WO2021051906A1 - 一种针对ii类vii型流行ndv株dhn3的感染性重组克隆方法 - Google Patents

一种针对ii类vii型流行ndv株dhn3的感染性重组克隆方法 Download PDF

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WO2021051906A1
WO2021051906A1 PCT/CN2020/096711 CN2020096711W WO2021051906A1 WO 2021051906 A1 WO2021051906 A1 WO 2021051906A1 CN 2020096711 W CN2020096711 W CN 2020096711W WO 2021051906 A1 WO2021051906 A1 WO 2021051906A1
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dhn3
plasmid
pbr322
gene
pxj40
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French (fr)
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陈瑞爱
黄梅
王楠楠
李延鹏
刘定祥
叶俊贤
罗琼
杨小云
董楠
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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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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • 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
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    • 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 present invention relates to the technical field of veterinary biological products, in particular to an infectious recombinant cloning method for the type II type VII epidemic NDV strain 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 NDV B1 strain were inserted between the NDV B1 strain, and a hybrid virus that was resistant to both NDV and H7 avian influenza virus (ref4) was successfully obtained.
  • the technical problem to be solved by this application is: how to develop an infectious recombinant cloned virus for the type II type VII epidemic NDV strain DHN3.
  • the purpose of the present invention is to provide an infectious recombinant cloning method for the type II type VII epidemic NDV strain DHN3, which can successfully clone a recombinant virus that is identical to the original virus strain without mutation.
  • an infectious recombinant cloning method for the type II type VII epidemic NDV strain DHN3 which includes the following steps:
  • Step 1 Construct a helper plasmid.
  • helper plasmids There are three kinds of helper plasmids, and the target fragments on the helper plasmid are NP gene, P gene, and L gene respectively;
  • Step 2 Construct a DHN3 full genome expression vector; recombine the DHN3 full genome obtained by artificial recombination into a vector plasmid to obtain a full genome expression vector; the sequence of the DHN3 full genome is shown in the sequence list SEQ ID NO 1;
  • Step 3 Co-transfect the three helper plasmids and the DHN3 whole genome expression vector into BHK-21 cells to obtain a viral solution containing the recombinant virus rDHN3;
  • the position of the NP gene in SEQ ID NO:1 in the sequence list is 1-1591nt; the position of the P gene in SEQ ID NO:1 in the sequence list is 1925-3109nt; the position of the L gene in SEQ ID NO:1 in the sequence list is 8166-15192nt .
  • the vector involved in the helper plasmid in the step 1 is the pXJ40 series plasmid or pcDNA3; the vector plasmid in the step 2 is the pBR322 series The plasmid or pACYC, pBAD series of plasmids.
  • Step 1 Construct helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L.
  • the target fragments of helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L are NP gene, P gene and L gene respectively, and the vector is pXJ40;
  • Step 2 Construct the whole genome expression vector pBR322-DHN3; recombine the artificially recombined DHN3 whole genome into the pBR322 plasmid to obtain the whole genome expression vector pBR322-DHN3; the whole DHN3 genome is the whole genome of the type II VII chicken Newcastle disease virus;
  • Step 3 Co-transfect BHK-21 cells with the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L, and the whole genome expression vector pBR322-DHN3 to obtain a viral solution containing recombinant virus rDHN3.
  • the step 2 specifically is:
  • Step 21 Establish a pBR322-Base vector; introduce a fragment capable of homologous recombination with the entire DHN3 genome into the pBR322 plasmid; the fragment has homology arms corresponding to the 3'end and 5'end of the DHN3 complete genome;
  • Step 22 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 L1, L2, L3, L4 and PD3 genes;
  • Step 23 Construct the whole DHN3 genome DHN3-A; digest the plasmid pBR322-PNP, plasmid pBR322-PDP, and plasmid pBR322-LPD3 to obtain gene fragments PNP, LPD3 and PDP, and pass the gene fragments PNP, LPD3 and PDP through T4 ligase Connect to get the whole DHN3 genome DHN3-A;
  • Step 24 construct a plasmid fragment with homology arms; use the pBR322-Base vector in step 21 as a template to perform PCR amplification to obtain a plasmid fragment with homology arms;
  • Step 25 Construct the whole genome expression vector pBR322-DHN3; perform homologous recombination of the plasmid fragment with homology arms in step 24 and the DHN3 whole genome DHN3-A in step 23 to obtain the plasmid pBR322 with DHN3 whole genome DHN3-A -DHN3;
  • 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 step 3 specifically is:
  • BHK-21 cells were co-transfected with a plasmid capable of expressing T7 RNA polymerase, auxiliary plasmids pXJ40-NP, pXJ40-P, pXJ40-L, and a genome-wide expression vector pBR322-DHN3; a viral solution containing recombinant virus rDHN3 was obtained.
  • the step 21 specifically is:
  • the step 22 specifically is:
  • Step 221 Preparation of pBR322 plasmid fragment: The pBR322 plasmid was digested with Hind3 and Nhe1, and then recovered by gel for use;
  • Step 222 preparing target fragments; the target fragments are NP, MINI, P, PD1, PD2, PD3, L1, L2, L3, L4 genes;
  • Step 223 Link the pBR322 plasmid fragment and the corresponding target fragment with recombinase to obtain the corresponding plasmid pBR322-PNP, plasmid pBR322-PDP, and plasmid pBR322-LPD3.
  • step 23 is:
  • Plasmid pBR322-PDP and plasmid pBR322-LPD3 were respectively digested with BtgZ1 to recover gene fragments LPD and PDP; vector pBR322-PNP was digested with BtgZ1 and Hind3 to recover gene fragments PNP;
  • the gene fragment LPD, gene fragment PDP, and gene fragment PNP were connected in vitro by T4 linkase to obtain DHN3 whole genome DHN3-A.
  • the method of the present invention can be successfully cloned from a recombinant virus with the same original virus strain without mutation.
  • the whole genome of DHN3 has a total of 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 generally available on the market, and the other is easy to introduce during PCR Random mutation. 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.
  • the present invention adopts the auxiliary plasmids pXJ40-NP, pXJ40-P and pXJ40-L, the whole genome expression vector pBR322-DHN3 to prepare the virus liquid, the main reason is: because the whole genome expression vector pBR322-DHN3 transfected into the cell is limited, It is not possible to synthesize enough structural and functional proteins in cells in a short period of time; co-transfection of helper genes can provide the necessary components for virus replication and assembly in a short period of time.
  • Figure 1 is an alignment diagram of the sequencing results of Example 1 of the present invention with all published NDV sequences.
