WO2021051907A1 - 一种全基因组表达载体pBR322-DHN3的制备方法 - Google Patents

一种全基因组表达载体pBR322-DHN3的制备方法 Download PDF

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WO2021051907A1
WO2021051907A1 PCT/CN2020/096713 CN2020096713W WO2021051907A1 WO 2021051907 A1 WO2021051907 A1 WO 2021051907A1 CN 2020096713 W CN2020096713 W CN 2020096713W WO 2021051907 A1 WO2021051907 A1 WO 2021051907A1
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plasmid
pbr322
dhn3
seq
fragment
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French (fr)
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陈瑞爱
黄梅
刘定祥
李延鹏
王楠楠
叶俊贤
罗琼
董楠
杨小云
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华农（肇庆）生物产业技术研究院有限公司
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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the technical field of veterinary biological products, in particular to a method for preparing the whole genome expression vector pBR322-DHN3.
  • Newcastle disease is a highly contagious and fatal disease caused by Newcastle Disease Virus (NDV), which mainly affects chickens, turkeys, wild birds and ornamental birds, commonly known as chicken plague. It is a highly contagious, acute and severe infectious disease. Humans occasionally become infected, manifested as conjunctivitis.
  • the World Health Organization (OIE) lists it as an infectious disease that must be notified, and the Ministry of Agriculture of my country also lists it as a type of animal disease that must be notified. Because of its rapid spread and the incidence and mortality rate can reach 100%, once it spreads, it will seriously harm the poultry industry and cause immeasurable losses.
  • vaccine immunization prevention is still a key measure to prevent and prevent Newcastle disease.
  • provision of an excellent, broad-spectrum, high-efficiency and inexpensive vaccine is the basis for preventing the disease.
  • Newcastle disease vaccines are divided into inactivated vaccines and live vaccines.
  • Live seedlings mainly include line I, line II (B1 strain), line III (line F), and line IV (Lasota strain).
  • the IV series live vaccine (Lasota strain) is a relatively good low-viral vaccine widely used at home and abroad, and its virulence and immunity are higher than the II series vaccine, and its safety is good.
  • the new generation of attenuated live vaccines currently favored by the market mainly include the Newcastle disease cloned live vaccine C/30 developed by the Netherlands and the IV line optimized cloned vaccine developed by American Pass.
  • the mesovirulence clone strain live vaccine of Newcastle disease (line I cloned vaccine) also showed the advantages of similar to the ordinary I line vaccine, but with milder virulence and stronger safety.
  • Chicken Newcastle Disease Virus belongs to the order of single-stranded negative-strand RNA and belongs to the genus of Paramyxovirus in the family Paramyxoviridae.
  • the virus has a double lipid layer envelope lined with a layer of M protein.
  • the outer membrane is coated with glycoproteins (HN and F) with fibrous protrusions to make the outer shape like a flower spike.
  • the capsule contains a long spiral nucleocapsid composed of a capsid protein and a negative-strand RNA.
  • Newcastle disease virus has 6 sets of genes, which are used to encode 6 viral proteins, namely (HN) glycoprotein with hemagglutinin and neuraminidase activity, (F) glycoprotein with fusion function, non-glycosylated internal Membrane protein (M), nucleocapsid protein (NP), phosphoprotein (P) and high molecular weight protein (L).
  • HN glycoprotein with hemagglutinin and neuraminidase activity
  • F glycoprotein with fusion function
  • M non-glycosylated internal Membrane protein
  • NP nucleocapsid protein
  • P phosphoprotein
  • L high molecular weight protein
  • the full length of the NDV gene is 15186nt to 15198nt.
  • NDV cDNA clones functionally mutate, replace, or insert foreign sequences into it will not hinder the replication, assembly and release of the virus.
  • the cDNA clone of NDV has been used in basic research and vaccine development.
  • NDV cDNA clones can be used as vectors to express antigen proteins of other pathogens to obtain multivalent vaccines against multiple pathogens.
  • Peeters et al. made base mutations at the cleavage point of the F gene to make the Latasa strain attenuated into a virulent (ref1); in 2004, Huang et al. exchanged the HN genes of the strength and attenuated strains and obtained new viruses with different virulence.
  • Strain (ref2) the HN genes of the strength and attenuated strains and obtained new viruses with different virulence.
  • Mebatson et al. used the S2 glycoprotein epitope gene of hepatitis virus to replace the dominant epitope of the NP protein of NDV, and successfully obtained a hybrid virus that is both anti-NDV and anti-hepatitis virus (ref3); in 2006, Man et al.
  • the HA gene of the H7 avian influenza virus and the PM gene of the NDVB1 strain were inserted between the NDVB1 strain and a hybrid virus (ref4) that was resistant to both NDV and H7 avian influenza virus was successfully obtained.
  • Ref4 hybrid virus
  • the whole genome of DHN3 is 15192nt. If the full-length cDNA is obtained by RT-PCR at one time, it is difficult to synthesize DNA fragments larger than 10k with DNA polymerases that are generally available in the market. It is easy to introduce random mutations during PCR.
  • the technical problem to be solved in this application is: how to successfully prepare the genome-wide expression vector pBR322-DHN3 and avoid random mutations.
  • the purpose of the present invention is to provide a method for preparing the whole genome expression vector pBR322-DHN3, which can successfully prepare the whole genome expression vector pBR322-DHN3 without random mutation.
  • the technical solution provided by the present invention is: a method for preparing the whole genome expression vector pBR322-DHN3, including the following steps:
  • Step 1 Establish a pBR322-Base vector, and introduce a fragment capable of homologous recombination with the whole genome of DHN3 into the vector pBR322; the fragment has homology arms corresponding to the 3'end and 5'end of the whole DHN3 genome;
  • Step 2 Construct a transition vector; the transition vector is plasmid pBR322-PNP, plasmid pBR322-PDP, plasmid pBR322-LPD3; the target fragment of plasmid pBR322-PNP includes NP, MINI and P genes; said plasmid pBR322-PDP
  • the target fragments include P, PD1 and PD2, PD3 genes; the target fragments of the plasmid pBR322-LPD3 include PD3 and L1, L2, L3, L4 genes;
  • Step 3 Construct the whole DHN3 genome DHN3-A; digest the plasmid pBR322-PNP, plasmid pBR322-PDP, and plasmid pBR322-LPD3 to obtain gene fragments PNP, PDP and LPD3, and pass the gene fragments PNP, PDP and LPD3 through T4 ligase Connect to get the whole DHN3 genome DHN3-A;
  • Step 4 Construct a plasmid fragment with homology arms; use the pBR322-Base vector in step 1 as a template to perform PCR amplification to obtain a plasmid fragment with homology arms;
  • Step 5 Construct the whole genome expression vector pBR322-DHN3; perform homologous recombination of the plasmid fragment in step 4 and the whole DHN3 genome DHN3-A in step 3 to obtain the plasmid pBR322-DHN3 with the whole DHN3 genome DHN3-A;
  • DHN3 genome-wide DHN3-A The sequence of DHN3 genome-wide DHN3-A is shown as SEQ ID NO: 1 in the sequence list;
  • the position of MINI gene in SEQ ID NO:1 in the sequence list is 1414-1949nt; the position of PD1 gene in SEQ ID NO:1 in the sequence list is 2935-4956nt; the position of PD2 gene in SEQ ID NO:1 in the sequence list is 4838-6454nt
  • the position of PD3 gene in SEQ ID NO:1 in the sequence list is 6261-8283nt; the position of L1 gene in SEQ ID NO:1 in the sequence list is 8166-10709nt; the position of L2 gene in SEQ ID NO:1 in the sequence list is 10174- 12299nt; the position of L3 gene is 12238-14433nt in SEQ ID NO:1 of the sequence list; the position of L4 gene is 14214-15192nt of SEQ ID NO:1 in the sequence list.
  • the specific method for constructing the pBR322-PNP plasmid in the step 2 is:
  • Step 211 Double-enzyme digestion of the pBR322 vector with Hind3 and Nhe1, and then use the gel to recover the pBR322 vector fragment for use;
  • Step 212 Use primers with homology arms and corresponding templates to amplify DNA fragments with homology arms under the action of a high-fidelity DNA polymerase, and recover them for use after verification by running a DNA gel;
  • the templates are plasmid pMD19-NP, plasmid pMD19-MINI, plasmid pMD19-P;
  • the primers corresponding to plasmid pMD19-NP are C-Sac11BtNhe-ST-R and C-NP-F;
  • the primers corresponding to plasmid pMD19-MINI are C-Mini-R and C-Mini-F;
  • the primers corresponding to plasmid pMD19-P are C-P-R and C-HindBt-P-F;
  • the plasmid pMD19-NP is the product of the NP gene cloned into the pMD19 vector;
  • the plasmid pMD19--MINI is the product of the MINI gene cloned into the pMD19 vector;
  • the plasmid pMD19-P is the product of the P gene cloned into the pMD19 vector;
  • primer C -Sac11BtNhe-ST-R as SEQ ID NO:1 in the sequence list;
  • primer C-NP-F as SEQ ID NO:1 in the sequence list;
  • primer C-Mini-R as SEQ ID NO:1 in the sequence list;
  • primer C-Mini -F is as SEQ ID NO:1 in the sequence list;
  • primer CPR is as SEQ ID NO:1 in the sequence list;
  • primer C-HindBt-PF is as SEQ ID NO:1 in the sequence list;
  • Step 213 Link the DNA fragment obtained in step 212 and the pBR322 vector fragment obtained in step 211 with a recombinase to obtain a pBR322-PNP plasmid.
  • the specific method for constructing the pBR322-PDP plasmid in the step 2 is:
  • Step 221 Double-enzyme digestion of the pBR322 vector with Hind3 and Nhe1, and then use the gel to recover the pBR322 vector fragment for use;
  • Step 222 Use primers with homology arms and corresponding templates to amplify DNA fragments with homology arms under the action of high-fidelity DNA polymerase, and recover them for use after verification by running DNA gel;
  • the templates are plasmid pMD19-P, plasmid pMD19-PD1, plasmid pMD19-PD2, pMD19-PD3;
  • the primers corresponding to plasmid pMD19-P are C-BtNhe-P-R and C-P-F;
  • the primers corresponding to plasmid pMD19-PD1 are C-PD1-R and C-PD1-F;
  • the primers corresponding to plasmid pMD19-PD2 are C-PD2-R and C-PD2-F;
  • the primers corresponding to plasmid pMD19-PD3 are C-PD3-R and C-HindBtg-PD3-F;
  • Step 223 Link the DNA fragment obtained in step 222 and the pBR322 vector fragment obtained in step 221 with a recombinase to obtain a pBR322-PDP plasmid.
