Preparation method of recombinant human albumin-uricase fusion protein

A uricase and protein technology, applied in the field of biotechnology genetic engineering, can solve the problems of restricting wide application and low microbial enzyme production

Inactive Publication Date: 2014-12-03
李殿明 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the microbial enzyme production is low, which limits its wide application.

Method used

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  • Preparation method of recombinant human albumin-uricase fusion protein
  • Preparation method of recombinant human albumin-uricase fusion protein
  • Preparation method of recombinant human albumin-uricase fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example Escherichia coli expression vector and the construction of expression strain

[0034] The designed coding genes of human serum albumin HSA and porcine uricase (UrATe oxidase of swine, pUO) were synthesized by Shanghai Handsome Biotechnology Company, and BamH I (5' end) and Nhe I were designed at both ends of the HSA gene fragment (3' end) restriction endonuclease sites, Nhe I (5' end) and Hind III (3' end) restriction endonuclease sites were designed at both ends of the pUO gene fragment respectively. Shanghai Handsome Biotechnology Co., Ltd. synthesized the gene fragment and cloned it into the pMD18T vector. After the sequence was correct, the E. coli containing the recombinant plasmid was sent back to the company.

[0035] Escherichia coli strains containing the coding genes of HSA and pUO were respectively inoculated into LB medium containing ampicillin, cultured overnight at 37°C with shaking, and the recombinant plasmid was extracted the next day according ...

Embodiment 2

[0038] Fermentation, purification and preparation of embodiment two engineering bacteria

[0039] Fermentation takes the strains of the basic seed bank for production, and after opening, mark 2 to 3 plates of seed medium (LB+Amp), pick 8 to 10 typical colonies on the plate after culture, store them after culture respectively, and then carry out recombinant uric acid respectively The determination of enzyme protein expression was repeated three times, and the one with the highest expression was selected for production and preparation of seed liquid. Pick a single bacterium and inoculate it in a test tube of LB liquid medium, culture and shake in a shaker at 32°C for 12 hours. Put the bacteria in the test tube into the shake flask with the inoculum amount of 1:100, and cultivate in the remote bed at 32°C until OD600=3, that is, the fermenter can be inoculated at a ratio of 10%. The fermentation medium is a modified LB medium, prepared with distilled water, which does not contai...

Embodiment 3

[0043] The mensuration of embodiment three enzyme activity

[0044] Referring to the method of Koyama et al. (1996), at 37°C, take 120 μmol / L uric acid solution dissolved in 3ml pH7.5 boric acid buffer solution, add an appropriate amount of uricase solution to a 1cm quartz cuvette, mix well, and place in UV -1601PC ultraviolet spectrophotometer, react accurately for 5 minutes, use boric acid buffer as blank, and measure the decrease of absorbance at 293nm. The activity unit is defined as the amount of enzyme required to catalyze 1 μmol of uric acid into allantoin within 1 min under the conditions. Its calculation formula is:

[0045] U / ml=(ΔA×VT×df) / (12.6×VE), in the formula: U=number of uricase activity units per ml; ΔA=optical density drop value at 293nm wavelength per minute; VT=total volume of reaction solution (ml); df=dilution factor; 12.6 is the micromolar extinction coefficient of uric acid at a wavelength of 293nm; VE=enzyme solution volume (ml).

[0046] Relative ...

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Abstract

The invention relates to a preparation method of a recombinant human albumin-uricase fusion protein drug. The recombinant human albumin-uricase fusion protein drug contains human albumin, pig uricase and a purification tag. The preparation method of the recombinant human albumin-uricase fusion protein drug has stable processes and is suitable for large-scale production. External enzyme activity detection and white mouse in-vivo preliminary pharmacodynamic experiments prove that the recombinant uricase can obviously reduce an internal uric acid concentration.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering and mainly relates to the preparation of uricase. Specifically, using gene recombination technology, human serum albumin (HSA) and uricase gene are connected in series and cloned into the vector, transformed into host bacteria, and after fusion expression, purification and preparation process, a recombinant uricase drug with ideal therapeutic effect is obtained. , for the treatment of hyperuricemia complicated by gout, nodules, renal insufficiency, organ transplantation and malignant diseases. Background technique [0002] Hyperuricemia is not only the direct cause of gout and related diseases, but also an independent risk factor for certain renal and cardiovascular diseases (Wu Xin et al., 2007). Therefore, reducing blood and tissue uric acid levels is a key link in the prevention and treatment of various uric acid-related diseases. The human body cannot synthesize uric acid oxi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21A61K38/44A61K47/48A61P19/06
CPCC12N9/0048A61K38/44A61K47/643C07K14/765C12Y107/03003
Inventor 李殿明蒲勤李毅齐春梅田春辉任百亮张导春刘甜甜顾富香
Owner 李殿明
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