  • Fig. 2 is a micrograph showing that BHK-21 cells infected with NDV will have obvious cell fusion phenomenon according to the first embodiment of the present invention
  • Figure 3 is a diagram of the relationship between the L gene and the sequencing plasmids pMD19-L1, pMD19-L2, pMD19-L3, and pMD19-L4 in Example 1 of the present invention;
  • Fig. 4 is an electrophoresis diagram of PCR products of the helper plasmid pXJ40-L in Example 1 of the present invention
  • Fig. 5 is an electrophoresis diagram of PCR products of the helper plasmid pXJ40-L in Example 1 of the present invention
  • Fig. 6 is a restriction electrophoresis diagram of the helper plasmid pXJ40-L in Example 1 of the present invention.
  • Figure 7 is a schematic diagram of the plasmid pXJ40-L of Example 1 of the present invention.
  • Figure 8 is a schematic diagram of the plasmid pXJ40-P of Example 1 of the present invention.
  • Figure 9 is a schematic diagram of the plasmid pXJ40-NP of Example 1 of the present invention.
  • Fig. 10 is a schematic diagram of a plasmid of the pBR322-Base vector of Example 1 of the present invention.
  • FIG. 11 is a schematic diagram of the plasmid pBR322-PNP of Example 1 of the present invention.
  • Figure 12 is a schematic diagram of the plasmid pBR322-PDP of Example 1 of the present invention.
  • Figure 13 is a schematic diagram of the plasmid pBR322-LPD3 of Example 1 of the present invention.
  • Fig. 14 is a schematic structural diagram of a full-length DHN3-A according to the first embodiment of the present invention.
  • Fig. 16 is a restriction electrophoresis diagram of the whole genome expression vector pBR322-DHN3 in Example 1 of the present invention.
  • FIG. 17 is a schematic diagram of a plasmid of the whole genome expression vector pBR322-DHN3 of Example 1 of the present invention.
  • FIG. 18 is a schematic diagram of a plasmid of the whole genome expression vector pBR322-DHN3 of Example 1 of the present invention.
  • Fig. 19 is a graph showing the growth characteristics of DHN3 virus, rDHN virus and blank control in Example 1 of the present invention.
  • Figure 20 is a PCR colony electrophoresis diagram of the pXJ40DE3 plasmid of Example 1 of the present invention.
  • 21 is a schematic diagram of a plasmid of the pBR322 plasmid fragment of Example 1 of the present invention.
  • Figure 22 is a schematic diagram of the plasmid pXJ40 of Example 1 of the present invention.
  • Figure 23 is a schematic diagram of the plasmid pXJ40-DE3 of 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, and TIAN prep Mini Plasmid Kit (DP103-03) was purchased from Tiangen Co., Ltd.
  • Gel Extraction Kit (D2500-02) was purchased from OMEGA; M-MLVRT (2641A) was purchased from TAKARA; RRI (2313A) was purchased from TAKARA; Random 6 Primer (3801) was purchased from TAKARA; Agarose (E0301) was purchased from TSINGK; 0.25% Trypsin-EDTA (25200-056), DMEM basic (C11995500BT) was purchased from Gibco; Lipofectamine LTX and Plus Reagent (15338-100) was purchased from Invitrogen; FBS (10099-141C) was purchased from Gibco; Premix-Taq (RR902A) was purchased from TAKARA; Pen Strep penicillin Streptomycin (15140-122) was purchased from Gibco; Primerstar GXL (R
  • 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
  • the NP, P and L genes were cloned into the pXJ40 vector to obtain the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L.
  • the pXJ40 plasmid contains CMV promoter and SV40 polyadenine A signal fragment, so foreign genes can be effectively transcribed under the action of cell DNA polymerase II, and then translated into proteins NP, P, and L used to assemble active virus particles protein.
  • the aforementioned sequencing plasmids pMD19-L1, pMD19-L2, pMD19-L3 and pMD19-L4 cover the complete DHN3 L gene, as shown in Figure 3 below.
  • the vector pXJ40 was digested with BamH1 and Pst1, and then recovered by gel purification.
  • the recombinant product was transformed into DH5 ⁇ cells and screened by single colony PCR.
  • the correct PCR product amplified was 2027bp.
  • Figures 4 and 5 prove 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: 26; primer P-L2-F see SEQ ID NO: 27; primer P-L2-R see SEQ ID NO: 28; primer P-L3-F see SEQ ID NO: 29; primer P-L3-R see SEQ ID NO: 30; primer P-L4-F see SEQ ID NO: 31; primer P-L4-Pst1-R see SEQ ID NO: 32.
  • pXJ40-NP pXJ40-P was constructed using a common ligase ligation transformation method, not the homologous recombination method used.
  • the pXJ40 plasmid was digested with EcoR I and Xho I, and the cut fragment (4288bp) was used as the plasmid fragment for constructing pXJ40-NP and pXJ40-P.
  • NP-PXJ-0F2/NP-PXJ-1591R2 Use NP-PXJ-0F2/NP-PXJ-1591R2 (see Table 9) to amplify NP fragments (R050, 60°C annealing temperature, 1min30s extension time).
  • P-PXJ-1083F2/P-PXJ-3080R2 was used to amplify the P gene (R050, annealing at 60°C, extension for 1min15s).
  • primer P-PXJ-0F2 and primer P-PXJ-1083F2 contain the restriction sequence of EcoR I; primer NP-PXJ-1591F2 and primer P-PXJ-3080R2 contain Xho I sequence.
  • the amplified product contains EcoR I and Xho I restriction sites at both ends.
  • the products amplified using these two pairs of primers are then double-enzyme digested with EcoR I and Xho I, so that the digested products will contain the sticky ends of EcoR I and Xho I.
  • the R050 high-fidelity enzyme was used to amplify each fragment, and the annealing temperature was 60°C.
  • the extension time calculation method and amplification system were calculated according to the previous R050 enzyme instructions.
  • Table 7 shows the digestion process of plasmid pXJ40 and the digestion process of NP and P gene amplification products.
  • Table 8 shows the ligation process between the digested products of NP and P genes and the digested products of pXJ40 plasmid. It should be pointed out that the PCR products and digestion products of NP and P genes and the digestion products of pXJ40 plasmid are all recovered by agarose gel electrophoresis before proceeding to the next step.
  • the P gene is inserted between EcoR1 and Xho1 of pXJ40; the NP gene is inserted between EcoR1 and Xho1 of pXJ40. See plasmid schematics 8 and 9.
