MUC1 (Mucins 1) and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) dual-gene co-expression recombinant vector and preparation method and application thereof

A GM-CSF, recombinant vector technology, applied in the field of recombinant vector and its preparation, can solve the problems of immune regulation, low anti-tumor ability, lack of immunotherapy targeting, etc., to promote T cell immune response, improve tumor immunity Therapeutic effect, the effect of enhancing the antibody response

Active Publication Date: 2015-07-15
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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  • MUC1 (Mucins 1) and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) dual-gene co-expression recombinant vector and preparation method and application thereof
  • MUC1 (Mucins 1) and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) dual-gene co-expression recombinant vector and preparation method and application thereof
  • MUC1 (Mucins 1) and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) dual-gene co-expression recombinant vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Obtaining MUC1 gene fragments containing specific enzyme cleavage sites

[0051] 1. Primer design

[0052] 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:

[0053] MUC1 upstream primer (as shown in SEQ ID NO:4 in the sequence listing):

[0054] 5'-GA AGATCT ATGACACCGGGCACCC-3' (the underlined part is the sequence of the Bgl II restriction site),

[0055] MUC1 downstream primer (as shown in SEQ ID NO:5 in the sequence listing):

[0056] 5'-TT GAATTC CTACAAGTTGGCAGAAGTGG-3' (the underlined part is the sequence of EcoR I restriction site).

[0057] 2. Obtain cDNA template

[0058] 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

[0065] Example 2: Construction of pIRES2-MUC1-EGFP recombinant vector

[0066] 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).

[0067] 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.

[0068] 1. D...

Embodiment 3

[0095] Example 3: Double PCR method to obtain GM-CSF gene fragments with restriction endonuclease cohesive ends

[0096] According to the GM-CSF gene sequence and the expected insertion multiple cloning site on the pIRES2-EGFP plasmid vector, design two pairs of primers with different lengths and sticky ends with restriction endonucleases; reverse transcribe with RNA extracted from CIK cells The cDNA of 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 GM-CSF gene fragments, wherein Both GM-CSF gene fragments have restriction endonuclease cohesive ends, which allow directional ligation of the GM-CSF gene fragments into the desired polyclonal of the plasmid vector without the need for restriction endonuclease digestion site.

[0097] Compared with the traditional PCR product cloning met...

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Abstract

The invention discloses an MUC1 (Mucins 1) and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) dual-gene co-expression recombinant vector. The MUC1 and GM-CSF dual-gene co-expression recombinant vector is characterized that an MUC1 gene, an IRES (Internal Ribosome Entry Sequence) and a GM-CSF gene are connected in sequence along the vector transcription direction, or the GM-CSF gene, IRES and MUC1 gene are connected in sequence along the vector transcription direction; a nucleotide sequence of the MUC1 gene is as shown in SEQ ID NO:1 in a sequence table; the nucleotide sequence of the GM-CSF 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 is utilized for connecting the MUC1 gene with the GM-CSF gene, and thus human polymorphic epithelial mucin and a granulocyte-macrophage colony-stimulating factor can be expressed through the same vector at the same time; the recombinant vector can be applied to immunogene therapy of tumor, enables the immune regulation effect of cytokines to be played, and can also produce specific anti-tumor effect for tumor 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] Cancer is a serious public health problem in the world. According to the data released by the International Agency for Research on Cancer, about 8 million people worldwide die of cancer every year. In the past 20 years, cancer in my country has shown a trend of younger age and higher morbidity and mortality in the "third line". The "2012 China Cancer Registration Annual Report" released by the National Cancer Registration Center shows that there are about 3.12 million new cancer cases each year, an average of 8,550 people per day, and 6 people are diagnosed with cancer every minute in the country. The national cancer mortality rate is 180.54 / 10 million, and 2.7 million deaths from cancer each year. [0003] Tumor immunotherapy...

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 曹毓琳左百乐栗炳南连杰林俊堂丰慧根
Owner HENAN HUALONG BIOLOGICAL TECH
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