Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for overexpression of IL-15 in porcine skeletal muscle myoblasts and application thereof

A technology for myoblasts and skeletal muscle, applied in the field of genetic engineering, can solve the problems of increased research cost, short half-life of IL-15, complicated operation, etc., to enhance the ability to resist oxidative damage, promote division and proliferation, and reduce the rate of apoptosis. Effect

Pending Publication Date: 2021-04-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Research on IL-15 is becoming more and more popular, but previous studies mostly used the method of directly adding IL-15 to increase its expression in cells. Due to the short half-life of IL-15, it is difficult to achieve the desired effect with a single treatment , so it needs to be added multiple times, the operation is cumbersome, and the research cost is increased
And there is no relevant report on the effect of IL-15 on porcine skeletal muscle myoblasts

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for overexpression of IL-15 in porcine skeletal muscle myoblasts and application thereof
  • Method for overexpression of IL-15 in porcine skeletal muscle myoblasts and application thereof
  • Method for overexpression of IL-15 in porcine skeletal muscle myoblasts and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Preparation of porcine IL-15 gene overexpression lentivirus

[0046] The porcine IL-15 gene sequence was obtained from NCBI, with accession number NM_214390.

[0047] 1. Construction of overexpression lentiviral vector

[0048] 1. Materials and methods

[0049] 1.1 Main reagents

[0050] As shown in Table 1

[0051] Table 1 Main reagent information

[0052]

[0053] 1.3 Main instruments and equipment

[0054] As shown in table 2.

[0055] Table 2 Main Instrument Information

[0056]

[0057]

[0058] 2. Carrier digestion

[0059] 2.1 Carrier information

[0060] The name of the vector used is GV492, the element sequence: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, which contains two cloning sites of BamHI / AgeI, and the vector map is as follows figure 1 shown. The control empty vector number is CON335.

[0061] 2.2 Digestion results

[0062] Plasmids extracted from bacteria have different migration rates due to different conformations such as supercoiled, ...

Embodiment 2

[0107] Overexpression of IL-15 in porcine skeletal muscle myoblasts

[0108] 1. Materials and equipment

[0109] The required experimental instruments are shown in Table 6, and the materials and reagents are shown in Table 7.

[0110] Table 6 Experimental Instrument Information

[0111]

[0112] Table 7 Experimental materials and reagent information

[0113]

[0114]

[0115] 2. Experimental procedures and results

[0116] 1. Isolation, culture and induction of porcine skeletal muscle satellite cells

[0117] 1.1 Isolation and culture of porcine skeletal muscle satellite cells

[0118] The piglets were bled to death from the cervical artery, and the uterine tissue was taken and placed in PBS containing P / S, and then 75% alcohol was used to sterilize the whole body of the piglets, and the thigh skeletal muscles were separated under aseptic conditions, and placed in PBS containing double antibodies. Rinse repeatedly 3-5 times, move to a petri dish, cut it into 1mm3...

Embodiment 3

[0179] Effect of IL-15 on Oxidative Damage of Porcine Skeletal Muscle Myoblasts

[0180] 1. Establishment of oxidative stress model

[0181] Cell viability was detected by MTT assay.

[0182] (1) Divide porcine skeletal muscle myoblasts into 1×10 4 cells / well were seeded in 96-well plate, at 37°C, 5% CO 2 Cultivate in the incubator for 24h;

[0183] (2) Remove the culture medium, add 0.1, 0.2, 0.4 and 0.8mM H 2 o 2 culture medium without adding H 2 o 2 As a control, 5 replicates in each group, and continue to culture for 1.5h;

[0184] (3) remove culture medium, PBS washes 2 times;

[0185] (4) Add 20 μL of 5g / L MTT solution to each well, and continue to incubate for 4 hours;

[0186] (5) Remove the supernatant, add 150 μL DMSO to each well, and shake for 10 minutes;

[0187] (6) Measure the OD value of each well with a microplate reader at a wavelength of 570 nm.

[0188] The result is as Figure 16 As shown, when establishing the oxidative stress model, the cell ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for overexpression of IL-15 in porcine skeletal muscle myoblasts and application thereof, and belongs to the field of gene engineering. According to the invention, the porcine IL-15 gene is transferred into a lentiviral vector, and then the lentiviral vector is transferred into the porcine skeletal muscle myoblasts, so that the IL-15 is successfully overexpressed in the porcine skeletal muscle myoblasts. The IL-15 overexpression method provided by the invention solves the problems that IL-15 needs to be added for multiple times and the steps are tedious in the traditional research process. The regulation and control effects of IL-15 overexpression on division and proliferation, cell apoptosis and oxidative damage resistance of porcine skeletal muscle myoblasts are proved, and a direction is indicated for further research of IL-15.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for overexpressing IL-15 in porcine skeletal muscle myoblasts and its application. Background technique [0002] With the improvement of animal husbandry in my country, especially the intensification of modern aquaculture, pigs are affected by more and more stress factors during growth, fattening and slaughter, which cause changes in the distribution of nutrients and free radical metabolism in the body. Stress will break the balance of oxidation and anti-oxidation in the body, lead to a sharp increase in free radicals in the body, cause irreversible oxidative damage to many important biomacromolecules in the body, lead to the destruction of cell structure and function, and ultimately affect animal health and meat quality ( Kerth, 2013). [0003] Interleukin-15 (interleukin-15, IL-15) is a pleiotropic cytokine discovered in 1994, which can regulate the proliferation, d...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N2740/16043C12N5/0658C12N2501/2315C07K14/5443C12N2740/15043
Inventor 郝月顾宪红邢明杰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com