WO2012071762A1 - 制备抗蓝耳病转基因猪的方法 - Google Patents

制备抗蓝耳病转基因猪的方法 Download PDF

Info

Publication number
WO2012071762A1
WO2012071762A1 PCT/CN2011/000274 CN2011000274W WO2012071762A1 WO 2012071762 A1 WO2012071762 A1 WO 2012071762A1 CN 2011000274 W CN2011000274 W CN 2011000274W WO 2012071762 A1 WO2012071762 A1 WO 2012071762A1
Authority
WO
WIPO (PCT)
Prior art keywords
transgenic
cell
shrna
cells
virus
Prior art date
Application number
PCT/CN2011/000274
Other languages
English (en)
French (fr)
Inventor
鲍永华
郭永臣
李秋燕
汤波
李宁
Original Assignee
北京济福霖生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京济福霖生物技术有限公司 filed Critical 北京济福霖生物技术有限公司
Publication of WO2012071762A1 publication Critical patent/WO2012071762A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to animal transgenic techniques, and in particular to a method of preparing transgenic pigs resistant to blue ear disease. Background technique
  • Porcine Reproductive and Respiratory Syndrome commonly known as blue ear disease
  • PRRSV Porcine Reproductive and Syndrome Virus
  • the disease was first discovered in the United States in 1987. In 2006, some of China's provincial pig herds developed highly pathogenic blue ear disease, and the virus showed a large variation compared with the classical blue ear virus. Strong pathogenicity.
  • RNA interference refers to the phenomenon that double-stranded RNA specifically induces silencing of homologous target gene expression, which is highly specific and can rapidly trigger post-transcriptional gene silencing. RNA interference is triggered by a double-stranded RNA consisting of 21-23 nucleotides.
  • Small interfering RNA siR A
  • siR A can be chemically synthesized in vitro or transcribed by an expression vector, 'it participates in the degradation of homologous target genes, Resisting virus invasion and inhibiting transposons will be powerful tools for gene function research, virus control, disease treatment and variety improvement.
  • RNAi can inhibit the replication of viral genes, prevent the assembly of virions and affect disease.
  • the interaction between toxic and host, the only prerequisite for RNAi for antiviral research is the known viral genomic sequence, and siRNA can rapidly, efficiently and specifically degrade homologous mRNA without any side effects, thus becoming an antiviral infection. Effective means. And has achieved remarkable results in anti-virus research.
  • FMDV Foot and mouth disease virus
  • Kahana et al. designed three specific siRNAs for all three conserved regions of FMDV, and the cell results showed almost 100% growth inhibition compared to the control.
  • Liu et al designed siRNA for the 5'NCR, VP4, Vpg, POL, 3, NCR in the conserved regions of the FMDV genome.
  • the titer of BMD221 cell line FMDV was reduced by 10-fold, 1000-fold 12 h after transfection, and this inhibition was Continue until the next 6d.
  • siRNA designed for the conserved region of the FMDV genome inhibits replication of the FMDV virus and is a novel strategy for the treatment of highly genetically altered FMDV viruses.
  • avian influenza virus H5N1 has caused widespread respiratory diseases in humans and poultry, but current vaccine immunization and drug treatment have limitations.
  • Zhou et al. designed specific siRNAs (pBabe-NP, pBabe-PA, pBabe-PBl) for the conserved regions of the influenza virus genome, and showed significant inhibition of influenza virus replication and proliferation in pregnant chicken eggs and BALB/c mice.
  • pBabe-NP inhibited the avian influenza virus and the specificity of colonization was the best.
  • RNAi technology provides a means of preventing and treating avian influenza viruses in humans and birds.
  • RNAi as a means of gene therapy can also be used for viral control of aquatic organisms.
  • IBD Infectious bursal disease
  • IBDV infectious bursal disease virus
  • Gao et al. designed three short interfering siRNAs (siVP1618, siVP11115, and siVP12571) for the VP1 gene in the conserved region of IBDV, and then transfected with IBDV.
  • the VERO cell line showed that siVP12571 is the most efficient site for controlling IBDV replication, and shRNA was designed at this site, and shRNA expression vector pEC2571-shRNA was constructed using murine U6 as a promoter, transfected with VERP infected with IBDV.
  • the results of the cells showed that the vector inhibited the replication of IBDV by 87.4%. This result indicates that DNA vector-mediated RNAi can effectively inhibit the replication of IBDV.
  • Swine fever is a highly contagious disease that has caused serious economic losses worldwide.
  • Xu et al. constructed siRNA targeting different regions of the swine fever virus (CSFV) Npro and NS5B genes. The results showed that cells transfected with siRNA reduced replication of the viral genome by 4-12 fold, and the three specific siRNAs designed to inhibit viral proliferation for 72 h-84 h.
  • CSFV swine fever virus
  • RNAi is effective against different gene sequences, and has strong specificity, high efficiency and convenient operation.
  • the emergence of RNA interference provides a new way of thinking about virus control. RNAi is of great significance in combating animal viruses, improving animal disease resistance and animal husbandry economics. Summary of the invention
  • the present invention first provides a transgenic cell comprising a DNA encoding shRNA which is a target gene interfering with ORFlb, ORF5, ORF6 or ORF7 of the blue ear virus.
  • the interfering target gene is the sequence shown in SEQ ID No. 1, 2, 3 and/or 4. More preferably, the DNA sequence encoding the shRNA is shown in SEQ ID No. 6 & 7, SEQ ID No. 8 & 9, SEQ ID No. 10 or 1 or SEQ ID No. 12 & 13.
  • the DNA sequence encoding the shRNA is cloned into a recombinant vector carrying the recombinase recognition sequence, and the host cell is transformed to obtain a transgenic cell.
  • the DNA encoding the shRNA described above is inserted into a ⁇ vector.
  • the above cells are mammalian somatic cells, such as fetal fibroblasts.
  • fetal fibroblasts For the preparation of anti-blue ear disease transgenic pigs, it is preferred to select porcine fetal fibroblasts.
  • the present invention provides a method for preparing a cloned embryo by transplanting a donor cell with the above-mentioned transgenic cell as a nucleus, and the isolated oocyte is a nuclear transfer recipient cell, and a cloned embryo is obtained by a nuclear transfer technique.
  • transgenic livestock were obtained by transferring the cloned embryo prepared by the above method into the uterus of the livestock by a non-surgical method for pregnancy.
  • the present invention also provides a shRNA which preferably has a significant inhibitory effect on the blue ear virus, and the DNA sequence encoding the shRNA is SEQ ID No. 6 & 7, SEQ ID No. 8 & 9, SEQ ID No. 10 & l l or SEQ ID No. 1 2 & 13.
  • the DNA sequence is cloned into an expression vector to obtain a recombinant vector encoding shRNA, thereby preparing a transgenic cell. The results indicate that these cells have significant anti-Blue Ear virus ability.
  • the present invention obtains a shRNA having significant inhibitory activity against the blue ear virus by screening small interfering RNA; and transforming the host cell by introducing the DNA encoding the shRNA into an expression vector,
  • the transgenic cells are obtained; the transgenic cells are used as nuclear donor cells, and the isolated oocytes are nuclear transfer recipient cells, and the cloned embryos are obtained by nuclear transfer technology; the cloned embryos are transferred into the livestock uterus by non-surgical methods. Pregnancy is performed to obtain transgenic livestock.
  • the transgenic pigs produced by the method of the invention have significant anti-blue ear disease ability.
  • FIG. 1 is a plasmid map of vector psiCHECK TM -2;
  • Figure 2 shows the results of PCR identification of ORF1B, 5, 6 and 7 ligated into pMD19-T Simple vector, wherein M is the DNA molecular weight standard, Lane 1 is the recombinant vector accessing ORF1B, and Lanes 2 and 3 are the recombinants accessing ORF5.
  • Vector, 4, 5 are recombinant vectors for accessing ORF6, and 6, 7 are recombinant vectors for accessing ORF7;
