Calreticulin-soluble programmed death receptor 1 fusion protein, and preparation method and purpose thereof

A programmed death and fusion protein technology, which is applied in the field of calreticulin-soluble programmed death receptor 1 fusion protein and its preparation and application, can solve the problems of not having tumor immunogenicity, etc., and achieve prolonged survival, growth inhibitory effect

Inactive Publication Date: 2013-01-30
CHINA THREE GORGES UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most antitumor drugs can induce tumor cell apoptosis but cannot cause membrane translocation and clustering of CRT, and such apoptotic tumor cells do not have tumor immunogenicity

Method used

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  • Calreticulin-soluble programmed death receptor 1 fusion protein, and preparation method and purpose thereof
  • Calreticulin-soluble programmed death receptor 1 fusion protein, and preparation method and purpose thereof
  • Calreticulin-soluble programmed death receptor 1 fusion protein, and preparation method and purpose thereof

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Embodiment Construction

[0045] The present invention will be described in detail through specific examples below.

[0046] This invention comprises the following steps successively:

[0047] 1. Construction and identification of recombinant eukaryotic expression plasmids

[0048] pcDNA3.1(+) / CRT Δ , pcDNA3.1(+) / sPD1 and pcDNA3.1(+) / CRT ⊿ - Construction of sPD1 eukaryotic expression plasmid

[0049] The target fragments of mCRT and mPD1 were obtained by PCR cloning using the cDNA of mouse lymphocytes as a template, and HindⅢ and XhoI restriction enzyme sites were introduced at the 5' ends of the upstream primer and downstream primer, respectively, to facilitate subsequent cloning operations. The PCR primers were designed as follows:

[0050] mCRT upstream primers:

[0051] GTCGAAGCTTAACCATGCTCCTTTCGGTGCCGCTC

[0052] Downstream primers:

[0053] CAAGCTCGAGTTAATGATGATGATGATGATGACCCCCTTCTGGGTGTATCCAGGTAC

[0054] mPD1 upstream primer:

[0055] CAGTAAGCTTATGTGGGTCCGGCAGGTAC

[0056] Downstream ...

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Abstract

The invention relates to a calreticulin-soluble programmed death receptor 1 fusion protein, and a preparation method and a purpose thereof. The invention provides a fusion protein CRTdelta-sPD1 of a mice C-zone-partially-amputated calreticulin and soluble programmed death receptor 1 extracellular domain; the fusion protein is subjected to eukaryotic expression, such that a cell line stably expressing CRTdelta-sPD1 is established; and the CRTdelta-sPD1 is secreted out of the cells by using secretory peptide. The CRTdelta-sPD1 fusion protein provided by the invention has activities of combining cell surface PD-L1 and promoting lymphocyte proliferation and killing. Also, the CRTdelta-sPD1 fusion protein can be activated in vivo, and can induce an immune system to perform specific killing against tumors. Therefore, tumor growth is inhibited, and tumor mice survival time is prolonged. The CRTdelta-sPD1 fusion protein can be adopted as a tumor immunotherapy medicine candidate, and can be used for treating diseases such as tumors.

Description

technical field [0001] The invention relates to the field of DNA recombination technology and protein medicine, belongs to the field of medical biotechnology, and specifically relates to a calreticulin-programmed death receptor 1 fusion protein, a DNA sequence encoding the fusion protein, a vector or a plasmid, and a host containing the DNA sequence A cell, a method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in diseases such as tumors. Background technique [0002] Calreticulin (CRT) is a highly conserved calcium-binding protein that belongs to the heat shock protein family. It mainly exists in the endoplasmic reticulum and has various biological functions. Immunogenic apoptosis mediated by CRT plays an important role in anti-tumor immunity. Studies have found that certain anti-tumor treatments can induce the rapid translocation of CRT from the intracellular to the membrane surface and cluster on the cell membrane, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/17A61K48/00A61P35/00A61P37/02
Inventor 刘朝奇
Owner CHINA THREE GORGES UNIV
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