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Selenium-containing recombinant human albumin preparation method

A technology of human serum albumin and sodium selenite, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of high production cost, poor selenium content, and low yield of human serum albumin, Achieve the effects of improving immunity, clear ingredients, and high yield

Active Publication Date: 2013-12-25
徐东
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of this, the present invention provides a method for preparing recombinant human serum albumin containing selenium, which solves the technical problems of high preparation cost, low yield and poor selenium content of human serum albumin in the prior art

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  • Selenium-containing recombinant human albumin preparation method
  • Selenium-containing recombinant human albumin preparation method
  • Selenium-containing recombinant human albumin preparation method

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preparation example Construction

[0015] The embodiment of the present invention provides a preparation method of recombinant human serum albumin containing selenium, comprising the following steps:

[0016] Step S01, constructing Pichia pastoris engineered bacteria;

[0017] The construction method adopts conventional technical means in the field of genetic engineering:

[0018] The rHSA derived from human hepatocytes was cloned by RT-PCR. After the amplified product was digested and recovered by electrophoresis, it was inserted into the multiple cloning site of the plasmid pPIC9K to construct the expression plasmid pPIC9K-rHSA. The positive clone was identified by double enzyme digestion and then transformed. Red yeast GS115, positive clones identified by PCR. details as follows:

[0019] Extraction of total RNA:

[0020] A. Prepare centrifuge tubes, pipette tips, mortars and related reagents for RNA extraction in advance:

[0021] 1. Soak a 1.5mL centrifuge tube, 10μL, 200μL and 1mL pipette tips in chl...

Embodiment 1

[0183] The preparation method of selenium-containing recombinant human serum albumin according to the embodiment of the present invention comprises the following steps:

[0184] 1. Construction of Pichia pastoris engineering bacteria

[0185] RNA was extracted from fresh human liver tissue, RT-PCR was amplified to obtain the rHSA target gene, the amplified product was connected to the cloning vector PMD-18T, and the plasmid PMD-18T-rHSA was constructed. pPIC9K-rHSA, positive clone identified by double enzyme digestion, transformed into Pichia pastoris GS115, positive clone identified by PCR;

[0186] 2. Fermentation culture

[0187] Pick a positive single colony and inoculate it in 50ml of YPD medium, incubate at 30°C for 15h, then inoculate 0.5% of the seed bacteria solution into a 500ml Erlenmeyer flask containing 100ml of BMGY medium, rotate at 200rpm, cultivate at 30°C, and use a mass of The concentration is 28% concentrated ammonia water to adjust the pH value to 6.0. A...

Embodiment 2

[0197] The preparation method of selenium-containing recombinant human serum albumin according to the embodiment of the present invention comprises the following steps:

[0198] 1. Construction of Pichia pastoris engineering bacteria

[0199] The construction of genetically engineered bacteria is the same as in Example 1

[0200] 2. Fermentation culture

[0201]Pick a positive single colony and inoculate it in 50ml of YPD medium, incubate at 30°C for 17h, then inoculate 1.0% of the seed bacteria solution into a 500ml Erlenmeyer flask containing 100ml of BMGY medium, rotate at 180rpm, cultivate at 30°C, and use a mass of The concentration was 28% concentrated ammonia water to adjust the pH value to 6.0. After 48 hours, the OD600 of the bacterial solution was detected to be 0.688. Add 0.5% analytical pure methanol first, then add 0.2% glycerol and 0.8% analytical pure methanol after 12h for induction. During the induction period, the culture temperature was 28°C, and the pH v...

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Abstract

The invention is suitable for the technical field of the gene engineering, and provides a selenium-containing recombinant human albumin preparation method. The method comprises the steps of constructing a Pichia pastoris engineering strain, carrying out fermentation culture, and purifying. The selenium-containing recombinant human albumin preparation method has the advantages of simple operation, low cost and high yield, a selenium-containing recombinant human albumin prepared through the method has a healthcare effect and overcomes a blood albumin security problem as an albumin supplement, and the selenium-containing recombinant human albumin inhibits cancers, improves the immunity, enhances the tissue restoration capability and has a great market potential as an organic selenium. Compared with present selenium nutriments, the selenium-containing recombinant human albumin has the advantages of clear components, high security, high organic purity and high nutritive value when the selenium-containing recombinant human albumin is sold in the market as a simple nutrition and healthcare product.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of selenium-containing recombinant human serum albumin. Background technique [0002] Human serum albumin is extremely rich in human plasma. It is the most used protein in the circulatory system, accounting for about 60% of the total plasma protein. The typical plasma concentration of HSA is 50g / L, and the osmotic pressure accounts for 80% of the total plasma. Normal human serum albumin level is 3.9-4.5g per 100mL. When the albumin level per 100mL of plasma is lower than 3g, systemic edema will occur. When the patient suffers from trauma, the brain pressure increases and the brain edema compresses the nerves. , albumin is the most important means of rescue. Therefore, human serum albumin is mainly used clinically for surgical blood transfusion and rehydration of critically ill patients, to treat traumatic shock, fever, edema, hypoalbuminemia and poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K1/36C07K1/18C07K1/30C07K1/34
Inventor 徐东杨艳群
Owner 徐东
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