WO2000063387A1 - Sequence genetique complete de la souche d'un vaccin contre le virus de l'anemie infectieuse equine et son application - Google Patents

Sequence genetique complete de la souche d'un vaccin contre le virus de l'anemie infectieuse equine et son application Download PDF

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WO2000063387A1
WO2000063387A1 PCT/CN2000/000096 CN0000096W WO0063387A1 WO 2000063387 A1 WO2000063387 A1 WO 2000063387A1 CN 0000096 W CN0000096 W CN 0000096W WO 0063387 A1 WO0063387 A1 WO 0063387A1
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gene
vaccine strain
infectious anemia
sequence
equine infectious
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PCT/CN2000/000096
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English (en)
French (fr)
Inventor
Yiming Shao
Rongxian Shen
Gang Chen
Kangzhen Yu
Pinliang Pan
Bin Jia
Yi Feng
Fei Xue
Wenhua Xiang
Xiujuan Fan
Xiaoling LÜ
Liping Zhao
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National Center For Aids Prevention And Control
Harbin Veterinary Research Institute, Chinese Academy Of Agricultural Science
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Application filed by National Center For Aids Prevention And Control, Harbin Veterinary Research Institute, Chinese Academy Of Agricultural Science filed Critical National Center For Aids Prevention And Control
Priority to DE60035680T priority Critical patent/DE60035680T2/de
Priority to AU39554/00A priority patent/AU3955400A/en
Priority to EP00918668A priority patent/EP1174507B1/en
Priority to US09/959,120 priority patent/US6987020B1/en
Publication of WO2000063387A1 publication Critical patent/WO2000063387A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the application of the full-length gene of equine infectious anemia virus donkey leukocyte attenuated vaccine strain, the DNA sequence of each functional gene and its encoded protein sequence, each functional gene and its protein product.
  • Equine Infectious Anemia Virus is the pathogen that causes equine infectious anemia and is the earliest virus discovered by humans. Horse infectious anemia was first discovered in France in 1843. It has caused great harm to the livestock industry for more than a century, and is therefore one of the diseases that scientists from all countries have been working hard to research and control. Since the 1960s, China has invested heavily in researching the biological characteristics of the virus. Isolate and cultivate strong virus strains that are significantly different from foreign strains for in vitro donkey leukocyte passage, and gradually domesticate the virus to make it non-destructive. Disease, but can cause animals to produce a stronger immune protection vaccine strain after vaccination. The vaccine strain has been produced since 1976 and has been widely used throughout the country in 1978. So far, 70 million horse donkeys have been vaccinated. Epidemic of the disease in China (Shen Rongxian et al., Research on Equine Infectious Anemia Immunity, Chinese Agricultural Science, No. 4 P1-15, 1979).
  • EIAV belongs to the genus Lentivirus of the retroviridae family. It shares many similarities with the human immunodeficiency virus (Human Immunodificiency Virus, HIV) belonging to the genus Lentivirus in terms of genomic structure, gene-encoding proteins, and gene regulation methods ( JMCoffm, The structure And Classfication of Retroviruses, The Retroviridae, Vol.1, pl9, edit by Jay A.
  • HIV Human Immunodificiency Virus
  • EIAV EIAV's reverse transcriptase, protease, dUTP enzyme, envelope glycoprotein and core protein
  • equine infectious anemia virus infects equine animals and causes acute onset of horse symptoms characterized by periodic fever, red blood cell anemia, and persistent viremia, 'Easy to observe and study, this virus has become an important model for studying lentiviral infection and pathogenic mechanisms and viral enzyme functions (RC Montelaro et al al Equine Retroviruses, in: vol. 2, p257).
  • the purpose of the present invention is to provide the full-length gene sequence of equine infectious anemia virus donkey leukocyte attenuated virus vaccine strain, the DNA sequence of each functional gene and its encoded protein sequence, and the application of each functional gene and protein product.
  • the present invention relates to the full-length DNA sequence of equine infectious anemia virus donkey leukocyte attenuated vaccine strain and its main structural genes (gag, pol, env) and regulatory genes (5, LTR, 3 'LTR ⁇ rev Gene, S2 gene, tat gene).
  • the full-length genome of the strain contains 8258 nucleotides and has the nucleotide sequence shown in SEQ ID NO: 1.
  • nucleotides 1-325 are 5 'LTR
  • nucleotides 7922-8258 are 3' LT
  • nucleotides 466-1926 are gag genes
  • nucleotides 1689- Position 5120 is the pol gene
  • nucleotides 5313-7904 is the env gene
  • nucleotides 365-462 are the first exon of the tat gene
  • nucleotides 5138-5276 are the second exon of the tat gene
  • nuclear Nucleotide 5454-5546 is the first exon of the rev gene
  • nucleotide 7250-7651 is the second exon of the rev gene
  • nucleotides 5287-5493 are the S2 gene.
  • the DNA sequence of the gag gene, the pol gene, the env gene, the rev gene, the tat gene, the S2 gene, and the corresponding deduced amino acid sequences are as shown in SEQ ID NOS: 2, 3, 4, 5, 6, 7, and SEQ ID NOS: 8 , 9, 10, 11, 12, and 13.
  • the full-length gene clone of the equine infectious anemia virus donkey leukocyte attenuated vaccine strain of the present invention has been deposited at the General Microbiology Center of the China Microbial Species Collection Management Committee on April 19, 1999, and the deposit number is CGMCC NO.0394, the deposit On April 19, 2000 it was converted into an international deposit under the Budapest Treaty on Internationally Recognized Microbial Deposits for Patent Procedures. It is well known to those skilled in the art that there may be sequencing errors in the sequences measured in SEQ ID NOs: 1-7 herein, so if this happens, the deposit of the present invention is decisive.
  • the genes of the vaccine strain are amplified by a standard PCR method, cloned into a plasmid vector, and then the DNA sequence is analyzed, and finally the full-length gene sequence of the virus is obtained.
  • the EIAV donkey leukocyte attenuated vaccine strain was integrated into the donkey leukocyte chromosome in the form of former viral DNA during in vitro donkey leukocyte replication.
  • the present inventors took the previous viral DNA as a material for amplifying viral genes.
  • chromosomal DNA was extracted from virus-infected donkey leukocytes, and the proviral DNA of the EIAV vaccine strain was amplified by PCR using this as a template.
  • the design of the amplification primers is based on the sequence of the international EIAV virulent strain, and the EIAV genes of each segment are used for pre-amplification. After several explorations, and based on the partial sequence information of the EIAV vaccine strain obtained by sequencing the obtained amplified fragments, the EIAV vaccine attenuated strain-specific primers were designed, the viral genes were amplified in stages and cloned into a plasmid vector, and further All genes were cloned and sequenced to obtain the full-length sequence of the viral genome and its main structural genes (gag gene, pol gene and env gene) and main regulatory genes (5, LTR, 3 'LTR, rev gene, S2 gene, tat gene, etc.).
  • the GCG software (Genetics Computer Group, Inc., Wisconsin, USA) was used to analyze the open reading frame of the full-length gene sequence to obtain the amino acid sequence of each structural gene and the protein encoded by the regulatory gene. Specific locations in long gene sequences.
  • the sequence of the obtained vaccine strain was compared with the sequence of the international standard strain (Wyoming strain, GeneBank Accession Number: AF028232) published by GenBank, and the nucleotide and amino acid homology were found.
  • the nucleotide homology is between 73.46-90.06%, among which the env gene, rev gene and S2 gene are significantly different from the international standard strains, with homology of 73.46%, 73.54% and 75.76%, respectively.
  • Amino acid sequence homology comparison with the corresponding sequence found in standard strains, outer membrane protein (Env protein) variant proteins and S2 and Rev proteins are large, the amino acid homology of 67.41 percent, respectively, 64.85% and 54.54% 0
  • the secondary structure of the protein encoded by each structural gene and regulatory gene of the vaccine strain was also predicted using GCG software. Comparison of the secondary structure of the Env and Tat proteins of the vaccine strain with the corresponding proteins of the international standard strains revealed significant differences.
  • the Env protein of the EIAV donkey leukocyte vaccine strain is different from the Env protein of the international standard virulent strain (AF028232) in various regions in the structure of ⁇ -helix, ⁇ -sheet, and corners. Among them, the number and position of the corner structures may be different. It is the main reason for the obvious difference in the secondary structure between the two. Secondary structure diagram of Tat protein of horse-borne poor donkey leukocyte vaccine strain There is an obvious hydrophobic group at the end.
  • the adjacent area is a ⁇ -sheet structure and forms a more concentrated hydrophilic group.
  • the amino end has four corner structures.
  • the secondary structure of the Tat protein of the international standard virulent strain (AF028232) The carboxyl end of the figure has no hydrophobic groups, and its adjacent area is a loose, irregularly curled structure, and there are two independent hydrophilic groups, and the amino end has a rich corner structure.
  • This difference in the high-level structure of proteins may be the basis for their functional differences, thus making them important candidate antigens for genetically engineered vaccines.
  • the corresponding protein genes of HIV and other lentiviruses can also be modified to study the possibility of using them as vaccine antigens.
  • the invention clarifies the full-length gene sequence of equine infectious anemia virus donkey leukocyte vaccine strain at home and abroad for the first time, as well as each structural gene and regulatory gene and each encoded protein, which will provide important guidance for the development of other lentivirus vaccines.
  • Nucleic acid diagnostic and serological diagnostic reagents for the development of major genes and their encoded proteins can be used for the diagnosis of EIAV infection, the differential diagnosis of EIAV wild virus infection and vaccination, and the development of genetically engineered vaccines.
  • the gene sequence of the present invention is helpful for the differential diagnosis of immunized horses and horses infected with American epidemic strains.
  • the full-length gene sequence of equine infectious anemia virus donkey leukocyte attenuated vaccine of the present invention can also be used to construct a gene transfer vector for gene therapy.
  • Equine infectious anemia virus cannot cause human disease, and constructing a gene transfer vector derived from the vaccine strain can not only overcome the defects such as low efficiency of gene transfer of murine leukemia virus-based gene transfer vectors and inability to transfect undivided cells, etc. , And there is better security.
  • FIG. 1 is an electrophoretic photograph showing the results of amplification of different templates by the primer set I of Example 2.
  • Lanes 1 and 2 in the figure are the results of amplification of total DNA from donkey leukocytes infected by the Chinese EIAV donkey leukocyte attenuated vaccine strain of the present invention (fragment) The size is about 200bp);
  • lane 3 is the DNA marker pBR322 / BstNI;
  • lane 4 is the DNA amplification result of the donkey leukocyte culture using the international standard strain Wyoming strain (no amplification phenomenon).
  • FIG. 2 is an electrophoretic photograph showing the amplification results of different templates by the primer set II of Example 2.
