CN106434649B - A kind of peptide nucleic acid of anti-avian infectious bronchitis virus and application - Google Patents

Abstract

The present invention relates to a kind of peptide nucleic acid of anti-avian infectious bronchitis virus, design two groups of anti sense nucleotide sequences of high specificity in the highly conserved region of gene of M and N protein for infectious bronchitis virus IBV, two groups of anti sense nucleotide sequences are specifically that M and the corresponding target gene of N protein are combined with IBV two key genes, and M and N protein are distributed in the different zones of IBV genomes, the stability and permeable membrane of antisensenucleic acids are improved.The sequence and above-mentioned target gene reverse complemental, according to base complementrity principle, anti sense nucleotide sequence is specifically combined with IBV (M and N protein) corresponding target gene, induction ribozyme equimolecular is degraded to target gene, block the transcription and expression of target gene, suppress IBV to replicate in host with assembling, to reach the purpose of prevention and control infective bronchitis disease.

Description

A kind of peptide nucleic acid of anti-avian infectious bronchitis virus and application

Technical field

Be used for the animal pharmaceuticals of prevention and control infectious bronchitis virus (IBV) the present invention relates to a kind of, more particularly, to containing There are the pharmaceutical composition of the anti sense nucleotide sequence, and its purposes in animal pharmaceuticals.

Background technology

Infective bronchitis (Infectious Brochitis, IB) refers to by infectious bronchitis virus (IBV) Cause a kind of acute, highly contagious disease of chicken, breathing, urogenital and the digestive system of main infringement chicken, infected chicken Death can be caused due to respiratory tract or renal lesions.IBV is that poultry B classes are infected as defined in International Office of Epizootics (OIE) and China One of disease.IBV susceptible animal is mainly chicken, and different cultivars is different to this sick sensitiveness with the chicken of strain, but all years The chicken in age all easily infects, especially the chicken of 3~26 week old easy infection the most.Can occur throughout the year, distribution is wide, spread speed It hurry up, morbidity and mortality are high.This disease is found in his state by Schalk and Hawn in nineteen thirty in northern section of the U.S. earliest, is initially considered that Main infringement chick, clinical manifestation is mainly that the respiratory tracts such as cough, sneeze, tracheae rale are characterized, but investigation display later should Disease shows as laying rate and declines 3%~10%, or even up to 25% in Growing Chicken and laying hen group also generally existing, laying hen, Simultaneously can not the quantity of incubation add 92%, incubation rate reduces 7%.Then many poison are being isolated in many areas in the world Strain, as Europe is separated to Dutch strain, Vac8 and VacG are separated in Australia.1972, Kuang Ronglu was proposed first in Guangzhou IB China presence and be successively separated to F strains (Foshan) and Hangzhou strain etc., then constantly have IB hair throughout the country It is existing, and there is variation phenomenon, cause between viral complicated clinical manifestation, each serotype only partially or completely without cross protection Effect, so that the preventing and treating to this disease brings very big difficulty, and brings huge economic losses to aviculture.

Pathogenic feature and traditional vaccine for IBV can not effectively control the disease etc., and researcher deploys the stream to IB Row disease learns investigation, and the parting of popular strain for implementing effective Blight control, cause of disease research and the evolution of research virus to having Significance.Current facing according to determinations such as the close preferendums, the major organs of infringement and caused clinical symptoms of viruses into tissues Bed parting has：Breathing pattern, reproduction channel type, kidney type, visible peristalsis visible intestinal peristalsis, Glandular Stomach Type and anomaly etc..Serotype analysis shows, whole world IBV It there are about cross-protection between more than 30 serotype, different serotypes poor, and emergence and the prevalence of variant, it is not ugly It is to cause the reason for immunity for chickens fails to go out this.

