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Humanized glycosylation modified hansenula polymorpha

A Hansenula yeast, glycosylated protein technology, applied in microorganism-based methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc., can solve the problem of Hansenula's human-derived glycosylation transformation has not yet been successful. cases, etc., to achieve the effect of eliminating antigenicity

Inactive Publication Date: 2013-08-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although Pichia pastoris has achieved great success, there have been no successful cases of human glycosylation in Hansenula

Method used

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  • Humanized glycosylation modified hansenula polymorpha
  • Humanized glycosylation modified hansenula polymorpha
  • Humanized glycosylation modified hansenula polymorpha

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the construction of recombinant plasmid A and engineering bacteria A

[0051] 1. Construction of recombinant plasmid A

[0052] The schematic diagram of the structure of recombinant plasmid pGNT (also known as recombinant plasmid A) is shown in figure 1 . The nucleotide sequence of the recombinant plasmid pGNT is shown in sequence 1 of the sequence listing. In sequence 1, the 1st to 6th nucleotides from the 5' end are the BglII restriction recognition sequence, the 7th to 541st nucleotides are the GAP promoter, the 548th to 1535th nucleotides are the UGnT gene, and the 1559th nucleotide The 1896th nucleotide is the AOX1TT termination sequence, the 1903rd to 2437th nucleotide is the GAP promoter, the 2438th to 2551st nucleotide is the MMN9 gene, the 2558th to 3781st nucleotide is the GnTI gene, the 3806th nucleotide Nucleotides 4143 to 4143 are AOX1TT termination sequences, nucleotides 4150 to 4684 are GAP promoters, nucleotides 4685 to 4825 are MNN2 gen...

Embodiment 2

[0107] Embodiment 2, the construction of recombinant plasmid B and engineering bacteria B

[0108] 1. Construction of recombinant plasmid B

[0109] The schematic diagram of the structure of recombinant plasmid PGAL2 (also known as recombinant plasmid B) is shown in figure 2 . The nucleotide sequence of the recombinant plasmid PGAL2 is shown in sequence 2 of the sequence listing. In sequence 2, the 6th to 11th nucleotides from the 5' end are the SnaBI restriction recognition sequence, the 12th to 616th nucleotides are the ALG3-HD fragment (homologous arm), and the 623rd to 1157th nucleotides It is the GAP promoter, the 1164th to 2231st nucleotide is the UGalE gene, the 2256th to 2593rd nucleotide is the AOX1TT termination sequence, the 2600th to 3600th nucleotide is the PMA1 promoter, the 3601st to 4659th nucleotide The acid is the UGT gene, the 4684th to 5021st nucleotide is the AOX1TT termination sequence, the 5028th to 7433rd nucleotide is the LEU2 gene, the 7440th to 7...

Embodiment 3

[0171] Embodiment 3, purification and identification of glycoprotein expressed by engineering bacteria

[0172] 1. Introduce the plasmid pHFMDZαL2-rGOD into the engineering strain A to obtain the recombinant strain H.pGNT-rGOD.

[0173] 2. The plasmid pHFMDZαL2-rGOD was introduced into the engineering strain B to obtain the recombinant strain H.pGAL-rGOD.

[0174] 3. The plasmid pHFMDZαL2-rGOD was introduced into Hansenula spp. Hpalg3Δalg11ΔpRft1 to obtain control bacteria.

[0175] 4. The recombinant bacteria H.pGNT-rGOD obtained in step 1, the recombinant bacteria H.pGAL-rGOD obtained in step 2, and the control bacteria obtained in step 3 were subjected to the following experiments:

[0176] (1) Induced expression of rGOD protein

[0177] ① Inoculate a single colony of engineering bacteria into a test tube containing 5mL SD medium, and incubate at 30°C for 48h; then transfer it to a 250mL Erlenmeyer flask containing 50mL YNG medium, and incubate at 30°C for 32 hours (30-35...

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Abstract

The invention discloses a humanized glycosylation modified hansenula polymorpha and provides a recombinant strain prepared by introducing recombinant plasmids A and B into hansenula polymorpha. The recombinant plasmid A comprises a UGnT gene expression cassette, an MMN9 and GnTI gene expression cassette, and an MNN2 and GnTII gene expression cassette; the UGnT gene is shown from position 548 to position 1,535 in a sequence 1; the MMN9 gene is shown from position 2,438 to position 2,551 in the sequence 1; the GnTI gene is shown from position 2,558 to position 3,781 in the sequence 1; the MNN2 gene is shown from position 4,685 to position 4,825 in the sequence 1; and the GnTII gene is shown from position 4,832 to position 5,899 in the sequence 1. The recombinant plasmid B comprises an MNN2 and GntII gene expression cassette, a UGT gene expression cassette and a UGalE gene expression cassette; the UGalE gene is shown from position 1,164 to position 2,231 in a sequence 2; and the UGT geneis shown from position 3,601 to position 4,659 in the sequence 2. The recombinant strain has great value for preparing humanized glycoproteins.

Description

technical field [0001] The present invention relates to human glycosylation modified Hansenula. Background technique [0002] More and more medical vaccines, therapeutic proteins, polypeptides, etc. can be produced through gene recombination technology, and play an important role in clinical treatment. Among them, glycoprotein drugs are the main body in the development of genetic engineering drugs, and their demand continues to grow rapidly. Proteins in glycoproteins are the main bearers of physiological functions, and sugar chains modify the functions of proteins. The realization of many protein functions is closely related to glycosylation modification. Sugar chains can change the conformation of proteins, leading to changes in protein function. Glycosylation plays an important role in protein folding, transportation, and localization, and participates in many important biological processes such as receptor activation and signal transduction. [0003] So far, mammalian...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12R1/78
Inventor 邱并生王辉
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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