  • Figure 3 shows the results of restriction enzyme digestion of ORF1B, 5, 6 and 7 into pMD19-T Simple vector
  • Figure 4 shows fluorescently labeled siRNA transfected MARC-145 cells
  • Figure 5 shows the inhibition rate of siRNA interference target to target gene expression
  • Figure 6 is a plasmid map of the pGenesil-1 vector
  • Figure 7 shows the results of restriction enzyme digestion of the DNA of Table 2-1 into pGenesil-1 vector
  • M is the DNA molecular weight standard
  • 1-4b is ORF1B-135, ORF1B-372, ORF 6-135 and ORF 6-, respectively.
  • Figure 8 is a map of pGenesil-1 + 2 + 3 + 4 vector
  • Figure 9 is a result of identification of the vector double digestion (BamHl+MM) of Figure 8.
  • Figure 10 is a map of the ⁇ vector
  • Figure 11 shows the results of double enzyme digestion of ⁇ connected to hU6+lB-372/6-135, where ⁇ is the DNA molecular weight standard;
  • Figure 12 shows the effect of cytopathic effect 72 h after challenge
  • Figure 13 shows the virus dilution of each group
  • Figure 14 shows shRNA interference with virus production in MARC-145 cells
  • Figure 15 shows the copy number of the viral gene in four time periods of 24-96h
  • Figure 16 shows the copy number of the 72h viral gene
  • Figure 17 shows the results of RT-PCR of PKR and OAS-1 24 h after pPNTIII lb-372 and pPNTIII 6-135 transfected cells, with the transfection concentrations of 50 nM and 100 nM from left to right in the OAS-1 group.
  • RT-PCR results of 150 nM pPNTIII lb-372 and 50 nM, 100 nM, 150 nM pPNTIII6-135 OAS-1 gene, PKR lane group from left to right were transfection concentrations of 50 nM, 100 nM, 150 nM pPNTIII lb- RT-PCR results of the PKR gene of 372 and 50 nM, 100 nM, and 150 nM of ⁇ 6-135, (the 3-actin lane group had a transfection concentration of 50 nM from left to right, respectively.
  • Figure 18 shows the results of Western blotting of M protein
  • Figure 19 shows pPNTIII-shRNA1B-372 and pPNTIII-shRNA6-135, which were digested by Aatll; lanes from left to right are DNA Marker, pPNTIII-shRNA1B-372 after digestion, electrophoresis results before enzymatic digestion, and pPNTIII- The electrophoresis result after shRNA6-135 digestion and before digestion;
  • Figure 20 shows the results of secondary PCR identification of pPNTIII-shRNA1B-372 and pPNTIII-shRNA6-135 positive cell clones, and M is the identification of different cell clones in other lanes of DNA Marker;
  • Figure 21 shows the results of PCR identification of positive cell clones integrating pPNTIII-4 shRNA combined with plasmids, and the identification of different cell clones in each lane;
  • Figure 22 is a transgenic cloned pig
  • Fig. 23 PCR amplification results of transgenic cloned pigs, from left to right, lanes 1-7 were PCR identification of 1-7 cloned pigs;
  • Figure 24 shows the cytopathic effect of fibroblasts from transgenic pigs 72 h after challenge, among which 1 is a transgenic negative pig and 3-7 is a transgenic positive pig;
  • Figure 25 shows the expression levels of viral gene lb in nine time periods from 12 to 120 h, of which 1 is a transgenic negative pig and 3-7 is a transgenic positive pig;
  • Figure 26 shows the results of western blot of lb protein, of which number 1 is a transgenic negative pig and number 3-7 is a transgenic positive pig.
  • Dual-luciferase reporter gene expression vectors psiCHECK TM -2 purchased from Promega (Cat C8021);
  • the Dual-Luciferase Reporter Assay System was purchased from Promega (Cat. No. El 910);
  • DharmaFECT Duo transfection reagent was purchased from Thermo (Cat. No. T-2010-01);
  • pMDTM 19-T Simple Vector was purchased from Takara Corporation (D 104);
  • shRNA expression vector pGenesile-1 carrier was purchased from Wuhan Jingsai Bioengineering Technology Co., Ltd.; Primer synthesis and sequencing were completed by Shanghai Biotech;
  • T4DNA ligase is a product of Biolabs; Taq enzyme and endonuclease are products of Takara
  • the plasmid purification and recovery kit is a product of Omega
  • the present invention selects the major structural protein genes (ORF5, 6, 7) and RNA polymerase gene (ORFlb) of the virus as interfering target genes.
  • the target sequence of siRNA should be selected among different strains. Conservative area.
  • the selection criteria are optimal for sequences that are identical between different strains. If this requirement is not met, at least one mismatch of at least 1 of 21 nucleotides in different strains is met, and the designed siRNA is known. There is no homology in any host gene.
  • siRNA interference sequences were designed for each target gene using Invitrogen's online software, a total of 20 (shown in Table 1-1), and the synthesis was completed by Shanghai Jima Pharmaceutical Technology Co., Ltd.
  • siRNAs sense antisense
  • 5-342 5 : ' -GCUACUCUUGUACCAGAUAdtdt-3 '5' -UAUCUGGUACAAGAGUAGCdtdt-3 '
  • 6-196 5 : ' -CCAGAUGCCGUUUGUGCUUdtdt-3 '5' -AAGCACAAACGGCAUCUGGdtdt-3 '
  • the psiCHECKTM-2 vector provides a quantitative and fast tool for initial optimization of RNAi.
  • the vector can monitor changes in expression of a target gene fused to a reporter gene (renilla luciferase).
  • the gene under study was cloned at the multiple cloning site.
  • the RNAi process initiated by the siRNA for the synthesis of the gene under investigation will result in cleavage of the fusion mRNA and its degradation.
  • the effect of RNAi was monitored by measuring the decrease in Renilla luciferase activity, and the map of the vector is shown in Figure 1.
  • the correct positive plasmid was identified by sequencing, digested with ⁇ w?I and Notl, and the digested product was subcloned into the psiCHECK-2 vector and the No-digested psiCHECK-2 vector, and finally the four plasmids containing the target gene were obtained: psiCHECK-2- Lb, psiCHECK-2-5, psiCHECK-2-6 and psiCHECK-2-7.
  • siRNA and psiCHECK-2 plasmid were co-transfected into MARC-145 cells.
  • the siRNA was co-transfected into the MARC-145 cells with the corresponding gene plasmid, and transfected with 96-well plate.
  • the transfection reagent was DharmaFECT Duo Transfection Reagent, 0.3 ul transfection reagent/well; lOOng plasmid/well; ⁇ siRNA/
  • the welL assay was set up with a blank control (transformed only to the plasmid), a negative control (random siRNA + plasmid), and transfected cells as shown in Figure 4.
  • Plasmid +T-222 24.83 ⁇ 3.75 The above experimental results show that 10 siRNAs interfere with the target gene by more than 60%, and include all targets of gene 1B and gene 6.
  • the construction of a single shRNA vector was based on the pGenesil-1 vector (as shown in Figure 6); the vector was first subjected to Bamm and Hindltt double digestion.
  • ORF1B-135 Top: ⁇ TCiiGGACATGCTCAAGGTTCAAttcaagagaTTGAACCTTGAGCATGTCCTTTTT ⁇ Bottom: ⁇ mAAAAGGACATGCTCAAGGTTCAAtctcttgaaTTGAACCTTGAGCATGTCCi;
  • ORF1B-372 Top: ⁇ iXCACATGAAGGCAAGTAATt tcaagagaATTACTTGCCTTCATGTGGTTTTT ⁇
  • Bot tom ⁇ TAAAAACCACATGAAGGCAAGTAATtc tct tgaaATTACTTGCCTTCATGTGGi;
  • ORF 6-135 Top: tcaagagaAAAGAAGTGCAACTACTGCTTTTT/i
  • Bot tom ⁇ rAAAAACCATAGAAACCTGGAAATTtctc t tgaaAATTTCCAGGTTTCTATGGi;
  • the multiple shRNA expression vectors were mediated by 1B-135, 1B-372, 6-135 and 6-169, and hU6, mU6, h7SK and hHl were used as promoters.
  • the vector is shown in Figure 8. 9 (1, 2, 3: Loxp+ORFlB-135+ ORF 1 B-372+ORF6-135+ ORF6-169 BamHl+Mlul).
  • a vector encoding a shRNA plasmid product was constructed, sequenced, and the correct product was selected. Subcloning was performed to construct a vector encoding multiple shRNA plasmid products. The construction of one vector encoding multiple shRNA plasmids was completed by Wuhan Jingsai Bioengineering Technology Co., Ltd. .
  • the vector p ⁇ containing the ⁇ site was selected (Fig. 10), and the correctly sequenced pGenesil-1 plasmid was digested with coRl and//mffll, and the target fragment was cleaved (promoter hU6+lB-372/6-135)
  • the EcoR1 and Hindlll double-cleavage sites inserted into the ⁇ vector were identified as shown in Figure 11.
  • a similar method was used to obtain plasmids ⁇ -shRNA-1 b-135, pPNT1II-shRNA-1 b-372, pPNT III-shRNA-6-135, pPNT1II-shRNA-6-169 and pPNTffl-4 shRNA, respectively.
