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.
CN105779490AInactive Publication Date: 2016-07-20BEIJING JIZHI XINCHUANG TECH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
BEIJING JIZHI XINCHUANG TECH
Publication Date
2016-07-20
Estimated Expiration
Not applicable · inactive patent

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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.
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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...

Claims

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