Recombination ferroportin adenovirus, preparation method and application thereof

A technology of ferroportin and adenovirus, which is applied in the field of recombinant human ferroportin adenovirus, can solve the problems that drugs cannot achieve the therapeutic effect, and achieve the effect of wide host range, simple process and low pathogenicity

Inactive Publication Date: 2011-05-18
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Many congenital or acquired iron-related diseases are related to FPN. Traditional drugs cannot achieve therapeutic effects, or can only relieve symptoms of patients. Therefore, there is an urgent need to develop new forms of drug delivery

Method used

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  • Recombination ferroportin adenovirus, preparation method and application thereof
  • Recombination ferroportin adenovirus, preparation method and application thereof
  • Recombination ferroportin adenovirus, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] ferroportin gene insertion recombination

[0075] As shown in Figure 1, pTrack-CMV is a shuttle plasmid of recombinant adenovirus. We connected the two ends of ferroportin gene with Bgl II and Sal I restriction sites, and added polyA tail as the termination signal region. Digest the ferroportin gene and the shuttle plasmid, and connect and transform them with T4 ligase. After screening, the correct clone is obtained and sent to the sequencing company for sequencing identification.

[0076] Ad-FPN structure: The recombinant human ferroportin adenovirus (Ad-FPN) vector lacks the E1 region, and cannot be packaged into mature virus particles in ordinary cells, which is a defective virus. The human ferroportin gene was the target gene. We inserted the human channel-type ferroportin gene into the genome of the virus, with double CMV as its promoter, green fluorescent protein (GFP) as its marker, and the downstream polyA tail as its termination signal region.

Embodiment 2

[0078] Preparation of recombinant human ferroportin adenovirus

[0079] The production process of recombinant human ferroportin adenovirus (Ad-FPN) includes: recombinant vector construction, vector linearization preparation, transfection of HEK293 cells, expanded culture, separation and purification, freeze-drying and packaging preparations. See the following detailed description and its production process flow chart (see Figure 2) for details.

[0080] 1. Obtaining the target gene ferroportin

[0081] Using human genome cDNA as a template, Pfu high-fidelity enzyme PCR amplification. PCR thermal amplification conditions were denaturation at 95°C for 5 minutes, 30 cycles at 95°C for 80 s, 58°C for 100 s, and 72°C for 120 s, and finally, incubation at 72°C for 5 min. The product is about 1710bp.

[0082] Its primer sequence is:

[0083] Upstream primer (Seq ID No.2):

[0084] CTCAC AGATCT ATGACCAGGG CGGGAGATCA

[0085] Downstream primer (Seq ID No.3):

[0086] TCAAT GTCGA...

Embodiment 3

[0177] Ad-FPN system infection and expression experiment

[0178] 1. Inoculate 1.5×10 C6 cells 7 cells in a 9cm culture dish (Corning), 37°C, 5% CO 2 Culture overnight;

[0179] 2. Change 5ml serum-free medium before infection;

[0180] 3. Add 0.5ul of the purified virus prepared in Example 2 to each petri dish;

[0181] 4. Culture 0h, 6h, 12h, 24h, 48h respectively

[0182] 5. Collect cells according to time points;

[0183] 6. Real-time PCR and western-blot quantitative detection of ferroportin expression at different time points;

[0184] 7. Statistical results.

[0185] The results are shown in Figures 3, 4, and 5. The expression of ferroportin mRNA reached 15 times that of the blank control group at about 12 hours, and continued to express.

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Abstract

The invention discloses a recombinant human ferroportin adenovirus (Ad-FPN) comprising ferroportin gene and adenovirus vector. The invention also discloses a preparation method and an application of the recombinant human Ad-FPN. The invention introduces the ferroportin gene into tissue cells of human body using deficient adenovirus, so that virus can express the ferroportin in patient body.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant human ferroportin adenovirus capable of autonomously expressing ferroportin in organisms by using a recombinant adenovirus vector to carry the human ferroportin gene, Its preparation method and application. Background technique [0002] In 1831, Fordisch proved that the blood iron content of patients with iron deficiency anemia was lower than that of healthy people, and confirmed that iron is one of the essential trace elements for the human body. Iron is widely involved in the metabolic process of the body, such as the synthesis of hemoglobin, myoglobin, hormones, and DNA, etc., and can also enhance the bactericidal and phagocytic functions of neutrophils, and maintain the proliferation, differentiation, and antibody production of T and B cells (Folin et al. 1991). For a long time, people's relationship between iron and health has focused on t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/12A61K48/00A61P3/00
Inventor 钱忠明葛啸虎汪程远
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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