CN110964746B - Method for promoting bovine skeletal muscle satellite cell myogenic differentiation by over-expressing PSMB5 - Google Patents
Method for promoting bovine skeletal muscle satellite cell myogenic differentiation by over-expressing PSMB5 Download PDFInfo
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Abstract
The invention relates to a method for promoting bovine skeletal muscle satellite cell myogenic differentiation by over-expressing PSMB5, which comprises the following steps: acquiring a target sequence including a bovine PSMB5 gene sequence; constructing an expression vector pcDNA3.1-PSMB 5; and c, transfecting the bovine skeletal muscle satellite cell with pcDNA3.1-PSMB 5: the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting bovine skeletal muscle satellite cells by using a liposome transfection reagent, so that the bovine skeletal muscle satellite cells overexpress PSMB5, and the in-vitro differentiation capacity of the bovine skeletal muscle satellite cells is improved by using the action of PSMB 5. The method utilizes PSMB5 to regulate and control the proliferation and myoblast differentiation of the bovine muscle satellite cells, can provide revelation for the research of ubiquitination process of muscle development and differentiation, and provides new thought and reference for clinical research and diagnosis and treatment of muscle development and differentiation and injury repair.
Description
Technical Field
The invention belongs to the technical field of biology and cell, and particularly relates to a method for promoting bovine skeletal muscle satellite cell myogenic differentiation by over-expressing PSMB 5.
Background
Skeletal muscle satellite cells (skeletal muscle satellite cells) are myogenic stem cells with proliferation and differentiation potential in skeletal muscle, usually exist between a myofibrillar sarcolemma and a basement membrane in a resting state, can be activated under certain conditions, proliferate and differentiate to form skeletal muscle cells, and play an important role in the growth, development and regeneration processes of the muscles after the birth of animals. The activation, proliferation and differentiation process of the muscle satellite cells are regulated and controlled by various factors, and the generation and regulation mode of the skeletal muscle satellite cells is known, so that the possibility of artificially controlling the formation of the muscle cells can be increased.
The proliferation and differentiation process of skeletal muscle involves the expression of multiple genes, signal path and network regulation, and the process is extremely complex. Studies have shown that the ubiquitin-proteasome system is involved in regulating many biological processes, such as cell cycle progression, cell proliferation and differentiation, and signaling. The 26S proteasome is an ATP-dependent proteolytic complex that degrades ubiquitinated substrates and is composed of a 20S core proteasome and 19S regulatory particles. 20S is a cylindrical structure and is composed of 4 ring stacks consisting of alpha subunits and beta subunits, 2 beta rings on the inner side have protease catalytic hydrolysis activity, beta 1, beta 2 and beta 5 are active subunits, have specific peptide cleavage sites and are key sites of the degradation protein of the whole ubiquitin-proteasome pathway. Among them, proteasome β 5 subunit (PSMB5) has chymotrypsin-like activity. PSMB5 can be transfected into cells or animals using expression vectors for studying proteasome function, typically the PSMB5 gene introduced into cells or animals using sequences comprising the CDS region of the PSMB5 gene. The PSMB5 is used for promoting the myogenic differentiation of the bovine muscle satellite cells, so that the method can provide reference for the utilization of the ubiquitin-proteasome system in the muscle development differentiation and injury repair, and provide new ideas and methods for the clinical research and diagnosis and treatment of the muscle development differentiation and injury repair.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the environmental stress problem in the prior art, and provides a method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by over-expressing PSMB5, the method utilizes PSMB5 to regulate and control the proliferation and the myogenic differentiation of bovine muscle satellite cells, can provide inspiration for the research of the ubiquitination process of muscle development and differentiation, and provides new thought and reference for the clinical research and diagnosis and treatment of muscle development and differentiation and injury repair.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for promoting bovine skeletal muscle satellite cell myogenic differentiation by overexpressing PSMB5, comprising the steps of:
acquiring a target sequence including a bovine PSMB5 gene sequence:
designing a pair of PCR primers according to the bovine PSMB5 gene sequence, respectively introducing XhoI enzyme cutting sites and XbaI enzyme cutting sites at the 5' ends of the two primers, and amplifying a CDS region sequence containing PSMB5 gene from a bovine genome by using the pair of primers, wherein the length is 792 bp;
constructing an expression vector pcDNA3.1-PSMB 5:
connecting the sequence obtained in the XhoI and XbaI double enzyme digestion step and a pcDNA3.1 vector by using ligase, inserting the sequence into XhoI and XbaI enzyme digestion sites of a plasmid vector pcDNA3.1, and constructing an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene;
and c, transfecting the bovine skeletal muscle satellite cell with pcDNA3.1-PSMB 5:
the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting bovine skeletal muscle satellite cells by using a liposome transfection reagent, so that the bovine skeletal muscle satellite cells overexpress PSMB5, and the in-vitro differentiation capacity of the bovine skeletal muscle satellite cells is improved by using the action of PSMB 5.
