Preparation method and application of AANAT and 14-3-3 protein compound

Abstract

The invention discloses a preparation method and application of an AANAT and 14-3-3 protein compound, belongs to the technical field of bioengineering, and solves the blank of difference comparison of the combining capacity of AANAT and 14-3-3 in fishes. Comprising the following steps: collecting zebrafish aanat and 14-3-3 gene family sequences; selecting a gene closely related to the synthesis of the coded melatonin, and performing codon optimization; constructing a prokaryotic expression vector by the target gene; recombinant protein expression and expression form determination; purifying the AANAT2 protein and including the complex; the AANAT2 protein is phosphorylated and is combined with 14-3-3 in vitro; respectively carrying out western blotting on the eluent and the magnetic beads; and measuring the relative gray value of the western blot strip, and comparing the binding capacity. The invention provides reference for deeply exploring the combining capacity difference between other 14-3-3 subtypes and phosphorylated AANAT (pAANAT), and provides guidance for subsequent research and development of small peptide drugs for regulating and controlling AANAT activity and further improving sleep.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically relates to a preparation method and application of an AANAT and 14-3-3 protein complex. Background Art

[0002] All kinds of organisms on the earth, from single-celled organisms to animals, plants and even humans, basically show circadian rhythm changes with a period of about 24 hours, and this phenomenon is called biological clock. As an important biological clock regulatory molecule, serotonin plays an indispensable regulatory role in various basic life processes such as sleep, growth, reproduction, development and immunity. Disruption of the biological clock can seriously damage the health of the body. Therefore, it is crucial to study the regulation mechanism of the biological clock and the regulation rules of the biological clock on various life processes.

[0003] The regulation of the vertebrate biological clock is closely related to the biosynthesis and metabolism of melatonin in the pineal gland of the brain. The change in the level of melatonin is positively correlated with the content of its key synthetic enzyme arylalkylamine N-acetyltransferase (AANAT). The increase in melatonin at night mainly depends on the activity of the 5-hydroxytryptamine → AANAT → melatonin synthesis pathway. The change in AANAT activity is usually strongly controlled by the binding of the cAMP-dependent dimer 14-3-3 protein. At night, the two phosphorylation sites T31 and S205 of AANAT promote the binding of 14-3-3 protein to AANAT. The formed complex can protect AANAT from the influence of protease hydrolysis and enable AANAT to continuously accumulate in the conversion of 5-hydroxytryptamine and melatonin synthesis. At the same time, the binding to 14-3-3 also increases the affinity of AANAT for arylalkylamine substrates such as 5-hydroxytryptamine and tryptamine (up to 10 times). During the day, the decrease in cAMP level leads to the dephosphorylation of AANAT, resulting in the dissociation of 14-3-3 from AANAT and then losing the protection of AANAT. Therefore, during the day, AANAT is hydrolyzed by protease and the synthesis and secretion of melatonin are reduced to extremely low levels. This phosphorylation and dephosphorylation cycle of AANAT is the main reason for the circadian rhythm change of melatonin production. However, at present, the research on comparing the binding ability differences between AANAT and 14-3-3 in fish is still blank.

[0004] Based on this, the present invention designs a preparation method and application of an AANAT and 14-3-3 protein complex to solve the above problems. Through in vitro experiments, the differences in the binding ability of three subtypes (γ1, γ2 and ζ) of 14-3-3 to phosphorylated AANAT2 are preliminarily demonstrated. It provides a strong reference for further exploring the differences in the binding ability between other subtypes of 14-3-3 and AANAT, and also provides guidance for the subsequent research and development of small peptide drugs for improving sleep. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the present invention provides a method for preparing a complex of AANAT and 14-3-3 protein and its application.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A method for preparing a complex of AANAT and 14-3-3 protein, comprising the following steps:

[0008] Step 1, collection of zebrafish aanat and 14-3-3 gene family sequences: Collect relevant data of the reported aanat and 14-3-3 gene families in zebrafish through databases such as GenBank and Ensembl. The zebrafish genome is used as a reference for searching the upstream and downstream regions of 14-3-3. Information on the members of the zebrafish aanat and 14-3-3 gene families collected is shown in Table 1 of Example 1.

[0009] Step 2, select the genes aanat2, 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ that are closely related to melatonin synthesis according to tissue distribution specificity, main physiological functions, and previous research results. These genes are genes without signal peptides after codon optimization in Escherichia coli K12.

[0010] Step 3, construct prokaryotic expression vectors for the genes aanat2, 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ:

[0011] The process for constructing the prokaryotic expression vector of the aanat2 gene is as follows: Using the method of overlapping PCR, design full-length splicing primers to synthesize the target gene aanat2; construct the synthesized aanat2 gene between the multiple cloning sites of the endonucleases NdeI and XbaI of the pCzn1 vector (6×His tag). The vector construction adopts the scheme of NdeI + optimized target sequence + stop codon + XbaI to obtain the aanat2-pCzn1 recombinant plasmid. Transfer the recombinant plasmid aanat2-pCzn1 into the DH5α cloning engineering strain, extract the plasmid by digestion, identify the positive clones by enzyme digestion and send them for sequencing to identify the correct strain.