Had a running nose quickly after young chicken infection IBV, tracheae rale, cough, sneeze, spiritual depressed, cold crowded heap, drinking-water Increase, respiratory symptom continues to disappear after 1~4d.Gradually aggravate in subsequent chicken group sudden onset, 2~3d, diseased chicken squeezes heap apocleisis, The white loose stool of row, body weight substantially mitigates, and cockscomb and skin are dimmed, and perianal sticks white watery stools, and the young chicken of infection occurs It is dead.For laying hen, there is egg production decline in group, part is resistance to cross chicken can gradually rehabilitation, the price of deed is low and influence incubation rate etc.. Initial infection cut open inspection, which can see, a large amount of mucus in tracheae and larynx, tunica mucosa tracheae is congested, and lungs are congested, and air bag is muddy.Afterwards Phase lesion is main in kidney, and the enlargement of diseased chicken kidney, pale, renal tubule and ureterectasia, the inside are full of a large amount of white lithates, Whole kidney appearance has many decorative patterns, in betel nut sample.Cutting kidney has the white lithate outflow of volume.Several cases peritonaeum, Also there is white uric acid mineralization on pericardium and pleura, degeneration change occurs for hen oviduct, intraperitoneal to have free yolk sample Material, ovary hyperemia or bleeding, what is had is in purple.

Avian infectious bronchitis virus (infectious bronchitis virus, IBV) category coronaviridae is coronal Tobamovirus, is single strand plus RNA virus, and virion is polymorphy, but most of slightly spherical, diameter about 90~200nm, is had Cyst membrane, cyst membrane derives from the Golgi membrane or rough surfaced endoplasmic reticulum (RER) film of host cell.There are long 20nm marshallings on cyst membrane surface It is shaft-like fine prominent, it is arranged radially, is about 20nm, end globulate has wider gap between fibre is prominent.Nucleocapsid is in long filament Shape, helical symmetry, 9~13nm of diameter.IBV is inactivated through 56 DEG C of 15min and 45 DEG C of 90min, extremely sensitive to ether and chloroform, is used 0.1% 4 DEG C of deoxysodium cholate effect 18h can make IBV lose infection ability completely.Potassium permanganate, 70% alcohol and 1% formal The disinfectants such as woods can kill virus in a few minutes at room temperature.

IBV genomes are single-stranded positive RNA, total length about 27kb.5 ' ends of genome have cap and 3 ' ends to have poly A tails Structure is fawned on, is divided into 6 regions, at least 10 ORF, IBV assignment of genes gene mapping order is 5 ' Cap-1a-1b-S-3a-3b-E- M-5a-5b-N-3′Poly A.IBV replicate when using strand RNA as template it is transcribed go out 6 kinds of subgenomic mRNA s, by by it is small to Big order is named as mRNA1~mRNA6.All mRNA have 3 ' common end nesting type structures, 64 nucleosides The targeting sequencing of acid.Wherein mRNA2, mRNA4, mRNA6 are feature monocistronic mRNA, are separately encoded IBV 3 kinds of structure eggs In vain, i.e. spike protein (spike, S), memebrane protein (membrane, M albumen), nucleocapsid protein (nucleocapsid, N egg In vain), mRNA1, mRNA3, mRNA5 contain more than one ORF, are feature polycistrons.6 independent genes are compiled The structural proteins and non-structural protein of code virus, have a bit of 9 nt consensus between each gene (CUUAACAAA), its effect may participate in the transcription of downstream gene, in the 5 ' cap sequences held followed by guide's sequence Row, are about 60~70nt, and the sequence plays important work as primer in guide's primer unique IBV transcribes synthesis mechanism pattern With.The discontinuity and the incomplete check and correction mechanism of RNA polymerase replicated due to IBV geneome RNAs, the partial volume of genome ten Easily occur point mutation, missing, insertion and homologous recombination between difference strain genomes etc. so that viral serotype, genotype, Structural proteins, immunogenicity etc. easily change.

M albumen is made up of 221 amino acid, as coded by mRNA4, with three transmembrane structures, can be with S protein knot Close, it is considered to be a key signal in coronavirus assembling process.M albumen is the most glycoprotein of content in IBV particles, 40%, the M albumen chief components cross-film 3 times of viral total protein is accounted for, across cyst membrane, most of medial surface in cyst membrane, The only glycosylated N-terminal of sub-fraction is exposed at outside film, and the N-terminal of M albumen has 2 glycosylation sites.M albumen is gathered in asperities In matter net and Golgi membrane, interacted with E protein, β-Actin, M albumen has control and mediate retroviral particle is from asperities Endoplasmic reticulum or Golgi membrane budding, interact, and nucleocapsid is attached on cyst membrane in Virus assemble with nucleocapsid Effect.The targeting signal of M albumen is located at its first transmembrane region and different from the cytoplasmic tail of E protein, be co-expressed M and E Albumen can form virion.The artificial mutation of A159 and K160 sites causes IBV infectious forfeiture in M albumen.Separately there is report Road is thought, the antigenic determinant of important immunogenicity, but its immuno-biology function, the especially albumen are there is on M albumen During viral pathogenesis effect and its with virus tissue tropism relation also need to further research confirm.

