CN109680001A - A kind of preparation method reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier - Google Patents
A kind of preparation method reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier Download PDFInfo
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Abstract
The present invention relates to the preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier, effectively solve the problem of that the interference of intramuscular fat cytolipin deposition MAT2A reaches treatment liver cancer medication.Method is, slow virus MAT2A interference carrier is constructed, slow virus MAT2A interference carrier is transfected into the culture medium of the 293T cell containing slow virus carrier, recombinant slow virus pLentiH1-MAT2AshRNA is packed, recombinant slow virus pLentiH1-MAT2AshRNA titer determination calculates virus titer；PLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell, detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis, and fat drips accumulation capacity reduces 48-52% in sh-MAT2A processing group.The method of the present invention is easy to operate, sensitive, reliable and stable, can significantly reduce the expression of MAT2A mRNA and albumen in fat cell, and lipid content level is also decreased obviously in fat cell, realizes the interference effect deposited to pig intramuscular fat cytolipin.
Description
Technical field
The present invention relates to pharmaceutical technology field, especially a kind of reduction intramuscular fat cytolipin deposition MAT2A gene is dry
Disturb the preparation method of carrier.
Background technique
Gene transfer vehicle of the slow virus carrier as a highly effective has obtained in basic and applied research field
It is widely recognized as.RNAi is can to inhibit a kind of tool that specific gene is expressed in normal bio body, and height is protected during evolution
The phenomenon that keeping, the efficient selective degradation of purpose mRNA is induced by double-stranded RNA (double-stranded RNA, dsRNA), benefit
It is mediated with RNA, specifically can block and reduce target gene.Slow virus carrier is as a kind of load for deriving from retrovirus
Body can infect division stage and nondividing phase cell, and efficiency of infection is high.It is mediated using RNA, specifically can block and reduce mesh
Gene expression, while the range of carrier infection cell expands, and the short-hairpin RNAs (shRNAs) of external source both may be used
Research for cell specific gene function, it may also be used for gene therapy.Research shows that there are two types of the adenosines of form for mammal
Methionine transferase, one is the MAT1A of liver specificity, encode MAT I/III；Another is non-liver specificity
MAT2A encodes MATII.Another gene-MAT2B, coding β regulate and control subunit, together with 2 catalytic subunit of α of MAT2A coding
Regulate and control the enzymatic activity of MATII.MATI/III is mainly expressed in liver cell and is maintain the differentiation state of these cells；
MATII is expressed in cell outside all livers, can't detect its expression in normal liver cell, but in the sharp of liver cancer cells
It lives or can be activated when dedifferenting state.In liver cancer cells, c-Myb, Sp1, the transcription factors such as NF- κ B and AP-1 can promote
Expression into the transcription of MAT2A promoter, while MAT2A is able to respond the processing in TNF-α.One as MafK of MAT2A
Transcription inhibition provides transmethylase together with chromatin control gene for SAMe.In addition to this, in the colon cancer of the mankind and
In liver cancer cells, the expression for the regulation Bal-2 that 2 ubiquitination of MAT α can be positive.In the liver cancer cells of rat, PPAR γ is negative to be adjusted
MAT2A gene expression when control is in quiescent condition liver cancer cells, but when turning to state of activation by quiescent condition, then can be with
Raise the expression of MAT2A gene.Meanwhile MAT2A can be specifically with H3K9 transmethylase SETDB1 interaction and in conjunction with MAT2B
Promote the H3K9 of COX-2 gene loci tri-methylated and inhibits COX-2 gene expression.Previous research are sent out using biochip technology
Existing MAT2B and pig intramuscular fat content are closely related, exist in the fat and musculature of lard type and bacon hogs
Significant differential expression, thus it is speculated that porcine intramuscular fat deposition may be influenced.Mainly collect about the research level of MAT2A gene at present
In in terms of, do not have relevant report in terms of Adipocyte Differentiation and its regulatory mechanism.This research passes through building
The slow virus interference carrier of MAT2A gene, reaching reduces intramuscular fat cytolipin deposition, for further research MAT2A regulation
What Adipocyte Differentiation and molecule mechanism realized treatment liver cancer determines technical foundation with medicine pad.
Summary of the invention
Intramuscular rouge is reduced it is an object of the invention to provide a kind of for above situation for the defect for solving the prior art
Fat cytolipin deposits the preparation method of MAT2A gene interference carrier, can effectively solve intramuscular fat cytolipin deposition MAT2A
Interference, reach treatment liver cancer medication the problem of.
