Construction and application of a cloning vector containing salmon mitochondrial genes

Abstract

The present invention provides a recombinant salmon mitochondrial gene (mDNA), the construction and application of its cloning vector. After vector construction, cell transformation, and proliferation and amplification, the recombinant salmon mitochondrial gene (mDNA) of the present invention can be used to prepare polydeoxyribonucleotides (PDRN) with higher purity and yield on a large scale, and the PDRN prepared by the recombinant mitochondrial DNA of the present invention has significantly better effects in anti-oxidation, anti-inflammation, improving cell proliferation, resisting ultraviolet damage, and improving cell repair ability than the PDRN prepared by wild-type mitochondrial DNA. In terms of the above effects, it is equivalent to the PDRN prepared by traditional salmon testes, providing a new idea or method for large-scale preparation or industrial production of polydeoxyribonucleotides, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the construction and application of a cloning vector containing salmon mitochondrial DNA (mDNA). Background Art

[0002] Nucleic acids are substances that store and transmit genetic information in the body. They are biological macromolecules composed of nucleotides linked by phosphodiester bonds. An accidental discovery by Italian fishermen promoted the research on salmon nucleic acids. Based on the experience of fishermen, the Italian company Mastelli extracted a mixture of nucleic acid fragments (50bp-500bp) from salmon semen / testis, which can significantly increase cell viability, inhibit the secretion of inflammatory factors, increase the synthesis of collagen and elastin, promote wound healing, and reduce scar formation. Subsequent studies have found that this nucleic acid mixture has a significant effect in improving microcirculation, promoting angiogenesis, anti-thrombosis and treating diabetic foot.

[0003] However, for a long time, researchers did not clearly know the active fragments in the mixture of nucleic acid fragments that can play the above-mentioned role. For this purpose, a series of exploratory studies have been carried out. The DNA in semen mainly comes from the nucleus and mitochondria of sperm. In order to ensure successful fertilization, the number of mitochondria in sperm is significantly increased, resulting in a significant increase in the proportion of mitochondrial DNA in the total DNA. Mitochondrial DNA plays an important role in increasing sperm activity and improving fertilization rates. In addition to some functional protein genes (ATP synthase, cytochrome oxidase, etc.), tRNA genes, and rRNA genes, mitochondrial DNA also contains the regulatory gene D-loop. Studies have found that D-loop plays an important role in maintaining the stability of mitochondrial genes and regulating the transcription / replication of target genes.

[0004] On the other hand, the shortage of salmon natural resources has brought unprecedented difficulties to obtaining high-quality male salmon semen or testes. Therefore, researchers have proposed the idea of ​​using genetic engineering technology to recombinant characteristic gene fragments of salmon or other species and clone and replicate them in vitro to replace natural materials such as salmon testes obtained from nature, but there are still many problems in the process. For example, whether the recombinant gene can be stably replicated at a high level, whether it contains effective fragments similar to traditional salmon sperm DNA, and whether the prepared nucleic acid fragment mixture has the same pharmacological activity. To this end, the present invention provides a recombinant salmon mitochondrial DNA (mDNA) vector construction, and uses it to clone recombinant salmon mitochondrial genes, and studies its pharmacological activity after separation and purification. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a recombinant salmon mitochondrial gene (mDNA), the construction and application of its cloning vector.

[0006] In the first aspect of the present invention, a recombinant salmon mitochondrial gene (mDNA) is provided, wherein the recombinant salmon mitochondrial gene has a human mitochondrial D-loop region gene sequence (gene numbered GenBank D84807.1) added before the NADH dehydrogenase subunit 3 and 4L genes of the wild-type salmon mitochondrial gene (gene numbered GenBank D63335.1), and the recombinant mitochondrial gene is 2725bp in total.

[0007] Further preferably, the nucleotide sequence of the wild-type salmon mitochondrial gene is as shown in SEQ ID NO.2.

[0008] Further preferably, the nucleotide sequence of the recombinant salmon mitochondrial gene is as shown in SEQ ID NO.1.

