WO2013189003A1 - J亚群禽白血病病毒的肽核酸及其应用 - Google Patents

J亚群禽白血病病毒的肽核酸及其应用 Download PDF

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WO2013189003A1
WO2013189003A1 PCT/CN2012/001674 CN2012001674W WO2013189003A1 WO 2013189003 A1 WO2013189003 A1 WO 2013189003A1 CN 2012001674 W CN2012001674 W CN 2012001674W WO 2013189003 A1 WO2013189003 A1 WO 2013189003A1
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nucleic acid
peptide nucleic
sequence
virus
alv
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French (fr)
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韩健宝
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Han Jianbao
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4808—Preparations in capsules, e.g. of gelatin, of chocolate characterised by the form of the capsule or the structure of the filling; Capsules containing small tablets; Capsules with outer layer for immediate drug release
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/11011—Alpharetrovirus, e.g. avian leucosis virus
    • C12N2740/11022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a peptide nucleic acid of a subgroup J avian leukosis virus and uses thereof.
  • Avian leukemia is a type of infectious disease caused by avian leukosis virus (ALV), which is mainly caused by hematopoietic malignant proliferation, including lymphocytic leukemia, erythroblastic leukemia, myeloblastic leukemia and myeloid leukemia.
  • ABV avian leukosis virus
  • LL avian lymphoblastic leukemia
  • a ⁇ J subgroup including reticuloendotheliosis virus All retroviruses that cause avian neoplastic diseases.
  • A, B, C, and D are exogenous viruses
  • E, F, G, H, and I are endogenous viruses.
  • the A and B subgroups are the most common exogenous virus subgroups of commercial laying hens (Laihang chicken), and the (D subgroups have extremely low infection rates, which are almost difficult to detect; E subgroups include ubiquitous low pathogenicity Sexual and non-pathogenic endogenous virus.
  • J subgroup was first isolated from commercial broiler chickens by colleagues such as Payne in 1988. J subgroup is an exogenous leukemia virus, which is endogenous. The recombinants of the E subgroup can be widely spread in the flocks by horizontal and vertical transmission.
  • ALV-J infection can cause multiple infections in chickens.
  • viruses can be detected simultaneously in individual diseased chickens, such as reticuloendotheliosis virus (REV), infectious bursal disease virus, and chicken infectious anemia virus.
  • REV reticuloendotheliosis virus
  • infectious bursal disease virus infectious bursal disease virus
  • chicken infectious anemia virus In the actual production process, people usually only pay attention to the impact of tumor formation on production, and often ignore its subclinical infection.
  • Immunosuppression may include lymphoid atrophy or hypoplasia, hypergamma gamma globulin increase, mitogen-induced reduction in blastogenesis, and decreased antibody response. Therefore, the loss of ALV to the poultry industry is mainly caused by its pre-neoplastic disease (immunosuppression), and the death caused by tumor in the later stage is only a superficial phenomenon of immunosuppression from quantitative to qualitative.
  • the clinical symptoms of sick chickens are mainly characterized by loss of appetite and progressive elimination; abnormal feathers, poor mental state of the diseased chickens; some chickens have swollen abdomen and can touch the swollen liver; cockscombs and flesh are pale, and some cocks are atrophied; Some heads, backs, chests, legs and wings can be seen in blood vesicles of 1 ⁇ 3 cm in size. They are brownish purple, soft in texture, have certain elasticity, and have clear boundaries with surrounding skin. Blood blister ruptures after blood rupture. The surrounding feathers are contaminated by large pieces of blood.
  • Immunosuppression refers to the phenomenon that the body's ability to respond to antigens is low or even absent due to various factors (such as nutrition, disease, stress, etc.). Among them, the immunosuppression caused by the virus is particularly serious, thereby forming immunosuppression.
  • ALV-J causes adult broiler myeloma, which results in high mortality and also reduces fertility (due to affecting the development of the cock). Due to the death and fertility of the hens, the hatching eggs are reduced, resulting in serious losses in the broiler breeding industry.
  • ALV is more important to damage immune organs, such as severe damage to the main immune organs such as chicken thymus and bursa, which reduces the function of immune organs and reduces the disease resistance of the body. , eventually leading to complications and secondary infections.
  • ALV-J is difficult to relieve immunosuppression, so that the flock does not respond to vaccine immunization or decline in response ability, resulting in immune failure, causing outbreaks of severe infectious diseases.
  • the virion is 80 ⁇ 100 nm in diameter and consists of an outer capsule and an internal electron-tight nucleocapsid.
  • the core structure consists of diploid RNA and nucleocapsid, reverse transcriptase, integrase, and protease. Face. There is a radial protrusion on the viral capsule with a diameter of about 8 ran.
  • the ALV genome is about 7. 2 kb in length and can be directly used as mRNA.
  • the ALV structural gene is gag-pol-env from the 5' end to the 3' end, encoding viral structural proteins, RNA-dependent DNA polymerases (reverse transcriptases), and envelope glycoproteins, respectively.
  • the gag gene encodes a viral internal non-glycosylated structural protein, including matrix proteins, proteases, capsids, and nucleocapsids. Among the ALV subgroups, these viral proteins are highly conserved and highly homologous, so-called group-specific antigens or
  • the GSAc pol gene encodes a viral reverse transcriptase and integrase to complete the process of integrating the virus into the pre-virus as well as the proviral DNA before integration into the cell chromosome.
  • the erw gene encodes a viral envelope glycosylation protein. These include the membrane surface glycoprotein subunit (SU) gp85 and the transmembrane glycoprotein subunit (TM) gp37.
  • Acute transformed ALV also carries a viral tumor gene (v2 0nC0gene ).
