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Construction method of rhil-12 expression system in cho cells

A eukaryotic cell, high-efficiency expression technology, applied in the direction of interleukin, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of unstable expression, low conversion rate, complicated operation, etc., to achieve simple operation, High conversion rate and high biological activity

Active Publication Date: 2020-06-30
GUANGDONG COOWAY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its complex operation, low conversion rate and unstable expression affect its industrial application

Method used

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  • Construction method of rhil-12 expression system in cho cells
  • Construction method of rhil-12 expression system in cho cells
  • Construction method of rhil-12 expression system in cho cells

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0019] Insert the gene fragment of SEQ ID NO.1 and SEQ ID NO.2 into the pcDNA3.1 (+) vector, the structure of the p35 expression unit is: SV40 enhancer-CMV promoter-SEQ ID NO.1, the structure of the p40 expression unit is: SV40 enhancer-CMV promoter-SEQ ID NO.2. A CHO cell line successfully expressing rhIL-12 was constructed using Gibco's CHO-S-SFM II for static culture, and then Sartorius BiosSTAT plus cell culture tank was used for batch culture and continuous culture. The content of rhIL-12 was detected by ELISA method, and the static culture The average concentration is 3.6μg / ml, and the average concentration of continuous culture in cell culture tank is 8.9μg / ml.

[0020] After the fermented culture medium is purified, the biological activity of the product is determined by the PBMC IFNγ induction method. Specifically, take fresh peripheral blood from healthy people, and separate lymphocytes. Prepare cell suspension with RPMI1640 basal culture medium containing 10% inac...

experiment example 2

[0022] Screen for suitable introns with expression-enhancing effects. An expression vector as described in Experimental Example 1 was constructed. The three introns were obtained according to the primers shown in Table 1, and the three introns were inserted into the p35 expression unit to form a structure of SV40 enhancer-CMV promoter-intron-SEQ ID NO.1.

[0023] A CHO cell line successfully expressing rhIL-12 was constructed and statically cultured with Gibco's CHO-S-SFM II, and then batch culture and continuous culture were carried out using Sartorius BiosSTAT plus cell culture tank, and rhIL-12 of different expression vectors were detected by ELISA content. The detection results are shown in Table 2. It can be known that the expression of rhIL-12 is most increased after the adh1 intron sequence is inserted.

[0024] Table 1 Introns and primers required for cloning introns

[0025]

[0026] Table 2 The expression level of rhIL-12 after inserting different introns

[0...

Embodiment 1

[0035] The p40cDNA and p35cDNA fragments were inserted into the pcDNA3.1(+) vector, and the adh1intron gene was inserted between the CMV promoter and the p35cDNA. The structure of the p35 expression unit is: SV40 enhancer-CMV promoter-adh1intron-p35cDNA, and the structure of the p40 expression unit is: SV40 enhancer-CMV promoter-p40cDNA. A CHO cell line successfully expressing rhIL-12 was constructed using Gibco's CHO-S-SFM II for static culture, and then Sartorius BiosSTAT plus cell culture tank was used for batch culture and continuous culture. The content of rhIL-12 was detected by ELISA method, and the static culture The average concentration is 6.7μg / ml, and the average concentration of continuous culture in cell culture tank is 16.9μg / ml.

[0036] After the fermented culture medium is purified, the biological activity of the product is determined by the PBMC IFNγ induction method. Specifically, take fresh peripheral blood from healthy people, and separate lymphocytes. ...

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Abstract

The invention provides a modified recombinant human interleukin-12 (rhIL-12) gene and also provides a construction method of a rhIL-12 expression system in CHO (Chinese Hamster Ovary) cells. According to the method provided by the invention, a genetic engineering means is utilized to artificially synthesize recombinant genes of recombinant human interleukin-12; and the recombinant gene sequence is inserted into a vector and is transformed into a host cell, and a high-efficiency expression rhIL-12 eukaryotic cell line is obtained.

Description

[0001] Technical field: the present invention relates to the field of preparation of a recombinant protein, and specifically discloses a method for constructing a rhIL-12 expression system in CHO cells. Background technique: [0002] Interleukin (interleukin, IL) is produced by a variety of cells and acts on a class of cytokines. It belongs to cytokines like blood cell growth factor. The two coordinate and interact with each other to complete hematopoietic and immune regulation functions. Interleukins play an important role in transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and inflammatory responses. [0003] Interleukin-12 (hereinafter referred to as IL-12), produced by antigen-presenting cells and B cells, is a pro-inflammatory cytokine in the form of a heterodimer, and is secreted extracellularly in this form. IL-12 can induce IFN-γ production, and it is also required for IFN-γ product...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/24C07K14/54C12N15/85C12N5/10
CPCC07K14/5434C12N15/85C12N2800/107C12N2800/22
Inventor 倪彦艳刘杰森刘璐瑜陈松彬李宇腾
Owner GUANGDONG COOWAY BIOTECH CO LTD
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