  • the plasmids pPNTlII-shRNA-lb-135, pPNTlII-shRNA-lb-372, pPNTlII-shRNA-6-135, pPNTlII-shRNA-6-169 and pPNTIII-4 shRNA was transfected into MARC-145 cells, transfected with 4.0 ug per well (6-well plate), three replicates per plasmid, plasmid pPNTlII-shRNA empty (no shRNA insertion) and pPNTlII-shRNA scrambled (express non-specific shRNA)
  • the transfection reagent was LipofectamineTM 2000.
  • the cells were infected with the 5th generation of JXWN strain.
  • the TCID50 of the strain was 10 4 ⁇ 5 , and each well was inoculated with PRRSV 0.35 MOI (6-well plate).
  • the morphology of the cells was observed under a microscope every day, 72 h after challenge. Virus-infected cells and transfected negative control cells began to show obvious reticular and cell-shedding lesions, while cells transfected with shRNA expression plasmid showed no obvious pathological effects. As shown in Figure 12.
  • MARC-145 cells 24h prior to infection seeded in 96-well culture plate MARC-145 cells (104 cells / well) were collected 3.1 experimental viral infection in cell culture medium for 72h, and dilution (10 -10_ 1 ()), seeded into 96 Orifice plates (100 ul/well), 8 wells per dilution. Parallel setting was performed only in the control group in which the virus was not transfected with shRNA and transfected with negative shRNA. There were five groups (1B-135+V, 1B-372+V, 6-135+V, 6-169+V, 4shRNA+V). ), the inoculation situation of each group is shown in Figure 13. The cytopathic effect was observed and monitored continuously for 7 days. The TCID50 calculation method was referred to the Reed-Munch method.
  • ⁇ 1 b- 135, ⁇ 1 b-372, ⁇ -135, ⁇ -169, ⁇ scrambled, ⁇ empty plasmids were transfected into 6-well plates, respectively.
  • the cells were subjected to PRRSV challenge test 4 h after transfection, and the non-transfected cells were subjected to the same virus infection treatment.
  • Primer and probe sequence sequences of the housekeeping gene ⁇ -actin, F, R and P represent forward primers, reverse primers and probes, respectively
  • FAM and TAMRA fluorescent labeling real-time PCR was performed in ABI Prism 7900HT detection system (Applied Biosystems, USA) using TaqMan universal PCR master mixture (Takara, Japan), reaction conditions were 50 ° C for 2 min; 95 ° C for 10 min; 95 °C 15 s, 60 °C 1 min, a total of 40 cycles.
  • the 1B-135 and 1B-372 targets also significantly reduced the expression of the 5, 6 and 7 genes while reducing the expression of the 1B gene, as shown in Figure 16.
  • Double-stranded RNA can be recognized by cells and induce the release of interferon in cells, which can be used as a signal by adjacent cells to initiate viral defenses, RNA-dependent protein kinases (PKR) and 2'-5' oligoadenosine Synthetase (OAS-1) can also be activated by binding to double strands, which in turn leads to mRNA degradation leading to translational inhibition.
  • PPKR RNA-dependent protein kinases
  • OAS-1 2'-5' oligoadenosine Synthetase
  • dsRNA (21-23nt) triggers RNAi and can prevent PKR and OAS-1 activation.
  • this experiment aims to detect the expression levels of PKR and OAS-1, and 293-FT as the host.
  • the cells and mRNA were transfected with pPNTIII lb-372 and ⁇ 6-135 (3 concentrations of 50 ⁇ , ⁇ , 150nM respectively) for 24h, and Figure 17 is the result of RT-PCR. With the increase of shRNA concentration, PKR And the transcript level of OAS-1 did not increase significantly
  • OAS-1 FAM-TTCCTGAAGCAGCGCCCCACC-TAMRA
  • Two efficient interfering target plasmids including pPNTIII-shRNA1B-372 and pPNTIII-shRNA6-135, were digested with Aatll (3°C at 37 °C), linearized, and recovered for the purpose of integrating linear target fragments into the genome. 19 is a single plasmid cut in two plasmids.
  • the linearized pPNTIII-shRNA1B-372, pPNTIII-shRNA6-135 and pPNTIII-4 shRNA were transfected into the fetal fibroblasts NDB1 and PCB1 of the Great White pig (NDB1F0, New American white ⁇ ; PCB1F0, New American Changbai?), G418 screening was performed for about 8 days, and the monoclonal spots were digested.
  • PCR analysis of the target gene integration was performed on the transgenic positive cell clones.
  • the amplification primers and PCR identification results were as follows:
  • the positive cells of pPNTIII-shRNA1B-372 and pPNTIII-shRNA6-135 were identified as Positive Sense primer: CTGTTCCACATACACTTCATT CT; Positive Antisense primer: CACAGATGCGTAAGGAGAAA, and the amplified length was 739 bp.
  • the amplification results showed that a total of 9 transgenic positive clones were obtained in the first batch of transfection (6-135: 1, 2, 3, 6, 20, 23, 46; 1B-372: 7, 35), which will be the first time.
  • the cell clone point positive for the PCR result was subjected to secondary PCR identification as shown in FIG.
  • the positive cell identification primer that integrates the pPNTIII-4shR A combined plasmid is MS2-sense: 5-CAGTTAGGGTGGGTTTCC-3; MS2-antisense: 5-GAAGATGGCTGTGAGG GA -3 , the amplification length was 784 bp, and all the cell clones screened were positive, as shown in FIG. 4. 3.
  • the preparation method of the transgenic pig can be carried out by a method such as microinjection, somatic cell cloning, or sperm carrier method.
  • the transgenic pig is preferably prepared by the somatic cell nuclear transfer method of the present invention, but is not limited to the somatic cell nuclear transfer method.
  • the transgenic positive cells are used as nuclear transfer donor cells, and the in vitro mature primordial sow oocytes are used as nuclear transfer recipient cells, and the nuclear transfer donor cells are transferred into the enucleated oocytes, and electrofused and electrofused.
  • Activated constructed into cloned embryos, selected clones with good morphology, and transferred to the uterus of natural estrus by non-surgical methods for pregnancy.
  • the non-surgical embryo transfer procedure is anesthesia with barbiturate, from the recipient
  • the sow's vagina is inserted into the cervical canal into a thick catheter with an outer diameter of 10 mm, and then a 5 cm diameter thin catheter is inserted into the inside of the thick catheter and into the uterus or one side of the uterine horn.
  • the cloned embryos were transplanted together with 2 liters of preservation solution through a thin tube, specifically carbon dioxide produced by dry ice, and the embryos were blown into the uterus through the catheter for 1-3 minutes. 30 days after embryo transfer B-mode ultrasound detects pregnancy or not. The date of transplantation, the number of transplanted embryos, etc. are shown in Table 4-1.
  • the cloned pig umbilical cord tissue was preserved, and then the genome was extracted.
  • the transfected RNAi vector was 1B-372, and PCR was performed on the cloned pig by PCR primer (1B-372) which identified the positive cell clone.
  • the target fragment size is 739 bp, and the result is shown in FIG. This result confirmed that the gene of interest was integrated into the genome of the cloned pig. A total of 5 transgenic positive pigs have been obtained.
  • transgenic piglets No. 1, No. 3-7)
  • 3-7 transgenic positive pigs (experimental pigs)
  • No. 1 transgenic negative pigs (control pigs)
  • Its ear tissue establishes a fibroblast cell line.
  • the challenge test was carried out on the fetal fibroblast cell line of pigs No. 1 and No. 3-7, respectively.
  • the infection strain generation, TCID50, and the inoculation amount per well (6-well plate, 0.35 MOI/well) were all in 3.1.
  • the cells were collected for 12-120 hours after virus infection, and the total RNA was extracted from each time period.
  • the primer probe and the operation procedure are described in 3.3.
  • the virus was infected for 72 hours, and the total protein of the cells was extracted, and then the western blot analysis of the lb protein was performed.
  • the No. 1 pig (transgenic negative) showed a distinct lb protein band, in contrast, 3 Cells of -7 pigs did not have a target band, as shown in Figure 26.
  • Industrial applicability The invention provides a preparation method for transgenic pigs against blue ear disease, and the transgenic pig obtained by the method has the remarkable anti-blue ear disease ability and can be widely used in animal husbandry.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Description

