Method for preparing infective hog cholera virus cDNA carrier and use thereof
A kind of swine fever virus and infectious technology, applied in the field of genetic engineering, can solve the problems of inactivated virus vaccine application, non-attenuated vaccine application, pathological changes, etc.
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Embodiment 1
[0041] Example 1 The cloning of the whole gene of classical swine fever virus and the construction of the expression vector
[0042] ①Dilute the freeze-dried virus of CSFV Shimen strain 10 times with normal saline (provided by China Veterinary Drug Control Institute, Beijing), and inoculate 2-month-old non-immune pigs (provided by Hubei Institute of Animal Husbandry and Veterinary Medicine) into the ear vein. 24 hours after inoculation, the body temperature of pigs reached 40°C, and clinical symptoms of classical swine fever appeared. When the body temperature turned from 42°C to drop on the 7th day, blood was collected from the anterior vena cava immediately, and EDTA anticoagulant was added as the experimental material.
[0043] ② Total RNA was isolated from anticoagulated whole blood with the RNA extraction kit produced by Promega. On the basis of the whole genome sequence of Shimen strain virus, 7 pairs of primers were designed, and 7 overlapping fragments were used to amp...
Embodiment 2
[0045] Embodiment 2 Confirmation of the whole genome expression vector of classical swine fever virus
[0046] The identification of the whole genome cloned plasmid pT7SM was carried out simultaneously by PCR amplification and enzyme digestion identification. Seven pairs of primers randomly distributed on the genome were selected for PCR identification, and three restriction enzymes, BamHI, KpnI and HindIII, were used for restriction endonuclease identification of the whole genome.
Embodiment 3
[0047] Example 3 Preparation of expression vector-deficient classical swine fever virus vector
[0048] Construction of Defective CSF Virus Deleting NS2 Gene Based on Obtaining Complete Gene Cloning of CSFV
[0049] ① Amplification of NS3 gene. In the genome of swine fever virus, the NS2 gene is closely connected with the NS3 gene. For the deletion of the NS2 gene, the PCR method was first used to amplify the downstream NS3 gene, and then the NS2 gene was excised by enzyme cutting behind the upstream P7 gene of the NS2 gene. , and fuse the NS3 gene in-frame to the downstream of P7. When amplifying the NS3 gene, pair the upstream primer NS3(+) with the downstream primer P3D of cDNA3 (as shown in the figure), use the plasmid pGEMT3-1 as a template, and use Pfu DNA polymerase to perform PCR amplification. The target product is in 3 The 'end contains the single restriction site BamHI in the middle of the genome. The Pfu PCR reaction system was prepared in an ice bath, 1ul of th...
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