Preparation and application of vaccine for curing tumor in positive carcino-embryonic antigen

A carcinoembryonic antigen and element technology, applied in the fields of biology and medicine, can solve the problem of no CEA-positive tumor therapeutic vaccine and other problems

Active Publication Date: 2006-02-01
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

[0006] However, there is no very effective CEA positive tumor therapeutic vaccine so far, therefore, there is an urgent need in this area to develop new effective CEA positive tumor therapeutic vaccines and preparation methods thereof

Method used

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  • Preparation and application of vaccine for curing tumor in positive carcino-embryonic antigen
  • Preparation and application of vaccine for curing tumor in positive carcino-embryonic antigen
  • Preparation and application of vaccine for curing tumor in positive carcino-embryonic antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Recombinant CEA 576-669 -Expression and purification of Hsp70L1 protein

[0074] 1.1 Cloning of human CEA cDNA:

[0075] Collect CEA-positive human colon cancer cells LS 174T (ATCC Number: CL-188), use total cell RNA extraction reagent Trizol (Invitrogen Company) to prepare cell total RNA, use AMV reverse transcriptase (Promega Company) to synthesize the first strand, and It is used as a template, and PCR oligonucleotide primers with the following sequences at the 5' and 3' ends are used to amplify to obtain a DNA fragment encoding CEA.

[0076] The 5' end oligonucleotide primer sequence used in the PCR reaction is:

[0077] 5'-CACGGTACCACCATGGAGTCTCCCTCGGCCCC-3' (SEQ ID NO: 1)

[0078] The primer contains an enzyme cutting site of KpnI restriction endonuclease, and behind the enzyme cutting site is a partial coding sequence of a translation initiator and CEA;

[0079] The 3' end primer sequence is:

[0080] 5'-CTCGTCGACTATCAGAGCAACCCCAACCAG-3' (SEQ ID NO...

Embodiment 2

[0116] Example 2 Preparation of a new therapeutic dendritic cell vaccine for CEA-positive tumors

[0117] 2.1 Isolation and culture of DC and T lymphocytes:

[0118] Take freshly isolated colon cancer patients (CEA + , HLA-A*0201 + ), peripheral blood mononuclear cells were separated by density gradient centrifugation of lymphocyte separation medium (Ficoll-Histopaque 1.077) (Sigma Company). Set at 37°C, 5% CO 2 Incubate the adherent monocytes for 2 hours in complete medium containing human recombinant GM-CSF (500 U / ml) (Schering Bauer) and human recombinant IL-4 (10 ng / ml) (Promega) 37 °C, 5% CO 2 Culture was carried out to obtain DCs derived from peripheral blood mononuclear cells. The non-adherent cells were suspended in RPMI 1640 medium with 5% fetal bovine serum, passed through a nylon wool column (incubated at 37° C. for 1 hour), and purified T lymphocytes were obtained.

[0119] 2.2 Sensitization of DC:

[0120] Collect DCs derived from peripheral blood mononucle...

Embodiment 3

[0122] Example 3 CEA 576-669 -Hsp70L1 fusion protein sensitized dendritic cells to induce CEA-specific T lymphocytes in vitro

[0123] 3.1 CTL killing assay:

[0124] 4 hours 51 The killing ability of CTL was detected by Cr release method. With SW480 cells (CEA + , HLA-A*0201 + ), LoVo cells (CEA + , HLA-A*0201 - ), T2 cells were used as target cells, respectively. Part of the T2 cells were suspended in 200 μl RPMI1640 medium without fetal bovine serum, and first mixed with 10 μg / ml CAP-1 (CEA 605-613 , YLSGANLNL, for CEA 576-669 An HLA-A*0201-restricted epitope contained in ) or an unrelated peptide Tyr 368-376 Incubate for 60 minutes. Add 200μCi 51 Cr (Amersham Pharmacia Company), placed in a 37°C water bath for 90 minutes, mixed gently every 15 minutes, and then washed thoroughly with medium to remove residual 51 Cr, used as the target cell for killing, was added to the corresponding prepared effector cells (CD8+ T cells from each group) according to three diffe...

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Abstract

A fusion protein CEA-Hsp70L1 of human carcino-embryonic antigen and heat shock protein 70L1, the DNA sequence for coding it, the carrier containing the DNA sequence, the host cell containing said carrier, the process for preparing said fusion protein by genetic engineering, and the composition containing the fusion protein are disclosed. A process for preparing the therapeutic vaccine of CEA positive cancer is also disclosed.

Description

technical field [0001] The present invention relates to the fields of biology and medicine. More specifically, the present invention relates to a fusion protein of human carcinoembryonic antigen (CEA) and human heat shock protein 70L1 (hereinafter referred to as Hsp70L1), a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, and a host cell containing the vector , a method for preparing the fusion protein by genetic engineering, and an application of the fusion protein in preparing CEA-positive tumor vaccines. Background technique [0002] Carcinoembryonic antigen (CEA) is a membrane-bound 180-200Kda intercellular adhesion glycoprotein, which is highly expressed on a considerable proportion of human tumor cell membranes, including more than 90% of colorectal cancer, gastric cancer, pancreatic cancer Cancer, 70% of non-small cell lung cancer and 50% of breast cancer, etc. These tumors are high-incidence tumors, and their malignancy is generally hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/08A61K39/00A61K48/00A61P35/00
CPCC07K2319/00A61K38/00A61K2039/6043C07K14/4748A61K39/0011A61K39/00A61P35/00A61K39/001182
Inventor 万涛吴艳峰周向阳李楠陈国友曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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