CN110042104B - Deep sea tubular worm AprA gene and expression method thereof - Google Patents

Abstract

A deep-sea tubular worm AprA gene and an expression method thereof relate to a deep-sea tubular worm AprA gene. A nucleotide sequence of the deep sea tubular worm AprA gene for coding a deep sea tubular worm autocrine propagation inhibiting protein. The protein sequence of the AprA gene of the deep-sea tubular worm is 56kDa of a protein coded by a reading frame. The molecular type of the deep-sea tubular worm AprA gene is DNA, and the sequence characteristics are as follows: the length is 1491bp, the type is nucleic acid, the strand is double-stranded, the topological structure is linear, and the nucleic acid sequence is marked as SEQ ID No. 1. The molecular type of the recombinant-expressed deep-sea tubular worm AprA is protein, and the sequence characteristics are as follows: the length is 476aa, the type is amino acid, and the sequence is represented as SEQ ID No. 3. The expression method comprises the following steps: constructing a recombinant expression vector of the deep-sea tubular worm AprA gene; and (3) determining the recombinant expression and protein activity of the AprA gene of the deep-sea tubular worm.

Description

Deep sea tubular worm AprA gene and expression method thereof

Technical Field

The invention relates to a deep sea tubular worm AprA gene, in particular to a deep sea tubular worm AprA (Lamelilabrachia luymesi Autocreatine promotion expression protein A) gene from a deep sea cold spring and an expression method thereof.

Background

The tubular worm Lamellibrachi Luymesi is collected from deep sea cold spring. The deep sea cold spring is a typical extreme environment, belongs to an ecological system of chemical energy, is different from a general ecological system which relies on photosynthesis to obtain energy, has a harsh environment, and is rich in toxic and harmful substances such as methane, heavy metal and the like^[1]. The cold spring biological system is a very direct mark for indicating the seabed cold spring, and the methane-oxidizing bacteria and the sulfate-reducing bacteria participate in the anoxic methane oxidation reaction of methane and sulfate ions in the cold spring fluid, so that a carbon source and energy are provided for the chemoautotrophic organisms, and the chemoautotrophic biological system becomes a primary producer of the cold spring ecological system. On the basis of the method, the bacterium mats, deep-sea bivalves (mussels and clams), worms (tube group worms and ice worms), polychaetes, starfishes, sea urchins, shrimps and other primary consumers are developed, wherein the tube worms only appear in the environment with low flow rate of the cold spring.

Many tissues or organs have inherent properties that allow growth to a specific size^[2]It is believed that many secreted factors in certain tissues at particular stages have the function of inhibiting cell proliferation, and that the identification of these secreted factors and the manner in which they function may beHelps to design drugs which can inhibit cell proliferation pathways, and provides possibility for simulating cancer tumor dormancy mode. Many secreted factors were initially found to function in regulating the size of specific tissues, but many have not been identified and are called chalones (chalones) because of their ability to inhibit proliferation or growth of secretory cells^[3]. The Autocrine proliferation inhibitor protein A (AprA) is a chalone, which is a kind of secretion factor produced by cells and can inhibit the proliferation of the cells under the condition of not influencing the survival of the cells^[4]And there is a lot of evidence that statins may function through G-protein mediated signal transduction pathways^[5]. Most studied about AprA is Dictyostelium, which secretes AprA of 60kDa, is part of an approximately 150kDa AprA factor complex that negatively regulates proliferation of Dictyostelium secreting cells, slows proliferation but does not stop proliferation, and reduces the number of multinucleated cells and increases spore production^[6]Cells lacking AprA show abnormal rapid proliferation, and the addition of recombinant AprA can relieve the phenomenon of rapid proliferation of AprA-null mutant strains^[7]. Inhibition of proliferation and activation of chemical repulsion by AprA requires the assistance of G-proteins, suggesting that AprA may be a ligand for GPCR^[8]。

Disclosure of Invention

The first object of the present invention is to provide a nucleotide sequence of the AprA gene of the deep-sea tubular worm.

