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Heat shock protein complex associated with cervical cancer and application of heat shock protein complex

A technology of heat shock proteins and complexes, applied in hybrid peptides, animal cells, drug combinations, etc., can solve problems such as low immune response

Inactive Publication Date: 2015-12-23
深圳市康尔诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, the technical problem to be solved by the present invention is to overcome the low immune response caused by immune editing of tumor cells, to induce the production of specific cytotoxic T lymphocytes (killer T cells, CTL) in patients with cervical cancer, and to utilize the specificity of CTL Killing ability to treat and prevent cervical cancer without damaging normal cells and tissues

Method used

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  • Heat shock protein complex associated with cervical cancer and application of heat shock protein complex
  • Heat shock protein complex associated with cervical cancer and application of heat shock protein complex
  • Heat shock protein complex associated with cervical cancer and application of heat shock protein complex

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1. Purification of gp96 protein from cervical cancer tissue

[0024] The gp96 complex was purified from tissues of HPV-positive and HPV-negative cervical cancer patients. Cervical cancer tissue was homogenized and centrifuged with 30% / 70% saturation (NH 4 ) 2 SO 4 Precipitate, after the precipitate is dissolved, use ConASepharose (GE Company) to perform affinity chromatography, use 8% α-methyl glucoside to elute the bound protein, and use Hitrap-Q (GE Company Chromatography System) as the glycoside eluent for anion Chromatography, after these three steps of purification, gp96 protein with >95% purity was obtained. The gp96 protein was identified by Western detection with gp96 / grp94 monoclonal antibody (Santa Cruz Company). Its purity was identified by SDS-PAGE, silver staining and reversed-phase HPLC.

Embodiment 2

[0025] Example 2. Release of non-covalently bound polypeptides from gp96 protein

[0026] Add the purified gp96 protein to trifluoroacetic acid (TFA) to make the final concentration reach 0.2% (pH about 2.0), then use ultrafiltration (molecular cut-off of 30kDa, Millipore Company) to separate the peptides, and perform MALDI-TOF mass spectrometry on the peptide mixture (ABI4700) analysis, the molecular weight of the polypeptide is mostly between 600-1200Da.

Embodiment 3

[0027] Example 3. Reverse-phase HPLC analysis of polypeptides

[0028] The peptide mixture was lyophilized and dissolved in solution A (0.065% TFA, 2% acetonitrile), loaded on reverse phase chromatography column C18 (SephasilpeptideC18; 5 μm particle size; 4.6X250mm, GE Company), from 0 to 65% solution B (0.05% TFA, 100% acetonitrile) for gradient elution at a flow rate of 1 mL / min, with detection at a wavelength of 214 nm. Compare HPV + with HPV - Tumor tissue HPLC pattern, found a HPV + HPV - Peptide peaks not present in tissue.

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Abstract

The invention discloses a heat shock protein complex associated with cervical cancer. The heat shock protein complex comprises heat shock protein and protein polypeptide associated with cervical cancer, wherein the heat shock protein is gp96 and HSP78; an amino acid sequence of a peptide fragment is any one of a sequence 1, a sequence 2 and a sequence 3 in a sequence table; the complex comprises a complex body formed by combining gp96, HSP78 and antigen peptides through noncovalent bonds, as well as fusion protein formed through connection of gp96 and HSP78 through covalent bonds. Therefore, the complex can be used for preparing a vaccine for treating and preventing cervical cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the invention discloses a heat shock protein complex capable of effectively activating papillomavirus (HPV) specific T cells and a preparation method thereof, and its application in the treatment and prevention of cervical cancer Applications. technical background [0002] Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today, and cervical cancer is one of the malignant tumors that seriously threaten women's lives and health. Together with breast cancer, it is called a female "double cancer" disease. According to statistics, there are about 530,000 new cases of cervical cancer and about 275,000 deaths each year worldwide, 85% of which occur in developing countries. In my country, there are about 100,000 new cases each year, accounting for about 5% of female tumors, and in recent years there has been an increase in regions and an ...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K19/00C12N5/0783A61K35/17A61P35/00
Inventor 黄锡坚张小俊章文乐
Owner 深圳市康尔诺生物技术有限公司
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