[0009] In a second aspect of the present invention, a biological material related to the recombinant salmon mitochondrial gene of the first aspect of the present invention is provided, wherein the biological material comprises at least one of the following 1)-4):

[0010] 1) An expression cassette containing the recombinant salmon mitochondrial gene according to the first aspect of the present invention;

[0011] 2) a recombinant vector containing the recombinant salmon mitochondrial gene according to the first aspect of the present invention;

[0012] 3) A recombinant microorganism or a recombinant cell containing the recombinant salmon mitochondrial gene according to the first aspect of the present invention;

[0013] 4) A recombinant microorganism or recombinant cell containing the recombinant vector described in 2).

[0014] Further preferably, in 2), the recombinant vector is an Escherichia coli plasmid vector, a phage vector, a COS plasmid vector, a phagemid vector, a shuttle plasmid, a eukaryotic cell cloning vector, etc. as a cloning vector;

[0015] More preferably, in 2), the recombinant vector uses the following vector as a cloning vector:

[0016] pBR322 series, pUC series, pGEM series, pBBR1 series, pET series, pACYC series, pSC101 series, colE1, λgt11, λGEM-11, EMBL3 / 4, pBluescripSK-, pAD10sacBⅡ, M13mp, C2XB, pJB8, , pAUR123, pPIC9K, pPIC3K, pPIczA, pSVK3 plasmid, PBV, pCA13, YEp, YRp, YAC, human adenovirus (Ad2 or Ad5), SV40 vector, BKV, human vaccinia virus, but not limited thereto;

[0017] More preferably, in 2), the recombinant vector uses the following preferred vector as a cloning vector:

[0018] pBR322, pUC18 / 19, pGEM-3Z, λGEM-11, pBluescripSK-, pAD10sacBⅡ, M13mp, C2XB, pJB8, pAUR123, pPIC9K, pYES2-T, pYAC3, Ad2, pcDNA3.1;

[0019] More preferably, in 2), the recombinant vector uses the following more preferred vector as a cloning vector:

[0020] pBR322, pUC18 / 19, pcDNA3.1, pYES2-T.

[0021] Further preferably, in 3) or 4), the recombinant microorganism is a recombinant Escherichia coli, a recombinant Bacillus subtilis, a recombinant Streptomyces, a recombinant yeast, a recombinant filamentous fungus, etc.;

[0022] More preferably, in 3) or 4), the recombinant microorganism is preferably recombinant HB101, recombinant DH5α, recombinant JM109, recombinant BL21, recombinant Pichia pastoris, recombinant Saccharomyces cerevisiae, but not limited thereto;

[0023] More preferably, in 3) or 4), the recombinant microorganism is more preferably recombinant HB101, recombinant DH5α, or recombinant brewer's yeast;

[0024] More preferably, in 3) or 4), the recombinant microorganism is most preferably recombinant DH5α or recombinant brewer's yeast INSCVI.

[0025] Further preferably, in 3) or 4), the recombinant cell is a recombinant insect cell, a recombinant mammalian cell, etc.;

[0026] More preferably, in 3) or 4), the recombinant cell is a recombinant CHO cell.

[0027] In the third aspect of the present invention, there is provided use of the recombinant salmon mitochondrial gene described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in the preparation of polydeoxyribonucleotides (PDRN).

[0028] In the fourth aspect of the present invention, a method for preparing a polydeoxyribonucleotide is provided, the method comprising the following steps: preparing the recombinant salmon mitochondrial gene described in the first aspect of the present invention by a genetic engineering method, dissolving it in a solvent, and under sterile conditions, after it is completely dissolved, using an ultrasonic DNA shearing device to shear the recombinant salmon mitochondrial gene to obtain a polydeoxyribonucleotide prepared from the recombinant salmon mitochondrial gene.

[0029] More preferably, the solvent is water.

[0030] Further preferably, the specific conditions for using an ultrasonic DNA shearing device to shear the recombinant salmon mitochondrial gene are 4000 joules / s for 60 seconds.

[0031] In the fifth aspect of the present invention, a polydeoxyribonucleotide prepared by the preparation method described in the fourth aspect of the present invention is provided.

[0032] In the sixth aspect of the present invention, there is provided use of the polydeoxyribonucleotide described in the fifth aspect of the present invention in the preparation of medicines and / or cosmetics.

[0033] More preferably, the medicine and / or cosmetic is an antioxidant medicine and / or cosmetic.

[0034] Further preferably, the medicine and / or cosmetic is an anti-inflammatory medicine and / or cosmetic.