制备抗蓝耳病转基因猪的方法
技术领域
本发明涉及动物转基因技术， 具体涉及一种制备抗蓝耳病的转基因猪的方 法。 背景技术
猪繁殖与呼吸综合症 ( Porcine Reproductive and Respiratory Syndrome , PRRS ), 俗称蓝耳病， 是由猪繁殖与综合症病毒 （PRRSV )所引起的一种传染 性疾病。该病于 1987年在美国首次发现， 2006年我国部分省巿猪群暴发高致病 性蓝耳病， 且该病毒与经典的蓝耳病病毒相比， 基因发生了较大的变异， 具有 很强的致病性。
在 PRRSV的大流行中 PRRS造成的经济损失早已经为人们所熟知。据估计 PRRS给美国养猪生产者造成的损失每年达 5.6亿美元 (Cho and Dee，2006)。 我国 是养猪大国， 几乎占世界养猪总量的一半。 随着猪集约化和规模化养殖的发展, 猪的传染性疾病发病率增高， 特别是近几年爆发的 "无名高热病" 等带来的损 失都是每年以亿元为计算单位， 给养猪业带来巨大损失， 极大地挫伤了养猪者 的积极性。 同时， 2007-2008年猪肉价格一涨再涨， 以至使猪肉价格创历史最高 记录， 极大地影响了人们生活。
目前在病毒防治中主要釆用疫苗， 许多学者对 PRRS 病毒进行了较深入的 研究， 并在此基础上研制了弱毒苗、 灭活苗和基因工程疫苗， 试图通过免疫途径 控制该病， 但是由于蓝耳病病毒能导致猪持续感染、.病毒血症及免疫性能低下， 至今还没有能有效地控制 PRRS的技术和措施。此外， PRRSV感染可引起机体的 细胞免疫功能低下， 而体内高水平的病毒特异性抗体又不足以清除病毒， 造成 病毒在猪体内的持续性感染 (Pirzadeh and Dea, 1998)，这给 PRRSV的防治带来很 大的困难， 也对现行的免疫策略和疫苗研制手段提出了严峻的挑战。 许多国家 包括美国已将蓝耳病列为危害最大和控制最难的猪传染病。
RNA干涉（ RNAi )是指双链 RNA特异性诱导同源靶基因表达沉默的现象， 它具有高度特异性， 能快速引发转录后基因沉默。 RNA干涉是由 21 ~ 23个核苷 酸组成的双链 RNA引发的， 小干涉 RNA(siR A)可以在体外化学合成， 也可通 过表达载体转录而成， '它参与降解同源靶基因、 抵御病毒入侵、 抑制转座子等， 将是基因功能研究、 病毒防治、 疾病治疗和品种改良等的有力工具。
研究证实， RNAi能够抑制病毒基因的复制、 阻止病毒粒子的装配及影响病 毒与宿主之间的相互作用， RNAi用于抗病毒研究的唯一前提是已知病毒基因组 序列， 且 siRNA能快速、 高效、 特异地降解同源 mRNA而不产生任何副作用'， 从而成为抗病毒感染的有效手段。 并在抗病毒研究方面已经取得了显著成效。
口蹄疫病毒 （FMDV )是感染偶蹄动物的危险病毒。 Kahana 等针对所有 FMDV的 3个保守区设计了 3个特殊的 siRNA, 细胞结果显示与对照相比， 几 乎显示了 100%的生长抑制作用。 Liu等针对 FMDV基因组的保守区域 5'NCR， VP4, Vpg, POL, 3，NCR设计了 siRNA, 转染后 12h使 BHK221细胞系 FMDV 的效价下降 10倍， 1000倍，且这种抑制作用可持续至转然后第 6d。针对 FMDV 基因组的保守区设计的 siRNA能抑制 FMDV病毒的复制，是治疗高基因变化的 FMDV病毒的新策略。
禽流感病毒 H5N1 已经在人类和禽类中引起了广泛的呼吸系统疾病， 但目 前的疫苗免疫和药物治疗都存在局限。 Zhou等针对流感病毒基因组的保守区域 设计了特异的 siRNA ( pBabe-NP、 pBabe-PA, pBabe-PBl ), 受孕鸡卵和 BALB/c 小鼠中显示了明显抑制流感病毒的复制增殖。其中 pBabe-NP抑制禽流感病毒增: 殖的特异性最好。 RNAi技术提供了预防和治疗禽流感病毒在人类和禽类中传染 的方法。
近年来， 由虹彩病毒引起的爬行动物、 两栖动物和鱼类疾病已在美洲、 欧 洲、 亚洲和澳洲等地普遍流行， 真鲷虹彩病毒 （RSIV ) 能引起海洋鱼类产生疾 病。 Dang等构建了靶向 RSIV中编码病毒衣壳蛋白基因 （MCP ) 的 siRNA， 在 转染感染了病毒的细胞后的 84h和 94h后， 对病毒 MCP基因表达的抑制达到 55.2%和..97.1%。 同时， 病毒的增殖也被抑制了， 表明针对 MCP基因的 siRNA 干扰作用能特异和有效阻止 RSIV的复制，是一种潜在的控制—病毒感染引起的疾 病的方法。 显示了 RNAi 作为一种基因治疗的手段也可用于水生生物的病毒防 治。
传染性法氏囊病（IBD )在鸡群中能引起严重的免疫抑制和高致死率， 进而 给家禽业带来巨大的经济损失。为研究 RNAi是否能有效抑制传染性法氏囊病毒 ( IBDV ) 的复制， Gao等针对 IBDV保守区域的 VP1基因设计了 3个短干扰 siRNA ( siVP1618, siVP11115, 和 siVP12571 ), 然后转染感染了 IBDV的 VERO 细胞系， 结果显示， siVP12571是最有效的控制 IBDV复制的位点， 并以该位点 设计 shRNA, 并用鼠 U6作为启动子构建了 shRNA表达载体 pEC2571-shRNA, 转染感染了 IBDV的 VERP细胞， 结果显示， 该载体对 IBDV复制的抑制率达 到 87.4%, 该结果表明 DNA载体介导的 RNAi能有效抑制 IBDV的复制。 猪瘟病是一种高度传染性疾病， 已经在全世界范围引起了严重的经济损失。
Xu等构建了靶向猪瘟病毒 （CSFV ) Npro和 NS5B基因的不同区域的 siRNA 。 结果显示， 转染了 siRNA的细胞能使病毒基因组的复制减少 4-12倍， 设计的 3 个特异的 siRNA抑制病毒增殖持续 72h-84h之久。
从当前的研究结果发现， RNAi针对不同基因序列都有效， 而且特异性强、 效率高， 操作方便。 RNA 干涉的出现为病毒防治提供了一种新的思路。 RNAi 在抵抗动物病毒， 提高动物抗病能力和畜牧业经济效益方面具有重大意义。 发明内容
本发明的目的是提供一种制备抗蓝耳病转基因猪的方法。
为实现上述目的， 本发明首先提供一种转基因细胞， 其含有编码以蓝耳病 病毒 ORFlb、 ORF5、 ORF6或 ORF7为干扰靶基因的 shRNA的 DNA。 优选所 述干扰靶基因为 SEQ ID No.l、2、3和 /或 4所示的序列。更优选，所述编码 shRNA 的 DNA序列如序列表 SEQ ID No.6 & 7、 SEQ ID No.8 & 9、 SEQ ID No.lO&ll或 SEQ ID No.l2&13所示。
通过上述编码 shRNA的 DNA序列克隆到带有重组酶识别序列的重组载体 中， 转化宿主细胞， 获得转基因细胞。 在本发明实施例中， 将上述编码 shRNA 的 DNA接入到 ρΡΝΤΠΙ载体中。
上述细胞为哺乳动物体细胞， 例如胎儿成纤维细胞。 对于制备抗蓝耳病转 基因猪而言， 优选选择猪胎儿成纤维细胞。
进一步本发明提供一种制备克隆胚胎的方法， 其以上述转基因细胞为核移 植供体细胞， 离体的卵母细胞为核移植受体细胞， 通过核移植技术获得克隆胚 胎。
进而， 通过将上述方法制备的克隆胚胎通过非手术法移入家畜子宫内进行 妊娠， 获得转基因家畜。
本发明还提供一种优选地对蓝耳病病毒具有显著抑制作用的 shRNA, 编码 该 shRNA的 DNA序列如序列表 SEQ ID No.6 & 7、 SEQ ID No.8 & 9、 SEQ ID No.lO&l l或 SEQ ID No.l2&13所示。 将所述 DNA序列克隆到表达载体可以获 得编码 shRNA的重组载体， 进而制备得到转基因细胞。 结果表明这些细胞具有 显著的抗蓝耳病病毒能力。
本发明通过筛选小干扰 RNA， 获得了对蓝耳病病毒具有显著抑制活性的 shRNA; 通过将编码所述 shRNA的 DNA导入表达载体， 进而转化宿主细胞， 获得了转基因细胞； 以所述转基因细胞为核移植供体细胞， 离体的卵母细胞为 核移植受体细胞， 通过核移植技术获得克隆胚胎； 将该克隆胚胎通过非手术法 移入家畜子宫内进行妊娠， 获得转基因家畜。 本发明方法制得的转基因猪具有 显著的抗蓝耳病能力。
附图说明
图 1为 psiCHECKTM-2载体的质粒图谱；
图 2为 ORFlB、 5 , 6和 7连接入 pMD19-T Simple载体的 PCR鉴定结果， 其中， M为 DNA分子量标准， 泳道 1为接入 ORF1B的重组载体， 泳道 2、 3 为接入 ORF5的重组载体， 4、 5为接入 ORF6的重组载体， 6、 7为接入 ORF7 的重组载体；
图 3为 ORFlB、 5， 6和 7连接入 pMD19-T Simple载体的酶切鉴定结果； 图 4显示的是荧光标记的 siRNA转染 MARC-145细胞；
图 5显示的是 siRNA干扰靶点对目标基因表达的抑制率；
图 6为 pGenesil-1载体的质粒图谱；
图 7为将表 2-1 中的 DNA克隆入 pGenesil-1载体的酶切鉴定结果， M为 DNA分子量标准， 1〜4分别是 ORFlB-135、ORFlB-372、ORF 6-135和 ORF 6-169; 图 8为 pGenesil- 1+2+3+4载体图谱；
图 9为图 8载体双酶切 （ BamHl+MM )鉴定结果；
图 10为 ρΡΝΤΠΙ载体图谱；
图 11为接入 hU6+lB-372/6-135的 ρΡΝΤΠΙ的双酶切鉴定结果， 其中 Μ为 DNA分子量标准；
图 12显示的是攻毒后 72h细胞病变效应；
图 13每组接种病毒稀释液情况；
图 14显示的是 shRNA在 MARC-145细胞中干扰病毒生产情况；
图 15显示的是 24-96h四个时间段的病毒基因的拷贝数；
图 16显示的是 72h病毒基因的拷贝数；
图 17显示的是 pPNTIII lb-372和 pPNTIII 6-135转染细胞后 24h的 PKR和 OAS-1的 RT-PCR结果，其中 OAS-1泳道组从左到右分别为转染浓度为 50nM、 100nM、 150nM的 pPNTIII lb-372和 50nM、 100nM、 150nM的 pPNTIII6-135的 OAS-1基因的 RT-PCR结果， PKR泳道组从左到右分别为转染浓度为 50nM、 100nM、 150nM的 pPNTIII lb-372和 50nM、 100nM、 150nM的 ρΡΝΤΠΙ6-135的 PKR基因的 RT-PCR结果， （3 -actin泳道组从左到右分别为转染浓度为 50nM、 ΙΟΟηΜ, 150nM的 { ΡΝΤΙΠ lb-372和 50nM、 100nM、 150nM的持家基因 β -actin 的 RT-PCR结果；
