CN110317831A - A kind of research method of the mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced - Google Patents
A kind of research method of the mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced Download PDFInfo
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Abstract
The present invention relates to cardiologic medical technical fields, and disclose a kind of research method of the mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced, it is grouped by cell, the acquisition of Ube3a gene, the packaging of Ube3a adenovirus, transfection and data analysis and etc. come observe Ube3a Iso induce myocardial hypertrophy during expression quantity situation of change, judge that Ube3a continuous cropping whether related with the generation of myocardial hypertrophy is used, then pass through the knockout of Ube3a gene, determine that specificity and Ube3a of the Ube3a in myocardial hypertrophy generating process are promotion or interception to myocardial hypertrophy；It is acted on by the overexpression of Ube3a gene, further clarifies the promotion or interception of Ube3a, and combine the state of development of cell after Ube3a knockout and overexpression, determine the causality between the effect of Ube3a and cardiac myocyte hypertrophy.Determine that Ube3a gene is able to suppress the cardiac myocyte hypertrophy of Iso induction by this research method, and MMP-9 albumen takes part in this molecular mechanism, provides new target gene for the prevention and treatment of myocardial hypertrophy.
Description
Technical field
The present invention relates to cardiologic medical technical field, specially a kind of be3a gene is in the myocardial hypertrophy that Iso is induced
The research method of mechanism of action.
Background technique
Ubiquitin-proteasome pathway the 1980s (the ubiquitin proteasome pathway, UPP)
Be the discovery that illustrate Intracellular proteolysis effect milestone event, UPP the cycle regulating of cell, DNA reparation, apoptosis,
Immune response, antigen processing, neurodevelopment, synaptic plasticity, muscular atrophy, hormone control, stress reaction and aging etc.
All play certain function.E3 ubiquitin ligase decides the high efficiency and accurate selectivity of UPP reaction.E3 ligase ginseng
With the normal cleavage of intracellular most albumen, regulate and control the synthesis of nucleus and cytoplasm internal protein and putting down for degradation
Weighing apparatus, once being hindered, body can generate a series of stress reaction, even result in the damage of tissue and organ.
E3 ubiquitin ligase is divided into three categories: HECT (homologous to E6AP carboxyl terminus) structure
Domain family, Ring structural domain family and U-box structural domain family.Only the HECT structural domain family of the mankind has more than 100 kinds of connections
Enzyme, Ube3a are a member therein.About Ube3a, studying most extensive, the most in-depth is to make one syndrome field in day.In fact
The relationship of Ube3a and other diseases is also appeared in the newspapers repeatly, is just reported early in J Veenstra-Vander Weele in 1999
The mutation of Ube3a gene can result in the generation of self-closing disease, the research constantly quilt of subsequent Ube3a and many tissues and organ disease
It confirms, these tissues include brain, mammary gland, cervix, lymph etc., but the research in terms of Ube3a and heart disease is seldom.
Ube3a coding region sequence overall length is about 2.6kb, is located at chromosome 15q11-q13, is divided into three transcripts, three kinds of codified pressures
Type albumen, respectively 98.9kDa, 101.1kDa and 101.7kDa albumen, and these three albumen all have the work of protein degradation
With.By to the relevant gene expression profile data of cardiovascular disease in Gene Expression Omnibus (GEO) database
The analysis and screening of Informatics Method are carried out, confirmation Ube3a and myocardial hypertrophy and heart failure very likely have relationship.
The certain E3 ligases of display have been reported and have differences expression in heart tissue, even belong to one with Ube3a
Other E3 ligase members of a family have been found to participate in the generation of regulation myocardial hypertrophy.In the E3 family of discovery earliest
Atrogin-1 albumen selective expression in cardiac muscle and striated muscle tissue, under the metabolism state in hungry or contraction of muscle,
The expression quantity of Atrogin-1 dramatically increases, this from side has confirmed Atrogin-1 in musculature, and there are certain effects.?