[0035] Further preferably, the medicine and / or cosmetic is a medicine and / or cosmetic that has the effects of increasing cell proliferation, resisting ultraviolet damage, and improving cell repair ability.

[0036] 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.

[0037] Compared with the prior art, the present invention has the following advantages and progress:

[0038] The present invention provides a cloning vector construction containing a recombinant salmon mitochondrial gene (mDNA) and its application. After the recombinant salmon mitochondrial gene (mDNA) of the present invention is used for vector construction, cell transformation, and proliferation and amplification, polydeoxyribonucleotides (PDRN) with higher purity and yield can be prepared on a large scale, and the PDRN prepared by the recombinant mitochondrial DNA of the present invention has significantly better effects in anti-oxidation, anti-inflammatory, improving cell proliferation, resisting ultraviolet damage, and improving cell repair ability compared to the PDRN prepared by wild-type mitochondrial DNA. In terms of the above-mentioned effects, the effect of PDRN prepared by traditional salmon testes is equivalent. The present invention provides a new idea or method for the large-scale preparation or industrial production of polydeoxyribonucleotides, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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.

[0040] Figure 1 :Electrophoresis of salmon mitochondrial DNA cloned in different vectors (unit: bp). M: DNA marker; 1: pBR322-recombinant mDNA, host E. coli; 2: pcDNA3.1-recombinant mDNA, host CHO; 3: pYES2-recombinant mDNA, host Saccharomyces cerevisiae INSCVI; 4: pBR322-wild-type mDNA, host E. coli; 5: pcDNA3.1-wild-type mDNA, host CHO; 6: pYES2-wild-type mDNA, host Saccharomyces cerevisiae INSCVI.

[0041] Figure 2 :Ultrasound electrophoresis of salmon mitochondrial DNA. M: DNA marker; 1: pBR322-recombinant mDNA, host E. coli; 2: pcDNA3.1-recombinant mDNA, host CHO; 3: pYES2-recombinant mDNA, host Saccharomyces cerevisiae INSCVI; 4: pBR322-wild-type mDNA, host E. coli; 5: pcDNA3.1-wild-type mDNA, host CHO; 6: pYES2-wild-type mDNA, host Saccharomyces cerevisiae INSCVI.

[0042] Figure 3:Effect of salmon mitochondrial DNA on cell activity under the action of H2O2. * indicates that compared with the H2O2 group, *P<0.05 indicates that there is a significant difference compared with the H2O2 group; **P<0.01 indicates that there is a very significant difference compared with the H2O2 group. # indicates that compared with the pBR322-recombinant mDNA group, #P<0.05 indicates that there is a significant difference compared with the pBR322-recombinant mDNA group. $ indicates that compared with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates that there is a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates that compared with the pYES2-recombinant mDNA group, &P<0.05 indicates that there is a significant difference compared with the pYES2-recombinant mDNA group.

[0043] Figure 4 :Effect of salmon mitochondrial DNA on SOD activity under the action of H2O2. * indicates a comparison with the H2O2 group, *P<0.05 indicates a significant difference compared with the H2O2 group; **P<0.01 indicates a very significant difference compared with the H2O2 group. # indicates a comparison with the pBR322-recombinant mDNA group, #P<0.05 indicates a significant difference compared with the pBR322-recombinant mDNA group. $ indicates a comparison with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates a comparison with the pYES2-recombinant mDNA group, &P<0.05 indicates a significant difference compared with the pYES2-recombinant mDNA group.

[0044] Figure 5 :Effect of salmon mitochondrial DNA on GSH activity under the action of H2O2. * indicates a comparison with the H2O2 group, *P<0.05 indicates a significant difference compared with the H2O2 group; **P<0.01 indicates a very significant difference compared with the H2O2 group. # indicates a comparison with the pBR322-recombinant mDNA group, #P<0.05 indicates a significant difference compared with the pBR322-recombinant mDNA group. $ indicates a comparison with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates a comparison with the pYES2-recombinant mDNA group, &P<0.05 indicates a significant difference compared with the pYES2-recombinant mDNA group.

