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SiRNA and recombination lentivirus from preventing hepcidin from regulating protein and uses thereof

A technology of recombinant lentivirus and regulatory protein, which is applied in the fields of molecular biology, biomedicine and genetic engineering to achieve the effect of inhibiting the expression of hepcidin-regulated protein in vivo, increasing the expression level, and increasing the level of hepcidin.

Inactive Publication Date: 2012-06-13
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

[0011] However, in order to apply RNA interference technology to clinical treatment, it is necessary to solve important problems such as the sustained expression of RNA interference fragments and the expression efficiency.

Method used

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  • SiRNA and recombination lentivirus from preventing hepcidin from regulating protein and uses thereof
  • SiRNA and recombination lentivirus from preventing hepcidin from regulating protein and uses thereof
  • SiRNA and recombination lentivirus from preventing hepcidin from regulating protein and uses thereof

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Experimental program
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Effect test

Embodiment 1

[0050] Based on the hepcidin regulatory protein (HJV) sequence (AK098165), and based on the human U6 sequence plus a 20bp U6 sequence linker and a PolyA termination message sequence. Add an XhoI restriction site to the 3' end, and send the synthetic sequence to Invitrogen Company. After screening, the HJV interference fragment sequence of the present invention with the most significant interference effect is obtained as follows:

[0051] Template strand (Seq ID No.3):

[0052] 5'-GTGGAAAGGACGAAACACCGTCAAAGGCTGCAGGAAGATTGTTGATATCCGCAATCTTCCTGCAGCCTTTGACTTTTTCTCGAGCTGGA-3'

[0053] Coding chain (Seq ID No.4):

[0054] 5'-TCCAGCTCGAGAAAAAGTCAAAGGCTGCAGGAAGATTGTTGATATCCGCAATCTTCCTGCAGCCTTTGACGGTGTTTCGTCCTTTCCAC-3'

Embodiment 2

[0056] PLT-HJVi system construction

[0057] Construction of recombinant PLT-HJVi plasmid as attached figure 2 , as shown in 3. The construction process is divided into three steps:

[0058] 1. Obtain the human U6 promoter;

[0059] a) Add 1ml SNET solution at room temperature to lyse 1X10 10 Human-derived cell line HEK293;

[0060] b) Draw the cell lysate from the mortar into a 2ml EP tube, add 40ul proteinase K and 2ul RNase;

[0061] c) Seal the EP tube, place the centrifuge tube horizontally in a 55-degree water bath, and slowly shake and digest for at least 3 hours;

[0062] d) Take out the EP tube, add 1ml of phenol-chloroform mixture, and gently invert 3 times. Shake slowly on a shaker for 30 minutes. (<60rpm);

[0063] e) Centrifuge for 5 minutes at the highest speed (13000rpm) of a desktop centrifuge at room temperature;

[0064] f) Carefully separate the aqueous phase into a new EP tube, add an equal volume of isopropanol, and gently invert to mix;

[0065...

Embodiment 3

[0121] Production process of recombinant lentivirus PLT-HJVi

[0122] 1. Inoculate 293T cells 1.5X10 7 cells in a 9cm culture dish (Coming), 37°C, 5% CO 2 Culture overnight;

[0123] 2. Change the fresh medium 20 minutes before transfection;

[0124] 3. Mix the PLT-HJVi plasmid with the lentiviral packaging plasmid P8.2 and P-VSV (both purchased from Invitrogen) in a ratio of 9:8:1;

[0125] 4. Mix 20 ul of the above plasmid mixture with 500 ul of DMEM medium and mark it as tube A. Take another 20 ul of liposome and mix it with 500 ul of 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;

[0126] 5. After 24 hours, add 7.5ml medium to continue culturing for 48 hours;

[0127] 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...

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Abstract

The invention discloses an RNA with little interference for inhibiting the expression of an iron regulating element regulatory protein (HJV). The RNA with little interference is in reverse complementation with the iron regulating element regulatory protein gene mRNA, and the length of the RNA is 19-27bp. The invention further discloses a DNA for coding the corresponding shRNA of the RNA with little interference and a recombined slow virus capable of expressing the shRNA. The RNA with little interference, the DNA capable of coding the shRNA and the recombined slow virus can effectively inhibitthe expression of the iron regulating element regulatory protein in vivo, improve the level of the iron regulating element and reduce the level of serum iron in vivo, thereby reaching the aim of curing diseases related to 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 lentivirus interference system (PLT-HJVi ), 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 and proteins, called RNA-induced silencing complex (RISC), which can recognize mRNA of the source sequence and cleavage of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N7/01A61K48/00A61P3/12
Inventor 钱忠明葛啸虎柯亚汪程远
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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