The MURF-1 albumen expressed in cardiac muscle cell can result in titin M and distinguish solution, and make myofilament thickening.It is ground based on above-mentioned
Study carefully, sight is transferred to myocardial hypertrophy Mechanism Study field by scientific research personnel.The volume of cardiac muscle cell is that have albumen synthesis and albumen
The relationship of degradation determines, and the normal protein degradation effect of body mainly completed by ubiquitination degradation pathway.It is myocardium thin
Calcineurin is as the response to cause of disease sexual stimulus in born of the same parents, can by conjunction with the α-actinine in the myocardium area Z- come
Promoting myocardial hypertrophy, Li et al. confirms that ubiquitination ligase E3 plays important regulating and controlling effect in myocardium protein metabolism earliest,
The paper ligatures building myocardial hypertrophy model by thoracic aorta, and the trangenic mice that discovery Atrogin-1 is overexpressed can subtract
Slow myocardial hypertrophy, Atrogin-1 are assembled into one kind with ubiquitination streptokinase activity by combining with Skp1, Cull and Rucl
SCFatrogin-1 complex living.Experiment can reinforce calcineurin to the downward of Atrogin-1 by siRNA
Activity promotes myocardial hypertrophy；The overexpression of Atrogin-1 reduces the level of calcineurin in transgenic mice, subtracts
Myocardial hypertrophy is delayed；Demonstrating Atrogin-1 by ubiquitination reaction is acted on by the ubiquitination to calcineurin
Horizontal to reduce its, the ubiquitination hydrolysis for showing that SCFatrogin-1 is mediated is able to suppress myocardial hypertrophy caused by lesion stimulation.
The murine double minute gene 2 (MDM2) of E3 family is most important for the endocardial cushion form generation during heart development, also grinds
Cardiac myocyte hypertrophy, generating process of the MDM2 in cardiovascular disease can be alleviated by E3 ubiquitinbond enzyme effect by studying carefully display MDM2
In have a decisive role.In addition, the MURF-1 in E3 connection enzyme family is overexpressed energy in new born rats cardiac muscle cell
The cell protein synthesis for inhibiting PKC ε to mediate, alleviates myocardial hypertrophy, and MURF-1 plays one in different signal paths
Fixed effect, such as Akt/mTOR access, illustrate ubiquitin protein regulation myocardial hypertrophy signal path may be intersect,
Synergistic effect.
Comprehensive current research report, we can speculate that Ube3a probably takes part in the generating process of myocardial hypertrophy,
And the accumulation of cardiac mast cells internal protein, the increase of volume, it is possible to which being obstructed with Ube3a ubiquitin protein degradation function has
It closes, it is suppressed that this effect of being obstructed may be embodied in Ube3a protein expression level, it is also possible to pass through certain methylations, glycosyl
The inhibition or activation of the modifications such as change, phosphorylation cause enzymatic vigor to decline.Therefore it is lured by observation Ube3a in Iso
The situation of change for leading expression quantity during myocardial hypertrophy judges that Ube3a continuous cropping whether related with the generation of myocardial hypertrophy is used, so
The knockout for passing through Ube3a gene afterwards determines specificity and Ube3a of the Ube3a in myocardial hypertrophy generating process to myocardium fertilizer
It is promotion or interception greatly；It is acted on by the overexpression of Ube3a gene, further clarifies the promotion or prevention of Ube3a
Effect, and combine Ube3a knock out and be overexpressed after cell state of development, determine Ube3a effect and cardiac myocyte hypertrophy it
Between causality.The new gene that myocardial hypertrophy occurs is participated in for discovery, provides new drug to myocardial hypertrophy is treated and prevented
Action target spot needs a kind of research method of the mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced thus.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the work the present invention provides a kind of Ube3a gene in the myocardial hypertrophy that Iso is induced
With the research method of mechanism, has the advantages that rigorous accurate, solve determining Ube3a gene in the myocardial hypertrophy that Iso is induced
Mechanism of action problem.