  • the virus contains five structural proteins, the virus-specific antigen capsid protein P27, the viral basement membrane protein P19, the nucleocapsid protein P12 involved in RNA processing and packaging, and the aspartase P15 involved in protein precursor cleavage. Unknown P10.
  • a variant of P27, P270, is present in the endogenous virus RAV-0 strain.
  • An antisense nucleic acid is a naturally occurring or synthetic nucleotide sequence complementary to a certain sequence of a target gene (mRNA or DNA), and the antisense nucleic acid is specifically associated with a viral target gene by base pairing Binding to form a hybrid molecule, thereby regulating the expression of the target gene at the level of replication, transcription and translation, or inducing RNase H to recognize and cleave the mRNA, thereby losing its function.
  • Antisense nucleic acids include antisense RNA and antisense DNA, which are easy to synthesize, simple in sequence design, easy to modify, high in selectivity, and strong in affinity.
  • antisense nucleic acid has revolutionized the field of pharmacology, that is, the new drug receptor mRNA through the new receptor binding method (Watson-Crick hybrid), triggering new drug receptors.
  • Post-body binding reactions (1) RNase H-mediated degradation of target RNA; (2) inhibition of DNA replication and transcription and post-transcriptional processing and translation. It can be said that antisense oligonucleotides (ODNs) therapy have higher specificity than traditional drug treatments.
  • ODNs antisense oligonucleotides
  • antisense nucleic acid drugs From the late 1970s to the present, during these 30 years, antisense nucleic acid drugs have gone out of the laboratory and entered practical clinical applications. Especially after the first antisense drug Foraivirsen was approved by the FDA, people were particularly concerned about antisense therapy.
  • the principle of antisense nucleic acid action is based on the principle of base pairing, and can participate in the regulation of related gene expression by base-pairing binding with target RNA.
  • the mode of action may be as follows: 1
  • the antisense RNA binds to the viral mRNA to form a complementary double strand to block the binding of the ribosome to the viral mRNA, thereby inhibiting the process of translation of the viral mRNA into a protein.
  • Antisense DNA can form a triple helix nucleic acid with a target gene, which regulates transcription of a gene by acting on a transcript, an enhancer and a promoter region that control gene transcription.
  • 3 The binding of antisense nucleic acid to viral mRNA blocks the transport of mRNA to the cytoplasm.
  • Antisense nucleic acids recognize the target gene through the principle of base pairing. From the theoretical analysis, the researchers take animal cells as an example. The chromosomes are about several billion pairs of bases, if 4 bases (A, The numbers of G, C and T) are roughly the same and are randomly distributed throughout the gene. According to statistical principles, antisense nucleic acids larger than 17 bases are unlikely to hybridize with non-target genes, so the length is more than 17 The binding of the base antisense nucleic acid molecule to the target gene can be said to be unique, thereby making the antisense nucleic acid highly specific.
  • antisense nucleic acids have very low toxicity in vivo, and although they remain in the living organism for a long time, they will eventually be degraded, which avoids the integration of foreign genes into the host chromosome in transgenic therapy. The danger on it.
  • antisense nucleic acid drugs have the advantages of high specificity, low efficiency and low side effects, and have shown good application value in inhibiting tumor growth and antiviral replication.
  • a number of drugs have entered the US and European markets, and more than 30 antisense nucleic acid drugs have undergone preclinical studies or development and have entered Phase I, II and III trials.
  • antisense nucleic acids Due to the large amount of exonuclease present in animals, antisense nucleic acids are rapidly degraded and lose their activity if not chemically modified. There are many methods for chemical modification of antisense nucleic acids. Commonly, there are thio-modified antisense nucleic acids and 2'-methoxy-modified antisense nucleic acids. At present, the most comprehensive research on thio-modified drugs can effectively resist the degradation of nucleases and promote the activity of nuclease Rase H. At present, this modification method has been successfully applied to clinical antisense nucleic acid drugs. However, these are only the first-generation antisense nucleic acid modification methods. With the development and advancement of technology, new modification pathways and methods are emanating to make antisense nucleic acids. The study entered the second and third generations, and the modification of peptide nucleic acids was the most interesting.
  • PNAs Peptide Nucleic Acids
  • the structural unit of the skeleton is N (2- Aminoethyl)-glycine, the base moiety is attached to the amino group N of the main backbone via a methylene carbonyl group. It is the second generation of antisense nucleic acids.
  • the peptide nucleic acid provided by the present invention is selected from any one or any of the following peptide nucleic acids:
  • the peptide nucleic acid may be a chitosan-modified peptide nucleic acid.
  • the peptide nucleic acid of the present invention is useful for the preparation of a drug against the subgroup J avian leukosis virus.
  • the active ingredient is the peptide nucleic acid.
  • the preparation is a colon-controlled release microcapsule preparation, a lyophilized preparation for injection or a water-soluble granule for oral administration.
  • the peptide nucleic acid preparations of the invention also contain a pharmaceutically acceptable carrier or excipient.
  • High temperature High temperature of circulating steam 105 ° C, sterilization for 20 minutes does not affect its biological activity.
  • the peptide nucleic acid of the invention has no toxic and side effects, no drug resistance, and can directly inhibit ALV-J replication, has good antiviral effect, and has no food safety problems such as drug residues.
  • J subgroup avian leukemia is a kind of infectious disease mainly caused by chicken caused by subgroup J avian leukosis virus (aivan leukosi s subgroup J, ALV-J), which mainly causes malignant proliferation of hematopoietic cells in chickens.
  • ALV-J subgroup J avian leukosis virus
  • the positive rate of virus in Chinese chickens showed a rapid upward trend, and the host range expanded.
