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Inference sequence capable of inhibiting diabetes mellitus type I CCR5 (CC-chemokine receptor 5) gene expression as well as recombinant vector and application thereof

A gene expression and recombinant vector technology, applied in the field of genetic engineering, can solve the problems of delaying the onset time and reducing the incidence of type I diabetes

Inactive Publication Date: 2014-09-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it has been reported that the incidence of type 1 diabetes in people with CCR5-delta32 mutations is reduced, or the onset time is delayed, but it is also reported that CCR5-delta32 mutations are not associated with the onset of TIDM

Method used

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  • Inference sequence capable of inhibiting diabetes mellitus type I CCR5 (CC-chemokine receptor 5) gene expression as well as recombinant vector and application thereof
  • Inference sequence capable of inhibiting diabetes mellitus type I CCR5 (CC-chemokine receptor 5) gene expression as well as recombinant vector and application thereof
  • Inference sequence capable of inhibiting diabetes mellitus type I CCR5 (CC-chemokine receptor 5) gene expression as well as recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Construction of pGenesil-1-CCR5-shRNA1 eukaryotic expression vector

[0059] 1.1 Required reagents

[0060] Restriction enzymes BamH I and EcoR I and corresponding Buffer, T4 ligase and its buffer, PCR product purification kit, and gel recovery kit were all purchased from TaKaRa Company (Dalian Bao Biological Engineering Co., Ltd.). Eukaryotic expression vector selection conventional RNAi vector

[0061] 1.2 Design, synthesis and screening of CCR5 specific shRNA.

[0062] In this experiment, the pGenesil-1siRNA expression vector provided by Wuhan Jingsai Company was used to produce shRNA. Expression of small hairpin RNA (short hairpin RNA, shRNA) in mammalian cells terminates transcription by adding a string (3 to 6) U. shRNA will be automatically processed into siRNA in cells, thereby triggering gene silencing or expression inhibition.

[0063] The sequence of Human CC chemokine receptor5 mRNA, complete cds (GenBank Accession No. U57840) is shown in SEQ ...

Embodiment 2

[0068] Embodiment 2: in vitro cell test

[0069] 2.1 Identification of pGenesil-1-CCR5-shRNA-1 inhibitory effect

[0070] The pGenesil-1-CCR5-shRNA-1 and pGenesil-1-CCR5-shRNA-2 and the negative control recombinant plasmids were respectively transfected with 200 μg / ml DEAE-dextran to mouse spleen T cells for 90 minutes, and the number of T cells was about at 10 7 about. 48 hours after transfection, the expression of CCR5 was detected by flow cytometry and RT-PCR. figure 2 It can be seen that siRNA1 can suppress the expression of CCR5 by more than 70%; siRNA2 can only suppress the expression of CCR5 by about 20%; therefore, the present invention screens out the pGenesil-1 with the best interference effect, that is, the most significant effect of inhibiting CCR5 expression. -CCR5-shRNA-1.

[0071] 2.2 Detection of cell cycle after T cell pGenesil-1-CCR5-shRNA-1 inhibits CCR5 expression

[0072] GFP transfected with 10 μg of CCR5-shRNA-1 plasmid collected under the above ster...

Embodiment 3

[0075] Embodiment 3: animal experiments

[0076] T cells transfected with pGenesil-1-CCR5-shRNA-1 and empty vector pGenesil-1 were sorted by flow cytometry to collect GFP positive cells, and untreated T cells were used as blank control, and 200 μl of PBS suspension was injected intraperitoneally Contains about 1×10 6 The T cells of the above-mentioned groups were adopted to the NOD.scid mice, the change of blood sugar in the NOD.scid mice of each group, and the insulitis were observed by tissue sections. The results found that the blood glucose in the control group was 10.7±2.1mmol / l in the fifth week, 9.8±1.3mmol / l in the empty vector group and 5.2±1.1mmol / l in the RNAi group, and the blood glucose in each group was 18.8±1.1mmol / l in the control group at the tenth week 3.1mmol / l, 19.7±2.4mmol / l in the empty vector group and 9.3±2.1mmol / l in the RNAi group; the RNAi group was significantly lower than the other two groups. One week after the adoption of T cells, it was found ...

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Abstract

The invention belongs to the technical field of genetic engineering and specifically relates to an inference sequence capable of inhibiting diabetes mellitus type I specificity-related gene expression as well as a recombinant vector thereof and an application of the recombinant vector in preparing a medicine for treating diabetes mellitus type I. The recombinant vector constructed by the inference sequence is used for transfecting mouse T lymphocytes, so that secretion of Th1 / Th2 cell factors can be affected, and therefore, the recombinant vector plays an important role in insulitis at a prediabetic state, and blocks a process of the diabetes mellitus type I. The invention provides a novel means for treating the diabetes mellitus type I.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an interference sequence capable of inhibiting the expression of specific related genes of type I diabetes and its recombinant vector, as well as its application in the preparation of drugs for treating type I diabetes. Background technique [0002] In the process of the body's immune response and the occurrence and development of diseases, a class of immune regulatory factor systems - chemokines and their receptors play an important role. Chemokines produce effects by binding to receptors on the cell surface, and their specific receptors belong to the seven transmembrane domain G protein-coupled receptors (GPCRS). Nearly 20 types have been discovered so far. Chemokines and their receptors are involved in a wide range of pathological and physiological processes. [0003] Chemokine receptor 5 (CC-cheokine receptor5, CCR5) is a specific receptor for CC subfa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/66A61K48/00A61P3/10
Inventor 王栋郭满盈孙瑜郭葆玉邱磊许家军杨向群黄会龙张冉弓雪莲李玉泉刘镇
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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