A histone acetyltransferase mutant and its application in the preparation of biomacromolecules

Abstract

The present invention provides a histone acetyltransferase mutant and its application in the preparation of biomacromolecules. Based on wild-type histone acetyltransferase, the present invention obtains a histone acetyltransferase mutant by site-directed mutagenesis, and verifies that the histone acetyltransferase mutant can further improve the vitality and lifespan of fish sperm compared with wild-type histone acetyltransferase. At the same time, the histone acetyltransferase mutant is used to treat fish testis, and PDRN is prepared after inducing testis cell maturation. The prepared PDRN has a higher extraction rate and purity than no treatment or treatment with wild-type histone acetyltransferase. The histone acetyltransferase mutant of the present invention provides a new idea for improving the vitality and lifespan of fish sperm and industrial preparation and production of PDRN, showing a wide range of application potential.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a histone acetyltransferase mutant and application thereof in the preparation of biological macromolecules. Background Art

[0002] Histone acetyltransferase (MOF), also known as KAT8 or MYST1, is a member of the histone acetyltransferase MYST family. It can specifically acetylate the 16th lysine residue (H4K16ac) of histone H4 and is an important regulatory factor for embryonic development and transcriptional activation. MOF plays a vital role in many physiological activities. It loosens the chromosome structure through acetylation, allowing various transcription factors to bind to their target genes and activate their transcription, thereby playing an important role in cell proliferation, differentiation, DNA replication, damage and repair. During spermatogenesis, histone acetylation regulates the development of spermatogenic cells in a specific way. Abnormal histone acetylation modification can cause abnormal spermatogenesis in male reproductive organs and cause spermatogenesis disorders. Studies have shown that the acetylation level of histone H4 in sperm cells of infertile men is significantly reduced.

[0003] PDRN (polydeoxyribonucleotide), a polydeoxyribonucleotide, is a long chain composed of two deoxypentyl ribose and phosphate molecules. It is a double-helix biomacromolecule formed by AT and GC base pairing. The long chain of pentose phosphate is arranged on the outside of the molecule, and the AT and CG base pairs are arranged on the inside. In 1952, the Italian Mastelli company extracted 50-500bp polydeoxyribonucleotides (PDRN) from salmon testicular cells based on the phenomenon that fishermen used salmon testes to treat wounds, and conducted relevant studies on its pharmacological activity. The results showed that 50-500bp PDRN from male salmon testicular cells significantly inhibited the synthesis of inflammatory factors by activating A2A receptors, increased cell activity, promoted tissue wound healing and effectively reduced scar formation. With the widespread application of PDRN in clinical practice, it has shown good effects in promoting angiogenesis, improving microcirculation, anti-embolism, and treating diabetic foot. Studies have shown that the base composition of salmon PDRN is similar to that of human DNA, and it will not cause allergic reactions after entering the human body. Toxicological studies have shown that within the physiological dose range, different administration methods will not cause the death of mice, and no pathological changes were found in tissues such as the liver, brain, lungs, skeletal muscle and heart. The quality of PDRN is determined by its raw materials, and high-quality PDRN comes from salmon semen or testis. However, due to the uneven quality of natural raw materials such as salmon semen or testis, it is very likely that ① the sperm quality in the semen does not meet the standards, including insufficient quantity and immature sperm; ② histones are tightly bound to nucleic acids and difficult to separate, resulting in the inability to obtain high-purity, high-activity and high-extraction rate PDRN using traditional extraction methods. Therefore, how to overcome the problems of the above-mentioned natural raw materials through technical means to improve the preparation quality and efficiency of PDRN is a core issue that needs to be solved urgently. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a histone acetyltransferase mutant and its use in improving fish sperm motility and / or lifespan and preparing PDRN.

[0005] In a first aspect of the present invention, a histone acetyltransferase (MOF) mutant is provided, wherein the amino acid sequence of the histone acetyltransferase (MOF) mutant is obtained by mutations A31V, D82V and R98K on the basis of the amino acid sequence of the wild-type histone acetyltransferase (MOF).