  • Lane 1 is DNA-labeled DL2000; lanes 2 and 3 are infected with donkey leukocytes by the Chinese EIAV donkey leukocyte attenuated vaccine strain of the present invention. Total DNA amplification result (fragment size about 190bp); lane 4 is the result of donkey leukocyte DNA amplification; lane 5 is the result DNA amplification result of donkey leukocyte culture of international standard strain Wyoming strain (no amplification phenomenon).
  • FIG. 3 is an electrophoretic photograph showing the amplification results of the primer set III of Example 2 for different templates.
  • lane 1 is a DNA marker pBR322 / BstNI
  • lane 2 is a DNA amplification of a donkey leukocyte culture using the international standard strain Wyoming strain. Increased results (no amplification phenomenon)
  • lanes 3 and 4 are the amplified DNA results of donkey leukocytes infected with the Chinese EIAV donkey leukocyte attenuated vaccine strain of the present invention (fragment size about 380bp).
  • FIG. 4 is an electrophoretic photograph showing the results of amplification of different templates by the primer set IV of Example 2.
  • Lanes 1 and 2 in the figure are the amplification results of total DNA from donkey leukocytes infected with the Chinese EIAV donkey leukocyte attenuated vaccine strain of the present invention (fragment) The size is about 220bp) ;
  • lane 3 is the DNA marker pBR322 / BstNI;
  • lane 4 is the DNA amplification result of the donkey leukocyte culture using the international standard strain Wyoming strain virus (no amplification phenomenon).
  • the invention will be further described below with reference to the drawings and embodiments.
  • Example 1 Equine infectious anemia virus (EIAV) donkey leukocyte attenuated vaccine strain full-length genome sequencing virus culture and cell genomic DNA extraction
  • Equine infectious anemia virus donkey leukocyte attenuated vaccine strain (Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences) was inoculated with healthy bovine leukocytes cultured at 37 ° C (lml per 10 7 cells) until cytopathic changes (CPE ) But when the cells do not begin to fall off, collect the virus, discard the supernatant, and use the Qiagen Genomenic DNA Kit (M13433, midi) according to the method provided in the instructions to extract the genomic DNA of the cell and use it as a template.
  • CPE cytopathic changes
  • the primers were designed according to the sequence of the EIAV Wyoming strain that has been published internationally.
  • the gp90 and 5'LTR genes were first amplified, and then the primers were designed based on the measured sequences to amplify multi-segment genes.
  • the clone was directly cloned into pGEM R- T Easy Vector (Promega) according to the method recommended in the instructions, transformed into E. coli DH5 cc, identified by EcoRI digestion, sequenced by ABI 377 DNA automatic sequencer, and sequenced by GCG software package to analyze each gene.
  • SEQ ID NO: 1 full-length viral genome sequence
  • SEQ ID NO: 2--7 were obtained.
  • the specific amplification method of each fragment is as follows-Fragment A (FA):
  • Amplified region 5'LTR (1-320) Amplification primers and methods:
  • Reaction system lOx buffer (Mg 2+ 2mM) 5 ⁇ 1, dNTP (10 mM) 1 ⁇ , LTR-F 1: (20 ⁇ ) 1 ⁇ 1, GRll: (20 ⁇ ) 1 ⁇ 1, Taq (promega) 1 ⁇ 1 (1 ⁇ m), template ll ( 200ng), ⁇ 2 041 ⁇ 1
  • Reaction conditions 95 ° C for 5 minutes, 30 cycles of 94 ° C for 1 minute, 52 ° C for 1 minute, 72 ° C for 1.5 minutes, and 72 ° C for 10 minutes
  • Reaction conditions 95 ° C for 5 minutes, 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, and 72 ° C for 10 minutes
  • Sequencing method T7 universal primer sequencing.
  • Amplification primers and methods are Amplification primers and methods:
  • reaction conditions are the same as in the first round of amplification of fragment A above.
  • Reverse primer EVENR2 5'-CAGATACTGAGGTTGTCTTCCT -3 'Reaction system and conditions:
  • Sequencing method First, use T7 and SP6 universal primers for sequencing to determine the insertion direction, and then design primers RTF1, RTR20, RTR40, and GF40 based on the detected sequences, and use the walking method to determine the entire sequence. For areas where it is difficult to find the ideal sequencing primer, subclones are constructed using restriction enzyme sites on the viral gene, and then the universal primers T7, SP6 and the designed primer RLR10 are used for sequencing.
  • the primer sequences used are as follows:
  • Forward primer F70 5'- CCAACAAGGAAGACAACCTC -3 'Reverse primer: R51 5'-AGACATAQTAQCGCTAQCAG -3'
  • the reaction system is the same as the first round of amplification of fragment A above, except that the primers used are F70 and R51.
  • Reaction conditions 95 ° C for 5 minutes, 30 cycles of 94 ° C for 1 minute, 52 ° C for 1 minute, 72 ° C for 2 minutes, and 72 ° C for 10 minutes
  • Sequencing method First, use T7 and SP6 universal primers for sequencing to determine the insertion direction, and then design primers LTR20 (5 '— GCCTTAATGCAACAGTC3') based on the detected sequence to determine the entire sequence.
  • Sequencing method T7, SP6 universal primers were used for sequencing.
  • Amplification primers :
  • Reverse primer LTR-R1 5'-CCCCCTCTAQATGTAQGATCTCGAACAGAC-3 '
  • the first round of amplification reaction system is the same as the first round of amplification of fragment A above, except The primers used were EF51 and LTR-Rl.
  • Reaction conditions 95 ° C for 5 minutes, 30 cycles of 94 ° C for 1 minute, 52 ° C for 1 minute, 72 ° C for 1.5 minutes, and 72 ° C for 10 minutes
  • the second reaction system was the same as the first round of amplification, except that the primers used were LTR-F1 and LTR-Rl, and the template was 5 microliters of the product of the first round of amplification.
  • Reaction conditions 95 ° C for 5 minutes, 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, and 72 ° C for 10 minutes
