A SETDB1 mutant and its application in the preparation of biomacromolecules

Abstract

The present invention is based on wild-type histone methyltransferase (SETDB1), obtains a SETDB1 mutant by site-directed mutagenesis technology, and verifies that the SETDB1 mutant can further improve the number, vitality and survival index of pig sperm compared with wild-type SETDB1 and the control group. At the same time, the SETDB1 mutant is used to treat boar semen / spermatocysts, and PDRN is prepared after inducing sperm cell maturation. The prepared PDRN has a higher extraction rate and purity than no treatment or treatment with wild-type SETDB1. The SETDB1 mutant of the present invention provides a new idea for improving the number, vitality, and survival index of sperm in mammals such as pigs 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 SETDB1 mutant and application thereof in the preparation of biological macromolecules. Background Art

[0002] Histone methylation plays an important role in multiple stages of spermatogenesis. SETDB1, as a histone methyltransferase, can catalyze the formation of monomethylation, dimethylation and trimethylation modifications on the ninth lysine of histone H3. H3K9 methylation is mainly involved in spermatogonial stem cell maintenance, meiosis and histone-protamine replacement. At the same time, the gene expression regulation mediated by H3K9me3 modification is crucial for cell fate determination and individual development. SETDB1, as a H3K9 methylation modification, participates in spermatogonial stem cell maintenance, meiotic prophase transition, meiotic chromosome pairing, synapsis and crossing over. Studies have shown that during germ cell development, knocking out Setdb1 will lead to reduced H3K9me3 levels, DNA damage, spermatocyte apoptosis, and spermatogenesis disorders.

[0003] PDRN (polydeoxyribonucleotide), a polydeoxyribonucleotide, is a long chain composed of two deoxypentylribose 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. Gentium Pharmaceuticals of Italy (acquired by Jazz Pharmaceuticals of the United States) uses porcine intestinal mucosa as raw material to obtain a polydeoxyribonucleotide mixture with pro-fibrinolytic properties. The average molecular weight is 13-20kDa (about 400~550bp). This polydeoxyribonucleotide has significant effects in protecting the liver, anti-inflammation, anti-thrombosis, promoting fibrinolysis, improving microcirculation and anti-ischemia. It was approved for marketing in the European Union in 2013 and approved for marketing in the United States by the FDA in 2016. The drug is mainly used in the treatment of sinusoidal obstruction syndrome (SOS) / veno-occlusive disease (VOD) in clinical practice. Scientific research shows that the genetic similarity between pigs and humans is as high as 97.7% to 98%. Genetic drugs produced with pig intestines as raw materials will not cause allergic reactions or damage the body after entering the human body. The extraction of polydeoxyribonucleotides using pig intestines as raw materials has the disadvantages of cumbersome extraction process, complex purification process, unstable mixture composition, and large molecular weight variation between batches, which seriously affects the quality and efficacy of polydeoxyribonucleotides. Boar semen or seminal vesicles have the characteristics of few impurities and rich nucleic acid content. Using them as raw materials to produce polydeoxyribonucleotides can simplify the extraction process and obtain high-purity products. However, due to the uneven growth and development of pigs, the use of semen or seminal vesicles as raw materials may have problems such as incomplete development of seminal vesicles, lack of or immature sperm cells in semen, and insufficient DNA copies. Therefore, how to overcome the problems of natural raw materials such as pig semen or seminal vesicles 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 shortcomings of the prior art, the object of the present invention is to provide a histone methyltransferase (SETDB1) mutant and its application in improving the sperm count, vitality, survival index of mammals such as pigs and preparing PDRN.

[0005] In a first aspect of the present invention, a SETDB1 mutant is provided, wherein the amino acid sequence of the SETDB1 mutant is obtained by causing V29T, G34S, G225R, V265S and L1218S mutations to occur on the basis of the wild-type SETDB1 amino acid sequence.

