CN114921483A - Gene for coding archizurin, engineering bacteria and biosynthesis method thereof - Google Patents

Abstract

The invention discloses a gene for coding archipellin, engineering bacteria and a biosynthesis method thereof, belonging to the technical field of genetic engineering. In particular to the construction of a gene engineering bacterium for producing SUMO-Argiline, the fermentation of the gene engineering bacterium for producing SUMO-Argiline, the purification and enzyme digestion of the SUMO-Argiline, and the application of the gene engineering bacterium. The invention constructs a recombinant plasmid pNY 326-SUMO-Argiline by taking pNY326 as a vector and converting a gene sequence shown in SEQ ID NO.1 into a short plasmidBacillus brevis (Brevibacillus）SP3And (3) constructing to obtain recombinant brevibacillus brevis, fermenting to produce recombinant SUMO-Argiline, purifying and carrying out enzyme digestion on the recombinant SUMO-Argiline, and finally obtaining the asirelin single product. The archirelin synthesized by the biological method gets rid of the pollution defect of an organic solvent used in chemical synthesis, so that the synthesis process becomes simple, the archirelin is a green production mode, and the practical application significance is great.

Description

Gene for coding archipellin, engineering bacterium and biosynthesis method thereof

Technical Field

The invention relates to a biosynthesis method of cosmetic polypeptide-argireline, belonging to the technical field of genetic engineering.

Background

Polypeptides are compounds in which amino acids are linked by peptide bonds, and are the same as proteins in the manner of linkage, and polypeptides containing less than 100 amino acids and proteins containing more than 100 amino acids are generally called. The cosmetic polypeptide refers to polypeptide products in the field of cosmetics.

Acetyl hexapeptide-8, also known as achirelin, is a high-quality wrinkle-removing cosmetic raw material, has high wrinkle-removing activity and small side effect, and has been applied to various high-end cosmetic series. The facial mask can locally block nerve transmission muscle contraction information, influence the skin sac nerve conduction, relax the facial muscles and achieve the aim of smoothing dynamic lines, static lines and fine lines; effectively reorganizes the elasticity of collagen, can increase the activity of elastin, relaxes the lines of the face, and smoothes wrinkles and improves laxity. Can be used in cosmetics as an anti-wrinkle component with excellent effect.

There are three main methods for producing and manufacturing cosmetic polypeptides, namely liquid phase synthesis, solid phase synthesis and recombinant fermentation. The liquid phase synthesis has soft ribs during the synthesis of long peptides, and is suitable for synthesizing a large amount of short peptide medicaments; the recombinant fermentation has great advantages in the synthesis of long peptides and recombinant proteins, and the large-scale preparation cost is low, but the initial investment capital and the time cost are high, and the introduction of unnatural amino acids and structural modification are difficult; the solid phase synthesis method is fast and flexible, is suitable for modifying medium-length and long-length peptides, and has the main defect of higher cost in amplification production.

The biological method for synthesizing the cosmetic polypeptide has the advantages of chemical synthesis of the polypeptide, low cost, low pollution and high yield, and can meet the requirement of large-scale application.

Disclosure of Invention

The first purpose of the invention is to provide a genetically engineered bacterium capable of synthesizing cosmetic polypeptide-Archerelin. The gene selected herein is in the form of a fusion protein (SUMO) + Argireline (Argireline), encoding an amino acid sequence of:

HHHHHHSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGHREQIGGEEMQRR。

the nucleotide sequence of the gene for coding SUMO-Argileline is shown in SEQ ID NO. 1.

The invention also provides a recombinant plasmid, which comprises a vector and the gene shown in SEQ ID No. 1.

In one embodiment of the invention, the genetically engineered bacterium uses pNY326 plasmid as a vector and uses Brevibacillus brevis (Brevibacillus brevis) ((TM))Brevibacillus) Is the host.

In one embodiment of the present invention, the genetically engineered bacterium uses pNY326 plasmid as a vector.

