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Blocking agent for inhibiting infection of High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

A syndrome virus and highly pathogenic technology, applied in the field of biology, can solve the problems of low cross-immunity protection of common strains, unsatisfactory vaccine immune effect, difficulties in HP-PRRSV prevention and treatment, etc., to expand the scope of research, Reduce the effect of inhibitory activity

Active Publication Date: 2018-12-25
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the immune effect of existing vaccines is not ideal, and the cross-immunity protection between HP-PRRSV and common strains of PRRSV is low, which brings great difficulties to the prevention and treatment of HP-PRRSV

Method used

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  • Blocking agent for inhibiting infection of High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus
  • Blocking agent for inhibiting infection of High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus
  • Blocking agent for inhibiting infection of High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Participate in the identification of PRRSV infection 14-3-3 subtype

[0052] In the early stage, we connected the PRRSV non-structural protein nsp2 gene to the eukaryotic expression vector, expressed it in 293T cells, collected the cellular proteins that might interact with nsp2 through GFP-Trap agarose beads, and found that the cellular protein 14 was verified by mass spectrometry and Western blot -3-3 interacts with nsp2 (Xiao et al, J Proteome Res. 2016, 15(5):1388-1401). In order to further study which subtype of 14-3-3 is involved in PRRSV infection, two subtypes of 14-3-3β and ε were selected according to the mass spectrometry results, and siRNAs targeting these two subtypes were designed. After reducing their expression, analysis Effects on HP-PRRSV infection.

[0053] According to the 14-3-3β and ε gene sequences published by NCBI (GenBank accession numbers: XM008009806.1, XM_017028039.2), primers were designed to amplify the 14-3-3β and 14-3-3ε g...

Embodiment 2

[0059] Example 2: Construction of 14-3-3 protein overexpression cell line and its effect on PRRSV infection

[0060] (1) 14-3-3 lentivirus construction

[0061] According to the 14-3-3 gene sequence registered in GenBank, design primers for amplifying different subtypes of 14-3-3, as follows:

[0062] 14-3-3β-leti: Forward primer 5'-CGGGATCCATGACAATGGATAAAAGTGAG-3'; Reverse primer 5'-CCCGAATTCTTAGTTCTCTCCCCTCCCCAG-3'.

[0063] 14-3-3ε-leti: forward primer 5'-CGGGATCCATGGATGATCGAGAGGATCTG-3'; reverse primer 5'-CCCGAATTCTCACTGATTTTCGTCTTCCAC-3'.

[0064] Respectively with BamH I, EcoR I restriction enzyme sites. Using the RNA from Marc145 cells as a template, the target fragment was cloned, and the target fragment digested by restriction enzymes was ligated with the lentiviral empty vector pwpxld overnight at 16°C. Transform into T1 competent, pick a single colony for PCR, enzyme digestion verification and sequence determination verification. A large number of extracted and ...

Embodiment 3

[0069] Example 3: Knockdown of 14-3-3ε on PAM and its effect on HP-PRRSV infection

[0070] In order to further verify the results, primary porcine lung macrophages (PAM) were used for verification. The 50-day-old SPF piglets were bled, their lungs were aseptically removed after the trachea was ligated, and the outer surface was washed with autoclaved PBS (1640 medium with 1 / 25 volume of PBS and 5× double antibody), and the pH7 .2 Infuse 30.0ml-50ml of PBS into the lungs from the trachea, gently pat the lung surface, recover the lavage fluid after 1-2 minutes, and repeat this process until the lavage fluid is clear. Gently blow the recovered bronchoalveolar lavage fluid with a straw to break up the cell clumps, filter through a single-layer sterile 100-mesh stainless steel sieve, collect all the lavage fluid, centrifuge at 1500r / min for 5-10min, and collect the precipitate. Wash twice with PBS containing 5× double antibody, each time gently mix and centrifuge. Then add an ap...

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Abstract

The invention discloses a blocking agent for inhibiting the infection of High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV). According to the blocking agent, a novel HP-PRRSV cell target protein called 14-3-3Epsilon protein and the inhibition targets of the receptor are found. The infection of HP-PRRSV could be significantly reduced by performing interference on the 14-3-3Epsilon gene at these inhibition targets or using inhibitor difopein of the 14-3-3Epsilon protein. These inhibition targets and difopein can be developed into a drug for preventing and curing PRRSVinfection so as to provide a brand-new idea for HP-PRRS research, prevention and cure and expand the scope of research. The invention has great significance in actual production.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a blocking agent for inhibiting highly pathogenic porcine reproductive and respiratory syndrome virus infection. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as PRRS, is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). The disease is mainly characterized by reproductive impairment in sows and dyspnea in piglets, causing huge economic losses to the swine industry. [0003] In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) appeared again. Compared with ordinary PRRSV, the NSP2 protein of HP-PRRSV lacked about 30 amino acids to cause mutations, resulting in enhanced virulence and ability to Cause higher morbidity and mortality, so the Ministry of Agriculture classified it as a Class A infectious disease in 2008. [0004] At present, the immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12Q1/6883C12N15/113
CPCA61K31/713A61P31/14C12N15/113C12N2310/14
Inventor 肖一红曹胜亮丛芳源刘思当丁国飞刘家琪
Owner SHANDONG AGRICULTURAL UNIVERSITY
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