图 18显示的是 M蛋白 western blot的结果；
图 19是 pPNTIII-shRNAlB-372和 pPNTIII-shRNA6-135 , 经 Aatll酶切图； 泳道从左到右分别为 DNA Marker, pPNTIII-shRNAlB-372酶切后、 酶切前的电 泳结果， 以及 pPNTIII-shRNA6-135酶切后、 酶切前的电泳结果；
图 20为整合 pPNTIII-shRNAlB-372和 pPNTIII-shRNA6-135阳性细胞克隆 的二次 PCR鉴定结果， M为 DNA Marker其它各泳道为不同细胞克隆点的鉴定 情况；
图 21为整合 pPNTIII-4shRNA联合质粒的阳性细胞克隆的 PCR鉴定结果， 各泳道为不同细胞克隆点的鉴定情况；
图 22为转基因克隆猪；
图 23转基因克隆猪 PCR扩增结果， 从左到右 1-7泳道分别是 1-7头克隆猪 个体的 PCR鉴定情况；
图 24为来自转基因猪个体的成纤维细胞攻毒后 72h细胞病变效应， 其中 1 号为转基因阴性猪， 3-7号为转基因阳性猪；
图 25为 12-120h九个时间段的病毒基因 lb的表达水平，其中 1号为转基因 阴性猪， 3-7号为转基因阳性猪；
图 26为 lb蛋白 western blot的结果， 其中 1号为转基因阴性猪， 3-7号为 转基因阳性猪。
具体实施方式
以下实施例进一步说明本发明的内容， 但不应理解为对本发明的限制。 在 不背离本发明精神和实质的情况下， 对本发明方法、 步骤或条件所作的修改或 替换， 均属于本发明的范围。
若未特别指明， 实施例中所用的技术手段为本领域技术人员所熟知的常规 手段， 所用试剂均可从商业途径获得。
材料: _ — MARC-145细胞购于北京信得威特科技有限公司技术中心；
双荧光素酶报告基因表达载体 psiCHECKTM-2购于 Promega公司 （货号 C8021 );
Dual-Luciferase Reporter Assay System购于 Promega公司 （货号 El 910)；
DharmaFECT Duo 转染试剂购于 Thermo公司 （货号 T-2010-01);
pMD™ 19-T Simple Vector购自 Takara公司 （ D 104 );
shRNA表达载体 pGenesile- 1载体购于武汉晶赛生物工程技术有限公司； 引物合成和序列测定由上海生工完成；
T4DNA连接酶为 Biolabs公司产品； Taq酶和核酸内切酶为 Takara公司产品
质粒纯化及回收试剂盒为 Omega公司产品；
酶切、 连接、 回收、 转化、 PCR扩增等常规分子生物学实验操作步骤详见《分子克隆 (第 三版)》。 实施例 1
1. siRNA干扰靶点的筛选
1. 1 RNA干扰靶基因的选择
近五年来，在我国流行的蓝耳病病毒 85%以上均属于 JXwn毒株型，该毒株 属于高致病性毒株。 本发明选择该病毒的主要结构蛋白基因 （ORF5 , 6, 7 )及 RNA聚合酶基因 （ORFlb )为干扰靶基因。
1. 2 siRNA序列的设计与合成
虽然以 JXwn毒株型为目标，但为实现所选干扰靶点利用的最大化，避免因 不同毒株间的序列差异而限制 RNAi效应， 因此， siRNA的靶序列尽量选择在不 同毒株间较为保守的区域。 选择标准以不同毒株间完全一致的序列为最佳， 如 果达不到该要求， 至少满足在不同毒株中 21个核苷酸只有至多 1个的错配， 所 设计的 siRNA与已知的任何宿主基因都没有同源性。
利用 Invitrogen公司在线软件针对每个目标基因设计 5条 siRNA干扰序列， 共 20条（如表 1-1所示）， 合成由上海吉玛制药技术有限公司完成。
表 1-1 siRNA序列
siRNAs sense antisense
lb-135 5' -GGACAUGCUCAAGGUUCAAdtdt-3 ' 5， -UUGAACCUUG AGCAUGUCCdtdt-3 ' lb-372 ' 5， -CCAC AUG A AGGC A AGU A AUdtdt-3 ' 5， -AUUACUUGCCUUCAUGUGGdtdt-3 ' lb-761 5， -GCCCUCUAGAUGAGGUGUUdtdt-3 ' 5' -AACACCUCAUCUAGAGGGCdtdt-3 ' lb-947 5' '-CCUUGCUCCCUACUUGUAAdtdt-3 ' 5， -UUACAAGUAGGGAGCAAGGdtdt-3 ' lb-1153 5' ' -GCAGGUACAACGCUGCAAUdtdt-3 ' 5' -AUUGCAGCGUUGUACCUGCdtdt-3 '
5-252 5' -GGUAUGUCUUGAGUAGCAUdtdt-3 ' 5' -AUGCUACUCAAGACAUACCdtdt-3 '
5-289 5' -GGCUGCGCUGAUUUGCUUUdtdt-3 ' 5' -A A AGCA A AUCAGCGCAGCCdtdt-3 '
5-295 5， -GCUGAUUUGCUUUGUCAUUdtdt-3 ' 5 '-AAUGACAAAGCAAAUCAGCdtdt-3
5-342 5: ' -GCUACUCUUGUACCAGAUAdtdt-3 ' 5' -UAUCUGGUACAAGAGUAGCdtdt-3 '
5-463 5' -CCUCAAGAGAGUUGUGCUUdtdt-3 ' 5' -AAGCACAACUCUCUUGAGGdtdt-3，
6-95 5， -GCACUUUGAGAGCACAAAUdtdt-3 ' 5' -AUUUGUGCUCUCAAAGUGCdtdt-3 '
6-135 5' -GCAGUAGUUGCACUUCUUUdtdt-3 ' 5， -AAAGAAGUGCAACUACUGCdtdt-3 '
6-169 5' -CC AUAG A A ACCUGG A A AUUdtdt-3 ' 5， -AAUUUCCAGGUUUCUAUGGdtdt-3，
6-196 5: ' -CCAGAUGCCGUUUGUGCUUdtdt-3 ' 5' -AAGCACAAACGGCAUCUGGdtdt-3 '
6-285 5， ' -GCAAAUGAUAACCACGCAUdtdt-3 ' 5， -AUGCGUGGUUAUCAUUUGCdtdt-3 '
7-56 5， -GCCAAAUGCUGGGUAAGAUdtdt-3 ' 5， -AUCUUACCCAGCAUUUGGCdtdt-3 '
7-66 5: ' -GGGUAAGAUCAUCGCCCAAdtdt-3 ' 5， -UUGGGCGAUGAUCUUACCCdtdt-3 ' 5 ' -CCCUCUAGCGACUGAAGAUdtdt-3 ' 5 '-AUCUUCAGUCGCUAGAGGGdtdt-3， 5， -GCAAUUGUGUCUGUCGUCGdtdt-3 ' 5， -CGACGACAG ACACAAUUGCdtdt-3， 5， -GAUCCAGACUGCCUUCAAUdtdt-3 ' 5 '-AUUGAAGGCAGUCUGGAUCdtdt-3， 5 '-UUCUCCGAACGUGUCACGUdtdt-3 ' 5 '-ACGUGACACGUUCGGAGAAdtdt-3 '
1. 3 psiCHECK- 2-1B, -5, -6, -7载体的构建
psiCHECK™-2载体为起始优化 RNAi提供定量而快速的工具。 该载体可以 监控与报告基因 （海肾萤光素酶） 融合的靶基因的表达变化。 所研究的基因要 克隆在多克隆位点上。 针对所研究基因合成的 siRNA起始的 RNAi过程将导致 融合 mRNA的切断及其降解。 通过测量海肾萤光素酶活性的降低来监控 RNAi 的效果， 该载体的图谱如图 1所示。
选择 psiCHECKTM-2载体上的;^ ol和 Notl酶切位点，完成载体双酶切备用。 克隆目标基因 1B， 5 , 6和 7的引物设计如表 1-2所示（这四个基因的 PCR 引物均设计有 οΙ和 Notl酶切位点， 表中斜体字母）， 先将 1B， 5 , 6和 7连 接入 pMD19-T Simple载体， 经 PCR和酶切鉴定（图 2和 3所示）， 片段大小均 与目标基因符合， 测序。
测序鉴定正确的阳性质粒，经^ w?I和 Notl酶切,酶切产物亚克隆入经 和 No l -酶切的 psiCHECK-2 载体， 最后获得含有目标基因的四种质粒： psiCHECK-2-lb, psiCHECK-2-5 , psiCHECK-2-6和 psiCHECK-2-7。
表 1-2 引物序列
引物名 序列 （5'-3，）
ORFlbF CCCrCG^GTCTGGCGACCCGATTACC
ORFlbR TTGCGGCCGCAGTTCTTGCCAGGACACC
ORF5F CCCrCG^GGCCGTTCTATCTTGCTGTG
ORF5R TTGCGGCCGCTGTTCCGCTGAAACTCTG
ORF6F CCCrC04GATGCTCTAAAGGTAAGTCGC
ORF6R TTGCGGCCGCGGTTTACCACTCCCTGCTT
ORF7F CCCrCG^GAACGGCAAGCAGCAAAAG
ORF7R TTGCGGCCGCACAGGGCACAAGTTCCAG
1. 4 siRNA和 psiCHECK-2质粒共转染 MARC- 145细胞。
siRNA与对应的基因的质粒共转染 MARC-145 细胞， 釆用 96孔板进行转 染实验，转染试剂为 DharmaFECT Duo Transfection Reagent, 0.3ul转染试剂 /well; lOOng plasmid/well; ΙΟΟηΜ siRNA/welL 实验设定空白对照 （只转质粒）， 阴性 对照 （随机 siRNA+质粒）， 转染细胞如图 4所示。
1. 5各干扰靶点对载体表达基因抑制效果的检测 转染 48h后， 通过萤火虫荧光素酶和海肾荧光素酶-双荧光素酶报告系统， 检测各干扰靶点对目标基因的抑制效果，经过标准化变换之后，计算每个 siRNA 的干扰效率， 结果如图 5所示， 干扰效率在 60%以下的未显示。
本实验设计三个重复组， 每组包含三个样品孔重复， 共计 9个样品重复， 结果显示组内组间重复性非常好（放弃 1/69*3孔),实验的标准差很小，所得到的 结果真实可靠， 可以反映 siRNA对靶点序列抑制的真实情况（表 1-3所示）。
表 1-3 siRNA的干扰效率
苹已点 干扰效率
Flasmid only 0