[0045] Figure 6:Effect of salmon mitochondrial DNA on MDA content under the action of H2O2. * indicates a comparison with the H2O2 group, *P<0.05 indicates a significant difference compared with the H2O2 group; **P<0.01 indicates a very significant difference compared with the H2O2 group. # indicates a comparison with the pBR322-recombinant mDNA group, #P<0.05 indicates a significant difference compared with the pBR322-recombinant mDNA group. $ indicates a comparison with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates a comparison with the pYES2-recombinant mDNA group, &P<0.05 indicates a significant difference compared with the pYES2-recombinant mDNA group.

[0046] Figure 7 :Effect of salmon mitochondrial DNA on cell proliferation under ultraviolet irradiation. * indicates that compared with the ultraviolet group, *P<0.05 indicates that there is a significant difference compared with the ultraviolet group; **P<0.01 indicates that there is a very significant difference compared with the ultraviolet group. # indicates that compared with the pBR322-recombinant mDNA group, #P<0.05 indicates that there is a significant difference compared with the pBR322-recombinant mDNA group. $ indicates that compared with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates that there is a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates that compared with the pYES2-recombinant mDNA group, &P<0.05 indicates that there is a significant difference compared with the pYES2-recombinant mDNA group.

[0047] Figure 8 :Effect of salmon mitochondrial DNA on EGF content under ultraviolet irradiation. * indicates that compared with the ultraviolet group, *P<0.05 indicates that there is a significant difference compared with the ultraviolet group; **P<0.01 indicates that there is a very significant difference compared with the ultraviolet group. # indicates that compared with the pBR322-recombinant mDNA group, #P<0.05 indicates that there is a significant difference compared with the pBR322-recombinant mDNA group. $ indicates that compared with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates that there is a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates that compared with the pYES2-recombinant mDNA group, &P<0.05 indicates that there is a significant difference compared with the pYES2-recombinant mDNA group.

[0048] Fig. 9:Effect of salmon mitochondrial DNA on type I collagen content under ultraviolet irradiation. * indicates a comparison with the ultraviolet group, *P<0.05 indicates a significant difference compared with the ultraviolet group; **P<0.01 indicates a very significant difference compared with the ultraviolet group. # indicates a comparison with the pBR322-recombinant mDNA group, #P<0.05 indicates a significant difference compared with the pBR322-recombinant mDNA group. $ indicates a comparison with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates a comparison with the pYES2-recombinant mDNA group, &P<0.05 indicates a significant difference compared with the pYES2-recombinant mDNA group.

[0049] Fig.10 :Effect of salmon mitochondrial DNA on IL-6 content under the action of lipopolysaccharide. * indicates that compared with the lipopolysaccharide group, *P<0.05 indicates that there is a significant difference compared with the lipopolysaccharide group; **P<0.01 indicates that there is a very significant difference compared with the lipopolysaccharide group. # indicates that compared with the pBR322-recombinant mDNA group, #P<0.05 indicates that there is a significant difference compared with the pBR322-recombinant mDNA group. $ indicates that compared with the pcDNA3.1-recombinant mDNA group, $P<0.05 indicates that there is a significant difference compared with the pcDNA3.1-recombinant mDNA group. & indicates that compared with the pYES2-recombinant mDNA group, &P<0.05 indicates that there is a significant difference compared with the pYES2-recombinant mDNA group. DETAILED DESCRIPTION

[0050] 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 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 in accordance with the general technical conditions described in the literature of the field or the relevant product instructions. The materials, reagents, etc. used in the embodiments, unless otherwise specified, can be obtained from commercial channels.

[0051] Example 1 Construction of an Escherichia coli cloning vector containing salmon mitochondrial gene (mDNA)

[0052] According to the recombinant salmon mitochondrial gene sequence (SEQ ID NO.1) and the wild-type salmon mitochondrial gene sequence (SEQID NO.2) (GenBank D63335.1), AvaⅠ and Hind Ⅲ restriction sites were added to both ends of the above genes respectively, and then they were synthesized by a gene company according to the method of chemical total synthesis. The pBR322 plasmid was digested with restriction endonucleases AvaⅠ and Hind Ⅲ, and then the synthesized salmon mitochondrial gene containing AvaⅠ and Hind Ⅲ restriction sites was connected with the pBR322 plasmid linearized by double restriction digestion to construct the cloning plasmids pBR322-recombinant mDNA and pBR322-wild-type mDNA of the recombinant salmon mitochondrial gene.

