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Engineering strain for producing recombinant human IL-1ra and preparation method and use thereof

A technology of engineering strains and strains, applied in the field of genetic engineering, can solve the problems of Escherichia coli toxicity, influence on drug safety, process condition control and expression level not as ideal as IPTG, so as to improve drug quality and safety, increase production and The effect of detection cost and reducing the pressure of subsequent purification

Active Publication Date: 2022-01-21
北京君泰联合科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, vectors and engineered strains with ampicillin resistance cannot meet the needs of drug production;
[0028] 2. Isopropyl-β-D-thiogalactoside (IPTG) is used as an inducer of protein expression. IPTG is expensive and increases production costs; it is toxic to Escherichia coli and affects the production of bacteria in the fermentation process; Toxic, if not completely removed, will affect the safety of the drug
Although some people are now studying the use of lactose as a substitute for IPTG, the control of process conditions and the amount of expression are not as ideal as IPTG

Method used

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  • Engineering strain for producing recombinant human IL-1ra and preparation method and use thereof
  • Engineering strain for producing recombinant human IL-1ra and preparation method and use thereof
  • Engineering strain for producing recombinant human IL-1ra and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Construction of Engineering Bacteria and Induced Expression of Target Protein

[0079] 1. Base sequence design of IL-1ra

[0080] The original gene sequence was selected as the vector construction.

[0081] The human peripheral blood cDNA library stimulated by PMA was used as a template for PCR amplification. The sequence of the synthetic hIL-1ra primer is shown in SEQ ID NO: 4-5.

[0082] The upstream primer is: 5'-CGACCCTCTGGGAGAAAATCC-3';

[0083] The downstream primer is: 5'-CTTAAGCTTACTCGTCCTCCTGGAAGTGG-3';

[0084] The PCR system is: 10XTE buffer 2μl; DNA polymerase 1-2μl; dNTP 1-2μl; primer 1-2μl; template DNA 1-2μl;

[0085] Taq DNA polymerase, thermocycling conditions: 90°C-95°C 3-5min, 1 cycle; 90°C-95°C 1min-2min, 40°C-60°C 2min-3min, 70°C-75°C 3min-4min, 30 -35 cycles; 70°C-75°C for 10-20min, 1 cycle. The IL-1ra PCR product was purified using the Magic CR purification kit from Promega, USA.

[0086] 2. Carrier transformation

[0087] 1) Cut...

Embodiment 2

[0143] 1. Gene sequence modification

[0144] Select the following mutation sites for genetic modification:

[0145]

[0146] Remove the start codon at the 5' end: atg; add the sequence at the 3' end: gcttagg, and form the restriction site of HandIII with the tail.

[0147] A new sequence is formed as shown in SEQ ID NO:7.

[0148] Due to the large number of mutation sites, the gene sequence was obtained by whole gene synthesis.

[0149] 2. Transformation of the vector, construction of the pKpL-3K-rhIL-1ra vector, transfer of the vector into host cells and engineered strains to induce expression of the target protein, the same as in Example 1.

[0150] Through the identification of the strain and the expression product, the results are as follows:

[0151] Westen Blot identified that the expression product can bind to IL-1ra antibody, which conforms to the immunological characteristics.

[0152] The N-terminal sequencing results showed that the modified amino acid seque...

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Abstract

The invention relates to the technical field of gene engineering, in particular to an engineering strain for producing recombinant human IL-1ra and a preparation method and use thereof. The preparation method of the engineering strain comprises the following steps: 1) selection and modification of a carrier: selecting an escherichia coli carrier pKpL-3a for modification; (2) constructing an rhIL-1ra expression plasmid: connecting a gene sequence of rhIL-1ra to the constructed empty carrier obtained in step (1); carrying out connection by using a DNA ligase; and directly transferring a connection product into competent cells to construct an engineering strain; and (3) construction of engineering strains and expression. The engineering strain constructed through the method has an expression quantity of 20%-40% or above, is free of mycoplasma infection and chlamydia infection, does not use penicillin or macrolide antibiotics, does not use IPTG as an inducer, and meets requirements of medicine production and requirements of large-scale and industrial production of proteins.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and specifically relates to an engineering strain for producing a recombinant human interleukin-1 receptor antagonist and a preparation method thereof. Background technique [0002] In 1985, Arend et al. discovered the first protein with natural antagonistic effect, which can antagonize the biological activity of interleukin 1 (hereinafter referred to as: IL-1), and it was named interleukin-1 receptor antagonist agent (hereinafter referred to as: IL-1ra). [0003] As a member of the interleukin-1 family (hereinafter referred to as: IL-1 family) and interleukin-1 subfamily (hereinafter referred to as: IL-1 subfamily), IL-1ra interacts with interleukin-1 receptor ( hereinafter referred to as: IL-1R) tightly combined, blocking the interaction between IL-1 (including interleukin-1α (hereinafter referred to as: IL-1α) and interleukin-1β (hereinafter referred to as: IL-1β)) and IL-1R Comb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/11C12N15/70C12N15/65C07K14/47C12R1/19
CPCC07K14/4703C12N15/70C12N15/65C12N2800/22
Inventor 王苗颖莫晓宁马大龙范中玉李强卢键赵建权雷苑李冬梅卢文娜
Owner 北京君泰联合科技有限公司
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