  • This example illustrates the application of the gene sequence of the present invention in detecting the pathogenic properties of EIAV, which is helpful for the differential diagnosis of immunized horses and horses infected with American epidemic strains.
  • the following primers for the gene sequence of the EIAV donkey leukocyte vaccine strain were designed and synthesized by comparing the entire gene sequence of the international standard strain Wyoming strain (Wyoming Genebank Accession: AF028232)- Primer name position sequence (5, 3, 3) Primer direction
  • Donkey leukocyte total DNA infected with the EIAV donkey leukocyte vaccine strain of the present invention and donkey leukocyte culture total DNA of the international standard strain Wyoming strain virus were used as templates, respectively.
  • Taq polymerase from PROMEGA was used to perform PCR. mMMgCl 2 5ul, dNTP (2.5mM) lul, primer 1 (20uM) lul, primer 2 (20uM) lul, Taq DNA polymerase 2u, H 2 034.6ul, template 2 ul.
  • Primer 1 and Primer 2 have the following different combinations:
  • Combination I CHF1369 + CHR1567 (expected amplified fragment size about 200bp)
  • Combination II CHF1369 + CHR1553 (expected amplified fragment size about 190bp)
  • Combination III CHF5129 + CHR5610 (expected amplified fragment size about 380bp)
  • Combination IV CHF1338 + CHR1572 (expected amplification Fragment size is about 220bp)
  • the reaction program is 95 ° C for 5 minutes, 30 cycles of 94 ° C for 40 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, and then 72. C for 10 minutes.
  • TGCTAGGCAA CTAAACCGCA ATATCCTGTA GTTCCTCTTG CGTTCCGCAT TTGTGACGH TTAAGTTCCT GTTTTTACAG TATATAAGTG CTTGTATTCT 200
  • GATATTGGCA CTATGAGACA AAAAATGGCA TTATTAGCCA AGGCACHCA AGCAGGATTA GCTGGTCCTA TGAAGGGAGG ⁇ GGGGGACCCT 1600
  • AAATACAGCA AAGAAAGAAC ACTTGGATTC CAAAAAGGTG TGAGAAAACT TGGGCTAAGG TAAAACATTG TCCAATGGAT TTATTATATG GTATAAATAA 6100
  • TTACAATCTC CAAAGTTTGC TCACTATAAT TGCACCATAA ATAATAAAAC AGAG MGG CGATGGCAAT TGGTAAAAAC ATCAGGCATC ACTCCTTTAC 1300

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Description

马传染性贫血病毒驴白细胞弱毒疫苗株的全长基因及其应用 发明领域
本发明涉及马传染性贫血病毒驴白细胞弱毒疫苗株的全长基因、 各功能基因的 DNA序列及其编码的蛋白质序列、 各功能基因及其蛋 白产物的应用。
发明背景
马传染性贫血病毒 (Equine Infectious Anemia Virus, EIAV)是引起 马传染性贫血的病原体， 是最早被人类发现的病毒。 马传染性贫血 于 1843 年首先在法国发现， 一个多世纪以来对畜牧业造成了巨大的 危害， 因而也是各国科学家一直在致力研究和控制的疾病之一。 中 国自六十年代起投入巨资对该病毒的生物学性状进行了研究， 分离 并培育出与国外毒株有明显差异的强毒株进行体外驴白细胞传代， 逐步驯化该病毒使之成为无致病力， 但可以使动物在接种之后产生 较坚强免疫保护的疫苗株， 该疫苗株自 1976年开始生产， 1978年在 全国大规模应用， 至今已接种 7000万匹马骡驴， 已经控制了该病在 我国的流行 (沈荣显等， 马传染性贫血免疫的研究， 中国农业科学， 第 4期 P1-15, 1979)。
EIAV属逆转录病毒科慢病毒属 (lentivirus), 与同属于慢病毒属的 人类免疫缺陷病毒 (Human Immunodificiency Virus, HIV)在基因组结 构，基因编码蛋白及基因调控方式等方面有许多相似之处（J.M.Coffm， The structure And Classfication of Retroviruses, The Retroviridae, Vol.1, pl9, edit by Jay A. Levy, Plenum Press), EIAV的逆转录酶、 蛋白酶、 dUTP酶、 包膜糖蛋白和核心蛋白的结构和功能与 HIV的相应组份亦 有很高的相似性， 而且马传染性贫血病毒感染马属动物后引起急性 发病的马症状特点为周期性发热、 红血球减少性贫血和持续性病毒 血症， '易于观察和研究， 因此该病毒成为研究慢病毒感染和致病机 制及病毒酶功能的重要模型 (R.C. Montelaro et， al Equine Retroviruses, in: vol.2, p257)。
对 EIAV病毒基因组的研究是在 70年代分子生物学技术发展并 得到广泛应用之后才兴起的。 现在 GenBank 中已发表的马传染性贫 血病毒的全长基因组序列均来源于美国标准强毒株 （Wyoming株） 和日本强毒株 （Goshim株）， 及由它们衍生得到的细胞培养适应株的 基因序列。 然而这些毒株均不是疫苗毒株。 中国研制成功的 EIAV弱 毒疫苗至今还是目前世界上唯一经过大规模应用、 长时间检验而被 证明是安全和有效的慢病毒疫苗 （R.C.Montelaro， et al. in: Vaccines against Retroviruses, Vol.4，P605 ， R.C.Montelaro et,al Equine Retroviruses, in: vol.2, P.257)。 但由于这种马传染性贫血疫苗毒株是由 经典路线制造出的， 其基因组序列尚未被阐明。 有必要进行这一方 面的工作， 从而为优化该疫苗、 制备基因工程疫苗提供有利的基础， 并使该疫苗模型对其他慢病毒疫苗研制具有指导作用。
因此， 本发明的目的在于提供马传染性贫血病毒驴白细胞弱毒疫 苗株的全长基因序列、 各功能基因的 DNA序列及其编码的蛋白质序 列及各功能基因及蛋白产物的应用。
发明概述
根据本发明的一个方面， 本发明涉及马传染性贫血病毒驴白细 胞弱毒疫苗株的全长 DNA序列及其主要结构基因 （gag, pol， env) 和 调控基因 (5， LTR, 3 ' LTR^ rev基因， S2基因， tat基因)的 DNA序列。 其中该毒株的全长基因组包含 8258个核苷酸， 具有如 SEQ ID NO: 1所示的核苷酸序列。 在该毒株的全长基因组中， 核苷酸 1-325位为 5' LTR, 核苷酸 7922-8258位为 3' LT , 核苷酸 466-1926位为 gag 基因， 核苷酸 1689-5120位为 pol基因， 核苷酸 5313-7904位为 env 基因， 核苷酸 365-462位为 tat基因第一外显子， 核苷酸 5138-5276位 为 tat基因第二外显子，核苷酸 5454-5546位为 rev基因第一外显子， 核 苷酸 7250-7651位为 rev基因第二外显子， 核苷酸 5287-5493位为 S2 基因。 gag基因、 pol基因、 env基因、 rev基因、 tat基因、 S2基因 的 DNA序列和相应的推导的氨基酸序列分别如 SEQ ID NOS: 2、 3、 4、 5、 6、 7以及 SEQ ID NOS: 8、 9、 10、 11、 12、 13所示。 本发 明的马传染性贫血病毒驴白细胞弱毒疫苗株的全长基因克隆已于 1999年 4月 19日保藏于中国微生物菌种保藏管理委员会普通微生物 中心， 保藏号为 CGMCC NO.0394, 该保藏物于 2000年 4月 19日转 成根据国际承认的用于专利程序的微生物保藏布达佩斯条约的国际 保藏。 本领域技术人员公知的是， 本文中 SEQ ID NO: 1—7所测得 的序列有可能存在测序误差， 因此， 若出现这种情况， 本发明的保 藏物是决定性的。
发明详述 本发明通过以下技术手段实现： 首先用标准的 PCR方法扩增该 疫苗株的基因， 分别克隆到质粒载体中再进行 DNA序列的分析， 最 后得到该病毒的全长基因序列。 EIAV驴白细胞弱毒疫苗毒株在体外 驴白细胞复制过程中以前病毒 DNA的形式整合到驴白细胞的染色体 上， 本发明人取此前病毒 DNA作为扩增病毒基因的材料。 首先从病 毒感染的驴白细胞中提取染色体 DNA， 并以此为模板用 PCR方法扩 增 EIAV疫苗毒株的前病毒 DNA。 扩增引物的设计是根据国际 EIAV 强毒株序列， 先用其各区段 EIAV基因进行预扩增。 经过多次摸索， 并根据对得到的扩增片段进行测序中获得的 EIAV疫苗株的部分序列 资料， 设计出 EIAV疫苗弱毒株特异性引物， 分段扩增病毒基因并克 隆到质粒载体上， 进而对全部基因进行克隆和序列测定， 获得了病 毒基因组全长序列和其主要结构基因（gag基因， pol基因和 env基因） 及主要调控基因 (5， LTR, 3' LTR,rev基因， S2基因， tat基因等)的序 列。
利用 GCG软件 (Genetics Computer Group, Inc., Wisconsin, USA) 对全长基因序列的开放读码框架进行分析， 得到各个结构基因及调 控基因所编码的蛋白质的氨基酸序列， 并推导出各基因在全长基因 序列中的具体位置。
将得到的疫苗株序列与 GenBank 所发表的国际标准株序列 (Wyoming株， GeneBank Accession Number: AF028232)进行核苷酸和 氨基酸同源性的比较， 结果发现， 各基因与国外标准野毒株的核苷 酸同源性在 73.46-90.06%之间， 其中 env基因、 rev基因和 S2基因与 国际标准株的差异较大， 同源性分别为 73.46%， 73.54%和 75.76%。 氨基酸序列与国际标准株相应序列的同源性比较结果发现， 外膜蛋 白 （Env蛋白） 及 Rev蛋白和 S2蛋白的变异均较大， 氨基酸同源性 分别为 67.41%, 64.85%和 54.54% 0
另外， 还利用 GCG软件对该疫苗株各结构基因和调控基因所编 码蛋白质的二级结构进行了预测。 疫苗株 Env和 Tat蛋白二级结构与 国际标准株的相应蛋白的二级结构的比较发现有显著差异。 EIAV驴 白细胞疫苗株的 Env蛋白与国际标准强毒株 （AF028232) 的 Env蛋 白在多个区域的 α螺旋、 β片层和转角等结构都有不同， 其中转角 结构的数量和位置的不同， 可能是导致了两者间二级结构有明显差 异的主要原因。 马传贫驴白细胞疫苗株的 Tat蛋白的二级结构图羧基 端有一明显的疏水基团， 其临近区域为 β片层结构并形成较集中的 亲水基团， 其氨基端有四个转角结构； 国际标准强毒株 （AF028232) 的 Tat蛋白的二级结构图的羧基端无疏水基团， 其临近区域为松散的 无规则卷曲结构， 并有两个独立的亲水基团， 其氨基端有丰富的转 角结构。 这种蛋白质高级结构的差异可能是它们功能差异的基础， 从而使其称为基因工程疫苗的重要候选抗原。 根据此原理， 也可对 艾滋病毒和其他慢病毒的相应蛋白基因进行改造， 以研究将其作为 疫苗抗原的可能性。
另外， 通过对氨基酸序列分析发现， 驴白细胞疫苗株的 Env蛋白 包含 19个潜在的糖基化位点， 国际标准强毒株 （AF028232) 的 Env 蛋白包含 23个潜在的糖基化位点。
