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Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use

A technology of Hansenula polymorpha and SARS virus, applied in recombinant DNA technology, gene therapy, antiviral agents, etc.

Inactive Publication Date: 2004-02-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is found that the Spike protein difference of all SARS virus isolates is only pure on S1, and S2 is highly conserved, and the difference of all SARS virus isolate proteins is only pure on S1, S2 is highly conserved, and the S2 of all current SARS virus isolates Genes are all the same

Method used

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  • Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use
  • Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use
  • Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use

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Embodiment 1

[0016] The following examples can make those skilled in the technical field understand the present invention more comprehensively, but do not limit the present invention in any way. The design of embodiment 1S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene

[0017] 1. Embed the H. polymorpha highly expressed gene codon usage table (Chinese patent application number: 03110441.X) described in Figure 1 into the DNAStar program

[0018] 2. According to the positions corresponding to the encoded amino acids of the SARS virus S gene (accession number: AY278554, CUHK-W1 strain) reported on Genbank, S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene encoding corresponding amino acid sequence S(1-1255AA), S1(17-640AA), S1-1(66-609AA), S1- 2(17-232AA), S1-3(258-572AA), S2(641-1247AA), S2-1(641-856AA), S2-2(883-1197AA), S2-3(1149-1183AA), S2-4(1149-1236AA), S2-5(787-1188AA); that is, SEQ ID NO: 12-22 were converted into corresponding nucleotide sequences respe...

Embodiment 2

[0020] Construction, transformation and screening of embodiment 2 recombinant expression vector

[0021] 1. Construction of recombinant expression vectors: using Hansenula polymorpha expression vectors pHMOXZ-A (intracellular expression), pHFMDHZ-A (intracellular expression), pHMOXZα-A (secretory expression), pHFMDHZα-A (secretory expression ) or any other eukaryotic expression vectors that can be integrated and expressed in Hansenula, construct optimized SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2 , S2-3, S2-4, S2-5 gene recombinant expression vector. Any of the above recombinant expression vectors can be highly expressed in Hansenula polymorpha.

[0022] 2. Transformation and screening of recombinant expression vectors: Transformation of Hansenula polymorpha by electroporation transformation method. Pick a single clone of Hansenula polymorpha NCYC495 in 5ml YPD liquid medium, culture overnight at 37°C

[0023] 3. Take 2ml of the bacterial liquid and add it to 200ml...

Embodiment 3

[0038] Example 3 Fermentation and induced expression

[0039] After the positive recombinant yeast clones containing multiple copies of the gene were cultured in YPD medium at 37°C for 12 hours, they were inoculated into fresh YPD fermentation medium (containing 1.5% glycerol) at a ratio of 1:20, and the fermentation temperature was maintained at 30-37°C. ℃, pH3-5, dissolved oxygen 20%, air flow rate 5-10L / min. 24 hours later when O 2 When the pressure rises sharply (prompting that the glycerol in the culture medium has been consumed), start feeding (YPD containing 50% glycerol), strictly control the feeding speed, so that the final concentration of glycerol in the fermentation broth is maintained between 0.05%-0.4% . After 24-48 hours of fermentation, it can be harvested. For the fermentation broth with intracellular expression, the cells were harvested by centrifugation at 12,000 rpm for 2 min at 4°C; for the fermentation broth with secretory expression, the supernatant w...

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Abstract

An expression of recombinant SARS virus gene in polymorphic Hanson yeast and its application are disclosed. In the procedure of industrially preparing the expressed product of SARS virus' genes S, S1and S2 and primary antigen epitope gene, the SARS virus' genes S, S1 and S2 and primary antigen epitope gene are redesigned according to the application method of the codon of high-expression gene for Hanson yeast. The high-expression product can be used as vaccine to prevent the atypical pneumatonitis caused by SARS virus. The designed gene can also be used for fusion expression with cholera toxin B subunit gene in Hanson yeast.

Description

technical field [0001] The invention belongs to the field of protein expression genetic engineering, in particular relates to the use of SARS virus recombinant S, S1, S2 and major epitope genes and the use of Hansenula polymorpha (Hansenula polymorpha) containing these genes as a cell factory for high-efficiency expression As well as the application of the expression product in the development of a vaccine for preventing SARS virus. Background technique [0002] The SARS virus is a newly emerged coronavirus that causes severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) in humans and is the culprit of atypical pneumonia. The viral genome is single-stranded positive-sense RNA. The RNA is about 30Kb in full length, the largest among all RNA viruses, and is infectious. The pathogenesis of SARS virus is still unclear. It has a wide range of natural hosts, can survive in the air and feces for a long time, and is mainly transmitted through the respiratory...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61K39/215A61K48/00A61P11/00A61P31/14C07K14/165C12N15/50C12N15/81
Inventor 邱并生宋厚辉李勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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