[0006] Further preferably, the wild-type SETDB1 amino acid sequence is numbered KAI4082513.1 in GeneBank, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Further preferably, the amino acid sequence of the SETDB1 mutant is as shown in SEQ ID NO.2.

[0008] In the second aspect of the present invention, a biological material related to the SETDB1 mutant described in the first aspect of the present invention is provided, wherein the biological material comprises at least one of the following 1)-5):

[0009] 1) A gene encoding the SETDB1 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 SETDB1 mutant described in the first aspect of the present invention in improving the sperm count, sperm motility and / or sperm survival index of a mammal, or in the preparation of a product that improves the sperm count, sperm motility and / or sperm survival index of a mammal.

[0020] Further preferably, the mammal is a porcine animal;

[0021] More preferably, the porcine animal is Duroc pig, Chenghua pig, Yorkshire pig, Landrace pig, Hampshire pig, Bozhong pig, Mashen pig, Ningxiang Hua pig, Songliao black pig, etc.;

[0022] More preferably, the porcine animal is a Yorkshire pig.

[0023] In the fourth aspect of the present invention, provided is the use of the SETDB1 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 mammalian semen or seminal vesicles with the SETDB1 mutant described in the first aspect of the present invention to induce sperm cell maturation and then prepare PDRN.

[0025] Further preferably, the mammal is a porcine animal;

[0026] More preferably, the porcine animal is Duroc pig, Chenghua pig, Yorkshire pig, Landrace pig, Hampshire pig, Bozhong pig, Mashen pig, Ningxiang Hua pig, Songliao black pig, etc.;

[0027] More preferably, the porcine animal is a Yorkshire pig.

[0028] Further preferably, the concentration of the SETDB1 mutant is 0.1-2 mg / mL;

[0029] More preferably, the concentration of the SETDB1 mutant is 0.5-1 mg / mL;

[0030] More preferably, the concentration of the SETDB1 mutant is 0.8 mg / mL.

[0031] Further preferably, the treatment time of the SETDB1 mutant is 1-24h;

[0032] More preferably, the treatment time of the SETDB1 mutant is 2-12 h;

[0033] More preferably, the treatment time of the SETDB1 mutant is 6 hours.

[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 methyltransferase (SETDB1), obtains a SETDB1 mutant by site-directed mutagenesis technology, and verifies that the SETDB1 mutant can further improve the number, vitality and survival index of pig sperm compared with wild-type SETDB1 and the control group. At the same time, the SETDB1 mutant is used to treat boar semen / spermatocysts, and PDRN is prepared after inducing sperm cell maturation. The prepared PDRN has a higher extraction rate and purity than no treatment or treatment with wild-type SETDB1. The SETDB1 mutant of the present invention provides a new idea for improving the number, vitality, and survival index of sperm in mammals such as pigs 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 SETDB1 and SETDB1 mutants (unit: KDa). M: protein marker; 1: wild-type SETDB1; 2: SETDB1 mutants.

[0039] Figure 2 :The effects of different treatments on the number of pig sperm. In the figure, * indicates that compared with the normal control group, *p<0.05, there is a significant difference compared with the normal group, **p<0.01, there is an extremely significant difference compared with the normal group; # indicates that compared with the wild-type SETDB1 group, #p<0.05, there is a significant difference compared with the wild-type SETDB1 group.

[0040] Figure 3 :Effects of different treatments on the motility of pig sperm. In the figure, * indicates that compared with the normal control group, *p<0.05, there is a significant difference compared with the normal group, **p<0.01, there is an extremely significant difference compared with the normal group; # indicates that compared with the wild-type SETDB1 group, ##p<0.01, there is an extremely significant difference compared with the wild-type SETDB1 group.