  • step 2 In the method for preparing the whole genome expression vector pBR322-DHN3, the specific method for constructing the pBR322-LPD3 plasmid in step 2 is:
  • Step 231 Double-enzyme digestion of the pBR322 vector with Hind3 and Nhe1, and then use the gel to recover the pBR322 vector fragment for use;
  • Step 232 Use primers with homology arms and corresponding templates to amplify DNA fragments with homology arms under the action of a high-fidelity DNA polymerase, and recover them for use after running DNA gel for verification;
  • the template is plasmid PMD19-PD3, plasmid pXJ40-L;
  • Plasmid PMD19-PD3, the corresponding primers are C-BtNhe-PD3-R and C-PD3-F;
  • Plasmid pXJ40-L the corresponding primers are C-L1-R, P-L2-R, P-L3-F, C-HindBtNot-L4-F;
  • Step 233 link the DNA fragment obtained in step 232 and the pBR322 vector fragment obtained in step 231 with a recombinase to obtain a pBR322-PDP plasmid.
  • the method for recovering the fragment PNP from the pBR322-PNP plasmid is: pBR322-PNP plasmid is double digested with BtgZ1 and Hind3 and then the fragment PNP is recovered;
  • the method of recovering fragment PDP from pBR322-PDP plasmid is: pBR322-PDP plasmid is digested with BtgZ1 and then recovered fragment PDP;
  • the method of recovering fragment LPD3 from pBR322-LPD3 plasmid is: pBR322-LPD3 plasmid is digested with BtgZ1 and then recovered fragment LPD3;
  • fragment PNP, fragment PDP, and fragment LPD3 are connected in vitro by high-concentration T4 linkase to obtain full-length DHN3-A.
  • step 1 is specifically:
  • the present invention divides DHN3 whole gene components into 3 fragments, which are respectively cloned into pBR322 vector.
  • the size of the fragment is determined based on the BtgZ1 restriction site it holds to facilitate subsequent in vitro DNA fragment ligation.
  • the three fragments were successfully cloned into the pBR322 vector, and the whole genome expression vector pBR322-DHN3 was obtained.
  • the preliminary identification was performed by Sac1 digestion.
  • the correct digestion product should be 1 8482bp, 1 7065bp, 1 3132bp, and 1 908bp , And a 32bp fragment.
  • Figure 6 shows that the digested product is consistent with the result of sequence inference.
  • the present invention solves the problem that large genomes are not easy to be artificially synthesized.
  • Fig. 1 is a structural diagram of the pBR322-Base vector of the first embodiment of the present invention.
  • Figure 2 is a schematic diagram of the structure of the pBR322-PNP plasmid of Example 1 of the present invention
  • Figure 3 is a schematic diagram of the structure of the pBR322-PDP plasmid of Example 1 of the present invention.
  • Figure 4 is a schematic diagram of the structure of the pBR322-LPD3 plasmid of Example 1 of the present invention.
  • FIG. 5 is an electrophoresis diagram of the plasmid A1-A3 PCR identification of the colony A in Example 1 of the present invention
  • Figure 6 is an electrophoresis diagram of plasmid A identification by restriction digestion in Example 1 of the present invention.
  • Figure 7 is a schematic diagram of the structure of the pBR322-DHN3 plasmid of Example 1 of the present invention.
  • Figure 8 is a schematic diagram of the structure of the pBR322-DHN3 plasmid of Example 1 of the present invention.
  • Fig. 9 is an electrophoresis diagram of the PCR product of the plasmid PXJ40-L identified by the primer PXJ40-F/P-L1-R in Example 1 of the present invention.
  • Fig. 10 is an electrophoresis diagram of the correct PCR product amplified by primers P-L3-F/P-L3-R in Example 1 of the present invention
  • Fig. 11 is an electrophoresis diagram of restriction digestion identification of plasmid pXJ40-L in Example 1 of the present invention.
  • Figure 12 is a schematic diagram of the cytopathic pathology of BHK-21 cells infected with NDV of the present invention.
  • Figure 13 is a diagram showing the alignment of the sequencing results of the present invention with all published NDV sequences.
  • FIG. 14 is a schematic diagram of the plasmid structure of the pBR322 plasmid fragment of Example 1 of the present invention.
  • FIG. 15 is a schematic diagram of the plasmid structure of the pXJ40-New plasmid in Example 1 of the present invention.
  • the THZ-100 electric heating constant temperature incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.
  • A17105653 clean bench was purchased from Suzhou Antai Air Technology Co., Ltd.
  • the DK-8D three-hole electrothermal constant temperature water tank was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.
  • the THZ-100 constant temperature incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.
  • Haier refrigerator type BCD-579WE was purchased from Haier.
  • DNA/RNA co-extraction kit (AP-MN-BF-VNA-250G) was purchased from AXYGEN;
  • TIANprep MiniPlasmidKit (DP103-03) was purchased from Tiangen Co., Ltd.;
  • GelExtractionKit (D2500-02) was purchased from OMEGA;
  • M-MLVRT 2641A was purchased from TAKARA;
  • RRI 2313A was purchased from TAKARA;
  • Random 6 primer (3801) purchased from TAKARA;
  • Agarose (E0301) was purchased from TSINGK;
  • LipofectamineLTXandPlusReagent (15338-100) was purchased from Invitrogen;
  • FBS (10099-141C) was purchased from Gibco;
  • Premix-Taq (RR902) was purchased from TAKARA;
  • PenStreppenicillin Streptomycin (15140-122) was purchased from Gibco;
  • Primerstar GXL (R050) was purchased from TAKARA;
  • BtgZ1 R0703S
  • T4DNALigase M0202M
  • restriction enzymes and linking enzymes were purchased from TAKARA.;
  • ClonExpressMultisOneStepCloningKit (C113); purchased from Novizan.
  • BL21(DE) was purchased from Merck;
  • the vector pMD19 was purchased from Takara, and pBR322 was purchased from NEB.
  • the virus contained in the allantoic fluid was plaque purified on BHK-21 cells, and the purified virus was named DHN3.
  • the specific operations are as follows:
  • Allantoic fluid was recovered from the viral DHN3 diluted 1:10 with DMEM culture medium into six gradients were prepared by infection of 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 - 6 Spare.
  • BHK-21 cells were cultured in a 6-well culture dish, and after the cells grew to 90% full, 200 ⁇ l of the infection solution was added, and 800 ⁇ l of DMEM culture solution was added to make the final volume of each well 1 ml. Shake gently and incubate at 37°C for 2h. Discard the incubation solution and wash with PBS twice. Add 2.5ml of agarose solution to each well. Observe the plaques after 4-6 days.
  • PCR products were purified and cloned into the pMD19 vector, and then sequenced using the primers (M13-F, M13-R) at both ends of the pMD19 vector.
  • a total of 10 plasmids cover the entire genome of DHN3, as detailed in Table 1.
  • Primer L1-F see SEQ ID NO: 2; primer L1-R see SEQ ID NO: 3; primer L2-F see SEQ ID NO: 4; primer L2-R see SEQ ID NO: 5; primer L3-F see SEQ ID NO: 6; primer L3-R see SEQ ID NO: 7; primer L4-F see SEQ ID NO: 8; primer L4-R see SEQ ID NO: 11; primer PD1-F see SEQ ID NO: 9; primer PD1-R see SEQ ID NO: 10; primer PD2-F see SEQ ID NO: 12; primer PD2-R see SEQ ID NO: 13; primer PD3-F see SEQ ID NO: 14; primer PD3-R see SEQ ID NO: 15; primer MINI-F see SEQ ID NO: 16; primer mini-r see SEQ ID NO: 17; primer PF see SEQ ID NO: 18; primer PR see SEQ ID NO: 19; primer NDV-ST-W R see SEQ ID NO: 20; primer NP-LB-R see
  • Step 1 In order to obtain the recombinant virus, it is first necessary to establish a basic plasmid pBR322-Base that can obtain single-stranded negative-strand RNA of the whole genome.
  • a T7 promoter, a T7 terminator and a HDVRibozyme were introduced into the pBR322 vector, so that they could finally be exogenous. Under the action of T7RNA polymerase, it can accurately synthesize DHN3 genome-wide negative-strand full-length RNA.
  • the 3'end HC1 of DHN3 was introduced downstream of the T7 promoter; the 5'end part of the DHN3 base HC2 was introduced upstream of HDV Ribozyme; bases
  • the position sequence of HC1 in the DHN3 genome sequence is 15192-15159 nt; the position of base HC2 in the DHN3 genome sequence is 141-1 nt.
  • DNA fragments containing T7 terminator, HDVRibozyme, HC2, HC1, and T7 promoters were artificially synthesized and combined into the pBR322 vector by homologous recombination to obtain pBR322-T7Pro-HDV_Ter, which is the basic plasmid pBR322-Base (plasmid diagram 1 ).
  • Step 2 Establish 3 sub-genome transition vectors: DHN3 complete genome with a total of 15192nt. If the full-length cDNA is obtained by RT-PCR at one time, one is difficult, (generally commercially available DNA polymerases are difficult to synthesize larger than 10k DNA fragments). Second, it is easy to introduce random mutations in the PCR process. Therefore, the whole DHN3 gene component was divided into 3 fragments, which were cloned into the pBR322 vector. The size of the fragment is determined based on the BtgZ1 restriction site it holds to facilitate subsequent in vitro DNA fragment ligation.
  • pBR322 vector was digested with Hind3 and Nhe1, and then recovered by gel for use.
  • Target gene fragments respectively use primers with homology arms and corresponding templates (see Table 5 below) to amplify DNA fragments with homology arms under the action of high-fidelity DNA polymerase, and run DNA glue to verify Recycle for spare.
  • Link the target fragment and pBR322 vector fragment with recombinase refer to Table 6. After bacterial transformation, single colony PCR preliminary selection, amplification of plasmid DNA, enzyme digestion of plasmid DNA, run DNA gel reselection, and finally sequence verification.
  • the target fragments used to construct the pBR322-PNP plasmid include NP, MINI and P.
  • the target fragments used to construct the pBR322-PDP plasmid include P, PD1 and PD2, PD3.
  • the target fragments used to construct the pBR322-LPD3 plasmid include L1, L2, L3, L4 and PD3.
  • Figure 2 is a schematic diagram of pBR322-PNP plasmid
  • Figure 3 is a schematic diagram of pBR322-PDP plasmid
  • Figure 4 is a schematic diagram of pBR322-LPD3 plasmid.
