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Method for producing Delta sleeping peptide by utilizing escherichia coli prokaryotic expression system

A technology of Escherichia coli and prokaryotic expression, applied in the field of microbiology, can solve the problems of low content of aspartic acid α-delta sleep peptide and impurity of delta sleep peptide, achieve obvious activity, solve product impurity, and avoid β The effect of isomers

Inactive Publication Date: 2010-09-01
LIAONING UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the above problems, the object of the present invention is to achieve a large amount of expression of delta sleep peptide through the Escherichia coli expression system, overcome the impurity of delta sleep peptide components synthesized by chemical methods, and the low content of 5-aspartic acid α-delta sleep peptide question

Method used

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  • Method for producing Delta sleeping peptide by utilizing escherichia coli prokaryotic expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 Construction of expression plasmid with delta sleep peptide gene

[0020] From the amino acid sequence of the delta sleep peptide, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu, the coding chain 5'-TGGGCTGGTGGTGACGCTTCCGGTGAA-3' of the delta sleep peptide gene was obtained by reverse translation. In order to optimize the expression, the genes encoding the delta sleep peptide all select codons preferred by the expression host Escherichia coli, and the plasmids and hosts are respectively selected from pET-28a(+) and Escherichia coli BL21(DE3). The specific implementation is as follows:

[0021] 1. Acquisition of delta sleep peptide gene:

[0022] According to the amino acid sequence of the delta sleep peptide (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu), the gene sequence of the delta sleep peptide can be known. In order to insert it into the expression plasmid, a protective base, EcoRI restriction site sequence and enterokinase recognition site sequence, add stop codon, Xh...

Embodiment 2

[0031] Example 2 Induced expression and purification of recombinant Delta sleep peptide

[0032] 1. Induced expression of recombinant Delta sleep peptide

[0033] Inoculate the recombinant Escherichia coli carrying the plasmid pET28a-dsip in 10ml LB medium containing Kan (Kanamycin, Kanamycine, Kan)) (40 μg / ml), cultivate for 12-16 hours, and press 2% Inoculation amount, inoculate the cultured bacterial liquid into 200ml LB medium containing Kan (40 μg / ml) for cultivation, and when the absorbance value of the bacterial liquid reaches 0.6, induce with 1 mM IPTG. In order to avoid the occurrence of inclusion bodies during the expression process, the present invention selects 30° C. as the induction temperature where the bacterial cell growth is not very active, and induces for 4-5 hours.

[0034] 2. Purification of recombinant Delta sleep peptide

[0035] After 4 hours of induced expression, the bacterial cells were collected by centrifugation at 8000 rpm for 5 minutes. Freeze ...

Embodiment 3

[0040] Example 3, using recombinant enterokinase to cleave recombinant Delta sleep peptide to obtain Delta sleep peptide.

[0041] The specific implementation is as follows: 100μl system

[0042] Fusion protein: 1~1.5mg / ml

[0043] Recombinant enterokinase: 2~3U

[0044] 10× buffer: 10 μl

[0045] Temperature (°C): 22°C

[0046] Time (hour): 8~16

[0047] The obtained Delta sleep peptide with histidine tag was cleaved with histidine tag by enterokinase in vitro. After the tag cleavage was completed, the Delta sleep peptide was passed through Ni-NTA affinity chromatography column and Ni affinity chromatography. The sleep peptide and 6×Histag are separated, because 6×Histag will bind to Ni ions, and the Delta sleep peptide flows through the Ni-NTA affinity chromatography column together with the solution to obtain the target product, namely the Delta sleep peptide.

[0048] Test results: After sequencing verification, the Delta sleep peptide with nine amino acid sequences s...

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Abstract

The invention relates to a method for producing a Delta sleeping peptide by utilizing an escherichia coli prokaryotic expression system. The adopted technical scheme is as follows: carrying out enzyme digestion on genes dsip of the obtained Delta sleeping peptide, then inserting into a vector plasmid pET28a, and constructing a plasmid pET28a-dsip; transferring the plasmid pET28a-dsip into escherichia coli BL21 competent cells, inoculating the obtained recombinant escherichia coli into an LB culture medium, using 1mM IPTG for inducing for 4-5h under the condition of 30 DEG C when the absorbance value of bacteria liquid is 0.6, and collecting bacterial cells in a centrifugal manner; using ultrasonic waves for breaking the bacterial cells, removing insoluble cell broken pieces and collecting supernatant liquid; and passing through an Ni-NTA affinity chromatography column twice, and obtaining the Delta sleeping peptide. The method can realize the mass expression of the Delta sleeping peptide through the escherichia coli expression system and solve the problem of impurity of a product formed by chemical synthesis and low content of active ingredients.

Description

technical field [0001] The invention relates to a method for producing Delta sleep peptide by using an Escherichia coli prokaryotic expression system, and belongs to the fields of microbiology, molecular biology and genetic engineering. Background technique [0002] 1 / 3 of human life is in sleep (sleep) state, the quality of sleep is related to the normal progress of various functional activities when waking up, and determines the degree of personal ability when waking up, while sleep disorders seriously affect the quality of life, decrease productivity. In the 2nd edition of the International Classification of Sleep Disorders in 2005, there are more than 90 sleep disorders listed, which are closely related to many physical diseases, mental disorders, brain dysfunction and other disciplines, and have a negative impact on each other. Sleep disorders reduce people's quality of life, affect social harmony, increase national health care expenditures, and cause huge annual econo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P21/02C07K7/06C07K1/22C12R1/19
Inventor 胡风庆代有金
Owner LIAONING UNIVERSITY
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