The technical solution that the present invention solves is a kind of reduction intramuscular fat cytolipin deposition MAT2A gene interference carrier
Preparation method, comprising the following steps:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM RNAi
Designer screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed, and it is double to be formed
Chain, being respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble(Sh-scramble is experiment contrast
Sequence；Control sequence does not have interference effect to any gene, and experiment has processing, must just have control, that is, pair compared
As；Sh-scramble is also transferred in cell, it is therefore an objective to which all processing are under same level), with processBamH
I andXhoPLenti-H1 carrier connection after I (TaKaRa) double digestion, converts DH5 α competent cell, picking positive colony
It extracts plasmid and carries out sequencing identification after digestion identification is correct, it is ensured that be inserted into the target sequence of carrier and the target gene of design synthesis
Sequence is consistent, obtains slow virus MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
2-4h in fresh culture medium is placed in the 293T cell that cell density is 85-95%, so that cell adapted, described training
Supporting base is that (DMEM is dulbecco's modified eagle to the dry powder of DMEM containing 0.5g in every 100mL PBS buffer solution
The abbreviation of medium), 10 mL(of fetal calf serum buys in Gbico company, the U.S.), 0.22g NaHCO3, 0.26gHepes；So
After transfected, when transfection, first configure two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are slow
(VSVG is a kind of envelope protein to viral+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier, and virus is exactly based on envelope protein
Identify that, subsequently into cell, full name is vesicular stomatitis virus glycoprotein G, the same below with host cell) it is dissolved in 1.5 mL
In serum-free DMEM culture medium without double antibody (serum-free DMEM culture medium without double antibody is referred to as Opti-DMEM culture medium, the same below),
It mixes, serum-free DMEM culture medium without double antibody is the dry powder of DMEM containing 0.4-0.6g (purchase in every 100 mL PBS buffer solution
Buy in Gbico company, the U.S.), 0.18-0.26g NaHCO3, 0.2-0.3gHepes, mix be made；B liquid, for dissolved with lipid
The solution of body: being dissolved in 1-2 mL serum-free DMEM culture medium without double antibody by 30-40 μ l X-tremeGENE HP DNA, is mixed
It is even to be made；By A liquid and B liquid, it is stored at room temperature 4-6min respectively, is uniformly mixed, then be stored at room temperature 18-22min, drops evenly training
It supports in ware, it is slight to shake, put incubator culture, 20-30h observes fluorescent protein expression situation, when 48h and 72h collects respectively
Clear liquid；By the vial supernatant of collection in 1800-2200rpm centrifugation 8-12min removal cell fragment to get the recombinant lentiviral of packaging
Viral pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.4-0.5 μm of filter membrane, is used
80000-120000r·min-1The virion that centrifugation 25-35min is concentrated；According to 8-12 times of volume with water dilution disease
Poison, with 10-2~10-6Concentration gradient infects 293T cell respectively, 3 repetitions of each gradient, and 35-40 DEG C, to be placed in volume be 5%
CO2Incubator culture 48h counts green fluorescence expression, and every hole counts the fluorescence number under 5 visuals field, by formula: virus drop
(pfumL-1)=GFP positive cell number × 0.01 mL of viral dilution multiple ÷ is spent, virus titer is calculated；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP (illustrates that virus infection efficiency is higher) when there is GFP expression in cell, collects rouge respectively
The RNA and albumen of fat cell detect the jamming effectiveness of MAT2A-shRNA using Realtime-PCR and Westernblot, with
Sh-scramble negative control is compared, MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 in fat cell
MRNA jamming effectiveness is 45%, 70% and 55% respectively, collects interference carrier (the MAT2A-shRNA2 interference of MAT2A-shRNA2
Efficiency highest, so MAT2A-shRNA2 is optimum jamming segment, i.e. MAT2A-shRNAp)；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier (abbreviation MAT2A-shRNA2) of lipidosis.
The preparation method of the MAT2A interference carrier of the present invention for reducing pig intramuscular fat cytolipin deposition is easily grasped
Make, is sensitive, it is reliable and stable, MAT2A mRNA in fat cell can significantly reduce by pLentiH1-MAT2A-shRNA slow virus
With the expression of albumen, and lipid content level is also decreased obviously in fat cell, is realized to pig intramuscular fat cytolipin
The interference effect of deposition reaches the purpose for the treatment of disease, is the innovation treated on disease medicament, and economic and social benefit is significant.
Specific embodiment
It elaborates with reference to embodiments with concrete condition to a specific embodiment of the invention.
The present invention in specific implementation, is realized by following embodiment.