  • Primer P-PXJ-1893F2 see SEQ ID NO: 33; primer P-PXJ-3080R2 see SEQ ID NO: 34; primer NP-PXJ-0F2 see SEQ ID NO: 35; primer NP-PXJ-1591R2 see SEQ ID NO: 36;
  • pXJ40 The vector plasmid pXJ40 is mentioned above and below in many places.
  • pXJ40 is a very widely used vector plasmid, which has abundant restriction sites, and pXJ40 is used in the construction of a large number of plasmids.
  • 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 plasmid schematic diagram is shown in Figure 22.
  • the pXJ40-NEW shown in Figure 22 is pXJ40, and its sequence is shown in the sequence table SEQ ID NO: 66 ; 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 plasmid structure diagram of Figure 22.
  • 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.
  • Channel 2 Purchased from Purutin Biotechnology (Beijing) Co., Ltd., the article number is pXJ40-flag.
  • Step 1 Prepare plasmid fragments: 2ug pXJ40Flag+1ul EcoR1+1ul Bsa1+5ulBuffer add water to 50ul, and incubate at 37°C for 2 hours; the 2998bp fragment is recovered by the gel for use;
  • Step 2 PCR amplification of fragments without Flag: Using 2ng pXJ40Flag as template, primers dFlagF:ACTATAGGGCGAATTCGGATCCAAGCTTCTCG/Bsa1R:TGAGCGTGGGTCTCGCGGT, high-fidelity DNA polymerase (TAKARA company R050) amplified 1322bp fragment (annealing temperature 60 degrees, extension time 1 minute 30 seconds), the glue is recycled for use.
  • TAKARA company R050 high-fidelity DNA polymerase amplified 1322bp fragment
  • Step 3 Use ClonExpress Multis One Step Cloning Kit (Novezin C113) by homologous recombination, the 2998bp fragment obtained in step 1 above is 50ng, the 1322bp fragment obtained in step 2 above is 22ng, buffer 4ul, enzyme 2ul, add water to 20ul ; Incubate at 37°C for 30 minutes.
  • Step 4 Transform the above-mentioned recombinant product into DH5 ⁇ (conventional method, TAKARA), culture and screen ampicillin.
  • TAKARA conventional method, TAKARA
  • the primer pXJ40R:AGCGGAAGAGTCTAGAGTCG was used for sequencing verification.
  • the plasmid containing the correct sequence is the pXJ40-New plasmid.
  • pBR322-Base a basic plasmid that can obtain single-stranded negative-strand RNA of the whole genome.
  • a T7 promoter, a T7 terminator, and a HDV Ribozyme were introduced into the pBR322 plasmid, so that they can finally be externalized. Under the action of source T7RNA polymerase, it can accurately synthesize DHN3 full-genome 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. (See Plasmid Diagram 10)
  • the DNA fragment containing the T7 terminator, HDVRibozyme, HC2, HC1, and T7 promoter was 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 10 ).
  • the whole genome of DHN3 has a total of 15192nt. If the full-length cDNA is obtained by RT-PCR at one time, it is difficult (generally commercially available DNA polymerases are difficult to synthesize DNA fragments larger than 10k), and the other is easy to use in the PCR process. Introduce random mutations in. 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 (as shown in Figure 21) was digested with Hind3 and Nhe1, and then recovered by gel for use.
  • Target gene fragments use primers with homology arms and corresponding templates (see Table 11 below) to amplify DNA fragments with homology arms under the action of high-fidelity DNA polymerase, and run DNA gel to verify Recycle for spare.
  • 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 11 is a schematic diagram of pBR322-PNP plasmid
  • Figure 12 is a schematic diagram of pBR322-PDP plasmid
  • Figure 13 is a schematic diagram of pBR322-LPD3 plasmid.
  • primer C-Sac11BtNhe-ST-R see SEQ ID NO: 37; for primer C-NP-F, see SEQ ID NO: 38; for primer C-Mini-R, see SEQ ID NO: 39; for primer C-Mini-F, see SEQ ID NO: 40; primer CPR see SEQ ID NO: 41; primer C-HindBt-PF see SEQ ID NO: 42; primer C-BtNhe-PR see SEQ ID NO: 43; primer CPF see SEQ ID NO: 44; primer C -PD1-R see SEQ ID NO: 45; primer C-PD1-F see SEQ ID NO: 46; primer C-PD2-R see SEQ ID NO: 47; primer C-PD2-F see SEQ ID NO: 48; See SEQ ID NO: 49 for primer C-PD3-R; See SEQ ID NO: 50 for primer C-HindBtg-PD3-F; See SEQ ID NO: 51 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 them according to the recombination system in Table 12 above, and then follow the steps of homologous recombination. Construct the corresponding plasmid.
  • the virus genome contains two BtgZI restriction sites. According to these two restriction sites, the whole virus gene component is divided into 3 large fragments. This means we construct PBR322-PNP, PBR322 -PDP, PBR322-LPD3 three large fragments contained in the three plasmids.
  • the entire viral 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 PD3 Therefore, when constructing PBR322-PNP, PBR322-PDP, and PBR322-LPD3 plasmids, when amplifying P fragment and PD3, they are 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, the template pXJ40-L was constructed
  • 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 was constructed
  • the primer C-HindBtNot-L4-F for the amplification of L3-4 contains the homology region required for the construction of the PBR322-DHN3 plasmid and a BtgZI digestion sequence, so that it can be
  • the viral genome sequence in PBR322-LPD3 was completely excised by BtgZI, and the excised fragments were 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 the above table 13 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.
  • DHN3-A fragment Recover fragment PNP, fragment PDP, and fragment LPD3 from pBR322-PNP plasmid, pBR322-PDP plasmid, pBR322-LPD3 plasmid, and ligate fragment PNP, fragment PDP, and fragment LPD3 to obtain the full length DHN3-A is spare.
  • the pBR322-PNP plasmid was double digested with BtgZ1 and Hind3 to recover the fragment PNP;
  • 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;
  • fragment PNP requires the cleavage of two enzymes Hind3 and BtgZI. It should be pointed out that these two enzyme digestion reactions are carried out separately, not at the same time. That is, use Hind3 enzyme digestion first, and then use BtgZI digestion after the agarose electrophoresis gel to recover the target product. Therefore, pBR322-PNP' in the above table refers to the product of Hind3 digestion.
  • the fragment PNP, fragment PDP, and fragment LPD3 are connected in vitro by high-concentration T4 linkase to obtain full-length DHN3-A.
  • Table 14 for specific parameters.
  • the plasmid structure of full-length DHN3-A is shown in Figure 14;
  • Fragment PNP, Fragment PNP, Fragment LPD3 are calculated and used according to the amount in the above table, and connected.
