | 2023-04-14 | 华中农业大学 | Application of preparation for activating PJA1 gene expression of pig in preparation of pig epidemic diarrhea virus infection resisting medicine |
| CN115957349B (en) * | 2022-10-08 | 2024-04-05 | 华中农业大学 | Application of preparation for activating PJA1 gene expression of pigs in preparation of medicines for resisting porcine epidemic diarrhea virus infection |
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