  • primer C-Sac11BtNhe-ST-R see SEQ ID NO: 24; for primer C-NP-F, see SEQ ID NO: 25; for primer C-Mini-R, see SEQ ID NO: 26; for primer C-Mini-F, see SEQ ID NO: 27; primer CPR see SEQ ID NO: 28; primer C-HindBt-PF see SEQ ID NO: 29; primer C-BtNhe-PR see SEQ ID NO: 30; primer CPF see SEQ ID NO: 31; primer C -PD1-R see SEQ ID NO: 32; primer C-PD1-F see SEQ ID NO: 33; primer C-PD2-R see SEQ ID NO: 34; primer C-PD2-F see SEQ ID NO: 35; See SEQ ID NO: 36 for primer C-PD3-R; See SEQ ID NO: 37 for primer C-HindBtg-PD3-F; See SEQ ID NO: 38 for primer C-BtNhe-PD3-R; See SEQ ID NO:
  • PBR322-PNP plasmid After the target fragments required to construct the PBR322-PNP plasmid, PBR322-PDP plasmid, and PBR322-LPD3 plasmid are amplified separately, mix according to the recombination system in Table 6 above, and then follow the steps of homologous recombination. Construct the corresponding plasmid.
  • the virus genome contains two BtgZ I restriction sites. According to these two restriction sites, the whole virus gene component is divided into 3 large fragments. This is the construction of PBR322-PNP. Three large fragments contained in the three plasmids of PBR322-PDP and PBR322-LPD3.
  • the whole virus gene composition is divided into 10 small fragments (NP, P, PD1, PD2, PD3, L1, L2, L3, L4), and the BtgZ I restriction site is just right at the P fragment and
  • PBR322-PNP, PBR322-PDP, PBR322-LPD3 plasmids when amplifying P fragment and PD3, it is referred to as P left, P right, PD3 left, and PD3 right.
  • the L protein is the nucleocapsid protein of the NDV virus
  • the plasmid PXJ40-L was first constructed as an auxiliary protein to rescue the virus . Therefore, one of the templates used when constructing the PBR322-LPD3 plasmid is PXJ40-L, which already includes the entire sequence of the L gene.
  • L1-2 refers to the continuous sequence of small fragments L1 and L2, which is amplified by primer C -L1-R, P-L2-R, constructed from template pXJ40-L
  • L3-4 refers to the continuous sequence of small fragments L3 and L4, which is composed of primers P-L3-F, C-HindBtNot-L4-F, template pXJ40-L); and it should be pointed out that the primer C-HindBtNot-L4-F used to amplify L3-4 contains the homology region and a BtgZ I digestion sequence required for the construction of the PBR322-DHN3 plasmid. Then, BtgZ I can cut the viral genome sequence in PBR322-LPD3 completely, and the cut fragments can be directly used for the construction of PBR322-DHN3 plasmid.
  • the enzymes used in the PCR amplification reaction involved in the construction of the PBR322-PNP plasmid, PBR322-PDP plasmid, and PBR322-LPD3 plasmid in Table 6 above are all PrimeSTAR GXL DNA Polymerase (R050, TAKARA), and the reaction system refers to the previous one. Narrated. According to the characteristics of this enzyme R050, the pre-denaturation temperature and time in the amplification reaction are 95°C, 3min, the denaturation temperature and time are 98°C, 10s, the annealing temperature and time are both 60°C, 15s, and the extension temperature The extension time at 68°C is appropriately selected according to the fragment size according to the ratio of 1000bp extension for 60s.
  • the reaction system is 30 ⁇ l.
  • kits used are all ClonExpressMultisOneStepCloningKit (C113, Novizan), which will not be repeated.
  • the 5'end of L1 also has a homology arm that is homologous to the pXJ40BamH1 end, and the 3'end of L4 has a homology arm that is homologous to the pXJ40Pst1 end.
  • the vector pXJ40 was digested with BamH1 and Pst1, and then recovered by gel purification. Add the above four fragments L1, L2, L3 and L4, and pXJ40 after double digestion with BamH1 and Pst1, and perform homologous recombination under the action of recombinase (ClonExpressMultisOneStepCloningKit). The recombinant product was transformed into DH5 ⁇ cells and screened by single colony PCR.
  • Figures 9-10 show that 8 colonies were detected, and 8 positive bacteria all produced PCR products of the correct molecular weight.
  • Primer P-L1-BamH1-F see SEQ ID NO: 45 in the sequence table; primer P-L1-R see SEQ ID NO: 46 in the sequence table; primer P-L2-F see SEQ ID NO: 47 in the sequence table; primer P- See SEQ ID NO: 48 in the sequence list for L2-R; see SEQ ID NO: 49 in the sequence list for primer P-L3-F; see SEQ ID NO: 49 in the sequence list for primer P-L3-R; see SEQ ID NO: 50 for primer P-L4-F in the sequence list.
  • List SEQ ID NO: 51; primer P-L4-Pst1-R see SEQ ID NO: 52 in the sequence list;
  • the R050 high-fidelity enzyme is used to amplify each fragment, and the annealing temperature is 60°C.
  • the extension time calculation method and amplification system are calculated according to the previous R050 enzyme instructions.
  • the vector plasmid pXJ40 used in the present invention is derived from the laboratory of Professor Liu Dingxiang from the Population Microbiology Center of South China Agricultural University.
  • the schematic diagram of the plasmid structure is shown in Figure 15.
  • the pXJ40-NEW shown in Figure 15 is pXJ40; its sequence is shown in the sequence table SEQ ID NO: 60; a person of ordinary skill in the art can obtain it by artificial sequence synthesis according to the sequence listing.
  • the laboratory of Professor Liu Dingxiang from the Colony Microbiology Center of South China Agricultural University can provide a vector plasmid as shown in the structure of the plasmid as shown in FIG.
  • the vector plasmid pXJ40 can also be obtained by conversion of other commercial plasmids in the field.
  • the commercial plasmid pXJ40-flag can be used to prepare the pXJ40 used in this example.
  • the modification principle is: The impact of; we obtained pXJ40-New from the commercial plasmid pXJ40-Flag after modification and removal of Flag.
  • pXJ40-flag is preserved in the laboratory of Professor Liu Dingxiang of the Population Microbiology Center of South China Agricultural University, and the laboratory of Professor Liu Dingxiang of the Population Microbiology Center of South China Agricultural University can provide pXJ40-flag plasmid; pXJ40-flag is published by Professor Liu Dingxiang There is a record in the literature, and the specific record is "pXJ40F(with a Flag tag)".
  • Channel 2 Purchased from Purutin Biotechnology (Beijing) Co., Ltd., the article number is pXJ40-flag.
  • S12 PCR amplification of fragments without Flag: using 2ng pXJ40Flag as a template, using primers dFlagF:actatagggcgaattcggatccaagcttctcg (see SEQ ID NO: 53 in the sequence list)/Bsa1R: tgagcgtgggtctcgcggt (see SEQ ID NO: 54 in the sequence list), high-fidelity polymerase (TAKARA Company R050) A 1322bp fragment was amplified by PCR (annealing temperature of 60 degrees, extension time of 1 minute and 30 seconds), and the gel was recovered for use.
  • Step 3 Recover fragment PNP, fragment PDP, and fragment LPD3 from pBR322-PNP plasmid, pBR322-PDP plasmid, and pBR322-LPD3 plasmid; ligate fragment PNP, fragment PDP, and fragment LPD3 to obtain full-length DHN3-A spare;
  • the pBR322-PNP plasmid was digested with BtgZ1 and Hind3 and then the fragment PNP was recovered;
  • the method of recovering fragment PDP from pBR322-PDP plasmid is: pBR322-PDP plasmid is digested with BtgZ1 and then recovered fragment PDP;
  • the method of recovering fragment LPD3 from pBR322-LPD3 plasmid is: pBR322-LPD3 plasmid is digested with BtgZ1 and then recovered fragment LPD3;
  • Fragment PNP, fragment PDP, and fragment LPD3 are prepared in accordance with the restriction digestion system and time in the above table; after the restriction is completed, a small amount can be used for gel electrophoresis to observe whether the restriction is complete, if not complete, add a little more enzyme appropriately Or extend the incubation time, or both;
  • the fragment PNP, fragment PDP, and fragment LPD3 are connected in vitro by high-concentration T4 linkase to obtain full-length DHN3-A. Refer to Table 10 for specific parameters.
  • Fragment PNP, Fragment PNP, Fragment LPD3 are calculated and used according to the amount in the above table, and connected.
  • Step 4 Using the above pBR322-Base plasmid as a template, make special primers containing homology arms matching bases (A2-F: atcggtagaaggttccctcaggttc (see SEQ ID NO: 56 in the sequence list); A2-R: ggtcctatagtgagtcgtattaatg (see SEQ in the sequence list) ID NO: 57)), use the materials provided by the Primerstar GXL kit and perform PCR amplification according to the method provided by the kit.
  • A2-F atcggtagaaggttccctcaggttc
  • A2-R ggtcctatagtgagtcgtattaatg
  • Step 5 Perform homologous recombination of the plasmid fragment pBR322-Base obtained in Step 4 with DHN3-A to obtain the whole genome expression vector pBR322-DHN3.
  • a recombination kit (ClonExpressMultisOneStepCloningKit, C113) purchased from Novezin. Take the above-mentioned plasmid fragment with homology arms and the above-mentioned DHN3 whole genome DNA fragment (that is, the above-mentioned in vitro ligation product, DHN3-A) for homologous recombination according to the method provided by the Novezin recombination kit. Refer to Table 11 for specific formula parameters.
  • Step 6 Transform the recombinant product into DH5a competent cells with specific primers (C-pBR322-R: gaaattgcatcaacgcatatagcgc (see SEQ ID NO: 58); C-NP-F: catctggttgcccttgcggcttgttc (see SEQ ID NO :59)) Perform single colony PCR to obtain 3 positive colonies A1-A3.
  • Figure 5 is the electrophoresis diagram of the plasmid A1-A3 of the colony A1.
  • the colony A1 was digested with Sac1 for preliminary identification.
  • the correct digestion product should be 1 8482bp, 1 7065bp, 1 3132bp, 1 908bp, and 1 32bp fragment; the result is shown in Figure 6,
  • Figure 6 is the plasmid
  • the electropherogram of A1 shows that the digested product is consistent with the result of sequence inference.
  • the 32bp fragment is too small to be displayed in the agarose gel.
  • the plasmid was named pBR322-DHN3.
  • This plasmid was sequenced again and amplified for use after verification.
  • FIG. 8 The schematic diagram of the plasmid structure of pBR322-DHN3 is shown in FIG. 8.
  • the present invention solves the problem that large genomes are not easy to be artificially synthesized, and avoids the occurrence of random mutations.