In the first step, the bovine PSMB5 Gene sequence is Gene ID 534640.
In the first step, the PCR primers are SEQ ID No.1 and SEQ ID No. 2.
The invention has the advantages and positive effects that:
1. the method utilizes PSMB5 to regulate and control the proliferation and myoblast differentiation of the bovine muscle satellite cells, and can provide a hint for the research of the ubiquitination process of the muscle development and differentiation.
2. The method for promoting the myogenic differentiation process of the bovine skeletal muscle satellite cells by changing the expression of the PSMB5, which is established by the invention, can effectively regulate and control the muscle development and differentiation process, can provide a certain reference for the research of the ubiquitin-proteasome system in the muscle development and differentiation and injury repair, and provides a new thought and reference for the clinical research and diagnosis and treatment of the muscle development and differentiation and injury repair.
3. The method constructs an expression vector of a bovine proteasome beta 5 subunit (PSMB5) gene, and introduces the gene into bovine skeletal muscle satellite cells through gene transfection, thereby promoting the myogenic differentiation capacity of the skeletal muscle satellite cells. Firstly, designing a pair of PCR primers according to a bovine PSMB5 gene sequence, respectively introducing XhoI enzyme cutting sites and XbaI enzyme cutting sites at the 5' ends of the two primers, amplifying a CDS region sequence containing PSMB5 gene from a bovine genome by using the pair of primers, wherein the length is 792bp, and inserting the sequence into a vector pcDNA3.1 by using the introduced enzyme cutting sites to construct an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene; then the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting the bovine skeletal muscle satellite cells, so that the bovine skeletal muscle satellite cells overexpress PSMB5, and the myogenic differentiation capacity of the bovine skeletal muscle satellite cells can be improved by utilizing the action of PSMB 5.
Drawings
FIG. 1 is a schematic structural view of a plasmid vector pcDNA3.1 used in the present invention;
FIG. 2 is a schematic structural diagram of pcDNA3.1-PSMB5, an overexpression PSMB5 vector constructed in the present invention;
FIG. 3 is a graph showing the effects of the expression level of PSMB5 in bovine skeletal muscle satellite cells after transfection of the PSMB5 overexpression vector (pcDNA3.1-PSMB5) for 48h and the untransfected overexpression vector (pcDNA3.1-NC) in the invention;
FIG. 4 is a picture of bovine skeletal muscle satellite cells transfected with PSMB5 overexpression vector (pcDNA3.1-PSMB5) and untransfected overexpression vector (pcDNA3.1-NC) in the present invention before and after differentiation induction, wherein GM represents the proliferation stage and DM represents the differentiation stage.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A method for promoting bovine skeletal muscle satellite cell myogenic differentiation by overexpressing PSMB5, comprising the steps of:
acquiring a target sequence including a bovine PSMB5 gene sequence:
designing a pair of PCR primers according to the bovine PSMB5 gene sequence, respectively introducing XhoI enzyme cutting sites and XbaI enzyme cutting sites at the 5' ends of the two primers, and amplifying a CDS region sequence containing PSMB5 gene from a bovine genome by using the pair of primers, wherein the length is 792 bp;
constructing an expression vector pcDNA3.1-PSMB 5:
connecting the sequence obtained in the XhoI and XbaI double enzyme digestion step and a pcDNA3.1 vector by using ligase, inserting the sequence into XhoI and XbaI enzyme digestion sites of a plasmid vector pcDNA3.1, and constructing an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene;
and c, transfecting the bovine skeletal muscle satellite cell with pcDNA3.1-PSMB 5:
the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting bovine skeletal muscle satellite cells by using a liposome transfection reagent, so that the bovine skeletal muscle satellite cells overexpress PSMB5, and the in-vitro differentiation capacity of the bovine skeletal muscle satellite cells is improved by using the action of PSMB 5.