[0041] Figure 4 :The effects of different treatments on the survival index of pig sperm. In the figure, * indicates that compared with the normal control group, *p<0.05 has a significant difference compared with the normal group, **p<0.01 has an extremely significant difference compared with the normal group; # indicates that compared with the wild-type SETDB1 group, ##p<0.01 has an extremely significant difference compared with the wild-type SETDB1 group. DETAILED DESCRIPTION

[0042] The present invention provides a histone methyltransferase 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 of the present invention. It should be pointed out that the 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 protection scope 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 art or the relevant product instructions.

[0043] Example 1 Construction of wild-type SETDB1 and SETDB1 mutant recombinant expression plasmids and expression in eukaryotic cells

[0044] According to the gene sequences of wild-type histone methyltransferase (SETDB1) and histone methyltransferase mutant (SEQ ID 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-SETDB1 of wild-type histone methyltransferase and the recombinant expression plasmid pSK1-SETDB1m of histone methyltransferase mutant. DH5α competent bacteria were conventionally transformed and amplified. After the plasmid was extracted, it was verified to be completely correct by sequencing.

[0045] The pSK1-SETDB1, pSK1-SETDB1m 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-SETDB1 and pAdTrack-CMV-SETDB1m. After sequencing confirmation, the above plasmids were linearized with PmeⅠ and homologously recombined with pAdEasy-1 in Escherichia coli BJ5183 to form pAdEasy-SETDB1 and pAdEasy-SETDB1m. Using the transfection kit Lipofectamine2000, the pAdEasy-SETDB1 and pAdEasy-SETDB1m 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 methyltransferase (SETDB1) and histone methyltransferase mutant (SETDB1m) were obtained. The molecular weight was about 140~170KDa detected by Western-blot, which was consistent with the expected molecular weight, indicating that the wild-type histone methyltransferase and histone methyltransferase mutant were successfully expressed in eukaryotic cells ( Figure 1 ).

[0046] The wild-type histone methyltransferase (SETDB1) amino acid sequence is shown in SEQ ID NO. 1. The histone methyltransferase mutant amino acid sequence is shown in SEQ ID NO. 2. The wild-type histone methyltransferase gene DNA sequence is shown in SEQ ID NO. 3. The histone methyltransferase mutant gene DNA sequence is shown in SEQ ID NO. 4.

[0047] Example 2 SETDB1 mutant can improve the number, motility and survival index of pig sperm

[0048] Accurately weigh 0.3g of glucose, 0.21g of sodium bicarbonate, 2.0g of sodium citrate dihydrate, 0.04g of sodium chloride, and 0.3g of sulfonamide, place them in a sterilized measuring cup, add 100mL of double distilled water, stir and dilute, adjust the pH to 6.35 by CO2, and filter the dilution through a 0.22um filter to obtain a sterile diluent. Select the immature seminal vesicle or semen of healthy male Yorkshire pigs that have just been slaughtered, remove the fascia of the seminal vesicle, grind and break it up, and dissolve it with sterile diluent (make up to 1L). The control group does not add any components to the diluent, and the experimental group adds 0.8mg / mL of wild-type SETDB1 or SETDB1 mutant to the above diluent, stir slowly and evenly, and let it stand at 17℃ for 6h. After standing, draw 50ul of diluted semen on the cell counting plate, cover it with a coverslip, observe it under a 400x microscope, and select five fields of view to calculate the number of sperm. The temperature was raised to 37°C, four fields of view were selected to observe the number of sperm in linear motion, and the sperm motility and survival index were calculated using the ten-level assessment method.

[0049] Sperm count test results Figure 2 The sperm motility test results are shown in Figure 3 The sperm survival index test results are shown in Figure 4 As shown. Figure 2 , Figure 3 , Figure 4 It can be seen that compared with the control group and wild-type SETDB1, the SETDB1 mutant can further significantly increase the number, sperm motility and survival index of sperm in mammals such as pigs.