[0012] The procedures for constructing prokaryotic expression vectors of 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ genes are as follows: Using the method of overlapping PCR, full-length splicing primers were designed to synthesize the target genes 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ. The target genes 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ were constructed between the multiple cloning sites of the endonucleases EcoRI and XhoI of the pGEX-4T-1 vector (GST tag). The vector construction adopted the scheme of EcoRI + target gene + TAA + XhoI, and the recombinant plasmids of 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 were obtained respectively. These recombinant plasmids were transferred into the DH5α cloning engineering strain. Through plasmid extraction, enzyme digestion identification, and screening of positive clones for sequencing, the correct strains were identified.

[0013] Step 4: Determination of recombinant protein expression and the form of recombinant protein expression: The expression plasmids aanat2-pCzn1, 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 after correct sequencing verification were inoculated monoclonally in LB broth medium and cultured overnight at 37°C, 200 rpm and 16°C, 200 rpm respectively. After plasmid extraction, they were transferred into the BL21 expression strain respectively to obtain the expression strains of aanat2-pCzn-BL21, 14-3-3γ1-pGEX-4T-1-BL21, 14-3-3γ2-pGEX-4T-1-BL21, and 14-3-3ζ-pGEX-4T-1-BL21. Under the conditions of 37°C and 16°C respectively, IPTG was used for induction expression. When inducing expression at 37°C, the culture was expanded at a ratio of 1:200 by volume and cultured at 200 rpm until the OD600 value of the bacterial liquid reached 0.6 - 0.8 in the logarithmic growth phase. 500 ml of the bacterial liquid was taken as the negative control, and IPTG was added to the remaining bacterial liquid to a final concentration of 0.5 mM, and the culture was continued for about 4 h. 200 ml was taken as the sample after induction. When inducing expression at 16°C, the culture was expanded at a ratio of 1:100 by volume and cultured at 200 rpm until the OD600 of the bacterial liquid reached 0.6 - 0.8. 1.0 mL was taken as the negative control. IPTG was added to the remaining bacterial liquid to a final concentration of 0.5 mM, the temperature was adjusted to 16°C and the culture was continued overnight (16 h). 1.0 mL was taken as the sample after induction. A small amount of the bacterial liquid was centrifuged at 12000 rpm for 2 min, the bacterial cell precipitate was rinsed twice with PBS, loading buffer was added, boiled at 100°C for 10 min and then centrifuged at 12000 rpm for 1 min, and 15% SDS-PAGE electrophoresis was used to analyze the expression of the supernatant and precipitate of the protein.

[0014] Set the cultured bacterial liquid before adding IPTG as the negative control, and culture the positive monoclonal aanat2-pCzn1, 14-3-3γ1-pGEX-4T1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 with BL21 as the expression engineering bacteria according to the aforementioned conditions. Collect 1.0 mL of bacterial liquid cells, resuspend the precipitate after lysis, ultrasonically disrupt on ice, centrifuge at 12000 rpm for 15 min, and take samples of the supernatant; take samples after resuspending the precipitate with the same amount of lysis solution, and analyze the expression of each protein by SDS-PAGE electrophoresis.

[0015] Step 5: Purify the AANAT2 protein and include the complex. Culture and collect the bacterial cells of all engineering strains under the optimized optimal induction expression conditions. The AANAT2 bacterial cells are resuspended in the binding / equilibration buffer (50 mmol / L NaH2PO4, 300 mmol / L NaCl, 10 mmol / L imidazole, pH 8.0), ultrasonically disrupt the bacterial cells, centrifuge at 12000 rpm for 15 min, dissolve the precipitate in the binding / equilibration buffer containing 8 M urea and centrifuge at high speed, filter the centrifuged supernatant through a 0.45 μm filter, and bind the filtrate to the Ni 2+ column; rinse with 3 column volumes of the washing buffer (50 mmol / L NaH2PO4, 300 mmol / L NaCl, 100 mmol / L imidazole, pH 8.0); elute the target protein with different concentrations of imidazole (150, 200, 250, 300, 500 mmol / L; pH 8.0), collect the eluate, and analyze the eluate by SDS-PAGE electrophoresis.

[0016] Furthermore, renature the inclusion bodies of the protein AANAT that cannot be soluble-expressed in the supernatant. After analysis by the above affinity chromatography purification, samples with relatively high protein purity and concentration are collected. Add them to the treated protein dialysis bag and slowly dialyze into the buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 4 mM GSH, 0.4 mM GSSG, 0.4 M L-Arginine) for renaturation at 4°C. After renaturation, the AANAT2 protein is finally dialyzed into the storage solution (50 mM Tris (pH 8.0), 150 mM NaCl, 10% Glycerol) solution for about 6-8 h. After the dialysis renaturation is completed, the supernatant is filtered through a 0.22 μm filter, aliquoted, and stored at 4°C.

