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Prokaryotic expression vector of tryptophan hydroxylase-2 gene

A tryptophan hydroxylase and prokaryotic expression technology, applied in the field of genetic engineering, can solve the problems of resource scarcity, limited utilization, and no recombinant expression of TPH-2 gene, and achieve low cost, easy operation, and convenient industrial production Effect

Inactive Publication Date: 2011-10-19
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] 5-Hydroxytryptophan is mainly found in Grifania simplicifolia, an African leguminous plant, with a content of up to 10% of dry weight. Due to its special medicinal effect and rarity, its resources are increasingly scarce , which greatly limits the use of
At present, in the field of molecular biology, the recombinant protein expressed by TPH-1 gene in Escherichia coli has been used to detect the activity of 5-hydroxytryptophan (Devanand Kowlessur, Seymour Kaufman, et al. Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme. Biochimica et Biophysica Acta,1999, 1434: 317-330.), but there is no report of recombinant expression of TPH-2 gene

Method used

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  • Prokaryotic expression vector of tryptophan hydroxylase-2 gene
  • Prokaryotic expression vector of tryptophan hydroxylase-2 gene
  • Prokaryotic expression vector of tryptophan hydroxylase-2 gene

Examples

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Effect test

Embodiment 1

[0025] A tryptophan hydroxylase-2 gene prokaryotic expression vector, the expression vector is an Escherichia coli expression vector containing the tryptophan hydroxylase-2 gene sequence.

[0026] The tryptophan hydroxylase-2 gene sequence encodes the amino acid sequence of sequence 1 in the sequence listing.

[0027] The sequence 1 is recorded in the UniProtKB (http: / / www.uniprot.org / ) database, and the information is: sp / Q8IWU9 / TPH2_HUMAN Tryptophan 5-hydroxylase 2 OS=Homo sapiens GN=TPH2 PE=1 SV=1.

[0028] The tryptophan hydroxylase-2 gene sequence is the nucleotide sequence of sequence 2 in the sequence listing.

[0029] The tryptophan hydroxylase-2 gene sequence is the TPH2_HUMAN sequence of NCBI number NM 173353.3 obtained after codon preference optimization of Escherichia coli.

[0030] The Escherichia coli expression vector is pET32(a+).

[0031] The construction method of the prokaryotic expression vector of the tryptophan hydroxylase-2 gene, the steps of the const...

Embodiment 2

[0038] This example is the specific method for constructing the prokaryotic expression vector of tryptophan hydroxylase-2 gene in Example 1. Such as figure 2 It is a flowchart of the method for constructing the prokaryotic expression vector of the tryptophan hydroxylase-2 gene.

[0039] The steps of this build method are:

[0040] A, obtaining the gene sequence of tryptophan hydroxylase-2;

[0041] a. The tryptophan hydroxylase-2 gene sequence is obtained from the TPH2_HUMAN sequence of NCBI No. NM 173353.3 after codon preference optimization in Escherichia coli, with a size of 1473bp and restriction endonucleases at both ends Restriction sites for BamH I and Hind III; the gene sequence of tryptophan hydroxylase-2 (TPH-2) was synthesized by Sanbo Yuanzhi Biotechnology Co., Ltd. and connected to the PMD18-T vector;

[0042] The tryptophan hydroxylase-2 gene sequence is the nucleotide sequence of sequence 2 in the sequence listing.

[0043]The optimization process is: optim...

Embodiment 3

[0100] This example is the preparation of the recombinant of the Escherichia coli expression vector containing the tryptophan hydroxylase-2 gene sequence in Example 2.

[0101] The prokaryotic expression vector of the tryptophan hydroxylase-2 gene is transformed into Escherichia coli E. coli BL21 (DE3) competent cells to obtain the prokaryotic expression recombinant of the tryptophan hydroxylase-2 gene.

[0102] The preparation method of the prokaryotic expression recombinant of the tryptophan hydroxylase-2 gene is as follows:

[0103] A. Prepare the Escherichia coli E.coli BL21(DE3) competent cells:

[0104] ⅰ. Streak the preserved strain on the LB plate and culture overnight at 37°C;

[0105] ⅱ. Pick a large and plump single colony and inoculate it in 4mL LB liquid medium, cultivate overnight at 37°C and 230rpm;

[0106] ⅲ. Transfer 200 μL of the overnight culture into a 250ml shake flask containing 30mL of LB liquid medium, and culture at 37°C and 200rpm until the OD600 i...

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Abstract

The invention belongs to the field of genetic engineering and in particular relates to an escherichia coli expression vector containing a tryptophan hydroxylase-2 gene sequence. The tryptophan hydroxylase-2 gene sequence is obtained by carrying out escherichia coli codon preference optimization on a TPH2_HUMAN sequence with a serial number of MM173353.3 in the NCBI (National Center of Biotechnology Information). The expression vector of escherichia coli is pET32 (a+). As the tryptophan hydroxylase-2 gene sequence is expressed by the expression vector of escherichia coli, an expression product, i.e., tryptophan hydroxylase-2, can be obtained within shorter time, and the required cost is relatively low; and the expression vector and an integral expression system are easy to operate and convenient for industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to an Escherichia coli expression vector containing tryptophan hydroxylase-2 gene sequence. Background technique [0002] 5-Hydroxytryptophan (5-Hydroxytrophan, 5-HTP) is a natural amino acid that has a strong antidepressant effect and is the only natural drug that can replace the existing Western medicine Prozac for the treatment of depression. , and has no toxic side effects. In addition, 5-HTP has central analgesic effects and has important effects on the function of the cardiovascular system. The melatonin and brain gold currently on the market are all transformed from 5-hydroxytryptophan. [0003] 5-Hydroxytryptophan is mainly found in Grifania simplicifolia, an African leguminous plant seed, with a content of up to 10% of dry weight. Due to its special medicinal effect and rarity, its resources are increasingly scarce , making its use extremely limited. There are curre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12R1/19
Inventor 袁其朋陈京顺孙新晓孙杰
Owner BEIJING UNIV OF CHEM TECH
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