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Signal peptide for improving secretion and expression of mammalian cell recombinant protein and its application

A mammalian, secretory expression technology, applied in signal peptides and related application fields to improve the secretion and expression of mammalian recombinant proteins, can solve the problems of high cost, long screening time, etc., to reduce time and cost, reduce production cost, The effect of broad application prospects

Active Publication Date: 2021-05-11
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, if you want to obtain a higher expression level, you need to screen multiple signal peptides individually for each secreted protein, and select a suitable signal peptide from them, which has the disadvantages of long screening time and high cost.

Method used

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  • Signal peptide for improving secretion and expression of mammalian cell recombinant protein and its application
  • Signal peptide for improving secretion and expression of mammalian cell recombinant protein and its application
  • Signal peptide for improving secretion and expression of mammalian cell recombinant protein and its application

Examples

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Effect test

Embodiment 1

[0036] In this example, 9 proteins were selected as verification proteins for the application effect of the signal peptide of the present invention. Under the same conditions, the signal peptide of the present invention and the signal peptide of the verification protein were used to compare the secretion and expression levels of the verification proteins in HEK293 cells. The specific operation is as follows:

[0037] 1. Construction of recombinant expression vector

[0038] First, construct a recombinant expression vector using the signal peptide of the present invention (SEQ ID No.1, see Table 1 for the specific sequence), named pHEK1.0: the amino acid sequence encoded by Shanghai Bioengineering Co., Ltd., such as SEQ ID No.21~SEQ The polynucleotide of ID No.29 (see Table 1 for the specific sequence), the sequence is SEQ ID No.39~SEQ ID No.47 (wherein the polynucleotide sequence encoding the signal peptide of the present invention is shown in SEQ ID No.39~SEQ ID No. The 1st ...

Embodiment 2

[0069] In this embodiment, Human CD73 protein is selected as the verification protein for the application effect of the signal peptide of the present invention in CHO cells. Under the same conditions, the signal peptide of the present invention and the signal peptide of the verification protein were used to compare and verify the secreted expression level of HumanCD73 protein in CHO cells. The specific operation is as follows:

[0070] 1. Construction of recombinant expression vector

[0071] First, a recombinant expression vector using the signal peptide of the present invention (SEQ ID No.1, see Table 1 for the specific sequence) is constructed: a polynuclear polynucleotide encoding the amino acid sequence SEQ ID No.29 (see Table 1 for the specific sequence) is synthesized by Shanghai Bioengineering Co., Ltd. Nucleotide, the sequence is SEQ ID No.47, a restriction endonuclease HindIII enzyme cutting site and a Kozak sequence are added at the 5' end; a restriction endonuclea...

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Abstract

The invention provides a signal peptide for improving secretion and expression of mammalian cell recombinant protein and application thereof. The present invention firstly provides a polypeptide as follows (a) or (b): (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID No.1; (b) a polypeptide consisting of the amino acid sequence shown in SEQ ID No.1 through substitution, deletion and / or Or a polypeptide with one or several amino acids added and having the same function as the polypeptide composed of the amino acid sequence shown in SEQ ID No.1. The polypeptide of the present invention can be used as a signal peptide to secrete and express the target protein in mammalian cells, which is conducive to improving the secretion and expression level of the target protein, and screening high-expression monoclonal cell lines, greatly reducing the screening of high-expression signal peptides for secreted proteins time and cost required.

Description

technical field [0001] The present invention relates to a signal peptide and its application, more specifically, to a signal peptide which can be used to improve the secretion and expression of mammalian cell recombinant protein and its related application. Background technique [0002] Recombinant protein drugs are bright pearls in biological drugs. With the advancement of drug production technology and the increase in drug demand, the types of recombinant protein drugs in my country are also constantly enriched. [0003] Systems for the production of recombinant proteins using mammalian cells have been established for a long time. Compared with lower eukaryotic cell expression systems or prokaryotic expression systems, mammalian cell expression systems can guide the correct folding of proteins, provide complex N-type glycosylation and accurate O-type glycosylation and other post-translational processing modifications function, so the expression product is closest to natur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/85C12N15/62C12N5/10
CPCC07K7/08C07K14/4725C07K14/54C07K14/5418C07K14/70503C07K14/70521C07K14/70596C07K14/7155C07K2319/02C07K2319/21C12N5/0603C12N5/0682C12N5/0686C12N15/85C12N2510/02C12N2800/107
Inventor 秦丽丽范华李明月
Owner ACROBIOSYSTEMS INC
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