  • NDV Newcastle disease virus

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Abstract

一种全基因组表达载体pBR322-DHN3的制备方法，包括如下步骤：步骤1：建立pBR322-Base载体；步骤2：构建pBR322-PNP质粒、pBR322-PDP质粒、pBR322-LPD3质粒；步骤3：回收片段PNP、片段PDP、片段LPD3；并将片段PNP、片段PDP，以及片段LPD3连接得到全长DHN3-A备用；步骤4：将步骤1所得pBR322-Base载体为模板，扩增、纯化后制得带同源臂的质粒片段；步骤5：将步骤4得到的质粒片段与步骤3得到的DHN3-A进行同源重组得到重组产物。

Description

一种全基因组表达载体pBR322-DHN3的制备方法 技术领域
本发明涉及兽用生物制品技术领域，特别涉及全基因组表达载体pBR322-DHN3的制备方法。
背景技术
新城疫是由新城疫病毒(NDV)引起的一种主要侵害鸡、火鸡、野禽及观赏鸟类的高度接触传染性、致死性疾病，俗称鸡瘟。属高度接触性、急性、烈性传染病。人类偶会感染，表现为结膜炎。世卫组织(OIE)将其列为必须通报的传染病，我国农业部也将其列为必须通报的一类动物疫病。因其传播速度快且发病和病死率均可达100％，一旦波及将严重危害家禽业,造成不可估量的损失。
新城疫的防预除了合理饲养，疫苗免疫预防仍是关键措施。除了建立建全正确的免疫程序，提供优良广谱高效廉价疫苗是预防该病的根本。
鸡新城疫疫苗分灭活苗和活苗。活苗主要有Ⅰ系，Ⅱ系(B1株)、Ⅲ系(F系)、Ⅳ系(Lasota株)。其中Ⅳ系活疫苗(Lasota株)是目前国内外广泛应用的较优良的低毒苗,其毒力与免疫性能高于Ⅱ系苗,安全性良好。随着克隆技术的应用新一代克隆苗正逐步上市。目前被市场青睐的新一代弱毒活苗主要有由荷兰研制的新城疫克隆活苗C/30和由美国通研制的Ⅳ系优化克隆苗。此外新城疫中等毒力克隆株活疫苗(I系克隆苗)也表现出与普通I系苗相似但毒力稍温和,安全性较强的优势。
据流行病学调查，NDV的流行基因型随时间和地理环境而变化。上世纪20-50年代主要流性I-IV型，70年代首次发现V型主要在南美和中美以致全欧洲。80年代出现VI型并盛行于中东，亚洲和欧洲。85年VII型开始蔓延，并与VIII型流行于世界多个国家并 在90年代导致在亚洲，非洲和中东的大流行。V-VIII型均为强毒。IX和X型尚局限在局部散发状态。由于针对IV型的疫苗得到了广泛应用，IV型NDV已得到良好控制。但针对其它基因型的疫苗尚欠缺从而均有流行的趋势。因此利用反向遗传克隆技术迅速开发针对实时流行株的高效，广谱，安全甚至多价的弱毒苗势在必行。此外，由于疫苗的广泛使用使疫检更加困难。因为无法甄别被感染鸡携带的是疫苗株还是野生毒株。通过重组方式可引入人工标签，使其能与野毒有效鉴别。
鸡新城疫病毒(NDV)属于单股负链RNA目，副粘病毒科副粘病毒属。该病毒具有一个双脂层囊膜，内衬一层M蛋白。膜外被具有纤突的糖蛋白(HN和F)包被使之外形呈花穗状。囊内含有由壳蛋白和一条负链RNA组成的长螺旋状核衣壳。新城疫病毒有6组基因，分别用于编码6个病毒蛋白，即具血凝素和神经氨酸酶活性的(HN)糖蛋白、具融合功能的(F)糖蛋白，非糖基化内膜蛋白(M)，核壳蛋白(NP)，磷蛋白(P)和高分子量蛋白(L)。NDV基因全长为15186nt到15198nt。
已有研究表明将NDV的cDNA克隆作人功突变，更换，或在其中插入外源序列并不会阻碍该病毒的复制，装配和释放。NDV的cDNA克隆已用于基础研究和疫苗开发。NDV的cDNA克隆可作为载体表达其它病原菌的抗原蛋白从而获得抗多种病原菌的多价疫苗。如1999年Peeters等将F基因裂解点作碱基突变使Latasa株弱毒变成了强毒(ref1)；2004年Huang等将强度和弱毒株的HN基因互换也获得了不同毒力的新毒株(ref2)。2002年，Mebatson等用肝炎病毒的S2糖蛋白抗原表位基因取代NDV的NP蛋白优势抗原表位，成功获得了既抗NDV又抗肝炎病毒的杂合病毒(ref3)；2006年，Man等将H7禽流感病毒的HA基因、插在NDVB1株的P-M基因之间也成功获得了既抗NDV又抗H7禽流感病毒的杂合病毒(ref4)。最近Abzeid等又成功将埃及当前流行的IBVS蛋白组装进重组NDV，获得了既针对原NDV又针对相应的IBV免疫保护的杂合病毒。(ref5)我国学者也用LaSota株为载体先后获得多个具二价功能的杂合病毒。如既抗NDV又抗IBDV5杂合病毒(ref6)；既抗NDV又抗H5N1的杂合病毒(ref7)；既抗NDV又抗鸡支原体TM1病毒的杂合病毒(ref8)。 由于NDV只有一个血清型，因此它的遗传性能相对稳定。虽然NDV的感染性cDNA克隆在国际上早有建立但国内基本上仅限于Latasa株，且基本局限在基础和临床的研究层面，这可能与国内疫苗生产单位的研发技术能力有关。
在实际应用过程中，DHN3全基因组共15192nt，如将全长cDNA一次性通过RT-PCR获得，一是困难，一般市场可买到的DNA聚合酶很难合成大过10k的DNA片段，二是容易在PCR过程中引入随机突变。
所以，本申请所要解决的技术问题是：如何成功制备全基因组表达载体pBR322-DHN3，并避免随机突变。
发明内容
本发明的目的在于提供一种全基因组表达载体pBR322-DHN3的制备方法，其能够成功制备全基因组表达载体pBR322-DHN3，无随机突变发生。
为了实现上述目的，本发明提供的技术方案是：一种全基因组表达载体pBR322-DHN3的制备方法，包括如下步骤：
步骤1：建立pBR322-Base载体，在pBR322载体上引入能够与DHN3全基因组进行同源重组的片段；所述片段上具有与DHN3全基因组的3’末端和5’末端对应的同源臂；
步骤2：构建过渡载体；所述过渡载体为质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3；所述质粒pBR322-PNP的目的片段包括NP、MINI和P基因；所述质粒pBR322-PDP的目标片段包括P，PD1和PD2,PD3基因；所述质粒pBR322-LPD3的目标片段包括PD3和L1,L2,L3,L4基因；
步骤3：构建DHN3全基因组DHN3-A；将质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3进行酶切得到基因片段PNP、PDP和LPD3，将基因片段PNP、PDP和LPD3通 过T4链接酶连接得到DHN3全基因组DHN3-A；
步骤4：构建带同源臂的质粒片段；将步骤1中的pBR322-Base载体为模板进行PCR扩增得到带同源臂的质粒片段；
步骤5：构建全基因组表达载体pBR322-DHN3；将步骤4中的质粒片段和步骤3中的DHN3全基因组DHN3-A进行同源重组得到具有DHN3全基因组DHN3-A的质粒pBR322-DHN3；
DHN3全基因组DHN3-A的序列如序列表SEQ ID NO：1；
MINI基因在序列表SEQ ID NO：1中位置为1414-1949nt；PD1基因在序列表SEQ ID NO：1中位置为2935-4956nt；PD2基因在序列表SEQ ID NO：1中位置为4838-6454nt；PD3基因在序列表SEQ ID NO：1中位置为6261-8283nt；L1基因在序列表SEQ ID NO：1中位置为8166-10709nt；L2基因在序列表SEQ ID NO：1中位置为10174-12299nt；L3基因在序列表SEQ ID NO：1中位置为12238-14433nt；L4基因在序列表SEQ ID NO：1中位置14214-15192nt。
在上述全基因组表达载体pBR322-DHN3的制备方法中，所述步骤2中pBR322-PNP质粒构建的具体方法为：
步骤211：将pBR322载体用Hind3和Nhe1作双酶切，然后经胶回收pBR322载体片段备用；
步骤212：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用；
其中，模板为质粒pMD19-NP、质粒pMD19-MINI、质粒pMD19-P；
质粒pMD19-NP对应的引物为C-Sac11BtNhe-ST-R和C-NP-F；
质粒pMD19-MINI对应的引物为C-Mini-R和C-Mini-F；
质粒pMD19-P对应的引物为C-P-R和C-HindBt-P-F；
其中，质粒pMD19-NP为NP基因克隆进pMD19载体中的产物；质粒pMD19--MINI为MINI基因克隆进pMD19载体中的产物；质粒pMD19-P为P基因克隆进pMD19载体中的产物；引物C-Sac11BtNhe-ST-R如序列表SEQ ID NO：1；引物C-NP-F如序列表SEQ ID NO：1；引物为C-Mini-R如序列表SEQ ID NO：1；引物C-Mini-F如序列表SEQ ID NO：1；引物C-P-R如序列表SEQ ID NO：1；引物C-HindBt-P-F如序列表SEQ ID NO：1；
步骤213：将步骤212所获得的DNA片段和步骤211所获得的pBR322载体片段用重组酶链接，得到pBR322-PNP质粒。
在上述全基因组表达载体pBR322-DHN3的制备方法中，所述步骤2中pBR322-PDP质粒构建的具体方法为：
步骤221：将pBR322载体用Hind3和Nhe1作双酶切,然后经胶回收pBR322载体片段备用；
步骤222：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用；
其中，模板为质粒pMD19-P、质粒pMD19-PD1、质粒pMD19-PD2、pMD19-PD3；
质粒pMD19-P对应的引物为C-BtNhe-P-R和C-P-F；
质粒pMD19-PD1对应的引物为C-PD1-R和C-PD1-F；
质粒pMD19-PD2对应的引物为C-PD2-R和C-PD2-F；
质粒pMD19-PD3对应的引物为C-PD3-R和C-HindBtg-PD3-F；
质粒pMD19-PD1为PD1基因克隆进pMD19载体中的产物；质粒pMD19-PD2为PD2基因克隆进pMD19载体中的产物；质粒pMD19-PD3为PD3基因克隆进pMD19载体中的产物； 引物C-BtNhe-P-R如序列表SEQ ID NO：1；引物C-P-F如序列表SEQ ID NO：1；引物为C-PD1-R如序列表SEQ ID NO：1；引物C-PD1-F如序列表SEQ ID NO：1；引物C-PD2-R如序列表SEQ ID NO：1；引物C-PD2-F如序列表SEQ ID NO：1；引物C-PD3-R如序列表SEQ ID NO：1；引物C-HindBtg-PD3-F如序列表SEQ ID NO：1；
