Recombinant porcine pseudorabies virus strain capable of simultaneously expressing PEDV variant S1 gene CS region and porcine IL-18 and application of recombinant porcine pseudorabies virus strain

A porcine pseudorabies virus, rprv-pedvs1-il18 technology, applied in the field of molecular biology, can solve the problems of antigenic variation and incomplete protection of infection, and achieve good genetic stability

Pending Publication Date: 2021-11-12
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antigenicity of the PRV epidemic strains has mutated, and commonly used attenuated vaccines such as Bartha K61 can no longer completely protect the infection of the PRV epidemic mutant strains

Method used

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  • Recombinant porcine pseudorabies virus strain capable of simultaneously expressing PEDV variant S1 gene CS region and porcine IL-18 and application of recombinant porcine pseudorabies virus strain
  • Recombinant porcine pseudorabies virus strain capable of simultaneously expressing PEDV variant S1 gene CS region and porcine IL-18 and application of recombinant porcine pseudorabies virus strain
  • Recombinant porcine pseudorabies virus strain capable of simultaneously expressing PEDV variant S1 gene CS region and porcine IL-18 and application of recombinant porcine pseudorabies virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Rescue of PEDV mutant strain S1 gene CS region and pig IL-18 recombinant porcine pseudorabies virus strain

[0054] 1 Materials and methods

[0055] 1.1 Materials

[0056] 1.1.1 Plasmids, cells and viruses

[0057] PRV universal transfer plasmid pG, three-gene deletion mutant strain of PRV epidemic variant rPRV HN2012-TK - / gE - / gI - (CCTCC NO.V201748), PEDV variant CH-HNKF-2016 (Accession No. KY649107) cDNA, CRISPR / Cas9 plasmid knockout vector (PX459-gRNA1-EZ-gRNA3-EGFP) were provided by Zhengzhou Key Experiment for the Prevention and Control of Swine Major Diseases The laboratory was constructed and preserved; pig testicular cells (ST cells) and Vero cells were passaged and preserved by the Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control.

[0058] 1.1.2 Main reagents

[0059] The cDNA Synthesis Kit (reverse transcription kit) was purchased from Nanjing Novozyme Company; the transfection reagent ZLip2 000 was purchased from Beij...

Embodiment 2

[0111] Embodiment 2: the biological characteristic of recombinant virus rPRV-PEDV S1-IL18

[0112] 1 Materials and methods

[0113] 1.1 Materials

[0114] 1.1.1 Cells, strains and plasmids

[0115] ST passage cells, PK-15 passage cells, IPEC passage cells, Vero passage cells, parent strain rPRV HN2012-TK - / gE - / gI - , PEDV mutant strain CH-HNKF-2016, and PRV popular mutant strain PRV / HN2012 were all preserved by the Zhengzhou Key Laboratory of Swine Major Disease Prevention and Control; the recombinant virus rPRV-PEDV S1-IL18 was constructed, purified and preserved in Experiment 1.

[0116] 1.1.2 Main reagents and instruments

[0117] Reagents and instruments were the same as in Experiment 1.

[0118] 1.2 Method

[0119] 1.2.1 Culture characteristics of recombinant virus on different cells

[0120] The recombinant virus rPRV-PEDV S1-IL18 was inoculated into ST, PK-15, Vero, and IPEC cells respectively, and the cytopathic changes were observed at 6 h, 12 h, 24 h, and ...

Embodiment 3

[0149] Example 3: Immunization test research of recombinant virus rPRV-PEDV S1-IL18 in pigs

[0150] 1 Materials and methods

[0151] 1.1 Materials

[0152] 1.1.1 Cells, strains and vaccines

[0153] ST cells, Vero passage cells, PEDV variant CH-HNKF-2016, PRV variant PRV / HN2012 (CCTCCNO.V201314), recombinant pseudorabies virus rPRV-PEDV S1 strain, the classification name is rPRV-PEDVS1, and the preservation number is: CCTCCNO: V201750, date of deposit: October 31, 2017, deposited in China Center for Type Culture Collection, address: China.Wuhan.Wuhan University. rPRV-PEDV S1-IL18 strain was constructed from Experiment 1; PEDV inactivated vaccine (PEDV-TGEV dual inactivated vaccine, containing PEDV AJ1102 strain) was purchased from Wuhan Keqian Company. PRV BarthaK61 attenuated vaccine was purchased from Zhejiang Nuobeiwei Biotechnology Co., Ltd.

[0154] 1.1.2 Experimental animals

[0155] 2-week-old healthy piglets were purchased from a pig farm in Henan.

[0156] 1.1.3 ...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a recombinant porcine pseudorabies virus strain capable of simultaneously expressing a PEDV variant S1 gene CS region and a porcine IL-18 and an application of the recombinant porcine pseudorabies virus strain. The classification name of the strain is rPRV-PEDV S1-IL18, the preservation number is CCTCC NO: V201740, the preservation date is October 31, 2017, and the strain is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The recombinant porcine pseudorabies virus strain is successfully obtained, can induce piglets to generate specific antibodies aiming at the PEDV and the PRV, and can protect the piglets from virulent lethal attacks of the PEDV and the PRV. The level of a PRV specific antibody generated by a recombinant strain group is higher than that of a recombinant virus rPRV-PEDV S1 group without porcine IL-18, and the recombinant strain group is expected to become a candidate strain of a novel porcine epidemic diarrhea and porcine pseudorabies bigeminy attenuated vaccine.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a recombinant porcine pseudorabies virus strain, in particular to a recombinant porcine pseudorabies virus strain simultaneously expressing the CS region of the S1 gene of a PEDV mutant strain and porcine IL-18 and an application thereof. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), the main clinical manifestations are vomiting, watery diarrhea, dehydration And suckling piglet mortality is high, more common in winter and spring cold season. The disease occurs worldwide, especially in Europe and Asia. Since the use of PEDV CV777 vaccine in pig farms in my country, PED has been well controlled. However, since December 2010, a new round of PED outbreaks have broken out in many pig farms in many cou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/50C12N15/38C12N15/85C12N15/66C12N15/65C12N15/24A61K38/20A61K39/245A61K39/215A61P31/14A61P31/22C12R1/93
CPCC12N7/00C07K14/005C12N15/85C12N15/66C12N15/65C07K14/54A61K38/20A61K39/12A61P31/14A61P31/22C12N2710/16021C12N2710/16034C12N2710/16052C12N2800/107A61K2039/5254A61K2039/70A61K2039/552A61K2039/55527C12N2770/20034
Inventor 郑兰兰陈红英崔建涛韩昊莹张远航陈曦艋
Owner HENAN AGRICULTURAL UNIVERSITY
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