N protein, N protein is one of major structural protein of coronavirus, is the constitutive protein of the nucleocapsid of virus, is one Phosphorylated protein is planted, is encoded by mRNA6, molecular weight is 46kD, phosphorylated molecular weight is 50kD, is made up of 409 amino acid , it is unique albumen being fully located in film, it is impossible to be glycosylated, deduced amino acid sequence analysis is found, wherein 17% is alkali Acidic amino acid, and 80% basic amino acid be in position it is conservative, usual gathering 66aa~88aa, 181aa~ 234aa, 334aa~regions of 373aa 3.The essential characteristic of 3 basic amine group acid regions of N protein is advantageously possible for itself and virus The combination of nucleic acid, to wrap up nucleic acid, and containing nuclear location and nuclear export signal regulation kytoplasm and karyon dynamic transport process, makes Virus genome RNA is easily assembled inside virion.In infection cell, N protein is expression quantity highest virus protein One of, it may have good immunogenicity, body can be induced to produce high-caliber antibody and mediating cytotoxicity T cell effect Should.Fan etc. obtains stable N protein N- ends (IBV-N29-160) amino acid residue segment, and (this region is similar In a SARS-CoV RNA calmodulin binding domain CaM), and its crystal structure is obtained, its crystal structure analysis is shown, the protein core It is made up of 5 antiparallel β lamellas with the beta hairpin structure of a positively charged and a hydrophobic plane, and infers this Core texture probably participates in combining viral RNA.Reed M L etc. have studied VeroV the and DF21 cells of IBV infection, hair One octapeptide (291-LQLDGLHL-298) rich in leucine of present N protein C-terminal plays a part of nuclear export signal, leads to Crossing amino acid replacement and lacking confirms that Leu296 and Leu298 is necessary to N protein is effectively exported from core.N protein is weight The immunogene wanted, plays an important role in the cellular immunity and humoral immunity for stimulating body.Research finds there is 3 in N protein Individual IBV specific b cells recognize epitope, respectively positioned at 175aa~241aa, 310aa~370aa and 360aa~409aa regions. ELISA detections show, earliest, potency also highest occurs in antibody that N protein is induced, points out N protein to have higher immunogene Property.

Antisensenucleic acids (antisense nucleic acid) is one section mutual with certain section of sequence of target gene (mRNA or DNA) The naturally occurring or artificial synthesized nucleotide sequence mended, antisensenucleic acids is specific with viral target base by base pairing mode Because combining to form hybrid molecule, so as to know in duplication, the expression of transcription and translation Level tune target gene, or induction RNase H Not and cut mRNA, and then lose its function.

Antisensenucleic acids includes antisense RNA (antisense RNA) and antisense DNA (antisense DNA), with synthesis side Just the features such as, sequences Design simply, is easily modified, selectivity is high, affinity is strong.Antisensenucleic acids as it is a kind of it is new it is antiviral, Antineoplastic, has started the revolution of an area of pharmacology, i.e., new drug receptor mRNA passes through novel receptor combination (Watson-Crick hybridization), new drug receptor is triggered to be reacted after combining：(1) the target RNA of RNase H mediations degraded； (2) processing and translation after DNA duplication and transcription and transcription etc. are suppressed.It may be said that ASON (ODNs) therapy ratio Traditional class of medications has higher specificity.From twentieth century end of the seventies till now, in this 30 years time, Antisense nucleic acid medicament has walked out laboratory, enters practical clinical.Particularly first antisense nucleic acid medicament After Fomivirsen is by FDA approval listings, people are particularly paid close attention to antisense therapy.

Antisensenucleic acids action principle is based on basepairing rule, can be by way of being combined with target RNA progress base pairings Participate in the regulation and control to related gene expression.Its mode of action may have：1. antisense RNA combines to form complementary double-strand with virus mRNA Ribosomes and virus mRNA combination are blocked, so as to inhibit viral mRNA translation into the process of protein.2. antisense DNA energy A kind of three chain nucleic acid (triple helix nucleic acid) is formed with target gene, it is by acting on control genetic transcription Transcripton, enhancer and promoter region, the transcription to gene regulates and controls.3. antisensenucleic acids and virus mRNA combination can Stop mRNA to cytoplasmic transport.4. cause mRNA is more easily recognized by nuclease to drop after antisensenucleic acids is combined with virus mRNA Solution, so as to greatly shorten mRNA half-life period.Above-mentioned four kinds of action pathway all can behave as suppression to viral gene expression or Regulation and control, and this regulation and control are high degree of specificity.

