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Method for preparing influenza hemagglutinin glycoprotein with animal cell glycosylation modification by using glycosyl engineering yeast

A hemagglutinin glycoprotein, glycosylation technology, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc.

Active Publication Date: 2019-08-23
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No report on preparation of influenza hemagglutinin glycoprotein polymer nanoparticles by yeast

Method used

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  • Method for preparing influenza hemagglutinin glycoprotein with animal cell glycosylation modification by using glycosyl engineering yeast
  • Method for preparing influenza hemagglutinin glycoprotein with animal cell glycosylation modification by using glycosyl engineering yeast
  • Method for preparing influenza hemagglutinin glycoprotein with animal cell glycosylation modification by using glycosyl engineering yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1, Construction of Yeast Transformed by Glycosyl Modification Pathways

[0131] (1) Construction of glycoengineering yeast strains knocked out of phosphomannosyltransferase gene, phosphate synthase gene and β-mannosyltransferase gene

[0132] 1. Construction of yeast strains with phosphomannosyltransferase gene inactivation

[0133] Yeast strain GJK02 with phosphomannosyltransferase gene inactivation is to introduce the DNA molecule shown in Sequence 1 into Pichia pastoris GJK01, and undergo homologous recombination with the homologous sequence in the GJK01 genome to knock out phosphomannose in the yeast genome transferase gene, resulting yeast;

[0134] 1. Construction of phosphomannosyltransferase gene inactivation vector

[0135] The knockout plasmid pYES2-pno1 for knocking out the mannosyltransferase (PNO1) gene is to insert the knockout mannosyltransferase (PNO1) gene fragment shown in Sequence 1 in the sequence listing into the KpnI and XbaI restriction ...

Embodiment 2

[0264] Example 2. Preparation of Influenza Virus Hemagglutinin Glycoprotein or Its Polymer Nanoparticles with Gal2GlcNAc2Man3GlcNAC2 Glycosyl Structure Modification

[0265] 1. H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by engineered yeast

[0266] (1), construction of recombinant expression vector

[0267] 1. According to the full-length amino acid sequence of influenza HA from Genbank (KC853766) (A / Hongzhou / 1 / 2013 (H7N9), the coding gene was optimized according to the principles of yeast preferred codons and high gene expression.

[0268] 2. Design and synthesize the following primers:

[0269] HA7-3: 5'-ATC GCGGCCGC The sequence underlined in TTAAATACAGATAGTACATCTCAT-3' is the recognition site for NotI digestion.

[0270] HA7-5: 5'-ATC TTCGAA The sequence underlined ACGATGAACACCCAAATACTGGTTTTC-3' is the NspV restriction enzyme recognition site.

[0271] 3. Using the optimized coding gene as a template, and using HA7-3 and HA7-5 as primer...

Embodiment 3

[0411] Example 3. Preparation of H7N9 influenza virus hemagglutinin glycoprotein polymer nanoparticles with GlcNAC2Man3GlcNAC2 glycosyl structure modification

[0412] 1. H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by engineered yeast

[0413] (1), the construction of the recombinant expression vector: the same as that of Example 2;

[0414] (2), construction and screening of recombinant yeast:

[0415] The recombinant vector pPICZα-HA7 was introduced into the Pichia engineering strain GJK07 constructed in (4) of Example 1 by the method of (2) of Example 2 to obtain the recombinant strain GJK07 / / pPICZα-HA7.

[0416] Screening and identification methods are the same as in Example 2 (two).

[0417] (3) Engineering yeast fermentation

[0418] The recombinant bacterium GJK07 / pPICZα-HA7 was fermented according to the method in (3) of Example 2 to obtain a homogenate.

[0419] (4) Purification and identification of influenza hemagglutinin glycoprotei...

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Abstract

The invention discloses a method for preparing influenza hemagglutinin glycoprotein with animal cell glycosylation modification by using glycosyl engineering yeast. The method provided by the present invention comprises the steps of: expressing the hemagglutinin HA gene of influenza virus in a yeast mutant to obtain recombinant yeast; culturing the recombinant yeast to prepare a fucose-free yeast with a mammalian glycoform structure influenza virus hemagglutinin glycoprotein. Experiments of the present invention prove that the vaccine prepared by the influenza hemagglutinin glycoprotein particles prepared by the method of the present invention can induce higher anti-influenza virus neutralizing antibodies, and avoid the problems that fungal glycosylation modifications may cause allergies and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing influenza hemagglutinin glycoprotein with glycosylation modification of animal cells by using glycosyl engineering yeast. Background technique [0002] Influenza hemagglutinin is one of the main proteins on the surface of influenza virus. It exists in the form of a trimer and is an important component of the surface of influenza virus involved in the adsorption and invasion of host cells. The neutralizing antibodies induced by it can block the virus on the surface of host cells. Adsorption and invasion, and thus are the main components of influenza vaccines. Influenza hemagglutinin mutates and reassorts quickly, and various new types of influenza occur frequently. After the outbreak of a new epidemic, the rapid development and production of vaccines is the key to epidemic control. [0003] Existing influenza vaccines are mainly obtained by culturing chicken em...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P21/00C07K14/11C12R1/84C12R1/78C12R1/645
CPCC07K14/11C12P21/00C12N1/145C12N1/165C12R2001/645C12R2001/78C12R2001/84
Inventor 刘波吴军巩新唱韶红王莎马清钧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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