The second purpose of the invention is to provide a method for constructing a brevibacillus brevis gene engineering bacterium for expressing fusion protein and Archerelin, which is based on an amino acid sequence shown by SUMO-Argiline and based on brevibacillus brevis (B) (A)Brevibacillus）SP3Optimizing the preferred codon to obtain a gene sequence shown in SEQ ID NO.1, connecting the gene sequence with a vector, and transforming the gene sequence into the brevibacillus brevis.

In one embodiment of the present invention, the Brevibacillus brevis (Brevibacillus brevis) ((B))Brevibacillus) Is composed ofB. choshinensis SP3。

In one embodiment of the invention, the method comprises the steps of: expanding machineThe gene shown in SEQ ID NO.1 is added, after enzyme digestion, the gene is connected to a vector pNY326, and the recombinant plasmid pNY 326-SUMO-Argiline is transformed and expressed into a host Brevibacillus brevis (a) (Brevibacillus）SP3Competent cells are coated with a selective plate culture medium to obtain the SUMO-Argiline-preparing engineering bacteria.

The third purpose of the invention is to provide a method for producing the Archimedes and fermenting by applying the genetic engineering bacteria.

In one embodiment of the invention, the medium is LB medium or 2SY medium.

In one embodiment of the invention, the method is to use genetically engineered bacterium Brevibacillus brevis (Bacillus brevis)Brevibacillus）SP3Single colonies of the-pNY 3326-SUMO-Argiline are inoculated in LB or 2SY liquid culture medium, cultured for 12-24h at 25-35 ℃ and at 220r/min at 150-.

In one embodiment of the invention, the bacterial liquid collected after fermentation is used for adsorbing the fusion protein onto a nickel column by using an affinity chromatography method, and then the fusion protein is eluted to obtain the SUMO-Argiline. Thereafter, the fusion protein was digested and SUMO was removed by affinity chromatography to give Archimedes (Argireline), which was lyophilized.

Has the beneficial effects that:

the invention constructs a recombinant plasmid pNY 326-SUMO-Argiline by taking pNY326 as a vector and transforming the gene sequence shown in SEQ ID NO.1 into brevibacillus brevis (aBrevibacillus）SP3And (3) constructing to obtain recombinant brevibacillus brevis, fermenting to produce recombinant SUMO-Argiline, purifying and carrying out enzyme digestion on the recombinant SUMO-Argiline, and finally obtaining the asirelin single product. The biological method for synthesizing the ayurrelin gets rid of the pollution defect of an organic solvent used for chemical synthesis, so that the synthesis process becomes simple, and the ayurrelin is a green production mode and has great practical and application significance.

Drawings

FIG. 1 is a restriction enzyme digestion verification diagram of pNY 326-SUMO-Argiline vector, wherein lane 1 is marker, and lane 2 is a band of SUMO-Argiline gene fragment; lane 3 is the band for the pNY326 vector. From left to right, lanes 1, 2 and 3 are shown.

FIG. 2 is a diagram showing the construction of the pNY 326-SUMO-Argiline vector.

FIG. 3 shows a recombinant strain Brevibacillus brevisSP3SDS-PAGE electrophoresis of shaking flask fermentation centrifugation supernatant of pNY 326-SUMO-Argiline, marker in lane 1 and SUMO-Argiline in lane 2. the obvious band at 19KDa in the figure can be seen, which indicates that SUMO-Argiline fusion protein can be obtained. From left to right, lanes 1, 2 and 3 are shown.

FIG. 4 purified SUMO-Argiline and its digested samples. Wherein, Lane 1 is marker, Lane 2 is SUMO-Argiline fusion protein; lane 3 is SUMO tag. From left to right, lanes 1, 2 and 3 are shown.