Embodiment 1
A kind of preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier of the present invention, including following step
It is rapid:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK- is utilized
iTTMRNAiDesigner screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed
Double-strand is formed, MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble are respectively designated as, with processBamHI andXhoPLenti-H1 carrier connection after I (TaKaRa) double digestion, converts DH5 α competent cell, and picking positive colony extracts
Plasmid carries out sequencing identification after digestion identification is correct, it is ensured that is inserted into the target sequence of carrier and the target-gene sequence of design synthesis
Unanimously, slow virus MAT2A interference carrier is obtained；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 90% to cell density is placed in 3h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10mL, 0.22g NaHCO3, 0.26gHepes；Then
It is transfected, when transfection, first configures two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are sick slowly
Malicious+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed,
Serum-free DMEM culture medium without double antibody is dry powder containing 0.5gDMEM in every 100 mL PBS buffer solution, 0.22g
NaHCO3, 0.26gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 35 μ lX-tremeGENE HP DNA
It is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 5min respectively, mixes
It closes uniformly, then is stored at room temperature 20min, drop evenly in culture dish, it is slight to shake, incubator culture is put, observes fluorescence egg for 24 hours
White expression, when 48h and 72h, collect supernatant respectively；By the vial supernatant of collection in 2000rpm centrifugation 10min removal
Cell fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.45 μm of filter membrane, is used
100000r·min-1The virion that centrifugation 30min is concentrated；According to 10 times of volume with water dilution viruses, with 10-4It is dense
Degree gradient infects 293T cell respectively, 3 repetitions of each gradient, and 37 DEG C, be placed in the CO that volume is 5%2Incubator culture 48h, system
Green fluorescence expression is counted, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP sun
Property cell number × viral dilution multiple ÷ 0.01mL, calculate virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP (illustrates that virus infection efficiency is higher) when there is GFP expression in cell, collects rouge respectively
The RNA and albumen of fat cell detect the jamming effectiveness of MAT2A-shRNA using Realtime-PCR and Westernblot, with
Sh-scramble negative control is compared, MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 in fat cell
MRNA jamming effectiveness is 45%, 70% and 55% respectively, collects interference carrier (the MAT2A-shRNA2 interference of MAT2A-shRNA2
Efficiency highest, so MAT2A-shRNA2 is optimum jamming segment, i.e. MAT2A-shRNAp)；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
Embodiment 2
A kind of preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier of the present invention, including following step
It is rapid:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM
RNAiDesigner screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed to be formed
Double-strand is respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, with processBamHI andXhoI (TaKaRa) double enzymes
PLenti-H1 carrier connection after cutting converts DH5 α competent cell, and picking positive colony extracts plasmid, and digestion identification is correct
Afterwards, sequencing identification is carried out, it is ensured that the target sequence for being inserted into carrier is consistent with the target-gene sequence of design synthesis, obtains slow virus
MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 85% to cell density is placed in 2h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10 mL, 0.22g NaHCO3, 0.26gHepes；So
After transfected, when transfection, first configure two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are slow
Viral+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed
Even, serum-free DMEM culture medium without double antibody is the dry powder of DMEM containing 0.4g in every 100 mL PBS buffer solution, 0.18g
NaHCO3, 0.2gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 30 μ l X-tremeGENE HP DNA
It is dissolved in 1 mL serum-free DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 4min respectively, mixes
Uniformly, then it is stored at room temperature 18min, dropped evenly in culture dish, it is slight to shake, incubator culture is put, 20h observes fluorescin
Expression, when 48h and 72h, collect supernatant respectively；The vial supernatant of collection is thin in 1800rpm centrifugation 12min removal
Born of the same parents' fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.4 μm of filter membrane, uses 80000r
min-1The virion that centrifugation 35min is concentrated；According to 8 times of volume with water dilution viruses, with 10-3Concentration gradient difference
Infect 293T cell, 3 repetitions of each gradient, 35 DEG C, be placed in volume be 5% CO2Incubator culture 48h counts green fluorescence
Expression, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP positive cell number
× viral dilution multiple ÷ 0.01mL calculates virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP (illustrates that virus infection efficiency is higher) when there is GFP expression in cell, collects rouge respectively
The RNA and albumen of fat cell detect the jamming effectiveness of MAT2A-shRNA using Realtime-PCR and Westernblot, with
Sh-scramble negative control is compared, MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 in fat cell
MRNA jamming effectiveness is 45%, 70% and 55% respectively, collects interference carrier (the MAT2A-shRNA2 interference of MAT2A-shRNA2
Efficiency highest, so MAT2A-shRNA2 is optimum jamming segment, i.e. MAT2A-shRNAp)；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
Embodiment 3