[0053] The DH5α strain was inoculated into LB medium and cultured at 37°C with shaking overnight to prepare competent cells. The DH5α cells containing the recombinant plasmid were obtained with the help of an electroporation instrument. The electroporated DH5α cells were screened with ampicillin and tetracycline to obtain DH5α cells containing the pBR322-recombinant mDNA plasmid and the pBR322-wild-type mDNA plasmid, respectively. They were inoculated into LB medium containing ampicillin and cultured at 37°C for 16-24 hours until there were enough positive clone cells. Centrifuge at 5000r / min to obtain wet bacterial pellets, add lysozyme solution to mix and shake, centrifuge at 11000r / min to collect the supernatant, add phenol-chloroform-isoamyl alcohol solution at a volume ratio of 3:1 to mix thoroughly, centrifuge at 7000r / min to collect the upper aqueous phase, add isopropanol, and centrifuge at 11000r / min to discard the supernatant, wash the precipitate with 70% ethanol, collect the precipitate by centrifugation, dissolve it in TE (Tris-EDTA, pH 8.0) solution, and add CsCl, EB ( Ethidium bromide) solution, mix thoroughly, put into ultracentrifuge tubes to ensure that each tube has the same density, balance and seal the tubes, centrifuge at 45000r / min and 20℃ for 16h, extract plasmid DNA under ultraviolet light, extract with CsCl-saturated isopropanol to remove EB in the plasmid, add cold anhydrous ethanol and place in a -20℃ refrigerator for 2h, centrifuge at 11000r / min for 30min, discard the supernatant, wash once with 70%, centrifuge and discard the supernatant, and obtain recombinant pure plasmid DNA and wild-type pure plasmid DNA respectively. The recombinant pure plasmid DNA and wild-type pure plasmid DNA were digested with the corresponding restriction enzymes and purified by agarose molecular sieve chromatography to obtain cloned target genes respectively. It was verified that their sequences were the same as those of the recombinant mDNA and wild-type mDNA. At the same time, gel electrophoresis was performed on them (see Figure 1 ).

[0054] Example 2 Construction of a eukaryotic cell cloning vector containing salmon mitochondrial gene (mDNA)

[0055] According to the recombinant salmon mitochondrial gene sequence (SEQ ID NO.1) and the wild-type salmon mitochondrial gene sequence (SEQID NO.2), NdeⅠ and XhoⅠ restriction sites were added to both ends of the above genes, and then synthesized by a gene company according to the method of chemical total synthesis. The pcDNA3.1 plasmid was digested with restriction endonucleases NdeⅠ and XhoⅠ, and then the synthesized salmon mitochondrial gene containing NdeⅠ and XhoⅠ restriction sites was connected with the pcDNA3.1 plasmid linearized by double restriction digestion under the action of T4 ligase to construct the cloning plasmids pcDNA3.1-recombinant mDNA and pcDNA3.1-wild-type mDNA of the salmon mitochondrial gene, which were transformed into DH5α clones and sequenced. After confirming the correct sequence, the Hieff Trans TM According to the instructions of the transfection reagent, add pcDNA3.1-recombinant mDNA and pcDNA3.1-wild-type mDNA to CHO, then add the transfection reagent, mix well, and place in a 5% CO237℃ incubator for culture; after incubation for 48~72 hours, add the resistance screening medium containing G418, and change the medium every other day. The cells that are suspended and not tightly connected to the bottom of the culture dish are removed by changing the medium, and the resistance cell culture is continued until it is determined that there are enough positive clone cells. Centrifuge at 3000r / min and 4℃ for 5min), discard the supernatant, add a certain amount of PBS, mix gently by blowing, freeze at -20℃ for 30min, and freeze at 37℃ Thaw, repeat this 3 times, collect the supernatant by centrifugation at 11000r / min, add phenol-chloroform-isoamyl alcohol solution at a volume ratio of 3:1 and mix thoroughly, collect the upper aqueous phase by centrifugation at 7000r / min, add isopropanol, 11000r / min, discard the supernatant, wash the precipitate with 70% ethanol, collect the precipitate by centrifugation, dissolve it in TE (Tris-EDTA, pH8.0) solution, add CsCl and EB (ethidium bromide solution) solution successively, mix thoroughly, put it into ultracentrifuge tubes to ensure that each tube has the same density, balance and seal the tubes, centrifuge at 45000r / min at 20℃ for 16h, extract plasmid DNA under ultraviolet light, extract EB in the plasmid with CsCl-saturated isopropanol, add cold anhydrous ethanol and place in a -20℃ refrigerator for 2h, centrifuge at 11000r / min for 30min, discard the supernatant, wash once with 70%, centrifuge and discard the supernatant, and obtain recombinant pure plasmid DNA and wild-type pure plasmid DNA respectively. The recombinant pure plasmid DNA and the wild-type pure plasmid DNA were digested with the corresponding restriction enzymes and purified by agarose molecular sieve chromatography to obtain cloned target genes. The sequences of the genes were verified to be identical to those of the recombinant mDNA and the wild-type mDNA. The genes were also subjected to gel electrophoresis (see Figure 1 ).

