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Recombinated multi shape ttansenula yeast, its structural method and application

A technology for Hansenula polymorpha and yeast is applied in the field of recombinant Hansenula polymorpha and its construction and application, which can solve the problem of difficulty in obtaining ideal transformants, inconvenience in large-scale industrial production, and low genetic stability. problems, to achieve the effect of maintaining physiological and biochemical characteristics, high industrial application value, and high genetic stability

Active Publication Date: 2007-11-07
TIANJIN HEMU JIANMIN BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low frequency of homologous recombination in Hansenula polymorpha, it is only possible to obtain a gene-blocking mutant strain using a gene-blocking method similar to that of S. Type strains are mostly obtained through chemical mutagenesis, and their prominent disadvantages are low genetic stability, difficulty in obtaining ideal transformants, and the possibility of multiple mutation effects, making the physiological and biochemical characteristics of cells significantly different from wild-type cells. Inconvenience for large-scale industrial production

Method used

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  • Recombinated multi shape ttansenula yeast, its structural method and application
  • Recombinated multi shape ttansenula yeast, its structural method and application
  • Recombinated multi shape ttansenula yeast, its structural method and application

Examples

Experimental program
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Embodiment 1

[0029] Example 1, Construction of Hansenula polymorpha (Hansenula polymorpha) HU-11 CGMCC No.1218 and detection of its biomass and biological activity

[0030] 1. Construction of recombinant plasmid p18HURA containing the orotidine-5-phosphate decarboxylase gene (HURA3) of Hansenula polymorpha

[0031] As shown in Figure 1, the construction process of the recombinant plasmid p18HURA is as follows:

[0032] 1. Acquisition of orotic acid-5-phosphate decarboxylase gene (HURA3) of Hansenula polymorpha

[0033] The primer sequences designed according to the reported HURA3 nucleotide sequence of Hansenula polymorpha are as follows:

[0034] Primer 1: 5′-CCA GGATCC TCAACATTTCCCTGAATAAT-3' (sequence 1 in the sequence listing) (the bases underlined are BamH I recognition sites)

[0035] Primer 2: 5′-CGA GAATTC TCACTAGTATTC CCGCGACT-3' (sequence 2 in the sequence listing) (the underlined part of the base is the EcoR I recognition site)

[0036] Using the total DNA of Hansenula pol...

Embodiment 2

[0062] Example 2, Intracellular expression of amylase gene in Hansenula polymorpha (Hansenula polymorpha) HU-11 CGMCCNo.1218

[0063] 1) The vector YIp5 (Struhl K, St inchcomb DT, et al. High-frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. ProcNatl Acad Sci USA, 1979, 76: 1035-1039) was used as the starting vector. Digest plasmid YIp5 and pHARS (Roggenkamp R, Hansen H, et al. Transformation of themethylotrophic yeast Hansenula polymorphabby autonomous replication and integration vectors. Mol Gen Genet, 1986, 202: 302-308) respectively with restriction endonuclease SalI, low melting point The 5.6kb linear YIp5 DNA fragment and the 0.5kb Hansenula polymorpha self-replicating sequence HARS were recovered by agarose gel electrophoresis, and the recovered DNA fragments were dissolved in TE buffer (10mM Tris-HCl, 1mM EDTA, pH8. 0), the ligation reaction was performed, and the ligation reaction system was: 3 μl deionized water, 1 μl 10× ligation buf...

Embodiment 3

[0071] Example 3, Secretory expression of amylase gene in Hansenula polymorpha (Hansenula polymorpha) HU-11 CGMCCNo.1218

[0072] 1) Design and synthesize primer amy3(5'-CC CTGCAG AATATGCAAATTTCAAAAGC-3 ', the underlined part is the PstI recognition site), and together with the primer amy2 in Example 2, the chromosome of Saccharomyces spp. Using DNA as a template, the PCR reaction was performed to amplify the α-amylase gene (AMY).

[0073] Design and synthesis of primer αP1(5′-TAA GAATTC AAAATGAGATTTCCTT-3′, the underlined part is EcoRI recognition site) and αP2 (5′-CGT CTGCAG CTCAGCTTCAGCCTCTCTT-3', the underlined part is the PstI recognition site), and the plasmid pMETαB (Invitrogen Company) was used as a template to perform PCR reaction to amplify about 0.3kb of the sequence MFα1s encoding the α-factor signal peptide of Saccharomyces cerevisiae.

[0074] The plasmid pHAM1 constructed in Example 2 was digested with the restriction endonuclease EcoRI, and the PCR amplif...

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Abstract

A recombinant polymorphic Hansenula with high genetic stability features that its orotidine-5-phosphate decarboxylase gene is blocked. Its configuring process includes such steps as configuring recombinant carrier, introducing it to the wild polymorphic Hansenula, and screening. It can be used for preparing the extrinsic protein.

Description

technical field [0001] The invention relates to a recombinant Hansenula polymorpha and its construction method and application, in particular to a recombinant Hansenula polymorpha and its construction method and its application in the production of exogenous proteins. Background technique [0002] Yeast, as a single-celled eukaryote, not only has the characteristics of fast growth and simple genetic manipulation of prokaryotes, but also has similar gene expression regulation and post-translational modification mechanisms as higher eukaryotes, so it is ideal for the production of eukaryotic active proteins expression system. Over the past 20 years, Saccharomyces cerevisiae has been widely used as the first eukaryotic gene expression system. However, this expression system also has many shortcomings in the application process, such as low expression level of foreign genes, low secretion level of expressed protein, excessive glycosylation of polypeptide chain, and unstable str...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/65C12R1/78
Inventor 何秀萍张博润汪和睦
Owner TIANJIN HEMU JIANMIN BIOTECHNOLOGY CO LTD
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