A kind of preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier of the present invention, including following step
It is rapid:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM RNAi
Designer screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed, and it is double to be formed
Chain is respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble, with processBamHI andXho I
(TaKaRa) the pLenti-H1 carrier connection after double digestion, converts DH5 α competent cell, and picking positive colony extracts plasmid,
After digestion identification is correct, sequencing identification is carried out, it is ensured that the target sequence for being inserted into carrier is consistent with the target-gene sequence of design synthesis,
Obtain slow virus MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 95% to cell density is placed in 4h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10 mL, 0.22g NaHCO3, 0.26gHepes；So
After transfected, when transfection, first configure two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are slow
Viral+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed
Even, serum-free DMEM culture medium without double antibody is the dry powder of DMEM containing 0.6g in every 100 mL PBS buffer solution, 0.26g
NaHCO3, 0.3gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 40 μ l X-tremeGENE HP DNA
It is dissolved in 2 mL serum-frees DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 6min respectively, mixes
Uniformly, then it is stored at room temperature 22min, dropped evenly in culture dish, it is slight to shake, incubator culture is put, 30h observes fluorescin
Expression, when 48h and 72h, collect supernatant respectively；The vial supernatant of collection is removed into cell in 2200rpm centrifugation 8min
Fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.5 μm of filter membrane, uses 120000r
min-1The virion that centrifugation 25min is concentrated；According to 12 times of volume with water dilution viruses, with 10-5Concentration gradient difference
Infect 293T cell, 3 repetitions of each gradient, 40 DEG C, be placed in volume be 5% CO2Incubator culture 48h counts green fluorescence
Expression, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP positive cell number ×
Viral dilution multiple ÷ 0.01mL calculates virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP (illustrates that virus infection efficiency is higher) when there is GFP expression in cell, collects rouge respectively
The RNA and albumen of fat cell detect the jamming effectiveness of MAT2A-shRNA using Realtime-PCR and Westernblot, with
Sh-scramble negative control is compared, MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 in fat cell
MRNA jamming effectiveness is 45%, 70% and 55% respectively, collects interference carrier (the MAT2A-shRNA2 interference of MAT2A-shRNA2
Efficiency highest, so MAT2A-shRNA2 is optimum jamming segment, i.e. MAT2A-shRNAp)；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
The MAT2A interference method of the present invention for reducing pig intramuscular fat cytolipin deposition is easy to operate, special, clever
It is quick, it is reliable and stable, MAT2A mRNA and egg in fat cell can significantly reduce by pLentiH1-MAT2A-shRNA slow virus
White expression, and lipid content level is also decreased obviously in fat cell, is realized and is deposited to pig intramuscular fat cytolipin
Interference effect, economic and social benefit is significant, by repeatedly test repeatedly achieve it is consistent as a result, related experiment data
It is as follows:
Test the building and identification of 1:MAT2A recombinant slow virus interference carrier
Utilize Invitrogen online software BLOCK-iT RNAi Designer (https: //www.rnaid esigner.
Invitrogen.com/rnaiexpress/ pig) is designedMATEach 3 shRNA of 2A gene, and the single-stranded few core of nothing to do with sequence
Nucleotide sequence is together in the raw work synthesis (table 1) in Shanghai.Double-stranded DNA is formed after annealing, with processBamHI andXhoI double digestion
LentiH1 carrier connection, conversion extract plasmid after, digestion is identified that successful plasmid send company to be sequenced, it is ensured that insertion carrier
Sequence with design synthesis target gene chain it is completely the same.It is identified,MATThe few nucleosides of the shRNA nothing to do with sequence of 2A gene
For acid without mutation, insertion is correct.
The shRNA sequential parameter of 1 pig MAT2A of table
Table 1 Design of shRNA targeting on porcine MAT2A gene
Test the packaging and titer determination of 2 pLentiHl-MAT2A slow virus interference carriers
293T cell density be 90% or so, and it is in good condition when, using X-tremeGENE-HP DNA transfection reagent with
Recombinant plasmid and packaging plasmid (CMV- Δ 8.9 and CMV-VSVG) transfect 293T cell together, observe green fluorescence egg after 48h
The expression of white (GFP).Cell state is good, and green fluorescence intensity is high, 90% or more cell expressing green fluorescent protein；After
Continuous culture, collects viral supernatants, and carry out centrifugal concentrating after transfected plasmids 48h and 72h.Using viral gradient dilution method
Measure virus titer.Virus titer result is respectively 7.31 × 107Pfu/mL, 6.70 × 107Pfu/mL and 7.0 × 107
Pfu/ mL meets the requirement of experiment for infecting Primary adipocyte.
Experiment 3: the effect experiment of pig intramuscular fat cell is infected.
Pig intramuscular fat cell is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscope after 48h
The expression of lower observation green fluorescent protein GFP illustrates that virus infection efficiency is higher when there is more GFP expression in cell；
The RNA and albumen for collecting fat cell respectively utilize the dry of Realtime-PCR and Western blot detection MAT2A-shRNA
Disturb efficiency；
Experiment shows compared with sh-scramble negative control, in fat cell MAT2A-shRNA1, MAT2A-shRNA2,
The mRNA jamming effectiveness of MAT2A-shRNA3 is 45%, 70% and 55% or so respectively, MAT2A-shRNA2 jamming effectiveness highest,
So MAT2A-shRNA2 is optimum jamming segment (carrier), i.e. MAT2A-shRNAp.