[0056] Example 3 Construction of a yeast cloning vector containing salmon mitochondrial gene (mDNA)

[0057] According to the recombinant salmon mitochondrial gene sequence (SEQ ID NO.1) and the wild-type salmon mitochondrial gene sequence (SEQID NO.2), ScaⅠ and BamH Ⅰ restriction sites were added to both ends of the above genes, respectively, and then synthesized by a gene company according to the method of chemical total synthesis, and the pYES2-T vector was digested with restriction endonucleases ScaⅠ and BamH Ⅰ, and then the synthesized salmon mitochondrial gene containing ScaⅠ and BamH Ⅰ restriction sites was connected with the pYES2-T vector linearized by double restriction digestion under the action of T4 ligase to construct the yeast efficient cloning vector pYES2-recombinant mDNA and pYES2-wild-type mDNA of salmon mitochondrial gene, and transformed into Escherichia coli TOP10 for proliferation and sequencing. After determining the correct sequence, pYES2-recombinant mDNA and pYES2-wild-type mDNA were transferred into brewer's yeast INSCVI by electroporation, and cultured continuously and screened for resistance until there were enough positive clone cells. Centrifuge at 1500 rpm for 5 min at room temperature to collect the cells, add yeast lysis buffer, phenol / chloroform / hexyl amyl alcohol (25:24:1) solution and acid-washed glass beads. Vigorously shake for 5 minutes, then centrifuge at 12000r / min at room temperature for 5 minutes, collect the supernatant, add phenol-chloroform-isoamyl alcohol solution at a volume ratio of 3:1 and mix thoroughly, collect the upper aqueous phase at 7000r / min, add isopropanol, centrifuge at 11000r / min, discard the supernatant, wash the precipitate with 70% ethanol, collect the precipitate by centrifugation, dissolve it in TE (Tris-EDTA, pH8.0) solution, add CsCl and EB (ethidium bromide solution) solution successively, mix thoroughly, put it into ultracentrifuge tubes to ensure that each tube has the same density, balance and seal the tubes, centrifuge at 45000r / min at 20℃ for 16h, extract plasmid DNA under ultraviolet light, extract EB in the plasmid with CsCl-saturated isopropanol, add cold anhydrous ethanol and place in a -20℃ refrigerator for 2h, centrifuge at 11000r / min for 30min, discard the supernatant, wash once with 70%, centrifuge and discard the supernatant, and obtain recombinant pure plasmid DNA and wild-type pure plasmid DNA respectively. The recombinant pure plasmid DNA and the wild-type pure plasmid DNA were digested with the corresponding restriction enzymes and purified by agarose molecular sieve chromatography to obtain cloned target genes. The sequences of the genes were verified to be identical to those of the recombinant mDNA and the wild-type mDNA. The genes were also subjected to gel electrophoresis (see Figure 1 ).