[0017] Step 6: Phosphorylate the purified AANAT2 protein and perform in vitro binding with three subtypes (γ1, γ2, and ζ) of 14-3-3.

[0018] Before binding to AANAT2, the 14-3-3 protein was pretreated. Specifically, the engineering strains expressing 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ proteins were respectively induced to express and ferment. After recovering the bacterial cells, they were ultrasonically disrupted on ice at a power of 350 watts, centrifuged to remove the precipitate, and the supernatant containing the target protein was reserved for use.

[0019] Furthermore, the in vitro binding technique between AANAT2 and the 14-3-3 protein adopted the glutathione-S-transferase fusion protein precipitation operation technique (GST pull-down), including magnetic bead preparation and equilibration, magnetic bead binding to the target protein, washing the magnetic beads to remove impurities, binding of the target protein-magnetic bead complex to the target protein, washing the magnetic beads again to remove impurities, and eluting the target protein.

[0020] Step 7: Perform Western blotting on the eluate and magnetic beads respectively. This includes preparing the SDS-PAGE electrophoresis gel, denaturing and electrophoresing the samples, blotting and transferring the samples to the membrane, and immunological detection. The experimental operation procedure of Western blotting refers to "Protein Electrophoresis Experimental Techniques" written by Guo Yaojun.

[0021] Step 8: Measure the relative gray values of the Western blotting bands and compare the binding abilities. Further, after completing the binding experiments between pAANAT2 and each subtype of 14-3-3, based on the obtained Western blotting detection results, use the software GelQuantNET (Version 1.8.2) to measure the relative gray values of each band and calculate the pAANAT2 / 14-3-3 ratio, and compare the differences in the binding abilities of each subtype of 14-3-3 to AANAT according to the gray values.

[0022] In the technical solution of this application, the GST pull-down experiment is a method for studying protein interactions in vitro, mainly used to verify whether two proteins can interact with each other or screen for unknown proteins that interact with known proteins. Its basic principle is to fuse the target protein with a GST (Glutathione-S-transferase) tag, affinity-solidify it on magnetic beads labeled with glutathione, and then use it as a support to affinity with the target protein. As a "bait protein", it is incubated with a solution containing the target protein, and the "capture protein" (target protein) that can interact with it can be captured. After eluting the binding complex with the eluent, SDS-PAGE electrophoresis analysis is performed to confirm whether the two proteins can interact with each other or screen for the corresponding target protein. Both the "bait protein" and the "capture protein" can be obtained by various methods such as cell crude lysates, purified proteins, expression systems, and in vitro expression systems.

[0023] Beneficial effects

[0024] The protein complex prepared by the present invention has the advantages of easy purification, good stability, high yield, low cost, etc., provides guidance for the further research and development of small peptide drugs that regulate AANAT activity and thus improve sleep, and can be used as a protein candidate for the research and development of small peptide drugs for improving sleep.

[0025] The present invention prepared expression engineering bacteria of aanat2-pCzn1-BL21, 14-3-3γ1-pGEX-4T1-BL21, 14-3-3γ2-pGEX-4T-1-BL21 and 14-3-3ζ-pGEX-4T-1-BL21. By culturing the above strains, a variety of proteins related to biological rhythm regulation can be obtained.

[0026] The present invention compared the differences in the binding ability of three subtypes of 14-3-3 to pAANAT. Multiple repeated experiments showed that whether it was the identification result of the pAANAT / 14-3-3 complex in the eluate or on the magnetic beads, the binding ability of 14-3-3γ1 and γ2 to pAANAT2 was stronger than that of ζ. Generally speaking, the binding ability of 14-3-3γ (whether γ1 or γ2) to pAANAT2 was about twice that of 14-3-3ζ. It provides a strong reference for further in-depth exploration of the differences in the binding ability of other subtypes of 14-3-3 to pAANAT. Brief Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is a schematic diagram of the construction of the aanat2-pCzn1 expression vector of the present invention.

[0029] Figure 2 It is a restriction enzyme digestion identification result diagram of the aanat2-pCzn1 recombinant expression plasmid of the present invention. In the figure, M represents Marker, Line1 is the plasmid before enzyme digestion, and Line 2 is the plasmid after enzyme digestion.

[0030] Figure 3 It is a sequence splicing and partial sequencing result diagram of the aanat2-pCzn1 clone verification of the present invention; the underlined sequence is the coding region of the aanat2 gene, and the gray-marked sequence is the restriction enzyme site.

[0031] Figure 4Schematic diagram of the construction of the 14-3-3-pGEX4T-1 expression vector of the present invention; among which (A) is the vector carrying the 14-3-3γ1 gene; (B) is the vector carrying the 14-3-3γ2 gene; (C) is the vector carrying the 14-3-3ζ gene.