Antisensenucleic acids is that practiced shooting gene is recognized by the principle of base pair complementarity, is analyzed from theory, research Personnel are by taking zooblast as an example, about tens pairs bases of its chromosome, if the number of 4 bases (A, G, C and T) is substantially It is identical, and the random distribution in whole gene, then according to Principle of Statistics, more than antisensenucleic acids and the non-target base of 17 bases Because the possibility of hybridization is little, so combination of the length more than the antisense nucleic acid molecule and target gene of 17 bases can be described as only One, so that antisensenucleic acids has the specificity of height.

Research shows that the gene of one copy in portion can produce 200-300 bar mRNA in the cell, and translating 100,000 has The protein molecule of bioactivity.Conventional medicament mainly acts on several on certain domain of the protein molecular of biological function Action site, in fact the structure of albumen is extremely complex, and in vivo, the space structure of activated protein is thousand changes again Ten thousand changes, control the dynamic and overall function of target molecule to be extremely difficult to reason with a limited number of kind of action site of conventional medicament The effect thought, thus it is not difficult to find out the limitation of conventional medicament.Tens to hundreds of albumen, antisensenucleic acids can be translated by mRNA MRNA level in-site it is direct to target gene regulate and control, the step is tens of to hundreds of equivalent to conventional medicament effect is exaggerated Times, it is seen that the regulation and control of antisensenucleic acids are extremely economical rationality.

Toxicologic study shows, antisensenucleic acids has very low toxicity in vivo, although its in vivo when retaining Between have with short, but final eliminations that will all be degraded, this avoid as in transgenosis therapy exogenous origin gene integrator to host's dyeing Danger on body.Compared with conventional medicament, antisense nucleic acid medicament has high specificity, and efficiency high and toxic side effect are low excellent Point, good application value has been shown in terms of tumour growth and antiviral duplication is suppressed.Existing multiple medicines enter at present Enter US and European market, separately also have more than 30 kinds of antisense nucleic acid medicament to be entered after carrying out the research or development of preclinical phase Ith, II and III phase tested.

Due to there is substantial amounts of exonuclease in animal body, if antisensenucleic acids is dropped soon without chemical modification Solution, loses activity.Have many methods to the chemical modification of antisensenucleic acids at present, common are thio-modification antisensenucleic acids and 2 '- Methoxyl group modification antisensenucleic acids etc..And the research of current thio-modification medicine is the most comprehensive, it is effective against the drop of nuclease Solution, while can promote Nuclease R ase H activity, current this method of modifying is successfully used for the antisense nucleic acid medicament of clinic. But these are also the method for modifying of first generation antisensenucleic acids, with the development and progress of technology, new modification approach and method It is developed so that the research of antisensenucleic acids enters second and third generation, the modification of wherein peptide nucleic acid is the most noticeable.

Peptide nucleic acid (peptidenucleic acids, PNAs), is a kind of brand-new DNA by skeleton of neutral amide bonds Analog, can sequence specific targeting in DNA big groove, the construction unit of its skeleton is N (2- amino-ethyls)-sweet Propylhomoserin, base portion is connected in the amino N of main framing by methylene carbonyl.For the second generation product of antisensenucleic acids.

Chitosan is unique alkaline polysaccharide in natural polysaccharide, with abundance, nontoxic, chemically reactive modified, biology The superior functional character such as compatibility and recyclability and unique molecular structure.Chitosan is used for new as Biodegradable material Type delivery system, curative effect of medication is greatly improved by changing method of administration, with control release, increase targeting, reduces thorn Swash and reduce toxic side effect and improve hydrophobic drug and pass through the action characters such as cell membrane, increase medicine stability.

