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
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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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