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System, method and application for saving swine enteric alphacoronavirus

A coronavirus, pig intestinal technology, applied in the system field of saving pig intestinal type A coronavirus, to achieve the effect of wide application prospects

Active Publication Date: 2019-11-19
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] At present, there is no report on the successful construction of the reverse genetic system of SADS-CoV / SeACoV at home and abroad

Method used

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  • System, method and application for saving swine enteric alphacoronavirus

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Embodiment 1

[0036] 1. Amplification of the target fragment of SADS-CoV / SeACoV full-length infectious clone

[0037] According to the gene sequence of porcine enteric alpha-coronavirus SeACoV CH / GD-01 / 2017 / P2 strain published on Genbank (GenBank accession no. MF370205, figure 1 ), the full length of the virus genome is 27155 nt, and the homology of the bat enteric A-coronavirus HKU2 found in the same area 10 years ago and infecting the Chinese horseshoe bat at the whole genome nucleic acid level is 94.9%. The cell infection supernatant sample of the cell stable passage strain SADS-CoV / SeACoV-P10, the Trizol method extracted the RNA and then reverse-transcribed to obtain cDNA, using 16 pairs of primers provided by the present invention (see Table 1 for the primer sequence), amplified to obtain the corresponding The 16 full-length genome fragments of SADS-CoV / SeACoV-P10 were cloned by pCR-Blunt vector, 3 single clone colonies were picked, plasmids were extracted, sequenced and spliced, and t...

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Abstract

The invention discloses a system, a method and application for saving swine enteric alphacoronavirus. The system comprises a recombinant transcription vector containing whole genome DNA of a swine enteric alphacoronavirus and helper plasmids containing SeACoV-N genes. For the system, the method and the application disclosed by the invention, an SADS-CoV / SeACoV reverse genetic system is successfully constructed, produced viruses can be subjected to serial passage, and reviving viruses and parental viruses have consistent biological characteristics. Establishment of the reverse genetic system ishindered by the instability and the toxicity of a virus genome sequence; and the saving method has a potential application value in reverse genetics research and vaccine creation of the swine coronavirus and has a wide application prospect in the aspects of virus gene function research and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a system, method and application for rescuing swine intestinal type A coronavirus. Background technique [0002] China is the world's largest pig-raising country, and large-scale breeding is developing rapidly. However, with the rapid development of large-scale pig farming, coronavirus infection has become more and more harmful to the pig industry. Therefore, strengthening the understanding of the infection and pathogenic mechanism of porcine coronavirus will provide new theoretical and technical support for the prevention and control of these diseases. [0003] Swine entericalphacoronavirus (SeACoV), also known as swine acute diarrhea syndrome virus (Swine acute Diarrhea Syndrome Coronavirus, SADS-CoV), was first discovered in South China in 2017, causing severe diarrhea in piglets. It is an evolutionary origin of bat HKU2- Novel coronavirus of CoV. Compared with HKU2, the S prot...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/00A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/552A61P31/14C12N7/00C12N15/85C12N2770/20034C12N2770/20052
Inventor 黄耀伟杨永乐王斌
Owner ZHEJIANG UNIV
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