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Recombinant pichia pastoris for producing human hyaluronidase PH20 and construction method of recombinant pichia pastoris

A technology of hyaluronidase and Pichia pastoris, which is applied in the field of genetic engineering, can solve problems such as inability to meet application requirements, and achieve the effects of reducing intraocular pressure, promoting diffusion and absorption, and increasing permeability

Pending Publication Date: 2022-06-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2016, some researchers used the plasmid pGAPZαC and the promoter P GAP Realized the constitutive expression of hPH20 in P. pastoris GS115, but the yield of hPH20 in the fermentation broth was only 2U / L, far from meeting the application requirements

Method used

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  • Recombinant pichia pastoris for producing human hyaluronidase PH20 and construction method of recombinant pichia pastoris
  • Recombinant pichia pastoris for producing human hyaluronidase PH20 and construction method of recombinant pichia pastoris
  • Recombinant pichia pastoris for producing human hyaluronidase PH20 and construction method of recombinant pichia pastoris

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Construction of hPH20 and its mutants

[0048] According to the hPH20 sequence reported by NCBI (Genbank accession number: NP_003108.2), the nucleotide sequence corresponding to the 36-511th amino acid sequence was optimized according to the Pichia pastoris codon preference. The optimized gene sequence SEQ ID NO .1 It was synthesized by Universal Biosystems (Anhui) Co., Ltd. and ligated between the EcoRI and Not I restriction sites of the expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-hPH20. Next, the truncation mutation primers △484C-F, △484C-R and △491C-F, △491C-R as shown in Table 1 were designed, using the plasmid pPIC9K-hPH20 as the template, using PCR technology, respectively construct the C deletion hPH20 Truncated mutants of amino acids after position 484 (that is, retaining position 483, truncating positions 484 and 484) and amino acids after position 491 (that is, retaining position 490, truncating position 491 and later) △484C an...

Embodiment 2

[0065] Example 2 Shake flask fermentation of hPH20 and its mutants

[0066] The recombinants P. pastoris-pPIC9K-hPH20, P. pastoris-pPIC9K-Δ484C, P. pastoris-pPIC9K-Δ491C, P. pastoris-pPIC9K-ap constructed in Example 1 2 -△491C, P.pastoris-pPIC9K-hl 28 -△491C and P. pastoris-pPIC9K-sumo-△491C were streaked on the YPD solid plate and placed in a 30°C constant temperature incubator until a single colony was grown. A single colony was inoculated into a 250 mL shake flask containing 50 mL of YPD liquid medium, and cultured overnight at 30° C. and 200 rpm to obtain a seed culture. The seed culture was transferred to a 250 mL shake flask containing 50 mL of BMGY medium at 10% (v / v) inoculum, and cultivated to OD at 30 °C and 200 rpm 600 When reaching 6, collect all the cells and wash them with 0.9% NaCl for 3 times, then transfer them to a 250 mL shake flask containing 50 mL of BMMY medium, and cultivate at 30 °C and 200 rpm for 96 h, supplemented with a final concentration of 1% (...

Embodiment 3

[0073] Example 3 Mutant AP 2 - △ 491C 3-L fermenter test

[0074] The recombinant P. pastoris-pPIC9K-ap constructed in Example 1 2 -△491C was streaked on the YPD solid plate and placed in a 30°C constant temperature incubator until a single colony was grown. A single colony was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD liquid medium, and cultured overnight at 30°C and 200 rpm. The seed culture was transferred to a 3-L fermentor containing 900 mL of BSM medium at a 10% (v / v) inoculum with initial parameter settings: temperature 30°C, pH 5.0, aeration 2.0 vvm and rotation speed 200 rpm. When the glycerol was exhausted and the dissolved oxygen in the fermentation broth rebounded, the glycerol mother liquor with a concentration of 500g / L containing 1.2% PTM1 by volume was added at a rate of 25mL / L / h, and the feed was fed for 10h. During the process, the ventilation volume was adjusted as 4.0vvm, the rotation speed was gradually increased to 800-900rpm ac...

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Abstract

The invention discloses recombinant pichia pastoris capable of producing human hyaluronidase PH20 and a construction method of the recombinant pichia pastoris, and belongs to the technical field of genetic engineering. According to the invention, an hPH20 mutant AP2-491C is constructed by optimizing a C-terminal structural domain of hPH20 and combining an N-terminal protein tag fusion strategy; induced expression of an hPH20 mutant in P.pastoris GS115 is realized by using an integrated plasmid pPIC9K and a strong promoter PAOX1, and the enzyme activity in a fermentation solution reaches 258.1 U / L after culture in a 3-L fermentation tank, which is the highest level of the preparation of hPH20 by fermentation of a microbial expression system at present. The mutant can hydrolyze macromolecular HA in body tissues and increase the permeability of tissue cell membranes, and has application value in clinical medicine aspects such as promotion of drug diffusion and absorption, enhancement of drug effects and reduction of intraocular pressure.

Description

technical field [0001] The invention relates to a recombinant Pichia pastoris producing human-derived hyaluronidase PH20 and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Hyaluronidase (Hyaluronidases, Hyals) is a general term for a class of enzymes that produce low molecular weight hyaluronic acid (HA). Hyals are widely used in medicine due to their ability to promote drug absorption, anti-tumor signaling, and lowering intraocular pressure. According to their different catalytic mechanisms and products, Hyals are divided into three categories: the first category is endo-β-N-acetylglucosaminidase (EC 3.2.1.35), which is mainly found in mammals, honeybees, snake venom, bee venom and lysozyme In body and other venoms, it belongs to the hyaluronate 4-glycanohydrolases family. It acts on the β-1,4 glycosidic bond of HA to generate tetrasaccharide (HA4) as the main hydrolysis product, and can also hydrolyze ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/81C12N1/19A61K38/47A61P27/02C12R1/84
CPCC12N9/2474C12N15/815C12N1/16A61P27/02C12N2800/60A61K38/00
Inventor 康振堵国成王淼庞博王阳
Owner JIANGNAN UNIV
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