Detailed Description

Example 1SP3Construction of-pNY 3326-SUMO-Argiline gene engineering bacteria

1. Gene

According to the amino acid sequence of SUMO-Argileline, the gene sequence shown in SEQ ID NO.1, Nanjing Kingsrei Biotech Co., Ltd was entrusted for synthesis. A pair of primers for amplifying the SUMO-Argiline gene was designed using DNAMAN, as follows:

an upstream primer: 5'-ATGGCTTTCGCTGCAGGATCCCATCACCATCACCATCAT-3'

A downstream primer: 5'-GAACCCGGGGTACCGAATTCTTAACGTCTCTGCATTTC-3'

2. PCR amplification of ALDH gene sequence fragment

The synthesized SUMO-Argiline gene was used as a template, PCR amplification was performed using upstream and downstream primers, and the amplification product was detected by 1% agarose gel electrophoresis, as shown in FIG. 1.

3. Construction and transformation of Gene expression vectors

The pNY326 vector is double-digested by restriction enzymes BamH I and EcoR I respectively, the product after digestion is recovered and purified, and then the vector fragment and the Novozan Clonexpress II One Step Cloning Kit are used for carrying out the purificationConnecting target gene fragments obtained by PCR amplification, and transferring a recombinant vector pNY 3326-SUMO-Argiline (shown in figure 2) into Brevibacillus brevis (Bacillus brevis)Brevibacillus）SP3Is expressed in (1).

EXAMPLE 2 preparation of recombinant SUMO-Argiline

Recombinant strainSP3The single colony of-pNY 3326-SUMO-Argiline is inoculated in a 2SY liquid culture medium, cultured overnight at 25-35 ℃ and 220r/min for about 16h, inoculated into the 2SY liquid culture medium according to the inoculum size of 1-5% (V/V), cultured at 25-35 ℃ and 220r/min for 48h, then a bacterial liquid is collected, the bacterial liquid is centrifuged in a low-temperature refrigerated centrifuge at 4 ℃ and 7000r/min for 10min, and a supernatant is collected, wherein the supernatant contains SUMO-Argiline fusion protein.

SDS-PAGE detection: 20 mu.L of the supernatant of the fermentation broth was aspirated and detected by SDS-PAGE (12% separation gel, 5% stacking gel), as shown in FIG. 3, it can be clearly seen that a distinct band at 19kDa in the figure is consistent with the SUMO-Argiline fusion protein, indicating that the SUMO-Argiline fusion protein can be obtained.

EXAMPLE 3 purification of recombinant SUMO-Argiline fusion proteins

The gene designed by the research carries HIS label, so the separation and purification of the fusion protein product are carried out by adopting a metal chelating affinity chromatography method. The fusion protein of the supernatant obtained in example 2 was adsorbed to a nickel column by affinity chromatography, followed by elution to obtain a SUMO-Argireline fusion protein. The selected fillers are: ni Focuse 6FF (TED). And (3) carrying out enzyme digestion on the eluent by SUMO enzyme, then loading the eluent on a column again for affinity chromatography, removing the SUMO label, and freeze-drying the collected asirelin. As shown in fig. 4.

Example 4SP3Practical application of-pNY 3326-SUMO-Argiline gene engineering bacteria

The strain SP3-pNY 3326-SUMO-Argiline genetic engineering bacteria constructed by the invention are used for batch fermentation in a fermentation tank to prepare the archipeline. The specific process is as follows;

1) preparing a seed solution: taking the frozen seedsSP3pNY 3326-SUMO-Argiline, inoculated into 2SY medium. The culture was carried out at 30 ℃ overnight at 180 r/min. The incubation time was 16 hours.

2) Culturing in a fermentation tank: the fermentation was carried out using a 10L fermenter. The fermentation tank culture medium is 2SY culture medium, the charging amount is 5L, and after the material preparation is finished, the sterilization is carried out at the sterilization temperature of 121 ℃ for 20 min. And then, cooling. The seeds were inoculated at 5% inoculum size, incubated at 30 ℃ and pH controlled at 7.0 using 10% phosphoric acid and ammonia. The fermentation time was 24 hours.