[0058] Test example: efficacy test of mitochondrial DNA

[0059] The positive cloned cells in Examples 1-3 (DH5α cells containing pBR322-recombinant mDNA, DH5α cells containing pBR322-wild-type mDNA, CHO cells containing pcDNA3.1-recombinant mDNA, CHO cells containing pcDNA3.1-wild-type mDNA, Saccharomyces cerevisiae INSCVI containing pYES2-recombinant mDNA, Saccharomyces cerevisiae INSCVI containing pYES2-wild-type mDNA) were proliferated and amplified, and the corresponding proliferated positive cloned cells were treated in the same manner as in Examples 1-3 to obtain 6 pure plasmid DNAs (pBR322-recombinant mDNA, pBR322-wild-type mDNA, pcDNA3.1-recombinant mDNA, pcDNA3.1-wild-type mDNA, pYES2-recombinant mDNA, pYES2-wild-type mDNA). After obtaining the 6 pure plasmid DNAs, they were respectively purified by using the corresponding AvaⅠ and Hind The plasmid DNA was digested with restriction endonucleases Ⅲ, NdeⅠ and XhoⅠ, ScaⅠ and BamHⅠ, and then passed through a gel chromatography column. The filtrate containing the target gene was collected, and sodium acetate at a final concentration of 0.3 M was added thereto, and the mixture was fully stirred and mixed. After the sodium acetate was fully dissolved, pre-cooled anhydrous ethanol was slowly added to the solution at a volume ratio of 1:1.5, and the mixture was precipitated for 30 min, centrifuged at 8000 rpm for 20 min, and the supernatant was discarded. The precipitate was rinsed once with 75% ethanol, and centrifuged at 6000 rpm for 15 min, discard the supernatant, get the product, freeze-dry and obtain 6 kinds of salmon mitochondrial DNA (3 kinds of recombinant salmon mitochondrial DNA, 3 kinds of wild salmon mitochondrial DNA), and use ultraviolet spectrophotometry to detect their purity and content as shown in Table 1; weigh 1g of mitochondrial DNA respectively, dissolve it in 100ml of injection water, under sterile conditions, after it is completely dissolved, use ultrasonic DNA fragmentation instrument (SCIENTZ18-A) to fragment the sample DNA (4000 joules / s for 60 seconds), and obtain PDRN (polydeoxyribonucleotide) prepared from 6 kinds of salmon mitochondrial DNA. The sample fragmentation effect is shown in Figure 2 .

[0060] Table 1. Purity and content of mitochondrial DNA in different host-vectors

[0061]

[0062] (1) Cell antioxidant test:

[0063] Human skin fibroblasts (HS27, purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum. When the cell density reached 2×10 6Afterwards, they were inoculated in 24-well plates and divided into 9 groups, including normal group, model group, a (Escherichia coli-recombinant mDNA group), b (CHO-recombinant mDNA group), c (yeast-recombinant mDNA group), d (Escherichia coli-wild type mDNA group), e (CHO-wild type mDNA group), f (yeast-wild type mDNA group), and g (PDRN group, salmon testis, purchased from Shanghai Saimengxin Biotechnology Co., Ltd.). When the cells grew to 80%, except for the normal group, the cells in the other groups were given 300uM H2O2, and then 100ul of 1% mitochondrial DNA aqueous solution from different hosts was added (the normal group and the model group were added with equal amounts of water), and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the MTT method was used to determine the effect of mitochondrial DNA of each group on cell activity; after 24 hours of treatment in the same way as above, the cells of each group were collected, and the kit was used to detect the effect of mitochondrial DNA of each group on SOD, GSH and MDA. Figure 3 , Figure 4 , Figure 5 and Figure 6 The experimental results show that the PDRN prepared by the recombinant mitochondrial DNA in Examples 1, 2, and 3 of the present invention has a significantly better antioxidant effect than the PDRN prepared by the wild-type mitochondrial DNA, and has a comparable antioxidant effect to the PDRN prepared by the traditional salmon testis.

[0064] (2) Cell UV repair efficacy test:

[0065] Human skin fibroblasts HS27 were used as experimental subjects. They were inoculated in 6-well plates and divided into 9 groups: normal group, model group, a (Escherichia coli-recombinant mDNA group), b (CHO-recombinant mDNA group), c (yeast-recombinant mDNA group), d (Escherichia coli-wild-type mDNA group), e (CHO-wild-type mDNA group), f (yeast-wild-type mDNA group), and g (PDRN group, salmon testis, purchased from Shanghai Saimengxin Biotechnology Co., Ltd.). When the cells grew to 80%, except for the normal group, the cells in the other groups were placed under ultraviolet irradiation with 0.2J / cm2UVA (365nm) for 60min every 24 hours. After each irradiation, 250ul of 1% mitochondrial DNA aqueous solution from different hosts was added (the normal group and the model group were added with equal amounts of water), and the cells were cultured in a 37°C, 5% CO2 incubator. After 48 hours, the relative cell proliferation rate was detected by CCK-8 method; the cell supernatant was collected and the content of type I collagen and epidermal growth factor was determined by ELISA kit. Figure 7 , Figure 8 and Fig. 9The experimental results show that the PDRN prepared from the recombinant mitochondrial DNA prepared in Examples 1, 2, and 3 of the present invention has significantly better effects in improving cell proliferation, resisting ultraviolet damage, and improving cell repair ability than the PDRN prepared from wild-type mitochondrial DNA, and is equivalent to the PDRN prepared from traditional salmon testis.

