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Optimized hIL-2 (human interleukin-2) genes as well as expression method and application thereof

A kind of interleukin and human technology, applied in the field of bioengineering, can solve the problems of easily broken disulfide bonds, inactivation, process amplification, etc., and achieve the effect of simple process, low cost and improved growth status

Active Publication Date: 2019-06-18
青岛红樱桃生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the heterologous expression of hIL-2 currently takes Escherichia coli as the host, and its expression form is inclusion body, which requires ultrasonic disruption of the cells and subsequent complex procedures such as denaturation and renaturation. This method is difficult to scale up the process
And the disulfide bond in the IL-2 structure is easy to be destroyed during the denaturation and refolding process. The correct disulfide bond is necessary for the biological activity of IL-2, so IL-2 expressed in E. coli is easy to be inactivated in the post-treatment

Method used

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  • Optimized hIL-2 (human interleukin-2) genes as well as expression method and application thereof
  • Optimized hIL-2 (human interleukin-2) genes as well as expression method and application thereof
  • Optimized hIL-2 (human interleukin-2) genes as well as expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Human interleukin-2 gene sequence optimization and vector construction

[0050] The present invention refers to the amino acid sequence (SEQ ID NO: 1) of the mature region of human interleukin-2hIL-2 (Genebank ID: AAH66254.1), and first encodes the original coding gene of hIL-2 (SEQ ID NO: 2) Codon optimization determined the codon-optimized gene sequence (SEQ ID NO: 3) of the present invention. Then, at the end of the codon-optimized gene sequence (SEQ ID NO: 3), a sequence was added to obtain a gene whose nucleotide sequence was shown in SEQ ID NO: 5. Compared with natural hIL- 2. An amino acid sequence is added to the N-terminal of the peptide chain, and its optimized amino acid sequence is shown in SEQ ID NO:4.

[0051] The protein encoded and expressed by the gene encoded by the sequence shown in SEQ ID NO: 2 is named hIL-2n; the protein encoded by the gene encoded by the gene shown in SEQ ID NO: 3 is named hIL-2c; the sequence is SEQ ID NO: 5 The expr...

Embodiment 2

[0063] Example 2: Transformation and high-copy screening of human interleukin-2 in Pichia pastoris

[0064] Recombinant expression plasmids phIL-2n, phIL-2c and phIL-2a were linearized with SalI digestion, and the linearized vectors were transformed into the expression host Pichia GS115 by electroporation transformation method, and hIL-2n was obtained by screening on the MD plate Transformants of , hIL-2c and hIL-2a expressing strains.

[0065] The grown transformants were picked out with a toothpick, and spotted on YPD plates containing different concentrations of geneticin, and the transformants highly resistant to geneticin were selected as high-copy recombinant transformants.

Embodiment 3

[0066] Example 3: Verification and induced fermentation of hIL-2 recombinant expression strains

[0067] Pick a single colony of the transformant and inoculate it in a YPD test tube with shaking for 16 hours, extract the genome as a template, and use AOX1 universal primers (3'AOX and 5'AOX) to amplify to verify whether the gene is correctly integrated into the transformant genome. After PCR, The target band was detected by agarose gel electrophoresis, and the obtained DNA was sequenced for verification.

[0068] Streak the verified recombinant strain on the YPD plate, culture it statically at 30°C for 72 hours, pick a single colony and inoculate it in BMGY medium, culture it with shaking at 30°C for 18 hours, and centrifuge to obtain the bacteria. Transfer an appropriate amount of bacteria into BMMY medium to make the concentration of bacteria reach OD 600 =1, continue shaking culture, and add 1% methanol of the culture volume every 24 hours. After 96 hours of induced expres...

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Abstract

The invention provides optimized hIL-2 (human interleukin-2) genes as well as an expression method and an application thereof. Human-derived IL-2 is transformed with a two-step optimization method including codon optimization and amino acid sequence optimization. The expression quantity of hIL-2 obtained after codon optimization is improved by 132.1% than that of a natural sequence in pichia pastoris, and the expression quantity after amino acid sequence optimization is further improved by 96.5%. The invention also provides an industrial high-density fermentation method of the hIL-2. The invention also provides the application of the hIL-2 in breeding of white feather broilers and weaned piglets. The hIL-2 improves the growth condition of the white feather broilers, reduces inflammatory factors in the white feather broilers and greatly improves immunity of the white feather broilers after being added to feed; the hIL-2 improves the feed intake and daily average weight gain of the weaned piglets and reduces diarrhea rate of the weaned piglets after being added to weaned piglet ration.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a class of optimized human interleukin-2 gene and its expression method and application. Background technique [0002] Interleukin (IL) is a kind of cytokine. As of December 2013, at least 38 interleukins have been discovered and recognized, which are named IL-1~IL-38 in sequence. Among them, interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is mainly produced by T cells (especially CD4+ T cells), and can stimulate a variety of cells such as T cells, B cells, NK cells, Macrophages and oligodendrocytes produce effects, and the most significant effect of IL-2 is to affect the growth of T lymphocytes. IL-2 can effectively improve the immune function of the body. It is the core substance in the immune regulation network of the body. It has good effects in anti-virus, anti-bacterial infection and inhibition of tumor cells, so it is widely used in clinical practice. [0...

Claims

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Application Information

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IPC IPC(8): C12N15/26C07K14/55C12N15/81C12N1/19A23K20/147C12R1/84
Inventor 肖志壮薛海曌王政吕伟于文君
Owner 青岛红樱桃生物技术有限公司
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