本发明在国内外首次阐明了马传染性贫血病毒驴白细胞疫苗株 的全长基因序列， 以及各个结构基因和调控基因及各自编码的蛋白 质， 将为其他慢病毒疫苗的研制提供重要的指导。 各主要基因及其 编码的蛋白质研制的核酸诊断和血清学诊断试剂可分别用于 EIAV感 染的诊断、 EIAV野毒株感染与疫苗株接种的鉴别诊断以及基因工程 疫苗的研制。 例如， 如下述实施例 2 所述， 本发明的基因序列有助 于对免疫马和美洲流行株感染马的鉴别诊断。
本发明的马传染性贫血病毒驴白细胞弱毒疫苗全长基因序列还可 用于构建进行基因治疗的基因转移载体。 马传染性贫血病毒不能引 起人类发病， 构建源于该疫苗株的基因转移载体， 不但可以克服以 往所用的鼠白血病病毒来源的基因转移载体的基因转移效率较低和 不能转染未分裂细胞等缺陷， 而且有较好的安全保障。
附图简述
图 1为示出实施例 2的引物组合 I对不同模板的扩增结果的电泳 照片， 图中泳道 1和 2为对本发明的中国 EIAV驴白细胞弱毒疫苗株 感染驴白细胞总 DNA扩增结果（片段大小约 200bp); 泳道 3为 DNA 标记 pBR322/BstNI; 泳道 4为以国际标准株 Wyoming株病毒驴白细 胞培养物 DNA扩增结果 (无扩增现象)。
图 2为示出实施例 2的引物组合 II对不同模板的扩增结果的电泳 照片， 图中泳道 1为 DNA标记 DL2000; 泳道 2和 3为对本发明的 中国 EIAV驴白细胞弱毒疫苗株感染驴白细胞总 DNA扩增结果 （片 段大小约 190bp); 泳道 4为以驴白细胞 DNA扩增结果； 泳道 5为以 国际标准株 Wyoming株病毒驴白细胞培养物 DNA扩增结果 (无扩增 现象)。
图 3为示出实施例 2的引物组合 III对不同模板的扩增结果的电 泳照片， 图中泳道 1为 DNA标记 pBR322/BstNI; 泳道 2为以国际标 准株 Wyoming株病毒驴白细胞培养物 DNA扩增结果 (无扩增现象)； 泳道 3和 4为对本发明的中国 EIAV驴白细胞弱毒疫苗株感染驴白细 胞总 DNA扩增结果 （片段大小约 380bp)。
图 4为示出实施例 2的引物组合 IV对不同模板的扩增结果的电 泳照片， 图中泳道 1和 2为对本发明的中国 EIAV驴白细胞弱毒疫 苗株感染驴白细胞总 DNA扩增结果 （片段大小约 220bp); 泳道 3为 DNA标记 pBR322/BstNI; 泳道 4为以国际标准株 Wyoming株病毒 驴白细胞培养物 DNA扩增结果 (无扩增现象)。 以下将参照附图和实施例进一步描述本发明。
实施例 1 马传染性贫血病毒 (EIAV)驴白细胞弱毒疫苗株全长 基因组测序 病毒培养和细胞基因组 DNA的提取
将马传染性贫血病毒驴白细胞弱毒疫苗株 （中国农业科学院哈尔 滨兽医研究所） 接种 100%大牛血清 37°C培养的健康驴白细胞 (每 107 细胞接毒 lml)， 至出现细胞病变 (CPE)但细胞未开始脱落时收毒， 弃 上清， 用 Qiagen Genomenic DNA Kit(M13433， midi)按说明书提供的 方法提取细胞基因组 DNA， 用作模板。
全基因的分段扩增、 克隆和测序
根据国际上已经发表的 EIAV Wyoming株的序列设计引物， 首 先扩增出 gp90和 5'LTR基因， 再根据测得序列设计引物， 扩增多片 段基因。按说明书推荐方法直接克隆到 pGEMR-T Easy Vector (Promega) 上， 转化大肠杆菌 DH5 cc， EcoRI酶切鉴定， ABI 377DNA自动测序 仪测序， 再用 GCG软件包连接序列， 分析各基因。 最后获得了病毒 基因组全长序列 （SEQ ID NO: 1 ) 及各基因序列 （SEQ ID NO: 2— 7)。 各片段具体扩增方法如下- 片段 A(FA):
扩增区域： 5'LTR (1-320) 扩增引物和方法：
第一轮
正向引物： LTR-F 1 5'-GCGCGCGAATTCTGTGGGGTTTTTATAQG-3' 反向引物： GRl l 5'-AACCTTGCTGCTATGGGAAT-3'
反应体系： lOx缓冲液 （Mg2+ 2mM) 5μ1， dNTP(10 mM) Ιμΐ, LTR-F 1： (20μΜ) 1μ1， GRll : (20μΜ)1μ1, Taq(promega) 1μ1(1ιι)， 模 板 l l(200ng)， Η2041μ1
反应条件： 95°C 5分钟， 30个循环的 94°C 1分钟， 52°C 1分 钟， 72°C 1.5分钟， 之后 72°C 10分钟
第二轮
正向引物： LTR-F 1 5'-GCGCGCGAATTCTGTGGGGTTTTTATAQG-3' 反向弓 I物： LTR-R1 5'-CCCCCTCTAQATGTAQGATCTCGAACAGAC-3' 反应体系与上述第一轮扩增相同， 只是所用引物分别为 LTR— F1 和 LTR— Rl， 模板为 5微升第一轮扩增的产物。
反应条件： 95°C 5分钟， 30个循环的 94°C 1分钟， 55°C 1分 钟， 72 °C 1分钟， 之后 72°C 10分钟
测序方法： T7通用引物测序。
(2) 片段 B(FB)
扩增区域 (115-1188)
扩增引物和方法：
正向弓 I物： 732 5'-ACCGCAATAACCGCATTTGTGACG-3' 反向引物： GRl l 5*-AACCTTGCTGCTATGGGAAT-3* 反应体系与上述片段 A 的第一轮扩增相同， 只是所用引物分别 为 732和 GR11。
反应条件与上述片段 A的第一轮扩增相同
测序方法： T7， SP6通用引物测序
(3)片段 C(FC)
扩增区域： （460-5254)
扩增引物- 第一轮
正向引物： P13 5'-GTAAGATGGGAGACCCTTTG -3' 反向引物： EVENRl 5'-ATGCTGACCATGTTACCCCTT-3' 第二轮 正向引物： P13
反向引物： EVENR2 5'-CAGATACTGAGGTTGTCTTCCT -3' 反应体系和条件：
使用 Expend™Long Template PCR System(Boehringer Mannheim) 按说明书推荐的反应体系和反应条件进行 (缓冲液使用缓冲液 3)
测序方法： 首先用 T7， SP6通用引物测序， 确定插入方向， 然 后根据测知的序列设计引物 RTF1， RTR20, RTR40和 GF40, 采用 步行法测出全部序列。 对于难于找到理想测序引物的区域， 利用病 毒基因上的限制性内切酶位点构建亚克隆， 再使用通用引物 T7， SP6 及所设计的引物 RLR10测序。
所用引物序列如下：
Τ7 5' TAA TAC GAC TCA CTA TAG GGA GA 3'
SP6 5' CAT ACG ATT TAG GTG ACA CTA TAG 3'
RTF1 5' CCT CGA GGG AGA TGC ATA TTT C 3'
RTR20 5' CCC TGA TCT CAT CCA TGT T 3'
RTR40 5' GGT ATA GTG AAA TAT GCA TCT CC 3'
GF40 5* AAG TGA GGG ACA TCC GGC T 3'
RLR10 5' CTG TCC TCC CAT ACT TTC 3'
(4)片段 D(FD)
扩增区域（5222-6715)
扩增引物
正向引物： F70 5'- CCAACAAGGAAGACAACCTC -3' 反向引物： R51 5'-AGACATAQTAQCGCTAQCAG -3' 反应体系与上述片段 A 的第一轮扩增相同， 只是所用引物分别 为 F70和 R51。
反应条件： 95°C 5分钟， 30个循环的 94°C 1 分钟， 52°C 1分 钟， 72°C 2分钟， 之后 72°C 10分钟
测序方法： 首先用 T7， SP6通用引物测序， 确定插入方向， 然 后 根 据 测 知 的 序 列 设 计 引 物 LTR20 ( 5 ' — GCCTTAATGCAACAGTC3 ' )， 测出全部序列。
(5)片段 E(FE)
扩增区域（6680-8147)
扩增引物- 正向弓 I物: EF51 5'-GCTACTGCTATTGCTGCTAQ'-3 反向弓 I物: LR3 5'-CTCAGACCGCAGAATCTGAGT-3'
反应体系和条件：
使用 Expend™Long Template PCR System(Boehringer Mannheim) 按说明书推荐的反应体系和反应条件进行 (缓冲液使用缓冲液 3)
测序方法： 用 T7， SP6通用引物测序。
(6)片段 F(FF)
扩增区域: 3'LTR(7937-8258)
扩增引物：
第一轮
正向引物： EF51 5'-GCTACTGCTATTGCTGCTAQ'-3
反向引物： LTR-R1 5'-CCCCCTCTAQATGTAQGATCTCGAACAGAC-3'
第二轮
正向引物： LTR-F 1 5'-GCGCGCGAATTCTGTGGGGTTTTTATAQG-3' 反向引物： LTR-R 1 5'-CCCCCTCTAQATGTAQGATCTCGAACAGAC-3' 第一轮扩增反应体系与上述片段 A 的第一轮扩增相同， 只是所 用引物分别为 EF51和 LTR— Rl。
反应条件： 95°C 5分钟， 30个循环的 94°C 1分钟， 52°C 1分钟， 72°C 1.5分钟， 之后 72°C 10分钟
第二轮反应体系与上述第一轮扩增相同， 只是所用引物分别为 LTR— F1和 LTR— Rl， 模板为 5微升第一轮扩增的产物。
反应条件： 95°C 5分钟， 30个循环的 94°C 1分钟， 55°C 1分 钟， 72°C 1分钟， 之后 72°C 10分钟
测序方法： T7通用引物测序。 实施例 2 EIAV弱毒疫苗株与美洲流行株的基因鉴别诊断
本实施例说明本发明的基因序列在检测 EIAV病原属性方面的应 用， 其有助于对免疫马和美洲流行株感染马的鉴别诊断。
根据本发明的马传染性贫血病毒驴白细胞疫苗株基因序列， 对 照国际标准株 Wyoming株全基因序列 （ Wyoming Genebank Accession Number: AF028232), 设计并合成以下针对 EIAV驴白细胞疫苗株基 因序列的引物- 引物名称 位置 序列 （5，一3，） 引物方向
CHF1369 1369 GGACATCCGGCTGATATAAC 正向
CHR1567 1567 GACCAGCTAATCCTGCTTGA 反向
CHR1553 1553 GCTTGAAGTGCCTTGGCTAA 反向
CHF5129 5129 TCAGGAATCACCACCAGTCAG 正向
CHR5610 5610 GTTGTTGCCTCTCATACCAC 反向
CHF1338 1338 AGACAGATTGCTGTCTC 正向
CHR1572 1572 CATAGGACCAGCTAATC 反向
分别以本发明的 EIAV驴白细胞疫苗株感染驴白细胞总 DNA、 国际标准株 Wyoming株病毒驴白细胞培养物总 DNA为模板， 采用 PROMEGA公司 Taq聚合酶，进行 PCR，反应体系为 10x缓冲液 5ul， 2.5mMMgCl2 5ul, dNTP(2.5mM) lul，引物 1 (20uM) lul,引物 2 (20uM) lul, Taq DNA聚合酶 2u， H2034.6ul, 模板 2 ul。
引物 1和引物 2 具有下列不同组合：
组合 I CHF1369+CHR1567 (预期扩增片段大小约 200bp) 组合 II CHF1369+ CHR1553 (预期扩增片段大小约 190bp) 组合 III CHF5129+CHR5610 (预期扩增片段大小约 380bp) 组合 IV CHF1338+CHR1572 (预期扩增片段大小约 220bp) 反应程序为 95°C 5分钟， 30个循环的 94°C 40秒， 50°C 30秒， 72°C30秒， 之后 72。C 10分钟。
用琼脂糖凝胶电泳分析结果， 可见上述不同引物组合对本发明 的 EIAV驴白细胞疫苗株感染驴白细胞总 DNA均可进行特异性扩增。 而在上述相同条件下， 以国际标准株 Wyoming 株病毒驴白细胞培养 物 DNA为模板进行 PCR扩增， 则无特异扩增现象。 参见附图 1， 2， 3禾口 4。 序列表
SEQ ID NO: 1的信息：
(a)序列特征：
长度： 8258bp
类型： 核酸
链性： 双链
拓扑结构： 线性
(b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株
(d)序列描述： SEQ ID NO: 1
TGTGGGGTH HATGAGGGG TTTTATAAAT GAHATAAGA GTAAAAAGAA GGGGGCTGAT GCTCTCATAA CCHGTATAA CCCAAAGGAC TAGCTCATGT 100
TGCTAGGCAA CTAAACCGCA ATATCCTGTA GTTCCTCTTG CGTTCCGCAT TTGTGACGH TTAAGTTCCT GTTTTTACAG TATATAAGTG CTTGTATTCT 200
GACAATTGGG CACTCAGATT CTGCGGTCTG AGTCCCTTCT CTGCTGGGCT AGACTAGCCT HGTAATAAA TATAATTCTC TGCTMGTCC CTGTCTCTAG 300
TTTGTCTTGT TTTCAAGATC TAACAGCTGG CGCCCGAACA GGGACCTGAG GGCGCAGACC CTGCCTGCTG AACCTGGCTG ATCATAGGAT CCCTAGGACA 400
GCAGAGGAGA ACTTACAGAA GTCHCTGGA GGTGHCCTG GCCACAACAC AGGAAGACAG GTAAGATGGG AGACTCTTTG ACATGGAGCA AAGCGCTCAA 500