(2) technical solution
To achieve the above object, the invention provides the following technical scheme: the cardiac muscle fertilizer that a kind of Ube3a gene is induced in Iso
The research method of big-and-middle mechanism of action, comprising the following steps:
(1) cell is grouped: cell is divided into Iso induction grouping and gene knockout and is overexpressed grouping.Iso induction point
Cell culture is spread disk using six orifice plates by group, and 2ml cell culture medium is added in every hole.2 μ l are added into cellular control unit culture medium
PBS is as placebo；Drug-induced group of the Iso Iso solution for being added 2 μ l 10mM, is made final concentration of 10 μM of the cell of Iso
Culture medium, successively cultivated according to time gradient 12h, for 24 hours, 48h.Gene knockout and overexpression, which are grouped, uses six for cell culture
Orifice plate spreads disk, and 2ml cell culture medium is added in every hole.2 μ l PBS are added as placebo to negative control group, knock out and cross table
2 μ l PBS are added as placebo up to the control group in group, final concentration of 10 μM of the cell training of Iso is made in other Iso induction groups
Base is supported, 12h, for 24 hours and 48h is successively cultivated according to time gradient.
(2) acquisition of Ube3a gene: after Preparatory work of experiment, the competent escherichia coli cell saved in -80 DEG C of refrigerators is taken out
And Ube3a plasmid is placed on ice to melt, then a clean EP pipe is taken to sequentially add 30ul competent cell and 3ul plasmid, with shifting
Liquid device blows and beats ice bath 15min after mixing repeatedly, and the EP pipe insertion water-bath kickboard and being put into rapidly after ice bath 15min is preheated in advance
The heat shock 45s into 42 DEG C of water-baths, then 2min on ice is put back to rapidly, 900 μ l LB liquid mediums are added into EP pipe, put
Enter 37 DEG C of shaking tables, 180rpm cultivates 1h, the turbidity of culture checked, in super-clean bench, according to the Bacillus coli cells of culture
Turbidity takes a certain amount of be equably added dropwise on the mycin LB solid medium of benzyl containing ammonia of good plate, to use spreading rod in advance
Bacterium solution is equably coated with and is opened, is placed in biochemical cultivation case and cultivates, after cultivating 14-18h, checks bacterium colony growing state, if
Colony density reaches 60%-80%, terminates growth, and the clean 15ml test tube of access branch is separately added into 4ml LB liquid into test tube
Body culture medium picks them separately different monoclonal colonies and is inoculated in different test tubes, and in 37 DEG C of shaking tables, 180rpm cultivates 12-16h
Left and right, mini-scale plasmid extracting.
(3) packaging of Ube3a adenovirus: experiment first passes through PCR amplification and obtains containing restriction enzyme digestion sites
Ube3a target gene template segments, then by restriction endonuclease by viral vectors double digestion, by T4 ligase by target fragment group
It is attached in adenovirus vector, building recombination Ube3a is overexpressed adenoviral plasmid.Pass through the cotransfection with skeleton plasmid, packaging
Ube3a is overexpressed adenopathy.
(4) transfect: transfection includes the transfection and adenovirus infection of siRNA.The transfection procedure of siRNA is synthesis
The siRNA powder processed sterile water of DEPC dissolves, and is configured to 20 μM of solution, is then calculated according to the cell hole count of transfection
SiRNA (the 3 μ l of about 7 μ l RNAiMAX and 6 μ l synthesis are added generally for the hole of 3.5cm diameter for the transfection liquid for needing to prepare
RNAi#1 and 3 μ l RNAi#2) solution.Adenovirus infection takes the adenovirus solution of packaged 1010 titre of 5 μ l or more direct
It is added in 2ml cell culture medium, adenovirus will voluntarily infected cell.
(5) data are analyzed: data are analyzed by using SPSS16.0, and the comparison between group is analyzed using independent samples t test,
Comparison between multiple groups examines (ANOVADunnett ' s post hoc) using single factor test Dunnett ' s t, and data result is with Mean
± SD indicates that p < 0.05 indicates statistically significant sex differernce.
Preferably, the adenovirus vector first carries out adenovirus vector construct, then carries out the amplification of adenovirus vector.