  • the vertical and horizontal transmission caused clinical and subclinical infections and caused large economic losses. So far, there is no effective prevention and control measures for this disease. It is particularly urgent to develop new technologies for preventing and treating ALV-J infection.
  • ALV-J invades the chicken's immune system, making the chickens infected with infection low.
  • the present invention is the first to combine peptide nucleic acid technology with antisense nucleic acid technology and to prevent and treat diseases caused by ALV-J virus infection.
  • ALV-J strain NS- XI I strain, from the laboratory of Nansen Veterinary Diagnostic Technology Research Center.
  • CEF cells Chicken embryo fibroblasts (CEF) prepared by conventional methods.
  • DF-1 cells From the laboratory of the Nansen Veterinary Diagnostic Technology Research Center.
  • the genome of ALV-J was retrieved from the GenBank database, sequence analysis was performed using biological software, and the conservation of sequence, G+C% content, base distribution characteristics were considered, and then the appropriate region was designed to design antisense nucleic acids.
  • the antisense nucleic acid sequences of the gp85 and P27 genes determined for the virus are as follows.
  • Gp85-l 5, - AGACUAAGGCAAAAAUCUGUU-3 ' ;
  • Gp85-2 5, - UAAAUCGGUGUUGUUAUCGCA-3 ';
  • Gp85-3 5, - ACGACUUAUUGAAAAACUCUC-3 ' ;
  • peptide nucleic acids are artificially synthesized, and the sequence of the peptide nucleic acid is as follows:
  • Gp85-3 5, - ACGACUUAUUGAAAAACUCUC-3 ';
  • Chitosan-peptide nucleic acid The peptide nucleic acid is modified with chitosan, and the modification method is various methods known in the art, such as the following literature:
  • DF-1 cells prepared in the pre-digestion stage, collected by centrifugation, counted in cells, adjusted to a cell concentration of 3 to 6 X 105 cells/ml with complete medium (DMEM + 5% fetal bovine serum + chelostine), The 24-well plate was cultured in a 37 ° C carbon dioxide incubator for 18-24 hours.
  • the density of the cells was observed under a microscope, and the cells were in a good state when the cells were 70 to 80% of the area of the well plate.
  • Aspirate the culture medium add 30 (M to be screened drugs (ie peptide nucleic acid) per well, 10 wells per drug. After 1 hour of incubation, add ⁇ ⁇ ⁇ (infection ratio is 0.01), after adsorption for 2 hours With nutrient solution After the unadsorbed virus was washed away, DMEM medium containing 4% FBS was added, and the culture was continued in a 37 ⁇ and 5% CO 2 atmosphere, and cytopathic changes were observed periodically after infection.
  • the infected cells were repeatedly freeze-thawed to release the virus, and the virus was detected as a sample.
  • a normal cell control group without virus and peptide nucleic acid was added, and a virus, a peptide-free nucleic acid-positive control group, a peptide-added nucleic acid drug, and a negative control group without a virus were added.
  • the supernatant in each of the above treatment groups was collected, and viral RNA was extracted using a viral total RNA extraction kit.
  • the obtained viral RNA was first reverse transcribed into cDNA, and then the corresponding primers were used to detect the virus content in the ALV-J treatment group.
  • the results of quantitative amplification were calculated by statistical software using the software to calculate the virus titer in each treatment group.
  • the inhibition effect was a multiple of the difference between the PNA group and the blank control group.
  • the present invention refers to primers provided by Huang et al., and quantitatively detects ALV-J by real-time PCR.
  • Primer 1 5, - TCAGGACCAAGGGCTTAC -3, ;
  • Blank control group Peptide nucleic acids gp85-1/3, P27-2/3 are preferred.
  • DF-1 cells were used as test subjects, 96-well plates, and 100 ⁇ l of 5000 cells per well.
  • the 0D test is the 0D value of the treated cells, and the 0D control is the untreated cell control tube 0D value, 0D.
  • the background is the cell-free medium control 0D value. Changes in cell proliferation or toxicity after treatment are expressed as a percentage of untreated controls.
  • a total of 200 AA commercial broilers were provided at the age of 1 day (provided by the Experimental Animal Farm Division of Nansen Veterinary Diagnostic Center, and tested negative for ALV-J).
  • 40 patients in the ALV-J infection group (test group) were subcutaneously inoculated with ALV-J infected cell culture medium 0. 2 mL/head in the 1 day old neck and 40 in the blank control group (control group) without any treatment.
  • Gp85- 1/3 + P27-2/3 peptide nucleic acid in equal mass ratio mixture dose group 25ppm, 50ppm, lOOppm
  • each group of feed and drinking water are prepared separately, do not cross .
  • the drug treatment group showed a difference in mortality caused by ALV-J infection at week 3 after viral infection, as shown in Table 4.