[0066] (3) Cell anti-inflammatory test:

[0067] Raw264.7 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured in RPMI1640 medium containing 10% fetal bovine serum until the cell density reached 2×10 6 Afterwards, they were inoculated in a 24-well plate and divided into 9 groups, including normal group, model group, a (Escherichia coli-recombinant mDNA group), b (CHO-recombinant mDNA group), c (yeast-recombinant mDNA group), d (Escherichia coli-wild type mDNA group), e (CHO-wild type mDNA group), f (yeast-wild type mDNA group), and g (PDRN group, salmon testis, purchased from Shanghai Saimengxin Biotechnology Co., Ltd.). When the cells grew to 80%, except for the normal group, the cells in other groups were treated with lipopolysaccharide, and then 100ul of 1% mitochondrial DNA aqueous solution from different hosts was added (the normal group and the model group were added with equal amounts of water), and the cells were cultured in a 37°C, 5% CO2 incubator. The cell supernatant was collected by centrifugation for 48 hours, and the IL-6 content was detected using an ELISA kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.). By Fig.10 The experimental results show that the PDRN prepared by the recombinant mitochondrial DNA prepared in Examples 1, 2, and 3 of the present invention has significantly better anti-inflammatory effects than the PDRN prepared by the wild-type mitochondrial DNA, and is equivalent to the PDRN prepared by the traditional salmon testis.

[0068] 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 recombinant salmon mitochondrial gene, characterized in that: The nucleotide sequence of the recombinant salmon mitochondrial gene is shown in SEQ ID NO.

1.

2. The biological material related to the recombinant salmon mitochondrial gene according to claim 1, characterized in that: The biological material includes at least one of the following 1)-4): 1) An expression cassette containing the recombinant salmon mitochondrial gene according to claim 1; 2) A recombinant vector containing the recombinant salmon mitochondrial gene according to claim 1; 3) A recombinant microorganism or recombinant cell containing the recombinant salmon mitochondrial gene of claim 1; 4) A recombinant microorganism or recombinant cell containing the recombinant vector described in 2).

3. The biomaterial according to claim 2, characterized in that In the above 2), the recombinant vector uses an Escherichia coli plasmid vector, a phage vector, a COS plasmid vector, a phagmid vector, a shuttle plasmid, or a eukaryotic cell cloning vector as a cloning vector.

4. The biomaterial according to claim 2, characterized in that In the above 3) or 4), the recombinant microorganism is a recombinant Escherichia coli, a recombinant Bacillus subtilis, a recombinant Streptomyces, a recombinant yeast or a recombinant filamentous fungus; and the recombinant cell is a recombinant insect cell or a recombinant mammalian cell.

5. Use of the recombinant salmon mitochondrial gene according to claim 1 or the biological material according to claim 2 in the preparation of polydeoxyribonucleotides.

6. A method for preparing a polydeoxyribonucleotide, characterized in that: The method comprises the following steps: preparing the recombinant salmon mitochondrial gene according to claim 1 by a genetic engineering method, dissolving it in a solvent, and under sterile conditions, after it is completely dissolved, using an ultrasonic DNA shearing device to shear the recombinant salmon mitochondrial gene to obtain a polydeoxyribonucleotide prepared from the recombinant salmon mitochondrial gene.

7. A polydeoxyribonucleotide prepared by the preparation method according to claim 6.

8. Use of the polydeoxyribonucleotide according to claim 7 in the preparation of medicines and / or cosmetics, characterized in that: The medicine and / or cosmetic is an antioxidant medicine and / or antioxidant cosmetic or an anti-inflammatory medicine and / or anti-inflammatory cosmetic.

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