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Method for extracting hyaluronidase from bovine spleen

A technology of hyaluronidase and spleen, which is applied in the biological field, can solve the problem of extracting less hyaluronidase, and achieve the effect of being suitable for large-scale production, simple purification steps, and increasing extraction rate

Inactive Publication Date: 2017-07-21
NANNING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The effect of hyaluronidase from different sources is not the same. At present, it has been reported in the literature that hyaluronidase has been extracted from bee venom, scorpion venom, sheep testis, cattle testis, and bovine liver. However, hyaluronidase has been extracted from bovine spleen. There are few reports on acidase

Method used

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  • Method for extracting hyaluronidase from bovine spleen
  • Method for extracting hyaluronidase from bovine spleen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: Extract hyaluronidase from bovine spleen

[0068] (1) Take 100g of fresh bovine spleen, remove the epidermal envelope, cut into small pieces, add 100g of 2.0% normal saline and soak for 25min, then add 250g of extract, mash it with a tissue grinder, and put it in a -8°C incubator In the medium, stir with a mechanical stirrer at a stirring speed of 550r / min. After 50min, stand at -3°C for 1h, take the supernatant, add 35g of sodium chloride, stir at 0°C for 30min, let stand for 1h, and centrifuge at 6000r / min for 15min. Take the supernatant, add 100g of salt, stir at 0°C for 50min, then let it stand for 9h, centrifuge at 9000r / min for 10min, and take the precipitate to obtain the crude product of hyaluronidase;

[0069] (2) Take the precipitate in step (1), put it into a dialysis bag, put it into 50g of citric acid-sodium hydroxide-hydrochloric acid buffer solution, dialyze at 5°C for 13h, centrifuge at 8000r / min for 20min, take the supernatant, and freeze ...

Embodiment 2

[0070] Embodiment 2: Extract hyaluronidase from bovine spleen

[0071] (1) Take 100g of fresh bovine spleen, remove the epidermal envelope, cut into small pieces, add 100g of 2.0% normal saline and soak for 30min, then add 250g of extract, mash it with a tissue grinder, and put it in a -8°C incubator In the medium, stir with a mechanical stirrer at a stirring speed of 550r / min. After 50min, stand at -1°C for 1h, take the supernatant, add 35g of sodium chloride, stir at 0°C for 40min, let stand for 1.5h, and centrifuge at 6000r / min for 15min. , take the supernatant, add 100g of salt, stir at 0°C for 55min, then let it stand for 9h, centrifuge at 9000r / min for 10min, and take the precipitate to obtain the crude product of hyaluronidase;

[0072] (2) Take the precipitate in step (1), put it into a dialysis bag, put it into 50g of citric acid-sodium hydroxide-hydrochloric acid buffer, dialyze at 6°C for 13h, centrifuge at 8000r / min for 20min, take the supernatant, and freeze Drie...

Embodiment 3

[0073] Embodiment 3: Extract hyaluronidase from bovine spleen

[0074] (1) Take 100g of fresh bovine spleen, remove the epidermal envelope, cut into small pieces, add 100g of 2.0% normal saline and soak for 35min, then add 250g of extract, mash it with a tissue grinder, and put it in a -8°C incubator In the medium, stir with a mechanical stirrer at a stirring speed of 550r / min. After 50min, let stand at 0°C for 1h, take the supernatant, add 35g of sodium chloride, stir at 0°C for 50min, let stand for 2h, centrifuge at 6000r / min for 15min, and take Add 100g of salt to the supernatant, stir at 0°C for 60min, then let it stand for 9h, centrifuge at 9000r / min for 10min, and take the precipitate to obtain the crude product of hyaluronidase;

[0075] (2) Take the precipitate in step (1), put it into a dialysis bag, put it into 50g of citric acid-sodium hydroxide-hydrochloric acid buffer, dialyze at 7°C for 13h, centrifuge at 8000r / min for 20min, take the supernatant, and freeze Dri...

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PUM

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Abstract

The invention provides a method for extracting hyaluronidase from a bovine spleen. The method comprises the following steps: soaking the bovine spleen in normal saline with a certain concentration, adding extracting liquid to extract, triturate and extract, centrifuging and taking supernatant, adding sodium chloride to remove impurities, adding salt to perform salting-out, and performing dialysis to prepare the refined hyaluronidase. The method is used for extracting the hyaluronidase from the bovine spleen, simplifies the steps, is easy to operate, is high in extraction rate, purity and enzyme activity of the hyaluronidase, and explores a new approach for extraction of the hyaluronidase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting hyaluronidase from bovine spleen. [0002] technical background [0003] Hyaluronidase (Hyaluronidase, HAase) is a mucopolysaccharide endonuclease that can reduce the activity of hyaluronic acid in the body and improve the liquid permeability of tissues. play an important role in. Hyaluronidase is widely found in mammals, invertebrates (crustaceans, insects, leeches), pathogenic bacteria (Candida, Streptomyce), bacteria and phages, and comes from different biological tissues A large number of hyaluronidases have been identified. HAase is widely used in the field of medicine. Its main function is to reduce the relative molecular weight of hydrolyzed hyaluronic acid (HA) while eliminating the viscosity of HA and enhancing the permeability of the body to foreign substances. In medicine, HAase can promote the diffusion of drugs in the body. Therefore, the import...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26
CPCC12N9/2474C12Y302/01035
Inventor 谭春萍
Owner NANNING UNIV
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