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Abstract

提供一种J亚群禽白血病病毒的肽核酸及其应用。该肽核酸的核酸序列选自于下列序列1-4中的任一项：序列1：5'AGACUAAGGCAAAAAUCUGUU-3'；序列2：5'-ACGACUUAUUGAAAAACUCUC-3'；序列3：5'-UAUAACCGUCUGUAGUUGGAC-3'；序列4：5'-ACAUAUUUGAUUAUCUCUCCU-3'。本发明的肽核酸无毒副作用，无耐药性，能特异性直接抑制ALV-J复制，抗病毒效果好，无药物残留。

Description

J亚群禽白血病病毒的肽核酸及其应用 技术领域
本发明涉及一种 J亚群禽白血病病毒的肽核酸及其应用。
背景技术
禽白血病是由禽白血病病毒 （avian lekosis virus, ALV) 引起的以造血细 胞恶性增生为主的一类传染病， 包括淋巴细胞性白血病， 成红细胞性白血病， 成 髓细胞白血病和髓细胞样白血病， 对养禽业危害最大的是禽淋巴细胞性白血病 ( lymphoid leukosis, LL) 。 根据病毒囊膜特性、 病毒中和实验、 宿主范围和 基因组的分子生物学特性， 现己将该病毒分类成 10个亚群： A〜J亚群， 其包 括了除网状内皮组织增殖病病毒以外引起禽类肿瘤性疾病的所有逆转录病毒。其 中 A、 B、 C、 D 为外源性病毒， E、 F、 G、 H、 I 为内源性病毒。 A、 B 亚群是商 业蛋鸡（来航鸡） 最为常见的外源性病毒亚群， 而 (、 D亚群感染率极低， 几乎 很难检测到； E 亚群包括普遍存在的低致病性和无致病性的内源性病毒。 J亚群 于 1988年， 由 Payne等同事首次从商品代肉用鸡中分离得到， J亚群是一外源 性白血病病毒，其与内源性 E亚群的重组体，可以通过水平和垂直传播在鸡群中 广泛散播，所有肉用型品系的鸡对 ALV-J都易感，但感染鸡的肿瘤发病率却有显 著差异， 蛋鸡虽可感染 ALV- J， 但自然感染时很少引起肿瘤； 随着养鸡生产规模 的不断增大， 疑似淋巴性白血病 J 亚群 ( ALV— J) 的发生更为广泛， 使发病鸡 群死淘率高， 给养鸡生产场带来严重的经济损失。
该病 1998年在世界范围内暴发， 给世界养禽业造成了沉重打击。 1999年， 杜岩等在国内首次从商品代肉鸡中分离并检测到 ALV-J病毒。 2000 年以后， J 亚群白血病在我国鸡群中全面暴发， 并呈现出宿主范围扩展，其垂直和水平传播 引起临床和亚临床感染而造成较大的经济损失。
禽白血病导致的经济损失主要有两个方面。 一是引起肿瘤， 导致鸡的死亡。 二是产生非肿瘤性疾病，造成免疫抑制造成的亚临床感染，表现为鸡的消瘦和贫 血， 耐受性病毒血症， 免疫抑制等， 由此严重影响养鸡生产。 ALV- J感染可造成 鸡多重感染，在个体病鸡中可同时检出几种不同的病毒， 如网状内皮增生症病毒 ( REV) 、 传染性法氏囊病毒、 鸡传染性贫血病毒等。 在实际生产过程中， 人们 通常只关注肿瘤形成对生产的影响，往往忽略其亚临床感染。免疫抑制可包括淋 巴器官萎缩或发育不全、高血症丙球蛋白增多、促有丝分裂剂诱导的胚细胞形成 减少以及抗体应答降低。 因此， ALV对养禽业造成的损失主要是由于其前期非肿 瘤性疾病（免疫抑制）引发的， 而后期由于肿瘤造成的死亡只是免疫抑制从量变 到质变的一个表面现象。
病鸡临床症状主要表现为食欲不振， 进行性消痩； 羽毛异常， 患病鸡精神状 态差； 部分鸡的腹部胀大， 可触摸到肿大的肝脏； 鸡冠及肉髯苍白， 有些鸡冠萎 缩； 有的头部、 背部、 胸部、 腿部及翅膀可见 1〜3 cm大小的血疱， 呈褐紫色， 质地柔软， 有一定的弹性， 与周围皮肤界限清楚， 血疱破裂后流血不止， 血疱周 边的羽毛被大片血迹污染。
免疫抑制是指由于受到各种因素（如营养、 疾病、 应激等） 的影响， 使得机 体对抗原应答能力低下甚至缺失的现象。其中由病毒引起的免疫抑制尤为严重从 而形成免疫抑制。 ALV- J引起成年肉鸡骨髓细胞瘤， 导致很高的死亡率， 还能使 繁殖力下降（由于影响种公鸡的发育）。 由于种母鸡的死亡和繁殖力下降， 使得 孵化用蛋减少，造成肉鸡饲养业的严重损失。 ALV 除了可引起原发感染引起鸡的 死亡， 更重要的是损害免疫器官， 如对鸡胸腺、法氏囊等主要免疫器官可造成严 重损伤， 使免疫器官功能降低， 使得机体的抗病力下降， 最终导致并发症和继发 感染。 另外， ALV- J 造成免疫抑制的解除很困难， 使鸡群对疫苗免疫不产生应答 或应答能力下降， 造成免疫失败， 引起烈性传染病暴发。
病毒粒子直径 80〜100 nm, 由外部的囊膜和内部的电子致密的核衣壳构成， 核心结构由二倍体 RNA和核衣壳、 反转录酶、 整合酶、 蛋白酶组成， 呈正二十面 体。 病毒囊膜上有放射状突起， 直径约为 8 ran。 ALV基因组全长约 7. 2 kb, 可 直接作为 mRNA。 ALV 结构基因从 5 ' 端至 3' 端顺序为 gag-pol- env， 分别编码 病毒结构蛋白、 RNA依赖的 DNA聚合酶 (反转录酶） 和囊膜糖蛋白。 gag基因编 码病毒内部非糖基化结构蛋白， 包括基质蛋白、 蛋白酶、 衣壳和核衣壳。 在 ALV 亚群间， 这些病毒蛋白十分保守， 具有高度的同源性， 即所谓的群特异性抗原或 GSAc pol 基因编码病毒的反转录酶和整合酶， 以完成作为细胞基因信息表达前 的病毒至前病毒以及前病毒 DNA整合进细胞染色体的过程。 erw基因编码病毒囊 膜糖基化蛋白。 包括膜表面糖蛋白亚单位 （SU) gp85和跨膜糖蛋白亚单位（TM) gp37。 SU是由 gp85基因编码的， 它含有病毒一受体决定簇， 决定禽白血病的亚 群特异性； TM负责将病毒转入细胞。结构基因两侧的长末端序列（ long terminal repeats, LTR ) , 与病毒 RNA的复制和翻译有关。 急性转化型 ALV还带有病毒 性肿瘤基因（v20nC0gene)。 该病毒含有 5 种结构蛋白即病毒群特异性抗原衣壳 蛋白 P27， 病毒基膜蛋白 P19， 参与 RNA 加工与包装的核衣壳蛋白 P12， 参与蛋 白前体剪切的天冬氨酸酶 P15,功能不详的 P10。内源性病毒 RAV-0株中存在 P27 的变异体即 P270。
