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Highly effective expressing method of interleukin-12

A high-efficiency expression technology of interleukin, which is applied in the field of expression of recombinant IL-12, can solve the problems of hindering the transcription of target genes, unbalanced expression of P40 and P35, and low efficiency, so as to achieve good effect and increase the effect of expression

Inactive Publication Date: 2004-01-14
UNIV OF SCI & TECH OF CHINA
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Problems solved by technology

The genes of P40 and P35 were respectively inserted into the same vector for co-transfection. Due to the low efficiency of co-transfection, this method caused great difficulties in the screening of clones expressing IL-12, and it was difficult to obtain high-expression clones, and P40 and p35 expression are seriously unbalanced
Constructing vectors containing P40 and P35 expression elements in a single plasmid, no matter how precisely balanced this method is, because the order of the two target genes on the vector is different, the target gene carried by the upstream promoter often hinders the downstream start when it is transcribed The transcription of the target gene carried by the child, the expression of P40 and P35 is also unbalanced
Construction of IL-12 single-chain eukaryotic expression vector for expression, although this method can balance the expression of the two chains, but because little is known about the spatial structure of IL-12, the addition of a linker may affect the two chains of IL-12. The folding and spatial structure of the chain affect its biological activity. There are different reports on the biological activity of IL-12 expressed by this method

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  • Highly effective expressing method of interleukin-12
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  • Highly effective expressing method of interleukin-12

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Experimental program
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Effect test

Embodiment 1

[0020] 1.1 Construction of pcDNA3 / p40 recombinant plasmid

[0021] Carry out the p40 gene of human IL-12 by PCR amplification with the plasmid T carrier / p40 that extracts as template, predict length 1003bp (such as figure 1 ), the parameters are: 94°C for 1 minute, 62°C for 1 minute, 72°C for 2 minutes, cycle 28 times, and the last time is extended for 7 minutes. The construction process of pcDNA3 / p40 recombinant plasmid (see figure 2 ): After the pcDNA3 plasmid was recovered and purified by double digestion with HindIII and BamHI, it was ligated with the same double digestion PCR product, and the product was transformed into competent Escherichia coli DH5α, and positive recombinants were screened. The positive recombinant plasmid was sequenced by Dalian Bao Biotechnology Co., Ltd., and the results showed that the p35cDNA sequence was consistent with the report.

[0022] 1.2 Construction of pEE14 / p35 recombinant plasmid

[0023] Carry out the p35 gene of human IL-12 by PCR...

Embodiment 2

[0027] The amplification of exogenous genes in mammalian cells is one of the important methods to improve the expression yield of exogenous genes in mammalian cells. Since the generation of natural P40 is much larger than that of P35, the glutamine synthetase (GS) gene of pEE14 is used as a dominant gene amplification selection marker in this embodiment to amplify the P35 gene with high efficiency, so as to balance the expression of P40 as much as possible . From Example 1, a clone with higher expression of IL-12 was selected for pressurized amplification. MSX was set at 100, 250, 500 μ M and other concentration gradients, and G418 was set at 600, 800, 1000, 1200 mg / L and other concentration gradients. Make various combinations with G418 concentration gradients, add the culture medium for pressurized amplification of the target gene, culture for 10-14 days, and replace the culture medium once in the middle. The surviving clones were picked, and the culture supernatant was use...

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Abstract

A biotechnological method for efficient expression of interleukin-12 includes such steps as inserting the cDNA of IL-12's P40 subunit coding region into pcDNA3 eukaryon expression vector and the cDNA of IL-12's subunit coding region into pEE14 eukaryon expression vector to respectively configure the eukaryon cell's recombinant plasmids pcDNA3 / p40 and pEE14 / p35, cotransfecting host cells, adding G418 and MSX to screen culture medium, picking survival positive clone, and pressure amplification to obtain high-expression engineered cell strain IL-12.

Description

1. Technical field [0001] The invention relates to a method for preparing interleukin-12 (IL-12) by biotechnology, in particular to a method for expressing recombinant IL-12, which belongs to the technical field of bioengineering. 2. Background technology [0002] IL-12 can promote the proliferation and killing activity of NK cells and T cells, induce the production of cytokines such as γ-interferon, and regulate the development of Th1 cells, so it has a good clinical application prospect. At present, it has entered the stage of clinical trials for anti-tumor, anti-viral diseases and treatment of asthma. Natural IL-12 is a heterodimeric cytokine mainly produced by macrophages and B cells, composed of two subunits, P40 and P35. The production of P40 in the cytoplasm is much greater than that of P35, but only the heterodimers of P40 and P35 linked by disulfide bonds have biological activity. [0003] Before the present invention, some researchers respectively inserted the ge...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N15/24
Inventor 田志刚刘文涛魏海明
Owner UNIV OF SCI & TECH OF CHINA
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