A kind of highly contagious disease caused by IBV is by IBV because of infective bronchitis, this is the most important disease of poultry One of disease, great economic loss is caused to domestic fowl farming.The current disease is widely current in worldwide, and harm is serious It is that the aviculture to the whole world causes huge economic loss.Because IBV clinical classifications are numerous, and Tobamovirus list underlying stock RNA diseases Poison, the factor such as gene mutation, restructuring and immune pressure may all cause avian infectious bronchitis virus to occur hereditary variation, give The sick prevention and control bring very big difficulty.The new technology of exploitation prevention and treatment IBV infection seems particularly urgent.Sense Contaminate the relevant disease triggered.The virus that the present invention is designed for the conservative region of two kinds of major protein genes of IBV (M and N protein) Specific Antisensedigonucleotsequence sequence, is synthesized, ASON is packed in peptide modification and chitosan, makes it using special process Possesses the curative effect of high-caliber bioavilability, stable physicochemical property and highly effective and safe.

The content of the invention

The present invention seeks to the present invention is designed for the conservative region of two kinds of major protein genes of IBV (M and N protein) The Antisensedigonucleotsequence sequence of virus-specific, is synthesized, ASON is packed in peptide modification and chitosan using special process, The curative effect for making it possess high-caliber bioavilability, stable physicochemical property and highly effective and safe.The present invention takes the lead in peptide nucleic acid skill Art combines with antisense technology.And applied to prevention and treatment IBV caused by infective bronchitis is by IBV There is provided the new technology that a kind of prevention and treatment IBV infects for highly contagious disease.

Present invention employs following technical scheme：A kind of peptide nucleic acid of anti-avian infectious bronchitis virus, is to be used to prevent Specificity antivirus peptide nucleic acid (ASON) animal pharmaceuticals of infectious bronchitis virus (IBV) are controlled, it is characterized in that Design the antisensenucleic acids sequence of high specificity in the highly conserved region of gene of M and N protein for infectious bronchitis virus IBV Row, and using special process be manually made peptide nucleic acid fragment (peptide nucleic acid is oligonucleotide mimetic, by first synthesizing peptide backbone, Then base is introduced.It is not to use modification approach), improve the stability and permeable membrane of antisensenucleic acids.

Two groups of anti sense nucleotide sequences, it is characterized in that two key genes, M and N protein that they can specifically with IBV Corresponding target gene is combined, and M and N protein are distributed in the different zones of IBV genomes, is below involved in the present invention one Series progression or its combination：

First group of anti sense nucleotide sequence M：

M-1：5’-UUAUAAAUAGAUUAUAUUCCU-3’

M-2：5’-AUAACACUAUCAUUUUCAGUA-3’

M-3：5’-AUUGUUGACCAUUUGUGAGGA-3’

Second group of anti sense nucleotide sequence N：

N-1：5’-AUGCAUUUCCAGAAGAACCAA-3’

N-2：5’-ACUUGAUCAACCUUAUAACCA-3’

N-3：5’-AGUAGUAAAUUCAAAUUUCAA-3’；

Described anti sense nucleotide sequence, it is the sequence preferably used to filter out M-2, M-3, N-1 and N-3.

The preferred anti sense nucleotide sequence filtered out, difference example 1 in mass ratio:(0.4~3):(0.4~3):(0.4~ 3) to M-2, M-3, N-1 and N-3 are matched, so as to form preferred sequence group.

Described antisensenucleic acids is prepared into peptide nucleic acid；By peptide nucleic acid solution and urocanic acid chitosan (UAC) solution etc. Volume is placed in 55 DEG C of constant temperature water bath 30min, and both are mixed on the mixer, and UAC-PNA nano molecular micro-capsules are prepared in coupling, And use it for preparing anti-IBV medicines.

The pharmaceutical acceptable carrier or the drug regimen of excipient used.