The second purpose of the invention is to provide a protein sequence of the AprA gene of the deep-sea tubular worm.

The third purpose of the invention is to provide an expression method of the AprA gene of the deep-sea tubular worm.

The nucleotide sequence of the deep sea tubular worm AprA (autocrine proliferation inhibitor protein A) gene for coding the deep sea tubular worm autocrine proliferation inhibitor protein is marked as AprA.

The protein sequence of the AprA gene of the deep-sea tubular worm is 56kDa of a protein coded by a reading frame, and the protein can inhibit the growth of cells.

The molecular type of the deep sea tubular worm AprA gene is DNA, and the sequence characteristics are as follows: the length is 1491bp, the type is nucleic acid, the strand property is double-stranded, the topological structure is linear, the nucleic acid sequence is marked as SEQ ID No.1, and the nucleic acid sequence is shown as follows:

the nucleotide sequence of the AprA gene of the deep-sea tubular worm is obtained by adopting the following method: comparing and splicing by using a transcriptome result to obtain that the length of a complete Open Reading Frame (ORF) is 1491bp, obtaining that 1-60 bp in the AprA ORF is a signal peptide sequence through sequence analysis, entrusting a certain company to synthesize a part of the AprA ORF not containing the signal peptide and recording the part of the AprA ORF as SEQ ID No.2, then expressing the AprA gene through prokaryotic recombination, determining the activity of the AprA protein, wherein the head and the tail of the synthesized sequence carry restriction enzyme sites EcoRI and NheI, and the SEQ ID No.2 is as follows:

the molecular type of the recombinant-expressed deep-sea tubular worm AprA is protein, and the sequence characteristics are as follows: is 476aa in length, is amino acid in type, has a sequence shown as SEQ ID No.3 and is shown as follows:

the protein of the deep sea tubular worm AprA is polypeptide with an amino acid sequence shown in SEQ ID No. 3; or a polypeptide which is formed by substituting, deleting or adding at least one amino acid residue in the amino acid sequence shown in SEQ ID No.3 and has similar functions with the amino acid sequence shown in SEQ ID No. 3.

The expression method of the AprA gene of the deep-sea tubular worm comprises the following steps:

1) constructing a recombinant expression vector of the deep-sea tubular worm AprA gene;

2) and (3) determining the recombinant expression and protein activity of the AprA gene of the deep-sea tubular worm.

The invention establishes a foundation for further application of the deep sea organism AprA gene by analyzing and synthesizing the deep sea tubular worm AprA gene, constructing prokaryotic recombinant expression plasmid, carrying out recombinant protein expression in escherichia coli and verifying the growth inhibition activity of the escherichia coli.

Drawings

FIG. 1 is an SDS-PAGE electrophoretogram of recombinant expression of the AprA gene from the tubular worm. In FIG. 1, M is a protein Marker; line 1: uninduced expression of a recombinant vector pET-His-AprA in a strain BL21(DE3) PLysS; line 2: inducible expression of the recombinant vector pET-His-AprA in the strain BL21(DE3) PLysS.

FIG. 2 is a plot of BL21(DE3) PLysS growth. In FIG. 2, the control group transformation plasmid was pET-His, and the experimental group transformation plasmid was pET-His-AprA.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the specific conditions not specified in the examples below were generally carried out according to conventional conditions such as those described in the molecular cloning laboratory manual (15, SammBruke, Lassel, Huang Petang (ed.), molecular cloning, A laboratory Manual, scientific Press, 2002, third edition) or according to the conditions recommended by the manufacturers of reagents or instruments.