反义核酸（antisense nucleic acid)是一段与靶基因 （mRNA或 DNA) 的某段 序列互补的天然存在或人工合成的核苷酸序列，反义核酸通过碱基配对方式特异 性的与病毒靶基因结合形成杂交分子，从而在复制、转录和翻译水平调节靶基因 的表达， 或诱导 RNase H识别并切割 mRNA, 进而使其功能丧失。
反义核酸包括反义 RNA (antisense RNA)和反义 DNA (antisense DNA) , 具有 合成方便、 序列设计简单、 容易修饰、 选择性高、 亲和力强等特点。 反义核酸作 为一种新的抗病毒、抗肿瘤药物， 掀起了一场药理学领域的革命， 即新的药物受 体 mRNA通过新受体结合方式（Watson- Crick杂交） 、 引发新的药物受体结合后 反应： （1 ) RNase H介导的靶 RNA的降解； （2) 抑制 DNA的复制和转录及转录 后的加工和翻译等。 可以说反义寡核苷酸（ODNs )疗法比传统的药物治疗手段有 更高的特异性。 从二十世纪 70年代末到现在， 在这三十年的时间中， 反义核酸 药物已走出实验室， 进入了实际临床应用。 特别是第一个反义核酸药物 Foraivirsen通过 FDA批准上市后， 人们对反义疗法尤为关注。
反义核酸作用原理基于碱基配对原则，可通过与靶 RNA进行碱基配对结合的 方式参与对相关基因表达的调控。 其作用方式可能有： ①反义 RNA与病毒 mRNA 结合形成互补双链阻断核糖体与病毒 mRNA的结合，从而抑制了病毒 mRNA翻译成 蛋白质的过程。②反义 DNA能与靶基因形成一种三链核酸 (triple helix nucleic acid) , 它通过作用于控制基因转录的转录子、 增强子和启动子区， 对基因的转 录进行调控。③反义核酸与病毒 mRNA的结合可阻挡 mRNA向细胞质的运输。④反 义核酸与病毒 mRNA结合后使得 mRNA更加易被核酸酶识别降解， 从而大大缩短 mRNA 的半衰期。 上述四种作用途径都可表现为对病毒基因表达的抑制或调控， 且这种调控是高度特异性的。
反义核酸是通过碱基互补配对的原理来识别所打靶的基因， 从理论来分析， 研究人员以动物细胞为例，其染色体大约有几十亿对碱基，如果 4个碱基（A、 G、 C和 T ) 的数目大致相同， 并在整个基因中随机分布， 那么按照统计学原理， 大 于 17个碱基的反义核酸与非靶基因杂交的可能性不大，所以长度超过 17个碱基 的反义核酸分子与靶基因的结合可以说是唯一的，从而使反义核酸具有高度的特 异性。
研究表明，在细胞内部一个拷贝的基因会产生 200-300条 mRNA， 翻译出 10 万个具有生物活性的蛋白质分子。传统药物主要作用于有生物功能的蛋白分子的 某结构域上的几个作用位点， 事实上蛋白的结构是非常复杂的， 且在生物体内， 活性蛋白的空间结构又是千变万化的，以传统药物有限的几种作用位点来控制靶 分子的动态的和整体的功能很难达到理想的效果，因而不难看出传统药物的局限 性。 由 mRNA可翻译出几十到几百个蛋白， 反义核酸在 mRNA水平对靶基因直接进 行调控，这个步骤相当于将传统药物作用放大了数十到数百倍， 可见反义核酸的 调控是极其经济合理的。
毒理学研究表明， 反义核酸在体内具有很低的毒性， 尽管其在生物体内的存 留时间有长有短，但最终都将被降解消除，这避免了如转基因疗法中外源基因整 合到宿主染色体上的危险性。 与传统药物相比， 反义核酸药物具有特异性强， 效 率髙和毒副作用低等优点，在抑制肿瘤生长和抗病毒复制等方面显现出了良好的 应用价值。 目前己有多个药物进入美国和欧洲市场， 另还有 30多种反义核酸药 物正在进行临床前期的研究或开发后进入了 I、 II和 III期实验。
由于动物体内存在大量的核酸外切酶， 反义核酸若不经过化学修饰，很快就 被降解， 失去活性。 目前对反义核酸的化学修饰有很多方法， 常见的有硫代修饰 反义核酸及 2 ' -甲氧基修饰反义核酸等。且目前硫代修饰药物的研究最为全面， 它可有效抵抗核酸酶的降解， 同时可促进核酸酶 Rase H的活性， 目前这种修饰 方法已成功用于临床的反义核酸药物。 但这些还只是第一代反义核酸的修饰方 法， 随着技术的发展与进步， 新的修饰途径与方法被幵发出来， 使得反义核酸的 研究进入了第二、 三代， 其中肽核酸的修饰最为引人关注。
肽核酸 （peptidenucleic acids, PNAs ) ， 是一种以中性酰胺键为骨架的全 新的 DNA类似物，可序列特异的靶向作用于 DNA的大沟槽， 其骨架的结构单元为 N (2-氨基乙基) -甘氨酸，碱基部分通过亚甲基羰基连接于主骨架的氨基 N上。为 反义核酸的第二代产品。
发明内容
本发明的目的是提供一种
本发明所提供的肽核酸， 选自如下任一种或任几种肽核酸：
a) 其核酸序列为序列表中的序列 1
