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Recombinant Pichia pastoris for producing small-molecular hyaluronic acids and construction method thereof

A Pichia pastoris and hyaluronic acid technology, applied in the field of recombinant Pichia pastoris and its construction, can solve the problems of expensive substrates, cumbersome steps, and low synthesis efficiency, and achieve the effects of reducing viscosity and easy purification and recovery

Active Publication Date: 2015-01-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, although it has been reported that HA oligosaccharides are prepared by de novo synthesis, it is difficult to realize the preparation and application of HA oligosaccharides due to many problems such as expensive substrates, cumbersome steps and low synthesis efficiency in chemical synthesis.

Method used

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  • Recombinant Pichia pastoris for producing small-molecular hyaluronic acids and construction method thereof
  • Recombinant Pichia pastoris for producing small-molecular hyaluronic acids and construction method thereof
  • Recombinant Pichia pastoris for producing small-molecular hyaluronic acids and construction method thereof

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Embodiment 1

[0037] Cloning of embodiment 1hasA gene and UDP-glucose dehydrogenase tuaD gene

[0038] The hasA gene used in the present invention is derived from Streptococcus zooepidemicus ATCC 35246 and the UDP-glucose dehydrogenase gene is derived from Bacillus subtilis. The Streptococcus zooepidemicus strain is inoculated with 5ml of M17 liquid medium and cultured at 37°C and 200rpm for 16h. Bacillus subtilis was inoculated in 5ml LB liquid medium and cultured at 37°C and 200rpm for 16h. Pichia pastorisGS115 was inoculated in 5ml YPD liquid medium, cultured at 200rpm and 30°C for 24h. Bacteria were collected separately, and the genomic DNA of the three strains was extracted using a bacterial genome extraction kit.

[0039] According to the published genome information sequence, design primers hasA-F / hasA-R, tuaD-F / tuaD-R respectively, use the extracted genomic DNA as a template, and use standard PCR amplification system and program to amplify and obtain hasA respectively and tuaD gen...

Embodiment 2

[0049] Construction of embodiment 2 recombinant plasmid PAPT9K

[0050] The amplified DNA fragments hasA, tuaD, GAP and TEF1 were used to fuse the promoter and the target gene by fusion PCR respectively. The specific operation procedure is as follows: take 2 ul of hasA and GAP fragments and mix them in a PCR tube, add 21 μl of sterile water and 25 μl of 2xsuper pfu Master Mix (Hangzhou Baosai Biotechnology Co., Ltd.), mix well, place in a PCR instrument, and run according to the following procedures: 94°C for 3min, [94°C for 30s, 50°C for 30s, 72°C for 1min]×10, 72°C for 5min. Immediately after self-fusion PCR without primers, add 1 μl of primers gap-F and hasA-R, mix well and run according to the following program: 94°C for 3 minutes, [94°C for 30s, 55°C for 30s, 72°C for 1min]×32,72 ℃ 5min. After the PCR product was subjected to 1% agarose gel electrophoresis, the target band was recovered by cutting the gel to obtain the GAP-hasA fragment.

[0051] Similarly, the TEF1-tu...

Embodiment 3

[0052] Embodiment 3 The strain construction of recombinant Pichia pastoris producing HA

[0053] After the recombinant plasmid PAPT9K was linearized with SalI, the Pichia pastoris GS115 host was electroporated according to the operation manual of Pichia pastoris, and positive recombinants were screened with MD plate (histidine-deficient selection marker). At the same time, in order to screen for hosts with multi-copy insertions, high-copy screening was performed on YPD plates containing different concentrations of antibiotic g418. The fermented recombinant strain used in the present invention is a positive recombinant strain screened out at 4 mg / ml, named PAPTGS115.

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Abstract

The invention discloses a recombinant Pichia pastoris for producing small-molecular hyaluronic acids and a construction method thereof, belonging to the technical field of bioengineering. Hyaluronic acid synthase hasA derived from Streptococcus zooepidemicus and UDP-glucose dehydrogenase tuaD derived from Bacillus subtilis are expressed in recombinant Pichia pastoris GS115 host to implement production of hyaluronic acids. Meanwhile, hyaluronidase derived from hirudo is integrated to the Pichia pastoris genome and subjected to secretion expression by constitutive promoters with different intensities; the secretion expression level of the hyaluronidase is controlled to prepare small-molecular hyaluronic acid products with different molecular weights; and the prepared products have different ranges of molecular weight, and have instruction and reference meanings for directly producing small-molecular hyaluronic acids within a specified range from microbes. The recombinant Pichia pastoris lays certain foundation for high-efficiency preparation of small-molecular hyaluronic acids, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a recombinant Pichia pastoris producing small molecule hyaluronic acid and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Hyaluronic acid (Hyaluronic Acid, HA) is a kind of polymer viscous polysaccharide. With its unique molecular structure and physical and chemical properties, it has good moisture retention, viscoelasticity, permeability and extensibility. It is currently found in nature. It is the substance with the best moisturizing properties, and has no immunogenicity and toxicity. It is widely used in cosmetics, food and medicine and other industries. In recent years, studies have found that low molecular weight HA (below 1×10 4 ) and hyaluronic acid oligosaccharides have unique biological functions. Low molecular weight HA (below 1×10 4 ) and oligomeric hyaluronic acid, exhibit very strong biological activity, have the functions of inhibiting tumor spre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P19/26C12R1/84
CPCC12N1/16C12N15/815C12P19/26C12N1/165C12R2001/84
Inventor 陈坚堵国成康振金鹏
Owner JIANGNAN UNIV
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