IBV genomes are single-stranded positive RNA, total length about 27kb.5 ' ends of genome have cap and 3 ' ends to have poly A tails Structure is fawned on, is divided into 6 regions, at least 10 ORF, IBV assignment of genes gene mapping order is 5 ' Cap-1a-1b-S-3a-3b-E- M-5a-5b-N-3′Poly A.IBV replicate when using strand RNA as template it is transcribed go out 6 kinds of subgenomic mRNA s, by by it is small to Big order is named as mRNA1~mRNA6.All mRNA have 3 ' common end nesting type structures, 64 nucleosides The targeting sequencing of acid.Wherein mRNA2, mRNA4, mRNA6 are feature monocistronic mRNA, are separately encoded IBV 3 kinds of structure eggs In vain, i.e. spike protein (spike, S), memebrane protein (membrane, M), nucleocapsid protein (nucleocapsid, N), and MRNA1, mRNA3, mRNA5 contain more than one ORF, are feature polycistrons.6 independent gene codes virus Structural proteins and non-structural protein, have a bit of 9 nt consensus (CUUAACAAA) between each gene, and it is made With that may participate in the transcription of downstream gene, in the 5 ' cap sequences held followed by leader, 60~70nt is about, should Sequence plays an important role as primer in guide's primer unique IBV transcribes synthesis mechanism pattern.Due to IBV geneome RNAs The discontinuity of duplication and the incomplete check and correction mechanism of RNA polymerase, genome very easily occur point mutation, missing, inserted Enter homologous recombination between different strain genomes etc. so that viral serotype, genotype, structural proteins, immunogenicity etc. Easily change.

Beneficial effect of the present invention：Peptide nucleic acid fragment is made using manual method in the present invention, improves the stability of antisensenucleic acids With permeable membrane.The sequence and above-mentioned target gene reverse complemental, according to base complementrity principle, anti sense nucleotide sequence specifically with IBV (M and N protein) corresponding target gene is combined, and induction ribozyme equimolecular is degraded to target gene, blocks target gene Transcription and expression, suppress IBV and are replicated in host and assembling, to reach the purpose of prevention and control infective bronchitis disease.This hair It is bright to further relate to the pharmaceutical composition containing the anti sense nucleotide sequence, and its purposes in animal pharmaceuticals.The nontoxic secondary work of the present invention With, have no drug resistance, can specificity directly suppress IBV duplications, antiviral effect is good, the food-safety problem such as no drug residue.For IBV Peptide nucleic acid extracorporeal antivirus effect result, antisensenucleic acids shows to the anti-IBV effects of external Vero cells through quantitative PCR detection result：M Special peptide nucleic acid, M-1, M-2, M-3 inhibiting rate is respectively:75%, 83% and 89%, it is seen that M-3 effects are preferable.N is specifically anti- Adopted peptide nucleic acid, in addition to N-2 DeGrains, N-1X and N-3 inhibiting rate are respectively 92% and 86%.But mixed during actual use The peptide nucleic acid of sequence is more preferable.

Embodiment：

The designed anti sense nucleotide sequence of invention：

Technical scheme

Main material and reagent

Virus stain

IBV strains：NS-IBV-57 plants, provided by the gloomy Disease Diagnosis of Veterinary Technical Research Center laboratory of nanmu.

Cell line

Vero cells：Shanghai cell institute of the Chinese Academy of Sciences.

The synthesis of antisensenucleic acids

The artificial synthesized of antisensenucleic acids is carried out using synthesis technique.

The modification of antisensenucleic acids

In order to obtain good practical application effect, peptide backbone is used to build and modify antisensenucleic acids to form stable physics and chemistry The peptide nucleic acid (PNA) of performance and particular space structure

The modification of peptide nucleic acid

Peptide nucleic acid is modified by trim of chitosan.

The formulation of peptide nucleic acid

1st, the peptide nucleic acid through modified is prepared into by the molten controlled-releasing microcapsule preparation of colon using controlled release pharmaceutical technology；

2nd, the peptide nucleic acid through modified is prepared into by injection freeze-dried powder using freeze-drying pharmaceutical technology；

3rd, using the peptide nucleic acid through modified is prepared into water-soluble granular for oral use by granulating process again after freezing.

Agent stability is analyzed

High temperature：105 DEG C of flowing steam high temperature, sterilizing in 20 minutes does not influence its bioactivity.

Extreme temperature：50 DEG C of storages do not influence its bioactivity in 6 months.

Room temperature：Storage does not influence its bioactivity in 24 months.

Low temperature：- 20 DEG C of storages do not influence its bioactivity in 48 months.

Embodiment is directed to anti-IBV peptide nucleic acid extracorporeal antivirus effect effect analysis

Go out IBV genome from GenBank database retrievals, be particularly the sequence of the IBV strains that China is reported in recent years Row, sequence analysis is carried out with biological software, considers the conservative of sequence, G+C% contents, base distribution feature, then Select suitable region design antisensenucleic acids, the anti sense nucleotide sequence of the last identified M and N genes for virus It is as follows.