[0032] Figure 5 Restriction enzyme digestion identification result diagram of the 14-3-3-pGEX4T-1 recombinant expression plasmid of the present invention; among which (A), (B), and (C) are the restriction enzyme digestion identification result diagrams of 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 respectively. In the figure, M represents Marker, Line 1 is the plasmid before digestion, and Line 2 is the plasmid after digestion.

[0033] Figure 6 Sequence splicing and partial sequencing result diagram of the 14-3-3-pGEX4T-1 clone verification of the present invention; among which (A), (B), and (C) are the sequence splicing and partial sequencing result diagrams of 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 respectively; the underlined sequences are the coding regions of the 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ genes respectively, and the gray-marked sequences are the restriction enzyme sites.

[0034] Figure 7 Expression result diagram of the AANAT2, 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ proteins of the present invention; among which, M is the protein marker: 10, 15, 25, 35, 40, 50, 70, 100, 140, 160 kDa (from bottom to top), C is the negative control, and the arrow indicates the target protein band. 1-3 are the expression conditions of AANAT2, 4-6 are 14-3-3ζ, 7-9 are 14-3-3γ1, and 10-12 are 14-3-3γ2 at 37°C; 13 is AANAT2, 14 is 14-3-3ζ, 15 is 14-3-3γ1, and 16 is 14-3-3γ2 at 16°C.

[0035] Figure 8 Protein immunoblotting result diagram of the pAANAT2 / 14-3-3 binding complex of the present invention; among which, A is the identification result of the eluate, and B is the direct electrophoresis identification result of the binding magnetic beads.

[0036] Figure 9 Model for comparing the binding and activation differences between the dimers (14-3-3γ)2 and (14-3-3ζ)2 and phosphorylated AANAT (pAANAT). Detailed implementation methods

[0037] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0038] The present invention will be further described below with reference to the embodiments.

[0039] Example 1 Collection and Selection of Zebrafish aanat and 14-3-3 Gene Family Sequences

[0040] In the present invention, we first collected relevant data of two reported aanat genes (aanat1a and aanat2) and 11 14-3-3 genes in zebrafish through the GenBank database (see Table 1).

[0041] Table 1 Information on Members of Zebrafish aanat and 14-3-3 Gene Families

[0042]

[0043]

[0044] The zebrafish genome version number: GRCz11 was used as a reference for searching the upstream and downstream regions of 14-3-3. In addition, it was found that AANAT1a is mainly related to visual function (dopamine acetylation), while AANAT2 is mainly expressed in the pineal gland and is more closely related to melatonin synthesis. In the present invention, AANAT2 was selected as the representative of the AANAT gene family for further construction of expression vectors and synthesis and purification of proteins. And 14-3-3, as a chaperone protein, binds to phosphorylated AANAT (pAANAT).

[0045] Figure 9It is a differential comparison model for the binding and activation of the dimers (14-3-3γ)2 and (14-3-3ζ)2 with phosphorylated AANAT (pAANAT). It can be seen that one (14-3-3ζ)2 can bind one pAANAT molecule (A), while one (14-3-3γ)2 can bind two pAANAT molecules (B). Their binding capacities differ by nearly 2-fold, which is convenient for further studying the differences in binding capacities. When a mutation occurs at one site (pT31 or pS205), the activation of (14-3-3)2 on pAANAT will be eliminated (the upper right two in A and B); only when both sites bind to (14-3-3)2, (14-3-3)2 shows complete activation of pAANAT in the complex. Therefore, in the present invention, for the 14-3-3 family, we select the γ and ζ subtypes of 14-3-3 as representatives to ensure that each gene is highly conserved and representative across species, and further carry out the construction of expression vectors and the synthesis and purification of proteins.

[0046] Example 2 Construction of Prokaryotic Expression Vector of Zebrafish aanat2 Gene

[0047] According to the reported zebrafish aanat2 gene sequence collected in GenBank, the corresponding amino acid sequence of the aanat2 gene was optimized using the codon online optimization website (http: / / www.jcat.de). The strain was selected as Escherichia coli K12 to obtain the gene sequence without a signal peptide. By using the method of overlapping PCR, full-length splicing primers were designed, and the target gene aanat2 was synthesized and ligated into the multiple cloning sites of the vector pCzn1; the obtained recombinant plasmid aanat2-pCzn1 was transferred into the DH5α cloning engineering strain, and positive clones were screened and sent for sequencing.

