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Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody

A Pichia pastoris and construction method technology, applied in the field of genetic engineering, can solve problems such as inconvenient production of pharmaceutical proteins, lack of eukaryotic protein modification and processing systems, and incorrect spatial conformation of endogenous protease degradation.

Inactive Publication Date: 2016-07-20
BEIJING JIZHI XINCHUANG TECH
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  • Claims
  • Application Information

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Problems solved by technology

However, prokaryotes lack the renaturation function of eukaryotic proteins, the lack of modification and processing systems for eukaryotic proteins, endogenous proteases degrade exogenous proteins with incorrect spatial conformations, and the periplasm contains a wide variety of endotoxins. and other characteristics have brought a lot of inconvenience to the production of pharmaceutical proteins

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  • Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody
  • Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody
  • Construction method of Pichia pastoris expressed by OCH1 defect anti-CD20 tetravalent antibody

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Embodiment

[0031] A method for constructing an OCH1-deficient anti-CD20 tetravalent antibody expressing Pichia pastoris, comprising the following steps:

[0032] 1. Construction of positive clone Δoch1 strain

[0033] 1) Construct fusion gene ΔOCH1

[0034] Two pairs of nucleotide primers OCH1-5F, OCH1-5R, OCH1-3F, OCH1-3R were designed according to the Pichia pastoris OCH1 gene sequence reported in Genebank (E12456). Using the genome of the X-33 strain as a template, the homology arm sequences at both ends of OCH1 were cloned. OCH1-5F, the product of OCH1-5R clone SEQ ID NO: 10; OCH1-3F, the sequence of the product of OCH1-3R clone is as SEQ ID NO: 11

[0035] Figure 4 : OCH1 left arm (5' end) and right arm (3' end) clone electrophoresis pattern, the reading frame sequence of about 1,200 bp is missing between the homologous arms on both sides, the size of the fusion fragment och1 is about 1600 bp, and the sequence is as SEQ ID NO: 12, so The open reading frame of the OCH1 gene was ...

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Abstract

The invention discloses a construction method of Pichia pastoris expressed by an OCH1 defect anti-CD20 tetravalent antibody. The method comprises the following steps: knocking out the alpha-1,6-mannosyl transferase OCH1 of a strain JC308, connecting reconstructed och1 having no expression ability and a screening label to pPICZalphaA, introducing the obtained pPICZalphaA to wild Pichia pastoris JC308, carrying out homologous recombination, screening to obtain an OCH1 defect strain denoted as detaoch1, connecting the sequence of a synthesized anti-CD20 tetravalent antibody with a Pichia pastoris expression plasmid pPIC9, and introducing the obtained product to the OCH1 defect strain detaoch1 to obtain the Pichia pastoris. The Pichia pastoris alpha-1,6-mannosyl transferase (OCH1) gene is knocked out to prevent formation of high-mannose carbohydrate chains, so glycosylation difference between yeast expression proteins and human natural proteins is shortened, and the safety of medicinal proteins is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a Pichia pastoris strain expressing an OCH1 gene-deficient anti-CD20 tetravalent antibody. Background technique [0002] Pichia pastoris expression system is currently one of the most successful exogenous protein expression systems. It has obvious advantages in methylation modification and other aspects, and has been widely used in the expression of foreign proteins. However, Pichia pastoris is not suitable for the expression of glycoproteins, and its expression product is high in mannose, which is easy to generate an immune response in the human body, which limits the use of Pichia pastoris as a host bacterium. The presence of α-1,6-mannosyltransferase encoded by the OCH1 gene is an important reason for the formation of high mannose. The knockout of the OCH1 gene can effectively block the post-translational high mannose modific...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P21/00C07K16/46C07K19/00C12R1/84
CPCC07K16/46C07K19/00C12N15/81C12P21/00C12N1/165C12R2001/84
Inventor 李云森朱婷婷王丽萍和运
Owner BEIJING JIZHI XINCHUANG TECH
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