Experiment 4: influence experiment of the interference MAT2A to pig intramuscular fat cell differentiation lipidosis.
After infecting the intramuscular Preadipocyte In Vitro 48h of pig with pLentiH1-MAT2AshRNA, differential medium induction is at rouge point
Change to maturation, after differentiation the 8th day, detected with oil red O lipid accumulation situation.Compared with sh-scramble control group, fat drips build up energy
Power significantly reduces in sh-MAT2A processing group, can reach 50% or so.
Experiment shows that the MAT2A gene interference of lentivirus mediated significantly suppresses the lipid accumulation of pig intramuscular fat cell.
It tests 5 pLentiHl-MAT2A/2B shRNA and infects the intramuscular Preadipocyte In Vitro effect experiment of pig
To detect packaging slow virus carrier to the jamming effectiveness of MAT2A gene in pig Primary adipocyte.Experiment will be viral
Carrier sh-scaramble (is set as compareing), and sh-MAT2A infects pig intramuscular fat cell, and 24 h observe green fluorescent protein
Expression, but infect efficiency is lower；There is the green fluorescent protein (GFP) of large area in intramuscular fat cell after 72h.It receives respectively
Collect RNA using the disturbed condition of Real-time qPCR detection sh-MAT2A, compared with sh-scramble control, sh-MAT2A-
2 interference effects are best, and jamming effectiveness achieves the desired results 70% or more.In order to further prove whether sh-MAT2A is right
MAT2A protein expression level has an impact, and is verified using West bolt experiment and analysis of protein.InterferenceMAT2A gene, MAT2A
Protein expression level reduces by 40% or so, reaches extremely significant level；These the result shows thatMAT2A slow virus interference carrier is built into
Function can carry out subsequent experimental.
6 interference MAT2A gene of experiment inhibits pig intramuscular fat cytolipin sedimentation experiment
The intramuscular Preadipocyte In Vitro of pig (sets transfection nonsense sequence after infecting the interference fragment sh-MAT2A 48h that slow virus carries
Sh-scramble is control group), MDI culture medium induction differentiation to maturation.The results show that with sh-scramble control group phase
Than fat drips accumulation capacity significantly reduces in sh-MAT2A processing group.Absorbance quantitative analysis results show,MAT2A gene
In the pig intramuscular fat cell of missing, the ability that fat drips are built up is substantially less than control group.
Experiment clearly shows that the MAT2A interference method of the present invention for reducing pig intramuscular fat cytolipin deposition is simple
Just, easy to operate, special, sensitive, it is reliable and stable, fat cell can significantly reduce by pLentiH1-MAT2A-shRNA slow virus
The expression of middle MAT2A mRNA and albumen, and lipid content level is also decreased obviously in fat cell, reachable 50% or so,
It realizes the interference effect deposited to pig intramuscular fat cytolipin, can be effectively used for preparation and pig intramuscular fat cytolipin is deposited
The drug of interference, has opened up the new way for the treatment of liver-cancer medicine, and economic and social benefit is significant.
Sequence table
<110>Xinyang College of Agriculture and Forestry
<120>a kind of preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 510
<212> mRNA
<213> Sus scrofa methionine adenosyltransferase 2A (MAT2A), mRNA
<400> 1
atgaacggac agctcaacgg cttccacgac gccttcatcg aggagggcac gttcctcttc 60
acgtccgagt ctgtagggga aggccaccca gataagattt gtgaccagat cagtgatgct 120
gtccttgatg cccaccttca gcaggaccct gatgccaaag tggcttgtga aactgttgct 180
aaaactggaa tgatccttct tgctggggaa attacatcca gagctgctgt tgactatcag 240
aaagtggttc gtgaaaccat taagcacata ggatatgatg attcttccaa agggtttgac 300
tacaagactt gtaatgtgct agtggccttg gagcagcagt caccagatat tgctcaaggt 360
gttcatcttg accggaatga ggaagacatt ggtgcaggag accagggttt gatgtttggt 420
tatgccactg atgaaactga ggaatgtatg cctttaacca ttgtcttagc acacaagctc 480
aatgccaaac tggctgaact acgccggaat ggcactttgc cttggttacg cccagattct 540
aaaactcaag tgactgtgca gtatatgcag gatcgaggtg ctgtgcttcc catcagagtc 600
cacacaattg ttatatctgt tcagcatgat gaagaagttt gtcttgatga gatgagagat 660
gccctaaagg agaaagtcat caaagcagtt gtacctgcaa aatatcttga tgaggataca 720
atctatcatc tacagccaag tggcagattt gttattggtg ggcctcaggg tgatgccggt 780
ttgactggtc ggaaaatcat tgtggacact tacggcggtt ggggagctca tggaggaggt 840
gccttttcag gaaaagatta caccaaggtg gaccgttcag ctgcttatgc tgctcgttgg 900
gtggcaaaat cccttgttaa aggaggtctg tgcagaaggg ttcttgttca ggtctcttat 960
gctattggag tatctcatcc attgtctatc tccattttcc attatggcac ctctcagaag 1020
agtgagagag agctattaga gattgtgaag aagaattttg acctccgccc tggggtcatt 1080
gtcagggatc tggatctgaa gaagccaatt tatcagagga ctgcagccta tggccacttt 1140
ggtagggaca gcttcccatg ggaagtgccc aaaaagctta agtattga 1188
Claims (5)
1. a kind of preparation method for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier, which is characterized in that including