Preferably, the bovine PSMB5 Gene sequence in the first step is Gene ID: 534640.
Preferably, the PCR primers in the first step are SEQ ID No.1 and SEQ ID No. 2.
The design idea of the invention is as follows:
designing a pair of PCR primers according to a bovine PSMB5 gene sequence, respectively introducing an XhoI enzyme cutting site and an XbaI enzyme cutting site at the 5' ends of the two primers, amplifying a CDS region sequence containing a PSMB5 gene from a bovine genome by using the pair of primers, wherein the length is 792bp, and inserting the sequence into a vector pcDNA3.1 by using the introduced enzyme cutting site to construct an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene; then the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting the bovine skeletal muscle satellite cells, so that the bovine skeletal muscle satellite cells over-express PSMB5 and the capability of promoting the adult muscle differentiation of the bovine skeletal muscle satellite cells is promoted.
Specifically, the method comprises the following steps:
a method for promoting bovine skeletal muscle satellite cell proliferation in vitro, comprising the steps of:
1. acquisition of a target sequence containing the PSMB5 gene sequence:
a pair of PCR primers is designed according to the bovine PSMB5 Gene sequence (Gene ID:534640), an XhoI enzyme cutting site and an XbaI enzyme cutting site are respectively introduced into the 5' ends of the two primers, and the pair of primers is used for amplifying a sequence containing a CDS region of a PSMB5 Gene from a bovine genome, wherein the length of the sequence is 792 bp.
The method comprises the following specific steps:
(1) and (4) PCR amplification. Designing a pair of specific primers containing XhoI and XbaI enzyme cutting sites, namely SEQ ID No.1, SEQ ID No.2, SEQ ID No. 1: 5' -CCGCTCGAGGCCACCATGGCGCTGGCCAGC GT-3' (bases underlined indicate XhoI cleavage sites), SEQ ID No. 2: 5' -TGCTCTAGATCAGTGG GTAGATCCACTATACT TG-3' (bases underlined indicate XbaI cleavage sites), and PCR was performed under the following conditions using the bovine genome as a template.
Reaction system: 25 μ L reaction system, 2 XEs Taq MasterMix 12.5 μ L, template DNA2 μ L, primer SEQ ID No.1(10 μ M)1 μ L, primer SEQ ID No.2(10 μ M)1 μ L, sterilized ultrapure water 8.5 μ L.
The reaction condition is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 45s, and circulating for 35 times; extension at 72 ℃ for 2 min.
And detecting the amplification product by agarose gel electrophoresis to obtain a CDS region sequence containing the PSMB5 gene, wherein the CDS region sequence is 792bp in length, an XhoI enzyme cutting site is introduced into the 5 'end of the CDS region sequence, and an XbaI enzyme cutting site is introduced into the 3' end of the CDS region sequence.
(2) And (6) recovering the glue. And (3) performing agarose gel electrophoresis on the amplified product, cutting the gel under an ultraviolet lamp, and recovering according to the operation instruction of the gel recovery kit.
2. Construction of expression vector pcDNA3.1-PSMB 5:
the PSMB5 obtained in the step 1 and the pcDNA3.1 vector are subjected to double enzyme digestion by XhoI and XbaI, and are connected by ligase, so that a target sequence is inserted into XhoI and XbaI enzyme digestion sites of a plasmid vector pcDNA3.1, and an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene is constructed.
The method comprises the following specific steps:
(1) the product is recovered by double digestion of the gel obtained in step 1 with XhoI and XbaI.
