Preparation method of recombinant human serum albumin containing selenium
The technology of human albumin and engineering bacteria is applied in the field of preparation of selenium-containing recombinant human albumin, can solve the problems of poor selenium content, low yield and high preparation cost of human albumin, and achieves clear composition and high yield , the effect of improving immunity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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 chlo...
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 a temperature of 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 2...
PUM
| Property | Measurement | Unit |
|---|---|---|
| capacitance | aaaaa | aaaaa |
| electrical resistance | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More