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Screening and identifying method for TLR vaccine adjuvants

A technology of vaccine adjuvant and identification method, which can be used in material inspection products, measuring devices, instruments, etc., and can solve problems such as unclearness

Active Publication Date: 2019-01-11
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking vaccinia virus as an example, vaccinia virus particles contain about 30 protein components. Related studies have identified a variety of virus surface antigen genes that function in the host immune response, but what mechanism do these antigen proteins activate the host immune response, and Whether vaccine adjuvants are required for immune protection is unclear

Method used

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  • Screening and identifying method for TLR vaccine adjuvants
  • Screening and identifying method for TLR vaccine adjuvants
  • Screening and identifying method for TLR vaccine adjuvants

Examples

Experimental program
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Embodiment 1

[0088] Experiment A: Lysis and pre-separation of virus, the operation steps are as follows:

[0089] VV-WR strain (VR-1354) was purchased from ATCC. Add 10 ml of PBS containing 1% Triton X-100 to dissolve 1 mg of VV virus pellet, and incubate at 37°C for 1 hour to extract the total protein.

[0090] In order to remove the effect of detergent, the total protein needs to go through the following dialysis step: add the lysate mixed with the total viral protein into the Slide-A-Lyzer dialysis card (2 kDa MWCO) (Thermo Scientific, Cat. No. 87719), and dialyze at 4°C overnight. The specific dialysis method is: dialyze in 1xPBS buffer solution at 4°C, and change the dialysate every 3 hours for a total of 3 times.

[0091] The mixture was centrifuged at 15,000 g for 30 min to remove insoluble precipitates.

[0092] The protein concentration of the mixture was determined by BCA method (Sanko, Cat. No. C503021-0500).

[0093] 250 μg of the mixture was ultrafiltered through 30 kD MWC...

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Abstract

The invention relates to the field of vaccines and discloses a screening and identifying method for TLR vaccine adjuvants. The method comprises the following steps: A) splitting and pre-separating viruses; B) separating specific virus sub-group proteins by SDS-PAGE and LC-MS identification; C) separating the virus sub-component proteins by means of an electroelution method; and D) establishing a 293T cell line which expresses B5R, D8L and K2L stably and testing vitality of a TLR2 ligand. The invention establishes a whole set of a method of screening and identifying vaccinia virus protein sub component with TLR2 ligand activity and identifies that the viral protein D8L has the TLR2 ligand activity and can be used for preparing anti-viral infection and anti-tumor TLR vaccine adjuvants.

Description

technical field [0001] The invention relates to the field of vaccines, and relates to a method for screening and identifying TLR vaccine adjuvants. Background technique [0002] The host's immune response mechanism in response to viral infection mainly recognizes relevant ligands in different viruses through pattern recognition receptors (PRRs), which are called pathogen-associated molecular patterns (PAMPs), including viral proteins, DNA, RNA, etc. Unlike viral DNA and RNA, viral proteins are primarily recognized by TLR family proteins, including TLR2 and TLR4. Innate immune cells, such as macrophages and DCs, both express TLR2 and TLR4, so they can be activated by related viruses and secrete a variety of inflammatory factors, leading to the activation of host immune responses. Our findings suggest that vaccinia virus (VV) can activate the TLR2-MyD88 signaling pathway in DCs (DCs) or macrophages, leading to increased secretion of proinflammatory cytokines and enhanced T ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/68
Inventor 杨文君其他发明人请求不公开姓名
Owner CARBIOGENE THERAPEUTICS CO LTD