MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof

A recombinant vector, IL-2 technology, used in gene therapy, recombinant DNA technology, and the introduction of foreign genetic material using vectors, can solve the problems of immune regulation, low anti-tumor ability, lack of immunotherapy targeting, etc. Enhance the expansion ability and cell viability, improve the effect of tumor immunotherapy, and expand the effect of anti-tumor immune effect

Active Publication Date: 2015-05-27
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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  • MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof
  • MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof
  • MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Obtaining MUC1 gene fragments containing specific enzyme cleavage sites

[0050] 1. Primer design

[0051] According to the nucleotide sequence of the MUC1 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:

[0052] MUC1 upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0053] 5'-GA AGATCT ATGACACCGGGCACCC-3' (the underlined part is the sequence of the Bgl II restriction site),

[0054] MUC1 downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0055] 5'-TT GAATTC CTACAAGTTGGCAGAAGTGG-3' (the underlined part is the sequence of EcoR I restriction site).

[0056] 2. Obtain cDNA template

[0057] RNA was extracted from human breast cancer cell MCF-7 by TRIzon method (TRIzon total RNA extraction kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number is CW0580), and r...

Embodiment 2

[0064] Example 2: Construction of pIRES2-MUC1-EGFP recombinant vector

[0065] Using restriction endonucleases Bgl II and EcoR I, digest the pIRES2-EGFP plasmid (the multiple cloning site of the plasmid contains Bgl II, EcoR I restriction sites) and the MUC1 gene fragment obtained in Example 1, respectively, to obtain Linearized pIRES2-EGFP vector after digestion and MUC1 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 to construct pIRES2-MUC1 -EGFP recombinant vector (such as image 3 shown).

[0066] Structural features of the pIRES2-EGFP plasmid (eg figure 2 Shown) It can be seen that after the MUC1 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, MUC1 and EGFP sequences were expressed separately under the same promoter.

[0067] 1. D...

Embodiment 3

[0093] Example 3: Double PCR method to obtain IL-2 gene fragment with restriction endonuclease sticky ends

[0094] According to the IL-2 gene sequence and the expected multi-cloning site on the pIRES2-EGFP plasmid vector, two pairs of primers with different lengths and sticky ends of restriction endonucleases were designed; reverse transcribed with RNA extracted from CIK cells The cDNA was used as a template, and the above-mentioned two pairs of primers were used for PCR amplification to obtain two PCR amplification products; after mixing the two PCR amplification products, denaturation and annealing were performed in turn to obtain four IL-2 gene fragments, wherein Both ends of the two IL-2 gene fragments have restriction endonuclease sticky ends, so that the IL-2 gene fragments can be directionally ligated to the intended polycloning of the plasmid vector without restriction endonuclease digestion in the site.

[0095] Compared with the traditional PCR product cloning meth...

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Abstract

The invention discloses an MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector which is sequentially connected with an MUC1 gene, an IRES (Internal Ribosome Entry Site) sequence and an IL-2 gene along the transcription direction of the vector, or is sequentially connected with the IL-2 gene, the IRES sequence and the MUC1 gene along the transcription direction of the vector, wherein the nucleotide sequence of the MUC1 gene is as shown in the sequence table SEQ ID NO:1; the nucleotide sequence of the IL-2 gene is shown in the sequence table SEQ ID NO:2; the nucleotide sequence of the IRES sequence is shown in the sequence table SEQ ID NO:3. According to the double-gene coexpression recombinant vector disclosed by the invention, the IRES sequence is adopted to connect the MUC1gene and IL-2 gene, human epithelium mucoprotein and human interleukin-2 can be simultaneously expressed in one same vector, the recombinant vector can be applied to gene immune treatment on tumors, and both the immunomodulatory effects of cell factors can be achieved, and specificity anti-tumor effects can be achieved in a targeted manner.

Description

technical field [0001] The present invention relates to a recombinant vector and its preparation method and application, in particular to a dual-gene co-expression recombinant vector and its preparation method and application. Background technique [0002] The purpose of tumor immunotherapy is not only to effectively activate and eliminate the immune response of tumor cells, but also to establish a long-term anti-tumor memory effect. Tumor immunotherapy includes two main strategies: nonspecific adjuvant immunotherapy and antigen-specific immunotherapy. Nonspecific adjuvant immunotherapy stimulates the immune system by modulating systemic or local concentrations of cytokines or co-stimulatory molecules. Antigen-specific immunotherapy, including antigen transport, expression and presentation, migration of antigen-activated T cells, and treatment with antigen-loaded DC cells, etc. [0003] Studies have shown that the expression of tumor-associated antigens and co-expression o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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