Small interfering RNA of fatty acid binding protein 4 and its application

A small interference and complex technology, applied in the field of biotechnology and medicine, can solve the problems of unseen intervention in the treatment and prevention of tuberculosis, and achieve the effect of promoting apoptosis

Active Publication Date: 2022-07-29
NINGXIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the prior art, there is no report on the prediction and diagnosis of tuberculosis by changes in the expression level o

Method used

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  • Small interfering RNA of fatty acid binding protein 4 and its application
  • Small interfering RNA of fatty acid binding protein 4 and its application
  • Small interfering RNA of fatty acid binding protein 4 and its application

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0083] Example 1: Changes in the expression of FABP4 after BCG infection of RAW264.7 cells

[0084]The cells were collected after 6h, 12h, 18h, and 24h of BCG infection (MOI 1:10) respectively, and the total protein of RAW264.7 cells was extracted. A negative control group (Negative Control, NC, wild RAW264.7 cells) was set in the experiment at the same time.

[0085] Specific steps are as follows:

[0086] a) Protein extraction: The protein extraction was carried out according to the operation steps of the whole protein extraction kit instructions (Keygen Biotechnology, Nanjing). The concentration of the extracted protein was detected by BCA kit (Thermo fisher, American), and 20 μg of sample was loaded into each well for subsequent Western-Blot detection;

[0087] b) Western-Blot detection: the separation gel concentration was selected as 12%. After electrophoresis, the proteins on the gel were electrotransferred to PVDF membrane (0.2 μm, BD company), and then blocked with 5...

Example Embodiment

[0089] Example 2: Observation of the expression of FABP4 in each treatment group by immunofluorescence

[0090] The experimental group was divided into four groups, namely Control group (RAW264.7 cells), BCG group (RAW264.7 cells infected with BCG), siRNA-FABP4 group (RAW264.7 cells transfected with siRNA-(A+B)) and siRNA -FABP4+BCG group (RAW264.7 cells were transfected with siRNA-(A+B) and then infected with BCG), in which the Control group and BCG group were transfected with siRNA-NC, and the siRNA-FABP4 group and siRNA-FABP4+BCG group were transfected with siRNA -FABP4, infected with or without BCG after 24h, treated by immunofluorescence after 12h, and then observed by confocal microscope.

[0091] Specific steps are as follows:

[0092] Cell crawl:

[0093] a) The day before infection, 5 × 10 per well 4 Cells were seeded on cell slides in twelve-well plates, 1 mL of DEME medium containing 10% FBS was added to each well, and the cells were incubated at 37°C, saturated ...

Example Embodiment

[0109] Example 3: Western-Blot detection of differences in the expression of FABP4 in each group

[0110] The setting of the experimental treatment group and the specific treatment time are the same as those in Example 2. After the treatment, the total protein of RAW264.7 cells in each treatment group was extracted, and the expression of FABP4 in the cells was detected by Western-Blot. Protein extraction, BCA quantification and Western-Blot specific The test operation was the same as that of Example 1. According to the steps described in the "Technical Method Description" section above, use the primary antibody (rabbit anti-rabbit FABP4 antibody, Thermo fisher, Prod#701158) to incubate overnight at 4°C, wash with the secondary antibody (goat anti-rabbit HRP enzyme-labeled secondary antibody, Proteintech) , SA00001-2) for Western-Blot detection. See the results image 3 , among which, the four columns from left to right are: Control group, BCG group, siRNA-FABP4 group and siR...

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Abstract

The present invention provides the use of fatty acid binding protein 4 (FABP4) in a kit for diagnosing and/or evaluating the prognosis of tuberculosis, and the preparation of small interfering RNA of the fatty acid binding protein 4 (FABP4) for preventing or treating tuberculosis use in medicines. The FABP4 of the present invention is highly expressed in BCG-infected macrophage RAW264.7 cells, and the small interfering RNA of FABP4 can inhibit the apoptosis function of macrophages, so the small interfering RNA of FABP4 can be used as a new drug target It is of great significance to the prevention and treatment of tuberculosis.

Description

technical field [0001] The invention relates to the field of biotechnology and medicine, in particular to an RNA interference sequence for inhibiting the expression level of human or mammalian FABP4 protein and a targeted interference technology for FABP4 based on the core sequence, in particular to an RNA for inhibiting the expression level of FABP4 protein Interfering sequences and their applications. Background technique [0002] RNA interference (RNAi) is guided by intracellular double-stranded small interfering RNA (siRNA), and the target mRNA with homologous sequence is identified by the principle of intracellular RNA interference, causing mRNA degradation or translation inhibition. The phenomenon of inhibiting or reducing the expression level of the mRNA-encoded protein is the core of which is a specific nucleic acid sequence with a length of about 21 nucleotides, which is collectively referred to as the core sequence below. RNAi is a gene silencing phenomenon that e...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61K48/00A61P11/00A61P31/06
CPCC12N15/113A61K31/713A61K48/005A61P11/00A61P31/06C12N2310/14
Inventor 邓光存马臣杰于嘉霖
Owner NINGXIA UNIVERSITY
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