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Selection method of signal peptide while foreign protein is expressed by Chinese hamster ovary cells and application thereof

A technology of ovary cells and Chinese hamsters, applied in the biological field, can solve the problems of cleavage of target protein amino acids, cleavage site offset, etc.

Active Publication Date: 2020-02-07
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the signal peptide is connected to the N-terminal of the foreign protein, the cleavage site will shift, and the amino acid of the target protein may be cleaved.

Method used

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  • Selection method of signal peptide while foreign protein is expressed by Chinese hamster ovary cells and application thereof
  • Selection method of signal peptide while foreign protein is expressed by Chinese hamster ovary cells and application thereof
  • Selection method of signal peptide while foreign protein is expressed by Chinese hamster ovary cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Informatics Analysis of Example 1 Signal Peptide Cleavage Site

[0022] Select 21-60AA, 141-160AA and 200-213AA of VP1 protein of type O foot-and-mouth disease (Gen Bank: QDJ95689.1) as dominant epitopes, and connect them rationally to construct a multi-epitope gene expression cassette. To prevent the formation of new epitopes after the ligation of two adjacent epitopes, a GGSSGG spacer sequence was introduced between the epitopes to ensure the correctness of the target antigenic site. The nucleotide sequences of signal peptides capable of effectively secreting and expressing foreign proteins were selected from the reported literature, and their nucleotide sequences were shown in Table 1, and they were connected with the multi-epitope gene expression cassette. The GGSSGG spacer and no GGSSGG spacer were introduced between the signal peptide and the multi-epitope gene expression cassette, and the amino acids of the recombinant protein were analyzed using analysis tools (...

Embodiment 2

[0023] Example 2 Construction of pCI-neo-O eukaryotic expression vector

[0024] 1. Synthesis of multi-epitope gene expression cassette

[0025] According to the gene sequence of O-type FMDV (Gen Bank: QDJ95689.1), 21-60AA, 141-160AA and 200-213AA on O-type FMDV VP1 were respectively selected as dominant epitopes, and each epitope was repeated twice in series, Linked through the linker sequence GGSSGG to form a new epitope cassette, the sequence of which is shown in SEQ ID: 01. And the gene sequence of the tandem epitope cassette was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis.

[0026] 2. The signal peptide is connected to the multi-epitope gene expression cassette

[0027] The selected 10 different secretory signal peptides were connected in series with the synthesized multi-epitope gene expression cassette by PCR method, among which Serum albumin preproprotein-F1 and Serum albumin preproprotein-F2 signal peptides were the same, but Serum albumin preprop...

Embodiment 3

[0030] Example 3 Recombinant protein accumulation analysis

[0031] 1. Transfection of recombinant plasmids into CHO cells

[0032] The pCI-neo-O recombinant plasmid was transfected into CHO cells according to the instructions of the Lipofectamine 3000 kit. After 8 hours of transfection, the high-glucose DMEM medium containing 10% serum was replaced, and the culture was continued.

[0033] 2. Western blot identification of recombinant epitope protein accumulation in CHO cell culture medium

[0034] 48 hours after transfection, the CHO cell culture medium was collected, mixed with PMSF to a final concentration of 0.001mol / L, centrifuged at 800r / min for 5min to remove cell debris, and the supernatant was taken for BCA protein quantification. With a total protein amount of 60 μg, it was transferred to NC membrane by SDS-PAGE. Block with 5% skim milk for 2 hours at room temperature, add rabbit anti-FMD virus type O serum, incubate overnight at 4°C; wash 5 times with PBST for 5 ...

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Abstract

The invention relates to a selection method of a signal peptide while foreign protein is expressed by Chinese hamster ovary cells and an application thereof, which belong to the technical field of biology. An information analysis technology is adopted; 29 kinds of high-secretory expression signal peptides and foreign protein tandem sequences commonly used in CHO cells are analyzed; finally, 10 signal peptides with proper analysis results are selected, 10 different secretory signal peptides are connected with the 5'end of the tandem epitope gene through a PCR method, eukaryotic expression vectors are constructed respectively, mammalian cell CHO is transfected, Western blot is adopted to detect the cumulative difference of recombinant epitope proteins in a CHO culture medium, and the resultproves that the cumulative difference of the proteins conforms to the selection result of the method, so that the feasibility of the method is proved.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and further relates to a signal peptide, in particular to a method for selecting a signal peptide when expressing a foreign protein with Chinese hamster ovary cells and its application. Background technique [0002] Chinese hamster ovary cell (CHO) is the first choice for the expression of medicinal bioactive macromolecules. CHO rarely secretes its own endogenous proteins, which is conducive to protein purification. Secretory expression is the main expression pathway of CHO, but the expression of many proteins (cytokines, etc.) is relatively low, and increasing the ability of cells to secrete can significantly promote the expression of recombinant proteins. [0003] In the process of expressing secretory proteins in CHO cells, a signal peptide composed of 15-30 hydrophobic amino acids will be synthesized at first, and the signal peptide will be recognized by the signal recognition particle (S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85G01N33/68C07K19/00
CPCC12N15/85G01N33/68C07K14/005C07K2319/02C12N2770/32151
Inventor 常惠芸孙振文邵军军张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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