miR-29 absorption sequence, absorption carrier and uses thereof

A technology of mir-29 and carrier, which is applied in the field of biotechnology and medicine, and can solve problems such as methods that have yet to be developed

Inactive Publication Date: 2012-12-26
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

[0008] Tuberculosis is already one of the most important public health problems in the world, but effective means

Method used

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  • miR-29 absorption sequence, absorption carrier and uses thereof
  • miR-29 absorption sequence, absorption carrier and uses thereof
  • miR-29 absorption sequence, absorption carrier and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1: IFN-γ expression in immune cells after Listeria (LM) and Bacillus Calmette-Guerin (BCG) infection in mice Up-regulated and miR-29a expression down-regulated

[0110] Culture, counting and infection of LM and BCG

[0111] The Listeria virulent strain (LM, Listeria monocytogenes of ATCC 19111, purchased by Shanghai Fuxiang Biotechnology Co., Ltd.) was cultured in BHI medium containing 5 μg / mL erythromycin. When the bacteria are in the logarithmic growth phase, the bacteria stock solution in the culture medium is diluted by a 10-fold gradient, and then spread on a BHI agar plate containing erythromycin. After incubating at 37°C overnight, the stock solution in the stock solution is calculated by the colony count Colony forming units (CFU).

[0112] Inject 5×10 intraperitoneally per mouse 4 CFU LM, after infection for a specified period of time, isolate mouse spleen cells and NK cells in them (labeled with DX5 magnetic beads and then sorted by magnetic beads).

[0113...

Embodiment 2

[0119] Example 2: miR-29 uptake carrier up-regulates the production of IFN-γ in EL4 and Th1 cells

[0120] A. Preparation of miR-29 absorption carrier

[0121] The miR-29 absorption vector (pGS29) is a new type of miRNA inhibitor that can absorb endogenous miR-29a, b, and c. Its core parts from 5'to 3'are: UBC promoter, green fluorescent protein eGFP And the miR-29 uptake sequence inserted downstream of the eGFP termination code, wherein the miR-29 uptake sequence is characterized by a 7-fold repetitive artificially synthesized sequence that is partially complementary to miR-29a.

[0122] The preparation of miR-29 absorption vector is as follows (the restriction endonuclease, Taq enzyme and pMD19-T vector used were all purchased from Takara):

[0123]

[0124] Contains 7 repeated miR-29 sponge inserts ( Picture 9 A-B) The following two chemically synthesized fragments are obtained after PCR reaction:

[0125] Fragment 1 (SEQ ID NO: 1):

[0126] 5’-TAACCGATTTTCTTGGTGCTATAACCGATTTTCTTGG...

Embodiment 3

[0186] Example 3: In NK cells and T cells of transgenic mice (GS29 mice) in which miR-29 absorbs the vector miR-29a expression down-regulated

[0187] Construction of GS29 mice

[0188] After the endonuclease Tth111 I linearized miR-29 to absorb the vector pGS29 and recover the DNA, we commissioned Shanghai Southern Model Biotechnology Development Co., Ltd. to carry out embryo microinjection. In brief, the transgenic mice are the male pronucleus of the fertilized egg through the foreign gene. Obtained by injection, donor fertilized eggs were taken from the first hybrid of C57BL / 6J (♀) and CBA (♂). During the preparation of the transgenic mice, we obtained a total of 385 injected eggs, which were transplanted into the fallopian tubes of 15 pseudo-pregnant female mice. Among them, 14 female mice were pregnant. The transplantation success rate was 93.33%. The mice were born. A total of 119 mice were detected by PCR of genomic DNA, and 5 mice were positive, with a positive rate of 4...

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Abstract

The invention relates to an miR-29 absorption sequence, an absorption carrier and uses thereof. The miR-29 absorption carrier carries expression cassettes 5'to 3' sequentially comprising the following elements: a) an II-type promoter; (b) an indication protein coding sequence; (c) an miR-29 absorption sequence which is repeated for 4 to 10 times and is a partially complementary or completely complementary sequence to miR-29; and (d) an optional enhancer sequence. The invention also provides application of the absorbent carrier and a pharmaceutical composition containing the carrier. The miR-29 absorption carrier can be used for preventing and treating inflammatory caused by intracellular bacterial infection, Th1-type reaction, tuberculosis and other diseases.

Description

Technical field [0001] The invention belongs to the fields of biotechnology and medicine. Specifically, the present invention relates to the preparation and application of miR-29 absorption carrier, especially the application in miR-29-related diseases, such as the effect, mechanism of action, implementation method and application in bacterial infection (especially intracellular infection). use. Background technique [0002] MicroRNAs (miRNAs) are non-coding microRNAs with a length of about 20-24 nt. They regulate target genes at the post-transcriptional level by inhibiting mRNA translation or promoting mRNA degradation (Bartel, DP, Cell. 2004, 116:281-297) . MiRNA controls almost every biological process, including cell differentiation, development, proliferation and apoptosis. In addition, the imbalance of miRNA expression level is also closely related to diseases such as tumors (Lu, J. et al., Nature. 2005, 435: 834-838; Li, QJ et al., Cell. 2007, 129: 147-161; Cheng, HY et...

Claims

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Application Information

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IPC IPC(8): C12N15/63A61K48/00A61P31/04A61P31/06A61P35/00A61P7/00A61P25/00A61P11/00A61P9/00A61P19/02A61P29/00A61P3/10A61P17/00A61P31/12A61P21/00
Inventor 曹雪涛马烽李楠徐胜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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