[0048] The specific scheme for constructing the prokaryotic expression vector of the aanat2 gene is as follows:

[0049] After translating the CDS sequence of the aanat2 gene into amino acids, the calculated protein molecular weight was 23.588 kd and the isoelectric point was 7.71. After codon optimization according to the prokaryotic protein expression system, it was constructed between the endonucleases NdeI (restriction site: CATATG) and XbaI (restriction site: TCTAGA) of the pCzn1 vector, fused with the N-His tag, and induced to express with IPTG. The vector construction used NdeI + optimized target sequence + stop codon + XbaI ( Figure 1 ). The enzyme digestion identification system is shown in Table 2. After loading the samples, gently mix them, centrifuge instantaneously for 5 - 10 seconds, react at 37 °C for 30 minutes, and the enzyme digestion results are shown in Figure 2 . The splicing result of the sequencing sequence of aanat2-pCzn1 clone verification and the comparison result of the intercepted partial sequence are shown in Figure 3。

[0050] Table 2 Enzyme digestion reaction system and reaction program of aanat2 gene

[0051]

[0052] From Figure 2 It can be seen that after enzyme digestion, two fragments with lengths of approximately 4.4 kb and 633 bp were obtained respectively, which were consistent with the expected sizes, indicating that the target gene had been successfully inserted into the expression vector.

[0053] Figure 3 The underlined sequence in the following is the coding region of the aanat2 gene, and the grayed-out sequence is the enzyme digestion site.

[0054] Example 3 Construction of prokaryotic expression vectors for zebrafish 14-3-3 (γ and ζ subtypes) genes

[0055] After codon optimization of the amino acid sequences corresponding to the 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ genes online, the full genes were synthesized (codon online optimization website: http: / / www.jcat.de, and the strain was selected as Escherichia coli K12 to obtain the gene sequence without signal peptide. Using the method of overlapping PCR, full-length splicing primers were designed, and the target genes 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ were synthesized and ligated into the multiple cloning site of vector pGEX-4T-1. The obtained recombinant plasmids 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 were transferred into the DH5α cloning engineering strain, and positive clones were screened and sent for sequencing.

[0056] The specific scheme for the construction of prokaryotic expression vectors for 14-3-3 (γ and ζ subtypes) genes is as follows:

[0057] After translating the CDS sequences of the 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ genes into amino acids, the protein molecular weights were calculated. 14-3-3γ1 was 28.234 kD, 14-3-3γ2 was 28.308 kD, and 14-3-3ζ was 27.962 kD. The isoelectric points of 14-3-3γ1 was 4.78, 14-3-3γ2 was 4.78, and 14-3-3ζ was 4.61. After codon optimization of the above genes according to the prokaryotic protein expression system, they were constructed between the endonucleases EcoR I and Xho I of vector pGEX-4T-1, fused with the N-GST tag, and induced to express by IPTG. The vector construction adopted the scheme of EcoR I + target gene + TAA + Xho I (see Figure 4 ). The enzyme digestion identification system is shown in Table 3. After loading the samples, gently mix them, centrifuge instantaneously for 5 - 10 seconds, and react at 37°C for 30 minutes. The enzyme digestion results are shown inFigure 5 。The splicing results of the sequencing sequences verified by 14-3-3-pGEX-4T-1 cloning and the comparison results of the intercepted partial sequences are shown in Figure 6 。

[0058] Table 3 Restriction Enzyme Reaction System and Reaction Program for 14-3-3 Gene

[0059]

[0060] It can be seen from Figure 5 that after digestion, two fragments with lengths of about 4.9 kbp and 750 bp were obtained from 14-3-3γ1-pGEX-4T-1, two fragments with lengths of about 4.9 kbp and 750 bp were obtained from 14-3-3γ2-pGEX-4T-1, and two fragments with lengths of about 4.9 kbp and 741 bp were obtained from 14-3-3ζ-pGEX-4T-1. The target bands were single and specific, and conformed to the expected size, indicating that the target gene had been successfully inserted into the expression vector.

[0061] Example 4 Expression of Recombinant Protein and Determination of Recombinant Protein Expression Form

[0062] Inoculate the correctly sequenced aanat2-pCzn1, 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1 and 14-3-3ζ-pGEX-4T-1 monoclonal colonies in LB broth medium and culture them at 37°C, 200 rpm and 16°C, 200 rpm respectively. When inducing expression at 37°C, expand the culture at a ratio of 1:200 by volume, culture at 200 rpm until the OD600 value of the bacterial liquid reaches 0.6 - 0.8 in the logarithmic growth phase. Take 500 ml of the bacterial liquid as the negative control, add IPTG to a final concentration of 0.5 mM in the remaining bacterial liquid, continue to culture for about 4 h, and take 200 ml as the sample after induction. When inducing expression at 16°C, expand the culture at a ratio of 1:100 by volume, culture at 200 rpm until the OD600 of the bacterial liquid is 0.6 - 0.8. Take 1.0 mL as the negative control. Add IPTG to a final concentration of 0.5 mM in the remaining bacterial liquid, adjust the temperature to 16°C and continue to culture overnight (16 h), and take 1.0 mL as the sample after induction. Pipette a small amount of the bacterial liquid, centrifuge at 12000 rpm for 2 min, wash the bacterial cell precipitate twice with PBS, add the loading buffer, boil at 100°C for 10 min, then centrifuge at 12000 rpm for 1 min, and analyze the protein expression by 15% SDS-PAGE electrophoresis. The results showed that under the conditions of 37°C and 16°C, effective expression engineering strains were obtained for AANAT2, 14-3-3ζ, 14-3-3γ1 and 14-3-3γ2 (see Figure 7 ).