5， - AGACUAAGGCAAAAAUCUGUU-3 ' ；
b ) 其核酸序列为序列表中的序列 2
5 ' - ACGACUUAUUGAAAAACUCUC-3 ' ；
c ) 其核酸序列为序列表中的序列 3
5， - UAUAACCGUCUGUAGUUGGAC-3 ' ；
d) 其核酸序列为序列表中的序列 4
5， - ACAUAUUUGAUUAUCUCUCCU-3 ' 。
其中， 所述肽核酸可以为经壳聚糖修饰的肽核酸。
本发明的肽核酸用于制备抗 J亚群禽白血病病毒的药物。
本发明的肽核酸制剂， 其活性成分为所述的肽核酸。
其中，所述制剂为结肠溶控释微囊制剂、注射用冻干制剂或口服用水溶性颗 粒剂。
本发明的肽核酸制剂还含有可药用载体或赋形剂。
本发明的肽核酸制剂的稳定性分析
高温： 流通蒸汽高温 105°C， 20分钟灭菌不影响其生物活性。
极端温度： 50'C存放 6个月不影响其生物活性。
室温： 存放 24个月不影响其生物活性。
低温： -2CTC存放 48个月不影响其生物活性。
本发明的肽核酸无毒副作用， 无耐药性， 能特异性直接抑制 ALV-J复制， 抗 病毒效果好， 无药残等食品安全问题。 具体实施方式
J 亚群禽白血病是由 J 亚群禽白血病病毒（aivan leukosi s subgroup J， ALV-J ) 引起的一种主要发生于鸡的传染病， 其主要引起鸡的造血细胞恶性增 生， 该病近年来在我国鸡群中病毒阳性率呈快速上升趋势， 并呈现出宿主范围扩 展，其垂直和水平传播引起临床和亚临床感染而造成较大的经济损失。到目前为 止，对该病尚无有效的防控措施， 开发预防和治疗 ALV-J感染的新技术显得尤为 迫切，另 ALV-J侵害鸡的免疫系统，使感染后鸡群的免疫力低下，引发免疫抑制， 一旦感染容易继发其它传染病。到目前为止， 尚未成功开发出可以预防该病的有 效疫苗， 本发明率先将肽核酸技术与反义核酸技术结合起来， 并应用于预防和治 疗 ALV-J病毒感染引发的相关疾病。
为此， 本发明采用了以下技术方案：
ALV-J毒株： NS- XI I株， 来自楠森兽医诊断技术研究中心实验室。
CEF细胞： 常规方法制备的鸡胚成纤维细胞 （CEF)。
DF-1细胞： 来自楠森兽医诊断技术研究中心实验室。
针对抗 ALV- J的肽核酸体外抗病毒效果分析
从 GenBank数据库检索出 ALV- J的基因组，用生物学软件进行序列分析，综合 考虑序列的保守性， G+C%含量， 碱基分布特点， 然后从中挑选出合适的区域设计 反义核酸， 最后所确定的针对病毒的 gp85和 P27基因的反义核酸序列如下。
gp85
gp85-l : 5， - AGACUAAGGCAAAAAUCUGUU-3 ' ；
gp85-2: 5， - UAAAUCGGUGUUGUUAUCGCA-3 ' ；
gp85-3 : 5， - ACGACUUAUUGAAAAACUCUC-3 ' ；
P27
P27-1 : 5， - AUAACUCUCAUUAGAUUCGUA-3 ' ；
P 27-2： 5， - UAUAACCGUCUGUAGUUGGAC-3 ' ；
P27-3 -. 5， - ACAUAUUUGAUUAUCUCUCCU-3 ' 。
人工合成如下肽核酸， 肽核酸的序列如下：
gp85
gP85-l : 5， - AGACUAAGGCAAAAAUCUGUU-3 ' ； gp85-2: 5， ― UAAAUCGGUGU翻画 CGCA- 3， ；
gp85-3: 5， - ACGACUUAUUGAAAAACUCUC-3 ' ；
P27
P27-1 : 5， - AUAACUCUCAUUAGAUUCGUA-3 ' ；
P 27-2： 5， - UAUAACCGUCUGUAGUUGGAC-3 ' ；
P27-3: 5， - ACAUAUUUGAUUAUCUCUCCU-3 ' 。
壳聚糖 -肽核酸：以壳聚糖修饰肽核酸,修饰方法为本领域中已知的多种方法 具体如下述文献：
Luessen H L, de leeuw B J, Lang emeyer M, et al . Mucoadhesive polymers in peroral peptide drug delivery. 0 . carbomer and chitosan impro ve t he abso rptio n of the pept ide drug buser elin in vivo [ J] . Pharm Res,
1996, 13 ( 11) ： 1 668- 1 172.
Kotze A F, Luessen H L, de Leeuw B J, et al . Compar ison o f the effect of differ ent chitosan salts and N- tr-I methyl chitosan chlo ride o n the permeability of intestinal epithelial cells [ J] . J Control Release, 1998, 51 ( 1) :35- 46.
T hanoo B C, Sunny M C, Jayakrishnan A. Cr osslinked chit osan micro spheres : prepa ratio n and ev aluatio n as a matr ix fo r the co ntr oiled release of pharmaceuticals [ J] . J PharmPharmacol, 1992, 44 ( 4) ： 283-286.