Following steps:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM RNAi
Designer screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed, and it is double to be formed
Chain is respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble, with processBamHI andXho I
(TaKaRa) the pLenti-H1 carrier connection after double digestion, converts DH5 α competent cell, and picking positive colony extracts plasmid,
After digestion identification is correct, sequencing identification is carried out, it is ensured that the target sequence for being inserted into carrier is consistent with the target-gene sequence of design synthesis,
Obtain slow virus MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
2-4h in fresh culture medium is placed in the 293T cell that cell density is 85-95%, so that cell adapted, described training
Supporting base is the dry powder of DMEM containing 0.5g, fetal calf serum 10 mL, 0.22g NaHCO in every 100mL PBS buffer solution3,
0.26gHepes；Then it is transfected, when transfection, first configures two kinds of liquid: A liquid, for the solution dissolved with plasmid, by step
(1) it is without double antibody to be dissolved in 1.5 mL serum-frees by+7.5 μ g Δ 8.9+5 μ gVSVG of 10 μ g slow virus MAT2A interference carrier prepared
It in DMEM culture medium, mixes, serum-free DMEM culture medium without double antibody is to contain 0.4- in every 100 mL PBS buffer solution
0.6g DMEM dry powder, 0.18-0.26g NaHCO3, 0.2-0.3gHepes, mix be made；B liquid, for dissolved with the molten of liposome
Liquid: being dissolved in 1-2 mL serum-free DMEM culture medium without double antibody by 30-40 μ l X-tremeGENE HP DNA, is mixed and is made；
By A liquid and B liquid, it is stored at room temperature 4-6min respectively, is uniformly mixed, then be stored at room temperature 18-22min, drops evenly in culture dish,
Slight concussion, puts incubator culture, and 20-30h observes fluorescent protein expression situation, when 48h and 72h collects supernatant respectively；It will
The vial supernatant of collection is in 1800-2200rpm centrifugation 8-12min removal cell fragment to get the recombinant slow virus of packaging
PLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.4-0.5 μm of filter membrane, is used
80000-120000r·min-1The virion that centrifugation 25-35min is concentrated；According to 8-12 times of volume with water dilution disease
Poison, with 10-2~10-6Concentration gradient infects 293T cell respectively, 3 repetitions of each gradient, and 35-40 DEG C, to be placed in volume be 5%
CO2Incubator culture 48h counts green fluorescence expression, and every hole counts the fluorescence number under 5 visuals field, by formula: virus drop
(pfumL-1)=GFP positive cell number × 0.01 mL of viral dilution multiple ÷ is spent, virus titer is calculated；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP collects the RNA and albumen of fat cell when there is GFP expression in cell respectively, utilizes
The jamming effectiveness of Realtime-PCR and Westernblot detection MAT2A-shRNA, compared with sh-scramble negative control,
In fat cell the mRNA jamming effectiveness of MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 be 45% respectively,
70% and 55%, collect the interference carrier of MAT2A-shRNA2；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
2. the preparation method according to claim 1 for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier,
Characterized by comprising the following steps:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK- is utilized
iTTMRNAiDesigner screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed
Double-strand is formed, MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble are respectively designated as, with processBamHI andXhoPLenti-H1 carrier connection after I (TaKaRa) double digestion, converts DH5 α competent cell, and picking positive colony extracts
Plasmid carries out sequencing identification after digestion identification is correct, it is ensured that is inserted into the target sequence of carrier and the target-gene sequence of design synthesis
Unanimously, slow virus MAT2A interference carrier is obtained；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 90% to cell density is placed in 3h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10mL, 0.22g NaHCO3, 0.26gHepes；Then
It is transfected, when transfection, first configures two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are sick slowly
Malicious+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed,
Serum-free DMEM culture medium without double antibody is dry powder containing 0.5gDMEM in every 100 mL PBS buffer solution, 0.22g
NaHCO3, 0.26gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 35 μ lX-tremeGENE HP DNA
It is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 5min respectively, mixes
It closes uniformly, then is stored at room temperature 20min, drop evenly in culture dish, it is slight to shake, incubator culture is put, observes fluorescence egg for 24 hours
