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Rapid purification method of apolipoprotein B

A technology of apolipoprotein and purification method, which is applied in the direction of preparation method of apolipoprotein and peptide, chemical instruments and methods, etc., can solve the problems of extended production cycle, cumbersome operation, low purification efficiency, etc., and achieve reduction of purification cost, operation Simple steps and high purification yield

Active Publication Date: 2012-11-07
广东派特埃尔生物科技有限公司
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  • Claims
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AI Technical Summary

Problems solved by technology

The high-speed gradient centrifugation method has the disadvantages of small processing capacity, low purification efficiency and high purification cost
High-performance liquid chromatography requires expensive equipment and skilled operators, and has the disadvantages of high purification costs
D-glucoside sulfate precipitation is currently a commonly used purification method, but this method has expensive reagents; cumbersome operation, long-term dialysis, increased purification costs, and prolongs the production cycle; the purified product has many degradation fragments and the purity is not high enough; and due to At present, the principle of the precipitation of apolipoprotein B by D-glucoside sulfate is not clear, and the precipitate cannot be dissociated again, that is, the obtained precipitate is an irreversible precipitate, and the physical and chemical properties of apolipoprotein B in the precipitation complex change, which seriously interferes with apolipoprotein Further purification of B

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  • Rapid purification method of apolipoprotein B
  • Rapid purification method of apolipoprotein B

Examples

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Embodiment 1

[0021] Example 1 Purification of apolipoprotein B.

[0022] Take 5mL of human plasma, add 5mL of pH 6.0 phosphate buffer to dilute at a volume ratio of 1:1, and mix well to obtain a diluted plasma sample; heat the mixed diluted plasma sample in the first water bath at 70°C, heat During this period, gently mix the diluted plasma sample once every 5 minutes, and after heating for 20 minutes, centrifuge the diluted plasma sample at 8000g for 15 minutes, take the supernatant, and separate the denatured impurity protein precipitation; Add 0.1mL Triton X-100, mix well, let it stand for 4 hours, place it in a second water bath at 55°C for heating, and gently mix the supernatant once every 5 minutes during the heating period, after heating for 20 minutes, Centrifuge the supernatant at 8000 g for 15 minutes, discard the supernatant, and collect the precipitate; wash the collected precipitate twice with 0.05 mol / L Tris-HCl buffer solution, and filter with suction to obtain purified ap...

Embodiment 2

[0024] Example 2 Purification of apolipoprotein B.

[0025] Take 5 mL of human plasma, add 300 μL of 1% D-glucoside sulfate solution, shake and mix. After the color of the serum turns light yellow, add 300 μL of 2 mol / L calcium chloride, mix by inverting repeatedly for 15 minutes, and then put it in a refrigerator at 4°C overnight. Then, centrifuge at 4000g for 30min at 4°C, discard the supernatant, dissolve the precipitate in 5mL of 12% sodium chloride, add pH7.4, 0.2mol / L Tris-HCl buffer 5mL and centrifuge After 10 min, the supernatant was discarded, and the obtained precipitate was washed once with 0.02 mol / L Tris-HCl; this step was repeated 3 times. The precipitate was dissolved in 10 mL of 1mol / L potassium oxalate, and centrifuged at 3000 g for 30 min at 4°C to remove the precipitate. The resulting solution was dialyzed in 1% sodium chloride and 1% barium chloride for 48 hours (4°C), the dialyzed solution was deprecipitated, and the obtained supernatant was put into 0...

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Abstract

The invention provides a rapid purification method of apolipoprotein B, comprising the following steps of: A) taking human plasma and adding a buffering solution to be diluted and uniformly mixed; B) placing an obtained diluted plasma sample into a first water bath with the temperature of 65-75 DEG C to be heated; centrifuging and taking liquid supernatant; C) adding a surfactant with the volume percentage of 0.5-3% into the liquid supernatant and uniformly mixing to obtain processed liquid supernatant; D) placing the treated liquid supernatant into a second water bath with the temperature of50-60 DEG C to be heated; centrifuging, removing the liquid supernatant and collecting sediments; and E) washing the sediments by using a Tris-HCl buffering solution with the concentration of 0.01-0.1 mol / L for two times, and filtering to obtain purified apolipoprotein B. The rapid purification method of the apolipoprotein B, disclosed by the invention, has the advantages of being simple in operation steps, simple in needed equipment and high in purity of a purified product, and effectively reduces the purification cost.

Description

technical field [0001] The invention relates to a purification method of apolipoprotein, in particular to a rapid purification method of apolipoprotein B. Background technique [0002] Apolipoprotein B is the main structural protein of low-density lipoprotein cholesterol (LDL-CHOL), accounting for about 97% of the total protein content of LDL-CHOL. The determination of apolipoprotein B can directly reflect the level of LDL-CHOL. Apolipoproteins combine with lipids to form micromicelle-like spheres, where apolipoproteins function to stabilize the sphere structure and transport lipids. The colloidal ball composed of apolipoprotein B and lipids is called low-density lipoprotein, and its diameter is 20 to 40 times that of ordinary protein. Apolipoprotein B is considered to be a risk factor for atherosclerosis, and the determination of its concentration provides a valuable indicator for the judgment and prediction of atherosclerosis and cardiovascular diseases, and has important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/775C07K1/14
Inventor 李光飞曹玉珍
Owner 广东派特埃尔生物科技有限公司
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