Novel PRRS virus receptor and block inhibitor thereof

A technology of receptor blocker, virus, applied in the field of biology

Inactive Publication Date: 2009-07-15
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there are other PRRSV cellular receptors that can bind or participate in binding to PRRSV-infected susceptible cells, causing PRRS

Method used

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  • Novel PRRS virus receptor and block inhibitor thereof
  • Novel PRRS virus receptor and block inhibitor thereof
  • Novel PRRS virus receptor and block inhibitor thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Discovery and identification of novel PRRSV cell receptors

[0019] A soluble protein was purified from MA-104 and PAM cells using a monoclonal antibody idiotype antibody that mimics PRRSV GP5 membrane antigen (Zhou EM, Xiao YH, Shi YF, et al.J.Virol.Methods, 2008, 149: 300 -308): 50 μg of soluble protein was separated by 7.5% SDS-PAGE, and the separated protein was hydrolyzed by pepsin, and the hydrolyzed fragment was separated by microcapillary high-pressure liquid chromatography, and the separated fragment was ionized by nano-electrospray, and analyzed by ion trapping mass spectrometry instrument analysis. The obtained hydrolyzed fragment sequence was compared with the GenBank amino acid sequence library, and the receptor protein was non-muscle myosin type II reconnected A (NMHCII-A).

Embodiment 2

[0020] Embodiment 2 NMHC II-A artificial polypeptide synthesis

[0021] The polypeptide sequence formulated according to the NMHC II-A reconnected carboxy-terminal sequence is synthesized by the solid-phase peptide synthesis method. The specific method is:

[0022] Mix and shake 2-Chlorotrityl Chloride Resin resin and DMF for 30-60 minutes to swell the resin; filter out DMF through sand core, add 3 times molar excess of the first amino acid at the C-terminal to connect with the resin, and then add 10 times molar excess of DIEA , finally add DMF to dissolve, shake for 30-60min; remove DMF, add 20% piperidine DMF solution, react for 5-15min; take more than a dozen resins, wash with ethanol three times, add ninhydrin, KCN, phenol solution each drop, Heating at 105°C-110°C for 5min to detect the reactants. Wash twice with DMF, methanol and DMF sequentially. Condensate the ligation product by one of the following methods.

[0023] Method a: Add a three-fold excess of protected a...

Embodiment 4

[0031] Example 4 Preparation of Anti-NMHC II-A Protein and Artificial Polypeptide Antibody

[0032] Protein and polypeptide-aluminum adjuvant preparation: NMHC II-A protein or polypeptide-KLH coupling product was dissolved in 0.005MPBS, pH 6.2 (final volume 10mL), slowly added potassium aluminum sulfate dissolved in 0.005M PBS (pH 6.2) ( The ratio is 50μg protein / 0.4mg aluminum), the pH is raised to 6.8-7.3, stirred for 2 hours, centrifuged at 800g for 10 minutes, the protein-aluminum adjuvant precipitate is washed once with normal saline, and dissolved in 0.1M PBS. Proteins and peptides can also be used with other adjuvants, such as Freund's adjuvant.

[0033] Immunization procedure: each mouse was immunized intraperitoneally with 50 μg of NMHC II-A protein or artificial polypeptide for a total of three times, and the serum of the mice was collected to detect and identify antibodies. Antibodies against NMHC II-A protein or artificial polypeptide can also be used in other ani...

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Abstract

The invention relates to a novel cell receptor of a PRRS virus, that is, a nonmuscle myosin heavy chain II-A (NMHC II-A) protein and an inhibitor blebbistati of nonmuscle myosin II which are taken as drugs for inhibiting a PRRS virus infected cell. The invention provides a method for inhibiting the PRRS virus infected cell by purified NMHC II-A protein and synthetic polypeptide and the inhibitor blebbistati thereof. The invention further provides an antibody generated by the NMHC II-A protein and the polypeptide. The NMHC II-A protein obtained by purification and the polypeptide obtained by a synthetic method and the blebbistatin thereof, and an anti-NMHC II-A protein and anti-polypeptide antibody have inhibition activity on the PRRS virus infected cell, and can be developed into the drugs for preventing and treating the PRRS virus infection.

Description

technical field [0001] The invention relates to the discovery of a novel PRRS virus cell receptor and the use of receptor proteins, polypeptides and derivatives thereof to prevent and treat PRRS virus infection, belonging to the field of biology. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as PRRS, is an acute infectious disease of pigs caused by PRRS virus (PRRSV) infection. PRRSV invades host cells first by binding to specific receptors on the cell membrane surface. Endocytosis is used to infect susceptible cells (Nauwynck, HJ, Duan X, Favoreel HW, etc. J. Gen. Virol. 1999, 80:297-305), such as MA-104 and PAM. Four independent but functionally related PRRSV cell receptors have been reported so far, they are sialoadhesin (Vanderheijden N, Delputte PL, Favoreel HW, etc.J.Virol.2003,77:8207- 8215), Heparinlike (Delputte PL, Vanderheijden N, Nauwynck, HJ, etc.J.Virol.2002, 76:4312-4321), Vimentin (Kim JK, Fahad AM, Shanmukha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705A61K38/08A61K38/10A61K38/17A61K39/395A61K31/437A61P31/12
CPCC07K16/10A61K38/00C07K14/4716C07K14/705A61K31/437A61P31/12A61P31/14
Inventor 周恩民
Owner SHANDONG AGRICULTURAL UNIVERSITY
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