[0050] Example 3 Preparation of PDRN using SETDB1 mutants

[0051] Accurately weigh 0.3g of glucose, 0.21g of sodium bicarbonate, 2.0g of sodium citrate dihydrate, 0.04g of sodium chloride, and 0.3g of sulfonamide, place them in a sterilized measuring cup, add 100mL of double distilled water, stir and dilute, adjust the pH value to 6.35 by CO2, and filter the dilution through a 0.22um filter to obtain a sterile diluent. Select the immature seminal vesicle or semen of healthy male Yorkshire pigs that have just been slaughtered, remove the fascia from the seminal vesicle, grind and break it up, and dissolve it with sterile diluent (make up to 1L). The control group does not add any components to the diluent, and the experimental group adds 0.8mg / mL of wild-type SETDB1 or SETDB1 mutant to the above diluent, stir slowly and evenly, and let it stand at 17℃ for 6h, and then prepare PDRN for the control group and the experimental group respectively.

[0052] The preparation method of PDRN is as follows:

[0053] 1) Take samples from the control group and experimental group, centrifuge to obtain 100 g of precipitate, add 600 mL of lysis buffer (0.02 mol / L LEDTA, 2 mol / L NaOH, 0.5% SDS), invert to mix, and keep at 90°C for 20 min.

[0054] 2) Cool down to 40℃, add 1mol / L Tris-HCl and 1mol / L NaCl (pH 8.0) in a volume equal to 1 / 2 of the reaction system in step 1), and mix by inversion.

[0055] 3) Add 1 mol / L HCl in an amount that is 1 / 5 of the volume of the reaction system in step 2) and mix by inversion.

[0056] 4) Centrifuge at 6000 rpm for 15 min at 0-4°C and collect the supernatant.

[0057] 5) Use a DNA fragmentor at 6000 J / s for 30 s.

[0058] 6) Add an equal volume of -20℃ precooled anhydrous ethanol, invert to mix, and place at -20℃ for 2h.

[0059] 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.

[0060] 8) The precipitate obtained in step 7) is suspended in physiological saline and dried in a vacuum freeze dryer to obtain polydeoxyribonucleotide.

[0061] Calculation of PDRN purity and extraction rate:

[0062] Extraction rate = PDRN product weight / semen or seminal vesicle weight;

[0063] 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.

[0064] The PDRN extraction rate and purity test results are shown in Table 1.

[0065] Table 1 Effects of different treatment conditions on the extraction rate of polydeoxyribonucleotides

[0066]

[0067] * indicates that compared with the normal control group, *p<0.05, there is a significant difference compared with the normal group, **p<0.01, there is a very significant difference compared with the normal group; # indicates that compared with the wild-type SETDB1 group, #p<0.05, there is a significant difference compared with the wild-type SETDB1 group.

[0068] As can be seen from Table 1, the PDRN prepared using the SETDB1 mutant has a significantly better extraction rate and higher purity than the control group and the wild-type SETDB1 group.

[0069] 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 SETDB1 mutant, characterized in that: The amino acid sequence of the SETDB1 mutant is shown in SEQ ID NO.

2.

2. A biological material related to the SETDB1 mutant according to claim 1, wherein the biological material is one of the following 1) to 5): 1) A gene encoding the SETDB1 mutant of 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 SETDB1 mutant according to claim 1 in the preparation of a product for improving the sperm count, sperm motility and / or sperm survival index of a mammal, characterized in that: The mammal is a suidae animal.

4. The use according to claim 3, characterized in that: The porcine animals are Duroc pig, Chenghua pig, Yorkshire pig, Landrace pig, Hampshire pig, Bozhong pig, Horse-body pig, Ningxiang spotted pig and Songliao black pig.

5. Use of the SETDB1 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: using the SETDB1 mutant of claim 1 to treat mammalian semen or seminal vesicles, inducing sperm cell maturation and then preparing polydeoxyribonucleotides; the mammal is a porcine animal.

7. The preparation method according to claim 6, characterized in that: The concentration of the SETDB1 mutant is 0.1-2 mg / mL.

8. The preparation method according to claim 6, characterized in that: The treatment time of the SETDB1 mutant is 1-24h.

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