Animal cell line and method for producing glycoprotein, glycoprotein and use thereof

一种动物细胞、细胞株的技术，应用在生物化学设备和方法、遗传修饰的细胞、引入外来遗传物质而修饰的细胞等方向，能够解决糖链含量高、减弱效率、治疗效率不高等问题，达到含量降低、稳定性和安全性优异、糖链均一性高的效果

Active Publication Date: 2021-12-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this cell line can produce N-glycoproteins with Man5-GlcNAc2 as the main structure, it cannot produce N-glycoproteins with Man9-GlcNAc2 and Man8-GlcNAc2 structures, or have mannose-6-phosphate structure of glycoproteins, and glycoproteins containing five mannose sugar chains cannot react with UDP-N-acetylglucosamine to form acidic sugar chains, thus weakening the binding efficiency of M6PR receptors
[0010] As another production method of high mannose type N-sugar chain glycoprotein, there is also production using α-1,2-mannosidase inhibitors such as kifunensine and deoxynojirimycin Glycoprotein method (Elbein, A.D.et al., J Biol Chem, 1990.265 (26): p.15599-605.), but the use of mannosidase inhibitors will cause the sugar chains to be in the form of M9, and if a long time Continuous use of inhibitors to culture cells will result in a high content of complex sugar chains in the obtained sugar chains, and the stability and safety of glycoproteins are not ideal
[0011] The heterogeneity of glycoproteins caused by the heterogeneity of sugar chains will have adverse effects on the production and application of glycoproteins
Since M6PR has specific recognition for glycoproteins, when some sugar chain structures in glycoproteins are not high-mannose or there is no sugar chain phosphorylated at position 6, the absorption efficiency of M6PR for drugs will be reduced. leading to ineffective treatment
In addition, when there is inhomogeneity in the sugar chain, the structure of the sugar chain may also cause the glycoprotein to be recognized by the body as a foreign antigenic substance, thereby causing an immune response

Method used

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  • Animal cell line and method for producing glycoprotein, glycoprotein and use thereof
  • Animal cell line and method for producing glycoprotein, glycoprotein and use thereof
  • Animal cell line and method for producing glycoprotein, glycoprotein and use thereof

Examples

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Effect test

Embodiment 1

[0109] [Example 1] Using the CRISPR-Cas9 (Clustered Regularly Interspaced ShortPalindromic Repeats) system to construct a double knockout cell line of the Golgi α-mannosidase gene (mammalian cell line: human embryonic kidney cell HEK293).

[0110] 1. Construction of knockout plasmids

[0111] Gene knockout using CRISPR-Cas9 technology often requires the design of a 20bp sequence fragment, and there must be a PAM site (NGG / NAG) behind the sequence fragment. In this experiment, the gene sequences of the two genes MAN1A1 / MAN1A2 to be knocked out were downloaded from NCBI (see SEQ ID NO: 44 and SEQ ID NO: 45, respectively). About the design of guide-RNA, in Michael Boutros lab's Target Finder ( http: / / www.e-crisp.org / E-CRISP / designcrispr.html ) to find the DNA sequence of the guide-RNA required to knock out the gene.

[0112] The two target sequences of MAN1A1 and the primer sequences used for each are:

[0113] MAN1A1KO1: AAAACCACGAGCGGGCTCTCAGG (serial number 1)

[0114]...

Embodiment 2

[0147] [Example 2] Analysis of sugar chains on the cell surface using a flow cytometer

[0148] After using the CRISPR / Cas9 system to knock out MAN1A1 / MAN1A2, two synthetic genes that control α-mannosidase in the Golgi apparatus, the sugar chains on the surface of the double knockout cell line will change to a certain extent. We confirmed this phenomenon with two different fluorescently labeled lectins. The lectin PHA-L4-FITC can recognize complex sugar chains on the cell surface, and the lectin ConA-FITC can recognize high-mannose sugar chains on the cell surface. The type of sugar chains on the surface of different cell lines can be compared by staining the cells with lectin. The specific method is as follows:

[0149] (1) Inoculate different cell lines in 6-well plates and wait for their growth to be 100%

[0150] (2) Remove the medium and wash once with 1ml of PBS

[0151] (3) Add 220ul Tryp / EDTA to digest the cells

[0152] (4) Add 1ml of fresh 10% FCS medium to harve...

Embodiment 3

[0166] [Example 3] Knockout of other genes related to α1,2-mannosidase

[0167] Introduce knockout plasmids of the two genes MAN1C1 and MAN1B1 into DKO cells (for the two knockout target sequences of the MAN1C1 gene and the corresponding primer sequences, see SEQ ID NO: 13-18, the two knockout target sequences of the MAN1B1 gene and the corresponding See sequence numbers 19-24 for the primer sequences respectively), and the plasmids introduced into the cells will express the sequences of Cas9 protein and target RNA. Cells were cultured for about ten days after transfection, and the cell genome was extracted. Because two target sequence sites are designed, the gene sequence on the chromosome will be shifted after knocking out the gene, and the inventors have confirmed the knockout of some genes (results shown in Figure 8, sequence numbers 29 and 30 are used for PCR testing of MAN1C1 Gene primers, sequence numbers 31 and 32 represent primers for PCR detection of MAN1B1 gene), a...

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Abstract

The present invention relates to animal cell strains and methods for producing glycoproteins, glycoproteins and uses thereof, and features of the animal cell strains of the present invention for producing glycoproteins having high mannose-type sugar chains as the main N-sugar chain structure In the cell line, at least two genes in the Golgi mannosidase gene and the endoplasmic reticulum mannosidase gene are disrupted or knocked out. According to the present invention, it is possible to obtain a glycoprotein with a greatly reduced complex-type sugar chain content and with high mannose-type N-sugar chains such as Man9-GlcNAc2 and Man8-GlcNAc2 as the main structure. The glycoprotein with high mannose-type sugar chain as the main N-sugar chain structure produced by the present invention can be used to produce biopharmaceutical molecules including lysosomal enzymes, antibodies, etc., so as to be used for treating various lysosomal storage diseases.

Description

technical field [0001] The present invention relates to animal cell strains and methods for producing glycoproteins, glycoproteins and uses thereof, in particular to animal cell strains for producing glycoproteins with high mannose sugar chains as the main N-sugar chain structure, The method for producing glycoprotein by using the animal cell line, the glycoprotein produced by the animal cell line and the application of the glycoprotein. [0002] technical background [0003] Glycoproteins are an important class of functional proteins in organisms. Structurally speaking, glycoproteins are complex sugars composed of branched oligosaccharide chains and polypeptide chains covalently connected. Divided into the following types: Asn residue binding type (also known as N-glycosidic bond type), O-Ser / Thr type, GPI anchor type and proteoglycan type. The present invention mainly relates to the production of glycoproteins (commonly known as N-sugars) having N-glycosidic bond type suga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N9/20C12N9/40C07K16/00A61K38/47A61K38/46A61P3/00
CPCA61K38/465A61K38/47C07K16/00C07K2317/14C12N9/20C12N9/2465C12N9/2488C12Y301/01003C12Y302/01022C12Y302/01024C12Y302/01113A61K38/46A61P3/00C12N15/09C12N5/10C12N2510/02
Inventor 藤田盛久金则成喜多岛敏彦张慧杰高晓冬
Owner JIANGNAN UNIV
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