M

M-1：5’-UUAUAAAUAGAUUAUAUUCCU-3’

M-2：5’-AUAACACUAUCAUUUUCAGUA-3’

M-3：5’-AUUGUUGACCAUUUGUGAGGA-3’

N

N-1：5’-AUGCAUUUCCAGAAGAACCAA-3’

N-2：5’-ACUUGAUCAACCUUAUAACCA-3’

N-3：5’-AGUAGUAAAUUCAAAUUUCAA-3’

Inhibition level of the peptide nucleic acid to viral target gene is detected using IBV virus-specifics quantitative RT-PCR, while with Viral titer determination experiment detects antiviral titre.

Day 1:

Bed board：The Vero cells of early-stage preparations are digested, are collected by centrifugation, cell count, with complete medium (DMEM+5% tires Cow's serum+mycillin) cell concentration is transferred to 3~6 × 10⁵Individual/ml, spreads 24 orifice plates, in 37 DEG C of CO2gas incubators Carry out culture 18-24 hours.

Day 2:

The density of micro- sem observation cell, when cell covers with the 50~70% of orifice plate area, and cell state is good.Inhale Nutrient solution is removed, 300 μ l medicines to be screened, each 10 holes of medicine are added per hole.After incubating 1 hour, 100 μ l IBV are added (1000PFU/mL), after adsorbing 2 hours, is washed away after unadsorbed virus with nutrient solution, adds the DMEM cultures containing 4%FBS Base, in 37 DEG C and 5%CO₂Continue to cultivate in environment, cytopathy is regularly observed after infection.72h after infection, infection cell is entered Row multigelation carries out Viral diagnosis with releasing virus as sample.Also set in experimentation and be not added with virus and peptide nucleic acid Normal cell controls group, plus virus, be not added with peptide nucleic acid positive controls and add peptide nucleic acid medicine, be not added with virus negative control Group.

Day 1-3:

Protective effect of the medicine to cell is observed, and result is judged.

Real-time PCR are quantitatively detected

The supernatant in above-mentioned each treatment group is collected, viral RNA is extracted with virus total RNA extracts kit.To acquisition Viral RNA carries out reverse transcription into cDNA first, then detects the viral level in IBV treatment groups respectively with the primer being directed to. Result after quantitative amplification, calculates virus titer in each treatment group, inhibition is with PNA groups and blank with statistics software The multiple of control group difference.

The primer that the present invention is provided with reference to (2010) such as Meir R, IBV is quantitatively detected using real-time PCR.

IBV specific detection primers

Primer 1:5′-ATGCTCAACCTTGTCCCTAGCA-3′

Primer 2:5’-TCAA-ACTGCGGATCATCACGT-3’

GAPDH is internal reference (Kuchipudi S V etc.)

Primer 1:5′-GAAGCTTACTGGAATGGCTTTCC-3′

Primer 2:5′-CGGCAGGTCAGGTCAACAA-3′

Reaction system (25 μ l)

Reaction condition：Reverse transcription, 42 DEG C of 5min, 95 DEG C of 10sec；

PCR is expanded：Cycle：40,95 DEG C of 5sec, 55 DEG C of 30sec, 72 DEG C of 30sec.

Reaction confirms Real Time One Step RT-PCR amplification curve and melt curve analysis after terminating, to ensure knot The specificity and reliability of fruit.

For IBV peptide nucleic acid extracorporeal antivirus effect result：

Quantitative PCR detection result shows, the special peptide nucleic acids of M, M-1, M-2, and M-3 inhibiting rate is respectively:75%, 83% He 89% (subordinate list 1), it is seen that M-3 effects are preferable.N specific antisense peptide nucleic acids, in addition to N-2 DeGrains, N-1X and N-3 suppression Rate processed is respectively:92% and 86% (subordinate list 1)

The antisensenucleic acids of subordinate list 1. is to the anti-IBV effects of external Vero cells

Drug regimen processing

Ibid, the M-2/3 filtered out for back, N-1/3 is preferred sequence to method.Then, in above these On experiment basis, the medicine of effective antivirus action to filtering out, which is combined, to be used, the antiviral effect after comparison combination With the difference between single medicine antiviral effect.After NS-IBV-57 plants of Vero cell infection IBV, it is separately added into for M's or N Genomic medicine is combined, while setting positive control and negative control and blank control, is detected with Real-time PCR methods, Viral suppression between each medication group of statistical analysis, determines the combination of medicine, such as subordinate list 2.

