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Hansenula polymorpha expression system, hansenula polymorpha construction method and application of hansenula polymorpha

A Hansenula yeast, expression system technology, applied in microorganism-based methods, biochemical equipment and methods, fermentation and other directions, can solve the problems of multiple mutation effects, unclear mutation sites, poor genetic stability, etc., and achieve low cost. , the effect of low reverse mutation rate and high biological expression

Active Publication Date: 2015-02-25
BEIJING MINHAI BIOTECH
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Problems solved by technology

Most of the current methods for obtaining auxotrophic yeasts are corresponding selection markers obtained by traditional methods such as chemical mutagenesis, hybridization or protoplast fusion. These methods have many obvious shortcomings, such as most of them are single point mutations, Easy to reverse mutations, multiple mutation effects, unclear mutation sites, poor genetic stability, difficult to obtain ideal strains

Method used

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  • Hansenula polymorpha expression system, hansenula polymorpha construction method and application of hansenula polymorpha
  • Hansenula polymorpha expression system, hansenula polymorpha construction method and application of hansenula polymorpha
  • Hansenula polymorpha expression system, hansenula polymorpha construction method and application of hansenula polymorpha

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Embodiment 1

[0046] The construction of embodiment 1 expression vector PMV-05

[0047] The expression vector PMV-05 of the present invention consists of 6 parts: promoter (MOX-P), terminator (MOX-T), replicon HARS, ura3 gene, ColE1 replicon, Amp resistance gene.

[0048] Using Hansenula ATCC26012 genomic DNA as a template, primers MOXP-F (see SEQ ID NO.3 for the sequence), MOXP-R (see SEQ ID NO.4); MOXT-F (see SEQ ID NO.5), MOXT-R (see SEQ ID NO.6); HARS-F (see SEQ ID NO.7), HARS-R (see SEQ ID NO.8); Ura3-F (see SEQ ID NO.9), Ura3-R (See SEQ ID NO.10) Perform PCR amplification to extract genes MOXP, MOXT, HARS, and Ura3. Using the PBR-SK plasmid (purchased from Treasure Bioengineering Dalian Co., Ltd., item number: D3050) as a template, primers Amp+ColE1-F (see SEQ ID NO.11), Amp+ColE1-R (see SEQ ID NO.12 ) for PCR amplification, and transfer the gene Amp+ColE1. The PCR reaction system was as follows: 10 μl of PCR buffer, 10 μl of dNTP, 1 μl of primer F, 1 μl of primer R, 1 μl of Taq DN...

Embodiment 2

[0050] Example 2 Acquisition and Stability Analysis of Uracil Auxotrophic Hansenula Cells

[0051] 1. Obtaining uracil-deficient Hansenula cells

[0052] The G418 resistance gene sequence was obtained from the Pichia pastoris expression vector pPIC9K (purchased from inritrogen), and the Hansenula Ura3 gene sequence was obtained from Gene bank. Primers P1, P2, P3, P4, P5, and P6 were designed according to the Ura3 and G418 gene sequences, and their nucleotide sequences are shown in SEQ ID NO.13-18, respectively.

[0053]Using the genomic DNA of Hansenula wild-type host strain ATCC26012 as a template, the 5' end gene fragment of Ura3 was obtained by PCR amplification with primers P1 and P2 respectively, and the 3' end gene fragment of Ura3 was obtained by PCR amplification with primers P5 and P6 ;Pichia pastoris expression vector pPIC9K was used as a template, and P3 and P4 were used as primers for PCR amplification to obtain the G418 gene fragment. The PCR reaction system is ...

Embodiment 3

[0064] Example 3 Expression of the gene encoding human papillomavirus type 16 L1 protein (HPV16L1-f) in uracil-deficient Hansenula

[0065] 1. Optimization of gene sequence encoding HPV16L1-f protein

[0066] According to the nucleotide sequence of the L1 protein of the highly pathogenic HPV16 strain popular in my country, the vector software was used to optimize the design of the HPV16L1 gene sequence according to the most preferred codons of Hansenula to improve its expression in Hansenula cells expression volume. The nucleotide sequence of the gene encoding HPV16L1-f protein provided by the present invention is a sequence without a yeast secretion signal peptide or a transcription termination signal recognized by yeast. The codons of the gene encoding HPV16L1-f protein in the present invention use the most preferred codons of Hansenula. For codon usage frequencies in Pichia angusta see http: / / www.kazusa.or.jp / codon / . In order to prevent the GC content of the translated...

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Abstract

The invention provides a hansenula polymorpha expression system, a hansenula polymorpha construction method and application of hansenula polymorpha. The expression system contains uracil auxotroph hansenula polymorpha AU-0501, of which the preservation number is CGMCC NO.7013. The uracil auxotroph hansenula polymorpha AU-0501 provided by the invention has the advantages of definite mutation site, low reverse mutation frequency, good hereditary stability, high biological expression quantity and the like, plays a significant role in researching and producing gene engineering vaccine, has the advantages of higher yield and low cost as compared with other eukaryotic expression systems adopted at present. The invention further provides an expression vector applied to the hansenula polymorpha expression system and a construction method of the expression vector. Two or more genes can be expressed simultaneously through the expression vector. Two target genes can be expressed in a hansenula polymorpha auxotroph cell at a high level without interference.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a Hansenula expression system, and further to a construction method and application of the Hansenula expression system. Background technique [0002] Yeast, as a low-level unicellular eukaryotic microorganism, not only has the characteristics of fast growth and simple genetic manipulation of prokaryotes, but also has the post-translational processing and modification functions of eukaryotes. It is an ideal expression system for the production of eukaryotic active proteins . As the first eukaryotic system for expressing foreign genes, Saccharomyces cerevisiae has been used for more than 30 years, and a large number of proteins have been successfully expressed. However, this expression system has many shortcomings in industrial production, such as strain instability, low expression level of foreign genes, low yield and secretion efficiency, and excessive protein glycosylation....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N15/81C12N15/09C12N15/66C12P21/00C12R1/78
Inventor 顾美荣宋琳琳孟凡童戚治国张凯泉魏文进刘建凯
Owner BEIJING MINHAI BIOTECH
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