[0063] To further determine the expression form of the recombinant protein, the bacterial culture solution before adding IPTG was set as the negative control, and the positive monoclonal aanat2-pCzn1, 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 with BL2 as the expression host bacterium were cultured under the above conditions. 1.0 mL of bacterial liquid cells were collected, the precipitate was resuspended after lysis, sonicated on ice, centrifuged at 12,000 rpm for 15 min, and the supernatant was sampled; the precipitate was resuspended with the same amount of lysis buffer and then sampled for SDS-PAGE electrophoresis to analyze the expression of each protein.

[0064] The results showed that after IPTG induction, the supernatant and precipitate were identified and analyzed by SDS-PAGE electrophoresis. Obvious high-level expression of exogenous protein bands appeared at about 23 kD in the precipitate of the positive recombinant aanat2-pCzn1-BL21 bacteria under the conditions of 16 °C and 37 °C, but not in the supernatant, indicating that the AANAT2 protein mainly exists in the form of inclusion bodies. For 14-3-3ζ, 14-3-3γ1, and 14-3-3γ2, the target proteins were expressed in both the supernatant and the precipitate regardless of whether the induction was carried out at 16 °C or 37 °C.

[0065] Example 5 Purification and Condition Optimization of AANAT2 Protein

[0066] The AANAT2 bacterial cells were resuspended in the binding / equilibration buffer (50 mmol / L NaH2PO4, 300 mmol / L NaCl, 10 mmol / L imidazole, pH 8.0), sonicated to break the bacterial cells, and centrifuged at 12,000 rpm for 15 min; the precipitate was dissolved in the binding / equilibration buffer containing 8 M urea and centrifuged at high speed. The centrifuged supernatant was filtered through a 0.45 μm filter, and the filtrate was bound to a Ni 2+ column pretreated with the corresponding binding / equilibration buffer; the column was rinsed with 3 column volumes of the washing buffer (50 mmol / L NaH2PO4, 300 mmol / L NaCl, 100 mmol / L imidazole, pH 8.0); the target protein was eluted with different concentrations of imidazole (150, 200, 250, 300, 500 mmol / L; pH 8.0), the eluate was collected, and the eluate was analyzed by SDS-PAGE electrophoresis. The recombinant protein AANAT2 purified by the Ni 2+ column showed the best elution effect at a 500 mM imidazole concentration as analyzed by SDS-PAGE.

[0067] Inclusion body protein renaturation: After analysis by affinity chromatography purification in the previous step, a sample with relatively high protein purity and concentration was collected. It was added to a treated protein dialysis bag and slowly dialyzed at 4°C into a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 4 mM GSH, 0.4 mM GSSG, and 0.4 M L-Arginine for renaturation. After renaturation, the AANAT2 protein was finally dialyzed into a storage solution of 50 mM Tris pH 8.0, 150 mM NaCl, and 10% Glycerol solution for about 6 - 8 h. After the dialysis renaturation was completed, the supernatant was filtered through a 0.22 μm filter, aliquoted, and stored at 4°C.

[0068] Example 6 In vitro binding of AANAT2 to 14-3-3

[0069] First, the purified AANAT2 was phosphorylated. The phosphorylation reaction system is shown in Table 4, and the reaction condition was incubation at 25°C for 1 hour.

[0070] Table 4 AANAT2 phosphorylation reaction system

[0071]

[0072] The phosphorylated AANAT2 (pAANAT2) was subjected to a glutathione-S-transferase fusion protein precipitation (GST-Pull down) with 14-3-3 for an in vitro binding experiment. Before binding, the 14-3-3 protein was pretreated. Specifically: The engineering strains expressing 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ proteins were induced to express and ferment respectively. After recovering the bacterial cells, they were ultrasonically disrupted on ice at a power of 350 watts, centrifuged to remove the precipitate, and the supernatant containing the target protein was reserved for use.

[0073] The operation process of glutathione-S-transferase fusion protein precipitation includes:

[0074] 1) Prepare magnetic beads

[0075] According to the purification requirements, appropriate amounts of GST purification magnetic beads (the magnetic beads account for 10% of the total volume) were aspirated, the magnetic beads were inverted and mixed evenly, and an appropriate amount of the magnetic bead suspension was aspirated with a pipette and added to a centrifuge tube. Then the centrifuge tube was inserted into the hole of the magnetic separator and left standing for about 1 min. After the solution became clear and transparent, the supernatant was aspirated and discarded with a pipette tip.