步骤223：将步骤222所获得的DNA片段和步骤221所获得的pBR322载体片段用重组酶链接，得到pBR322-PDP质粒。
在上述全基因组表达载体pBR322-DHN3的制备方法中，所述步骤2中pBR322-LPD3质粒构建的具体方法为：
步骤231：将pBR322载体用Hind3和Nhe1作双酶切，然后经胶回收pBR322载体片段备用；
步骤232：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用；
其中，模板为质粒PMD19-PD3、质粒pXJ40-L；
质粒PMD19-PD3，对应的引物为C-BtNhe-PD3-R和C-PD3-F；
质粒pXJ40-L，对应的引物为C-L1-R、P-L2-R、P-L3-F、C-HindBtNot-L4-F；
质粒PMD19-PD3为PD3基因克隆进pMD19载体中的产物；质粒pXJ40-L为L基因克隆进pXJ40载体中的产物；引物C-BtNhe-PD3-R如序列表SEQ ID NO：1；引物C-PD3-F如序列表SEQ ID NO：1；引物C-L1-R如序列表SEQ ID NO：1；引物P-L2-R如序列表SEQ ID NO：1；引物P-L3-F如序列表SEQ ID NO：1；引物C-HindBtNot-L4-F如序列表SEQ ID NO：1；
步骤233：将步骤232所获得的DNA片段和步骤231所获得的pBR322载体片段用重组酶链接，得到pBR322-PDP质粒。
在上述全基因组表达载体pBR322-DHN3的制备方法中，所述步骤3中，从pBR322-PNP质粒回收片段PNP的方法为：pBR322-PNP质粒用BtgZ1和Hind3双酶切后回收片段PNP；
从pBR322-PDP质粒回收片段PDP的方法为：pBR322-PDP质粒用BtgZ1单酶切后回收片段PDP；
从pBR322-LPD3质粒回收片段LPD3的方法为：pBR322-LPD3质粒用BtgZ1单酶切后回收片段LPD3；
所述片段PNP、片段PDP、片段LPD3通过高浓度T4链接酶作体外连接，获得全长DHN3-A。
在上述全基因组表达载体pBR322-DHN3的制备方法中，所述步骤1具体为：
在pBR322质粒上分别引入1个T7启动子，1个T7终止子以及1个HDV Ribozyme；T7启动子下游引入DHN3的3’末端的部分碱基HC1；在HDV Ribozyme的上游引入DHN3的5’末端部分碱基HC2；其中，碱基HC1在DHN3全基因组序列中的位置顺序为15192-15159nt；碱基HC2在DHN3全基因组序列中的位置顺序为141-1nt。
本发明的有益效果是：
本发明将DHN3全基因组分成3个片段，将其分别克隆到pBR322载体。片段的大小是基于它所持有的BtgZ1酶切位点来确定的，以利于随后的体外DNA片段连接。
通过3个片段成功克隆到pBR322载体，得到了全基因组表达载体pBR322-DHN3，通过Sac1酶切作初步鉴定，正确的酶切产物应是1个8482bp,1个7065bp,1个3132bp,1个908bp,和1个32bp的片段。结果如图6，显示该酶切产物与序列推断的结果一致。
本发明通过上述的设计，解决了大基因组不易人工合成的难题。
附图说明
图1为本发明的实施例一的pBR322-Base载体的结构图示。
图2为本发明的实施例一的pBR322-PNP质粒的结构图示；
图3为本发明的实施例一的pBR322-PDP质粒的结构图示；
图4为本发明的实施例一的pBR322-LPD3质粒的结构图示；
图5为本发明的实施例一的菌落A的质粒A1-A3PCR鉴定的电泳图；
图6为本发明的实施例一的质粒A酶切鉴定的电泳图；
图7为本发明的实施例一的pBR322-DHN3的质粒的结构图示；
图8为本发明的实施例一的pBR322-DHN3的质粒的结构图示；
图9为本发明的实施例一的引物PXJ40-F/P-L1-R鉴定质粒PXJ40-L的PCR产物的电泳图；
图10为本发明的实施例一的引物P-L3-F/P-L3-R扩增出出正确的PCR产物的电泳图；
图11为本发明的实施例一的质粒pXJ40-L的酶切鉴定电泳图。
图12为本发明NDV感染BHK-21细胞，细胞病变示意图。
图13为本发明的测序结果与已发表的所有NDV序列比对的图。
图14为本发明的实施例一的pBR322质粒片段的质粒的结构图示；
图15为本发明的实施例一的pXJ40-New质粒的质粒的结构图示。
具体实施方式
下面结合具体实施方式说明本发明：但是可以理解这些具体的实施方式只是用于说明本发明，而不是对本发明的限制。本领域的技术人员完全可以在本发明的启示下，对本发明的的具体实施方式或者技术特征进行改进，但这些经过改进或替换的技术方案，仍属于本发明的保护范围。
实施例一
一、仪器和试剂
在描述本发明的方法前，对本发明主要仪器及试剂试剂的来源进行具体描述：
主要仪器及试剂
THZ-100型电热恒温培养箱购自上海一恒科学仪器有限公司。
A17105653型洁净工作台购自苏州安泰空气技术有限公司。
DK-8D型三孔电热恒温水槽购自上海一恒科学仪器有限公司。
THZ-100型恒温培养箱购自上海一恒科学仪器有限公司。
BCD-579WE型海尔冰箱购自海尔。
5424R型离心机；EppendorfPCR仪；Thermo1300SERIESA2生物安全柜，均购自Eppendorf。
DNA/RNA共提试剂盒(AP-MN-BF-VNA-250G)购自AXYGEN；
TIANprepMiniPlasmidKit(DP103-03)均购自天根有限公司；
GelExtractionKit(D2500-02)购自OMEGA；
M-MLVRT(2641A)购自TAKARA；
RRI(2313A)购自TAKARA；
Random 6 primer(3801)购自TAKARA；
Agarose(E0301)购自TSINGK；
0.25％Trypsin-EDTA(25200-056)，DMEMbasic(C11995500BT)购自Gibco；
LipofectamineLTXandPlusReagent(15338-100)购自Invitrogen；
FBS(10099-141C)购自Gibco；
Premix-Taq(RR902)购自TAKARA；
PenStreppenicillinStreptomycin(15140-122)购自Gibco；
PrimerstarGXL(R050)购自TAKARA；
BtgZ1(R0703S)，T4DNALigase(M0202M)购自NEB；
其它限制性内切酶，链接酶均购自TAKARA.；
ClonExpressMultisOneStepCloningKit(C113)；购自诺维赞。
BL21(DE)购自Merck公司；
载体pMD19购自Takara，pBR322购自NEB。
二、病毒分离，纯化，测序和分析归类
(a)为了开发一株针对目前流行的NDV重组疫苗，我们首先从养鸡场采集病料NDV(QTCF1)，病料中添加适量生理盐水后碾磨充分，离心12000rpm 10分钟，然后取 上清通过0.4μm滤器过滤，将滤液中添加1％SP双抗后不经稀释，直接接种到鸡胚绒膜尿囊腔中，添加量为100μl/枚；
后进一步在SPF鸡胚的绒膜尿囊腔接种繁殖，待鸡胚死后收集尿囊液。
(b)将此尿囊液所含病毒在BHK-21细胞上进行噬斑纯化，纯化后的病毒命名为DHN3。具体操作如下：
使用双蒸水配置3％琼脂糖溶液，高压后置4℃备用。使用前用微波炉加热2～3分钟融化并按3％琼脂糖2ml+2％FBS DMEM 24ml配制成琼脂糖液，将其置40℃干浴待用。
配置2％FBS+DMEM+1％SP抗生素培养液备用。
将从尿囊液回收到的DHN3病毒用DMEM培养液按1：10稀释成6个梯度，制得感染液10 -1，10 -2，10 -3，10 -4，10 -5，10 -6备用。
将BHK-21细胞置6孔培养皿中培养，待细胞长至90％满后分别加入上述感染液200μl，另补DMEM培养液800μl，使每孔终体积为1ml。轻轻摇匀后置37℃孵育2h。弃去孵育液，再用PBS洗2遍。每孔加入琼脂糖液2.5ml.4-6天后观察噬斑。
挑选1个大小适中噬斑分别感染BHK-21细胞，待细胞病变明显(NDV感染的BHK-21细胞会发生明显的细胞融合现象时(如图12)提取细胞总RNA。
以这个总RNA为模板，用非特异性引物(Random 6 primer(3801))作逆转录，获得第一条cDNA。
再分以此cDNA为模板用特异性引物和ExTaq酶作PCR(其中特异性引物如下附表1中的各质粒对应的引物，PCR的反应条件见表2、表3和表4)；
分别将这些PCR产物纯化后克隆进pMD19载体，再用pMD19载体两端的引物 (M13-F，M13-R)分别测序。总共10个质粒覆盖DHN3全基因组，详细见附表1。
表1用于检测DHN3序列的质粒表
Figure PCTCN2020096713-appb-000001
引物L1-F见SEQ ID NO：2；引物L1-R见SEQ ID NO：3；引物L2-F见SEQ ID NO：4；引物L2-R见SEQ ID NO：5；引物L3-F见SEQ ID NO：6；引物L3-R见SEQ ID NO：7；引物L4-F见SEQ ID NO：8；引物L4-R见SEQ ID NO：11；引物PD1-F见SEQ ID NO：9；引物PD1-R见SEQ ID NO：10；引物PD2-F见SEQ ID NO：12；引物PD2-R见SEQ ID NO：13；引物PD3-F见SEQ ID NO：14；引物PD3-R见SEQ ID NO：15；引物MINI-F见SEQ ID NO：16；引物mini-r见SEQ ID NO：17；引物P-F见SEQ ID NO：18；引 物P-R见SEQ ID NO：19；引物NDV-ST-W R见SEQ ID NO：20；引物NP-LB-R见SEQ ID NO：21；引物M13-F见SEQ ID NO：22；引物M13-R见SEQ ID NO：23。
表2 PCR的反应条件
Figure PCTCN2020096713-appb-000002
表3 PCR的反应条件中的扩增引物表
Figure PCTCN2020096713-appb-000003
表4 PCR的反应条件中不同酶的应用配方表
Figure PCTCN2020096713-appb-000004
备注：
1.R050PCR反应程序