  • This plasmid fragment and DHN3-A were homologously recombined to obtain the whole genome expression vector pBR322-DHN3.
  • a recombination kit (ClonExpress Multis One Step Cloning Kit, C113) purchased from Novezin. Take the above-mentioned plasmid fragment with homology arms and the above-mentioned DHN3 whole genome DNA fragment (ie 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 15 for specific formula parameters.
  • the recombinant product was transformed into DH5a competent cells.
  • C-pBR322-R C-pBR322-R: GAAATTGCATCAACGCATATAGCGC
  • C-NP-F C-NP-F: CATCTGGTTGCCCTTGCGGCTTGTTC
  • 3 positive colonies A1-A3 were obtained, as shown in Figure 15.
  • primer C-pBR322-R see SEQ ID NO: 59
  • primer C-NP-F see SEQ ID NO: 60.
  • the colony A1 was amplified and cultured and plasmid DNA was extracted.
  • the A1 plasmid was digested with Sac1 for preliminary identification.
  • the correct digested product should be 1 8482bp, 1 7065bp, 1 3132bp, 1 908bp, and 1 32bp fragment.
  • the result is shown in Figure 16, which 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.
  • the plasmid structure diagram of plasmid pBR322-DHN3 is shown in Figure 17; the plasmid structure diagram of plasmid pBR322-DHN3 is shown in Figure 18.
  • BHK-21 cells were placed in a 30mm petri dish and cultured overnight to reach 80-90% full.
  • After 4 days place the petri dish in the -20 refrigerator for 2 freeze-thaw cycles, collect the cells and culture solution in a sterile centrifuge tube, centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant. Then 100 ⁇ l of the supernatant (containing rDHN3) was continued to infect BHK-21 cells, and cell fusion lesions were visible after 2-3 days.
  • the virus rDHN3P1 can be inoculated with SFP chicken embryos, and the allantoic fluid can be collected to obtain the virus rDHN3.
  • transfection steps involve the dosage of each substance in the transfection solution as follows:
  • Transfection solution includes A solution and B solution
  • Liquid A Opti-MEM 150ul
  • Transfection was performed according to the method provided by Lipofectamine LTX and Plus Reagent (15338-100), Invitrogen.
  • N There is HN on the surface of the Newcastle disease virus envelope, which can adsorb red blood cells and cause agglutination of red blood cells in some animals. When the concentration of Newcastle disease virus is high, it can further cause red blood cell lysis. According to this characteristic of Newcastle disease virus, this experiment can be used to detect whether there is Newcastle disease virus in the solution to be tested, and the content of the virus.
  • the following red blood cell coagulation experiment proves that the above rDHN3 recombinant virus has the same blood coagulation function as the mother strain. The highest coagulation titer of both is 28 .
  • the pXJDE3 plasmid involved above is as follows:
  • the pXJ40DE3 plasmid is a T7 RNA polymerase (DE3) expression plasmid.
  • the DE3 gene was obtained from E. coli BL21 (DE3) (Merck) by using primers BamH1-DE3-F/BamH1-DE3-R. After homologous recombination, it was inserted into the BamH1 site of plasmid pXJ40; the plasmid structure diagram of pXJ40DE3 plasmid is shown in Figure 23.
  • Step 1 Extract E. coli BL21 (DE3) DNA: conventional bacterial DNA extraction methods.
  • Step 2 PCR preparation of DE3 fragment: using E. coli BL21 (DE3) bacterial DNA as a template, using high-fidelity DNA polymerase (GXL) to pass primers:
  • BamH1-DE3-F actcactatagggcgaattcggatccgccatgaacacgattaacatcgc;
  • BamH1-DE3-R taagatctggtaccgagctcctgcagttacgcgaacgcgaagtccgactc for amplification.
  • GXL enzyme (R050), annealing temperature 60°C, extension time 2 minutes 36 seconds. The product was verified by gel electrophoresis and purified for later use.
  • Step 3 Preparation of the vector: pXJ40 was digested with Pst I and BamH I, and then recovered by the gel for use. Refer to Table 16 for the specific formula and process.
  • Step 4 Recombinate the DE3 fragment into the pXJ40 vector.
  • the specific process and formula are shown in Table 17.
  • Step 5 Transform the recombinant product into DH5 ⁇ by culturing on a plate containing 50ug/ul ampicillin. On the next day, 8 colonies were selected from the plate for PCR verification.
  • the rescued virus rDHN3 was used to infect BHK-21 cells through chicken embryos for 3 times, and the total RNA of the cells was extracted when the cytopathic changes were obvious.
  • FIG. 19 which is divided into three columns, A, B, and C.
  • Column A represents the morphology of BHK-21 cells after 12 hours of infection with DHN3 virus
  • column B represents the morphology of BHK-21 cells after 12 hours of infection with rDHN3
  • the arrow in the figure indicates the fusion lesion caused by DHN3 or rDHN3 infection of BHK-21 cells
  • DHN3 refers to the virus after virus isolation, blind transmission, and plaque purification on BHK-21 cells; this virus is involved in the sequencing and analysis and classification steps.
  • rDHN3 refers to a recombinant virus, that is, a virus rescued by recombinant cloning in the text.
  • both the recombinant virus rDHN3 and its parent virus DHN3 can infect BHK-21 to form cell fusion.
