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BCG polysaccharide nucleic acid extractive and preparation method thereof

A BCG polysaccharide nucleic acid and BCG technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and drug combinations, can solve the problems of unstable process, long production cycle, operator hazards, etc. The effect of high degree of process automation, shortened production cycle and reduced batch-to-batch variation

Active Publication Date: 2012-05-30
HUNAN SIQI BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process has its own insurmountable shortcomings: (1) The process is manual, and the technology is primitive and backward, resulting in high residues of BCG protein and phenol in the refined BCG polysaccharide nucleic acid extract, which affects the product (2) The process is unstable, resulting in large differences between batches of refined BCG polysaccharide nucleic acid extracts; (3) The production cycle is long and the production efficiency is low; (4) The feed liquid is easily polluted during the production process, and bacteria The number often exceeds the specified range, resulting in a large number of unqualified batches, which affects product quality and cost; (5) Since the production process requires the use of high-concentration phenol, the dialysis process is manual and has a long production cycle, so the harm caused to the operator bigger

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  • BCG polysaccharide nucleic acid extractive and preparation method thereof
  • BCG polysaccharide nucleic acid extractive and preparation method thereof
  • BCG polysaccharide nucleic acid extractive and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 The cultivation and harvest of BCG

[0028] 1. Bacterial culture: Dissolve the strains preserved in liquid cryogenic temperature (BCG strain D2PB302 for the preparation of BCG in China, China National Institute for the Control of Pharmaceutical and Biological Products) at room temperature, inoculate in potato Sutong culture medium, and culture continuously at 37°C for 14-20 days ; or after continuous cultivation at 37°C for 15 days, transfer to improved liquid Sutong medium, and continuous cultivation at 37°C for 14-20 days.

[0029] Wherein, the preparation method of potato Sutong culture medium can be:

[0030](1) Take and wash fresh potatoes (1), pierce them into cylinders with a piercer, and cut them into 4 cm long slopes with a knife; rinse the potato slopes with running drinking water for 1 hour, and then rinse the potato slopes with purified water; Wash the slanted block with Sutong medium, take 20ml of Sutong medium, and add it to a 100ml steriliz...

Embodiment 2

[0040] The preparation of embodiment two BCG polysaccharide nucleic acid mixture

[0041] 1. Cell crushing and thermal phenol treatment: Add the collected cells to purified water at a ratio of 10:1, crush the cells with a tissue mash homogenizer (12000rpm / min), 3 min x 3 times, and pound the cells Then add 0.5-2.0 times the volume of hot phenol (30-100° C.) of the crushed bacteria suspension, and keep warm for 30 minutes to 1 hour while stirring at a low speed.

[0042] 2. Extraction of BCG polysaccharide nucleic acid mixture: Precipitate the hot phenolic mixture naturally for 1 to 10 days, absorb the supernatant, centrifuge at high speed in a tube centrifuge, and filter the supernatant through a 0.45um sterile filter to obtain BCG polysaccharide nucleic acid mixture.

Embodiment 3

[0043] Example 3 Preparation of Purified BCG Polysaccharide Nucleic Acid Extract

[0044] 1. Refining BCG polysaccharide nucleic acid extract by dialysis

[0045] Manually put the BCG polysaccharide nucleic acid mixture into a dialysis bag (cut-off molecular weight > 5000 Daltons) and dialyze in 100 times the volume of purified water for 7 to 10 days, replace the purified water in the dialysis pool every day, and take samples from the dialysis bag for testing every day Phenol residues. Add appropriate amount of ethanol to the dialyzed BCG polysaccharide nucleic acid mixture to make the alcohol content 60-85%, and after natural precipitation for 1-10 days, the precipitate is fully stirred with absolute ethanol, centrifuged and washed 3 times. Then wash and centrifuge with ether for 3 times, put it in a desiccator for drying, and dry for 2 to 5 days to obtain the refined BCG polysaccharide nucleic acid extract.

[0046] 2. Purification of BCG Polysaccharide Nucleic Acid Extr...

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Abstract

Provided is a BCG polysaccharide nucleic acid extractive. In the extractive, the moisture content is less than 10% (w / w), the polysaccharide content is 70-78% (w / w), the nucleic acid content is 12-20% (w / w), the residual BCG bacterial protein content is 0-0.5% (w / w), the phenol residue is zero and the number of the mixed bacterium is 0-20 per gram. Also provided is a preparation method of BCG polysaccharide nucleic acid extractive, which comprises the following steps: culturing the BCG, disrupting the culture with physical methods to obtain BCG polysaccharide nucleic acid suspension, treatingthe BCG polysaccharide nucleic acid suspension with hot phenol method and high speed centrifugation to obtain BCG polysaccharide nucleic acid mixture, isolating the BCG polysaccharide nucleic acid extracting solution from the BCG polysaccharide nucleic acid mixture by gel column chromatography, and purifying the BCG polysaccharide nucleic acid extracting solution to obtain BCG polysaccharide nucleic acid extractive.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a polysaccharide nucleic acid extract and a preparation method thereof. Background technique [0002] BCG is bovine attenuated tuberculosis, non-pathogenic, and immunogenic. It is one of the most widely used vaccines in the world to replace the initial infection of tuberculosis with BCG inoculation to obtain immunity against tuberculosis. one. [0003] In 1882, Robert successfully isolated Mycobacterium tuberculosis and determined the pathogenic bacteria of tuberculosis. In 1908, Calnette and Guerin of the Pasteur Institute in France successfully bred a strain of attenuated Mycobacterium bovis and named it Bacillus Calmette-Guerin (BCG). In 1921, Weill-Halle successfully applied the bacteria to the human body to prevent and treat tuberculosis. In 1971, the Changsha Research Group for the Prevention and Treatment of Tracheitis first used the dead...

Claims

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Application Information

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IPC IPC(8): C12N15/00A61K35/74
CPCA61K31/7052A61K31/715A61K35/74A61P11/00A61P11/02A61P11/06A61P11/10A61P17/00A61P31/00A61P37/02A61P37/08
Inventor 宁云山关继峰
Owner HUNAN SIQI BIOPHARM