Preferably, the adenovirus infection first carries out the packaging that Ube3a is overexpressed adenovirus, and step includes recombinant adenovirus
A large amount of preparations of toxin grain, then cell culture and plasmid transfection are carried out, receive malicious (P1), freeze thawing and virus amplification and secondary receipts poison
(P2)。
Preferably, 250 μ l Opti- are added in the transfection of the siRNA into streaming pipe respectively first in Biohazard Safety Equipment
MEM serum free medium, then half is separately added into 7 μ l RNAiMAX transfection reagents, and it is molten that the other half is separately added into 6 μ l siRNA
Liquid, then the siRNA solution mixed is added in the Opti-MEM culture medium containing transfection reagent, after mixing, in biology
15min or so is placed in safety cabinet.The culture medium in 3.5cm diameter culture hole is finally replaced, about 1.5ml DMEM cell is added
Culture medium.The transfection reagent mixed is added in cell culture well, is placed in cell incubator and cultivates.It can when necessary
Secondary knockout is carried out to be transferred to siRNA again.
(3) beneficial effect
Compared with prior art, the effect machine the present invention provides a kind of Ube3a gene in the myocardial hypertrophy that Iso is induced
The research method of system, have it is following the utility model has the advantages that by cell grouping, the acquisition of Ube3a gene, Ube3a adenovirus packaging,
Transfection and data analysis and etc. come observe Ube3a Iso induce myocardial hypertrophy during expression quantity situation of change,
Judge that Ube3a continuous cropping whether related with the generation of myocardial hypertrophy is used, then passes through the knockout of Ube3a gene, determine Ube3a in the heart
Specificity and Ube3a in myohypertrophia generating process are promotion or interception to myocardial hypertrophy；Pass through Ube3a gene
Overexpression effect further clarifies the promotion or interception of Ube3a, and cell after combination Ube3a knockout and overexpression
State of development determines the causality between the effect of Ube3a and cardiac myocyte hypertrophy.
Detailed description of the invention
Fig. 1 is the expression for the cardiac muscle cell center myohypertrophia marker molecules that Ube3a of the present invention is knocked out with Iso difference induction time
Variation diagram.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
A kind of research method of the mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced, comprising the following steps:
(1) cell is grouped: cell is divided into Iso induction grouping and gene knockout and is overexpressed grouping.Iso induction point
Cell culture is spread disk using six orifice plates by group, and 2ml cell culture medium is added in every hole.2 μ l are added into cellular control unit culture medium
PBS is as placebo；Drug-induced group of the Iso Iso solution for being added 2 μ l 10mM, is made final concentration of 10 μM of the cell of Iso
Culture medium, successively cultivated according to time gradient 12h, for 24 hours, 48h.Gene knockout and overexpression, which are grouped, uses six for cell culture
Orifice plate spreads disk, and 2ml cell culture medium is added in every hole.2 μ l PBS are added as placebo to negative control group, knock out and cross table
2 μ l PBS are added as placebo up to the control group in group, final concentration of 10 μM of the cell training of Iso is made in other Iso induction groups
Base is supported, 12h, for 24 hours and 48h is successively cultivated according to time gradient.
(2) acquisition of Ube3a gene: the LB liquid that suitable distilled water prepares premix is added in Preparatory work of experiment in triangular flask
And solid medium, LB fluid nutrient medium, LB solid medium, EP pipe and pipetting tip are put into autoclave sterilization pot
Moist heat sterilization.Sterilized LB liquid and solid medium are put into super-clean bench, after temperature reduction, ammonia benzyl mycin is added,
The LB culture medium for blending together one thousandth ammonia benzyl, then pours into solid medium in culture dish, and every ware 15ml or so is placed in ultra-clean
Make its solidification in platform.Other sterilized articles are put into drying in thermostatic drying chamber, with after the sprinkling of 75% alcohol after being dried overnight
After being put into bacterium operation super-clean bench middle-ultraviolet lamp irradiation 30min Preparatory work of experiment, the Escherichia coli sense saved in -80 DEG C of refrigerators is taken out
It is placed on ice to melt by state cell and Ube3a plasmid, then a clean EP pipe is taken to sequentially add 30ul competent cell and 3ul
Plasmid blows and beats ice bath 15min after mixing with pipettor repeatedly, puts the EP pipe insertion water-bath kickboard after ice bath 15min and rapidly
Enter heat shock 45s in the water-bath for be preheated in advance 42 DEG C, then put back to 2min on ice rapidly, 900 μ l LB liquid are added into EP pipe
Body culture medium, is put into 37 DEG C of shaking tables, and 180rpm cultivates 1h, the turbidity of culture checked, in super-clean bench, according to culture
Bacillus coli cells turbidity takes a certain amount of be equably added dropwise in the mycin LB solid medium of benzyl containing ammonia of good plate in advance
On, bacterium solution is equably coated with spreading rod and is opened, is placed in biochemical cultivation case and cultivates, after cultivating 14-18h, checks that bacterium colony is raw
Long situation terminates growth if colony density reaches 60%-80%, and the clean 15ml test tube of access branch is distinguished into test tube
4ml LB liquid medium is added, picks them separately different monoclonal colonies and is inoculated in different test tubes, in 37 DEG C of shaking tables,
180rpm cultivates 12-16h or so, mini-scale plasmid extracting.