[0006] Further preferably, the amino acid sequence of the wild-type histone acetyltransferase (MOF) is numbered AAH37773.1 in GeneBank, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Further preferably, the amino acid sequence of the histone acetyltransferase (MOF) mutant is as shown in SEQ ID NO.2.

[0008] In the second aspect of the present invention, a biomaterial related to the histone acetyltransferase (MOF) mutant described in the first aspect of the present invention is provided, wherein the biomaterial comprises at least one of the following 1)-5):

[0009] 1) A gene encoding the histone acetyltransferase (MOF) mutant according to the first aspect of the present invention;

[0010] 2) an expression cassette containing the coding gene described in 1);

[0011] 3) a recombinant vector containing the coding gene described in 1);

[0012] 4) A recombinant microorganism or recombinant cell containing the coding gene described in 1);

[0013] 5) A recombinant microorganism or recombinant cell containing the recombinant vector described in 3).

[0014] Further preferably, in said 1), the nucleotide sequence of the coding gene is as shown in SEQ ID NO.4;

[0015] Further preferably, in said 3), the recombinant vector uses pSK1 vector as an expression vector;

[0016] Further preferably, in 4) or 5), the recombinant microorganism is recombinant Escherichia coli DH5α;

[0017] Further preferably, in 4) or 5), the recombinant cell is a recombinant animal or human cell;

[0018] More preferably, in 4) or 5), the recombinant cell is a recombinant HEK293 cell.

[0019] In the third aspect of the present invention, there is provided the use of the histone acetyltransferase (MOF) mutant described in the first aspect of the present invention in improving the sperm motility and / or lifespan of fish, or in the preparation of a product for improving the sperm motility and / or lifespan of fish.

[0020] Further preferably, the fish is salmonids;

[0021] More preferably, the salmonid fish is rainbow trout, trout, Taimen or salmon;

[0022] More preferably, the salmonid fish is salmon.

[0023] In the fourth aspect of the present invention, provided is the use of the histone acetyltransferase (MOF) mutant described in the first aspect of the present invention in the preparation of PDRN.

[0024] In the fifth aspect of the present invention, a method for preparing PDRN is provided, the method comprising the following steps: treating fish testis with the histone acetyltransferase (MOF) mutant described in the first aspect of the present invention to induce testis cell maturation and then prepare PDRN.

[0025] Further preferably, the fish is salmonids;

[0026] More preferably, the salmonid fish is rainbow trout, trout, Taimen or salmon;

[0027] More preferably, the salmonid fish is salmon.

[0028] Further preferably, the concentration of the histone acetyltransferase (MOF) mutant is 0.1-10 mg / mL;

[0029] More preferably, the concentration of the histone acetyltransferase (MOF) mutant is 0.5-5 mg / mL;

[0030] More preferably, the concentration of the histone acetyltransferase (MOF) mutant is 1 mg / mL.

[0031] Further preferably, the treatment time of the histone acetyltransferase (MOF) mutant is 0.5-5h;

[0032] More preferably, the treatment time of the histone acetyltransferase (MOF) mutant is 1-2.5 h;

[0033] More preferably, the treatment time of the histone acetyltransferase (MOF) mutant is 1.5 h.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0035] Compared with the prior art, the present invention has the following advantages and progress:

[0036] The present invention is based on wild-type histone acetyltransferase, and a histone acetyltransferase mutant is obtained by site-directed mutagenesis, and it is verified that the histone acetyltransferase mutant can further improve the vitality and lifespan of fish sperm compared to wild-type histone acetyltransferase. At the same time, the histone acetyltransferase mutant is used to treat fish testis, and PDRN is prepared after inducing testicular cell maturation. The prepared PDRN has a higher extraction rate and purity than not being treated or being treated with wild-type histone acetyltransferase. The histone acetyltransferase mutant of the present invention provides a new idea for improving the vitality and lifespan of fish sperm and industrial preparation and production of PDRN, showing a wide range of application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 : Western-blot analysis results of wild-type histone acetyltransferase and histone acetyltransferase mutant (unit: KDa). M: protein marker; 1: wild-type histone acetyltransferase; 2: histone acetyltransferase mutant.