Figure PCTCN2020096713-appb-000005
Figure PCTCN2020096713-appb-000006
2.RR902 PCR反应程序
Figure PCTCN2020096713-appb-000007
3.本发明中所涉及到的PCR反应体系的配制及反应程序的设定均按照上述方法进行。
在PCR过程中只有退火温度和延伸时间会根据具体引物和片段不同进行调整，其他均不发生变化。下面将不再详述PCR反应的过程。
通过NCBI Blast软件将测序结果与已发表的所有NDV序列比对，其结果显示如图13。结论是此DHN3新城疫病毒全基因组全长15192nt,属II类VII型NDV。
为了保证序列准确无误，上述实验从病毒感染到测序结果分析反复重做了3次。其测序结果均相同，证实由这个噬斑获得的是一株纯化的DHN3病毒株。
DHN3全基因组的序列见序列表SEQ ID NO：1。
三、全基因组表达载体pBR322-DHN3的制备
下面具体描述本发明的方法：一种全基因组表达载体pBR322-DHN3的制备方法，
包括如下步骤：
步骤1：为获得重组病毒，首先需建立1个能获得全基因组单股负链RNA的基础质粒pBR322-Base。
为了获得较高的转录水平且保证由此转录的RNA不含任何外源核糖核酸，在pBR322载体上分别引入1个T7启动子，1个T7终止子以及1个HDVRibozyme，使之最终能在外源的T7RNA聚合酶的作用下准确合成DHN3全基因组负链全长RNA。
为了方便将DHN3全基因组通过同源重组的方法整合到pBR322载体上，在T7启动子下游引入DHN3的3’末端的HC1；在HDV Ribozyme的上游引入DHN3的5’末端部分碱基HC2；碱基HC1在DHN3全基因组序列中的位置顺序为15192-15159nt；碱基HC2在DHN3全基因组序列中的位置顺序为141-1nt。
含T7终止子、HDVRibozyme、HC2、HC1、T7启动子的DNA片段通过人工合成，并通过同源重组的方式组合到pBR322载体上获得pBR322-T7Pro-HDV_Ter，即基础质粒pBR322-Base(质粒示意图1)。
步骤2：建立3个亚基因组过渡载体：DHN3全基因组共15192nt，如将全长cDNA一次性通过RT-PCR获得，一是困难，(一般市场可买到的DNA聚合酶很难合成大过10k的DNA片段)，二是容易在PCR过程中引入随机突变。因此将DHN3全基因组分成3个片段，将其分别克隆到pBR322载体。片段的大小是基于它所持有的BtgZ1酶切位点来确定的，以利于随后的体外DNA片段连接。
具体构建方法如下：
A.制备质粒片段：将pBR322载体用Hind3和Nhe1作双酶切，然后经胶回收备用。
B.制备目的基因片段：分别用带同源臂的引物和相应的模板(见下表5)在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用。
将目的片段和pBR322载体片段用重组酶链接，参考表6。经细菌转化，单菌落 PCR初选，扩增质粒DNA，酶切质粒DNA后跑DNA胶复选，最后测序验证。
用于构建pBR322-PNP质粒的目的片段包括NP,MINI和P。
用于构建pBR322-PDP质粒的目的片段包括P，PD1和PD2，PD3。
用于构建pBR322-LPD3质粒的目的片段包括L1，L2，L3,L4和PD3。
以上3个中间质粒分别图2-4所示，其中，图2为：pBR322-PNP质粒示意图；图3为：pBR322-PDP质粒示意图；图4为pBR322-LPD3质粒示意图。
表5目的片段所涉及的模板和引物表
Figure PCTCN2020096713-appb-000008
Figure PCTCN2020096713-appb-000009
引物C-Sac11BtNhe-ST-R见SEQ ID NO：24；引物C-NP-F见SEQ ID NO：25；引物C-Mini-R见SEQ ID NO：26；引物C-Mini-F见SEQ ID NO：27；引物C-P-R见SEQ ID NO：28；引物C-HindBt-P-F见SEQ ID NO：29；引物C-BtNhe-P-R见SEQ ID NO：30；引物C-P-F见SEQ ID NO：31；引物C-PD1-R见SEQ ID NO：32；引物C-PD1-F见SEQ ID NO：33；引物C-PD2-R见SEQ ID NO：34；引物C-PD2-F见SEQ ID NO：35；引物C-PD3-R见SEQ ID NO：36；引物C-HindBtg-PD3-F见SEQ ID NO：37；引物C-BtNhe-PD3-R见SEQ ID NO：38；引物C-PD3-F见SEQ ID NO：39；引物C-L1-R见SEQ ID NO：40；引物P-L2-R:见SEQ ID NO：41；引物P-L3-F见SEQ ID NO：42；引物C-HindBtNot-L4-F见SEQ ID NO：43；
表6各质粒的构建配方表
Figure PCTCN2020096713-appb-000010
备注：
1.在分别扩增完构建PBR322-PNP质粒,PBR322-PDP质粒,PBR322-LPD3质粒所需要的目的片段后，根据上表6中的重组体系进行混合，之后按照同源重组的步骤进行操作，构建相应质粒。
2.由PBR322-DHN3质粒构建策略可知，病毒基因组中包含两个BtgZ Ⅰ酶切位点，根据这两个酶切位点将病毒全基因组分成3个大片段，此即我们构建PBR322-PNP,PBR322-PDP,PBR322-LPD3三个质粒中所包含的三个大片段。同时在对病毒基因组进行基础测序时有将病毒全基因组分成10个小片段(NP,P,PD1,PD2,PD3,L1,L2,L3,L4),BtgZ Ⅰ酶切位点刚好处于P片段和PD3片段上，故在构建PBR322-PNP,PBR322-PDP,PBR322-LPD3质粒时扩增P片段和PD3时以P左，P右，PD3左，PD3右来代称。
3.由于L蛋白是NDV病毒的核衣壳蛋白，所以在完成质粒PMD19-L1,PMD19-L2,PMD19-L3,PMD19-L4后首先构建了质粒PXJ40-L，用作拯救病毒的辅助性蛋白。故而在构建PBR322-LPD3质粒时使用的模板之一是PXJ40-L，此质粒已经包括了L基因的全部序列。
在构建PBR322-LPD3过程中将L基因分作2段扩增，称之为L1-2,L3-4,(其中，L1-2是指小片段L1和L2的连续的序列，其由引物C-L1-R、P-L2-R，模板pXJ40-L构建得到，L3-4是指小片段L3和L4的连续序列，其由引物P-L3-F、C-HindBtNot-L4-F，模板pXJ40-L构建得到)；并且需要指出的是在扩增L3-4的引物C-HindBtNot-L4-F中包含构建PBR322-DHN3质粒时所需的同源区以及一个BtgZ Ⅰ酶切序列，这样便可以使BtgZ Ⅰ将PBR322-LPD3中的病毒基因组序列完整切下，并且切下的片段直接用于PBR322-DHN3质粒的构建。
4.上表6中PBR322-PNP质粒,PBR322-PDP质粒,PBR322-LPD3质粒的构建过程中所涉 及到的PCR扩增反应使用到的酶全部为PrimeSTARGXLDNAPolymerase(R050,TAKARA),反应体系参考前面所述。根据此酶R050的特性，在扩增反应中预变性温度和时间均为95℃，3min,变性温度和时间为98℃，10s,退火温度和时间使用的均为60℃，15s,延伸的温度均为68℃延伸时间则根据片段大小按照1000bp延伸60s的比例适当选择。反应体系均为30μl。
5.本发明中涉及到使用同源重组方法的实验中，使用的试剂盒均为ClonExpressMultisOneStepCloningKit(C113，诺维赞)，将不再赘述。
上表中，模板质粒pXJ40-L的构建方法为：
通过设计4对带同源重组序列的特异性引物(具体见表7)，分别以pMD9-L1，pMD9-L2，pMD9-L3和pMD9-L4为模板，扩增出能用于将L完整序列克隆进pXJ40载体的片段L1，L2,L3和L4。
L1的5’末端同时带有与pXJ40BamH1端同源的同源臂，L4的3’末端则带有与pXJ40Pst1端同源的同源臂。
将载体pXJ40用BamH1和Pst1双酶切，再通过胶纯化回收。将上述4个片段L1，L2,L3和L4和BamH1和Pst1双酶切后的pXJ40加在一起，在重组酶(ClonExpressMultisOneStepCloningKit)的作用下作同源重组。将重组产物转化进DH5α细胞，经单菌落PCR筛选。用引物PXJ40-F(pXJ40-F:gcaacgtgctggttattgtg，见SEQ ID NO：44)/P-L1-R(P-L1-R:ggacagttgactcattgctaacata，见SEQ ID NO：46)扩增出正确的PCR产物为2110bp，参考图9；用引物P-L3-F/P-L3-R扩增出正确的PCR产物为2027bp，参考图10。
图9-10显示证明检测8个菌落，8个阳性菌均产生了正确分子量的PCR产物。
进一步将其中一株菌培养扩增，提取质粒DNA。再经BamH1和Pst1双酶切，正确的酶 切产物应为6230bp和5081bp。实验结果如下图11所示，证实该质粒含正确分子量的DNA片段。
表7用于建立pXJ40-L的PCR引物
Figure PCTCN2020096713-appb-000011
引物P-L1-BamH1-F见序列表SEQ ID NO：45；引物P-L1-R见序列表SEQ ID NO：46；引物P-L2-F见序列表SEQ ID NO：47；引物P-L2-R见序列表SEQ ID NO：48；引物P-L3-F见序列表SEQ ID NO：49；引物P-L3-R见序列表SEQ ID NO：50；引物P-L4-F见序列表SEQ ID NO：51；引物P-L4-Pst1-R见序列表SEQ ID NO：52；
表8质粒pXJ40-L构建配方表
Figure PCTCN2020096713-appb-000012
备注：1.构建PXJ40-L质粒时扩增各片段时使用的是R050高保真酶，其退火温度均为60℃，延伸时间计算方法及扩增体系均按照前面R050酶的说明计算。
2.鉴定质粒PXJ40-L时，使用引物PXJ40-F和P-L1-R鉴定，使用的是RR902酶。配制反应液体系反应程序及延伸时间计算均按照前面所述，此对引物的退火温度为57℃。
