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Process for extracting and purifying crucian IgM

A crucian carp technology, applied in the field of extraction and purification of biological immunoglobulin, can solve the problems of low product purity, inability to separate protein samples, low purification efficiency and quality

Inactive Publication Date: 2011-01-26
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

Generally speaking, the purity of the product obtained by the salting out method is not high, and the purification efficiency and quality are extremely low; ion exchange chromatography is often unable to obtain a relatively good product due to the similar physical properties such as the isoelectric point between the separated sample and other compounds. For pure target samples, this method is mainly suitable for the initial purification of crude protein extracts; gel filtration chromatography uses the porosity of the gel to separate according to the size of solvent molecules, so protein samples with similar molecular weights cannot be separated by this method

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  • Process for extracting and purifying crucian IgM
  • Process for extracting and purifying crucian IgM
  • Process for extracting and purifying crucian IgM

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Embodiment Construction

[0020] Experimental methods not indicating specific conditions in the following examples are usually in accordance with conventional methods, such as the conditions described in the Protein Technology Handbook (Wang Jiazheng, Fan Ming Editor-in-Chief. Science Press, 2000), or according to the conditions suggested by the manufacturer .

[0021] 1. Extraction and purification of crucian carp IgM

[0022] (1) Preparation of crucian carp serum

[0023] Wipe the body surface of crucian carp with 75% alcohol cotton for disinfection, and then collect blood from the tail vein. Add it into a sterilized test tube, put it in a 37°C incubator for 1 hour, and then let it stand overnight at 4°C to fully analyze the serum. The next day, centrifuge at 3000g / min at 4°C for 20min, draw the supernatant with a micropipette, aliquot into 1.5mL EP tubes, and store at -20°C for later use.

[0024] (2) Saturated ammonium sulfate fractional precipitation

[0025] Dilute 5 mL of crucian carp serum ...

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Abstract

The invention relates to a process for extracting and purifying crucian IgM, which is a method for separating the crucian IgM in crucian blood serum with other compounds so as to obtain high-purity crucian IgM and comprises the steps of: preparing the crucian blood serum; coarsely extracting the crucian blood serum by using a fractional precipitation method with 33% to 50% of saturated ammonium sulfate; further extracting the crucian IgM from separated sediment extracts of the saturated ammonium sulfate of the crucian blood serum by using a Macro-prephighQ strong anion exchange column; further finely separating and purifying a sample obtained by ion exchange chromatography through Sephacryl TMS-300 chromatographic column; and purifying 5mL of crucian blood serum (with a protein concentration of 60mg / mL) to obtain 4.4mgIgM. The yield is 1.4%. The purity is more than 96% by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) detection and analysis of a protein scanner.

Description

technical field [0001] The invention relates to a method for extracting and purifying biological immunoglobulin, in particular to a process for extracting and purifying crucian carp IgM. Background technique [0002] The immune system of animals is gradually developed in the process of germline evolution, among which fish is the first animal to appear immunoglobulin (Ig), and its immune system is relatively primitive. Although the immune system of fish is relatively primitive and low-level, compared with invertebrates, it has made an important breakthrough in its evolution and has formed a relatively complete immune system including non-specific immune response and specific immune response. Like other vertebrates, the fish immune system includes three categories: immune tissue, immune cells and humoral immune factors. A large number of literatures prove that fish can carry out humoral and cellular immune responses, and immunoglobulin is the most important mediator in fish-s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/06C07K1/36C07K1/30C07K1/18C07K1/16
Inventor 刘永杰陆承平焦大为
Owner NANJING AGRICULTURAL UNIVERSITY
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