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CCA gene vaccine, and its establishing method and use for preparing vaccine for preventing and treating tumour

A gene vaccine and tumor vaccine technology, applied in gene therapy, antitumor drugs, pharmaceutical formulations, etc., can solve problems such as unsatisfactory effects, killing tumors, and inability to achieve specificity, and achieve good preventive effects.

Active Publication Date: 2005-05-04
中山大学中山医学院科技开发中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment of CEA tumors (carcinoembryonic antigen-positive tumors, such as about 90% of gastrointestinal malignant tumors and pancreatic cancer, more than 80% of non-small cell lung cancer, and about 50% of breast cancer) mainly adopts chemotherapy, radiotherapy and biological therapy. Immunotherapy, etc., but because the purpose of specifically killing tumors cannot be achieved, the effect is not satisfactory. The number of deaths due to CEA tumors is more than 60% of all tumor deaths every year. It is a common tumor that seriously endangers human health.
[0004] At present, there is no report on the use of gene vaccines with synchronous expression of cytokines in CEA tumors for the prevention and treatment of CEA tumors

Method used

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  • CCA gene vaccine, and its establishing method and use for preparing vaccine for preventing and treating tumour
  • CCA gene vaccine, and its establishing method and use for preparing vaccine for preventing and treating tumour

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1.1 Extraction and identification of total RNA from CEA tumor tissue: according to Invitrogen Concert TM Cytoplasmic RNA Reagent is a one-step method for extracting total cellular RNA. The total RNA extract was identified by 1% formaldehyde gel electrophoresis, and the OD was measured with a UV spectrophotometer 260 and OD 280 value and stored at -70°C for future use.

[0019] 1.2 Primer synthesis and RT-PCR amplification of cDNA encoding human CEA: The full-length cDNA sequence of M17303 published by GenBank was used as a template for primer design, and EcoR I and Not I restriction endonuclease sites were introduced respectively, and the GeneFisher online Screen and analyze the GC ratio, Tm value, hairpin structure, motif structure and specificity of the designed PCR primers through molecular biology software PCRDESN and Oligo5.0, and synthesize the following primers:

[0020] Sense primer: 5'GTCGAATTCAGACAGCAGAGACCATGGA-3'

[0021] Antisense primer: 5'AAGCGGCCGCA...

Embodiment 2

[0058] Example 2 After in vitro transfection with CHO (Chinese hamster ovary cell line), the expression of CEA antigen and hIL-2 was detected

[0059] 2.1 CHO cell recovery, culture, and liposome transfection CHO cells taken out of liquid nitrogen were quickly placed in a 37°C water bath to thaw and recover, centrifuged at 2000rpm for 5min, discarded the supernatant, and added RPMI 1640 cells containing 10% newborn calf serum Medium (without antibiotics), 37°C and 5% CO 2 cultivated in. Digest the adherent monolayer cells that grow to more than 85% with 0.025% trypsin, add the culture medium and blow the cells, centrifuge at 1000rpm for 10min, count the cells and pass them in a 6-well plate for 24h after dilution, and then add about 4μg of the recombinant plasmid pVCEA2 (mixed in 250 μl of serum-free medium) and 10 μl of lipofectAMINE2000 (mixed in 250 μl of serum-free medium) were gently mixed and transfected into CHO cells. At the 24th and 48th hour, 200 μl of the culture ...

Embodiment 3 Embodiment 1

[0072] The CEA antibody level detection of the immunized mice of the CEA gene vaccine of embodiment 3 embodiment 1

[0073] 3.1 Experimental method: Secondary male BALB / C mice, 6-8 weeks old, were randomly divided into five groups, with 6 mice in each group.

[0074] I pVAX1 control group

[0075] II pVCEA experimental group (pVAX1-CEA-hIL-2)

[0076] III pVCEA2 experimental group (i.e. the genetic vaccine that embodiment 1 obtains)

[0077] IV mixed experimental group (CEA-rV+pVCEA2)

[0078] V compound experimental group (pVCEA2+pV35-I-40)

[0079] Before each immunization of the mice, inject 0.25% bupivacaine into each 50 μl of the quadriceps femoris of the two hind legs, and then inject 50 μg of the plasmid on each side after 24 hours (the medicine for the first immunization injection in the mixed group is CEA-rV, Dosage is 5×10 6 PFU, pVCEA2 for the second and third times; 50 μg for each component in the compound group). On the 25th day and the 45th day, they were i...

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Abstract

The present invention relates to a CEA gene vaccine, its construction method and application in preparation of vaccine for preventing and curing tumor. It is characterized by that it utilizes eukaryotic cell expression plasmid to construct the carcion-embryonic antigen (CEA) gene vaccine with interleukin 2 coordinate expression. The detection shows that in the external cell it can implement coordinate expression of CEA and IL-2 protein molecule, and the animal model tests show that the CEA gene vaccine has the action of preventing and curing carcion-embryonic antigen positive tumor, and in the body it has not immunotoxic effect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the construction of a gene vaccine for carcinoembryonic antigen-positive tumors and its application in preparing and preventing tumor vaccines. Background technique [0002] DNA vaccine (DNA vaccine), also known as gene vaccine (Gene vaccine), its essence is to clone the gene fragment encoding foreign protein on the eukaryotic plasmid expression vector, and directly inoculate the body to express the foreign protein in the cell and induce the body to produce Specific cellular and humoral immune responses to prevent and treat diseases. Because it can overcome some of the defects of traditional vaccines, can simultaneously induce the body to produce specific cellular and humoral immune responses, and has the advantages of stable properties, it is considered to be the next-generation vaccine after attenuated vaccines, inactivated vaccines and genetically engineered subunit vaccines...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K48/00A61P35/00C12N15/70C12N15/79C12P19/34
CPCY02A50/30
Inventor 罗超权黄爱强
Owner 中山大学中山医学院科技开发中心
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