The antisense peptide nucleic acid of the various concentrations of subordinate list 2. is to the anti-IBV effects of external Vero cells

Cytotoxicity experiment：

1) detection object is made with Vero cells, 96 orifice plates add 100 microlitres of 5000 cells per hole.Drug concentration (0.02, 0.1,0.5,1,5,10 μm), each gradient sets 3 repeating holes, separately sets untreated cell control, cell-free medium control.

2) after processing terminates, per hole, every 100 μ l culture mediums are added continues to be incubated in 10 μ l MTT Stock, 37 DEG C of incubators 4 hours.Also MTT Stock are added after replaceable 100 μ l fresh serum-free medias again.

3) culture medium is sucked, 100 μ l MTT lytic agents are added per hole, keeps liquid volume in each hole consistent.

4) OD absorbances are determined in 570nm and is compared calculating.Note：It can be determined not at 699nm for accurate consideration The absorbance OD values of the MTT of reduction in itself, then use OD₅₇₀Subtract OD₆₉₉。

5) result judges：Cell is bred or toxicity=100%x (OD_Experiment- OD_Background)/(OD_Control- OD_Background)。

OD experiments are the OD values for receiving processing cell, and OD controls are untreated cell control tube OD values, and OD backgrounds are without thin Born of the same parents' culture medium compares OD values.Cell propagation or toxicity change are expressed as the percentage of untreated control after processing.

Testing result shows, non-toxic for IBV peptide nucleic acids.

IBV peptide nucleic acid antiviral effect in chicken embryo：

180 pieces of the SPF chicken embryos of 10 ages in days are taken, 6 groups, every group 30 pieces, every piece of 200 μ of IBV (5000PFU/mL) are randomly divided into L, peptide nucleic acid consumption is 3 μ g/ embryos, each sample set 3 it is parallel, put 37 DEG C of incubators and continue to hatch, during which observing idiosome does not have meat Eye lesions visible, collects chick embryo allantoic liquid after inoculation 24h, and quantitative RT-PCR detection IBV virus multiplication inhibiting rates see attached list 3：

Anti- IBV effect of the antisense peptide nucleic acid of the various concentrations of subordinate list 3. in chicken embryo

Experimental animal infection and raising：

(department of nanmu gloomy Disease Diagnosis of Veterinary center experimental animal plant provides healthy 17 age in days SPF chick totally 300, after testing IBV is negative) it is randomly divided into 6 groups.Wherein IBV infected groups (test group) 50, attack malicious mode, 100 μ l/ are only using collunarium；Blank Control group (control group) totally 50, does not make any processing.Three drug dose groups (25ppm, 50ppm, 100ppm), using drinking-water Administering mode, strict by group isolated rearing, the feed of each group individually prepares with drinking-water, not intersected.

After IBV infection, observation in detail day by day counts morbidity number and death toll, Computation immunity protection to 15day after poison is attacked Rate.Sample is gathered simultaneously, total serum IgE is extracted, and carries out the recall rate of IBV in RT-PCR detections, analysis each group, such as subordinate list 4.

The anti-IBV of the of table 4 peptide nucleic acid clinical drug experiment

Final preferred dosage is 50~100ppm.

Claims (2)

1. a kind of peptide nucleic acid of anti-avian infectious bronchitis virus, it is characterized in that for infectious bronchitis virus IBV M Two groups of anti sense nucleotide sequences of high specificity, two groups of anti sense nucleotide sequences are designed with the highly conserved region of gene of N protein It is specifically that M and the corresponding target gene of N protein are combined with IBV two key genes, and M and N protein are distributed in IBV genes The different zones of group：

First group of anti sense nucleotide sequence M

M-2：5’-AUAACACUAUCAUUUUCAGUA-3’

M-3：5’-AUUGUUGACCAUUUGUGAGGA-3’

Second group of anti sense nucleotide sequence N

N-1：5’- AUGCAUUUCCAGAAGAACCAA-3’

N-3：5’- AGUAGUAAAUUCAAAUUUCAA-3’.

2. the peptide nucleic acid of anti-avian infectious bronchitis virus according to claim 1, it is characterized in that the antisensenucleic acids Sequence, difference example 1 in mass ratio: 0.4~3: 0.4~3:0.4 ~ 3 couple of M-2, M-3, N-1 and N-3 are matched, so as to be formed Sequence group.