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Recombinant human hyaluronidase, preparation method thereof and adopted polyethylene glycol covalently modified compound and method

A technology of human hyaluronidase and polyethylene glycol, applied in the field of biomedicine, can solve problems such as prolongation of half-life, and achieve the effects of low production cost, high stability and good stability

Active Publication Date: 2015-07-01
杭州北斗生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The pharmacokinetic properties of PEG-modified hyaluronidase also changed greatly, and the half-life was greatly extended

Method used

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  • Recombinant human hyaluronidase, preparation method thereof and adopted polyethylene glycol covalently modified compound and method
  • Recombinant human hyaluronidase, preparation method thereof and adopted polyethylene glycol covalently modified compound and method
  • Recombinant human hyaluronidase, preparation method thereof and adopted polyethylene glycol covalently modified compound and method

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Experimental program
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Effect test

Embodiment 1

[0097] Embodiment 1, the preparation of recombinant human hyaluronidase

[0098] The preparation method of recombinant human hyaluronidase comprises the following steps: (1), the acquisition of human hyaluronidase gene; (2) the construction and transformation of expression vector Pichia engineering bacteria: the synthetic human hyaluronidase The cDNA fragment is inserted into the restriction site of the expression vector, and then directly transformed into the Pichia host bacterium; (3), screening, cultivation, and induced expression of positive clones: the positive clones are selected and cultured on the medium, and induced Target protein expression; (4), separation and purification of fusion protein: collect the Pichia pastoris fermentation broth, centrifuge to obtain the supernatant of the fermentation broth, and then purify the supernatant of the fermentation broth by chromatography; (5) crudely purified The protein was further separated and purified.

[0099]The recombin...

Embodiment 2

[0105] Embodiment two, the activity determination of recombinant human hyaluronidase

[0106] The amount of reducing sugar produced by hydrolysis of hyaluronic acid was measured by 3,5-dinitrosalicylic acid colorimetric method. The reaction system is 1 mL, and 2 mg / mL of hyaluronic acid is prepared with pH 5.5, 50 mM citric acid-disodium hydrogen phosphate buffer solution, and 900 μL of hyaluronic acid solution and 100 μL of the test sample are added to the reaction system. React at 38°C for 20 minutes, immediately boil in water for 5 minutes to terminate the reaction, centrifuge to remove the denatured protein precipitate; use the DNS method to measure the equivalent of reducing sugar produced (equivalent to the reducing power of an equivalent amount of glucose), and calculate the activity of the test product. The specific activity of the enzyme was calculated from the protein concentration. The specific activity of the purified human hyaluronidase is greater than 100,000 U / ...

Embodiment 3

[0107] Embodiment three, different molecular weight polyethylene glycols modify human hyaluronidase

[0108] 1. Preparation of modified products

[0109] Purified Pichia pastoris expresses human hyaluronidase with a purity greater than 95%, a concentration of 2.0 mg / ml, using a G-25 column to exchange the buffer, and preparing an equilibrium buffer of 0.1 mol / L Na 2 PO 4 , 20mmol / L NaCl, flow rate 10ml / min, load the sample after the column effluent reaches equilibrium, and collect the protein peak. Monomethoxy PEG propionate with different molecular weights was added to the hyaluronidase with a different buffer system at a mass ratio of 10:1, and the reaction was terminated after stirring at room temperature for 2 hours for modification. Use a 50KD ultrafiltration membrane to ultrafilter the hyaluronidase PEG reaction mixture against 20mmo / L PB, pH 7.0 buffer solution to remove free PEG, which is the hyaluronidase PEG modified stock solution.

[0110] SDS-PAGE analysis was ...

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Abstract

The invention relates to the field of biological medicines and in particular relates to recombinant human hyaluronidase, a preparation method thereof and an adopted polyethylene glycol covalently modified compound and method. The invention aims at providing the recombinant human hyaluronidase and a preparation method thereof. The preparation method of the recombinant human hyaluronidase comprises the steps of integrating a human hyaluronidase gene into pichia pastoris engineering bacteria, fermenting and purifying, so that a large number of high-activity and high-purity recombinant human hyaluronidase can be obtained. The invention also aims at providing a recombinant human hyaluronidase-polyethylene glycol modified compound and a preparation method thereof, wherein the recombinant human hyaluronidase-polyethylene glycol modified compound has high activity, low immunogenicity and good stability.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant human hyaluronidase, a preparation method thereof, and a covalently modified compound and method using polyethylene glycol. Background technique [0002] Hyaluronidase (HAase) mainly specifically hydrolyzes hyaluronic acid, widely exists in eukaryotes and prokaryotes, and participates in many important biological processes in animals, such as cell division, connection between cells, reproduction It is an important physiologically active substance for cell activity, DNA transfection, embryo development, repair of injured tissue, and proliferation of normal cells and tumor cells. It was first discovered by Duran Reynals in 1928 and called "diffusion factor", and officially named hyaluronidase by Chain and Duthie in 1940. [0003] Its mechanism of action is to reduce the viscosity of hyaluronic acid by catalyzing the hydrolysis of hyaluronic acid, thereby improving tissue p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/81C12N9/96C12R1/84
CPCC12N9/2474C12N9/96C12Y302/01035
Inventor 刘翔张加慧封小燕刘沐荣
Owner 杭州北斗生物技术有限公司
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