GAAGTTAGAG AAGGTGACGG TACAAGGGTC TCAAAAGCTA ACTAGTGGTA ACTGTAATTG GGCGCTGAAT TTGGTGGACT TATTCCATGA CACCAATHT 600
GGTAAAGAAA AAGACTGGCA ATTAAGGGAC GTCATTCCAT TGTTAGAGGA CGTTTCCCAG ACGTTGTCAG GACAAGAGAG AGAGGCATH GAAAAAACTT 700
GGTGGGCAAT AGCTGCCGH AAGATGGGCT TACAAATTAA TACTGTGAAT GATGCAAAAA CAACATHTC TATATTAAAA GCCAAGTTTG AAAGAAAGAC 800
TGCAAATAAT ACCAAAAAGC AGTCTGAGCC CGAGGAAGAA TACCCAATAA TGATTGATGG GGCTGGAAAC AGAAACTTTC GGCCAHAAC ACCCAGAGGA 900
TATACTACCT GGGTAAATAC TATACAGCAA AACAATCTCT TAAATGAAGC TAGTGTGAAT TTAHTGGTA TTTTATCAGT AGACTGTACT TCTGAGGAAA 1000
TGAATGCAH TTTGGATGTA GTACCAGGAC AAGCAGGACA AAAACAAGTA CTATTGGATA ATCHGATAA GAHGCAGAA GAATGGGATC GTAGGCACCC 1100
GTTGCCAAAT CCTCCATTAG TGGCACCACC ACAAGGGCCT ATTCCCATGA CAGCAAGGTT CATTAGGGGA TTGGGAGTTC CTAGAGAAAG ACAGATGAAA 1200
CCTGCTTTTG ATCAGTTTAG ACAAACHAT AGACAATGGA T TAGAAGC AATGACAGAA GGGATAAA TMTGATTGG GAAACCCAAA GCGCAAAATA 1300
TTAGGCAAGG ACCCAAAGAA CCCTATCCAG AGTTTATAGA CAGATTGCTG TCTCAGATM AAAGTGAGGG ACATCCGGCT GATATAACTA AATTCCTGAC 1400
AGACACTTTA ACTATTCAGA ATGCTAATGA TGAATGCAAA AATGCTATGA GACATTTGAG GCCAGAAGAT ACTTTAGMG AGAAAATGTA TGCATGTAGA 1500
GATATTGGCA CTATGAGACA AAAAATGGCA TTATTAGCCA AGGCACHCA AGCAGGATTA GCTGGTCCTA TGAAGGGAGG ΑΑΤΑΤΠΑΑΑ GGGGGACCCT 1600
TAGGGGCGAA GCAGACATGT TATAATTGTG GAAAACCAGG ACATTTTTCT AGTCAATGTA AAGCACCTAA AATATGTTTT AAGTGCAAAC AGCCAGGACA 1700
THCTCAAAA CAATGTATAA ATGCTCCAAA AAACGGGAAA CAAGGGGCTC AGGGGAGGCC CCAGAAACAA ACTTTCCCTG TGCAGAAGGA GTCAATGAAC 1800
AAAACACAAA AAGAGGAGAA ACAGCAAGGG ACCTTATATC CAGATTTAAG TCAGATGAAA CAGGAATACA AGATCAAGGA AGAGGAAAAT CAAGAGGATC 1900
TCAATCTGAA CAGTTTGTGG GAGTMCTTA TAATTTAGAA AAGAGACCAA CTACAATAGT OTGATTAAT GACACACCCT TAAATGTATT GHGGACACA 2000
GGAGCAGACA CATCAGTACT AACTATTGCA CATTGTAATA GGTTAAAGTA TGGAGGAAGA AAATATCAAG GTACAGGTAT TGHGGGGTT GGAGGTAATG 2100
TAGAAACATT TTCCACTCCT GTTACAGTGA AAAAGAAAGG AAAACAAAH AAAACTAGAA TGTTAGTAGC AGATATCCCA GTTACTATTT TGGGGCGAGA 2200
TATTCHCAA GAAHAGGCG CACAATTACT AATGGCTCAA CTTTCAAAAG AAATAACCCC AAGAGAAATT AAATTAAAAA CAGGCACAGT AGGGCCTAAG 2300
GHCCCCAAT GGCCACTTAC TAAAGAGAAG TTGTTAGGTG CTAAAGAAAT AGTCAAAAAA TTGTTGGATG AAGGTAAAAT ATCAGAAGCC AGTGATGATA 2400
ATCCHATAA HCTCCTATA HTGTAATAA AAAAGAAATC TGGAAAGTGG AGAHATTGC AAGAHTAAG AGAGHAATT AAGGGTGGTA CAAGTAGAAC 2500
TGAAATATCC AGAGGATTAC CTCATCCAGG GGGAHAATT AAATGTAATC ATATGACAGT ATTAGATATT GGAGATGCAT ATTTCACTAT ACCATTAGAT 2600
CCAAAGTTTA GACAATATAC AGCATTTACT GTGCCATCCA HAATCATCA GGAACCAGAT AAAAGATATG TGTGGAATTG CTTGCCACM GGTTTTGTGT 2700
TAAGTCCATA CATATATCAA AAAACAHAC AGGACATAH ACAAGCnn AGAGAAAGGC ATCCAGATGT ACAATTATAT CAATATATGG ATGATTTATT 2800
CATTGGGAGT AATGAATCTA AAAGACAACA TAAGGAACTA GTAGAAGAAT TAAGAGCTAT TCTTTTAGAA AAGGGCTTTG AGACGCCTGG GGATAAATTG 2900
CAGGAAGAAG CACCCTATAA TTGGCTGGGA TATCAACHA GTCCAGGCAA GGAAAGTA CAAAAGATGC AAHAGAATT GGTAAAAGAG CCAACATTAA 3000
ATGATGTGCA AAAATCAAAG GGAAATATAA CATGGATGAG CTCAGGGGH CCTGGAHAA CAGTGAAGCA AATAGCTGCT ACCACTAAAG CHGCTTAGA 3100 TTTAAATCAT AAAGGTAGTA GGACCAGAGA AGCCCAAAAA GACHAGAGG AAAHAHAA AAGTHCAGA AGCTCAGGAT TCCCATATTA TAACCCAGAA 3200
GAAGAAGTAA TCTGTGAGAT TGAAATTACT AAAAATTATG AGGCTACTTA TATAATAAAA CAGTCTCAAG GAATAHGTG GGCAGGAAAG AAAAHATGA 3300
GGGCTAATAA AGGATGGTCC GCAGCAAAAA ATCTAATGH ATTGTTACAA CATGTAGCCA CAGAAAGTAT TGHAGAAn GGAACATGTC CAAAATHAA 3400
AGTACCTTH ACTAAAGAAC AAGTCAAATG GGAAATGGAA AAGGGATGGT ATTATTCATG GCTACCAGAC ATGGTATA CACATCAAGT TGTTCATGAT 3500
GATTGGAGAC TGAAATTAGT AGAGCAACCA ACATCTGGTA TAACAAHTA TACTGATGGG GGTAAACAGA ATGAAGAAGG AG GCAGCT TATGTGACTA 3600
GTAATGGGAA AACTAAACAA AAAAGGHAG GGCCTGTTAC TCATCAAACT GCTGAGAGGA TAGCMTACA AATGGCATTA GAAGATACTG AAGAGACATT 3700
GGTAAATATA GTAACTGATA GHACTACTG TTGGAAAAAT ATTACAGAAG GATTAGGGH AGAAGGACCA GACAGCCCCT GGTGGCCAAT AATTCAAAAT 3800
ATTAGGGCTA AAGAAATGGT TTATTTTGCT TGGGTACCAG GTCACAAAGG AATATATGGC AATCAATTGG CAGATGAGGC TACTAAAATA ACAGAGGAAA 3900
TTATGHAGC ATATCAAGGC ACACAGATTA GGGAAAAAAG AGATGAAGAT GCAGGGTATG ATTTGTGTAT TCCTTATGAC ATAATGATAC CTGTCTCTGA 4000
GACAAAAGTT ATACCCACAG ATGTAAAAAT ACAGGTACCT CACAAATGH HGGATGGGT AACTGGTAAG TCATCAATGG CTMGCMGG ATTAHAATC 4100
AATGGGGGAA TAAHGATGA AGGATACACA GGTGAAATAC AGGTAAHTG TACTAATAH GGAAAGAGTA ACATGAAACT CAGGGAAGGA CAAAAGTTTG 4200
CACAATTAAT, CATATTACAG CATCGATCAA ATGATAAACA AATCTGGGAT GAAAATAAAA CATCTCAAAG GGGAGATAAA GGGTTTGGAA GCACAGGTAT 4300
ATTTTGGGTA GAGAATATCC AAGAGGCGCA AGATGAACAT GAAAAHGGC ATACATCTCC AAAGATATTG GCAAAAAGAT ATGGGTTACC A GACTGTA 4400
GCTAAACAGA TAACTCMGA ATGCCCTCAT TGTACTAAAC AAGGATCTGG ACCAGCAGGT TGTGTAATGA GATCTCCTAA TCATTGGCAG GCTGATTGTA 4500
CACATTTAGA AAACAGGGTA ATAATGACAT TTGTAGAGTC TAATTCAGGA TACATTCATG CTACTCTATT GTCCAAAGAA AATGCCTTGT GTCCTTCATT 4600
GGCTATT G GAATGGGTGA GGTTATHTC TCCTAAATCT TTACATACAG ACAATGGTAC TAATTTTGTG GCAGAGTCAG TAGCAAATCT GHGAAAHC 4700
CTGAAGGTGA CACATACTAC AGGAATACCT TATCACCCAG AGAGCCAAGG CATTGTGGAA AGAGCAAACA GGACAnAAA AGAAAGAAH AAAAGTCATA 4800
GAGGAAATAC TCAGACACTT GMGCAGCAT TACAACTTGC TCTCA ACT TGTAACAAAG GGAGGGAAAG TATGGGAGGA CAAACTCCAT GGGAAGTATT 4900
TATTACTAAT CAGGCTCAAA CAATACATGA AGAACTTHA TTACAACAAG CACAATCTTC TAAAAAATTT TGTTTTTATA AAATTCCTGG TGAGCATAAT 5000
TGGAAGGGGC CCACCAGAGT GTTGTGGAAA GGTGATGGAG CAGTAGTGGT CAATGATGAG GAAAAAGGAA TAATTGCTGT GCCTTTMCC AGGACTAAAT 5100
TATTAATAAG ACCAAATTGA GCAHGTTTC AGGAATCACC ACCAGTCAGC TATCATTGTC AACTGTGTTT CCTGAGATCA TTGGGAATTG ACTACCTTGA 5200
CAGCTCGCTG AAGAAGAAGA ACAAACAAAG ACAGAAGGCC ATCAGGGAGG AAGACAACCT CAGTATCTTG TTATAAGGH TGGTGTATGG GATTATTTGG 5300
TAAAGGGGTA ACATGGTCAG CATTACATTC TATGGGGGTA TCCCAGGGGG AATATCAACC CCTATCACCC AACAAACAGA ATCAACAGAC ACACAGAAAG 5400
GGGATCATAT GGTATATCAA CCCTATTGTT ATAATGATAG CCATAAAGAA GAAATGGCAG AGACAAGAGA CACAAGATAC CAAGAAGAAA TGAACCGGAA 5500
AGAAGATAAA GAAGATAAAA GAAAGAATAA CTGGTGGAAG ATAGGTATGT TCTTATTGTG TCTG AGAG ATCACTGGAG GATTCCTCTG GTGGTATGAG 5600
AGGCAACAAC ATTCATATTA TATAAGATTG GHACAATAG GAGGTAGACT GAATGGTTCA GGAATGACTA GTGCCATAAA ATGTTGGGGT TCATTTCCTG 5700
