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Application of PHB2 gene or protein inhibitor in preparation of anti-porcine circovirus type 2 drugs

A porcine circovirus and inhibitor technology, applied in antiviral agents, applications, genetic engineering and other directions, can solve the problems of few research reports, the interaction network between PCV2 infection and host cells is not very clear, and achieve the effect of inhibiting replication

Active Publication Date: 2020-01-21
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the interaction network between PCV2 infection and host cells is not very clear, and there are relatively few reports on the interaction between PCV2 and host cells

Method used

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  • Application of PHB2 gene or protein inhibitor in preparation of anti-porcine circovirus type 2 drugs
  • Application of PHB2 gene or protein inhibitor in preparation of anti-porcine circovirus type 2 drugs
  • Application of PHB2 gene or protein inhibitor in preparation of anti-porcine circovirus type 2 drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1.1 PCV2d infection of PK-15 cells

[0056] PK-15 cells without PCV1 / PCV2 contamination were seeded in 6-well cell culture plates, and the cells were cultured in DMEM medium containing 10% NBCS (newborn calf serum) until the cell fusion rate was 60%. Use TCID50=1×10 5.66 After the PK-15 cells were infected with PCV2d / mL, the cells in each well were collected at 3, 6, 12, 24 and 48 hours respectively, as the experimental group; the cells without any treatment were marked as 0h, as the control group, and each time point was set 3 duplicate holes.

[0057] 1.2 Detection of PCV2 proliferation in PK-15 cells by indirect immunofluorescence

[0058] After infecting PK-15 cells with PCV2d using the above method, the cells were fixed with 4% paraformaldehyde for 20 min at 0.5, 1, 12, 24 and 36 h, respectively, and then the cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min. After washing with PBS three times, block in PBS containing 3% BSA (bovine serum...

Embodiment 2

[0075] PK-15 cells were grown into a monolayer and transfected with an RNA interference fragment targeting the PHB2 gene. The interference fragment is PHB2siRNA-1, and the target sequence of PHB2siRNA-1 is shown in SEQ ID NO: 1, which was transfected into PK-15 cells. qPCR was used to detect the RNA interference efficiency of PHB2 gene. After 24 hours of transfection, discard the medium, wash with PBS 3 times, infect the cells with PCV2 for 1 hour, discard the medium, wash 3 times with PBS, replace with fresh medium and continue culturing for 48 hours. After the cells were lysed to calculate the protein concentration, β-actin was used as an internal reference, anti-β-actin (mouse) and rabbit-derived polyclonal antibodies against PCV2Cap were respectively used as primary antibodies, and corresponding HRP-labeled anti-mouse and anti-rabbit antibodies were used As a secondary antibody, a Western blot test was performed to detect changes in PCV2Cap protein.

Embodiment 3

[0077] PK-15 cells were grown into a monolayer and transfected with an RNA interference fragment targeting the PHB2 gene. The interference fragment is PHB2siRNA-2, and the target sequence of PHB2siRNA-2 is shown in SEQ ID NO: 2, which was transfected into PK-15 cells. qPCR was used to detect the RNA interference efficiency of PHB2 gene. After 24 hours of transfection, discard the medium, wash with PBS 3 times, infect the cells with PCV2 for 1 hour, discard the medium, wash 3 times with PBS, replace with fresh medium and continue culturing for 48 hours. After the cells were lysed to calculate the protein concentration, β-actin was used as an internal reference, anti-β-actin (mouse) and rabbit-derived polyclonal antibodies against PCV2Cap were respectively used as primary antibodies, and corresponding HRP-labeled anti-mouse and anti-rabbit antibodies were used As a secondary antibody, a Western blot test was performed to detect changes in PCV2Cap protein. And adopt IFA test me...

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Abstract

The invention discloses application of a PHB2 gene / protein inhibitor in preparation of anti-porcine circovirus type 2 (PCV2) drugs. According to the application of the PHB2 gene / protein inhibitor in preparation of the anti-PCV2 drugs, the proliferation changes of PCV2 in PK-15 cells are detected by an indirect immunofluorescence test, the dynamic changes of PHB2 mRNA are detected in PCV2-infectedPK-15 cells, and the expression of PCV2 Cap protein is detected in PK-15 cells transfected by the PHB2 gene / protein inhibitor. The results indicate that PHB2 is efficiently expressed in both normal and PCV2-infected PK-15 cells. PCV2 can significantly affect PHB2 transcription in PK-15 cells, PHB2 has a regulating effect on PCV2 virus replication, down-regulation of PHB2 expression, namely, PHB2 gene knock-out or protein expression down-regulation, can obviously inhibit PCV2 replication, thus the invention has important significance for elucidating PCV2 pathogenic mechanisms and studying new antiviral drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a PHB2 gene or protein inhibitor in the preparation of anti-porcine circovirus type 2 drugs. Background technique [0002] Porcine circovirus (porcine circovirus, PCV) belongs to Circoviridae Circovirus genus, is a non-enveloped circular single-stranded DNA virus (diameter 17nm). PCV1, PCV2 and PCV3 have been identified in pig herds. Among them, PCV2 clinical or subclinical infection is considered to be the main cause of porcine circovirus diseases (porcine circovirus diseases and porcine circovirus-associated diseases, PCVD / PCVAD). There are differences in the incidence and severity of PCVAD in different farms, and the disease is becoming more and more complex. Wasting syndrome (PMWS) and porcine respiratory disease complex (PRDC) can also cause reproductive disorders, seriously endangering the global breeding industry. PCV2Cap protein (Capsidprotein) is the on...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K31/713A61P31/20C12N15/12C12N15/113
CPCA61K31/7088A61K31/713A61P31/20C07K14/4702C12N15/113C12N2310/14C12N2310/531
Inventor 王乃东蒋一凡邹亚文余婉婷张佳鑫何庆
Owner HUNAN AGRICULTURAL UNIV
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