In order to achieve the purpose, the invention adopts the following technical scheme, which comprises the following specific steps:

1. synthesis and sequence analysis of deep sea tubular worm Lamellibrachi Luymesi AprA gene

As the sequence analysis shows that the complete Lamellibranchia lumesesi AprA reading frame is 1491bp long and can encode 496aa, but 1-60 bp is a signal peptide sequence, a company can be entrusted to directly synthesize a part of AprA ORF which carries enzyme cutting sites at two ends and does not contain signal peptide, the enzyme cutting sites are EcoRI and NheI, the synthesized AprA gene and pET-His are subjected to double enzyme cutting respectively, enzyme cutting products of the AprA gene and the pET-His are connected overnight after purification and are converted into Top10, 2-3 positive colonies are picked after colony PCR and are subjected to shake bacteria culture to extract plasmids and sequence.

A double enzyme digestion system:

purification of the digestion product (Gel recovery Kit QIAquick Gel Extraction Kit):

1) carrying out agarose gel electrophoresis on all enzyme digestion products, cutting the enzyme digestion products to obtain gel blocks containing target bands, and putting the gel blocks into an EP tube;

2) 3 volumes (0.1mg ═ 100 μ L) of Buffer QG were added separately;

3) dissolving the glue in 50 deg.C water bath for 10 min;

4) adding isopropanol with one time volume and mixing uniformly;

5) transferring the liquid into 13000g of a centrifugal column for centrifugation for 1min, and discarding the liquid;

6) adding 500 μ L of Beffer QG, 13000g, separating for 1min, and discarding the liquid;

7) adding 750 μ L Beffer PE, 13000g, separating for 1min, and discarding the liquid;

8)13000g is separated for 2min, and the liquid is thoroughly removed;

9) placing the centrifugal column in a collecting pipe, standing for 2-3 min at room temperature, and dripping 30 mu L H to the center of the centrifugal column₂Dissolving O for 2min, and centrifuging for 2 mi.

Carrier connection:

ligation was carried out overnight at 14 ℃.

Ligation product conversion competence:

1) adding the ligation product into an EP tube with competent Top10, mixing uniformly, and ice-cooling for 25 min;

2) heat shock at 42 deg.c for 90 sec and fast ice bath for 2 min;

3) adding 500 μ L LB culture medium, and shake culturing at 37 deg.C for 45 min;

4) 100. mu.L of the culture broth was pipetted and spread evenly on ampicillin (resistance gene carried by pET-His) plates, and cultured overnight at 37 ℃ in an inverted state.

Positive clones were selected by colony PCR:

1) single colony picking from plate using sterilized toothpick transfer to preinjection H₂O in numbered PCR tubes and streaked on new ampicillin resistant plates;

2) and (5) colony PCR.

20 μ L system:

plasmid extraction (plasmid extraction kit)

Spin Miniprep Kit)：

1) Collecting thallus by centrifuging (8000rpm/min,3 min);

2) adding 250 mu LBuffer P1 at 4 ℃ to resuspend the thalli;

3) adding 250 mu L of Buffer P2, and slightly overturning for 4-6 times to crack the thalli;

4) adding 350 mu L of Buffer N3, slightly overturning to mix uniformly, and slightly standing to separate out protein;

5)13000g, centrifugating for 10min to make the protein fully precipitate;

6) sucking the clear liquid, transferring the clear liquid to a micro centrifugal tube, centrifuging for 1min at 13000g, and discarding the liquid;

7) adding 500 μ L Buffer PB for washing, centrifuging at 13000g for 1min, and discarding the liquid;

8) adding 750 μ L Buffer PE for washing, centrifuging at 13000g for 1min, and discarding the liquid;

9) centrifuging at 13000g for 2min to completely remove the liquid, and slightly standing;

10) the column was placed in a clean collection tube and 30. mu. L H was added to the center₂13000g is centrifuged for 2min after O is dissolved for 2 min.

2. Expression and functional verification of deep sea tubular worm Lamellibrachi luymesi AprA protein

And (3) converting the sequencing-correct plasmid pET-His-AprA obtained in the steps into BL21(DE3) PLysS, selecting a positive colony for induced expression, simultaneously taking a parallel without an inducer as a control, and identifying the expression of the target protein AprA by SDS-PAGE electrophoresis (see figure 1).