Plasmid + scrambled siRMA 3.44士 1.12
Plasmid +1B-135 88.90 ±0.78
Plasmid -lB-372 88.90 ±1.20
Plasmid +1B-761 73.90 ±1.55
Plasmid +1B-947 64. lOzbl.09
Plasmid +1B-1153 60.50 + 1.23
Plasmid +5-252 21.01 ±0.97
Plasmid +5-289 10.90 ±2.98
Plasmid +5-295 S.39zbl.59
Plasmid +5-342 43.30 ±3.42
Plasmid +5-463 48.69 ±0.88
Plasmid 92.60 ±4.50
Plasmid +6-135 97.80士 2.34
Plasmid +6-169 95.50土 1.63
Plasmid +6-196 86.00士 3 58
Plasmid +6-285 87.50 ±0.98
Plasmid +7-56 7.90d l.08
Plasmi d +7-66 17.34土 3- 86
Plasmid +7-153 13.87土 1.68
Flasmid +7-204 9.07±1.21
Plasmid +T-222 24.83 ±3.75 上述实验结果表明， 10条 siRNA对目标基因的干扰效率在 60%以上， 并且 囊括基因 1B和基因 6的全部靶点。
最后选择抑制效率较好的四个靶点 1B-135 ( 89%), 1B-372 (89%), 6-135 (98%), 6-169 (96%)， 进行 sh NA表达载体的构建。
2. shRNA表达载体的构建
2.1 single shRNA表达载体 pGenesil- 1的构建
单个 shRNA载体的构建以 pGenesil-1载体为骨架（如图 6所示；)，首先将该 载体进行 Bamm和 Hindltt双酶切处理。
合成 4对编码短发夹 RNA ( shRNA ) 的 DNA单链序列 （如表 2-1所示）， 退火成 dsDNA, 克隆入经 Bamm和 Hindm双酶切后的 pGenesil- 1载体， 酶切 鉴定如图 7所示。
表 2-1编码 shRNA的 DNA单链序列
名称 编码 shRNA的 DNA单链序列 （5' -3')
ORF1B-135 Top: ^TCiiGGACATGCTCAAGGTTCAAttcaagagaTTGAACCTTGAGCATGTCCTTTTT^ Bottom: ^^mAAAAGGACATGCTCAAGGTTCAAtctcttgaaTTGAACCTTGAGCATGTCCi;
ORF1B-372 Top: ^^iXCACATGAAGGCAAGTAATt tcaagagaATTACTTGCCTTCATGTGGTTTTT^
Bot tom: ^^TAAAAACCACATGAAGGCAAGTAATtc tct tgaaATTACTTGCCTTCATGTGGi;
ORF 6-135 Top:
Figure imgf000011_0001
tcaagagaAAAGAAGTGCAACTACTGCTTTTT/i
Bot tom: ^^OTAAAAAGCAGTAGTTGCACTTCTTTtctc t tgaaAAAGAAGTGCAACTACTGC^
ORF 6-169 Top: ^^CiXCATAGAAACCTGGAAATTt tcaagagaAATTTCCAGGTTTCTATGGTTTTT l
Bot tom: ^^rAAAAACCATAGAAACCTGGAAATTtctc t tgaaAATTTCCAGGTTTCTATGGi;
2. 2 multiple shRNA表达载体的构建
multiple shRNA表达载体以 1B-135 , 1B-372, 6-135 , 6-169为干扰靶点， 以 hU6, mU6, h7SK, hHl为启动子， 该载体如图 8所示， 酶切鉴定如图 9所 示 （ 1 , 2 , 3 : Loxp+ORFlB-135+ ORF 1 B-372+ORF6- 135+ ORF6-169 BamHl+Mlul )。
首先分别构建一个载体编码一条 shRNA质粒产品， 测序， 挑选测序正确的 产品，亚克隆构建一个载体编码多条 shRNA质粒产品，一条载体编码多条 shRNA 质粒的构建由武汉晶赛生物工程技术有限公司完成。
2.3. pPNTIII载体的构建
选择含有 Ιοχρ位点的载体 ρΡΝΤΠΙ (图 10 ), 将测序正确的 pGenesil-1质粒 进行 coRl和// mffll双酶切， 将切下的目标片段（启动子 hU6+lB-372/6-135 ) 插入到 ρΡΝΤΠΙ载体的 EcoRl和 Hindlll双酶切位点， 酶切鉴定如图 11所示。 类似的方法分别获得质粒 ρΡΝΤΠΙ-shRNA- 1 b- 135、 pPNTlII-shRNA- 1 b-372、 pPNT III-shRNA-6-135、 pPNTlII-shRNA-6-169及 pPNTffl-4shRNA。
3 shRNA对病毒基因抑制效果研究
3. 1. 细胞病变效应 （CPE )
为了研究 shRNA对细胞病变效应 （CPE, cytopathic effect ) 的影响， 将质 粒 pPNTlII-shRNA-lb-135、 pPNTlII-shRNA-lb-372 , pPNTlII-shRNA-6-135 , pPNTlII-shRNA-6-169及 pPNTIII-4shRNA转染 MARC- 145细胞，每孔转染 4.0ug ( 6孔板），每个质粒三个重复，质粒 pPNTlII-shRNA empty (无 shRNA插入）和 pPNTlII-shRNA scrambled (表达非特异 shRNA)为阴性对照， 并做相同处理， 转 染试剂为 Lipofectamine™ 2000。 转染 5小时后 , 细胞感染 JXWN毒株第 5代， 该毒株的 TCID50为 104·5, 每孔接种 PRRSV 0.35 MOI ( 6孔板）， 每天显微镜下 观察细胞形态， 攻毒后 72h, 病毒感染细胞以及转染阴性对照细胞开始出现明显 的拉网状和细胞脱落的病变，而转染 shRNA表达质粒的细胞未见明显病变效应， 如图 12所示。
3. 2. 病毒滴度 ( Tissue culture infectious dose, TCID50 )检测
感染前 24h在 96孔培养板接种 MARC-145细胞（ 104细胞 /孔）， 收集 3.1实 验中病毒感染 72h的细胞培养液， 并进行倍比稀释（10 -10_1() )， 接种到 96孔板 (100 ul/孔)， 每个稀释度接种 8个孔。 平行设定只接种病毒未转染 shRNA以及 转染阴性 shRNA的对照组， 共五组（ 1B-135+V, 1B-372+V, 6-135+V, 6-169+V, 4shRNA+V )，每组接种情况如图 13所示。 观察细胞病变，连续监测 7天， TCID50 计算方法参考 Reed-Munch法。
结果表明： 4shRNA ( TCID50为 101 5 )、 1B-372 ( TCID50为 101 7 )、 1B-135 ( TCID50为 102·3 )、 6-135 ( TCID50为 104 14 )及 6-169 ( TCID50为 10427 ) 转 染组中病毒滴度比病毒对照组（TCID50为 104' 5 )分别降低了 1000倍、 600倍、 150倍、 2.3倍及 1.7倍。 该结果证实了的 shRNAs能有效抑制 MARC-145细胞 中病毒复制， 如图 14所示。
3. 3. 反转录及 Real-time PCR分析
为了检测 shRNAs能否在 MARC-145细胞中有效抑制病毒基因 mRNA表达， ρΡΝΤΠΙ 1 b- 135、 ρΡΝΤΠΙ 1 b-372、 ρΡΝΤΠΙό- 135、 ρΡΝΤΠΙό- 169、 ρΡΝΤΠΙ scrambled、 ρΡΝΤΠΙ empty质粒分别转染 6孔板细胞， 转染后 4h进行 PRRSV攻毒实验， 非转染细胞施行同样的病毒感染处理。
感染后 24-96h，提取各时间段细胞总 RNA，操作过程参照 Rneasy Micro Kit (Qiagen, USA) , 少量的污染的 DNA通过 RNase-Free DNase Set (Qiagen, USA)除 去， 根据 Promega反转录程序， 总体系 25ul, 42 °C 反应 1 h， 上海生工合成 Taq-Man探针和引物 （如表 3-1所示）。
表 3-1 引物和探针序列
引物或探针名 序列 （5'-3')
bRT-lbF ACCAGACCATGCTCGACATG
bRT-lb CAGGGAATTGCAGCGTTGTA
bRT-lbP FAM-ACGTGCCGGTTCAACGTTCCAGC-TAMRA bRT-5F CTCACCACCAGCCATTTCCT
bRT-5R CAAGACATACCGCCCGTGAT
bRT-5P FAM-TGGTCTGGCCACTGTGTCCA-TAMRA
bRT-6F CGGGCTTTCATCCGATTG
b T-6R ATGTGCCGTTGACCGTAGTG
bRT-6P FAM-ACCACGCATTTGTCGTCCGGC-TA RA
"RT-7F CCGGAGAAGCCCCATTTC
bRT-7R CAGACACAATTGCCGCTCACT bRT-7P FAM-TGACGTCAGGCATCACTTTACCC-TAMRA
cp-actinF CGGGCTTTCATCCGATTG
°P-actinR ATGTGCCGTTGACCGTAGTG
cP-actinP FAM-ACCACGCATTTGTCGTCCGGC-TAMRA b PRRSV lb， 5， 6, 7的引物和探针序列
。 持家基因 β -actin的引物和探针序列序列， F， R和 P 分别代表正向引物， 反向引物和探 针
GCGGCCGC, Notl 酶切位点； CTCGAG, Xhol 酶切位点； cc 和 tt， 保护性碱基
FAM和 TAMRA, 荧光标记物 荧光定量 PCR在 ABI Prism7900HT检测系统 (Applied Biosystems, USA)运 行， 使用 TaqMan universal PCR master 混合物（Takara, Japan), 反应条件为 50°C 2 min; 95 °C 10 min; 95 °C 15 s, 60 °C 1 min, 共 40个循环。
