The rna, recombinant and its application for inhibiting human ferroportin

A technology of ferroportin and recombinants, which is applied in the field of retrovirus interference system and drug development and preparation of iron metabolism-related diseases, can solve problems such as inability to achieve therapeutic effects, achieve treatment of iron metabolism-related diseases, and reduce serum iron levels , the effect of inhibiting expression

Inactive Publication Date: 2011-12-07
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Many congenital or acquired iron-related diseases are related to FPN1. Traditional drugs cannot achieve therapeutic effects, or can only relieve symptoms of patients. Therefore, there is an urgent need to develop new drug delivery forms

Method used

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  • The rna, recombinant and its application for inhibiting human ferroportin
  • The rna, recombinant and its application for inhibiting human ferroportin
  • The rna, recombinant and its application for inhibiting human ferroportin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] According to the human ferroportin (FPN1) sequence (NM_000617.1), the template strand and coding strand were designed. Add BglII and XhoI restriction sites to the 5' end respectively, and send the sequence to Invitrogen Company for synthesis. After screening, the FPN1 interference fragment sequence of the present invention with the most significant interference effect is obtained as follows:

[0058]Template strand (Seq ID No.3):

[0059] 5'-GATCCCC AGACTATAATGATAACACT TTCAAGAG AGTGTTATCA TTATAGTCT TTTTTTA-3'

[0060] Coding chain (Seq ID No.4):

[0061] 5’-AGCTTAAAAA AGACTATAATGATAACACT TCTCTTGA AGTGTTATCATTATAGTCT GGGG-3'

Embodiment 2

[0063] Retro-FPN1i system construction

[0064] 1. Anneal the synthesized FPN1 interference fragments (Seq ID No.3 and Seq ID No.4). Specific steps are as follows:

[0065] 1. Establish an annealing system

[0066] 5ul 10x annealing buffer

[0067] 2nmole template chain Seq ID No.3

[0068] 2nmole coding chain Seq ID No.4

[0069] Make up to 50ul with triple distilled water.

[0070] 2. Put it into the PCR instrument, 95°C for 5 minutes, 75°C for 5 minutes, 55°C for 5 minutes, 42°C for 5 minutes, and 37°C for 15 minutes, then gradually decrease the temperature to room temperature.

[0071] 3. Make 12% 1x TAE polyacrylamide glue, take 10ul of the above annealed product and run the glue for 200V1h. The annealed bands were observed by silver staining. Store returned products at -80°C.

[0072] 3. Enzyme cutting (XbaI and XhoI) retroviral vector ( figure 1 shown), after gel purification and recovery, T4 ligase was used to ligate the annealed product at room temperature f...

Embodiment 3

[0091] Production process of recombinant retrovirus Retro-FPN1i

[0092] 1. Inoculate Phoenix cells 1.5X10 7 cells in a 9cm culture dish (Corning), 37°C, 5% CO 2 Culture overnight;

[0093] 2. Change the fresh medium 20 minutes before transfection;

[0094] 3. Mix 20ul of the above-mentioned Retro-FPN1i with 500ul of DMEM medium and mark it as tube A. Take another 20ul of liposome and mix it with 500ul DMEM medium and mark it as tube B. Mix tube A and tube B, place at room temperature for 30 minutes, gently add the mixture to the culture medium of the 9cm petri dish in step 1, and incubate overnight;

[0095] 5. After 24 hours, add 7.5ml medium to continue culturing for 48 hours;

[0096] 6. Collect the cell supernatant and filter it with a 0.45um filter. The cells can be expanded and cultured, and the virus supernatant can be collected continuously.

[0097] 7. Measure the titer of the virus supernatant, adjust the cell titer to 1000VP / ml with PH8.0 Tris-HCl.

[0098] 8...

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Abstract

The invention discloses a short-interfering RNA for inhibiting human ferroportin from being expressed. The invention further discloses a DNA for encoding the corresponding shRNA of the short-interfering RNA and a recombinant capable of expressing the RNA. The short-interfering RNA, the DNA and the recombinant can effectively inhibit the expression of the human ferroportin inside body, as well as the release and the catabolism of iron and adjust the serum iron level inside body, thereby achieving the purpose of curing the related diseases of iron metabolism.

Description

technical field [0001] The invention relates to the technical fields of molecular biology, biomedicine and genetic engineering, in particular to the construction of a retrovirus interference system (Retrovirus) for the gene of human ferroportin (FPN1) by utilizing retrovirus (Retrovirus)-mediated RNA interference technology. -FPN), and apply it to the drug development and preparation of diseases related to iron metabolism. Background technique [0002] RNAi refers to the phenomenon that when a double-stranded RNA homologous to an endogenous mRNA coding region is introduced into a cell, the mRNA is degraded and gene expression is silenced. [0003] The process of RNA interference mainly has two steps: (1) long double-stranded RNA is cut into short double-stranded RNA of 21-23 base pairs by cell-derived double-stranded RNA-specific nuclease, called small interfering RNA ( small interfering RNA, siRNA). (2) Small interfering RNA forms a complex with some cell-derived enzymes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/867A61K31/713A61K31/7088A61K48/00A61P3/00C12N15/113
Inventor 钱忠明葛啸虎柯亚
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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