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A-type Seneca virus full-length infectious cDNA clone as well as construction method and application thereof

A construction method, infectious technology, applied in the direction of recombinant DNA technology, positive-sense single-stranded RNA virus, virus, etc.

Active Publication Date: 2021-02-02
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the full-length infectious cDNA clone of Seneca virus type A SVA / HN / 11 / 2017 Lanshouyan

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  • A-type Seneca virus full-length infectious cDNA clone as well as construction method and application thereof
  • A-type Seneca virus full-length infectious cDNA clone as well as construction method and application thereof
  • A-type Seneca virus full-length infectious cDNA clone as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] 1. Primer Design

[0085] According to the measured whole genome sequence of the SVA / HN / 11 / 2017 Lanshouyan strain, and referring to the single restriction enzyme site in the vector M-pSK, 8 primers covering the full length of the SVA genome were designed ( Table 1), synthesized by Jinweizhi Biotechnology Co., Ltd. In order to distinguish the rescued virus from the parental virus, two silent mutations were introduced into the primers SmF and SmR as molecular tags (an XhoI restriction site was introduced and an XmaI restriction site was eliminated at the same time).

[0086] Table 1 Primers for constructing HN / 11 / 2017 full-length infectious cDNA clone

[0087]

[0088]

[0089] Note: The restriction endonuclease sites in the primers are underlined; the T7 promoter sequence is italicized.

[0090] 2. Construction of SVA full-length cDNA recombinant plasmid

[0091] The RNA of the virus sample SVA / HN / 11 / 2017 Lanshouyan virus strain was extracted according to the in...

Embodiment 2

[0097] 1. Virus rescue

[0098] After the identified correct recombinant plasmid pSVA-HN was cleaved with Not I enzyme, Lipofectamine was used to TM 2000 mediated transfection of BSR / T7 cells grown to 80% to 90% full, while setting normal cells as a control. 5 hours after transfection, add 2 mL of GMEM medium containing 10% fetal bovine serum, and place at 37°C, 5% CO 2 Cells were harvested after continuing to culture in the incubator for 72 h. After repeated freezing and thawing for 3 times, they were continuously passaged on PK-15 cells until the typical cytopathic effect appeared, and the collected virus samples of each passage were stored at -70°C for future use. The rescued recombinant virus was named rSVA-HN.

[0099] Virus rescue results

[0100] The Not I-lined recombinant plasmid pSVA-HN was transfected into BSR / T7 cells, and the transfected supernatant was collected 72 hours later, and was continuously passaged on PK-15 cells until obvious cytopathic effects appe...

Embodiment 3

[0115] 1. Plaque phenotype test

[0116] The 6th generation rescued virus rSVA-HN and the parent virus SVA / HN / 11 / 2017 Lanshouyan were diluted 10 times respectively, and the viruses of different dilutions were inoculated on the PK-15 cells with a monolayer (200 μL / well, 6-well plate) at 37°C, 5% CO 2 Incubate in a constant temperature incubator for 1 hour, during which time, gently shake once every 10 minutes to make the liquid infiltrate the cell surface. Then add 2 mL of tragacanth gum mixture (1 part of 1.2% tragacanth gum, 1 part of 2×MEM, 1% serum) to each well and store at 37°C, 5% CO 2 Cultivate statically in a constant temperature incubator, discard the culture medium after 48 hours, add pre-cooled fixative solution (50% formaldehyde + 50% acetone) and fix at -20°C for 30 minutes, stain with crystal violet (2g / L) at room temperature for 1 hour, wash with water The plaque phenotype was observed after washing.

[0117] 2. One-Step Growth Curve of Rescued Virus

[011...

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Abstract

The invention provides an A-type Seneca virus SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences full-length infectious cDNA clone as well as a construction methodand application thereof, and belongs to the technical field of viruses. On the basis of carrying out whole genome determination on SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences, a T7 promoter sequence is introduced to the 5' end of a genome cDNA sequence, a Not I restriction enzyme cutting site sequence is introduced to the 3' end, meanwhile, an Xho I restriction enzyme cutting site is introduced to a 2B gene, the Xma I restriction enzyme cutting site is eliminated, and recombinant plasmids containing SVA / HN / 11 / 2017 Lanzhou Veterinary Institute of Chinese Academy of Agricultural Sciences full-length cDNA are constructed. The invention further discloses application of the virus obtained on the basis of infectious cDNA cloning rescue in preparationof an A-type Seneca virus labeled vaccine. The infectious cDNA clone and the preparation method provided by the invention provide an effective platform for deep development of basis and application research of SVA, and have important scientific application value.

Description

technical field [0001] The invention belongs to the technical field of viruses, and in particular relates to a full-length infectious cDNA clone of Seneca virus type A SVA / HN / 11 / 2017 Lanshouyan and its construction method and application. Background technique [0002] Senecavirus A (SVA) is a non-enveloped, single-stranded positive-sense RNA virus that causes vesicular lesions in pigs. The clinical symptoms caused by SVA infection include blisters and ulcers on the pig nose, oral epithelium and hoof crown, resulting in lameness. In young piglets, it can cause lethargy, diarrhea, neurological symptoms and acute death. The mortality rate of piglets is as high as (40% to 80%), and the mortality rate of piglets aged 4 to 7 days is as high as (0 to 30%). The clinical symptoms caused by SVA infection are similar to the lesions caused by foot-and-mouth disease, swine vesicular disease, vesicular stomatitis and swine herpes, and it is difficult to distinguish them clinically. [0...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/63A61K39/125A61P31/14
CPCC07K14/005C12N15/63A61K39/12A61P31/14C12N2770/32022C12N2770/32034Y02A40/70
Inventor 马雪青孙普李平花卢曾军刘在新曹轶梅白兴文付元芳张婧李坤
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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