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A method for optimizing the nucleotide sequence and efficient soluble expression of recombinant human interleukin-2

A technology of interleukin and nucleotide sequence, which is applied in the field of optimizing the nucleotide sequence and efficient soluble expression of recombinant human interleukin-2, can solve the mismatch, contribute to the solubility of interleukin-2, and step Complicated problems, to achieve the effect of reducing the formation of inclusion bodies, reducing the formation of inclusion bodies, and reducing the growth temperature

Active Publication Date: 2019-04-09
SHANGHAI HUAXIN HIGH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology screens the renaturation method of inclusion bodies, but the steps are still complicated, there are still mismatch defects, and it does not contribute to the improvement of the solubility of interleukin-2

Method used

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  • A method for optimizing the nucleotide sequence and efficient soluble expression of recombinant human interleukin-2
  • A method for optimizing the nucleotide sequence and efficient soluble expression of recombinant human interleukin-2
  • A method for optimizing the nucleotide sequence and efficient soluble expression of recombinant human interleukin-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Codon Optimization of Recombinant Human Interleukin-2 Nucleotide Sequence

[0047] The nucleotide sequence of recombinant human interleukin-2 is based on NCBI (LOCUS: NM_000586), the signal peptide sequence is removed, and the free energy and stability of the secondary structure of the mRNA are considered while the amino acid sequence is kept unchanged. sequence, etc., replace and optimize the gene sequence using the preferred codons in E.coli, and then carry out the whole gene synthesis.

[0048] The recombinant human interleukin-2 nucleotide sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the specific sequence is shown in SEQ ID NO:1; the synthesized nucleotide sequence is 405bp in length, and it was constructed in a cloning vector for sequencing verification correct. Compared with the natural human interleukin-2 gene (specific sequence such as SEQ ID NO: 2), the recombinant human interleukin-2 nucleotide sequence synthesized by t...

Embodiment 2

[0049] The construction of embodiment two recombinant expression vectors

[0050] Carriers and reagents: The vector pET21a and strain DH5α were donated by Professor Tan Xiaoli of Jiangsu University; restriction enzymes and DNA markers were purchased from Dalian Bao Biological Engineering Co., Ltd.

[0051] pET21a includes ampicillin selection marker, inducible promoter Ptac, multiple cloning site. The cloning vector with 405bp optimized rhIL-2 sequence and the pET21a plasmid were double digested with NdeI and XhoI respectively, and the two fragments were ligated with T4 ligase to obtain the pET21a / rhIL-2 plasmid, and rhIL-2 was inserted into pET21a Among the multiple cloning sites of , the upstream is Ptac. The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock, clones were selected on LB plates containing ampicillin, plasmids were prepared in small quantities, and positive clones were screened by double enzyme digestion and sequencing i...

Embodiment 3

[0053] Example 3: Screening, acquisition and induction culture of highly efficient soluble expression engineered bacteria

[0054] (1) The recombinant plasmid obtained in Example 2 was transformed into a host bacterium, ie, Escherichia coli BL21 (DE3), which is the engineering bacterium of the present invention, and positive transformants were obtained by screening on an ampicillin solid medium plate.

[0055] (2) Select a positive transformant, add 50ml of LB culture medium, shake culture overnight at 37°C in a 250ml shake flask to mid-late logarithm, that is, overnight activation, inoculate 50ml 2× at 1:100 (v / v) In YT liquid medium (containing 0.1mg / ml ampicillin), culture on a shaker at 37°C until OD600=0.6;

[0056] (3) Add the inducer IPTG to make the final concentration 1mM; culture under different conditions (A, 37°C shaker culture for 4h; B, 25°C shaker culture for 6h; C, 15°C shaker culture for 20h); The cells were collected by centrifugation, the supernatant was di...

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Abstract

The invention discloses an optimized nucleotide sequence of recombinant human interleukin-2. The nucleotide sequence is optimized according to preferred codons of Escherichia coli under the circumstance of not changing amino acid coding sequence. The nucleotide sequence is as shown in SEQ ID NO:1, or is complementary nucleotide sequence. Furthermore, expression vector and engineering bacteria of the nucleotide sequence are constructed, and recombinant human interleukin-2 is expressed in the engineering bacteria. The expressed recombinant human interleukin-2 accounts for 10-30% of total mycoprotein, and soluble expression amount accounts for 3-15% of total expression amount. The amount of recombinant human interleukin-2 inclusion body is reduced, expression amount of soluble protein is raised, and conditions are created for mass production of soluble recombinant human interleukin-2.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for optimizing the nucleotide sequence of recombinant human interleukin-2 and its high-efficiency soluble expression. Background technique [0002] Interleukin-2 (IL-2), a glycoprotein secreted by white blood cells, can mediate the interaction between cells and play a regulatory role in cell activation, proliferation and differentiation. IL-2 is the core substance in the body's immune regulation network, and it has synergistic and antagonistic effects with other cytokines to complete the balance regulation of the body's immune function; it can stimulate the activation and proliferation of NK cells, CTL, and LAK cells, and can also promote T lymphocytes, NK cells produce interferon, tumor necrosis factor and so on. Therefore, as a cytokine with a wide range of biological activities, IL-2 is widely used in the treatment of diseases such as anti-virus, anti-ba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/26C12N15/70C12N1/21C12R1/19
CPCC07K14/55C12N15/70C12N2800/101C12N2800/22
Inventor 刘惠李冠英钟京谕张德宝李海红毛明川吉田昌利
Owner SHANGHAI HUAXIN HIGH BIOTECH
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