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GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) and MART-1 (Melanoma Antigen Recognized By T-Cells 1) dual-gene co-expression recombinant vector and preparation method and application thereof

A technology of GM-CSF and MART-1, which is applied in gene therapy, recombinant DNA technology, and the use of vectors to introduce foreign genetic material, etc., can solve the problems of lack of immunotherapy targeting, immune regulation, and low anti-tumor ability. Achieve good melanoma immunotherapy effect, promote T cell immune response, and enhance the body's immune regulation ability

Active Publication Date: 2014-02-26
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current GM-CSF gene immunotherapy method only locally increases the expression level of GM-CSF protein in the body, its immune regulation effect and anti-tumor ability are small, and it lacks the targeting of immunotherapy

Method used

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  • GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) and MART-1 (Melanoma Antigen Recognized By T-Cells 1) dual-gene co-expression recombinant vector and preparation method and application thereof
  • GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) and MART-1 (Melanoma Antigen Recognized By T-Cells 1) dual-gene co-expression recombinant vector and preparation method and application thereof
  • GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) and MART-1 (Melanoma Antigen Recognized By T-Cells 1) dual-gene co-expression recombinant vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Obtaining GM-CSF Gene Fragments Containing Specific Restriction Sites

[0071] 1. Primer design

[0072] According to the nucleotide sequence of the GM-CSF gene (as shown in SEQ ID NO: 1 in the sequence listing) and the expected insertion multiple cloning site on the pIRES2-EGFP plasmid vector, design specific primers as follows:

[0073] GM-CSF upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0074] 5'-CG GAATTC ATGTGGCTGCAGA-3' (the underlined part is the EcoR I restriction site sequence),

[0075] GM-CSF downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0076] 5'-TT GGATCC TCACTCCTGGACTGGCT-3' (the underlined part is the sequence of the restriction site of BamH I).

[0077] 2. Obtain cDNA template

[0078]TRIzon method was used to extract RNA from CIK cells (Cytokine-Induced Killer, cytokine-induced killer cells) or mononuclear cells isolated from human peripheral blood (TRIzon total RNA extraction kit was p...

Embodiment 2

[0085] Example 2: Construction of pIRES2-GM-CSF-EGFP recombinant vector

[0086] Using restriction endonucleases EcoR I and BamH I, digest the pIRES2-EGFP plasmid (the multiple cloning site of the plasmid contains EcoR I and BamH I restriction sites) and the GM-CSF gene fragment obtained in Example 1, respectively , to obtain the linearized pIRES2-EGFP vector after digestion and the GM-CSF gene sequence after digestion; use T4 DNA ligase system for ligation reaction, incubate at 22°C for 30 minutes, and then inactivate at 70°C for 5 minutes, Construct the pIRES2-GM-CSF-EGFP recombinant vector (such as image 3 shown).

[0087] Structural features of the pIRES2-EGFP plasmid (eg figure 2 Shown) It can be seen that after the GM-CSF gene is inserted into the multiple cloning site of the pIRES2-EGFP plasmid, it is located upstream of the plasmid vector's own sequence IRES (such as image 3 shown), that is, the GM-CSF and EGFP sequences were expressed separately under the same p...

Embodiment 3

[0115] Example 3: Double PCR method to obtain the MART-1 gene fragment with restriction endonuclease cohesive ends

[0116] According to the MART-1 gene sequence and the expected insertion multiple cloning site on the pIRES2-EGFP plasmid vector, two pairs of primers with different lengths and sticky ends of restriction endonucleases were designed; RNA extracted from melanoma cell A375 was reversed The transcribed cDNA was used as a template, and the above two pairs of primers were used for PCR amplification to obtain two PCR amplification products; the two PCR amplification products were mixed and then denatured and annealed sequentially to obtain four MART-1 gene fragments , where the two ends of the two MART-1 gene fragments have restriction endonuclease cohesive ends, so that the MART-1 gene fragments can be directionally connected to the plasmid vector without restriction endonuclease digestion. in the multiple cloning site.

[0117] Compared with the traditional PCR prod...

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Abstract

The invention discloses a GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) and MART-1 (Melanoma Antigen Recognized By T-Cells 1) dual-gene co-expression recombinant vector. The GM-CSF and MART-1 dual-gene co-expression recombinant vector is characterized that a GM-CSF gene, an IRES (Internal Ribosome Entry Sequence) and an MART-1 gene are connected in sequence along the vector transcription direction, or the MART-1 gene, IRES and GM-CSF gene are connected in sequence along the vector transcription direction; a nucleotide sequence of the GM-CSF gene is as shown in SEQ ID NO:1 in a sequence table; the nucleotide sequence of the MART-1 gene is as shown in SEQ ID NO:2 in the sequence table; the nucleotide sequence of the IRES is as shown in SEQ ID NO:3 in the sequence table. According to the dual-gene co-expression recombinant vector, the IRES sequence is utilized for connecting the GM-CSF gene with the MART-1 gene, and thus human melanoma differentiation antigen and the granulocyte-macrophage colony-stimulating factor can be expressed in the same vector at the same time; the recombinant vector can be applied to immunogene therapy of melanoma, enables the immune regulation effect of cytokines to be played, and can also produce specific anti-tumor effect for malignant melanoma in a targeting way.

Description

technical field [0001] The invention relates to a recombinant vector and its preparation method and application, in particular to a double-gene co-expression recombinant vector and its preparation method and application. Background technique [0002] Melanoma (Melanoma), also known as malignant melanoma (Malignant Melanoma, MM), is a malignant tumor derived from neural crest melanocytes. It is a malignant tumor with the highest mortality rate among surface tumors. In recent years, the incidence and corresponding mortality of melanoma are increasing rapidly, and the age of onset is getting earlier and earlier, which seriously threatens human health. [0003] The traditional treatment of melanoma is a comprehensive treatment including surgery, chemotherapy, and immunotherapy. At present, the only effective treatment method is surgical resection of the tumor when the tumor thickness is less than 1 mm, but due to the high degree of invasion and metastasis of melanoma and resis...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66A61K48/00A61P35/00
Inventor 左百乐曹毓琳栗炳南林俊堂丰慧根连杰
Owner HENAN HUALONG BIOLOGICAL TECH
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