Cell strain capable of expressing glucocerebrosidase with high mannose content as well as preparation method and applications of cell strain

Abstract

The invention discloses a preparation method of a cell strain capable of expressing glucocerebrosidase with high mannose content. The preparation method comprises the following steps: (1) carrying outlipofection transfection, namely, aiming at the sequence of a GNT1 gene, designing a sgRNA sequence guiding cutting of endonuclease, recombining the sgRNA sequence into a knock-out vector, thus obtaining synthetic plasmids with coexpression of sgRNA and endonuclease, and transfecting a cell pool stably expressing glucocerebrosidase by adopting the synthetic plasmids through a liposome transfection method; (2) carrying out cloning by adopting a limiting dilution method, namely, screening out monoclonal cells, and carrying out enlarged culturing; (3) carrying out mutation cloning screening on the target gene, namely, acquiring monoclone with the GNT1 gene knocked out through cell PCR sequencing, and thus the cell strain capable of expressing glucocerebrosidase with high mannose content is obtained. The cell strain can stably express glucocerebrosidase, meanwhile, the content of the mannose is obviously improved, the binding capacity with mannose receptor is effectively improved, the enzyme activity capacity is improved, and the purpose of increasing the efficacies is achieved.

Description

technical field [0001] The present invention relates to the field of biopharmaceuticals, in particular to a method for expressing a target protease in CHO (Chinese Hamster Ovary) cells and modifying the glycoform of the product after knocking out the target gene using the gene editing technology CRISPR. Background technique [0002] Deficiency of each enzyme in lysosomes will lead to the accumulation of a specific biomacromolecule in lysosomes that cannot be degraded normally. The common result is that lysosomes swell subsequently, cells become bloated and abnormal, and cell functions are seriously affected, eventually leading to a disease called lysosomal storage disease (Lysosomal storage disease, LSD). Glucocerebrosidosis, a type of LSD, is a family disease of glucose and lipid metabolism. Due to the lack of glucocerebrosidase, a large amount of glucocerebroside is deposited in the liver, spleen, bones, lungs and brain tissues. Accumulates in monocyte-macrophages, eventu...

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Problems solved by technology

[0004] However, the cell line used in the existing method is not high in the content of mannose glycoforms in the glucocerebrosidase, the enzyme activity is relatively poor, and the drug effect is not ideal enough.

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