GGTGTAGGCC ATHACTAAC TATTTCAGTT ATGAGACTAA TAGGACTGTT AGTAGAGATA ATAATACTGC TACTCTGHA GATACHATC AAAGAGAAAT 5800
AACAAACATA TACAGGACAT CTTGTGTGGA TAGTGATCAC TGTCAAGAAT ATAAATGTAA GCAAGTACAG TTGAAAAAGA ACAGCAATAA CATTATAATG 5900
AATAATTGTA GTAACAATAG GTGTGAAGAG TTTTGGGGGT TTAGCTGGn AGAATGTAAT CAGACAGAAA ATGCAATAAC TATATTGGTC CCAGAAATAG 6000
AAATACAGCA AAGAAAGAAC ACTTGGATTC CAAAAAGGTG TGAGAAAACT TGGGCTAAGG TAAAACATTG TCCAATGGAT TTATTATATG GTATAAATAA 6100
AATAAGAATG TGTGTCCAAC CTCCATTCTT TTTGTTTAAA CAGAATGATA CTTCTAATAA TACTAATATT CTCAGTAAH GTGGACCTTT AGTATTTCTT 6200
GGAATATTTG AGGACAATAA GGCAGCAATC CAGAATGGGA GTTGCACTCT TCACAGGACA AATATTAACA GGCCAGATTA TAGTGGATTT TACCAAGTGC 6300
CTATATTTTA TATATGCACC TTGACAGGAT TTCAAAGHG TAATAATGGA TCAATAATTA GTATAM AT GTATGAGTCT AATAATGTTC AATACTTGTT 6400
ATGCAATACT AGTAATACTA ATAGTACCAA TAATGCTAAT GTCTCTTGTG TGGTACAAAG TTTTGGAGTG ATAGGACAGG CACATGTGGC AHGCCCAGA 6500
AAAAATAAGA GGTTACAATC TCCAAAGTH GCTCACTATA ATTGCACCAT AAATAATAAA ACAGAGTTAA GGCGATGGCA ATTGGTAAAA ACATCAGGCA 6600
TCACTCCTTT ACCCATTTCC TCTACAGCTA ATACTGGATT AGTCAGACAC AAGAGAGACT HGGTATATC TGCTATAATA GCTGCCATTG TAGCTGCTAG 6700
TGCTATTGCT GCTAGTGCTA CTATGTCTTA TATCGCTTTG ACAGAAGTCA ACAAATTAGA TAGTGTACAA AATCATACTT TTGAAGTAGA GAACAATACT 6800
ATCAATAACA TAGAGHAAC AGAAGAGCAA AHCATATAT TATATGCTAT GGTTCTCCAA ACACATGCAG ATGTTCAATT GTTAAAAGAA CAACAAAAGA 6900
TTGAGGAAAC ATTTAATTTA AHGGATGTA TAGAAAGATC ACATACATH TGTCATACTG GACATCCCTG GAATGAATCA TGGGGTCAGT TAAATGATTC 7000
TACACAGTGG GATGACTGGG TAGATAAGAT GGAAAATTTA AATCATGATA TATTAACAAC ACTTCATACT GCTAGAAATA ATCTAGAACA ATCTATGATA 7100
ACTTTCAATA CACCTGACAG TGTAGCACAA TTTGGAAAAA ATATTTGGAG TCATATTGCA AATTGGATTC CTAGATTAGG AGCTTCCATA ΑΠΑΑΑΤΑΤΑ 7200
TAGTGTTGAT ATTACHATA TATGTGTTAC TAACCTCTGC ACCTAAGATC CTCAGAGGCC TCTTGACAAC GATGAGTGGT GCAGGATCCT CCGCCAGTCG 7300
CTACCTGAAG AAAAGATACC ATCACAAACA TGCATCGCGA GGAGACATCT GGGCCCAGGT CCAATATCAT GCGTACCTGG CAGACGAGAC TCATGGCTCA 7400
GGGGACAAGT CCAACATGCG GMGCTCTCC AGGAACAACT GGAATGGCGA ATCAGAGGAG TACAACAGAC GACAAAAAAA TTGGAAAAAG HAHAAAGA 7500
GATCTGGAGA GAATTACAAT ACACACGAAG ACAACATGGG GACTATGGGT CGTHGGTGA CTACCGCCGC CGAGAAGAAG AACGTCGGGG TGAATCCTCA 7600 CCAAGGGTCC TTMCCCTGG AGATTCAAAG CAAAGGAGGA AACATCTATG ACTGTTGCAT TAAGGCTCAA GMGGMCTC HGCTATTCC TTGCTGTGGC 7700 TTCCCACTAT GGCCGTTTTG GGGACTTATA ATCATATTAG AACGCTTGH GGGATATGGG CHCGGGAAA TTGCAAAAAT TATAATGATT CTAGGGAAAG 7800 GACTMGTAT AATAATTACA GGATTAAGAA AATTATGTGA nATAHGGG AAAATGCTAA ATCCAGCTAC ATCTCATGTA ACAATGCCTC AATATGATGT 7900 TTAGAAAAAC AAGGGGGGAA CTGTGGGATT AATATAAGAT TCTTATAAGT GAATATGAAA GTTGCTGATG CTCTCAAGTT GCTGATGCTC TCATAACCTT 8000 ATGACTAGCT CATGTTGCCA GGCAACTGAA CTGTGATAAC CTTTTGTTCC TCATTATAGT TCCGCTTTTG TATAGTTCCG CTTTTGTGAC GCGHAAGTT 8100 CCTGTTTTTA CAGTATATAA GTGCTTATAT TCTGACATTT GGTCACTCAG ATTCTGCGGT CTGAGTCCCT TCTCTGCTGG GCTAGACTAG CCTTTGTMT 8200 AAATATAATT CTCTGCTAAG TCCCTGTCTC TAGTTTGTCT TGTTTTCAAG ATCTAACA 8258
SEQ ID NO: 2的信息:
(a)序列特征：
长度 1461bp 链性： 双链
拓扑结构： 线性
(b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 gag基因
(d)序列描述： SEQ ID NO: 2
ATGGGAGACT CTTTGACATG GAGCAAAGCG CTCAAGAAGT TAGAGAAGGT GACGGTACAA GGGTCTCAAA AGCTMCTAG TGGTAACTGT AATTGGGCGC 100
TGAATTTGGT GGACTTATTC CATGACACCA ATTTTGGTM AGAAAAAGAC TGGCAATTAA GGGACGTCAT TCCATTGTTA GAGGACGTTT CCCAGACGTT 200
GTCAGGACAA GAGAGAGAGG CAmGAAAA AACTTGGTGG GCAATAGCTG OTTAAGAT GGGCTTACAA ATTAATACTG TGAATGATGC AAAAACAACA 300
TTTTCTATAT TAAAAGCCAA GTTTGAAAGA AAGACTGCAA ATAATACCAA AAAGCAGTCT GAGCCCGAGG AAGAATACCC AATAATGATT GATGGGGCTG 400
GAAACAGAAA CTTTCGGCCA TTAACACCCA GAGGATATAC TACCTGGGTA AATACTATAC AGCAAAACAA TCTCTTAAAT GAAGCTAGTG TGAATTTAn 500
TGGTATT™ TCAGTAGACT GTACTTCTGA GGAAATGAAT GCATTTTTGG ATGTAGTACC AGGACAAGCA GGACAAAAAC AAGTACTATT GGATAATCTT 600
GATAAGATTG CAGAAGAATG GGATCGTAGG CACCCGTTGC CAAATCCTCC ATTAGTGGCA CCACCACAAG GGCCTATTCC CATGACAGCA AGGTTCA™ 700
GGGGATTGGG AGTTCCTAGA GAAAGACAGA TGAAACCTGC TTTTGATCAG TTTAGACAAA CTTATAGACA ATGGATAATA GAAGCAATGA CAGAAGGGAT 800
AAAAATAATG ATTGGGAAAC CCAAAGCGCA AAATATTAGG CAAGGACCCA AAGAACCCTA TCCAGAGTTT ATAGACAGAT TGCTGTCTCA GATAAAAAGT 900
GAOGGACATC CGGCTGATAT AACTAAATTC CTGACAGACA CTTTAACTAT TCAGAATGCT AATGATGAAT GCAAAAATGC TATGAGACAT TTGAGGCCAG 1000
AAGATACTTT AGAAGAGAAA ATGTATGCAT GTAGAGATAT TGGCACTATG AGACAAAAAA TGGCATTATT AGCCAAGGCA CTTCAAGCAG GATTAGCTGG 1100
TCCTATGAAG GGAGGAATAT TTAAAGGGGG ACCCTTAGGG GCGAAGCAGA CATG ATAA TTGTGGAAAA CCAGGACATT TTTCTAGTCA ATGTAAAGCA 1200
CCTAAAATAT GTTTTAAGTG CAAACAGCCA GGACATTTCT CAAAACAATG TAGAAATGCT CCAAAAAACG GGAAACAAGG GGCTCAGGGG AGGCCCCAGA 1300
AACAAACTTT CCCTGTGCAG AAGGAGTCAA TGAACAAAAC ACAAAAAGAG GAGAAACAGC AAGGGACCH ATATCCAGAT TTAAGTCAGA TGAAACAGGA 1400
ATACAAGATC AAGGAAGAGG AAAATCAAGA GGATCTCAAT CTGAACAGTT TGTGGGAGTA A 1461
SEQ ID NO: 3的信息:
(a)序列特征：
长度： 3432bp
类型： 核酸
链性： 双链
拓扑结构： 线性 (b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 pol基因
(d)序列描述： SEQ ID NO: 3
ACAGCCAGGA CATTTCTCAA AACAATGTAT AAATGCTCCA AAAAACGGGA AACAAGGGGC TCAGGGGAGG CCCCAGAAAC AAACTTTCCC TGTGCAGAAG 100
GAGTCAATGA ACAAAACACA AAAAGAGGAG AAACAGCAAG GGACCTTATA TCCAGATTTA AGTCAGATGA AACAGGAATA CAAGATCAAG GAAGAGGAAA 200
ATCAAGAGGA TCTCAATCTG MCAGTTTGT GGGAGTAACT TATAATTTAG AAAAGAGACC AACTACAATA GTCTTGATTA ATGACACACC CTTAAATGTA 300
TTG GGACA CAGGAGCAGA CACATCAGTA CTAACTATTG CACATTGTAA TAGGTTAAAG TATGGAGGAA GAMATATCA AGGTACAGGT ATTGTTGGGG 400
TTGGAGGTAA TGTAGAAACA TTTTCCACTC CTGTTACAGT GAAAAAGAAA GGAAAACAAA AAAACTAG AATGTTAGTA GCAGATATCC CAGmCTAT 500 mGGGGCGA GATATTCTTC MGAATTAGG CGCACAATTA CTAATGGCTC AACTTTCAAA AGAAATAACC CCAAGAGAAA TTAAATTAAA AACAGGCACA 600
GTAGGGCCTA AGGTTCCCCA ATGGCCACTT ACTAAAGAGA AGnGTTAGG TGCTAAAGAA ATAGTCAAAA M GTTGGA TGAAGGTAAA ATATCAGAAG 700
CCAGTGATGA TAATCCnAT AATTCTCCTA TATTTGTAAT AAAAAAGAAA TCTGGAAAGT GGAGATTATT GCAAGATTTA AGAGAGTTAA TTAAGGGTGG 800
TACAAGTAGA ACTGAAATAT CCAGAGGATT ACCTCATCCA GGGGGAHAA TTAAATGTAA TCATATGACA GTATTAGATA TTGGAGATGC ATATTTCACT 900
ATACCAHAG ATCCAAAGTT TAGACAATAT ACAGCAHTA CTGTGCCATC CAHMTCAT CAGGAACCAG ATAAAAGATA TGTGTGGAAT TGCTTGCCAC 1000