Por tero A, ReraunanLo pez C, Cr iado M T, et al . Reacety lated chito san micr ospher es fo r contro lied deliver y of ant-i micro bial agents t o t he gastr ic mucosa [ J] . J Microencapsul, 2002, 19 ( 6) ： 797- 809. 采用 ALV-J病毒特异性定量 RT- PCR检测肽核酸对病毒靶基因的抑制程度， 同时运用病毒毒价测定实验检测抗病毒的滴度。
Day 1 :
铺板： 消化前期准备的 DF- 1 细胞， 离心收集， 细胞计数， 用完全培养基 (DMEM+5%胎牛血清 +青链霉素）将细胞浓度调到 3〜6 X 105 个 /ml , 铺 24孔板， 于 37°C二氧化碳培养箱中进行培养 18-24小时。
Day 2 :
显微镜观察细胞的密度， 待细胞长满孔板面积的 70~80%时， 且细胞状态良 好。 吸去培养液， 每孔加入 30(M待筛选药物（即肽核酸） ， 每个药物 10个孔。 温育 1小时后， 加入 ΙΟΟμΙ ΑΙ Μ (感染比为 0. 01 ) ， 吸附 2小时后， 用营养液 洗去未吸附的病毒后， 再加入含 4%FBS的 DMEM培养基， 在 37Ό和 5%C02环境中 继续培养， 感染后定时观察细胞病变。 感染后 72h， 将感染细胞进行反复冻融以 释放病毒， 以此为样本进行病毒检测。实验过程中还设不加病毒和肽核酸的正常 细胞对照组， 加病毒、 不加肽核酸阳性对照组和加肽核酸药物、 不加病毒的阴性 对照组。
Day 3-5 :
观察药物对细胞的保护效应， 并对结果进行评判。
Real-time PCR定量检测
收集上述各处理组中的上清液， 用病毒总 RNA提取试剂盒提取病毒 RNA。 对 获得的病毒 RNA首先进行反转录成 cDNA, 然后运用针对的引物分别检测 ALV-J 处理组中的病毒含量。定量扩增后的结果，运用统计学软件计算出各处理组中病 毒滴度， 抑制效果以 PNA组与空白对照组差异的倍数。
本发明参考 Huang等提供的引物， 釆用 real- time PCR定量检测 ALV-J。
ALV-J特异性检测引物
引物 1 : 5， - TCAGGACCAAGGGCTTAC -3， ；
引物 2 : 5， - CTGCCGCTATAACCGTCTG -3' 。 β -actin 为内参
Actin-F: 5， - TCCCTGTATGCCTCTGGTC -3， ；
Actin-R: 5， - TCTCTCTCGGCTGTGGTGG -3， 。 反应体系 (25 μ ΐ )
试剂 用量 (ul )
2 X One-Step SYBR RT-PCR Buffer 12. 5
Ex Taq T M HS 0. 5
PrimeScript T M RT Enzyme Mix II 0. 5
PCR 正向引物 0. 5
PCR 反向引物 0. 5
总 讓 2 无 RNase dH20 8. 5
总里 25 反应条件
反转录 组
42 °C 5 min
95 °C 10 sec
PCR扩增
Cycle: 40
95 °C 5 sec
60 °C 30 sec
反应结束后确认 Real Time One Step RT-PCR的扩增曲线和融解曲线， 以确 保结果的特异性与可靠性。
针对 ALV- J的肽核酸体外抗病毒结果 .
定量 PCR检测结果显示， 除 gp85-2效果不明显外， gp85- 1， gp85_3的抑制率 分别为： 78 %和83% (表 1) ； 除 P27- 1效果不明显外， P27- 2和 P27-3的抑制率分 别为： 73%和 77% (表 1) 。
表 1. 核酸对体外 MARC- 145细胞抗 PRRSV效果
病毒抑制率
组别
72h
gp85-l组
gp85-2组
gp85-3组
用
P27-1组
P27-2组
P27-3组
病毒对照组
阴性对照组
空白对照组 肽核酸 gp85- 1/3， P27-2/3为优选。
药物组合处理
针对前一步所筛选出的。然后， 在前面的这些实验基础上， 对筛选出的有效 抗病毒作用的药物进行组合使用，比较组合后的抗病毒效果与单药抗病毒效果之 间的差异。 DF- 1细胞感染 ALV- J NS-X11株后， 分别加入针对 gp85或 P27的基 因药物组合， 同时设阳性对照和阴性对照及空白对照， 用 Real- time PCR方法进 行检测， 统计分析各用药组之间病毒抑制率， 方法同上， 如表 2。
表 2. 不同浓度的肽核酸对体外 DF-1细胞抗 ALV-J效果 病毒抑制率
72h
gp85-l/3组 79%
感染
P27-2/3组 81%
用药组
gp85-l/3组 + P27-2/3组 88%
病毒对照组 - 阴性对照组 - 空白对照组 - 细胞毒性实验
1)以 DF-1细胞作检测对象， 96孔板，每孔加 100微升 5000个细胞。 gp85_l、 gp85- 3、 P27- 2和 P27- 3肽核酸浓度 (0. 02， 0. 1， 0. 5， 1， 5， lO m) ， 每个 梯度设置 3个重复孔， 另设未处理细胞对照、 无细胞培养基对照。
2)处理结束后， 每孔每 100 μΐ培养基加入 Ιθμΐ MTT Stock, 37 °C培养箱 内继续孵育 4小时。 也可更换 100 μΐ新鲜无血清培养基后再加 MTT Stockc
