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Preparation of ST2 antigen and encoding DNA molecule special for ST2 antigen

A DNA molecule and DNA sequence technology, applied in the field of genetic engineering, can solve problems such as reducing the premature termination of transcription, and achieve the effect of reducing the possibility of premature termination of transcription and increasing the amount of prokaryotic expression.

Inactive Publication Date: 2017-07-07
北京市华信行生物科技有限公司
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  • Abstract
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Problems solved by technology

[0007] One of the technical problems to be solved by the present invention is to construct a recombinant vector expressing ST2 by optimizing the ST2 DNA sequence, and propose a preparation method of ST2 antigen, which solves the problem of hairpin structure caused by excessive local GC content in the natural ST2 gene sequence At the same time, it reduces the possibility of premature termination of transcription caused by excessive local AT content, 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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  • Preparation of ST2 antigen and encoding DNA molecule special for ST2 antigen
  • Preparation of ST2 antigen and encoding DNA molecule special for ST2 antigen
  • Preparation of ST2 antigen and encoding DNA molecule special for ST2 antigen

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Embodiment 1

[0034] Embodiment 1, optimization of ST2 coding gene

[0035]The ST2 natural sequence has several defects that are not conducive to protein expression: 1. The GC content is 41%, the AT content deviates from 50%, there are many AT-rich regions, and potential transcription occurs in the coding region of the protein to be expressed The possibility of initiation and early termination (transcription initiation 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) is very high. 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, GGG), etc.,...

Embodiment 2

[0040] The preparation of embodiment 2, ST2 antigen

[0041] 1. Construction of expression vector

[0042] The artificially synthesized and optimized ST2 coding gene sequence 1 in Example 1 was digested with Xho I and Nhe I to replace the prokaryotic expression vector pET-28a(+) (Merck Millipore, catalog number 69864, figure 1 ) between the XhoI / NheI fragments to obtain pET-28a(+)-ST2 (after optimization).

[0043] The ST2 coding gene before artificial synthesis and optimization in Example 1 was artificially synthesized and optimized in Example 3, and the ST2 coding gene was double digested with Xho I and Nhe I to replace the Xho I / Nhe I of the prokaryotic expression vector pET-28a (+) Between the fragments, pET-28a(+)-ST2 (before optimization) was obtained.

[0044] The recombinant plasmid pET-28a(+)-ST2 (after optimization) or the recombinant plasmid pET-28a(+)-ST2 (before optimization) were identified by Xho I and Nhe I double enzyme digestion, the results are as follows ...

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Abstract

The invention discloses a preparation of an ST2 antigen and an encoding DNA molecule special for the ST2 antigen. A new ST2 encoding gene is obtained through optimization, a recombinant expression ST2 antigen is obtained through a gene engineering means, the blank in existing ST2 recombinant expression is solved, an ST2 natural DNA sequence is optimized, the problem that a hairpin structure is generated due to the fact that the local GC content in the natural ST2 gene sequence is too high is solved, meanwhile, the probability that transcription ends in advance due to the fact that the local AT content in the natural ST2 gene sequence is too high is reduced, all amino acid codons are high-frequency codons, beneficial for high-level expression, in an escherichia coli system, and the prokaryotic expression amount of the ST2 antigen is increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the preparation of ST2 antigen and its special coding DNA molecule. Background technique [0002] The human ST2 gene is about 40kb, located on human chromosome 2q12, and can encode a soluble protein (sST2) and a transmembrane protein (ST2L), the transcription of which is regulated by different promoters. There are four known subtypes of ST2: sST2, ST2L, ST2V and ST2LV. ST2V and ST2LV are two splice forms of ST2L, ST2L includes an extracellular domain (3 consecutive immunoglobulin motifs), a transmembrane segment and a Toll / IL-1 receptor (TIR) ​​intracellular structure domain; while sST2 lacks the transmembrane and intracellular domains and has only a unique C-terminal sequence consisting of 9 amino acids. [0003] ST2 is a member of the interleukin-1 (interleukin-1, IL-1) receptor superfamily, with transmodel (ST2L) and soluble (sST2) forms, mainly expressed in Th2 ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/715
CPCC07K14/7155C12N15/70C12N2800/22
Inventor 张明程陈庆全
Owner 北京市华信行生物科技有限公司
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