White expression, when 48h and 72h, collect supernatant respectively；By the vial supernatant of collection in 2000rpm centrifugation 10min removal
Cell fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.45 μm of filter membrane, is used
100000r·min-1The virion that centrifugation 30min is concentrated；According to 10 times of volume with water dilution viruses, with 10-4It is dense
Degree gradient infects 293T cell respectively, 3 repetitions of each gradient, and 37 DEG C, be placed in the CO that volume is 5%2Incubator culture 48h, system
Green fluorescence expression is counted, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP sun
Property cell number × viral dilution multiple ÷ 0.01mL, calculate virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP collects the RNA and albumen of fat cell when there is GFP expression in cell respectively, utilizes
The jamming effectiveness of Realtime-PCR and Westernblot detection MAT2A-shRNA, compared with sh-scramble negative control,
In fat cell the mRNA jamming effectiveness of MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 be 45% respectively,
70% and 55%, collect the interference carrier of MAT2A-shRNA2；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
3. the preparation method according to claim 1 for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier,
Characterized by comprising the following steps:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM
RNAiDesigner screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed to be formed
Double-strand is respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, with processBamHI andXhoI (TaKaRa) double enzymes
PLenti-H1 carrier connection after cutting converts DH5 α competent cell, and picking positive colony extracts plasmid, and digestion identification is correct
Afterwards, sequencing identification is carried out, it is ensured that the target sequence for being inserted into carrier is consistent with the target-gene sequence of design synthesis, obtains slow virus
MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 85% to cell density is placed in 2h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10 mL, 0.22g NaHCO3, 0.26gHepes；So
After transfected, when transfection, first configure two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are slow
Viral+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed
Even, serum-free DMEM culture medium without double antibody is the dry powder of DMEM containing 0.4g in every 100 mL PBS buffer solution, 0.18g
NaHCO3, 0.2gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 30 μ l X-tremeGENE HP DNA
It is dissolved in 1 mL serum-free DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 4min respectively, mixes
Uniformly, then it is stored at room temperature 18min, dropped evenly in culture dish, it is slight to shake, incubator culture is put, 20h observes fluorescin
Expression, when 48h and 72h, collect supernatant respectively；The vial supernatant of collection is thin in 1800rpm centrifugation 12min removal
Born of the same parents' fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.4 μm of filter membrane, uses 80000r
min-1The virion that centrifugation 35min is concentrated；According to 8 times of volume with water dilution viruses, with 10-3Concentration gradient difference
Infect 293T cell, 3 repetitions of each gradient, 35 DEG C, be placed in volume be 5% CO2Incubator culture 48h counts green fluorescence
Expression, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP positive cell number
× viral dilution multiple ÷ 0.01mL calculates virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP collects the RNA and albumen of fat cell when there is GFP expression in cell respectively, utilizes
The jamming effectiveness of Realtime-PCR and Westernblot detection MAT2A-shRNA, compared with sh-scramble negative control,
In fat cell the mRNA jamming effectiveness of MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 be 45% respectively,
70% and 55%, collect the interference carrier of MAT2A-shRNA2；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
4. the preparation method according to claim 1 for reducing intramuscular fat cytolipin deposition MAT2A gene interference carrier,
Characterized by comprising the following steps:
(1) building of slow virus MAT2A interference carrier:
According to pig MAT2A gene order in GenBank, Invitrogen company online software BLOCK-iT is utilizedTM RNAi
Designer screens 3 shRNA target sequences and unrelated control sequence respectively, and the single-stranded oligonucleotide of synthesis is annealed, and it is double to be formed
Chain is respectively designated as MAT2A-sh1, MAT2A-sh2, MAT2A-sh3, Sh-scramble, with processBamHI andXho I
(TaKaRa) the pLenti-H1 carrier connection after double digestion, converts DH5 α competent cell, and picking positive colony extracts plasmid,
After digestion identification is correct, sequencing identification is carried out, it is ensured that the target sequence for being inserted into carrier is consistent with the target-gene sequence of design synthesis,