AprA is a cell proliferation-inhibiting protein with autocrine effect, originally found in bifidobacterium cells, and it inhibits the proliferation of bifidobacterium without affecting the survival of bifidobacterium, and it only slows down but does not prevent cell proliferation. To confirm whether or not the Lamellibranchia luymesi AprA also has the function of inhibiting cell proliferation, pET-His transformed BL21(DE3) PLysS was used as an experimental control and pET-His-AprA transformed BL21(DE3) PLysS was cultured by shaking and adding an inducer, and OD of the inoculum was measured every 1 hour from inoculation of 0₄₅₀Until 6h (see fig. 2).

Inducing expression:

1) picking positive colonies, and performing shake culture at 37 ℃ in a liquid culture medium overnight;

2) performing scale-up culture at a ratio of 1: 40;

3) determination of bacterial liquid OD by spectrophotometer₄₅₀The inducer isopropylthio- β -D-galactoside (IPTG) was added at 0.6 to a final concentration of 1 mM;

4) and after continuing culturing for 4-5 h, centrifuging 1ml of bacterial liquid to obtain a bacterial precipitate, adding a proper amount of protein loading buffer solution, and boiling the sample in water bath at 100 ℃ to perform SDS-PAGE electrophoresis.

SDS-PAGE electrophoresis:

1) preparing glue: the concentration of the separation gel is 10 percent, and the concentration of the concentrated gel is 4 percent;

2) sample preparation: carrying out induced expression on the obtained sample, centrifuging for 5min at 14,000 Xg, and sampling 5-10 mu L;

3) electrophoresis: firstly, carrying out electrophoresis at 100V, increasing the voltage to 160V after the bromophenol blue enters the separation gel (about 30min), and stopping electrophoresis when the bromophenol blue just runs out of the gel;

protein glue dyeing:

1) soaking the protein gel obtained by SDS-PAGE electrophoresis in about 10ml Coomassie brilliant blue, and performing low-speed shaking staining for 1 h;

2) taking out the protein glue, soaking in clear water, shaking at medium speed, and decolorizing, wherein water is required to be changed for many times.

AprA protein activity exploration:

1) respectively picking pET-His-AprA and BL21(DE3) PLysS positive colonies transformed by pET-His to shake and culture in a liquid culture medium at 37 ℃ for overnight;

2) performing scale-up culture at a ratio of 1: 40;

3) the OD of the bacterial suspension was measured every 1h with a spectrophotometer at 0h from the start of the scale-up₄₅₀Until 6 h;

4) the resulting data were plotted in a growth curve.

The nucleotide sequence of the AprA gene is obtained by transcriptome analysis and comparison, the sequence is simply analyzed, and the protein sequence coded by the AprA gene of the tubular worm is predicted. The pET-His vector is used for constructing recombinant plasmid suitable for prokaryotic expression, recombinant protein expression is carried out in Escherichia coli expression strain BL21(DE3) plysS, and the exogenous recombinant protein has leakage expression and inhibits the growth of the strain.

Claims (4)

1. An isolated deep-sea tubular worm AprA gene, which is characterized in that the gene codes a deep-sea tubular worm autocrine propagation inhibiting protein, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. The isolated AprA gene from a deep-sea tubular worm according to claim 1, wherein the signal peptide has the sequence shown in SEQ ID No. 2.

3. An isolated deep-sea tubular worm AprA protein, characterized in that the amino acid sequence thereof is shown in SEQ ID No. 3.

4. The method for expressing the AprA gene of the isolated deep-sea tubular worm according to claim 1, which comprises the following steps:

1) constructing a recombinant expression vector of the deep-sea tubular worm AprA gene;

2) and (3) determining the recombinant expression and protein activity of the AprA gene of the deep-sea tubular worm.

Images