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Encoding gene and preparation method of interleukin-6 (IL-6) as well as application of encoding gene

A technology for interleukin and encoding gene, which is applied in the field of encoding gene of interleukin-6 and its preparation, can solve problems such as reducing premature termination of transcription, and achieves the effect of reducing the possibility of premature termination of transcription and increasing prokaryotic expression.

Inactive Publication Date: 2018-07-13
北京市华信行生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a coding gene of interleukin-6, which solves the problem of hairpin structure caused by excessive local GC content in the natural IL-6 gene sequence, and reduces the problem of excessive local AT content at the same time. The possibility of premature termination of transcription, and the codons for all amino acids are high-frequency codons that are conducive to high-level expression in the E. coli system

Method used

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  • Encoding gene and preparation method of interleukin-6 (IL-6) as well as application of encoding gene
  • Encoding gene and preparation method of interleukin-6 (IL-6) as well as application of encoding gene
  • Encoding gene and preparation method of interleukin-6 (IL-6) as well as application of encoding gene

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Experimental program
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Embodiment 1I

[0027] The optimization of embodiment 1 IL-6 coding gene

[0028] The natural sequence of the coding gene of IL-6 has the following defects that are unfavorable for protein expression: 1. There are more local AT-rich regions, and potential transcription initiation and early termination (transcription initiation) occur in the coding region of the protein to be expressed. It is very likely that the start and early termination will lead to shortened mRNA length or unwanted RNA fragments, which is not conducive to the complete expression of the target protein). 2. The local GC content is high (>70%), which is easy to form a local hairpin structure, which is not conducive to the smooth progress of genetic information transmission processes such as replication, transcription, and translation. 3. The codons of individual amino acids belong to the low-frequency codons of the E. coli expression system, such as R (AGA, AGG), P (CCC), I (ATA), L (CTA), G (GGA), etc., which are not conduc...

Embodiment 2I

[0031] Preparation of Example 2 IL-6 Protein

[0032] 1. Construction of expression vector

[0033] The artificially synthesized optimized IL-6 coding gene sequence SEQ ID NO.1 in Example 1 was double digested with Xho I and Nhe I to replace the prokaryotic expression vector pET-30a(+) (Merck Millipore, catalog number 69909) The fragment between Xho I / Nhe I was used to obtain pET-30a(+)-IL-6 (after optimization).

[0034] Replace the Xho I / Nhe I of the prokaryotic expression vector pET-30a (+) after the artificially synthesized optimized IL-6 coding gene sequence SEQ ID NO.3 in Example 1 is double digested with Xho I and Nhe I Fragment, pET-30a(+)-IL-6 (before optimization) was obtained.

[0035] Recombinant plasmids pET-30a(+)-IL-6 (after optimization) and pET-30a(+)-IL-6 (before optimization) were identified by XhoI and NheI double enzyme digestion, the results are as follows figure 1 As shown, where M: DL5000Marker; 1: double enzyme digestion of pET-30a(+)-IL-6 (after op...

Embodiment 3

[0049] Example 3 Comparison of IL-6 Protein Differences Prepared Before and After IL-6 Gene Sequence Optimization

[0050] 1. Protein expression

[0051] The protein concentration was detected by ultraviolet spectrophotometry, the OD value of IL-6 protein at 280nm was detected, and the concentration of IL-6 was calculated by combining the extinction coefficient of IL-6 protein. Determination of IL-6 protein expression in 200mL LB medium, wherein the protein concentration of IL-6 protein (after DNA optimization) is 2.3mg / mL, the volume is 7.9mL, a total of 18.17mg; IL-6 protein (before DNA optimization) The protein concentration is about 1.9mg / mL, the volume is 7.8mL, and the total is 14.82mg. It can be seen that the expression level of IL-6 protein after DNA optimization is much higher than that before DNA optimization.

[0052] 2. The local GC content in the natural IL-6 gene sequence is too high and the hairpin structure appears

[0053] The GC content of local sequences ...

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Abstract

The invention discloses an encoding gene of interleukin-6 (IL-6). The encoding gene is any one or more of (a)-(c): (a) a nucleotide sequence shown in SEQ ID NO. 1; (b) a nucleotide sequence which hasat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the nucleotide sequence shown in the SEQ ID NO. 1 and is capable of encoding proteins with the same functions; (c) a nucleotide sequence which is hybridized with the nucleotide sequence defined by (a) or (b) under stringent conditions and is capable of encoding proteins with the same functions. By optimizing the coding gene of the IL-6, the problem that a hairpin structure appears since the local GC content in a natural IL-6 gene sequence istoo high is solved, the possibility of early termination of transcription due to overhigh local AT content in the natural IL-6 gene sequence is reduced at the same time; furthermore, all amino acid codons are high frequency codons, beneficial to high level expression, in an escherichia coli system, so that the prokaryotic expression of the IL-6 is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the coding gene of interleukin-6, its preparation method and application. Background technique [0002] Interleukin-6, IL-6 for short, is a glycoprotein with a molecular weight of about 26kD. Its protein precursor consists of 212 amino acid residues, of which 27 amino acids are its leader peptide. The mature IL-6 protein consists of 185 amino acid residues, and the molecular weight of the unglycosylated protein is about 20kDa. [0003] IL-6 and its receptors are related to inflammatory diseases. It is a multifunctional inflammatory cytokine, a key component of inflammatory mediator network, and plays an important role in inflammatory response. IL-6 is closely related to the occurrence and development of a variety of tumors. It affects the progression of tumors by intervening in cell adhesion and motility, thrombus formation, expression of tumor-specific a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/24C12N15/70G01N33/68G01N33/53
CPCC07K14/5412C12N15/70C12N2800/22G01N33/53G01N33/68
Inventor 陈庆全封冰冯婷婷
Owner 北京市华信行生物科技有限公司
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