[0076] 2) Equilibrate the magnetic beads

[0077] Remove the centrifuge tube from the magnetic separator, add binding buffer with the same volume as the original suspension, pipette up and down 5 - 10 times using a pipette tip, and place the centrifuge tube in the hole of the magnetic separator for about 1 min. After the solution becomes clear and transparent, aspirate and discard the supernatant using a pipette tip. Repeat the washing operation 2 times.

[0078] 3) Binding of magnetic beads to target protein

[0079] Add the target protein sample or lysate containing GST tag to the freshly prepared magnetic beads and mix well.

[0080] Place the centrifuge tube on a mixer and incubate at room temperature for about 30 min.

[0081] 4) Washing of magnetic beads

[0082] Place the centrifuge tube in the hole of the magnetic separator and let it stand for about 1 min. After the solution becomes clear and transparent, aspirate the supernatant using a pipette tip and retain the sample for detection. Add washing buffer with 2 - 3 times the volume of the original suspension to the centrifuge tube, pipette up and down 5 - 10 times using a pipette tip, place the centrifuge tube in the hole of the magnetic separator, let it stand for about 1 min. After the solution becomes clear and transparent, aspirate and discard the supernatant using a pipette tip. Repeat the above steps 2 times to obtain the target protein - magnetic bead complex.

[0083] 5) Binding of target protein - magnetic bead complex to the target protein:

[0084] Add the sample containing the target protein to the prepared target protein - magnetic bead complex and mix well. Place the centrifuge tube on a mixer and incubate at room temperature for 30 min.

[0085] 6) Washing of magnetic beads again

[0086] Place the centrifuge tube in the hole of the magnetic separator for about 1 min. After the solution becomes clear and transparent, aspirate the supernatant using a pipette tip and retain the sample for measurement. Add washing buffer with about 2 times the volume of the original suspension to the centrifuge tube, pipette up and down 5 - 10 times using a pipette tip, place the centrifuge tube in the hole of the magnetic separator, let it stand for about 1 min. After the solution becomes clear and transparent, aspirate and discard the supernatant using a pipette tip. Repeat the above steps 2 times. After the target protein binds to the target protein, it is captured by the magnetic beads from the mixed system.

[0087] 7) Elution of the target protein:

[0088] Add an elution buffer with the same volume as the original suspension to the above centrifuge tube, and pipette about 5 times. Then place it on a vertical mixer at room temperature or gently invert the centrifuge tube by hand. After about 5 - 10 minutes, let it stand in the hole of the magnetic separator for about 1 minute. When the solution becomes clear and transparent, use a pipette to aspirate the supernatant and collect the eluted fraction to obtain the complex of the target protein and the desired protein.

[0089] To obtain more samples, repeat the elution operation once, collect the eluted fractions separately, and save them for subsequent detection.

[0090] Example 7 Identification by Western Blot and Comparison of the Binding Ability of pAANAT2 / 14 - 3 - 3

[0091] After completing the binding experiments of pAANAT2 and each subtype of 14 - 3 - 3, perform Western Blot on the samples. The operation procedure of the Western Blot experiment refers to "Protein Electrophoresis Experimental Techniques" written by Guo Yaojun. The results of the Western Blot detection of the samples are as Figure 8 shown.

[0092] Further use the software GelQuantNET Version 1.8.2 to measure the relative gray values of each band and calculate the pAANAT2 / 14 - 3 - 3 ratio as shown in Table 5.

[0093] Table 5 Western Blot Gray Value Ratio of the pAANAT2 / 14 - 3 - 3 Binding Complex Note: The numerical values of the eluate identification results correspond one - to - one with the Figure 8 sequence numbers of Band A; the numerical values of the direct electrophoresis identification results of the magnetic beads correspond one - to - one with the Figure 8 sequence numbers of Band B.

[0094] The results in Table 5 and Figure 8 show that the 14 - 3 - 3γ subtype has a stronger ability to bind AANAT2 than the 14 - 3 - 3ζ subtype.

[0095] The present invention only shows the representative results of one experiment. Multiple repeated experiments all show that whether it is the identification results of the pAANAT / 14 - 3 - 3 complex in the eluate or on the magnetic beads, the 14 - 3 - 3γ1 and γ2 have a stronger ability to bind pAANAT2 than ζ. Generally speaking, the ability of 14 - 3 - 3γ (whether γ1 or γ2) to bind pAANAT2 is about twice that of 14 - 3 - 3ζ.

[0096] The protein complex prepared by the present invention has the advantages of easy purification, good stability, high yield, low cost, etc., provides guidance for the further research and development of small peptide drugs that regulate AANAT activity and thus improve sleep, and can be used as a protein candidate for the research and development of small peptide drugs for improving sleep.

