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Human-derived muramidase protein fermentation method

A fermentation method and lysozyme technology, which are applied in the fields of genetic engineering and fermentation engineering, can solve the problems of high price, difficulty in ensuring no pollution from viruses and other harmful substances, and unstable product quality.

Pending Publication Date: 2016-11-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the world's largest and most famous sigma-aldrich biological / chemical reagent company (http: / / www.sigma-aldrich.com) sells genetically engineered human lysozyme (activity ≥ 100ku / mg), priced at 478.53 yuan / g, price Expensive, only used by various research institutions
In addition, the extraction of lysozyme from biological materials has its inherent defects. First, it is limited by the source of biological materials; Cause unstable product quality

Method used

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  • Human-derived muramidase protein fermentation method
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  • Human-derived muramidase protein fermentation method

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0152] Preparation of Pichia Competent Cells: Pick a single yeast colony and inoculate it in 50mL of YPD culture medium, culture it with shaking at 30°C until OD600=1.0-2.0. Centrifuge at 5000rpm for 5min at room temperature to collect the cells. 8×10 8 The cells were resuspended in 10 mL solution A (100 mM LiCl, 10 mM DTT, 0.6 M sorbitol and 10 mM Tris-HCl, pH 7.5), and allowed to stand at room temperature for 30 min. Centrifuge at 5000rpm at 4°C for 5min to collect the precipitate, then resuspend with 1.5mL of ice-cold 1M sorbitol, centrifuge to collect the precipitate. Repeat the above steps three times. Finally, resuspend with 500 μL of ice-cold 1M sorbitol, and put it on ice for later use.

[0153] Electrotransformation of Pichia pastoris competent cells: take 80 μL (about 10 μL) of yeast competent cells resuspended in ice-cold 1M sorbitol 10 cells / mL) were mixed with 3 μg of linearized DNA, and placed on ice for 5 minutes (note: the volume of the plasmid should be co...

Embodiment 1

[0184] Example 1 Bioinformatics analysis of human c-type lysozyme The mRNA sequence encoded by Homo sapiens lysozyme (LYZ) is derived from NCBI accession number: NM_000239.2. A preferred sequence is shown in SEQ ID NO 5. For the results of the conserved site analysis between human c-type lysozyme (LYZ) and some animal c-type lysozyme / a-whey protein, see figure 2 . The isoelectric point analysis of human c-type lysozyme and some animal c-type lysozyme / a-lactalbumin is shown in Table 1.

[0185] Prediction of mature protein isoelectric point and molecular weight of H-LYZ and animal LYZ / a-whey protein with ExPasy. According to the prediction results, the PI of human c-type lysozyme protein is 9.28, the PI of a-lactalbumin is lower than that of other proteins, the PI of bovine lysozyme protein is neutral, and the rest of lysozyme are alkaline, of which turkey The PI of lysozyme protein was the highest.

[0186] Table 1 Prediction results of isoelectric point and molecular wei...

Embodiment 2

[0188] Expression of embodiment 2LYZ gene in Pichia pastoris

[0189] 2.1 Construction of expression vector pPIC9K-LYC6

[0190] Use the universal primers 5'AOX1 and 3'AOX1 of the pPIC9K vector to carry out PCR to identify positive clones. The size of the positive clones is about 890bp, and the empty vector is about 460bp. The specific results are as follows figure 1 shown.

[0191] After the pPIC9K-LYC6 (LYC6 is LYZ) recombinant plasmid was linearized and electroporated into Pichia pastoris, white single colonies could grow on the MD plate without histidine. Pick a single colony for PCR verification. Using LYC6-F and LYC6-R as primers, a specific band at about 430bp is a positive clone, indicating that the target gene has been integrated into the Pichia pastoris genome.

[0192] Phenotype screening results of transformants, almost all transformants are Mut + The phenotype is consistent with the expected result of the plasmid integration method. The clones with uniform gro...

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Abstract

The invention belongs to the field of genetic engineering and fermentation engineering, and provides a human-derived muramidase protein fermentation method. Seed fermentation liquor of human-derived muramidase is added into a fermentation tank for high-density fermentation; high-cell-density fermentation includes methyl alcohol induction, tryptone addition and glycerinum supplement; a BSM fermentation medium is adopted in high-density fermentation of the fermentation tank and is inoculated with seed culture liquor of 5%. The highest activity of human-derived muramidase obtained through the method is 151600 U / ml and 127300 U / ml, the highest protein expression quantity reaches 1.65 g / L, and the batch has high stability. The amplification result is ideal, and practical reference base is provided for industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, in particular, the invention relates to the application of a production process for fermenting human lysozyme protein. Background technique [0002] Infectious diseases have always been a major threat to human health and life. At present, antibiotics and synthetic antibacterial drugs are still the most important treatment for bacterial infections. However, after entering the 21st century, the continuous emergence of multi-drug resistant bacteria makes the treatment of infectious diseases difficult, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), penicillin-resistant The emergence of various super-resistant bacteria such as Streptococcus pneumoniae (PRSP), pan-drug-resistant (PDR) Pseudomonas aeruginosa, and Acinetobacter baumannii. Therefore, it is urgent to find and develop antibacterial drugs that are different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12R1/84
Inventor 江松敏余龙余文博于颖曹立环陶建军唐丽莎杨鲜梅赛音
Owner FUDAN UNIV
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