Obtain slow virus MAT2A interference carrier；
(2) recombinant slow virus pLentiH1-MAT2AshRNA is packed:
Slow virus MAT2A interference carrier prepared by step (1) is transfected into the culture medium of the 293T cell containing slow virus carrier
In, recombinant slow virus pLentiH1-MAT2AshRNA is packed, method is:
The 293T cell for being 95% to cell density is placed in 4h in fresh culture medium, so that cell adapted, the culture medium is
The dry powder of DMEM containing 0.5g in every 100 mL PBS buffer solution, fetal calf serum 10 mL, 0.22g NaHCO3, 0.26gHepes；So
After transfected, when transfection, first configure two kinds of liquid: A liquid, for the solution dissolved with plasmid, the 10 μ g prepared by step (1) are slow
Viral+7.5 μ g Δ 8.9+5 μ gVSVG of MAT2A interference carrier is dissolved in 1.5 mL serum-frees DMEM culture medium without double antibody, is mixed
Even, serum-free DMEM culture medium without double antibody is the dry powder of DMEM containing 0.6g in every 100 mL PBS buffer solution, 0.26g
NaHCO3, 0.3gHepes, mix be made；B liquid, for the solution dissolved with liposome: by 40 μ l X-tremeGENE HP DNA
It is dissolved in 2 mL serum-frees DMEM culture medium without double antibody, mixes and be made；By A liquid and B liquid, it is stored at room temperature 6min respectively, mixes
Uniformly, then it is stored at room temperature 22min, dropped evenly in culture dish, it is slight to shake, incubator culture is put, 30h observes fluorescin
Expression, when 48h and 72h, collect supernatant respectively；The vial supernatant of collection is removed into cell in 2200rpm centrifugation 8min
Fragment to get packaging recombinant slow virus pLentiH1-MAT2AshRNA supernatant；
(3) recombinant slow virus pLentiH1-MAT2AshRNA titer determination:
Recombinant slow virus pLentiH1-MAT2AshRNA supernatant prepared by step (2) is crossed into 0.5 μm of filter membrane, uses 120000r
min-1The virion that centrifugation 25min is concentrated；According to 12 times of volume with water dilution viruses, with 10-5Concentration gradient difference
Infect 293T cell, 3 repetitions of each gradient, 40 DEG C, be placed in volume be 5% CO2Incubator culture 48h counts green fluorescence
Expression, every hole counts the fluorescence number under 5 visuals field, by formula: virus titer (pfumL-1)=GFP positive cell number ×
Viral dilution multiple ÷ 0.01mL calculates virus titer；
4) pLentiH1-MAT2AshRNA infects the detection of pig intramuscular fat cell:
Pig intramuscular fat cell 48h is infected respectively with pLentiH1-MAT2A-shRNA slow virus, in fluorescence microscopy microscopic observation
The expression of green fluorescent protein GFP collects the RNA and albumen of fat cell when there is GFP expression in cell respectively, utilizes
The jamming effectiveness of Realtime-PCR and Westernblot detection MAT2A-shRNA, compared with sh-scramble negative control,
In fat cell the mRNA jamming effectiveness of MAT2A-shRNA1, MAT2A-shRNA2, MAT2A-shRNA3 be 45% respectively,
70% and 55%, collect the interference carrier of MAT2A-shRNA2；
5) detection of the interference carrier MAT2A-shRNA2 to pig intramuscular fat cell differentiation lipidosis:
After pLentiH1-MAT2A-shRNA infects the intramuscular Preadipocyte In Vitro 48h of pig, differential medium induction is broken up 8 days at rouge
To maturation, detected with oil red O lipid accumulation situation, compared with sh-scramble control group, fat drips accumulation capacity is at sh-MAT2A
It is significantly reduced in reason group, reaches 48-52%, the MAT2A gene interference of as lentivirus mediated significantly inhibits pig intramuscular fat cell
The MAT2A gene interference carrier of lipidosis.
5. the reduction intramuscular fat cytolipin deposition MAT2A gene interference of any one of claims 1 or 2-4 the method preparation
Carrier reduces the application in intramuscular fat cytolipin deposition of medicament in preparation.
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039342A (en) * | 2015-08-06 | 2015-11-11 | 李家平 | siRNA capable of inhibiting MAT2A genetic expression and application of siRNA |
Non-Patent Citations (5)
| Title |
|---|
| CUNZHEN ZHAO ET AL.,: ""MAT2A promotes porcine adipogenesis by mediating H3K27me3 at Wnt10b locus and repressing Wnt/β-catenin signaling"", 《BBA-MOLECULAR AND CELL BIOLOGY OF LIPIDS》 * |
| QUN WANG ET AL.,: ""Inhibition of hepatocelluar carcinoma MAT2A and MAT2beta gene expressions by single and dual small interfering RNA"", 《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》 * |
| TAO ZHANG ET AL.,: ""Overexpression of methionine adenosyltransferase II alpha (MAT2A) in gastric cancer and induction of cell cycle arrest and apoptosis in SGC-7901 cells by shRNA-mediated silencing of MAT2A gene"", 《ACTA HISTOCHEMICA》 * |
| 杨永青等: ""慢病毒载体介导shRNA干扰IL-6Rα基因表达削弱IL-6对猪脂肪细胞分化的影响"", 《中国生物化学与分子生物学报》 * |
| 赵存真等: ""过表达MAT2A基因促进猪肌内脂肪细胞分化的研究"", 《中国畜牧兽医》 * |
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