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NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine

A eukaryotic expression vector, DNA vaccine technology, applied in recombinant DNA technology, DNA/RNA fragments, applications, etc., can solve problems such as T cell clone apoptosis

Inactive Publication Date: 2008-01-02
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only dual-signal co-stimulation can effectively activate specific T cells, and the lack of the second signal causes T cell clone apoptosis and makes tumor cells immune escape

Method used

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  • NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine
  • NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine
  • NDA vaccine eucaryon expression carrier and application in preparation of gene vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the construction of DNA vaccine vector pVAX1-IRES

[0081] 1. PCR amplification and purification of RES coding sequence

[0082] First, design its PCR amplification primers according to the IRES coding sequence in the pIRES-neo vector (the position of the IRES sequence is 1295-1880bp), and add a restriction internal to the upstream primer IRES-F for PCR amplification of the IRES coding sequence Dicer EcoR V, Cla I, BstB I, Pvu I, and Psi I recognition sites, restriction enzymes BssH II, Swa I, Pac added to the downstream primer IRES-R for PCR amplification of the IRES coding sequence I, Nsi I and Xho I recognition sites, specific primer sequences are as follows: IRES-F (upstream primer): 5'-CCGATATCGATTCGAACGATCGTTATAACTAGGGCGGCCAATTCCGCC-3'IRES-R (downstream primer): 5'-GCCTCGAGCGCGCATTTAAATTAATTAATGCATTGGCTGCAGGAATTATCCCG-3'. After the primers are synthesized, use sterilized distilled water to prepare a 20 μmol / L use solution, aliquot them, and store th...

Embodiment 2

[0151] Example 2, Verification of the expression function of the eukaryotic DNA vaccine vector pVAX1-IRES

[0152] In order to check whether the eukaryotic DNA vaccine vector pVAX1-IRES constructed in Example 1 has the function of co-expressing two genes at the same time, the red fluorescent protein gene was first amplified from the red fluorescent protein expression vector pDsRed1-N1 (Clontech Company) DsRed1 (EF 517319), the enhanced green fluorescent protein gene EGFP (EF 587314) was amplified from the enhanced green fluorescent protein expression vector pEGFP-N1 (Clontech Company), and then the amplified DsRed1 gene fragment and EGFP gene fragment The upstream and downstream of the IRES coding sequence were respectively inserted into the pVAX1-IRES vector to obtain recombinant expression plasmids pVAX1-IRES-DsRed1, pVAX1-IRES-EGFP, and pVAX1-DsRed1-IRES-EGFP. These three recombinant eukaryotic expression plasmids were transiently transfected into 293T human embryonic kidne...

Embodiment 3

[0255] Example 3, Construction and eukaryotic expression of multi-target compound antigen anti-tumor gene vaccine

[0256] 1. Design of antigen complex 2PAG fusion gene

[0257] Survivin mainly stimulates cellular immune response. Survivin mRNA can produce three isoforms through different cutting methods in vivo: Survivin, Survivin-2B and Survivin-EX3, among which the 80th-88th amino terminal of Survivin-2B Anti-tumor vaccines with amino acid residues as specific targets have entered phase II clinical trials. A large number of literature investigations found that the T cell antigen epitope of Survivin-2B is mainly concentrated between amino acid residues 5-28 and 80-121 from the amino terminal, so these two regions were selected as the multi-target complex of the present invention. Antigen is one of the targets of anti-tumor gene vaccine. Find the nucleotide sequence (134-205, 359-513) corresponding to the above two regions in human Survivin 2B (NM-001012271) from Genebank, ...

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Abstract

The invention discloses a eucaryon expression vector of a DNA vaccine and the application. The eucaryon expression vector of DNA vaccine is a recombination pVAX1 eucaryon expression vector which is got by adding the internal ribosome in pVAX1 vector skeleton and entering the site coded sequence. The eucaryon expression vector may show the wide spectrum curing function for breast cancer, pancreatic cancer, restocarcinoma and multi-type malignancy solid tumor, which has good humoral immunity and cell-mediated immunity effect, and inhibits the growth of the tumor.

Description

technical field [0001] The invention relates to a vector and its application, in particular to a DNA vaccine eukaryotic expression vector and its application in preparing an anti-tumor isogenic vaccine. Background technique [0002] At present, malignant tumors have become one of the most important killers that threaten human health. Therefore, it is of great significance to strengthen the research on the prevention and treatment of malignant tumors. Currently, surgical resection, chemotherapy, and radiotherapy are still the main treatments for malignant tumors. Although the traditional surgical treatment method can improve the survival rate of patients, the recurrence rate is still more than half after 5 years after surgery. In addition to severe toxic side effects of chemotherapy and radiotherapy, many tumors such as kidney cancer are insensitive or resistant to it. Tumor metastasis and recurrence are important factors affecting the prognosis of patients, and they are al...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/62C12N15/12A61K48/00A61P35/00
Inventor 于继云阎瑾琦贾锐王浩张亮刘宁
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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