Preparation of flounder erythrocyte outer membrane protein

A technology of flounder red blood cells and outer membrane proteins, which is applied in the collection and processing of fish red blood cells and the preparation of red blood cell outer membrane proteins, can solve the problems of less red blood cell immunity, lack of in-depth analysis and identification of red blood cell immune molecules, and unknown mechanism of action, etc.

Inactive Publication Date: 2009-06-24
张振冬
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These works have been carried out most in humans and other mammals and birds, but there are few reports and insufficient understanding of erythrocyte immunity in fish
[0006] 2. The research foundation of the immune mechanism of fish red blood cells is weak
[0007] The mechanism of the immune function of fish red blood cells is still unknown, and the important molecules and modes of action involved in the immune response

Method used

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  • Preparation of flounder erythrocyte outer membrane protein
  • Preparation of flounder erythrocyte outer membrane protein
  • Preparation of flounder erythrocyte outer membrane protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The specific operation steps are:

[0036] (1) Blood was collected from the tail veins of four experimental flounder with a body length of about 20 cm using a 5 mL disposable sterile syringe, and the peripheral blood of the flounder was mixed with Arbori anticoagulant at a ratio of 1:1. Collect the anticoagulated blood and store it in an ice box.

[0037] (2) Separation of erythrocytes in peripheral blood of flounder with specific gravity of 1.08-1.09 lymphocyte separation medium. The anticoagulated blood was diluted 1:1 with 0.1M PBS buffer, mixed with lymphocyte separation medium in a ratio of 1:2, and centrifuged horizontally at 3000rpm for 15min at 4°C. After centrifugation, the peripheral blood cells were divided into 4 layers, and the separation liquid, white blood cells and plasma in the upper layer were carefully sucked away, and the red blood cell pellet in the bottom layer was centrifuged and washed 3 times with 0.1M PBS buffer at 4°C, and the supernatant was...

Embodiment 2

[0045] The specific operation steps are:

[0046] (1) Blood was collected from the tail veins of four experimental flounders with a body length of about 20 cm using a 5 mL disposable sterile syringe, and mixed with the peripheral blood of the flounder at a ratio of 1:1.5 with Alfred anticoagulant. Collect the anticoagulated blood and store it in an ice box.

[0047] (2) Separation of erythrocytes in peripheral blood of flounder with specific gravity of 1.08-1.09 lymphocyte separation medium. The anticoagulated blood was diluted 1:1 with 0.1M PBS buffer, mixed with lymphocyte separation medium in a ratio of 1:2, and centrifuged horizontally at 3000rpm for 15min at 4°C. After centrifugation, the peripheral blood cells were divided into 4 layers, and the separation liquid, white blood cells and plasma in the upper layer were carefully sucked away, and the red blood cell pellet in the bottom layer was centrifuged and washed 3 times with 0.1M PBS buffer at 4°C, and the supernatant...

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Abstract

The invention relates to a method for preparing red cell outer membrane protein of left- -eyed flounders, Paralichthys olivaceus, comprising the following steps: blood is taken from the caudal vein of the left-eyed flounder; the Alsever gelation inhibitor and the peripheral blood of the left-eyed flounder are mixed; the blood is diluted by PBS buffer solution, mixed with separating medium and centrifugated at the temperature of 4 DEG C, and the deposit formed by the red cells at the bottom is centrifugally washed at the temperature of 4 DEG C by the PBS buffer solution to remove supernatant fluid; hypotonic buffer solution is added and kept at the temperature of 4 DEG C to fully swell the red cell so that the membrane is ruptured; the deposit of the red cell membrane of the left-eyed flounder is centrifugally collected at the temperature of 4 DEG C; the hypotonic buffer solution is added and then the deposit of the red cell membrane is scattered by a whirlpool mixer and centrifugally washed at the temperature of 4 DEG C; membrane protein lysis solution is added and stirred to fully cleave the red cell membrane of the left-eyed flounder; after centrifugating at the temperature of 4 DEG C, the supernate is the extracting solution of the red cell membrane protein of the left-eyed flounder; after being subpackaged, the extraction solution is preserved in an refrigerator at the temperature of minus 80 DEG C. Compared with the prior art, the method is simpler and more efficient, thereby not only ensuring the high efficiency of the preparation of membrane protein, but also maintaining the biological activity of the membrane protein.

Description

technical field [0001] The invention relates to the technical field of collection and processing of fish erythrocytes and preparation of erythrocyte outer membrane protein. Background technique [0002] Flounder is commonly known as Yapian and Piankou in my country. It is an excellent species, grows fast, and has delicious meat. It is one of the excellent marine fish breeding species in my country. At present, with the development of the flounder aquaculture industry and the expansion of the aquaculture scale, various diseases of the aquaculture flounder frequently break out, causing huge economic losses. Fish have a relatively complete immune system, which can defend against the invasion of various pathogens through specific and non-specific immunity. Peripheral blood cells are one of the important lines of defense in fish non-specific immune defense system 1，2 . However, there is no definite report on the immune function of fish erythrocytes and erythrocyte immune molec...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/46C07K14/705
Inventor 张振冬王淑芬邹亚男张伟
Owner 张振冬
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