3.本发明所用的载体质粒pXJ40来源于华南农业大学的群体微生物中心刘定祥教授实验室，其质粒的结构示意图如图15，图15所示的pXJ40-NEW即为pXJ40；其序列见序列表SEQ ID NO：60；本领域普通技术人员可根据该序列表通过人工序列合成方法得到。华南农业大学的群体微生物中心刘定祥教授实验室可提供如图15所示的质粒的结构图示的载体质粒。
同时，载体质粒pXJ40也可以通过本领域其他商业化的质粒转换得到，具体来说，采用商品质粒pXJ40-flag可以制备得到本实施例所用的pXJ40，其改建原理为：为了避免Flag对病毒蛋白功能的影响；我们从商品化的质粒pXJ40-Flag经过改建去除Flag获得pXJ40-New。
pXJ40-flag的来源渠道有至少以下两种；
渠道一、实验室保存；pXJ40-flag保存于华南农业大学的群体微生物中心刘定祥教授实验室，华南农业大学的群体微生物中心刘定祥教授实验室可提供pXJ40-flag质粒；pXJ40-flag在刘定祥教授发表的文献中有记载，具体记载为“pXJ40F(with a Flag tag)”，该文献出处为：Journal of virology,Dec.2009,p.12462-12472，《Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells》作者廖瑛、王晓星、刘定祥。
渠道二、从普如汀生物技术(北京)有限公司购买，货号为pXJ40-flag。
具体制备步骤为:
S11：制备质粒片段：2ug pXJ40Flag+1ul EcoR1+1ul Bsa1+5ulBuffer加水至50ul，37℃孵育2小时；胶回收2998bp片段备用；
S12：PCR扩增无Flag片段：以2ng pXJ40Flag为模板，用引物dFlagF:actatagggcgaattcggatccaagcttctcg(见序列表SEQ ID NO：53)/Bsa1R:tgagcgtgggtctcgcggt(见序列表SEQ ID NO：54)，高保真DNA聚合酶(TAKARA公司R050)通过PCR扩增1322bp片段(退火温度60度，延伸时间1分30秒)，胶回收备用。
S13：通过同源重组的方法用ClonExpress Multis One Step Cloning Kit(诺维赞C113)，上述步骤1得到的2998bp片段50ng,上述步骤2得到的1322bp片段22ng,buffer 4ul,酶2ul，加水至20ul；37℃孵育30分钟。
S14：将上述重组产物转化进DH5α(常规方法，TAKARA)，氨苄青霉素培养筛选。常 规制备质粒后用引物pXJ40R:agcggaagagtctagagtcg(见序列表SEQ ID NO：55)进行测序验证。含正确序列(无Flag序列)者为pXJ40-New质粒，其质粒的结构图示如图15。
步骤3：从pBR322-PNP质粒、pBR322-PDP质粒、pBR322-LPD3质粒上酶切回收片段PNP、片段PDP，以及片段LPD3；并将片段PNP、片段PDP，以及片段LPD3连接得到全长DHN3-A备用；
具体来说，参考表9，pBR322-PNP质粒用BtgZ1和Hind3双酶切后回收片段PNP；
从pBR322-PDP质粒回收片段PDP的方法为：pBR322-PDP质粒用BtgZ1单酶切后回收片段PDP；
从pBR322-LPD3质粒回收片段LPD3的方法为：pBR322-LPD3质粒用BtgZ1单酶切后回收片段LPD3；
表9各质粒片段的回收配方和工艺参数表
Figure PCTCN2020096713-appb-000013
备注：
1.片段PNP,片段PDP,片段LPD3的制备按照上表中的酶切体系及时间操作；酶切结束后，可取少量作凝胶电泳观察酶切是否完全，如不完全，需适当多加一点酶或延长孵育时间，或二者兼备；
2.片段PNP的制备需要Hind3和BtgZ Ⅰ两个酶的切割。需要指出的是这两个酶切反应是分别进行的，不是同时进行。即先使用Hind3酶切，琼脂糖电泳胶回收到目的产物后再使用BtgZ Ⅰ酶切。所以上表中的PBR322-PNP’指的是Hind3酶切后的产物。
3.制备含0.03％全式金染色液的0.6％琼脂(TsingKe,TSJ001)凝胶。取0.6微克琼脂加100毫升TAE缓冲液置微波炉加热1-2分钟至琼脂完全融化，待冷却至50℃左右加全式金染色液(按1：3000倍稀释)混匀后铺胶。注意染色剂加多了可能抑制DNA片段的链接。将上述的酶切产物全部经上述的凝胶电泳分离。将凝胶置一清洁的玻璃平板上，用一次性不锈钢刀片将正确的目的片段切下回收。用GelExtractionKit(D2500-02，OMEGA)按试剂盒提供的方法进行纯化。
所述片段PNP、片段PDP、片段LPD3通过高浓度T4链接酶作体外连接，获得全长DHN3-A。具体参数参考表10。
表10 PBR322-DHN3质粒的连接配方表
Figure PCTCN2020096713-appb-000014
备注：
1.片段PNP,片段PNP,片段LPD3按照上表的量计算使用，并连接。
2.将连接产物直接进行试剂盒
(MiniBESTDNAfragmentpurificationkitVersion4.0，TAKARA，9761)纯化，纯化后 的产物跑琼脂糖电泳胶看是否连接完全。
步骤4：用上述pBR322-Base质粒为模板，使含同源臂配对碱基的特殊引物(A2-F：atcggtagaaggttccctcaggttc(见序列表SEQ ID NO：56)；A2-R：ggtcctatagtgagtcgtattaatg(见序列表SEQ ID NO：57))，用PrimerstarGXL试剂盒提供的材料并按照试剂盒提供的方法进行PCR扩增。
用上述纯化试剂盒(MiniBESTDNAfragmentpurificationkitVersion4.0，TAKARA，9761)，按试剂盒提供的方法进行对本步骤的PCR扩增产物进行纯化，制得带同源臂的质粒片段。
步骤5：将步骤4得到的质粒片段pBR322-Base与DHN3-A进行同源重组得到全基因组表达载体pBR322-DHN3。
具体来说，通过使用购自诺维赞重组试剂盒(ClonExpressMultisOneStepCloningKit，C113)来进行。取上述带同源臂的质粒片段、上述DHN3全基因组DNA片段(即上述体外连接产物，DHN3-A)按诺维赞重组试剂盒提供的方法进行同源重组，具体配方参数可参考表11。
表11质粒片段与DHN3-A同源重组的配方表
试剂 用量
DHN3-A 200ng
pBR322-Base 91.02ng
5×CE MultiS Buffer 4μl
Exnase MultiS 2μl
ddH 2O up to 20μl
步骤6：将重组产物转化入DH5a感受态细胞，用特异性引物(C-pBR322-R：gaaattgcatcaacgcatatagcgc(见序列表SEQ ID NO：58)；C-NP-F：catctggttgcccttgcggcttgttc(见序列表SEQ ID NO：59))作单菌落PCR，获得3个阳 性菌落A1-A3，参考图5，图5为菌落A1的质粒A1-A3的电泳图。
将菌落A1用Sac1酶切作初步鉴定，正确的酶切产物应是1个8482bp,1个7065bp,1个3132bp,1个908bp,和1个32bp的片段；结果如图6，图6为质粒A1的电泳图,显示该酶切产物与序列推断的结果一致。32bp片段太小无法在琼脂电泳胶中显示。命名该质粒为pBR322-DHN3。
将此质粒再次测序，验证无误后扩增备用。
pBR322-DHN3的质粒的结构图示如图7所示；
pBR322-DHN3的质粒的结构图示如图8所示。
通过上述描述可以得知，本发明解决了大基因组不易人工合成的难题，避免了随机突变发生。
引用文献：
1.Peeters B.P.H,etal 1999.Rescue of Newcastle disease virus from cloned cDNA:evidence of that cleavability of the fusion protein is a major determinant for virulence.Journal of virology 1999,73(6):5001-9.
2.Huang,et al.The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence.Journal of virology 2004,788(8):4176-84.
3.Metabson et al.Newcastle disease virus(NDV)marker vaccine:An immunodominant epitope on the nucleoprotein gene of NDV can be deleted or replaced by a foreign epitope.Journal of virology 2002,76(20):10138-46.
4.Man-Seong.P,etal Engineered viral vaccine constructs with dual specificity:Avian Influenza and Newcastle disease.Proceedings of the National Academy of Sciences.103(21):8203-8206.
5.Abozeid,et al.Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains  circulating in Egypt.Vet Res(2019)50:12
6.葛金英 等2008，表达传染性法氏囊病毒VP2基因的重组新城疫LaSota弱毒疫苗株的构建。中国农业科学200841(1):243-251.
7.Chen H,Bu.Z.Development and application of avian of influenza vaccines in china.(J)Current Topics in Microbiology&immunology.2009,333(333):153-162.
8.胡海霞 表达C亚型禽肺病毒糖蛋白和融合蛋白的新城疫病毒的二价苗的构建及免力的评价(D)吉林大学2011
9.Patti,et al.Comparison of Viral Shedding following Vaccination with Inactivated and Live Newcastle Disease Vaccines Formulated with Wild-Type and Recombinant Viruses.