AAGGTTTTGT GTTAAGTCCA TACATATATC AAAAAACAH ACAGGACATA TTACAAGCH TTAGAGAAAG GCATCCAGAT GTACAAHAT ATCAATATAT 1100
GGATGATTTA TTCATTGGGA GTAATGAATC TAAAAGACAA CATAAGGAAC TAGTAGAAGA ATTAAGAGCT ATTcrnTAG AAAAGGGCTT TGAGACGCCT 1200
GGGGAT AT TGCAGGAAGA AGCACCCTAT AATTGGCTGG GATATCAACT TAGTCCAGGC AATTGGAAAG TACAAAAGAT GCAATTAGAA TTGGTAAAAG 1300
AGCCAACATT AAATGATCTG CAAAAATCAA AGGGAAATAT AACATGGATG AGCTCAGGGG TTCCTGGATT AACAGTGAAG CAAATAGCTG CTACCACTAA 1400
AGGTTCCTTA GATTTAAATC ATAAAGGTAG TAGGACCAGA GAAGCCCAAA AAGACTTAGA GGAAATTATT AAAAGTTTCA GAAGCTCAGG ATTCCCATAT 500
TATAACCCAG AAGAAGAAGT AATCTGTGAG ATTGAAATTA CTAAAAATTA TGAGGCTACT TATATAATAA AACAGTCTCA AGGAATATTG TGGGCAGGAA 1600
AGAAAAmT GAGGGCTAAT AAAGGATGGT CCGCAGCAAA AAATCTAATG TTATTGTTAC AACATGTAGC CACAGAAAGT ATTGTTAGAA TTGGAACATG 1700
TCCAAAATTT AAAGTACCTT HACTAAAGA ACAAGTCAAA TGGGAAATGG AAAAGGGATG GTATTATTCA TGGCTACCAG ACATGGTATA TTCACATCAA
GTTGTTCATG ATGAHGGAG ACTGAAATTA GTAGAGCAAC CAACATCTGG TATAACAATT TATACTGATG GGGGTAAACA GAATGAAGAA GGAGTTGCAG 1900
CTTATGTGAC TAGTAATGGG AAAACTAAAC AAAAAAGGTT AGGGCCTGTT ACTCATCAAA CTGCTGAGAG GATAGCAATA CAAATGGCAT TAGAAGATAC 2000
TGAAGAGACA TTGGTAAATA TAGTAACTGA TAGTTACTAC TGTTGGAAAA ATAHACAGA AGGA AGGG HAGAAGGAC CAGACAGCCC CTGGTGGCCA 2100
ATAATTCAAA ATATTAGGGC TAAAGAAATG GTTTATTTTG CTTGGGTACC AGGTCACAAA GGAATATATG GCAATCAAH GGCAGATGAG GCTACTAAAA 2200
TAACAGAGGA AATTATGTTA GCATATCAAG GCACACAGAT TAGGGAAAAA AGAGATGAAG ATGCAGGGTA TGAmGTGT ATTCCTTATG ACATAATGAT 2300
ACCTGTCTCT GAGACAAAAG TTATACCCAC AGATGTAAAA ATACAGGTAC CTCACAAATG TTTTGGATGG GTAACTGGTA AGTCATCAAT GGCTAAGCAA 2400
GGATTATTAA TCAATGGGGG AATAATTGAT GAAGGATACA CAGGTGAAAT ACAGGTAA TGTACTAATA GGAAAGAG TAACATGAAA CTCAGGGAAG 2500
GACAAAAGTT TGCACAATTA ATCATATTAC AGCATCGATC AAATGATAAA CAAATCTGGG ATGAAAATAA AACATCTCAA AGGGGAGATA AAGGGTTTGG 2600
AAGCACAGCT ATATTTTGGG TAGAGAATAT CCAAGAGGCG CAAGATGAAC ATGAAAA G GCATACATCT CCAAAGATAT TGGCAAAAAG ATATCGGHA 2700
CCATTGACTG TAGCTAAACA GATAACTCAA GAATGCCCTC A GTACTAA ACAAGGATCT GGACCAGCAG GTTGTGTAAT GAGATCTCCT AATCATTGGC 2800
AGGCTGATTG TACACATTTA GAAAACAGGG TAATAATGAC ATTTGTAGAG TCTAATTCAG GATACATTCA TGCTACTCTA TTGTCCAAAG AAAATGCCTT 2900
GTGTCCTTCA TTGGCTATTT TGGAATGGGT GAGGTTATTT TCTCCTAAAT CTTTACATAC AGACAATGGT ACTAATTTTG TGGCAGAGTC AGTAGCAAAT 3000
CTGTTGAAAT TCCTGAAGGT GACACATACT ACAGGAATAC CmTCACCC AGAGAGCCAA GGCATTGTGG AAAGAGCAAA CAGGACA A AAAGAAAGAA 3100
TTAAAAGTCA TAGAGGAAAT ACTCAGACAC TTGAAGCAGC ATTACAACTT GCTCTCATTA CTTGTAACAA AGGGAGGGAA AGTATGGGAG GACAAACTCC 3200
ATGGGAAGTA TTTATTACTA ATCAGGCTCA AACAATACAT GAAGAACTTT TATTACAACA AGCACAATCT TCTAAAAAAT TTTGTTnTA TAAAATTCCT 3300
GGTGAGCATA ATTGGAAGGG GCCCACCAGA GTGTTGTGGA AAGGTGATGG AGCAGTAGTG GTCAATGATG AGGAAAAAGG AATAATTGCT GTGCCTTTAA 3400
CCAGGACTAA ATTATTAATA AGACCAAAH GA 3432
SEQ ID NO: 4的信息:
(a)序列特征： 长度： 2592bp
类型： 核酸
链性： 双链
拓扑结构： 线性
(b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 env基因
(d)序列描述： SEQ ID NO- 4
ATGGTCAGCA TTACATTCTA TGGGGGTATC CCAGGGGGAA TATCAACCCC TATCACCCAA CAAACAGAAT CAACAGACAC ACAGAAAGGG GATCATATGG 100
TATATCAACC CTATTGTTAT AATGATAGCC ATAAAGAAGA AATGGCAGAG ACAAGAGACA CAAGATACCA AGAAGAAATG AACCGGAAAG AAGATAAAGA 200
AGATAAAAGA AAGAATAACT GGTGGAAGAT AGGTATGTTC TTATTGTGTC TGHAGAGAT CACTGGAGGA TTCCTCTGGT GGTATGAGAG GCAACAACAT 300
TCATATTATA TAAGATTGGT TACAATAGGA GGTAGACTGA ATGGTTCAGG AATGACTAGT GCCATAAAAT G GGGGTTC ATTTCCTGGG TGTAGGCCAT 400
TTACTAACTA TTTCAGTTAT GAGACTAATA GGACTGTTAG TAGAGATAAT AATACTGCTA CTCTGTTAGA TACTTATCAA AGAGAAATAA CAAACATATA 500
CAGGACATCT TGTGTGGATA GTGATCACTG TCAAGAATAT AAATGTAAGC AAGTACAGTT GAAAAAGAAC AGCAATAACA TTATAATGAA TAAHGTAGT 600
AACAATAGGT GTGAAGAGn TTGGGGGTTT AGCTGGTTAG AATGTAATCA GACAGAAAAT GCAATAACTA TATTGGTCCC AGAAATAGAA ATACAGCAAA 700
GAAAGAACAC Γ TTCCA AAAAGGTGTG AGAAAACTTG GGCTAAGGTA AAACATTGTC CAATGGATTT ATTATATGGT ATAAATAAAA TAAGAATGTG 800
TGTCCAACCT CCATTCTTTT TGTHAAACA GAATGATACT TCTAATAATA CTAATATTCT CAGTAATTGT GGACCTTTAG TATTTCTTGG AATATTTGAG 900
GACAATAAGG CAGCAATCCA GAATGGGAGT TGCACTCTTC ACAGGACAAA TATTAACAGG CCAGAHATA GTGGATTTTA CCAAGTGCCT ΑΤΑΤΤΠΑΤΑ 1000
TATGCACCTT GACAGGATTT CAAAGHGTA ATAATGGATC AATAA AGT ATM™TGT ATGAGTCTAA TAATGTTCM TACTTGHAT GCAATACTAG 1100
TAATACTAAT AGTACCAATA ATGCTAATGT CTCTTGTGTG GTACAAAGTT πθΟΑ Τ ΑΤ AGGACAGGCA CATGTGGCAT TGCCCAGAAA AAATAAGAGG 1200
TTACAATCTC CAAAGTTTGC TCACTATAAT TGCACCATAA ATAATAAAAC AGAG MGG CGATGGCAAT TGGTAAAAAC ATCAGGCATC ACTCCTTTAC 1300
CCATTTCCTC TACAGCTAAT ACTGGATTAG TCAGACACAA GAGAGACTTT GGTATATCTG CTATAATAGC TGCCATTGTA GCTGCTAGTG CTAHGCTGC 1400
TAGTGCTACT ATGTCTTATA TCGCTTTGAC AGAAGTCAAC AAAHAGATA GTGTACAAAA TCATACTTTT GAAGTAGAGA ACAATACTAT CAATAACATA 1500
GAGTTAACAG AAGAGCAAAT TCATATATTA TATGCTATGG TTCTCCAAAC ACATGCAGAT GTTCAATTGT TAAAAGAACA ACAAAAGATT GAGGAAACAT 1600
TTAATTTAAT TGGATGTATA GAAAGATCAC ATACATTTTG TCATACTGGA CATCCCTGGA ATGAATCATG GGGTCAGTTA AATGAHCTA CACAGTGGGA 1700
TGACTGGGTA GATAAGATGG AAAATTTAAA TCATGATATA AACAACAC TTCATACTGC TAGAAATAAT CTAGAACAAT CTATGATAAC TTTCAATACA 1800
CCTGACAGTG TAGCACAATT TGGAAAAAAT ATTTGGAGTC ATATTGCAAA HGGATTCCT AGATTAGGAG CTTCCATAAT TAAATATATA GTGTTGATAT 1900
TACTTATATA TGTGTTACTA ACCTCTGCAC CTAAGATCCT CAGAGGCCTC TTGACAACGA TGAGTGGTGC AGGATCCTCC GCCAGTCGCT ACCTGAAGAA 2000
AAGATACCAT CACAAACATG CATCGCGAGG AGACATCTGG GCCCAGGTCC AATATCATGC GTACCTGGCA GACGAGACTC ATGGCTCAGG GGACAAGTCC 2100
AACATGCGGA AGCTCTCCAG GAACAACTGG AATGGCGAAT CAGAGGAGTA CAACAGACGA CAAAAAAAH GGAAAAAGH AHAAAGAGA TCTGGAGAGA 2200
ATTACAATAC ACACGAAGAC AACATGGGGA CTATGGGTCG TTTGGTGACT ACCGCCGCCG AGAAGAAGAA CGTCGGGGTG AATCCTCACC AAGGGTCCTT 2300
AACCCTGGAG AHCAAAGCA AAGGAGGAAA CATCTATGAC TGTTGCATTA AGGCTCAAGA AGGAACTCTT GCTATTCCTT GCTGTGGCTT CCCACTATGG 2400
CCGTTTTGOG GACTTATAAT CATATTAGAA CGCTTCTTGG GATATGGGCT TCGGGAAATT GCAAAAATTA TAATGAHCT AGGGAAAGGA CTAAGTATAA 2500
TAATTACAGG ATTAAGAAAA HATGTGAn ATATTGGGAA AATGCTAAAT CCAGCTACAT CTCATGTAAC AATGCCTCAA TATGATGTTT AG 2592
SEQ ID NO: 5的信息:
(a)序列特征：
长度： 495bp
类型： 核酸
链性： 双链