Real-time PCR结果表明： 随时间的增加， 未转染 shRNA的攻毒组的病毒 lb和 6基因的表达量增加， 而转染 shRNAlb-135和 lb-372的攻毒组在攻毒后 24h就产生了抑制效果, 在攻毒后 72h， 与对照组相比， lb-135, lb-372, 6-135, 6-169使病毒表达量分别减少了 99.4%, 99.5%, 82%, 54%。 图 15为 24, 48, 72, 96h四个时间段的病毒基因的表达情况。
另外， 1B-135和 1B-372两个靶点在减少 1B基因表达的同时， 也明显减少 了 5， 6和 7基因的表达， 如图 16所示。
3. 4. RNAi特异性检测
双链 RNA能被细胞识别并诱发细胞中干扰素的释放，后者能被临近细胞作 为一种信号， 进而启动病毒防御措施， 依赖 RNA的蛋白激酶（PKR )及 2' -5' 寡腺甘酸合成酶（OAS-1 ) 也能通过结合双链而被激活， 进而导致 mRNA降解 以致翻译抑制。 研究认为 PKR和 OAS-1的激活需要至少 30nt长的序列。
dsRNA ( 21-23nt )引发 RNAi并能避免 PKR和 OAS-1的激活， 为排除干扰 素系统的抗病毒活性，本实验以检测 PKR和 OAS-1的表达水平为目标，以 293-FT 为宿主细胞， mRNA为转染 pPNTIII lb-372和 ρΡΝΤΠΙ6-135 ( 3个浓度分别为 50ηΜ, ΙΟΟηΜ, 150nM ) 24h后的细胞裂解物， 图 17为 RT-PCR检测结果， 随 shRNA浓度浓度的增加， PKR和 OAS-1的转录本水平未明显增加
随后， 进行了 PKR和 OAS-1转录本的实时荧光定量 PCR检测， 表 3-2为 Taqman探针序列， real-time PCR结果显示最高浓度的 shRNA ( 150nM )确实增 加了这两个转录本的表达， 然而， 与对照相比， 无统计学意义。 该结果证实了 PRRSV基因表达减少是 RNAi引起的， 并非干扰素相关的防御机制的作用。
表 3-2 Taqman探针序列
名称 探针序列 5'-3'
OAS-1 FAM-TTCCTGAAGCAGCGCCCCACC-TAMRA
PKR FAM-TCAGCAGGTTTCTTCATGGAGGAA-TAMRA β-actin FAM-TCTGGCGGCACCACCATGT-TAMRA
3. 5病毒蛋白翻译的抑制
为了研究 shRNA表达载体对病毒蛋白翻译的抑制效果， 对 pPNTlII-shRNA 质粒转染后 72 h的细胞提取总蛋白， 然后执行 M蛋白的 western blot分析， 病 毒感染细胞， 阴性对照 （包括 ρΡΝΤΠΙ negative和 ρΡΝΤΠΙ empty ) 细胞组均呈 现大小为 18-19 kDa的 M蛋白带， 对比之下， 转染 shRNA表达质粒（ ρΡΝΤΠΙ lb-135, pPNTlIIlb-372, ρΡΝΤΠΙ6-135, ρΡΝΤΐΠ6-169, pPNTlH4-shRNA ) 的细胞 组没有目标带出现， 如图 18所示。
4. 体细胞核移植生产抗 PRRSV转基因猪
4. 1. RNAi载体 ρΡΝΤΠΙ的单酶切、 线性化
两 个 高 效 的 干 涉 靶 点 质 粒 ， 包 括 pPNTIII-shRNAlB-372 和 pPNTIII-shRNA6-135, 经 Aatll酶切 （37°C酶切 3h )， 线性化， 回收， 目的是线 性目标片段整合到基因组， 图 19为两质粒单酶切图。
4. 2. 转 RNAi载体阳性细胞的筛选与鉴定
将 线 性 化 的 pPNTIII-shRNAlB-372 、 pPNTIII-shRNA6-135 及 pPNTIII-4shRNA转染大白猪的胎儿成纤维细胞系 NDB1和 PCB1 ( NDB1F0,新 美系大白 δ ; PCB1F0, 新美系长白？）， 进行大约 8d左右的 G418筛选， 消化 单克隆点， 对转基因阳性的细胞克隆点进行目的基因整合的 PCR检测， 扩增引 物及 PCR鉴定结果如下：
整合 pPNTIII-shRNAlB-372和 pPNTIII-shRNA6-135的阳性细胞鉴定引物为 Positive Sense primer: CTGTTCCACATACACTTCATT CT; Positive Antisense primer: CACAGATGCGTAAGGAGAAA , 扩增长度为 739bp。 扩增结果显示第 一批转染共获得了 9个转基因阳性克隆点 ( 6-135: 1， 2, 3 , 6, 20, 23 , 46; 1B-372: 7， 35 ), 将第一次 PCR结果阳性的细胞克隆点进行二次 PCR鉴定， 如 图 20所示。
整合 pPNTIII-4shR A 联合质粒的阳性细胞鉴定引物为 MS2-sense： 5-CAGTTAGGGTGGGTTTCC-3 ； MS2-antisense : 5-GAAGATGGCTGTGAGG GA -3 , 扩增长度为 784bp, 筛选的全部细胞克隆点均为阳性， 如图 21所示。 4. 3. 转基因猪制备
转基因猪制备方法可以釆用显微注射法、 体细胞克隆法、 精子载体法等方 法。 本发明优选体细胞核移植法制备转基因猪， 但不限于体细胞核移植法。
以上述转基因阳性细胞为核移植供体细胞， 以体外成熟的初情期前母猪卵 母细胞为核移植受体细胞， 将核移植供体细胞移入去核的卵母细胞， 经电融合 与激活， 构建成克隆胚胎， 挑选形态优良的克隆胚胎用非手术法移入自然发情 的经产母猪子宫内进行妊娠,非手术法胚胎移植步骤为用无巴比妥略作麻醉后， 从受体母猪阴道向子宫颈管道插入外径 10毫米的粗导管， 然后将直径 5亳米的 细导管插入粗导管内侧， 伸入到子宫体或一侧子宫角。 将克隆胚胎连同 2 亳升 保存液一起通过细导管移植进去， 具体是用干冰产生出来的二氧化碳经过导管 将胚胎吹入到子宫内 1-3分钟。 胚胎移植后 30天 B型超声波检测妊娠与否。 移植日期， 移植胚胎数等见表 4-1所示。
表 4-1核移植情况
耳号 胚胎移植曰期 发情曰期 核移植曰期 体细胞系 移植胚胎数
2064 09.10.21 09.10.21早 09.10.20 NDB 1-6- 135-46 391
2040 09.10.22 09.10.22早 09.10.21 PCB1-1B-372-35 416
2033 09.10.27 09.10.27 09.10.26 PCB 1-1B-372-35 259
2445 09.10.29 09.10.29 09.10.28 PCB1-1B-372-7/35 290
2100 09.10.30 09.10.30 09.10.29 NDB 1-6- 135-20/23 396
PCB 1-1B-372-7
2098 09.10.31 09.10.31 09.10.30 NDB1-6-135-20 418
PCB1-1B-372-35
2116 09.11.2 09.11.1下 09.10.31 PCB1-1B-372-35/7 368
2054 09.11.2 09.11.2 09.11.1 PCB1-1B-372-7 355
2460 09.11.3 09.11.3早 09.11.2 PCB1-1B-372-7 372
2163 09.11.5 09.11.4 09.11.3 NDB 1-6-135-3 289
2159 09.11.5 09.11.5 09.11.4 NDB 1-6- 135-1 358
2015 09.11.7 09.11.7 09.11.5/6 NDB1-6-135-1 500
2444 09.11.12 09.11.12 - 09.11.11 NDB 1-6- 135-2/5 343
2174 09.11.13 09.11.13 09.11.12 NDB1-6-135-8/15 361
2032 09.11.16 09.11.16 09.11.15 NDB1-6-135-3/5/8/46 325
2086 09.11.18 09.11.18 09.11.17 NDB1-6-135-38 255
2407 09.11.20 09.11.20 09.11.18/19 NDB 1-6- 135-41 381 . 4.克隆猪出生状况
2010年 4月 24、 25日， 共出生 7头克隆猪， 健康状况良好， 克隆猪图片见 —―. —- , 丄 Λ / υ υ L/ /
22所示。
4. 5 转基因克隆猪阳性检测
保留克隆猪脐带组织， 然后提取其基因组， 根据表 4-1的记录可知， 转染的 RNAi载体是 1B-372 , 利用鉴定阳性细胞克隆的 PCR引物 (1B-372)对克隆猪进 行 PCR检测， 目标片段大小为 739bp, 结果如图 23所示。 该结果证实目的基因 整合到克隆猪的基因组中。 目前共获得转基因阳性猪 5头。
5. 阳性转基因克隆猪的抗病能力检测
5. 1. 构建稳定表达猪繁殖与呼吸综合症特异性 shRNA转基因猪胎儿成纤维细胞 系
对生产的转基因猪仔（ 1号， 3-7号），其中 3-7号为转基因阳性猪（实验猪）， 1号为转基因阴性猪 （对照猪）， 在不超过 1 日龄时分别取其耳组织， 建立成纤 维细胞系。
5.2. 细胞感染实验
分别对 1号， 3-7号猪来源的胎儿成纤维细胞系， 进行攻毒实验， 感染毒株 代次、 TCID50、 以及每孔接种量（6孔板， 0.35 MOI/孔）均与 3.1中所述一致， 镜下观察细胞形态， 攻毒后 72h， 对照 1号猪来源的成纤维细胞出现明显细胞毒 症状（拉网状和细胞脱落）， 而 3-7号猪来源的胎儿成纤维细胞未见明显病变效 应， 如;图 24所示。 -
5.3. lb基因表达水平分析
对 1号、 3-7号猪来源的细胞， 分别收取病毒感染后 12-120h的细胞， 提取 各时间段细胞总 RNA, 引物探针以及操作过程见 3.3所述。
Real-time PCR结果如图 25所示： 随时间增加， 1号攻毒组细胞的病毒 lb 基因的表达量增加， 而 3-7号在攻毒后 24h就产生了抑制效果， 在攻毒后 72h， 与对照组 1号相比， 3-7号细胞的病毒 lb基因表达分别减少了 61.19%, 66.21%, 58.07%, 54.80%, 76.35%。
5.4 lb蛋白翻译水平检测
对 1号、 3-7号猪来源的细胞， 病毒感染 72h， 提取细胞总蛋白， 然后执行 lb蛋白的 western blot分析， 1号猪（转基因阴性）呈现明显的 lb蛋白带， 对比 之下， 3-7号猪的细胞没有目标带出现， 如图 26所示。 工业实用性 本发明提供了抗蓝耳病转基因猪的制备方法， 通过该方法获得的转基因猪 具有显著的抗蓝耳病的能力， 可以在畜牧业上广泛使用。

