Recombinant human HNP gene liposome complex, preparation and use thereof

A technology of liposome complex and liposome, which is applied in the field of gene therapy, can solve the problems of little effect, achieve the effect of prolonging the therapeutic effect, reducing the cost of treatment, and inhibiting tumor angiogenesis

Active Publication Date: 2011-05-11
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effectiveness of the introduction of apoptosis genes and angiogenesis inhibitory genes has been confirmed by most studies, but the effect must depend on high transfection efficiency and sustained inhibition. The advantage of using immune genes is that only part of the transfection is required. Tumor immunity can be induced by infecting tumor cells, but it has little effect when the tumor burden is large. Therefore, an ideal strategy for gene therapy is to obtain ideal candidate genes with dual or multiple effects.

Method used

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  • Recombinant human HNP gene liposome complex, preparation and use thereof
  • Recombinant human HNP gene liposome complex, preparation and use thereof
  • Recombinant human HNP gene liposome complex, preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Construction of recombinant vector of the present invention

[0041] In this example, the RT-PCR method was used to amplify HNPs fragments from the total RNA of human lymphocytes, and the fusion gene was cloned into the target vector by means of molecular cloning. Because HNP1 and HNP2 have a high degree of similarity, the Gene Bank of NCBI is currently designated as the same gene , that is HNP1, so the present invention cloned and constructed the HNP1 mature peptide and HNP3 mature peptide fusion gene recombinant plasmid, taking HNP1 as an example:

[0042] Design and synthesize RT-PCR primers according to the human HNP1 gene sequence, insert the PCR product directly into the cloning vector-T vector (purchased from Promega Company), and use the primer sequence as follows:

[0043] Full-length HNP1 upstream primer (SEQ ID NO.6) (full-HNP1-forward):

[0044] 5'-ATAGGATCCGCCATGAGGACCCTCGC CATCCTTG-3'.

[0045] Full-length HNP1 downstream primer (SEQ ID NO.7...

Embodiment 2

[0058] Example 2 Preparation of the liposome complex of the recombinant carrier of the present invention

[0059] First, the pSec-HNP1 and pSecTag2B vector plasmids were extracted and quantified using the endotoxin-removing mass plasmid extraction kit from QIAGEN.

[0060] Then the liposome complex of HNP1 mature peptide recombinant vector was prepared as follows.

[0061] Obtain liposomes: accurately weigh DOTAP 58mg, cholesterol Chol 32mg (mol / mol=1: 1), in a 100ml rotary evaporator, add 20ml chloroform to dissolve, and then rotate on a rotary evaporator for 45 minutes to remove chloroform, After drying in a vacuum dryer for 12 hours, take it out, add a certain amount of 5% glucose solution, ultrasonicate the probe (with a power of 400W) at 60 degrees Celsius for 10 minutes, and continue to incubate the obtained liquid at 60 degrees Celsius for 1 hour to obtain the product.

[0062] Preparation of lyophilized powder injection: Mix cationic liposomes and HNP1 recombinant pla...

experiment example 3

[0065] Experimental example 3. In vitro expression ability and antitumor activity of the recombinant vector of the present invention

[0066] 1. Detection of recombinant vector expression of the present invention:

[0067] The recombinant plasmid was transfected into A549 cells, and 48 hours later, the cells were collected to extract RNA for RT-PCR. Only the cells transfected with the pSec-HNP1 plasmid could amplify the HNP1 mature peptide gene fragment, while those transfected with the simple pSecTag plasmid, and those without The RNA of the transfected A549 cells could not amplify the target band ( image 3 A).

[0068] In order to detect the secretion of HNP1 and the expression level of HNP1 in the supernatant, the cell supernatants were collected 24 and 48 hours after transfection, and detected by ELISA. The results showed that the amount of HNP1 in the supernatant 24 hours after transfection was about 102 ng / ml, and the amount of HNP1 mature peptide 48 hours after trans...

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Abstract

The invention relates to a recombinant human HNP gene liposome complex, the preparation method and the usage thereof, which belong to the gene therapy field. The utility model solves the technical problem that a new tumor gene therapeutic drug is provided. The eukaryotic expression vector contains coding genes which can express human alpha-defensin mature peptide, and can be prepared into liposome complex. The eukaryotic expression vector and the liposome complex thereof can express in a tumor and excrete human alpha-defensin mature peptide with cytotoxic activity, thus plays an obvious role of anti-tumor effect, the default that the activity of the human alpha-defensin mature peptide is easy to be influenced by serum protein and calcium ion concentration is avoided, thus the invention provides a new choice for the gene therapy of the tumor.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a recombinant vector of the alpha-defensin (HNP) mature peptide coding sequence, a liposome complex, and a preparation method and application thereof. Background technique [0002] Human alpha defensins (α-defensins) are cationic active polypeptides (Human neutrophil peptides, HNPs) derived from the azurophilic blue granules of human neutrophils, with a molecular weight of 4-5KD and containing six conserved cysteine amino acid residues, forming three typical intramolecular disulfide bonds (Ganz and Lehrer 1995). There are four kinds of α-defensins (HNPs) discovered so far, but there are three main functions: HNP1, HNP2, and HNP3. The gene structure can be roughly divided into three parts, encoding signal peptide, precursor peptide and mature peptide respectively. Taking HNP1 as an example, a polypeptide of 94 amino acid residues (prepro-HNP1) was first synthesized. After the signal p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N15/861C12N15/12A61K48/00A61K9/127A61K9/19A61K47/14A61P35/00
Inventor 魏于全王永生杨莉
Owner SICHUAN UNIV
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