3) The fermentation broth was centrifuged and the supernatant was collected. And (4) putting the supernatant on a nickel column, and performing affinity chromatography. Then, elution is performed, and the eluate is collected. And (3) carrying out enzyme digestion on the eluent by SUMO enzyme, then loading the eluent on a column again for affinity chromatography, removing the SUMO label, and freeze-drying the collected asirelin.

Examples 5-7 batch Experimental data

Fermentation and purification were carried out according to the procedure of example 4, in three consecutive batches, with the following data:

examples	Collecting the supernatant of the fermentation broth	Yield of fusion protein (g/L)	Yield of archiruine (g/L)
				Example 5	5	1.7	0.119
Example 6	7	1.5	0.105
				Example 7	6	1.6	0.112

Although the present invention has been described with reference to the preferred embodiments, it should be understood that the invention is not limited thereto, and that various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention.

Sequence listing

<110> Shandong Baijiawei Biotech Co., Ltd

<120> gene and engineering bacteria for coding archipellin and biosynthesis method thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 351

<212> DNA

<213> Brevibacterium (Brevibacillus)

<400> 1

catcaccatc accatcatag cgactccgaa gtaaaccaag aagccaaacc tgaagtaaaa 60

cctgaagtga aacctgaaac acatattaat cttaaagtta gcgatggctc aagcgaaatt 120

ttctttaaaa ttaagaaaac aacaccgctg agacgcctta tggaagcatt tgcgaaaaga 180

caaggcaaag aaatggattc tctgagattt ctgtacgatg gaattcgcat ccaagcagat 240

cagacaccgg aagatctgga tatggaagat aacgatatca tcgaagctca tagagaacaa 300

attggcggac atcgcgaaca gatcggcgga gaagaaatgc agagacgtta a 351

Claims (10)

1. The gene for coding SUMO-Argiline is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A recombinant plasmid comprising a vector and the gene of claim 1.

3. The recombinant plasmid of claim 2, wherein the pNY326 plasmid is used as a vector.

4. A genetically engineered bacterium producing SUMO-Argileline, said genetically engineered bacterium comprising the recombinant plasmid of claim 2 or 3.

5. The genetically engineered bacterium producing SUMO-Argileline of claim 4, wherein the genetically engineered bacterium is Brevibacillus brevis (Brevibacillus brevis.) (Brevibacillus）。

6. Use of the genetically engineered bacterium of claim 4 or 5 for the preparation of ayorelin.

7. A method for preparing the genetically engineered bacterium of any one of claims 4 to 5, wherein the gene represented by SEQ ID No.1 is ligated to a vector and transformed into Brevibacillus brevis.

8. The method of claim 7, wherein the method comprises the steps of: amplifying the gene shown in SEQ ID NO.1, connecting to the vector pNY326 double restriction enzyme fragment to obtain recombinant plasmid pNY 3326-SUMO-Argiline, transforming the recombinant plasmid pNY 3326-SUMO-Argiline to express host Brevibacillus brevis (B)Brevibacillus) SP3 cells, and obtaining the SUMO-Argiline gene engineering bacteria.

9. A method for the biosynthesis of SUMO-Argiline, which comprises fermenting the SUMO-Argiline with the genetically engineered bacterium according to any one of claims 4 to 5.

10. The method of claim 7, wherein the culture medium is LB culture medium or 2SY culture medium; the method is to inoculate the single colony of the genetically engineered bacteria of any claim 4 to 5 in LB or 2SY liquid culture medium, and obtain seed liquid after culturing at 25 to 35 ℃ and 220r/min at 150 to 24 h; inoculating into LB or 2SY liquid culture medium according to the inoculation amount of 1-5% (V/V), culturing at 25-35 deg.C and 220r/min for 12-24h, collecting bacterial liquid, centrifuging, collecting supernatant, and performing affinity chromatography to obtain SUMO-Argileline. Carrying out enzyme digestion on the SUMO-Argiline, and carrying out affinity chromatography for further removing the SUMO to obtain an archipeline pure product.

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