Claims

权 利 要 求 书 KHP 10314625.2
1、 一种转基因细胞， 其含有编码以蓝耳病病毒 ORFlb、 ORF5、 ORF6 或 ORF7为干扰靶基因的 shRNA的 DNA。
2、 根据权利要求 1所述的转基因细胞， 其特征在于所述干扰靶基因为 SEQ ID No.l、 2、 3和 /或 4所示的序列。
3、根据权利要求 2所述的转基因细胞，其特征在于所述编码 shRNA的 DNA 序列为：
ORF1B-135 Top:
Figure imgf000018_0001
tcaagagaTTGAACCTTGAGCATGTCCTTTTT/f
Bot tom: /iCCTTAAAAAGGACATGCTCAAGGTTCAAtctct tgaaTTGAACCTTGAGCATGTCCi?
ORF1B-372 Top: ^fCtCCACATGAAGGCAAGTAATt tcaagagaATTACTTGCCTTCATGTGGTTTTT^
Bot tom: ^CiTTAAAAACCACATGAAGGCAAGTAATtc tct tgaaATTACTTGCCTTCATGTGGi;
Figure imgf000018_0002
Bo 11 om: ^CCrmAAAAGCAGTAGTTGCACTTCTTT tctc t t gaaAAAGAAGTGCAACTACTGC G
或
Figure imgf000018_0003
Bot tom: ^CCJTAAAAACCATAGAAACCTGGAAATTtc tct tgaaAATTTCCAGGTTTCTATGGC
4、 根据权利要求 1〜3任一项所述的转基因细胞， 其特征在于所述细胞为哺 乳动物体细胞。
： 5、 根据权利要求 4所述的转基因细胞， 其特征在于所述细胞为胎儿成纤维 细胞。
6、 一种制备克隆胚胎的方法， 其以权利要求 1~5任一项所述的转基因细胞 为核移植供体细胞， 离体的卵母细胞为核移植受体细胞， 通过核移植技术获得 克隆胚胎。
7、 一种制备抗蓝耳病转基因家畜的方法， 其是将权利要求 6所述方法制备 的克隆胚胎通过非手术法移入家畜子宫内进行妊娠， 获得抗蓝耳病转基因家畜。
8、 根据权利要求 7所述的方法， 其特征在于所述家畜为猪。
9、 一种编码 shRNA的 DNA， 其核苷酸序列为：
ORF1B-135 Top: ^fCCGGACATGCTCAAGGTTCAAt tcaagagaTTGAACCTTGAGCATGTCCTTTTT i
Bot tom: /fCCfrAAAAAGGACATGCTCAAGGTTCAA tctct t gaaTTG AACCTTG AGCATGTCC C
0RF1B-372 Top: i¾7-(7 :CACATGAAGGCAACTAATt tcaagagaATTACTTGCCTTCATGTGGTTTTT/i
Bot tom: /iCC7TAAAAACCACATGAAGGCAAGTAATtctct tgaaATTACTTGCCTTCATGTGGC ORF 6-135 Top: 6¾7¾ GCAGTAGTTGCACTTCTTTUcaagagaAAAGAAGTGCMCTACTGCTTTTT^
Bottom: ^CCr7AAAAAGCAGTAGTTGCACTTCTTTtctcttgaaAAAGAAGTGCAACTACTGCi7 或
Figure imgf000019_0001
Bottom: (CCrrAAAAACCATAGAAACCTGGAAATT tctctt gaaAATTTCCAGGTTTCTATGG G
PCT/CN2011/000274 2010-12-01 2011-02-22 制备抗蓝耳病转基因猪的方法 WO2012071762A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201010576467A CN102115731B (zh) 2010-12-01 2010-12-01 制备抗蓝耳病转基因猪的方法
CN201010576467.2 2010-12-01

Publications (1)

Publication Number Publication Date
WO2012071762A1 true WO2012071762A1 (zh) 2012-06-07

Family

ID=44214657

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/000274 WO2012071762A1 (zh) 2010-12-01 2011-02-22 制备抗蓝耳病转基因猪的方法

Country Status (2)

Country Link
CN (1) CN102115731B (zh)
WO (1) WO2012071762A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102876699B (zh) * 2012-09-27 2013-10-23 浙江大学 靶向基因shRNA干扰猪蓝耳病病毒增殖的转基因细胞系的建立方法
CN114774468B (zh) * 2022-04-20 2022-12-20 温氏食品集团股份有限公司 一种等位基因分子标记及抗蓝耳病猪群体组建方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101603035A (zh) * 2009-05-18 2009-12-16 中国兽医药品监察所 猪繁殖与呼吸综合征病毒嵌合重组疫苗株及其生产活疫苗的方法
CN101748125A (zh) * 2008-12-11 2010-06-23 中国科学院动物研究所 用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用
EP2230246A1 (en) * 2009-03-19 2010-09-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Arterivirus glycoprotein 5 virus-like particles

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144108B (zh) * 2007-08-24 2010-05-26 深圳太太基因工程有限公司 一种用于检测高致病性猪蓝耳病病毒核苷酸片段的引物和探针序列
CN101205539B (zh) * 2007-12-14 2010-11-24 中国农业科学院上海兽医研究所 高致病性猪蓝耳病病毒重组质粒和遗传工程疫苗
CN101307305B (zh) * 2008-04-30 2010-06-16 中国动物疫病预防控制中心 一种猪繁殖与呼吸综合征病毒弱毒株及免疫原性物质和疫苗
CN101831412A (zh) * 2010-03-30 2010-09-15 洛阳普莱柯生物工程有限公司 一种大规模生产猪蓝耳病病毒的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101748125A (zh) * 2008-12-11 2010-06-23 中国科学院动物研究所 用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用
EP2230246A1 (en) * 2009-03-19 2010-09-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Arterivirus glycoprotein 5 virus-like particles
CN101603035A (zh) * 2009-05-18 2009-12-16 中国兽医药品监察所 猪繁殖与呼吸综合征病毒嵌合重组疫苗株及其生产活疫苗的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI, GUANGMING ET AL.: "Effective suppression of replication of porcine reproductive and respiratory syndrome virus by adenovirus-mediated small interfering RNAs targeting ORFl b, 5 and 7 genes", JOURNAL OF VIROLOGICAL METHODS, vol. 157, no. 1, 20 January 2009 (2009-01-20), pages 40 - 46, XP025994043, DOI: doi:10.1016/j.jviromet.2008.12.001 *
QIU, HUA-JI ET AL.: "Structural and functional aspects of viral proteins of porcine reproductive and respiratory syndrome virus", PROGRESS IN VETERINARY MEDICINE, vol. 21, no. 2, 30 June 2000 (2000-06-30), pages 23 - 26 *
YANG, DONG-SHAN ET AL.: "Human pro-insulin transgenic calf derived from somatic cell nuclear transfer", ZOOLOGICAL RESEARCH, vol. 28, no. 4, 15 August 2007 (2007-08-15), pages 409 - 416 *

Also Published As

Publication number Publication date
CN102115731B (zh) 2012-10-17
CN102115731A (zh) 2011-07-06

Similar Documents

Publication Publication Date Title
WO2016110214A1 (zh) 一种抗蓝耳病克隆猪的制备方法
JP2019193660A (ja) ブタ生殖器呼吸器症候群ウイルス耐性動物
Li et al. RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs
AU2008250973B2 (en) Treatment and prevention of influenza
AU2017344936A1 (en) Swine comprising modified CD163 and associated methods
WO2009150431A1 (en) Vector
CN110951745A (zh) Cd163突变基因及其在抑制或阻断猪产生抗体的方法和应用
CN118773199A (zh) 通过对cd163靶向灭活来改善猪类健康的方法
CN113416768B (zh) Prkra基因作为靶点在抑制小反刍兽疫病毒复制中的应用
CN116445454B (zh) 一种用于培育抗tgev感染的猪品种的成套系统及其应用
WO2012071762A1 (zh) 制备抗蓝耳病转基因猪的方法
CN113151195A (zh) 一种猪繁殖与呼吸综合征嵌合重组疫苗株及其应用
CN108753739B (zh) 表达猪瘟病毒e2蛋白的重组伪狂犬病毒毒株及其制备方法和应用
CN109679952B (zh) 一种抗PRRSV感染的猪源miR-c89及其应用
CN102559762A (zh) 抗口蹄疫病毒RNAi转基因家畜的制备方法
CN109694867B (zh) 一种DICER1基因及其siRNA的应用
CN116769016B (zh) pAPN突变体和用于pAPN基因定点修饰的组合物及应用
CN111304199B (zh) 抗猪繁殖与呼吸综合征病毒介导肺损伤的miRNA和应用
CHEN et al. Construction of multiple shRNA vectors targeting PEDV and TGEV and production of transgenic SCNT porcine embryos in vitro
AU2021105546A4 (en) MiRNA against lung injury caused by porcine reproductive and respiratory syndrome virus and its application
CN102174572A (zh) 猪体细胞转基因克隆质粒pEGFP-N1-shRNA及其构建方法
Söllner An experimental approach of an in vivo pathogen genome targeting strategy to generate African swine fever resistant pigs
Yu et al. Zona pellucida blocks BAV304a adenoviral infection in bovine oocytes
CN102703504B (zh) 能抑制口蹄疫病毒的shRNA转基因重组质粒及其应用
邓守龙 et al. RNAi Combining Sleeping Beauty Transposon System Inhibits Ex Vivo Expression of Foot-and-Mouth Disease Virus VP1 in Transgenic Sheep Cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11845965

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11845965

Country of ref document: EP

Kind code of ref document: A1