  • NDV Newcastle disease virus

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Abstract

属于兽用生物制品技术领域，公开了一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法，包括如下步骤：步骤1：构建辅助质粒，所述辅助质粒为三种，辅助质粒上的目标片段分别为NP基因、P基因、L基因；步骤2：构建DHN3全基因组表达载体；将人工重组得到的DHN3全基因组重组入载体质粒中得到全基因组表达载体；DHN3全基因组的序列如序列表SEQ ID NO 1；步骤3：将三种辅助质粒和DHN3全基因组表达载体共转染BHK-21细胞，得到含重组病毒rDHN3的病毒液；NP基因在序列表SEQ ID NO：1中位置为1-1591nt；P基因在序列表SEQ ID NO：1中位置为1925-3109 nt；L基因在序列表SEQ ID NO：1中位置为8166-15192nt。该方法可以成功的克隆出与原病毒株完全相同、无突变的重组病毒。

Description

一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法 技术领域
本发明涉及兽用生物制品技术领域，特别涉及针对II类VII型流行NDV株DHN3的感染性重组克隆方法。
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NDV B1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IBV S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。
所以，本申请所要解决的技术问题是：如何开发出针对II类VII型流行NDV株DHN3的感染性重组克隆病毒。
发明内容
本发明的目的在于提供一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法，该方法可以成功的克隆出于原病毒株完全相同、无突变的重组病毒。
为了实现上述目的，本发明提供的技术方案是：一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法，包括如下步骤：
步骤1：构建辅助质粒，所述辅助质粒为三种，辅助质粒上的目标片段分别为NP基因、P基因、L基因；
步骤2：构建DHN3全基因组表达载体；将人工重组得到的DHN3全基因组重组入载体质粒中得到全基因组表达载体；DHN3全基因组的序列如序列表SEQ ID NO 1；
步骤3：将三种辅助质粒和DHN3全基因组表达载体共转染BHK-21细胞，得到含重组病毒rDHN3的病毒液；
NP基因在序列表SEQ ID NO：1中位置为1-1591nt；P基因在序列表SEQ ID NO：1中位置为1925-3109nt；L基因在序列表SEQ ID NO：1中位置为8166-15192nt。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤1中的辅助质粒所涉及的载体为pXJ40系列的质粒或pcDNA3；所述步骤2中的载体质粒为pBR322 系列的质粒或pACYC、pBAD系列的质粒。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，包括如下步骤：
步骤1：构建辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L，辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L的目标片段分别为NP基因、P基因、L基因，载体为pXJ40；
步骤2：构建全基因组表达载体pBR322-DHN3；将人工重组得到的DHN3全基因组重组入pBR322质粒中得到全基因组表达载体pBR322-DHN3；所述DHN3全基因组为II类VII型鸡新城疫病毒的全基因组；
步骤3：将辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L、全基因组表达载体pBR322-DHN3共转染BHK-21细胞，得到含重组病毒rDHN3的病毒液。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤2具体为：
步骤21：建立pBR322-Base载体；在pBR322质粒上引入能够与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22：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L1，L2，L3,L4和PD3基因；
步骤23：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、LPD3和PDP，将基因片段PNP、LPD3和PDP通过T4链接酶连接得到DHN3全基因组DHN3-A；
步骤24：构建带同源臂的质粒片段；将步骤21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25：构建全基因组表达载体pBR322-DHN3；将步骤24中的带同源臂的质粒片段和步骤23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；
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。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤3具体为：
用能表达T7RNA聚合酶的质粒与辅助质粒pXJ40-NP、pXJ40-P、pXJ40-L、全基因组表达载体pBR322-DHN3共转染BHK-21细胞；得到含重组病毒rDHN3的病毒液。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤21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。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤22具体为：
步骤221：制备pBR322质粒片段：将pBR322质粒用Hind3和Nhe1作双酶切，然后 经胶回收备用；
步骤222：制备目标片段；所述目标片段为NP、MINI、P、PD1、PD2、PD3、L1、L2,L3,L4基因；
步骤223：将pBR322质粒片段和对应的目标片段用重组酶链接得到对应的质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3。
在上述针对II类VII型流行NDV株DHN3的感染性重组克隆方法中，所述步骤23为：
将质粒pBR322-PDP、质粒pBR322-LPD3分别用BtgZ1单酶切后回收基因片段LPD和PDP；载体pBR322-PNP用BtgZ1和Hind3双酶切回收基因片段PNP；
将基因片段LPD、基因片段PDP、基因片段PNP通过T4链接酶作体外连接，获得DHN3全基因组DHN3-A。
本发明的有益效果在于：
本发明的方法可以成功的克隆出于原病毒株完全相同、无突变的重组病毒。
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。
本发明采用辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L、全基因组表达载体pBR322-DHN3进行病毒液的制备，其主要原因在于：因为转染进细胞的全基因组表达载体pBR322-DHN3有限，在短期内不能在细胞内合成足够的结构和功能蛋白；通过共转染辅助基因可在短期内提供用于病毒复制和组装所必须的成份。
附图说明
图1为本发明的实施例一的测序结果与已发表的所有NDV序列比对图。
图2为本发明的实施例一的(NDV感染的BHK-21细胞会发生明显的细胞融合现象的显微图；
图3为本发明的实施例一的L基因与测序质粒pMD19-L1，pMD19-L2，pMD19-L3和pMD19-L4的关系图；
图4为本发明的实施例一的辅助质粒pXJ40-L的PCR产物的电泳图；
图5为本发明的实施例一的辅助质粒pXJ40-L的PCR产物的电泳图；
图6为本发明的实施例一的辅助质粒pXJ40-L的酶切电泳图；
图7为本发明的实施例一的质粒pXJ40-L的质粒示意图；
图8为本发明的实施例一的质粒pXJ40-P的质粒示意图；
图9为本发明的实施例一的质粒pXJ40-NP的质粒示意图；
图10为本发明的实施例一的pBR322-Base载体的质粒示意图；
图11为本发明的实施例一的质粒pBR322-PNP的质粒示意图；
图12为本发明的实施例一的质粒pBR322-PDP的质粒示意图；
图13为本发明的实施例一的质粒pBR322-LPD3的质粒示意图；
图14为本发明的实施例一的全长DHN3-A的结构示意图；
图15为本发明的实施例一的阳性菌落A1-A3的电泳图；
图16为本发明的实施例一的全基因组表达载体pBR322-DHN3的酶切电泳图；
图17为本发明的实施例一的全基因组表达载体pBR322-DHN3的质粒示意图；
图18为本发明的实施例一的全基因组表达载体pBR322-DHN3的质粒示意图；
图19为本发明的实施例一的DHN3病毒、rDHN病毒和空白对照样的生长特性图；
图20为本发明的实施例一的pXJ40DE3质粒的PCR的菌落电泳图；