[0039] Figure 2 :The effects of different treatments on fish sperm motility. In the figure, * indicates that compared with the normal control group, **p<0.01, there is a very significant difference compared with the normal group; # indicates that compared with the wild-type MOF group, ##p<0.01, there is a very significant difference compared with the wild-type MOF group.

[0040] Figure 3 :The effects of different treatments on the life span of fish sperm. In the figure, * indicates that compared with the normal control group, **p<0.01, there is a very significant difference compared with the normal group; # indicates that compared with the wild-type MOF group, ##p<0.01, there is a very significant difference compared with the wild-type MOF group. DETAILED DESCRIPTION

[0041] The present invention provides a histone acetyltransferase mutant and its application in the preparation of biomacromolecules. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and are carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0042] Example 1 Construction of wild-type histone acetyltransferase and histone acetyltransferase mutant recombinant expression plasmids and their expression in eukaryotic cells

[0043] According to the gene sequences of wild-type histone acetyltransferase and histone acetyltransferase mutant (SEQID NO.2, SEQ ID NO.4), XbaⅠ and Hind Ⅲ restriction sites were added at both ends respectively, and then synthesized by a gene company according to the method of chemical total synthesis. Then, they were connected with the pSK1 plasmid linearized by double enzyme digestion to construct the recombinant expression plasmid pSK1-MOF of wild-type histone acetyltransferase and the recombinant expression plasmid pSK1-MOFm of histone acetyltransferase mutant. DH5α competent bacteria were conventionally transformed and amplified. After the plasmid was extracted, it was verified by sequencing that it was completely correct.

[0044] The pSK1-MOF, pSK1-MOFm plasmids and pAdTrack-CMV plasmids were double-digested with XbaⅠ and Hind Ⅲ, respectively, and electroporated and amplified in DH10B competent bacteria to construct pAdTrack-CMV-MOF and pAdTrack-CMV-MOFm. After sequencing confirmation, the above plasmids were linearized with PmeⅠ and homologously recombined with pAdEasy-1 in Escherichia coli BJ5183 to form pAdEasy-MOF and pAdEasy-MOFm. Using the transfection kit Lipofectamine2000, the pAdEasy-MOF and pAdEasy-MOFm plasmids linearized with PacⅠ were introduced into HEK293 cells, respectively, and observed under a fluorescence microscope 7-15 days after transfection. Pure virus liquid was obtained by gradient centrifugation, and HEK293 cells were further infected with the virus liquid. After 72 hours, the cells were collected, lysed, centrifuged, and the supernatant was taken. After passing through the chromatography column, active wild-type histone acetyltransferase (MOF) and histone acetyltransferase mutant (MOFm) were obtained. The molecular weight was about 45~65KDa detected by Western-blot, which was consistent with the expected molecular weight, indicating that the wild-type histone acetyltransferase and histone acetyltransferase mutant were successfully expressed in eukaryotic cells ( Figure 1 ).

[0045] Wild-type histone acetyltransferase amino acid sequence:

[0046] MAAQGAAAAVAAGTSGVAGEGEPGPGENAAAEGTAPSPGRVSPPTPARGEPEVTVEIGETYLCRRPDSTWHSAEVIQSRVNDQEGREEFYVHYVGFNRRLDEWVDKNRLALTKTVKDAVQKNSEKYLSELAEQPERKITRNQKRKHDEINHVQKTYAEMDPTTAALEKEHEAITKVKYVDKIHIGNYEIDAWYFSPFPEDYGKQPKLWLCEYCLKYMKYEKSYRFHLGQCQWRQPPGKEIYRKSNISVYEVDGKDHKIYCQNLCLLAKLFLDHKTLYFDVEPFVFYILTEVDRQGAHIVGYFSKEKESPDGNNVACILTLPPYQRRGYGKFLIAFSYELSKLESTVGSPEKPLSDLGKLSYRSYWSWVLLEILRDFRGTLSIKDLSQMTSITQNDIISTLQSLNMVKYWKGQHVICVTPKLVEEHLKSAQYKKPPITGGWGAAVCRGRWGSVSMWTGRSQGLLIAVT (SEQ ID NO.1).