(3) packaging of Ube3a adenovirus: experiment first passes through PCR amplification and obtains containing restriction enzyme digestion sites
Ube3a target gene template segments, then by restriction endonuclease by viral vectors double digestion, by T4 ligase by target fragment group
It being attached in adenovirus vector, adenovirus vector will first carry out adenovirus vector construct, then carry out the amplification of adenovirus vector, wherein
Obtained adenovirus vector plasmid is first transformed into competent escherichia coli cell DH5 α by the amplification of adenovirus vector, using containing ammonia
The LB liquid medium of benzyl mycin is small to shake amplification, and the viral vectors after conversion applies plate, chooses Dan Ke to the bacterium colony after the screening of ammonia benzyl
It is grand, the amplification of monoclonal bacterial strain is carried out, shakes bacterium 14h by 37 DEG C, 200rpm, bacterium colony PCR identification, primer sequence are carried out to bacterium solution
Using carrier universal primer, stripe size can 200bp bigger than genetic fragment or so.
(4) building recombination Ube3a is overexpressed adenoviral plasmid.By the cotransfection with skeleton plasmid, packs Ube3a and cross table
Up to adenopathy.250 μ l Opti- are added in the wherein transfection procedure of siRNA into streaming pipe respectively first in Biohazard Safety Equipment
MEM serum free medium, then half is separately added into 7 μ l RNAiMAX transfection reagents, and it is molten that the other half is separately added into 6 μ l siRNA
Liquid, then the siRNA solution mixed is added in the Opti-MEM culture medium containing transfection reagent, after mixing, in biology
15min or so is placed in safety cabinet.The culture medium in 3.5cm diameter culture hole is finally replaced, about 1.5ml DMEM cell is added
Culture medium.The transfection reagent mixed is added in cell culture well, is placed in cell incubator and cultivates, it can when necessary
Secondary knockout is carried out to be transferred to siRNA again.
(5) transfect: transfection includes the transfection and adenovirus infection of siRNA.The transfection procedure of siRNA is synthesis
The siRNA powder processed sterile water of DEPC dissolves, and is configured to 20 μM of solution, is then calculated according to the cell hole count of transfection
SiRNA (the 3 μ l of about 7 μ l RNAiMAX and 6 μ l synthesis are added generally for the hole of 3.5cm diameter for the transfection liquid for needing to prepare
RNAi#1 and 3 μ l RNAi#2) solution.Adenovirus infection takes the adenovirus solution of packaged 1010 titre of 5 μ l or more directly to add
Enter into 2ml cell culture medium, adenovirus will voluntarily infected cell.
(6) data are analyzed: data are analyzed by using SPSS16.0, and the comparison between group is analyzed using independent samples t test,
Comparison between multiple groups examines (ANOVA Dunnett ' s post hoc) using single factor test Dunnett ' s t, data result with
Mean ± SD indicates that p < 0.05 indicates statistically significant sex differernce.