图21为本发明的实施例一的pBR322质粒片段的的质粒示意图；
图22为本发明的实施例一的质粒pXJ40的质粒示意图；
图23为本发明的实施例一的质粒pXJ40-DE3的质粒示意图。
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、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；Eppendorf PCR仪；Thermo1300 SERIES A2生物安全柜，均购自Eppendorf。
DNA/RNA共提试剂盒(AP-MN-BF-VNA-250G)购自AXYGEN，TIAN prep Mini Plasmid Kit(DP103-03)均购自天根有限公司。Gel Extraction Kit(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),DMEM basic(C11995500BT)购自Gibco；Lipofectamine LTX and Plus Reagent(15338-100)购自Invitrogen；FBS(10099-141C)购自Gibco；Premix-Taq(RR902A)购自TAKARA；Pen Strep penicillin Streptomycin(15140-122)购自Gibco；Primer star GXL(R050)购自TAKARA；BtgZ1(R0703S)，T4 DNA Ligase(M0202M)购自NEB；其它限制性内切酶，链接酶均购自TAKARA.ClonExpress Multis One Step Cloning Kit(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2)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 PCTCN2020096711-appb-000001
Figure PCTCN2020096711-appb-000002
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 PCTCN2020096711-appb-000003
表3 PCR的反应条件中的扩增引物表
Figure PCTCN2020096711-appb-000004
表4 PCR的反应条件中不同酶的应用配方表
Figure PCTCN2020096711-appb-000005
备注：
1.R050PCR反应程序
Figure PCTCN2020096711-appb-000006
2.RR902 PCR反应程序
Figure PCTCN2020096711-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(如上表中所述)，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1。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序列见序列表SEQ ID NO：1。
三、构建重组病毒rDHN3
3.1 建立为获得重组病毒所需的质粒
3.1.1 构建辅助质粒
将NP，P和L基因，分别克隆进pXJ40载体获得辅助质粒pXJ40-NP，pXJ40-P和pXJ40-L。pXJ40质粒含CMV启动子和SV40多聚腺嘌呤A信号片段，因此外源基因可在细胞DNA聚合酶II作用下被有效转录，然后翻译成用以组装活性病毒粒子的蛋白NP,P,和L蛋白。
具体方法阐述如下：
构建辅助质粒pXJ40-L
前述测序质粒pMD19-L1，pMD19-L2，pMD19-L3和pMD19-L4覆盖了完整的DHN3的L基因，如下图3所示。
通过设计4对带同源重组序列的特异性引物(具体可见附表5)，分别以pMD19-L1，pMD19-L2，pMD19-L3和pMD19-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个片段和BamH1/Pst1双酶切后的载体pXJ40加在一起，在重组酶(ClonExpress Multis One Step Cloning Kit(C113))的作用下作同源重组，得到重组产物。
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)/P-L1-R(P-L1-R:GGACAGTTGACTCATTGCTAACATA)扩增出正确的PCR产物为2110bp；用引物P-L3-F/P-L3-R扩增出正确的PCR产物为2027bp.图4和图5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；
引物PXJ40-F见SEQ ID NO：24；引物P-L1-R见SEQ ID NO：25；
进一步将其中一株菌培养扩增，提取质粒DNA。再经BamH1/Pst1双酶切，正确的酶切产物应为6230bp和5081bp。实验结果如下图6所示，证实该质粒含正确分子量的DNA片段，质粒pXJ40-L示意图如图7。
表5 用于建立pXJ40-L的PCR引物
Figure PCTCN2020096711-appb-000008
Figure PCTCN2020096711-appb-000009
引物P-L1-BamH1-F见SEQ ID NO：26；引物P-L2-F见SEQ ID NO：27；引物P-L2-R见SEQ ID NO：28；引物P-L3-F见SEQ ID NO：29；引物P-L3-R见SEQ ID NO：30；引物P-L4-F见SEQ ID NO：31；引物P-L4-Pst1-R见SEQ ID NO：32。
表6 质粒pXJ40-L构建配方表
Figure PCTCN2020096711-appb-000010
备注：
1.对于另外两个辅助性质粒pXJ40-NP,pXJ40-P的构建使用的是普通连接酶连接转化的方法，并非使用的同源重组的方法。
2.pXJ40-NP,pXJ40-P质粒的构建
2.1 使用EcoR Ⅰ和Xho Ⅰ双酶切pXJ40质粒，将切下的片段(4288bp)作为构建pXJ40-NP,pXJ40-P的质粒片段。
2.2 使用NP-PXJ-0F2/NP-PXJ-1591R2(见表9)扩增NP片段(R050,60℃退火温度，1min30s延伸时间)。使用P-PXJ-1083F2/P-PXJ-3080R2扩增P基因(R050,退火60℃，延伸1min15s)。扩增NP,P基因的两对引物中引物P-PXJ-0F2和引物P-PXJ-1083F2中含有EcoR Ⅰ的酶切序列；引物NP-PXJ-1591F2和引物P-PXJ-3080R2序列中含有Xho Ⅰ序列。这样，使用这两对引物分别扩增NP和P基因，扩增出的产物在两端便分别含有EcoR Ⅰ和Xho Ⅰ酶切位点。将使用这两对引物扩增出的产物再用EcoR Ⅰ和Xho Ⅰ进行双酶切，这样酶切后的产物便含有EcoR Ⅰ和Xho Ⅰ的粘性末端。
2.3将含有EcoR Ⅰ和Xho Ⅰ粘性末端的线性化pXJ40片段和分别含有EcoR Ⅰ和Xho Ⅰ的NP,P片段的酶切产物做连接转化，挑菌验证。如此便构建成了质粒pXJ40-NP,pXJ40-P。
3.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。
4.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℃。
下表7为质粒pXJ40的酶切过程及NP,P基因扩增产物的酶切过程。另外表8为NP,P基因的酶切产物与pXJ40质粒的酶切产物的连接过程。需要指出的是NP,P基因的PCR产物和酶切产物以及pXJ40质粒的酶切产物都是经过了琼脂糖电泳胶回收后再进行下一步操作。
表7 质粒pXJ40的酶切过程及NP,P基因扩增产物的酶切过程参数表
Figure PCTCN2020096711-appb-000011
表8 NP,P基因的酶切产物与pXJ40质粒的酶切产物的连接过程参数表
Figure PCTCN2020096711-appb-000012
P基因插在pXJ40的EcoR1和Xho1之间；NP基因插在pXJ40的EcoR1和Xho1 之间。见质粒示意图8和9。
表9用于建立pXJ40-P,pXJ40-NP的引物
Figure PCTCN2020096711-appb-000013
引物P-PXJ-1893F2见SEQ ID NO：33；引物P-PXJ-3080R2见SEQ ID NO：34；引物NP-PXJ-0F2见SEQ ID NO：35；引物NP-PXJ-1591R2见SEQ ID NO：36；
上文以及下文多处提及载体质粒pXJ40,pXJ40是一个应用非常广泛的载体质粒，其具有丰富的酶切位点，在大量的质粒构造过程中采用pXJ40。
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，其质粒示意图如图22，图22所示的pXJ40-NEW即为pXJ40，其序列见序列表SEQ ID NO：66；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22所示的质粒结构图的载体质粒。
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:
步骤1：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；
