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Small ribonucleic acid interference molecule for specifically inhibiting NK (Natural Killer) cell receptor KIR3DL1 and application of small ribonucleic acid interference molecule

A small nucleic acid interference and NK cell technology, applied in the field of biomedicine, can solve the problems of good therapeutic drugs and difficult to improve the curative effect, and achieve the effect of less toxic side effects and strong activity

Inactive Publication Date: 2013-02-06
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The scientific community generally believes that by using small nucleic acid interference technology, it is possible to produce promising therapeutic drugs for the treatment of cancer and infectious diseases, which are difficult to improve the efficacy of conventional technology

Method used

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  • Small ribonucleic acid interference molecule for specifically inhibiting NK (Natural Killer) cell receptor KIR3DL1 and application of small ribonucleic acid interference molecule
  • Small ribonucleic acid interference molecule for specifically inhibiting NK (Natural Killer) cell receptor KIR3DL1 and application of small ribonucleic acid interference molecule
  • Small ribonucleic acid interference molecule for specifically inhibiting NK (Natural Killer) cell receptor KIR3DL1 and application of small ribonucleic acid interference molecule

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Experimental program
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Effect test

Embodiment 1

[0053] Synthesis of siRNA that specifically inhibits the expression of the natural killer cell receptor KIR3DL1. In the present invention, the specific natural killer cell receptor KIR3DL1 mRNA is searched for in the gene bank, the genbank accession number is NM 013289, and the AA+N19+UU sequence or AA+N19 sequence (N19 is Any 19 mRNA nucleotide sequences), then calculate the G / C ratio in the selected 19-23nts mRNA base sequence to be between 30% and 70%, and then use Blast to search the EST gene library for the 19-23nts mRNA sequence to confirm The targeted gene is unique, and 19-23 antisense RNAs are designed, and there are two deoxyribonucleic acid dTdT at the 3' end of each strand to form a single-strand suspension, and a double-strand siRNA is synthesized.

[0054] On this basis, based on K4 with the best effect, by using cholesterol, or / and thiolation, or / and methylation at the 5' or 3' end of the siRNA sense strand or antisense strand, each strand 3 There are two deoxy...

Embodiment 2

[0060] Preparation of peripheral blood NK cells from HIV / AIDS patients. Peripheral venous blood from HIV / AIDS patients was collected, anticoagulated with EDTA-2K, and peripheral blood mononuclear cells (PBMC) were separated with lymphocyte separation medium (GE Healthcare, Sweden) within 6 hours after collection. After isolation, trypan blue (Sigma, USA) staining was used to determine the cell viability >95%. Put the separated PBMC into 1ml of RPMI-1640 (Inivitrogen, USA) containing 10% fetal bovine serum (Inivitrogen, USA), and add IL-2 (PeproTech, USA) and 50ng / ml final concentration of 200U / ml at the same time. PMA (Phorbol 12-Myristate 13Acetate) (Alexis Biochemicals, Switzerland), at 37°C, containing 5% CO 2 The incubator cultured for 24 hours to expand the human NK cells in the PBMC. for the following experiments.

Embodiment 3

[0062] The detection method for the inhibition of KIR3DL1 expression in NK cells by unmodified siRNA was established. The present invention uses LipofectAMINE TM 2000 (Lipo 2000) liposomes (Inivitrogen, USA) were used to transfect the KIR3DL1-siRNA fragments of K1-K5 described in Table 1, and the silencing effect was determined 72 hours after transfection. All operations are carried out according to the instructions. The siRNA powder was dissolved in DEPC water to prepare a 20nM stock solution.

[0063] Before the transfection operation, the NK cells expanded above were inoculated into 24-well plates with DMEM medium (Inivitrogen, USA) containing 10% fetal bovine serum, penicillin and streptomycin, and each well was inoculated with 5×10 5 cells at 37°C in 5% CO 2 incubator for 4 hours. Discard the original medium, wash once with DMEM medium without fetal bovine serum, penicillin, and streptomycin, and add 500 μl of DMEM medium without fetal bovine serum, penicillin, and st...

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Abstract

The invention discloses a small ribonucleic acid interference molecule for specifically inhibiting an NK (Natural Killer) cell receptor KIR3DL1 and an application of the small ribonucleic acid interference molecule. The genbank login number of the mRNA of the targeted KIR3DL1 of the small ribonucleic acid interference molecule for specifically inhibiting the NK cell receptor KIR3DL1 is NM_013289. A large amount of experiments show that the siRNA fragment can remarkably inhibit the expression of the receptor KIR3DL1 of an NK cell membrane, and the siRNA subjected to various chemical modifications has the same effect of inhibiting the expression of the receptor KIR3DL1. The expression level of IFN-gamma and TNF-alpha cell factors in an NK cell can be remarkably increased after the expression of the receptor KIR3DL1 is inhibited. Meanwhile, the killing activity of the NK cell can be remarkably enhanced after the siRNA is transfected. Therefore, the small ribonucleic acid interference molecule for specifically inhibiting the NK cell receptor KIR3DL1 can be applied to preparation of anti-aids drugs.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a small nucleic acid interference molecule for specifically inhibiting NK cell receptor KIR3DL1 and application thereof. Background technique [0002] AIDS is a new infectious disease that became popular in the 1980s. Since the first AIDS patient was discovered in the United States in 1981, AIDS has been widely distributed in more than 210 countries and regions on five continents. The UNAIDS / WHO "2009 Global AIDS Epidemic Trends" report pointed out that by the end of 2009, there were currently about 3,340 people in the world. million people living with HIV / AIDS, of which 2.6 million were newly infected in 2009, and 1.8 million people died of AIDS-related diseases. AIDS has become a disease that seriously endangers human life and health. Since 2009, AIDS has been the number one cause of death from statutory infectious diseases in my country. Due to the continuous emergence and prevalence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/18
Inventor 羊海涛傅更锋还锡萍徐金水陈国红徐晓琴胡海洋郭宏雄邱涛王小亮张之刘晓燕闫红静丁萍丁建平
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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