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IL-2 and MART-1 dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector

A technology of MART-1 and IL-2, which is applied in the field of recombinant vector and its preparation, can solve the problems of immune regulation, low anti-tumor ability, lack of targeting of immunotherapy, etc., and achieve enhanced amplification ability and cell viability, Improve the effect of melanoma immunotherapy and expand the effect of anti-tumor immune effect

Active Publication Date: 2014-02-05
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current IL-2 gene immunotherapy method only locally increases the expression level of IL-2 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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  • IL-2 and MART-1 dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector
  • IL-2 and MART-1 dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector
  • IL-2 and MART-1 dual-gene co-expression recombinant vector as well as preparation method and application of recombinant vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Obtaining IL-2 Gene Fragments Containing Specific Restriction Sites

[0071] 1. Primer design

[0072] According to the nucleotide sequence of the IL-2 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] IL-2 upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0074] 5'-CCG GAATTC ATGTACAGGATGCAACTCC-3' (the underlined part is the EcoR I restriction site sequence), IL-2 downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0075] 5'-CGC GGATCC TCAAGTCAGTGTTGAGATGATGC-3' (the underlined part is the restriction site sequence of BamH I).

[0076] 2. Obtain cDNA template

[0077]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 purchased fro...

Embodiment 2

[0084] Example 2: Construction of pIRES2-IL-2-EGFP recombinant vector

[0085] 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 IL-2 gene fragment obtained in Example 1, respectively , to obtain the linearized pIRES2-EGFP vector and the IL-2 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-IL-2-EGFP recombinant vector (such as image 3 shown).

[0086] Structural features of the pIRES2-EGFP plasmid (eg figure 2 shown), it can be seen that after the IL-2 gene is inserted into the multiple cloning site of the pIRES2-EGFP plasmid, it is located upstream of the self-sequence IRES of the plasmid vector (such as image 3 shown), that is, IL-2 and EGFP sequences were expressed separately under the same promoter.

[0087] 1. Dou...

Embodiment 3

[0113] Example 3: Double PCR method to obtain the MART-1 gene fragment with restriction endonuclease cohesive ends

[0114] 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.

[0115] Compared with the traditional PCR prod...

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Abstract

The invention discloses an IL-2 and MART-1 dual-gene co-expression recombinant vector, which is sequentially linked with an IL-2 gene, an IRES sequence and an MART-1 gene in a vector transcription direction, or is sequentially linked with an MART-1 gene, an IRES sequence and an IL-2 gene in a vector transcription direction, wherein nucleotide sequence of the IL-2 gene is represented in SEQ ID NO: 1 in a sequence list, nucleotide sequence of the MART-1 gene is represented in SEQ ID NO: 2 in the sequence list, and nucleotide sequence of the IRES sequence is represented in SEQ ID NO: 3 in the sequence list. The dual-gene co-expression recombinant vector, which links the IL-2 gene and the MART-1 gene through the IRES sequence, can simultaneously express a human melanin differentiation antigen and interleukin-2; the recombinant vector, in immunogene therapy of melanin, not only can develop an immune regulating function of a cell factor but also can generate a specific anti-tumor effect aiming at the melanin.

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): C12N15/85C12N15/66A61K48/00A61P35/00
Inventor 栗炳南丰慧根左百乐林俊堂曹毓林
Owner XINXIANG MEDICAL UNIV
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