拓扑结构： 线性 (b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 rev基因
(d)序列描述： SEQ ID NO: 5
ATGGCAGAGA CAAGAGACAC AAGATACCAA GAAGAAATGA ACCGGAAAGA AGATAAAGAA GATAAAAGAA AGAATAACTG GTGGAAGATA GGTCCTCAGA 100 GGCCTCTTGA CAACGATGAG TGGTGCAGGA TCCTCCGCCA GTCGCTACCT GAAGAAAAGA TACCATCACA AACATGCATC GCGAGGAGAC ATCTGGGCCC 200 AGGTCCAATA TCATGCGTAC CTGGCAGACG AGACTCATGG CTCAGGGGAC AAGTCCAACA TGCGGAAGCT CTCCAGGAAC AACTGGAATG GCGAATCAGA 300 GGAGTACAAC AGACGACAM AAAAHGGAA AAAGHATTA AAGAGATCTG GAGAGAAHA CAATACACAC GAAGACAACA TGGGGACTAT GGGTCGTTTG 400 GTGACTACCG CCGCCGAGAA GAAGAACGTC GGGGTGAATC CTCACCAAGG GTCC AACC CTGGAGATTC AAAGCAAAGG AGGAAACATC TATGA 495
SEQ ID NO: 6的信息：
(a)序列特征：
长度： 237bp
类型： 核酸
链性： 双链
拓扑结构： 线性
(b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 tat基因
(d)序列描述： SEQ ID NO: 6
C'IGCTGAACC TGGCTGATCA TAGGATCCCT AGGACAGCAG AGGAGAACT'I" ACAGAAGTCT TCTGGAGGTG TTCCTGGCCA CAACACAGGA AGACAGGTAC 100 CACCAGTCAG CTATCATTGT CAACTGTGTT TCCTGAGATC ATTGGGAATT GACTACCTTG ACAGCTCGCT GAAGAAGAAG AACAAACAAA GACAGAAGGC 200 CATCAGGGAG GAAGACAACC TCAGTATCTT GTTATAA 237
SEQ ID NO: 7的信息：
(a)序列特征：
长度： 207bp
类型： 核酸
链性： 双链
拓扑结构： 线性
(b)分子类型： DNA (基因组）
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 s2基因
(d)序列描述： SEQ ID NO: 7
ATGGGATTAT '!TGGTAAAO; GGTAACATGC. TCAGCATTAC ATTCTATGGG GGTATCCCAG GGGGAATATC AACCCCTATC ACCCAACAAA CAGAATCAAC 100 AGACACACAG AAAGGGGATC ATATGGTATA TCAACCCTAT TGTTATAATG ATAGCCATAA AGAAGAAATG GCAGAGACAA GAGACACAAG ATACCAAGAA 200 GAAATGA 207 SEQ ID NO: 8的信息：
(a)序列特征：
长度： 486个氨基酸
类型： 氨基酸
链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 gag蛋白
(d)序列描述： SEQ ID NO: 8
Figure imgf000018_0001
EKQQGTLYPDLSQMKQEYKIKEEENQEDLNLNSLWE
SEQ ID NO: 9的信息：
(a)序列特征：
长度： 1143个氨基酸
类型： 氨基酸
链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 pol蛋白
(d)序列描述： SEQ ID NO: 9
KLLIRPN
SEQ ID NO: 10的信息：
序列特征：
长度： 863个氨基酸
类型： 氨基酸
链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 env蛋白
(d)序列描述： SEQIDNO: 10
Figure imgf000019_0001
TSHVTMPQYDV
SEQIDNO: 11的信息:
(a)序列特征：
长度 164个氨基酸 链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒辽系野毒株 rev蛋白
(d)序列描述： SEQIDNO: 11 RRGESSPRVLNPGDSKQR KHL
SEQIDNO: 12的信息:
(a)序列特征：
长度 78个氨基酸 链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 tat蛋白
(d)序列描述： SEQIDNO: 12
JADHF DNLSILL
SEQIDNO: 13的信息
(a)序列特征：
长度 68个氨基酸 链性：
拓扑结构： 线性
(b)分子类型： 蛋白质
(c)来源： 马传染性贫血病毒驴白细胞疫苗株 s2蛋白
(d)序列描述： SEQIDNO: 13

Claims

权利要求
1、 马传染性贫血病毒驴白细胞疫苗株的全长前病毒基因组序列， 包括 SEQ ID ΝΟ:1所示的序列。
2、 如权利要求 1所述的序列， 其中所述的全基因组包括位于全基因组 核苷酸 1-325位的 5 ' LTR, 位于全基因组核苷酸 7922-8258位的 3 ' LTR, 位于全基因组核苷酸 466-1926位的 gag基因， 位于全基因组核苷酸 1689- 5120位的 pol基因， 位于全基因组核苷酸 5313-7904位的 env基因， 位于 全基因组核苷酸 365-462 位的 tat基因第一外显子， 位于全基因组核苷酸 5138-5276位的 tat基因第二外显子， 位于全基因组核苷酸 5454-5546位的 rev基因第一外显子， 位于全基因组核苷酸 7250-7651位的 rev基因第二外 显子， 位于全基因组核苷酸 5287-5493位的 s2基因。
3、 马传染性贫血病毒驴白细胞疫苗株 gag基因编码的氨基酸序列， 其包括如 SEQ ID NO: 8所示的序列。
4、 马传染性贫血病毒驴白细胞疫苗株 pol 基因编码的氨基酸序列， 其包括如 SEQ ID NO: 9所示的序列。
5、 马传染性贫血病毒驴白细胞疫苗株 env基因编码的氨基酸序列， 其包括如 SEQ ID NO: 10所示的序列。
6、 马传染性贫血病毒驴白细胞疫苗株 rev基因编码的氨基酸序列， 其包括如 SEQ ID NO: 11所示的序列。
7、 马传染性贫血病毒驴白细胞疫苗株 tat基因编码的氨基酸序列， 其 包括如 SEQ ID NO: 12所示的序列。
8、 马传染性贫血病毒驴白细胞疫苗株 s2基因编码的氨基酸序列， 其 包括如 SEQ ID NO: 13所示的序列。
9、 权利要求 1一 8所述的序列在制备基因工程亚单位疫苗， 基因突变 和缺失疫苗， DNA疫苗和诊断试剂中的应用。
10、 权利要求 1一 8所述的序列在制备用于基因治疗的马传染性贫血病 毒基因转移系统中的应用。
11、 引物序列， 选自如下序列：
(a) GGAC ATCCGGCTGATATAAC，
(b) GACCAGCTAATCCTGCTTGA,
(c) GCTTGAAGTGCCTTGGCTAA，
(d) TCAGGAATCACCACCAGTCAG，
(e) GTTGTTGCCTCTC ATACC AC，
° 丄 工) OV TOO V工
e3I0IOIDOXIVOVDVOV(j)
.8C£9/00 OAV
PCT/CN2000/000096 1999-04-21 2000-04-21 Sequence genetique complete de la souche d'un vaccin contre le virus de l'anemie infectieuse equine et son application WO2000063387A1 (fr)

Priority Applications (4)

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DE60035680T DE60035680T2 (de) 1999-04-21 2000-04-21 Komplette gensequenz des esel-leukozyten-impfstoffstammes des equinen infektiösen anämievirus und ihre anwendung
AU39554/00A AU3955400A (en) 1999-04-21 2000-04-21 The full gene sequence of the donkey leukocyte vaccine strain of the equine infectious anemia virus and their application
EP00918668A EP1174507B1 (en) 1999-04-21 2000-04-21 The full gene sequence of the donkey leukocyte vaccine strain of the equine infectious anemia virus and their application
US09/959,120 US6987020B1 (en) 1999-04-21 2000-04-21 Full-gene sequence of the donkey leukocyte vaccine strain of the equine infectious anemia virus and their application

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CN99105852.6 1999-04-21

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WO2002020050A2 (en) * 2000-09-09 2002-03-14 Akzo Nobel N.V. Eiav p26 deletion vaccine and diagnostic
WO2002020049A3 (en) * 2000-09-09 2003-07-03 Akzo Nobel Nv Eiav chimeric vaccine and diagnostic

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* Cited by examiner, † Cited by third party
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CN101705246B (zh) * 2007-04-29 2012-06-27 中国农业科学院哈尔滨兽医研究所 慢病毒基因转移载体、其制备方法和应用

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WO1992000987A1 (en) * 1990-07-12 1992-01-23 President And Fellows Of Harvard College Primate lentivirus vaccines
CN1134458A (zh) * 1996-04-03 1996-10-30 中国农业科学院哈尔滨兽医研究所 马传染性贫血病驴白细胞弱毒株及其培育方法
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Publication number Priority date Publication date Assignee Title
WO2002020050A2 (en) * 2000-09-09 2002-03-14 Akzo Nobel N.V. Eiav p26 deletion vaccine and diagnostic
WO2002020049A3 (en) * 2000-09-09 2003-07-03 Akzo Nobel Nv Eiav chimeric vaccine and diagnostic
WO2002020050A3 (en) * 2000-09-09 2003-08-28 Akzo Nobel Nv Eiav p26 deletion vaccine and diagnostic

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DE60035680T2 (de) 2008-04-30
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EP1174507A1 (en) 2002-01-23
ES2288848T3 (es) 2008-02-01
AU3955400A (en) 2000-11-02
EP1174507A4 (en) 2004-03-24
CN1173036C (zh) 2004-10-27
EP1174507B1 (en) 2007-07-25
ATE368117T1 (de) 2007-08-15

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