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Application of Mstn recombinant protein in screening of epinephelus coioides

A kind of technology of grouper and recombinant protein, applied in the field of genetic engineering

Pending Publication Date: 2021-11-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods of using mstn for active immunization are mainly concentrated in animal husbandry, such as cattle, sheep, pigs, etc. In the field of aquatic products, especially in the cultivation of grouper, there are few reports about mstn

Method used

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  • Application of Mstn recombinant protein in screening of epinephelus coioides
  • Application of Mstn recombinant protein in screening of epinephelus coioides
  • Application of Mstn recombinant protein in screening of epinephelus coioides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: In this embodiment, a method for analyzing the subcellular localization of Mstn of the oblique-banded grouper is disclosed, and the specific steps are as follows:

[0053] 1.1 Collection of fish tissue samples

[0054] The grouper was anesthetized with eugenol, the fish body was dissected, and muscle tissue samples were collected and placed in RNase-Free EP tubes filled with 1mL RNA protection solution (Omega), and stored in a -80°C refrigerator.

[0055] 1.2 Total RNA extraction

[0056] (1) All scissors and tweezers are sterilized with 75% ethanol, wrapped in tinfoil, sterilized in an autoclave, and the RNA tips used are brand new boxed tips, and pre-cooled in a high-speed centrifuge at 4°C.

[0057] (2) Take the sample out of the -80°C refrigerator, thaw it on ice, take the RNase-Free EP tube and add 600 μL reagent reagent (Invitrogen), then put the sample into it, crush the sample tissue block with a grinding rod, and then add 400 μL Reagent reage...

Embodiment 2

[0152] Example 2 Study on the Effect of Mstn Recombinant Protein on the Downstream of Mstn Signaling Pathway and Related Genes

[0153] 2.1 Primary culture of muscle cells

[0154] (1) Scissors, tweezers, blades, etc. are all autoclaved after being wiped with 75% alcohol, and all experimental equipment is sterilized by ultraviolet light for 30 minutes;

[0155] (2) Wipe the fish body surface with 75% alcohol, cut out a rectangle with a blade, tear off the skin, and remove the upper muscle;

[0156] (3) Take the muscle and place it in a petri dish, and wash it three times with L15 culture solution added with 10% double antibody;

[0157] (4) When washing for the third time, cut the tissue into tissue pieces of about 1 mm 3 , suck off the culture medium, and wash again;

[0158] (5) Transfer the tissue blocks into the culture bottle according to a certain interval, and put 20-30 blocks in total;

[0159] (6) Gently turn the culture bottle over, pay attention not to let the sm...

Embodiment 3

[0196] Example 3 Study on the Effect of Mstn Recombinant Protein on Muscle Cell Proliferation

[0197] 3.1 Primary culture of muscle cells

[0198] Same as the muscle cell primary culture method in Example 2.

[0199] 3.2 Incubate primary muscle cells with Mstn recombinant protein

[0200] The cell suspension was inoculated in five 96-well plates (100 μL / well), treated with 1000 nM Mstn protein after 24 hours, the control group was treated with the same volume of PBS, the blank group was not treated, and each group had 8 repetitions;

[0201] The cell suspension was inoculated in a 6-well plate (1.6 mL / well), treated with 1000 nM Mstn protein 24 hours later, and the control group was treated with the same volume of PBS, with 3 replicates in each group.

[0202] 3.3 Detection of cell proliferation by CCK-8 method

[0203] (1) After the Mstn recombinant protein was treated for 1d, 2d, 3d, 4d, and 5d, the medium in the 96-well plate was replaced with a fresh medium;

[0204] ...

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Abstract

The invention relates to application of mstn recombinant protein in screening of epinephelus coioides. The application comprises the following steps: S1, performing Mstn subcellular localization analysis on the epinephelus coioides; analyzing the influence of the Mstn recombinant protein on the downstream of the mstn signal channel and related genes; and analyzing the influence of the Mstn recombinant protein on muscle cell proliferation.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of a Mstn recombinant protein in the screening of groupers. Background technique [0002] Myostatins gene (myostatins, mstn) is a negative regulatory gene that inhibits the proliferation and growth of muscle cells. It is a member of the TGF-β family, so it is also called gdf-8. Studies have shown that the inhibition of gene expression or mutation inactivation can block the inhibitory effect of Mstn on muscle cell proliferation and growth, cause extensive muscle proliferation and significantly reduce body surface fat deposition, resulting in a significant increase in the number of muscle fibers and weight gain. The most characteristic phenomenon of muscle hypertrophy is the so-called "double muscle trait". [0003] Prepare the mstn genetically engineered vaccine and immunize with the vaccine, and then use your own immune system to produce anti-Mstn antibodies fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C07K14/475C12N15/85C12N15/12G01N15/14
CPCC12Q1/6851C07K14/475C12N15/85G01N15/14C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 杨慧荣夏俊杨炎杨金增
Owner SOUTH CHINA AGRI UNIV
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