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Recombinant adenovirus with fusion protein gene and preparation method and application thereof

A technology of recombinant adenovirus and fusion protein, used in biochemical equipment and methods, fusion polypeptides, gene therapy, etc.

Inactive Publication Date: 2011-08-17
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that this scheme mostly adopts individualized treatment schemes, that is, the HSP-peptide complex used for immunization needs to be derived from autologous tumor cells

Method used

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  • Recombinant adenovirus with fusion protein gene and preparation method and application thereof
  • Recombinant adenovirus with fusion protein gene and preparation method and application thereof
  • Recombinant adenovirus with fusion protein gene and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. AdCEA 576-669 Design and preparation of hsp70L1

[0100] In this experiment, restriction enzymes and Taq enzymes were purchased from NEB Company; PQE 30 and host strain M15 were purchased from Qiagen Company; Escherichia coli BJ5183 and pShuttle-CMV pre-transfected with AdEasy-1 were purchased from Strategen Company; LS174T was purchased from ATCC .

[0101] Induction of dendritic cells derived from human monocytes: Take 200ml of anticoagulated blood from healthy volunteers, apply Ficoll1.077 density gradient centrifugation, and obtain human peripheral blood mononuclear cells; separate human monocytes by immunomagnetic beads, in short, Will 2×10 8 Human peripheral blood mononuclear cells were incubated with 200 μl CD14 immunomagnetic beads at 4°C for 15 minutes; human peripheral blood mononuclear cells combined with immunomagnetic beads were placed in a magnetic field, negative cells were washed away, and positive cells were washed out from the column...

Embodiment 2

[0118] Example 2. Recombinant AdCEA 576-669 Infection and expression of hsp70L1 in human monocyte-derived dendritic cells in vitro

[0119] In this experiment, RPMI1640 medium and Ficoll1.077 were purchased from PAA Company; human GM-CSF and human IL-4 were purchased from Peprotech Company; CD14 immunomagnetic beads, MS column and separation rack were purchased from Miltenyl Biotec Company; AdLacZ was purchased from Strategen Company .

[0120] The method for inducing human monocyte-derived dendritic cells (MoDC) is as described in Example 1.

[0121] Resuspend the collected MoDC in RPMI 1640 medium containing 10% FBS, and adjust the concentration to 10 7 cells / ml. According to MOI=1000:1, use recombinant AdCEA 576-669 hsp70L1 (prepared as in Example 1) infected MoDC. After 24 h, the cells were collected, total RNA was extracted, and RT-PCR (CEA 576-669 : Reaction parameters: 94°C, 30 seconds, 54°C, 30 seconds, 72°C, 30 seconds, 35 cycles followed by 72°C extension...

Embodiment 3

[0126] Example 3. Recombinant AdCEA 576-669 Gene modification of hsp70L1 promotes maturation and activation of human MoDC

[0127] Antibodies in this example were purchased from B.D.Pharmingen; CFSE was purchased from Calbiochem; CD3 immunomagnetic beads were purchased from Miltenyl Biotech; prokaryotic protein CEA 576-669 hsp70L1 was purified and preserved by our laboratory using conventional methods (picked and transformed with pQE30-CEA 576-669 The M15 bacteria of hsp70L1 plasmid, after induced expression, its expression exists in the form of inclusion body in the cell; the inclusion body is washed and dissolved, and the CEA is purified by metal ion chelation column and DEAE column chromatography 576-669 hsp70L1 fusion protein, dissolved in PBS after dialysis, stored at -80°C).

[0128] Freshly isolated human peripheral blood CD14 + Monocytes (Mo) with 10 6 The cells / ml were cultured in RPMI1640 medium containing 10% FBS, human GM-CSF (500 U / ml), and human IL-4 (1...

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Abstract

The invention relates to a recombinant adenovirus with a fusion protein gene and a preparation method and application thereof. Particularly, the invention relates to the recombinant adenovirus with coding sequences of a heat shock protein and a fusion protein of a carcinoembryonic antigen and the preparation method and the application thereof. An E1 region and an E3 region are deleted from a genome of the adenovirus and an expression box of the coding sequence of the fusion protein is inserted at the position of the E1 and / or E3 region. The expression box sequentially comprises a promotor sequence, the coding sequence of the fusion protein and a tailing signal sequence, wherein the heat shock protein is selected from Hsp70 or Hsp70L1. The recombinant adenovirus can be used for efficiently transferring and expressing a mediated gene (particularly tumor) and has wide application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a human carcinoembryonic antigen 576-669 (CEA 576-669 ) and heat shock protein 70-like molecule 1 (hsp70L1) fusion gene recombinant adenovirus and its preparation method and application. Background technique [0002] Heat shock protein (HSP) is a kind of protein that is highly conserved in biological evolution and widely exists in prokaryotes and eukaryotes. Its expression increases after cells are stressed. HSP acts as a chaperone molecule in cells and participates in the processing of antigens and the formation of MHC-antigen peptide complexes. [0003] In recent years, the obvious adjuvant-like effect of the HSP family has attracted the attention of academic circles at home and abroad. Studies have shown that HSP can be released outside the cell after the cells are stressed, and induce important physiological processes such as the maturation and activation of antigen-prese...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N5/10C12N15/62A61K48/00A61K39/00A61K35/14A61P35/00
CPCA61K39/00A61K39/0011C07K14/47C07K14/4748C07K2319/00C12N5/0639
Inventor 刘书逊曹雪涛韩超峰
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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