Reaction system: 30 μ L of the reaction system, 14 μ L of ultrapure water, 10 × Buffer 2 μ L, PSMB 5: 12. mu.L, XhoI enzyme 1. mu.L, XbaI enzyme 1. mu.L.
Reaction conditions are as follows: 20min at 37 ℃ and 5min at 85 ℃;
purifying the enzyme digestion product, and operating according to the instruction of a product purification kit;
(2) the pcDNA3.1 vector was double digested with XhoI and XbaI.
Reaction system: 20. mu.L of the reaction system, 11. mu.L of ultrapure water, 10 XBuffer 2. mu.L, plasmid pcDNA3.1: 5. mu.L, XhoI enzyme 1. mu.L, XbaI enzyme 1. mu.L.
Reaction conditions are as follows: 1h at 37 ℃ and 5min at 80 ℃.
Purifying the enzyme digestion product, and operating according to the instruction of a product purification kit;
(3) connecting the two types of glue to recover the enzyme digestion product.
Reaction system: 20 μ L of the reaction system, 5 μ L of the product recovered from the target sequence, 5 μ L of the product recovered from plasmid PcDNA3.1, 1 μ L of T4DNALigase, 2 μ L of T4 ligase buffer, and 7 μ L of ultrapure water.
Reaction conditions are as follows: 4h at 22 ℃.
(4) Escherichia coli DH 5. alpha. was transformed, the transformed product was spread evenly on a solid medium containing ampicillin, cultured at 37 ℃ for 24 hours, and the single clone was picked up and cultured in 5ml of a liquid medium containing benzyl amine at 37 ℃ for 12 hours with shaking.
(5) PCR of bacterial liquid
Reaction system: 20 mu L of reaction system, 1 mu L of bacterial liquid in the step 4, 0.2 mu L of primer SEQ ID No.1(10 mu M), 0.2 mu L of primer SEQ ID No.2(10 mu M), 10 mu L of 2x Es Taq MasterMix and 8.6 mu L of sterilized ultrapure water.
The reaction condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 69 ℃ for 30s, and extension at 72 ℃ for 24s, and circulating for 30 times; extension at 72 ℃ for 2 min.
The correct ligation was confirmed by further sequencing to obtain the expression vector pcDNA3.1-PSMB5 (shown in FIG. 2).
3. pcDNA3.1-PSMB5 transfected bovine skeletal muscle satellite cells:
culturing bovine skeletal muscle satellite cells into a six-well plate one day before transfection to ensure that the cells can reach about 70-80% full in the next day, and replacing each well with 2ml of fresh serum cell culture solution without double antibody before transfection;
the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting bovine skeletal muscle satellite cells by using a liposome transfection reagent, and the transfection operation is carried out according to the instruction of the liposome transfection reagent, so that the bovine skeletal muscle satellite cells overexpress PSMB 5.
The method comprises the following specific steps:
4. And (3) detecting the transfection effect:
(1) qRT-PCR detection of PSMB5 expression levels after transfection.
Detecting the transfection level 48h after transfection, extracting total RNA of untransfected muscle satellite cells and transfected muscle satellite cells by using an RNA rapid extraction kit, and extracting total RNA by using All-in-OneTMThe First-Strand cDNA Synthesis Kit reverse transcribes RNA into First Strand cDNA,
qRT-PCR reaction System: 10. mu. mol/L of the forward primer, 10. mu. mol/L of the reverse primer, 2.0. mu.L of 5-fold diluted cDNA, 10. mu.L of 2 × All-in-OneTMqPCR Mix, RNase-free water supplemented the system to 20. mu.L.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 15s, and 40 cycles; the preparation temperature range of the dissolution curve is 65-95 ℃,0.5 ℃/time and 6 s/time.
As a result: as shown in FIG. 3, the expression level of PSMB5 in the transfected muscle satellite cells is 38.9 times higher than that in the untransfected muscle satellite cells 48h after transfection, which indicates that pcDNA3.1-PSMB5 is successfully transfected and improves the expression level of the target gene.
(2) Effect of PSMB5 on myogenic differentiation of myosatellite cells after transfection.