[0047] Wild-type histone acetyltransferase gene DNA sequence:

[0048]

[0049] Histone acetyltransferase mutant amino acid sequence:

[0050] MAAQGAAAAVAAGTSGVAGEGEPGPGENAAVEGTAPSPGRVSPPTPARGEPEVTVEIGETYLCRRPDSTWHSAEVIQSRVNVQEGREEFYVHYVGFNRKLDEWVDKNRLALTKTVKD AVQKNSEKYLSELAEQPERKITRNQKRKHDEINHVQKTYAEMDPTTAALEKEHEAITKVKYVDKIHIGNYEIDAWYFSPFPEDYGKQPKLWLCEYCLKYMKYEKSYRFHLGQCQWRQP PGKEIYRKSNISVYEVDGKDHKIYCQNLCLLAKLFLDHKTLYFDVEPFVFYILTEVDRQGAHIVGYFSKEKESPDGNNVACILTLPPYQRRGYGKFLIAFSYELSKLESTVGSPEKPL SDLGKLSYRSYWSWVLLEILRDFRGTLSIKDLSQMTSITQNDIISTLQSLNMVKYWKGQHVICVTPKLVEEHLKSAQYKKPPITGGWGAAVCRGRWGSVSMWTGRSQGLLIAVT (SEQ ID NO.2).

[0051] Histone acetyltransferase mutant gene DNA sequence:

[0052]

[0053] Example 2 Histone acetyltransferase mutants can improve sperm motility and lifespan in fish

[0054] The immature testes of male salmon aged 1.5 years were selected, gently ground and broken up, and diluted with artificial seminal plasma. The formula of artificial seminal plasma is: NaCl 7.41g, KCl 2.78g, CaCl2•2H2O 0.2g, MgCl2•6H2O0.28g, NaHCO3 0.18g, dissolved in distilled water and fixed to 1L, pH value adjusted to 8.4. The control group did not add any components to the diluent, and the experimental group added histone acetyltransferase mutant or wild-type histone acetyltransferase at a concentration of 1mg / mL to the diluent, stirred well, and placed at 6℃ for 1.5h, and then samples were taken to detect sperm motility and sperm lifespan.

[0055] Sperm motility test results Figure 2 The sperm life span test results are shown in Figure 3 As shown. Figure 2 , Figure 3 It can be seen that histone acetyltransferase mutants can further improve the vitality and lifespan of fish sperm compared with the control group and wild-type histone acetyltransferase.

[0056] Example 3 Preparation of PDRN using histone acetyltransferase mutants

[0057] The immature testes of male salmon aged 1.5 years were selected, gently ground and broken up, and diluted with artificial seminal plasma. The formula of artificial seminal plasma is: NaCl 7.41g, KCl 2.78g, CaCl2•2H2O 0.2g, MgCl2•6H2O0.28g, NaHCO3 0.18g, dissolved in distilled water and fixed to 1L, pH value adjusted to 8.4. The control group did not add any components to the diluent, and the experimental group added histone acetyltransferase mutant or wild-type histone acetyltransferase at a concentration of 1mg / mL to the diluent, stirred well, and placed at 6℃ for 1.5h, and then PDRN was prepared for the control group and the experimental group respectively.

[0058] The preparation method of PDRN is as follows:

[0059] 1) Take samples from the control group and experimental group, centrifuge to obtain 100 g of precipitate, add 600 mL of lysis buffer (0.05 mol / L LEDTA, 1 mol / L NaOH, 1 wt% SDS), invert to mix, and keep at 90°C for 20 min.

