Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof

A dectin-1, heat shock protein technology, applied in the field of lectin receptor Dectin-1 and heat shock protein Hsp60 polymer, can solve the problem of not being identified

Inactive Publication Date: 2008-01-02
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, more and more evidence shows that there is a phagocytosis receptor of Hsp60 on the surface of antigen presenting cells, and this receptor plays an important biological function, but it has not been identified yet

Method used

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  • Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof
  • Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof
  • Dectin-1 and heat shock protein Hsp60 polymer and coding nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The cDNA of human Hsp60 and the cDNA of P550 were respectively connected to the pGEX-4T-1 vector by molecular biology techniques, transformed into DH5α, and the positive clones screened by ampicillin were purified by plasmids and sequenced to prove that the sequences of the selected clones were correct. Named pGEX-4T / Hsp60, pGEX-4T / P540. Transform BL21 Escherichia coli with pGEX-4T / Hsp60 and pGEX-4T / P540 plasmids respectively, pick clones and inoculate them into LB culture medium, and culture them at 37°C. induced expression. Cells were harvested and lysed 4 hours after induction of expression. According to the GST purification manual, purify the target protein by GST beads. The purified product was identified as the target protein Hsp60 and P540 by SDS-PAGE and Western blot.

Embodiment 2

[0043] The extracellular segment (aa112-247) cDNA of human Dectin-1 was inserted into the pGBKT7 vector with EcoRI and BamHI sites through molecular cloning technology, and then transformed into DH5α. Positive clones were screened by kalamycin, and single-clonal colonies were selected and extracted. Plasmids were identified by sequencing. The results showed that the cDNA sequence of the extracellular segment (aa112-247) of human Dectin-1 was consistent with the reading frame of the vector. The vector is called pGBKT7 / CTLD. The pGBKT7 / CTLD and pGADT7 vectors were co-transformed into AH109 yeast and screened on the medium. The results showed that pGBKT7 / CTLD had no self-activation phenomenon.

[0044] According to the yeast two-hybrid operation manual of Clontech Company, pGBKT7-CTLD was used to screen the cDNA library of human thymus. Select positive clones that can grow in SD-Ade / -His / -Leu / -Trp / X-a-Gal medium and turn blue, and perform plasmid isolation and sequencing on the ...

Embodiment 3

[0054] The cDNA of human Dectin-1 is cloned into the vector according to the reading frame of the pCDNA3.1 / Myc vector by molecular biology techniques. Among them, the cDNA stop codon of human Dectin-1 is deleted, and the Dectin-1 / Myc fusion protein can be produced. Sequencing showed that the constructed plasmid was correct and named pMyc-Dectin-1. HEK293T cells were transfected with pMyc-Dectin-1, and the cells were collected 48 hours later, and the cells were lysed in the lysate for immunoprecipitation experiments. Proceed as follows:

[0055] 1. The prokaryotic expressed Hsp60 in Example 1 was incubated with GST sepharose 4B beads in rotation for 2 hours at 4°C, and washed once with cold PBS.

[0056] 2. The beads from step 1 were then incubated with the above-mentioned cell lysate at 4°C for 4 hours, then centrifuged, washed the beads three times with cold lysis buffer, added loading buffer and boiled for 10 minutes for immunoblotting.

[0057] The results showed that Hs...

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PUM

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Abstract

The invention discloses an agglutinin acceptor Dectin-1 and heat shock protein Hsp60 polymer and its coded nucleic acid molecule of peptide and application in the molecular biological domain, which is characterized by the following: the Hsp60 activates tyrosine kinase Syk with Dectin-1 connecting zone interacted with Dectin-1 in the immune sediment and cell stream, which guides the maturity of antigen submit cell to secretory cell factor to adjust immune system; The invention provides the application with Hsp60 and its Dectin-1 connecting zone as vaccine adjuvant, adjusting immune function and self-immunne disease to treat chronic infectious arthritis at least.

Description

technical field [0001] The present invention relates to the field of molecular biology. Specifically, it relates to polymers of lectin receptor Dectin-1 and heat shock protein Hsp60, nucleic acid molecules encoding these polypeptides and applications thereof. Background technique [0002] The Dectin-1 gene is located on chromosome 12p13 and belongs to type II lectin receptor, which is divided into three parts: intracellular, transmembrane and extracellular. The extracellular segment contains a single C-type lectin-like domains (CTLD) sequence and does not contain a Ca2+ binding site, and the C-type lectin-like sequence contains a QPD motif (which can recognize galactose and N-acetylgalactosamine); the intracellular segment contains an immunoreceptor tyrosine-based activation-like motif (ITAM). Due to the different cleavage sites of Dectin-1, two main functional receptor forms and some non-functional small polypeptide forms exist. The two functional receptors are named α, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61P37/02C12N15/12A61K48/00C12Q1/04G01N33/68
Inventor 顾建新闻玉梅谢建辉陈晓宁郭亮
Owner FUDAN UNIV
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