[0097] The present invention prepared expression engineering bacteria of aanat2-pCzn1-BL21, 14-3-3γ1-pGEX-4T-1-BL21, 14-3-3γ2-pGEX-4T-1-BL21 and 14-3-3ζ-pGEX-4T-1-BL21. By culturing the above strains, various proteins related to biological rhythm regulation can be obtained.

[0098] The present invention compared the differences in the binding ability of three subtypes of 14-3-3 to pAANAT, providing a strong reference for further in-depth exploration of the differences in the binding ability of other subtypes of 14-3-3 to pAANAT.

[0099] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an AANAT and 14-3-3 protein complex, characterized in that, It includes the following steps: Step 1: Select the aanat2 gene and the 14-3-3 gene related to melatonin synthesis coding, and for the 14-3-3 gene, select the 14-3-3γ1, 14-3-3γ2, and 14-3-3ζ genes for codon optimization; Step 2: Construct prokaryotic expression vectors for the aanat2 gene and the 14-3-3 gene; Construct the aanat2 gene between the multiple cloning sites of the endonucleases NdeI and XbaI of the pCzn1 vector to obtain the aanat2-pCzn1 expression plasmid containing the nucleotide sequence shown in SEQ ID No1; Construct the three subtypes γ1, γ2, and ζ of the 14-3-3 gene between the multiple cloning sites of the endonucleases EcoRI and XhoI of the pGEX-4T-1 vector to obtain the 14-3-3γ1-pGEX-4T-1, 14-3-3γ2-pGEX-4T-1, and 14-3-3ζ-pGEX-4T-1 expression plasmids containing the nucleotide sequences shown in SEQ ID No2, SEQ ID No3, and SEQ ID No4 respectively; Step 3: Recombinant protein expression to obtain AANAT2 protein and 14-3-3 protein; Step 4: Purify the AANAT2 protein and include the complex; Step 5: Phosphorylate the AANAT2 protein to obtain the pAANAT2 protein and perform in vitro binding with the 14-3-3 protein.

2. The preparation method of the AANAT and 14-3-3 protein complex according to claim 1, wherein In Step 3, transfer the obtained expression plasmids into the DH5α cloning engineering bacteria respectively. After sequencing verification is correct, transfer the expression plasmids into the BL21 expression strain to obtain the expression engineering bacteria of aanat2-pCzn1-BL21, 14-3-3γ1-pGEX-4T-1-BL21, 14-3-3γ2-pGEX-4T-1-BL21, and 14-3-3ζ-pGEX-4T-1-BL21, and use IPTG to induce the expression of the target protein.

3. The preparation method of the AANAT and 14-3-3 protein complex according to claim 2, characterized in that, In step 4, the AANAT2 protein purification steps are as follows: The AANAT2 bacterial cells are resuspended in a buffer to precipitate, the bacterial cells are sonicated, centrifuged, the precipitate is dissolved in a buffer containing urea and centrifuged at high speed, the centrifuged supernatant is filtered, and the filtrate is bound to a Ni 2+ column pre-treated with the corresponding buffer; washed with the washing buffer for 3 column volumes; the target protein is eluted with imidazole, and the eluate is collected.

4. The method for preparing the AANAT and 14-3-3 protein complex according to claim 3, wherein In Step 4, perform inclusion body renaturation on the AANAT protein that cannot be solubly expressed in the supernatant: Add the AANAT2 protein into a protein dialysis bag and slowly dialyze it into the buffer for renaturation. After renaturation, the AANAT2 protein is finally dialyzed into the storage solution. After the dialysis renaturation is completed, filter to obtain the supernatant.

5. The preparation method of the AANAT and 14-3-3 protein complex according to claim 4, characterized in that, In Step 5, perform in vitro binding of the phosphorylated AANAT2 protein and the 14-3-3 protein through the glutathione-S-transferase fusion protein precipitation technique.

6. The method for preparing the AANAT and 14-3-3 protein complex according to claim 5, wherein Before Step 1, it also includes the collection of zebrafish aanat gene and 14-3-3 gene family sequences: Collect the data of the reported aanat and 14-3-3 gene families in zebrafish through the GenBank and Ensembl databases.

7. The method for preparing the AANAT and 14-3-3 protein complex according to claim 6, wherein It also includes Step 6: Perform Western blot on the eluate and magnetic beads respectively.

8. The method for preparing the AANAT and 14-3-3 protein complex according to claim 7, characterized in that, It also includes Step 7: Based on the obtained Western blot detection results, measure the relative gray values of each band and calculate the gray value ratio of the pAANAT2 protein / 14-3-3 protein, and compare the differences in the binding ability of each subtype of the 14-3-3 protein and the AANAT protein.

9. An AANAT and 14-3-3 protein complex prepared by the preparation method of the AANAT and 14-3-3 protein complex according to any one of claims 1 to 8.

10. Use of an AANAT and 14-3-3 protein complex according to any one of claims 1 to 8 in the preparation of a drug for improving sleep.

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