3)吸去培养基， 每孔加入 Ιθθμΐ MTT溶解剂， 保持各孔内液体体积一致。
4)在 570nm测定 0D吸光度并进行比较计算。注意： 为准确考虑可在 699 nm 处测定未还原的 MTT本身的吸光度 0D值， 然后用 0D570减去 0D699。
5)结果判断： 细胞增殖或毒性 = 100% X (0D实验一 0D本底） I (0D对照 一 0D本底）。
0D实验是接受处理细胞的 0D值， 0D对照是未处理细胞对照管 0D值， 0D 本底是无细胞培养基对照 0D值。 处理后细胞增殖或毒性变化表示为未处理对照 的百分数。
检测结果表明， 针对 ALV-J的肽核酸 gp85- 1、 gp85- 3、 P27-2和 P27- 3无毒 性。 实验动物感染及饲养
健康 1日龄 AA系商品代肉鸡共 200只（楠森兽医诊断中心实验动物养殖场司提 供， 经检测 ALV- J阴性）随机分为 5组。 其中 ALV- J感染组 (试验组) 40只， 于 1日龄 颈部皮下接种 ALV- J感染细胞培养液 0. 2mL/只； 空白对照组 (对照组)共 40只， 不 作任何处理。 gp85- 1/3 + P27-2/3肽核酸的等质量比混合物剂量组（25ppm，50ppm， lOOppm) ， 采用饮水给药方式， 严格隔离饲养， 各组的饲料与饮水均单独准备， 不交叉。
ALV- J感染后， 于不同的时间点采集鸡血， 取血清， 用 ELISA检测血清中病毒 阳性率， 如表 3。
表 3. 试验各组 ALV- J存在于血清中的阳性情况 时间 （天） 1 3 5 7 9
25 (ppm) + + + ― ―
感 染
50 (ppm) + + - - - 给药组
100 (ppm) + - - - 一 感染不给药组 + + + + + 空白对照组 - 一 ― - - 最终优选的给药剂量为 50〜100ppm。
病毒感染后肉鸡生长情况
对照组未出现死亡现象
病毒感染后第 1 周混感组就出现死亡
各药物处理组在病毒感染后第 3 周对 ALV- J 感染引起的死亡率表现出差 异， 如表 4。
同时检测肉鸡的临床表现、 增重、 免疫器官指数及死亡率测定： ①临床表 现、体重、免疫器官指数。每天观察鸡群发病及生长情况， 各组在 7 、 14 、 21 、 28 、 35 、 49d 称量体重。 每周随机剖杀各病毒感染组鸡 5只， 对照组 3只， 分 别于 7 、 21 、 35 、 49d 时取胸腺、 脾脏和法氏囊称重， 并按如下方法计算免 疫器官指数:免疫器官指数 =免疫器官重量 (g)/鸡活体重 (kg) 。 ②死亡率统计。 记录每天自然死亡鸡只，统计各组死亡率,并剖检、 观察病变。 ③数据统计分析。 表 4. ALV- J感染后对肉鸡不同日龄体重的影响 日龄 ALV-J感染组 空白对照组
25 50 100
7 149±32 136±32 129±32 128 ±23
14 362 ±49 375±51 392±38 350 ±38
21 502 ±55 542 ±54 532 ±55 560 ±54
28 650±75 700±85 670±65 708 ±65
35 998 ±89 1088 ±89 996 ±92 1260±59
49 1590±120 1688±140 1650±130 2020±45 病毒感染后对鸡免疫器官指数的影响， ALV - J感染组， 5周后中枢免疫器 官 (法氏囊和胸腺） 指数显著和极显著低于对照组， 表 5。
表 5. ALV-J感染后对肉鸡免疫器官指数之间关系 日龄 ALV-J感染组 +药物混合组 空白对照组
胸腺 2.9±0.32 2.86±0.35
7 法氏囊 1.29±0.22 1.65±0.4 脾 0.9±19 1.15±0.2 胸腺 3.5±0.36 3.66 ±0.34
21 法氏囊 2.33 ±0.23 2.65 ±0.42 脾 1.1±0.17 2.55 ±0.29 胸腺 2.9±0.33 4.86±0.55
35 法氏囊 1.33±0.31 1.65±0.4 脾 1.5±0.27 1.23±0.32 胸腺 3.1±0.46 5.26±0.17
49 法氏囊 0.93土 0.18 1.35±0.4 脾 1.21±0.23 1.25±0.3

Claims

O 2013/189003 权 利 要 求 书
1.一种肽核酸， 选自如下任一种或任几种肽核酸：
a)其核酸序列为序列表中的序列 1
5 ' - AGACUAAGGCAAAAAUCUGUU-3 ' ；
b ) 其核酸序列为序列表中的序列 2
5， - ACGACUUAUUGAAAAACUCUC-3 ' ；
c ) 其核酸序列为序列表中的序列 3
5 ' - UAUAACCGUCUGUAGUUGGAC-3 ' ；
d )其核酸序列为序列表中的序列 4
5， - ACAUAUUUGAUUAUCUCUCCU-3 ' 。
2.根据权利要求 1所述的肽核酸， 其特征在于， 所述肽核酸为经壳聚糖修饰 的肽核酸。
3.权利要求 1或 2所述的肽核酸在制备用于抗 J亚群禽白血病病毒的药物中的 应用。
4.一种肽核酸制剂， 其活性成分为权利要求 1-2中任一所述的肽核酸。
5.根据权利要求 4所述的肽核酸制剂， 其特征在于， 所述制剂为结肠溶控释 微囊制剂、 注射用冻干制剂或口服用水溶性颗粒剂。
6.根据权利要求 4或 5所述的肽核酸制剂，其特征在于，还含有可药用载体或 赋形剂。
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