[0060] 2) Cool down to 40℃, add 1 mol / L Tris-HCl (pH 8.0) in an amount equal to 1 / 2 of the volume of the reaction system in step 1), and mix by inversion.

[0061] 3) Add 2 mol / L HCl, the volume of which accounts for 1 / 5 of the volume of the reaction system in step 2), and mix by inversion.

[0062] 4) Centrifuge at 6000 rpm for 15 min at 0-4°C and collect the supernatant.

[0063] 5) Use a DNA fragmentor at 6000 J / s for 30 s.

[0064] 6) Add 0.5 mol / L NH4Ac solution, the volume of which accounts for 1 / 10 of the volume of the reaction system in step 5), and then add an equal volume of -20℃ precooled anhydrous ethanol, invert to mix, and place at -20℃ for 2h.

[0065] 7) Centrifuge at 10,000 rpm for 15 min at 0-4°C, discard the supernatant, add 70% ethanol, pipette gently to make the precipitate float, invert gently several times, then centrifuge at 10,000 rpm for 15 min, discard the supernatant, and remove the remaining ethanol with a pipette.

[0066] 8) The precipitate obtained in step 7) is suspended in physiological saline and dried in a vacuum freeze dryer to obtain PDRN.

[0067] Calculation of PDRN purity and extraction rate:

[0068] Extraction rate = PDRN product weight / testis weight;

[0069] The PDRN product was dissolved in physiological saline and the absorbance at 260 nm and 280 nm was measured using a spectrophotometer. The purity of the polydeoxyribonucleotide in the product was calculated according to the following formula: purity = OD260 / OD280.

[0070] The PDRN extraction rate and purity test results are shown in Table 1.

[0071] Table 1 Effects of different treatment conditions on PDRN extraction rate

[0072]

[0073] * indicates that compared with the normal control group, **p<0.01, there is a very significant difference compared with the normal group; # indicates that compared with the wild-type histone acetyltransferase group, ##p<0.01, there is a very significant difference compared with the wild-type histone acetyltransferase group.

[0074] As can be seen from Table 1, the PDRN prepared using the histone acetyltransferase mutant has a significantly better extraction rate and higher purity than the control group and the wild-type histone acetyltransferase group.

[0075] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A histone acetyltransferase mutant, characterized in that: The amino acid sequence of the histone acetyltransferase mutant is shown in SEQ ID NO.

2.

2. A biological material related to the histone acetyltransferase mutant according to claim 1, wherein the biological material is one of the following 1) to 5): 1) A gene encoding the histone acetyltransferase mutant according to claim 1; 2) an expression cassette containing the coding gene described in 1); 3) a recombinant vector containing the coding gene described in 1); 4) A recombinant microorganism containing the coding gene described in 1); 5) A recombinant microorganism containing the recombinant vector described in 3).

3. Use of the histone acetyltransferase mutant according to claim 1 in the preparation of a product for improving the motility and / or lifespan of fish sperm, characterized in that: The fish is salmonids.

4. The use according to claim 3, characterized in that: The salmonid fish is rainbow trout, trout, Taimen or salmon.

5. Use of the histone acetyltransferase mutant according to claim 1 in the preparation of polydeoxyribonucleotides.

6. A method for preparing a polydeoxyribonucleotide, characterized in that: The method comprises the following steps: treating fish testis with the histone acetyltransferase mutant according to claim 1, inducing testis cell maturation and then preparing polydeoxyribonucleotides.

7. The preparation method according to claim 6, characterized in that: The fish is salmonids.

8. The preparation method according to claim 6, characterized in that: The concentration of the histone acetyltransferase mutant is 0.1-10 mg / mL.

9. The preparation method according to claim 6, characterized in that: The treatment time of the histone acetyltransferase mutant is 0.5-5h.

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