Correlation of TLR4 with in-vitro binding of NY-ESO-1 and DC

A NY-ESO-1, cell technology, applied in the field of biomedicine, can solve problems such as affecting the natural history of tumors

Inactive Publication Date: 2017-05-31
宁波美丽人生医药生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, the reduction of spontaneous NY-ESO-1 antigen in melanoma suggests that immune responses against NY-ESO-1 may influence the natural history of tumors

Method used

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  • Correlation of TLR4 with in-vitro binding of NY-ESO-1 and DC
  • Correlation of TLR4 with in-vitro binding of NY-ESO-1 and DC
  • Correlation of TLR4 with in-vitro binding of NY-ESO-1 and DC

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] NY-ESO-1 protein can form multimeric structures even in loading buffer in the presence of regular concentrations of β-mercaptoethanol.

[0066] Experiments have found that NY-ESO-1 proteins obtained from bacteria and mammalian cells can form multimeric structures even in loading buffers with conventional concentrations of β-mercaptoethanol ( figure 1 A, B).

Embodiment 2

[0068] The polymerization reaction of NY-ESO-1 is mediated by its intermolecular disulfide bonds.

[0069] Cysteine ​​in amino acid residues 75, 76, 78, 152 and 165 were replaced with serine. Three types of NY-ESO-1 with amino acid substitutions were: ESOcs1 with cysteine-serine substitutions at positions 75, 76, and 78, ESOcs2 with substitutions at positions 152 and 165, and ESOcs3 with mutations at all five positions. Purify these proteins and use Western blot to compare the oligomerization state between them ( figure 1 B). The results showed that the wild-type protein clearly showed the presence of dimers, tetramers and even multimers of 130 kDa. ESOcs1 and ESOcs2 showed mainly monomer and dimer structures, while the result of ESOcs3 showed mainly monomer. This experiment shows that the polymerization of NY-ESO-1 is due to the disulfide bonds between its molecules.

Embodiment 3

[0071] NY-ESO-1 binds to human and mouse immature dendritic cells in vitro and is related to its own polymeric structure.

[0072] Purify these proteins and use Western blot to compare the oligomerization state between them ( figure 1 B). The results showed that the wild-type protein clearly showed the presence of dimers, tetramers and even multimers of 130 kDa. ESOcs1 and ESOcs2 showed mainly monomer and dimer structures, while the result of ESOcs3 showed mainly monomer. This experiment shows that the polymerization of NY-ESO-1 is due to the disulfide bonds between its molecules. NY-ESO-1 present in cancer cells was also analyzed by Western blot for its protein aggregation in the loading buffer containing conventional concentrations of β-mercaptoethanol, and it was found that NY-ESO-1 mainly manifested as trimers and tetramers Dimeric and monomeric forms are rarely found (mutation 1C). Experiments show that NY-ESO-1 binds to human and mouse immature dendritic cells in vit...

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Abstract

The invention relates to the field of biological medicine, in particular to a correlation of cell surface TLR4 and in-vitro binding of NY-ESO-1 and a DC (dentritic cell). The inventor of the invention discovers that NY-ESO-1 protein can form a poly structure even in a loading buffer with the conventional concentration of beta-mercaptoethanol; a polymerization reaction of NY-ESO-1 is mediated by an intermolecular sulfide bond of NY-ESO-1; the in-vitro binding of NY-ESO-1 with immature human and mouse dendritic cells is related to a polymerization structure of NY-ESO-1; TLR4 influences in-vitro binding of NY-ESO-1 and the DC in marrow; the binding of NY-ESO-1 with the immature human and mouse dendritic cells can also be achieved by a calprotectin effect; the oligomerization of a polymer reduces the binding capacity of TLR4 and NY-ESO-1; a polymer structure of NY-ESO-1 and TLR4 in a host participate in immunoglobulin antibody response; and Art v1 (wtArt) and CA9 gene immunogenicity is improved by NY-ESO-1 gene fusion expression. The cell surface TLR4 is confirmed to be correlated with in-vitro binding of NY-ESO-1 and the DC.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the association between cell surface TLR4 and NY-ESO-1 and DC cells in vitro. Background technique [0002] It has been postulated that innate immune system receptors may be involved in the initial autoimmune response to tumor-specific antigens (TAAs). Necrotic tumor cells release endogenous factors or damage-associated pattern molecules such as heat shock proteins, high mobility group box B1 (HMGB-1), and uric acid. These damage-patterning molecules alert the innate immune system via CD91, receptor for advanced glycation end products, TLR2 / TLR4, and interleukin-1 (IL-1) receptor, respectively. TAA itself is generally regarded as a bystander of the above-mentioned "danger signal", acting as an adjuvant in the initial stage of the anti-tumor immune response. According to this example, the spontaneous immune response is caused by new peptides produced by mutations in 11,000 genes prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/47
Inventor 田晓丽
Owner 宁波美丽人生医药生物科技发展有限公司
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