After 48h of transfection, the untransfected muscle satellite cells and the transfected muscle satellite cells were cultured by replacing the differentiation medium containing 2% horse serum, and the proliferation and induced differentiation effects of the muscle satellite cells were observed.
On day 3 after the addition of the differentiation medium, the differentiation of the transfected group muscle satellite cells is obvious, and thick myotubes are formed, while the untransfected group does not have obvious thick myotubes and only has sporadic small myotubes, as shown in fig. 4, the fact that the over-expression of PSMB5 can promote the myogenic differentiation of the bovine skeletal muscle satellite cells is demonstrated.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Sequence listing
<110> Tianjin college of agriculture
<120> a method for promoting bovine skeletal muscle satellite cell myogenic differentiation by overexpressing PSMB5
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA/RNA
<213> primer 1(Unknown)
<400> 1
ccgctcgagg ccaccatggc gctggccagc gt 32
<210> 2
<211> 34
<212> DNA/RNA
<213> primer 2(Unknown)
<400> 2
tgctctagat cagtgggtag atccactata cttg 34
Claims (2)
1. A method of promoting bovine skeletal muscle satellite cell myogenic differentiation by overexpressing PSMB5, comprising: the method comprises the following steps:
acquiring a target sequence including a bovine PSMB5 gene sequence:
designing a pair of PCR primers according to the bovine PSMB5 gene sequence, respectively introducing XhoI enzyme cutting sites and XbaI enzyme cutting sites at the 5' ends of the two primers, and amplifying a CDS region sequence containing PSMB5 gene from a bovine genome by using the pair of primers, wherein the length is 792 bp;
constructing an expression vector pcDNA3.1-PSMB 5:
connecting the sequence obtained in the XhoI and XbaI double enzyme digestion step and a pcDNA3.1 vector by using ligase, inserting the sequence into XhoI and XbaI enzyme digestion sites of a plasmid vector pcDNA3.1, and constructing an expression vector pcDNA3.1-PSMB5 of the bovine PSMB5 gene;
and c, transfecting the bovine skeletal muscle satellite cell with pcDNA3.1-PSMB 5:
the obtained expression vector pcDNA3.1-PSMB5 is used for transfecting bovine skeletal muscle satellite cells by adopting a liposome transfection reagent, so that the bovine skeletal muscle satellite cells overexpress PSMB5, and the in-vitro differentiation capacity of the bovine skeletal muscle satellite cells is improved by utilizing the action of PSMB 5;
the PCR primers in the step are SEQ ID No.1 and SEQ ID No. 2.
2. The method of promoting bovine skeletal muscle satellite cell myogenic differentiation by overexpressing PSMB5 according to claim 1, wherein: the bovine PSMB5 gene sequence in the step is GenEID 534640.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710387A (en) * | 2013-12-25 | 2014-04-09 | 天津农学院 | Method for facilitating in vitro multiplication of bovine skeletal muscle satellite cell |
| CN107287242A (en) * | 2017-07-14 | 2017-10-24 | 天津农学院 | A kind of method of promotion bovine muscle satellite cell myogenic differentiation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710387A (en) * | 2013-12-25 | 2014-04-09 | 天津农学院 | Method for facilitating in vitro multiplication of bovine skeletal muscle satellite cell |
| CN107287242A (en) * | 2017-07-14 | 2017-10-24 | 天津农学院 | A kind of method of promotion bovine muscle satellite cell myogenic differentiation |
Non-Patent Citations (1)
| Title |
|---|
| Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation;Ziyou Cui et a;《Mol Cell Biol》;20140131;第34卷(第1期);第107页右栏最后一段至第108页左栏第一段 * |
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Application publication date: 20200407 Assignee: Keyu Xincheng (Tianjin) Technology Development Co.,Ltd. Assignor: TIANJIN AGRICULTURAL University Contract record no.: X2023120000035 Denomination of invention: A method of promoting myogenic differentiation of bovine skeletal muscle satellite cells by overexpressing PSMB5 Granted publication date: 20220517 License type: Common License Record date: 20230927 |
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