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Chinese isolated strain of bovine enterovirus, and construction and application of infectious cDNA clone thereof

A technology of enteroviruses and isolates, applied in the field of bovine enteroviruses, to achieve the effect of easy preservation

Active Publication Date: 2012-11-14
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In 1959, Moll and Davis isolated BEV for the first time, and then other countries also reported the isolation of BEV, but bovine enterovirus has not yet been isolated in China

Method used

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  • Chinese isolated strain of bovine enterovirus, and construction and application of infectious cDNA clone thereof
  • Chinese isolated strain of bovine enterovirus, and construction and application of infectious cDNA clone thereof
  • Chinese isolated strain of bovine enterovirus, and construction and application of infectious cDNA clone thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Isolation and identification of bovine enterovirus Chinese isolate BHM26

[0041] 1 Experimental method

[0042] 1.1 Disease material treatment

[0043] Freshly collected feces samples were made into a 1:10 (W / V) feces suspension with PBS, vortexed, freeze-thawed three times, 3000r / min, and centrifuged at 4°C for 10min. After sterilizing by filtration with a pore filter, store in a -70°C refrigerator for later use.

[0044] 1.2 Virus isolation and culture

[0045] Wash the MA104 cells that have grown into a monolayer three times with PBS, inoculate the treated fecal samples into the MA104 monolayer cells, absorb at 37°C for 1 h, add serum-free DMEM, and inoculate in 5% CO 2 Culture at 37°C under conditions, and observe the cytopathic changes every day

[0046] (CPE). If there is still no CPE after 1 week of cell culture, or if the cells show 50%-80% CPE after inoculation, the cell culture is repeatedly frozen and thawed 3 times, and harvested. In the sam...

Embodiment 2、2

[0103] Construction and identification of embodiment 2, type 2 bovine intestinal rescue virus

[0104] 1. Construction of full-length infectious cDNA clones

[0105] Genomic RNA of bovine enterovirus was extracted from the cell culture of bovine enterovirus BHM26 strain by Trizol method. Then oligo(dT)-15 was used for reverse transcription, and the reverse transcription product was used as a template, and the primers in Table 2 were used to amplify the three overlapping fragments A, B and C covering the full length of the genome. By PCR amplification, a T7 RNA polymerase promoter sequence was introduced at the 5' end of the viral genome, and a Bgl II restriction enzyme site was introduced at the 3' end of the viral genome. Using site-directed mutagenesis, C is mutated to T at the 4074nt position of the viral genome, so that CAGCTG (PvuII) at this position becomes TAGCTG, and the PvuII restriction site is eliminated as a molecular marker. The three fragments were sequentially...

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Abstract

The invention relates to a Chinese isolated strain of bovine enterovirus, and constructions and applications of infectious cDNA clone thereof. Isolated strain of bovine enterovirus capable of stably generating CPE on MA104 cells can be separated from bovine diarreal stool samples, with a microbial preservation number of CGMCC No. 4782. Whole-length infectious cDNA clone is constructed from the isolated strain. B family gene type 2 bovine enterovirus rescue virus is obtained by in-vitro transcribing the cDNA and transfectioning cells. The isolated strain of bovine enterovirus, the constructed whole-length infectious cDNA clone and the rescue virus thereof can be used for researches of gene structures and functions of bovine enterovirus, can be used as vaccine vectors of bovine enterovirus, or can be prepared into vaccines or reagents capable of preventing or treating bovine enterovirus.

Description

technical field [0001] The present invention relates to bovine enterovirus strains and infectious cDNA clones thereof, in particular to Chinese isolates of bovine enteroviruses. The present invention also relates to the construction and application of infectious cDNA clones of the Chinese isolates of bovine enteroviruses, belonging to bovine enteroviruses. Enterovirus field. Background technique [0002] Bovine Enterovirus (BEV) is a member of the genus Enterovirus in the Picornaviridae family. Virus particles are spherical, icosahedral, without envelope, and the diameter of the complete virus particle is about 25-30nm. The genome is a single-stranded positive-strand RNA, consisting of about 7500 nucleotides (nt), which can be directly used as mRNA to encode and translate a polyprotein, and then undergo a series of degradation to produce four structural proteins (VP1, VP2, VP3 and VP4) and seven non-junction proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The capsid of BEV is i...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61K39/125A61K48/00A61P31/14C12Q1/70C12N15/41C07K14/085C12N15/66C12N15/63C12R1/93
Inventor 于力常继涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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