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Immune preparation method of antituberculous polypeptide

An anti-tuberculosis and immune enhancer technology, applied in the fields of immunomedicine and biopharmaceuticals, can solve the problems of unstable immune effect, unfavorable industrial production, low specific immune effect, etc., to improve the specific immune effect, reduce the preparation cost, The effect of increasing production

Active Publication Date: 2013-04-24
广东龙帆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At home and abroad, purified protein derivative (PPD) or BCG single immunization is often used to prepare anti-tuberculosis polypeptides, and the immune effect is unstable, which leads to the problem of low specific immune effect
And the extraction of its peptides is mostly separated and purified by reversed-phase chromatography, and there are also a few reports of separation and purification by affinity chromatography (antigen adsorption column chromatography separation and purification). The separation and purification of specific effective components by affinity chromatography is relatively Fast and simple steps, but requires a large amount of soluble antigen to amplify the corresponding antibody, which is difficult to complete in a short period of time and is not conducive to industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 20 mg of BCG, 10 mg of incomplete Freund's adjuvant, 3 mg of lecithin, and 2 mg of chitosan were mixed and stirred for 40 minutes under ultrasonic waves to prepare BCG liposomes. Select 50-60 kg healthy pigs, subcutaneously inject BCG liposome, inject once every 10 days, and inject 4 times continuously. Healthy pigs immunized with BCG were slaughtered. Take the pig spleen and lymph nodes, remove the fat and mucous membranes, and obtain the unprocessed material. Add physiological saline with a weight ratio of 1:2 to the untreated material, and mash it with a tissue mashing homogenizer to obtain a homogenate. After repeated freezing and thawing at minus 20 degrees for 4 times, it is then ultrafiltered with a 5000 Dalton ultrafiltration membrane, and the obtained filtrate is the crude anti-tuberculosis polypeptide. After the leukocyte adhesion inhibition test, the leukocyte adhesion inhibition rate was 37.1%, and then separated and purified by cross-linked Sephadex G-15 c...

Embodiment 2

[0021] 25 mg of BCG, 15 mg of incomplete Freund's adjuvant, 4 mg of lecithin, and 3.5 mg of chitosan were mixed and stirred under ultrasonic waves for 60 minutes to prepare BCG liposomes. Select 60-70 kg healthy pigs, subcutaneously inject BCG liposome, inject once every 10 days, and inject 5 times continuously. Healthy pigs immunized with BCG were slaughtered. Take the pig spleen and lymph nodes, remove the fat and mucous membranes, and obtain the unprocessed material. Add physiological saline with a weight ratio of 1:3 to the untreated material, and mash it with a tissue mashing homogenizer to obtain a homogenate. After repeated freezing and thawing at -30°C for 5 times, ultrafiltration with a 6000 Dalton membrane is performed, and the obtained filtrate is crude anti-tuberculosis polypeptide. After the leukocyte adhesion inhibition test, the leukocyte adhesion inhibition rate was 33.9%, and then separated and purified by cross-linked Sephadex G-15 chromatography column, fre...

Embodiment 3

[0023] 15 mg of BCG, 8 mg of incomplete Freund's adjuvant, 2 mg of lecithin, and 1.6 mg of chitosan were mixed and stirred for 60 minutes under ultrasonic waves to prepare BCG liposomes. Select 40-50 kg healthy pigs, subcutaneously inject BCG liposome, inject once every 10 days, and inject 3 times continuously. Healthy pigs immunized with BCG were slaughtered. Take the pig spleen and lymph nodes, remove the fat and mucous membranes, and obtain the unprocessed material. Add physiological saline with a weight ratio of 1:2 to the untreated material, and mash it with a tissue mashing homogenizer to obtain a homogenate. After repeated freezing and thawing at minus 30 degrees for 3 times, the filtrate was ultrafiltered with a 5000 Dalton membrane. The obtained filtrate was the crude anti-tuberculosis polypeptide. The leukocyte adhesion inhibition rate was 35.2% as detected by the leukocyte adhesion inhibition test. Then, the cross-linked Sephadex G-15 chromatographic column is used...

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Abstract

The invention relates to an immune preparation method of antituberculous polypeptide. The method is characterized by comprising the following steps of carrying out subcutaneous injection of bacillus calmette guerin vaccine lipidosome on healthy pigs so as to carry out immune response, wherein the bacillus calmette guerin vaccine lipidosome is prepared by mixing bacillus calmette guerin vaccine with an immune intensifier; taking spleens and lymph glands of the immune healthy pigs, and removing the fat and mucosa to obtain objects to be treated; adding normal saline into the objects to be treated, smashing and preparing homogenate; repeatedly freezing and melting the homogenate at minus 20 to minus 30 DEG C and carrying out ultrafiltration by using an ultrafiltration film so as to obtain the filtrate as a primary antituberculous polypeptide product; and further carrying out white blood cell adhesion inhibition experiments to detect that the white blood cell adhesion inhibition rate meets the requirements of range values of 33.9-38%, subsequently carrying out crosslinked glucan gel chromatography separation and purification, and freeze-drying so as to obtain the antituberculous polypeptide. The preparation process is simple, convenient and controllable; and the white blood cell adhesion inhibition rate of the prepared antituberculous polypeptide can be 33.9-38%.

Description

technical field [0001] The invention relates to an immune preparation method of an anti-tuberculosis polypeptide, belonging to the fields of immunomedicine and biopharmaceuticals. Background technique [0002] Tuberculosis is a chronic infectious disease caused by mycobacteria conjugating bacteria that seriously endangers people's health. At present, about 2 billion people are infected worldwide. my country is one of the hardest-hit areas of tuberculosis, and the annual death toll is more than twice the sum of deaths from other infectious diseases. Anti-tuberculosis chemical drug therapy has a good effect on controlling tuberculosis, but there are problems such as long time of taking drugs and easy to produce drug resistance. [0003] The anti-tuberculosis polypeptide can transmit the specific cellular immunity information of Mycobacterium tuberculosis, and has the function of regulating and enhancing the body's specific anti-tuberculosis infection cellular immunity. When ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12
Inventor 吴建国邓兆群张儒林杨小钢刘君炎刘胜武屈雪菊谭秋萍
Owner 广东龙帆生物科技有限公司
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