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Recombinant mIL-22BP vector, and liposome complex and preparation methods and application thereof

A technology of IL-22BP, liposome complex, applied in the directions of botanical equipment and methods, biochemical equipment and methods, liposome delivery, etc., can solve the problem of liposomes with no mouse IL-22BP gene. Compounds, etc.

Inactive Publication Date: 2019-08-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no related reports on liposome complexes of murine IL-22BP gene

Method used

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  • Recombinant mIL-22BP vector, and liposome complex and preparation methods and application thereof
  • Recombinant mIL-22BP vector, and liposome complex and preparation methods and application thereof
  • Recombinant mIL-22BP vector, and liposome complex and preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of mIL-22BP gene expression plasmid

[0042] 1. Acquisition of mIL-22BP gene

[0043] 1) According to the coding sequence of the mIL-22BP gene (Gene ID: 237310, NM_178258) in the NCBI (National Center for Biotechnology Information) database (sequence shown in SEQ ID NO.1), synthesize a full-length mIL cDNA plasmid of -22BP gene sequence.

[0044] 2) Design and synthesize corresponding primers

[0045] Upstream primer: 5'-GGGAAAGCTTATGATGCCTAAGCATTGCCTTTCTAGG-3', (SEQ ID NO.4)

[0046] The design introduces a Hind III restriction site;

[0047] Downstream primer 5'-GGGATCTAGATCATGGAATGTGCACACATCTCTCC-3', (SEQ ID NO.5)

[0048] The Xbal I restriction site was designed and introduced, the DNA fragment of mIL-22BP was obtained by PCR amplification, and the fragment was recovered by agarose gel electrophoresis to obtain the mIL-22BP gene fragment.

[0049] 2. Cloning construction of pVAX1-mIL-22BP plasmid

[0050] 1) Use Hind III and Xbal I restr...

Embodiment 2

[0052] Example 2 Preparation of recombinant mIL-22BP carrier-liposome complex

[0053] 1. Preparation of DOTAP-Chol cationic liposomes

[0054] The cationic lipid DOTAP and cholesterol (Chol) were mixed at a molar ratio of 1:1, and the mixture was dissolved in chloroform, and placed on a rotary evaporator at 60°C for 120 minutes to form a film. The formed film was taken out and dissolved in 5% glucose solution, then shaken in a water bath at 60°C for 20 minutes, and the mixture was ultrasonically disrupted at 0°C at 400w for 10 minutes to obtain a 5 mg / ml cationic liposome suspension.

[0055] 2. Preparation of mIL-22BP gene expression plasmid

[0056] The pVAX1-mIL-22BP plasmid was transformed into DH5α E. coli competent cells and spread on LB plates containing kanamycin resistance. After 24 hours, the clones were picked and placed in 3 mL of LB medium containing kanamycin for shaking overnight, and the pVAX1-mIL-22BP plasmid recombinant plasmid was extracted the next day. ...

Embodiment 3

[0059] Example 3 Recombinant mIL-22BP carrier liposome complex anti-tumor test

[0060] In order to study the anti-tumor effect of the pVAX1-mIL-22BP plasmid cationic liposome complex in vivo, a mouse model of peritoneal metastases of colon cancer was established in the peritoneal cavity of Balb / C mice (6-8 weeks old, female). Specifically, the CT26 mouse colon cancer cells cultured in vitro were digested with trypsin, and the volume was fixed in 1640 medium without serum and antibiotics, and 2.5×10 5 cells, after 3 days of cell inoculation, began to carry out random grouping treatment (every group of 5) as follows;

[0061] A) blank control group: 5% glucose solution;

[0062] B) simple cationic liposome group;

[0063] C) pVAX1-mIL-22BP treatment group: the cationic liposome / pVAX1-mIL-22BP complex was placed in 5% glucose solution.

[0064] Treat by intraperitoneal injection, the plasmid DNA cationic liposome complex ratio is as follows: plasmid DNA (5 μ g): cationic lipo...

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Abstract

The invention belongs to the field of tumor gene therapy, and specifically relates to a recombinant mouse mIL-22BP vector and preparation methods and application of a liposome complex thereof. The invention, in view of the problem that a liposome complex having the murine IL-22BP gene has not been reported so far, provides the recombinant murine IL-22BP vector and the liposome complex thereof. Thevector contains a gene encoding mouse IL-22BP protein and can be used for expressing mouse IL-22BP protein in eukaryotic cells; after vector is combined with a cationic liposome to form the complex,the complex can well play a role of resisting colon cancer tumor tissue growth, has a tumor growth resisting effect, can be used for preparing drugs for diagnosis or treatment of tumors, and providesa new choice for tumor treatment.

Description

technical field [0001] The invention belongs to the field of tumor gene therapy, and specifically relates to a recombinant mIL-22BP carrier and its liposome complex, as well as their preparation method and application. Background technique [0002] Gene therapy is one of the important means of tumor biotherapy, and several gene therapy products have played a role in the clinical treatment of tumors. As an important part of gene therapy drugs, therapeutic genes used for tumor treatment achieve anti-tumor therapeutic effects through mechanisms such as inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and regulating anti-tumor immunity. To further find and develop genes with good therapeutic effects is an important issue that still needs to be solved in tumor gene therapy. [0003] Interleukin is recognized as one of the most important regulators of innate immunity and adaptive immunity related diseases. Interleukin-22 (Interleukin-22, IL-22) is mainly secr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/66C12N15/88A61K38/17A61K9/127A61P35/00
CPCC07K14/7155C12N15/85C12N15/88A61K38/1709A61K9/127A61P35/00
Inventor 门可段醒妹魏于全
Owner SICHUAN UNIV
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