RNA and recombinant for inhibiting human DMT1 protein and application thereof

A recombinant and transporter technology, applied in the fields of biomedicine, genetic engineering, and molecular biology, can solve problems such as interfering with the normal function of DMT1, inhibit iron absorption and metabolism, treat iron metabolism-related diseases, and reduce serum iron levels Effect

Inactive Publication Date: 2009-09-23
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

The fourth transmembrane region is an important functional region, if a mutation occurs, it will seriously interfere with the normal function of DMT1
Previous pathological studies have shown that the substantia nigra is the area of ​​abnormal excessive iron deposition in PD patients. Therefore, some researchers speculate that the uncontrolled expression of DMT1 may be one of the many reasons for the excessive deposition of iron in the brain of some NDs.

Method used

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  • RNA and recombinant for inhibiting human DMT1 protein and application thereof
  • RNA and recombinant for inhibiting human DMT1 protein and application thereof
  • RNA and recombinant for inhibiting human DMT1 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] According to the human divalent metal ion transporter (DMT1) 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 DMT1 interference fragment sequence of the present invention with the most significant interference effect is obtained as follows:

[0056] Template strand (Seq ID No.3):

[0057] 5'-GATCCCC CTACCTGGATCCAGGAAAT TTCAAGAGA ATTTCCTGG ATCCAGGTAG TTTTTTA-3'

[0058] Coding chain (Seq ID No.4):

[0059] 5’-AGCTTAAAAA CTACCTGGATCCAGGAAAT TCTCTTGAA ATTTC CTGGATCCAGGTAG GGG-3'

Embodiment 2

[0061] Retro-DMT1i system construction

[0062] 1. Anneal the synthesized DMT1 interference fragments (Seq ID No.3 and Seq ID No.4). Specific steps are as follows:

[0063] 1. Establish an annealing system

[0064] 5ul 10x annealing buffer

[0065] 2nmole template chain Seq ID No.3

[0066] 2nmole coding chain Seq ID No.4

[0067] Make up to 50ul with triple distilled water.

[0068] 2. Put it into the PCR machine, 95°C for 5 minutes, 75°C for 5 minutes, 55°C for 5 minutes, 42°C for 5 minutes, 37°C for 15 minutes, and then gradually reduce the temperature to room temperature.

[0069] 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 the annealed product at -80°C.

[0070] 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 for...

Embodiment 3

[0089] Production process of recombinant retrovirus Retro-DMT1i

[0090] 1. Inoculate Phoenix cells 1.5×10 7 cells in a 9cm culture dish (Corning), 37°C, 5% CO 2 Culture overnight;

[0091] 2. Change the fresh medium 20 minutes before transfection;

[0092] 3. Mix 20ul of the above-mentioned Retro-DMT1i 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;

[0093] 5. After 24 hours, add 7.5ml medium to continue culturing for 48 hours;

[0094] 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.

[0095] 7. Measure the titer of the virus supernatant, adjust the cell titer to 1000VP / ml with PH8.0 Tris-HCl.

[0096] 8...

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Abstract

The invention discloses a short-interfering RNA for inhibiting human divalent metal ion transporter (DMT1) 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 divalent metal ion transporter inside body as well as the absorption and the catabolism of iron and reduce 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 human divalent metal ion transporter (Divalent metal transporter-1, DMT1) gene using retrovirus-mediated RNA interference technology The retrovirus interference system Retro-DMT1i, and applied 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-...

Claims

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Application Information

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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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