Avian Diseases,Vol.53,No.1(Mar.,2009),pp.39-49
Jacqueline et al.Sequence variation in the Newcastle disease virus genome.Virus Research 116(2006)168–184
10.Mark G.et al.RNA-dependent RNA polymerase gene analysis of worldwide Newcastle
disease virus isolates representing different virulence types and their phylogenetic relationship with other members of the paramyxoviridae.Virus Research 104(2004)71–80
11.Masaji Mase,etal.Phylogenetic Analysis of Newcastle Disease Virus Genotypes
Isolated in Japan JOURNAL OF CLINICAL MICROBIOLOGY,Oct.2002,p.3826–3830
Hyuk-Joon Kwon,et al.Molecular epidemiology of Newcastle disease in Republic of Korea.Veterinary Microbiology 95(2003)39–48
12.ZHILIANG WANG,et al.Genotyping of Newcastle Disease Viruses Isolated from  2002 to 2004 in China Ann.N.Y.Acad.Sci.1081:228–239(2006)
13.Francisco Perozo,et al.Biological and Phylogenetic Characterization of a Genotype VII
Newcastle Disease Virus from Venezuela:Effificacy of Field Vaccination.Journal of Clinical Microbiology p.1204–1208
Eun-Kyoung Lee et al.Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea.Veterinary Microbiology 134(2009)241–248。

Claims (6)

  1. 一种全基因组表达载体pBR322-DHN3的制备方法，其特征在于：包括如下步骤：
    步骤1：建立pBR322-Base载体，在pBR322载体上引入能够与DHN3全基因组进行同源重组的片段；所述片段上具有与DHN3全基因组的3’末端和5’末端对应的同源臂；
    步骤2：构建过渡载体；所述过渡载体为质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3；所述质粒pBR322-PNP的目的片段包括NP、MINI和P基因；所述质粒pBR322-PDP的目标片段包括P，PD1和PD2,PD3基因；所述质粒pBR322-LPD3的目标片段包括PD3和L1,L2,L3,L4基因；
    步骤3：构建DHN3全基因组DHN3-A；将质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3进行酶切得到基因片段PNP、PDP和LPD3，将基因片段PNP、PDP和LPD3通过T4链接酶连接得到DHN3全基因组DHN3-A；
    步骤4：构建带同源臂的质粒片段；将步骤1中的pBR322-Base载体为模板进行PCR扩增得到带同源臂的质粒片段；
    步骤5：构建全基因组表达载体pBR322-DHN3；将步骤4中的质粒片段和步骤3中的DHN3全基因组DHN3-A进行同源重组得到具有DHN3全基因组DHN3-A的质粒pBR322-DHN3；
    DHN3全基因组DHN3-A的序列如序列表SEQ ID NO：1；
    MINI基因在序列表SEQ ID NO：1中位置为1414-1949nt；PD1基因在序列表SEQ ID NO：1中位置为2935-4956nt；PD2基因在序列表SEQ ID NO：1中位置为4838-6454nt；PD3基因在序列表SEQ ID NO：1中位置为6261-8283nt；L1基因在序列表SEQ ID NO：1中位置为8166-10709nt；L2基因在序列表SEQ ID NO：1中位置为10174-12299nt；L3基因在序列表SEQ ID NO：1中位置为12238-14433nt；L4基因在序列表SEQ ID NO：1中位置14214-15192nt。
  2. 根据权利要求1所述全基因组表达载体pBR322-DHN3的制备方法，其特征在于： 所述步骤2中pBR322-PNP质粒构建的具体方法为：
    步骤211：将pBR322载体用Hind3和Nhe1作双酶切，然后经胶回收pBR322载体片段备用；
    步骤212：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用；
    其中，模板为质粒pMD19-NP、质粒pMD19-MINI、质粒pMD19-P；
    质粒pMD19-NP对应的引物为C-Sac11BtNhe-ST-R和C-NP-F；
    质粒pMD19-MINI对应的引物为C-Mini-R和C-Mini-F；
    质粒pMD19-P对应的引物为C-P-R和C-HindBt-P-F；
    其中，质粒pMD19-NP为NP基因克隆进pMD19载体中的产物；质粒pMD19--MINI为MINI基因克隆进pMD19载体中的产物；质粒pMD19-P为P基因克隆进pMD19载体中的产物；引物C-Sac11BtNhe-ST-R如序列表SEQ ID NO：1；引物C-NP-F如序列表SEQ ID NO：1；引物为C-Mini-R如序列表SEQ ID NO：1；引物C-Mini-F如序列表SEQ ID NO：1；引物C-P-R如序列表SEQ ID NO：1；引物C-HindBt-P-F如序列表SEQ ID NO：1；
    步骤213：将步骤212所获得的DNA片段和步骤211所获得的pBR322载体片段用重组酶链接，得到pBR322-PNP质粒。
  3. 根据权利要求1所述全基因组表达载体pBR322-DHN3的制备方法，其特征在于：所述步骤2中pBR322-PDP质粒构建的具体方法为：
    步骤221：将pBR322载体用Hind3和Nhe1作双酶切,然后经胶回收pBR322载体片段备用；
    步骤222：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带 同源臂的DNA片段，经跑DNA胶验证后回收备用；
    其中，模板为质粒pMD19-P、质粒pMD19-PD1、质粒pMD19-PD2、pMD19-PD3；
    质粒pMD19-P对应的引物为C-BtNhe-P-R和C-P-F；
    质粒pMD19-PD1对应的引物为C-PD1-R和C-PD1-F；
    质粒pMD19-PD2对应的引物为C-PD2-R和C-PD2-F；
    质粒pMD19-PD3对应的引物为C-PD3-R和C-HindBtg-PD3-F；
    质粒pMD19-PD1为PD1基因克隆进pMD19载体中的产物；质粒pMD19-PD2为PD2基因克隆进pMD19载体中的产物；质粒pMD19-PD3为PD3基因克隆进pMD19载体中的产物；引物C-BtNhe-P-R如序列表SEQ ID NO：1；引物C-P-F如序列表SEQ ID NO：1；引物为C-PD1-R如序列表SEQ ID NO：1；引物C-PD1-F如序列表SEQ ID NO：1；引物C-PD2-R如序列表SEQ ID NO：1；引物C-PD2-F如序列表SEQ ID NO：1；引物C-PD3-R如序列表SEQ ID NO：1；引物C-HindBtg-PD3-F如序列表SEQ ID NO：1；
    步骤223：将步骤222所获得的DNA片段和步骤221所获得的pBR322载体片段用重组酶链接，得到pBR322-PDP质粒。
  4. 根据权利要求1所述全基因组表达载体pBR322-DHN3的制备方法，其特征在于：所述步骤2中pBR322-LPD3质粒构建的具体方法为：
    步骤231：将pBR322载体用Hind3和Nhe1作双酶切，然后经胶回收pBR322载体片段备用；
    步骤232：分别用带同源臂的引物和相应的模板在高保真DNA聚合酶的作用下扩增带同源臂的DNA片段，经跑DNA胶验证后回收备用；
    其中，模板为质粒PMD19-PD3、质粒pXJ40-L；
    质粒PMD19-PD3，对应的引物为C-BtNhe-PD3-R和C-PD3-F；
    质粒pXJ40-L，对应的引物为C-L1-R、P-L2-R、P-L3-F、C-HindBtNot-L4-F；
    质粒PMD19-PD3为PD3基因克隆进pMD19载体中的产物；质粒pXJ40-L为L基因克隆进pXJ40载体中的产物；引物C-BtNhe-PD3-R如序列表SEQ ID NO：1；引物C-PD3-F如序列表SEQ ID NO：1；引物C-L1-R如序列表SEQ ID NO：1；引物P-L2-R如序列表SEQ ID NO：1；引物P-L3-F如序列表SEQ ID NO：1；引物C-HindBtNot-L4-F如序列表SEQ ID NO：1；
    步骤233：将步骤232所获得的DNA片段和步骤231所获得的pBR322载体片段用重组酶链接，得到pBR322-PDP质粒。
  5. 根据权利要求1所述全基因组表达载体pBR322-DHN3的制备方法，其特征在于：所述步骤3中，从pBR322-PNP质粒回收片段PNP的方法为：pBR322-PNP质粒用BtgZ1和Hind3双酶切后回收片段PNP；
    从pBR322-PDP质粒回收片段PDP的方法为：pBR322-PDP质粒用BtgZ1单酶切后回收片段PDP；
    从pBR322-LPD3质粒回收片段LPD3的方法为：pBR322-LPD3质粒用BtgZ1单酶切后回收片段LPD3；
    所述片段PNP、片段PDP、片段LPD3通过高浓度T4链接酶作体外连接，获得全长DHN3-A。
  6. 根据权利要求1所述全基因组表达载体pBR322-DHN3的制备方法，其特征在于：所述步骤1具体为：
    在pBR322质粒上分别引入1个T7启动子，1个T7终止子以及1个HDV Ribozyme；T7启动子下游引入DHN3的3’末端的部分碱基HC1；在HDV Ribozyme的上游引入DHN3的5’末端部分碱基HC2；其中，碱基HC1在DHN3全基因组序列中的位置顺序为15192-15159nt； 碱基HC2在DHN3全基因组序列中的位置顺序为141-1nt。
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Publication number Priority date Publication date Assignee Title
CN110564766A (zh) * 2019-09-20 2019-12-13 华农（肇庆）生物产业技术研究院有限公司 一种全基因组表达载体pBR322-DHN3的制备方法
CN112048484A (zh) * 2020-07-27 2020-12-08 华南农业大学 一株表达传染性法氏囊强毒株vp2蛋白的基因ⅶ型新城疫重组病毒和疫苗

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103451198A (zh) * 2013-08-20 2013-12-18 中国农业科学院哈尔滨兽医研究所 溶瘤型新城疫病毒D90株的全长感染性 cDNA及其构建方法和应用
CN104962526A (zh) * 2013-09-30 2015-10-07 中国农业科学院兰州兽医研究所 Vii型新城疫病毒l基因突变的减毒疫苗株及其制备方法
CN108220227A (zh) * 2017-12-27 2018-06-29 华农（肇庆）生物产业技术研究院有限公司 一种通过全悬浮传代细胞系悬浮培养新城疫病毒的方法
US20180280455A1 (en) * 2013-03-14 2018-10-04 Icahn School Of Medicine At Mount Sinai Newcastle Disease Viruses and Uses Thereof
CN110564766A (zh) * 2019-09-20 2019-12-13 华农（肇庆）生物产业技术研究院有限公司 一种全基因组表达载体pBR322-DHN3的制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1293195C (zh) * 2005-09-02 2007-01-03 中国农业科学院哈尔滨兽医研究所 新城疫LaSota疫苗株反向遗传操作系统及其应用
CN101205544B (zh) * 2007-08-07 2010-12-08 中国人民解放军第四军医大学 肿瘤靶向性重组新城疫病毒及其构建方法
CN103525777B (zh) * 2013-07-01 2016-05-18 中国农业科学院兰州兽医研究所 Vii型新城疫病毒l基因突变的减毒疫苗株及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180280455A1 (en) * 2013-03-14 2018-10-04 Icahn School Of Medicine At Mount Sinai Newcastle Disease Viruses and Uses Thereof
CN103451198A (zh) * 2013-08-20 2013-12-18 中国农业科学院哈尔滨兽医研究所 溶瘤型新城疫病毒D90株的全长感染性 cDNA及其构建方法和应用
CN104962526A (zh) * 2013-09-30 2015-10-07 中国农业科学院兰州兽医研究所 Vii型新城疫病毒l基因突变的减毒疫苗株及其制备方法
CN108220227A (zh) * 2017-12-27 2018-06-29 华农（肇庆）生物产业技术研究院有限公司 一种通过全悬浮传代细胞系悬浮培养新城疫病毒的方法
CN110564766A (zh) * 2019-09-20 2019-12-13 华农（肇庆）生物产业技术研究院有限公司 一种全基因组表达载体pBR322-DHN3的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG YUAN-YUAN, SHAO MENG-YU, YU XIAO-HUI, ZHAO JING, ZHANG GUO-ZHONG: "Molecular characterization of chicken-derived genotype VIId Newcastle disease virus isolates in China during 2005–2012 reveals a new length in hemagglutinin–neuraminidase", INFECTION, GENETICS AND EVOLUTION, ELSEVIER, AMSTERDAM, NL, vol. 21, 1 January 2014 (2014-01-01), NL, pages 359 - 366, XP055793224, ISSN: 1567-1348, DOI: 10.1016/j.meegid.2013.12.003 *

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