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Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof

A technology of BVDV-2 and a construction method, which is applied in the field of construction of infectious cDNA clones of swine-derived BVDV-2 strains, can solve the problem of no swine-derived BVDV-2 infectious clones and the like

Inactive Publication Date: 2017-01-11
YANGZHOU UNIV
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Problems solved by technology

Although, many infectious clones of BVDV isolates have been successfully constructed at present, but these are all for bovine BVDV isolates, and no porcine BVDV-2 infectious clones have been constructed so far

Method used

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  • Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof
  • Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof
  • Method for constructing swine-borne BVDV-22 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof

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Experimental program
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Embodiment Construction

[0028] 1. Materials

[0029] 1.1 Viruses, cells, bacteria and vectors

[0030]The porcine BVDV-2 strain SH-28 was isolated and identified in this experiment[Tao Jie, et al. Whole genome sequence and genetic analysis of porcine bovine viral diarrhea virus SH-28 isolate. Chinese Journal of Veterinary Medicine, 2013,33( 3): 321-325.]. The genetic engineering bacteria E. Coli HB101 and the medium-copy cloning vector pBR322 were purchased from TAKARA Company, and the low-copy cloning vector pACYC184 was purchased from NEB Company. The MDBK cell line was donated by Professor Bello, University of Pennsylvania, USA.

[0031] 1.2 Reagents and antibodies

[0032] DNA Ligation Kit LONG and pMD 19T Simple vectors were purchased from TaKaRa Company; various restriction endonucleases were purchased from NEB Company; Expand long template PCR system was purchased from Roche Company; trypsin, DMEM medium and Opti-MEM medium were purchased from From Gibco Company; QIAprep○R Spin Miniprep Ki...

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Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to swine-borne BVDV-2 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof. The swine-borne BVDV-2 strain infectious cDNA clone contains swine-borne BVDV-2 seed virus full-length cDNA, T7 RNA (ribonucleic acid) polymerase promoters are inserted in 5' terminals of the swine-borne BVDV-2 seed virus full-length cDNA, and SbfI restriction enzymes are inserted in 3' terminals of the swine-borne BVDV-2 seed virus full-length cDNA. The invention further discloses a plasmid pASH28 with the swine-borne BVDV-2 strain infectious cDNA. The plasmid is linearized and then is subjected to in-vitro transcription to obtain RNA, MDBK (Madin-Darby bovine kidney) cells are transfected by the plasmid, and bovine viral diarrhea viruses can be successfully rescued. The swine-borne BVDV-2 strain infectious cDNA clone, the application and the plasmid have the advantage that the swine-born BVDV-2 strain infectious cDNA clone can be applied to research on functional difference between different animal-borne BVDV-2 proteins and also can be used for genetically modifying and preparing high-titer attenuation BVDV vaccine.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products. In particular, it relates to a construction method and application of a porcine BVDV-2 strain infectious cDNA clone. Background technique [0002] The host spectrum of BVDV is very wide. In addition to infecting cattle, it can also cause disease in pigs, sheep, deer, camels and various wild animals. But it wasn't until 2012 that the World Organization for Animal Health (OIE) listed the disease as one of the notifiable diseases. [0003] Pigs infected with BVDV do not show clinical symptoms, or have symptoms similar to classical swine fever, which not only brings great difficulties to the prevention and control of classical swine fever, but also makes the prevention and control of BVDV even more difficult. At present, the current situation of BVDV infection in domestic pig herds is relatively serious and has become a problem that cannot be ignored. During 2005 to 2009, We...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/00C12R1/93
CPCC12N7/00C12N15/85C12N2770/24321C12N2770/24351C12N2800/107
Inventor 朱国强陶洁王建业朱礼倩张信军夏芃芃孟霞羊扬
Owner YANGZHOU UNIV
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