步骤2：PCR扩增无Flag片段：以2ng pXJ40Flag为模板，用引物dFlagF:ACTATAGGGCGAATTCGGATCCAAGCTTCTCG/Bsa1R:TGAGCGTGGGTCTCGCGGT，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。
步骤3：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。
步骤4：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进行测序验证。含正确序列(无Flag序列)者为pXJ40-New质粒。
3.1.2 建立pBR322-Base载体
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，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。(见质粒示意图10)
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10)。
3.1.3 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(如图21)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11)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。
C.将目的基因片段和pBR322质粒片段用重组酶链接。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11-13所示，其中，图11为：pBR322-PNP质粒示意图；图12为：pBR322-PDP质粒示意图；图13为pBR322-LPD3质粒示意图。
表11.目的片段所涉及的模板和引物表
Figure PCTCN2020096711-appb-000014
Figure PCTCN2020096711-appb-000015
引物C-Sac11BtNhe-ST-R见SEQ ID NO：37；引物C-NP-F见SEQ ID NO：38；引物C-Mini-R见SEQ ID NO：39；引物C-Mini-F见SEQ ID NO：40；引物C-P-R见SEQ ID NO：41；引物C-HindBt-P-F见SEQ ID NO：42；引物C-BtNhe-P-R见SEQ ID NO：43；引物C-P-F见SEQ ID NO：44；引物C-PD1-R见SEQ ID NO：45；引物C-PD1-F见SEQ ID NO：46；引物C-PD2-R见SEQ ID NO：47；引物C-PD2-F见SEQ ID NO：48；引物C-PD3-R见SEQ ID NO：49；引物C-HindBtg-PD3-F见SEQ ID NO：50；引物C-BtNhe-PD3-R见SEQ ID NO：51；引物C-PD3-F见SEQ ID NO：52；引物C-L1-R见SEQ ID NO：53；引物P-L2-R:见SEQ ID NO：54；引物P-L3-F见SEQ ID NO：55；引物C-HindBtNot-L4-F见SEQ ID NO：56；
表12 各质粒的构建配方表
Figure PCTCN2020096711-appb-000016
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12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BtgZⅠ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),BtgZⅠ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BtgZⅠ酶切序列，这样便可以通过BtgZⅠ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13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。
3.1.4 构建全基因组表达载体pBR322-DHN3
制备DHN3-A片段：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13，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13 各质粒片段的回收配方和工艺参数表
Figure PCTCN2020096711-appb-000017
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BtgZⅠ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BtgZⅠ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14。全长DHN3-A的质粒结构如图14所示；
表14 pBR322-DHN3质粒的连接配方表
Figure PCTCN2020096711-appb-000018
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.将连接产物直接使用试剂盒(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。
制备带同源臂的质粒片段：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:ATCGGTAG AAGGTTCCCTCAGGTTC；A2-R:GGTCCTATAGTGAGTCGTATTAATG)，用Primer star GXL试剂盒提供的材料并按照试剂盒提供的方法进行PCR扩增。引物A2-F见SEQ ID NO：57；引物A2-R见SEQ ID NO：57；
用上述纯化试剂盒(Mini BESTDNA fragment purification kit Version4.0，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。
将此质粒片段与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(ClonExpressMultis One Step Cloning Kit，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15。
表15 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
将重组产物转化入DH5a感受态细胞。用特异性引物C-pBR322-R(C-pBR322-R：GAAATTGCATCAACGCATATAGCGC)；C-NP-F(C-NP-F：CATCTGGTTGCCCTTGCGGCTTGTTC)作单菌落PCR，获3个阳性菌落A1-A3，如图15。引物C-pBR322-R见SEQ ID NO：59；引物C-NP-F见SEQ ID NO：60。
将菌落A1作扩增培养并提取质粒DNA。将A1质粒用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16，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。
质粒pBR322-DHN3质粒结构图如图17；质粒pBR322-DHN3质粒结构图如图18。
3.1.5 拯救病毒。
将BHK-21细胞置30mm培养皿培养过夜使其达80-90％满。用pXJ40DE3(DE3表达T7RNA聚合酶。)与pBR322-DHN3,pXJ40-NP，pXJ40-P和pXJ40-L共转染。4天后将培养皿置-20冰箱冻融2次后将细胞和培养液收集至无菌离心管，离心12000rpm 10分钟，收集 上清。再将此上清(含rDHN3)100μl继续感染BHK-21细胞，2-3天后可见细胞融合病变。继续培养1天后将培养皿置-20冰箱冻融2次，再收集离心12000rpm 10分钟，收集上清。为加速病毒扩增，可将病毒rDHN3P1接种SFP鸡胚，搜集尿囊液获取病毒rDHN3。
上述转染步骤涉及转染液各物质用量如下：
转染液包括A液和B液
A液：Opti-MEM    150ul
     LTX         15ul
Figure PCTCN2020096711-appb-000019
按Lipofectamine LTX and Plus Reagent(15338-100)，Invitrogen提供的方法进行转染。
新城疫病毒囊膜表面有HN，可吸附红细胞引起部分动物红细胞凝集。当新城疫病毒浓度高时，还可以进一步导致红细胞溶解。根据新城疫病毒的这个特性，可通过这个实验来检测待检溶液中是否有新城疫病毒，以及病毒的含量。我们将上述方法拯救出的第一代rDHN3在SFP鸡胚扩增后搜集尿囊液作为待检样品，通过下面红细胞凝结实验证明以上rDHN3重组病毒具备与母毒株相同的凝血功能。二者的最高凝血效价均为2 8。
具体测试过程如下：
1.将待检样品用生理盐水按1：10做系统稀释；
2.将96孔板中每孔加入25μl生理盐水；
3.将不同稀释度的病毒液分别加入到对应的孔中，25μl/孔(每一行为一个病毒，最后一排不添加病毒液做对照用)；
4.每孔加入25μl 1％鸡红细胞；
5. 15-20分钟后观察血凝情况。
上文涉及的pXJDE3质粒具体如下：
pXJ40DE3质粒为T7RNA聚合酶(DE3)表达质粒。DE3基因通过使用引物BamH1-DE3-F/BamH1-DE3-R从大肠杆菌BL21(DE3)(Merck公司)获得。再经同源重组将其插入到质粒pXJ40的BamH1位点中；pXJ40DE3质粒质粒结构图如图23。
pXJ40DE3质粒制备步骤
步骤1：提取大肠杆菌BL21(DE3)DNA：常规细菌DNA提取方法。
步骤2：PCR制备DE3片段：以大肠杆菌BL21(DE3)细菌DNA为模板，利用高保真DNA聚合酶(GXL)通过引物：
BamH1-DE3-F：actcactatagggcgaattcggatccgccatgaacacgattaacatcgc；
BamH1-DE3-R：taagatctggtaccgagctcctgcagttacgcgaacgcgaagtccgactc进行扩增。
引物BamH1-DE3-F见SEQ ID NO：61；
引物BamH1-DE3-R见SEQ ID NO：65；
GXL酶(R050),退火温度60℃，延伸时间2分36秒。产物经凝胶电泳验证后纯化备用。
步骤3：制备载体：将pXJ40用Pst Ⅰ，BamH Ⅰ双酶切后经胶回收备用，具体配方和工艺参考表16。
表16 载体pXJ40的双酶切配方工艺表
pXJ40 1μg
Quick Cut Pst Ⅰ 1μl
Quick Cut BamH Ⅰ 1μl
10×QuickCut Buffer 2μl
ddH20 up to 20μl
温度 37℃
时间 3h
步骤4：将DE3片段重组进pXJ40载体，具体工艺和配方如下表17。
表17 DE3片段重组进pXJ40载体的工艺配方表
Figure PCTCN2020096711-appb-000020
步骤5：将重组产物转化入DH5α中，通过在含50ug/ul氨苄的平板培养。次日从平板上挑选8个菌落进行PCR验证。
验证引物为pXJ40-F:GCAACGTGCTGGTTATTGTG；DE3-R:GAAGTCCGACTCTAAGATGTCACG,使用ExTaq酶(RR902),退火温度57℃，延伸2’42”(目的片段大小为2723bp).结果如图20:8个菌有3个强阳性(L1，L5，L7)1个弱阳性(L4)。将L1扩大培养后提取质粒，测序验证无误。引物DE3-R见SEQ ID NO：62；
四、鉴定rDHN3
4.1 基因稳定性
用上述拯救出的病毒rDHN3再经鸡胚传代3次后感染BHK-21细胞，待细胞病变明显时分别提取细胞总RNA。
用特异性引物作逆转录获得第一条cDNA.再以此cDNA为模板用特异性引物(JF-F:CCTTGCAGCTGCAGGAATTG；JF-R:GCTCTATACAGTATGAGGTGTCAAG)和DNA聚合酶作PCR。将此PCR产物纯化后用引物(JF-F:CCTTGCAGCTGCAGGAATTG)测序。其结果显示与原毒株无差异。说明在病毒拯救的过程中DHN3F基因是稳定的，无自然突变发生。引物JF-F见SEQ ID NO：63；引物JF-R见SEQ ID NO：64。
4.2 重组病毒rDHN3感染BHK-21细胞出现的融合病变情况
参考图19，分为A、B、C三列，其中，A列代表DHN3病毒感染BHK-21细胞12h后形态；B列为rDHN3感染BHK-21细胞12h后细胞的形态；C列为BHK-21细胞未感染病毒12h后的形态。
图中箭头所指出为DHN3或rDHN3感染BHK-21细胞出现的融合病变
DHN3是指病毒分离，盲传并在BHK-21细胞上进行噬斑纯化后的病毒；测序和分析归 类步骤中涉及的均为此病毒。
rDHN3是指重组病毒，即文中通过重组克隆拯救出的病毒。
通过图20可看出，重组病毒rDHN3和它的母体病毒DHN3均能感染BHK-21形成细胞融合。
此外我们也比较了rDHN3和DHN3的生长特性和毒力，均未发现二者有明显差异。
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Claims (8)

  1. 一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于，包括如下步骤：
    步骤1：构建辅助质粒，所述辅助质粒为三种，辅助质粒上的目标片段分别为NP基因、P基因、L基因；
    步骤2：构建DHN3全基因组表达载体；将人工重组得到的DHN3全基因组重组入载体质粒中得到全基因组表达载体；DHN3全基因组的序列如序列表SEQ ID NO 1；
    步骤3：将三种辅助质粒和DHN3全基因组表达载体共转染BHK-21细胞，得到含重组病毒rDHN3的病毒液；
    NP基因在序列表SEQ ID NO：1中位置为1-1591nt；P基因在序列表SEQ ID NO：1中位置为1925-3109nt；L基因在序列表SEQ ID NO：1中位置为8166-15192nt。
  2. 根据权利要求1所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于：所述步骤1中的辅助质粒所涉及的载体为pXJ40系列的质粒或pcDNA3；所述步骤2中的载体质粒为pBR322系列的质粒或pACYC、pBAD系列的质粒。
  3. 根据权利要求1所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于：包括如下步骤：
    步骤1：构建辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L，辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L的目标片段分别为NP基因、P基因、L基因，载体为pXJ40；
    步骤2：构建全基因组表达载体pBR322-DHN3；将人工重组得到的DHN3全基因组重组入pBR322质粒中得到全基因组表达载体pBR322-DHN3；所述DHN3全基因组为II类VII型鸡新城疫病毒的全基因组；
    步骤3：将辅助质粒pXJ40-NP、pXJ40-P和pXJ40-L、全基因组表达载体pBR322-DHN3 共转染BHK-21细胞，得到含重组病毒rDHN3的病毒液。
  4. 根据权利要求3所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于：所述步骤2具体为：
    步骤21：建立pBR322-Base载体；在pBR322质粒上引入能够与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22：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L1，L2，L3,L4和PD3基因；
    步骤23：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、LPD3和PDP，将基因片段PNP、LPD3和PDP通过T4链接酶连接得到DHN3全基因组DHN3-A；
    步骤24：构建带同源臂的质粒片段；将步骤21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25：构建全基因组表达载体pBR322-DHN3；将步骤24中的带同源臂的质粒片段和步骤23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；
    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。
  5. 根据权利要求4所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于，所述步骤3具体为：
    用能表达T7RNA聚合酶的质粒与辅助质粒pXJ40-NP、pXJ40-P、pXJ40-L、全基因组表达载体pBR322-DHN3共转染BHK-21细胞；得到含重组病毒rDHN3的病毒液。
  6. 根据权利要求4所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于，所述步骤21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。
  7. 根据权利要求4所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于，所述步骤22具体为：
    步骤221：制备pBR322质粒片段：将pBR322质粒用Hind3和Nhe1作双酶切，然后经胶回收备用；
    步骤222：制备目标片段；所述目标片段为NP、MINI、P、PD1、PD2、PD3、L1、L2,L3,L4基因；
    步骤223：将pBR322质粒片段和对应的目标片段用重组酶链接得到对应的质粒pBR322-PNP、质粒pBR322-PDP、质粒pBR322-LPD3。
  8. 根据权利要求4所述针对II类VII型流行NDV株DHN3的感染性重组克隆方法，其特征在于，所述步骤23为：
    将质粒pBR322-PDP、质粒pBR322-LPD3分别用BtgZ1单酶切后回收基因片段LPD和PDP；载体pBR322-PNP用BtgZ1和Hind3双酶切回收基因片段PNP；
    将基因片段LPD、基因片段PDP、基因片段PNP通过T4链接酶作体外连接，获得DHN3全基因组DHN3-A。
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