(7) Ube3a strikes the loose degree mutation analysis that low cardiac muscle cell induces with Iso: by RNAiMAX transfection reagent
Ube3a siRNA#1 and siRNA#2 are transferred to H9C2 cell and primary cardiomyocytes respectively, Ube3a strike low.Then
Cell culture medium is replaced, final concentration of 10 μM of Iso solution is added and carries out myocardial hypertrophy induction.Use qPCR method and Western
Blot method respectively detects Ube3a in H9C2 cell and primary cardiomyocytes and myocardial hypertrophy marker molecules, and GAPDH is
Reference gene and albumen, A figure and B figure are respectively that H9C2 cell and primary cardiomyocytes are knocked out by siRNA, intracellular Ube3a
With the mRNA of myocardial hypertrophy marker molecules with the variation of Iso inducing action；C figure and D figure are respectively H9C2 cell and the primary heart
The protein expression level of myocyte intracellular Ube3a and myocardial hypertrophy marker molecules ANP after siRNA is knocked out is with Iso
The variation of inducing action.SiUbe3a indicates that transfected sequences are Ube3a siRNA sequence, and it is messy code poly that Neg expression, which is transferred to sequence,
The negative control siRNA of nucleotide sequence.* indicate with negative control group there are statistical significant difference, # indicate with
For the Ube3a knockout group for not having Iso to induce there are significant difference, S indicates there is significant difference with 12h group, ￠ indicate with for 24 hours
Group has significant difference, p < 0.05.The histogram protein band of C figure and D figure being averaged after Quantity One analysis
Value, setting control group are 100, other groups are the ratio of control group.
Although have been shown and by Ube3a siRNA knockout, Ube3a table in H9C2 cell and primary cardiomyocytes
80% or more is all reduced up to amount, the induction with Iso to the Ube3a cardiac muscle cell knocked out, the transcriptional level of ANP and β-MHC
It sharply increases, when Iso induces 48h, the transcriptional level of ANP has reached 10 times of control group or more in cardiac muscle cell, β-MHC
Transcriptional level be also above 8 times；Expressing quantity also the luring with Iso of ANP in the cardiac muscle cell that Ube3a is knocked out simultaneously
It leads and increases sharply, when Iso induction time is 48h, the expressing quantity of ANP has also been above 3.5 times.The heart that Ube3a is knocked out
In myocyte, no matter myocardial hypertrophy marker molecules, in transcription or in translation skill, expression quantity is all significantly larger than simultaneously
Between section Iso induce normal myocardial cells.These results imply that the Ube3a gene in cardiac muscle cell may have inhibition Iso to lure
The myocardial hypertrophy effect led.
The embodiment of the present invention is described, for the ordinary skill in the art, it is possible to understand that do not departing from this
These embodiments can be carried out with a variety of change, modification, replacement and modification in the case where the principle and spirit of invention, it is of the invention
Range is defined by the appended claims and the equivalents thereof.
Claims (5)
1. a kind of research method of mechanism of action of Ube3a gene in the myocardial hypertrophy that Iso is induced, which is characterized in that including
Following steps:
(1) cell is grouped: cell is divided into Iso induction grouping and gene knockout and is overexpressed grouping, and Iso induction grouping will
Cell culture spreads disk using six orifice plates, and every hole is added 2ml cell culture medium, 2 μ l PBS are added into cellular control unit culture medium
As placebo；Drug-induced group of the Iso Iso solution for being added 2 μ l 10mM, is made final concentration of 10 μM of the cell culture of Iso
Base, successively cultivated according to time gradient 12h, for 24 hours, 48h, gene knockout and be overexpressed grouping by cell culture using six orifice plates paving
2ml cell culture medium is added in disk, every hole, and 2 μ l PBS are added to negative control group and are used as placebo, in knockout and overexpression group
2 μ l PBS are added as placebo in control group, and final concentration of 10 μM of the cell culture medium of Iso is made in other Iso induction groups, according to
Time gradient successively cultivates 12h, for 24 hours and 48h；
(2) acquisition of Ube3a gene: after Preparatory work of experiment, take out in -80 DEG C of refrigerators the competent escherichia coli cell that saves and
Ube3a plasmid is placed on ice to melt, then a clean EP pipe is taken to sequentially add 30ul competent cell and 3ul plasmid, uses liquid relief
Device blows and beats ice bath 15min after mixing repeatedly, and the EP pipe insertion water-bath kickboard and being put into rapidly after ice bath 15min is preheated in advance
Heat shock 45s in 42 DEG C of water-bath, then 2min on ice is put back to rapidly, 900 μ l LB liquid mediums are added into EP pipe, are put into
37 DEG C of shaking tables, 180rpm cultivates 1h, checks the turbidity of culture, muddy according to the Bacillus coli cells of culture in super-clean bench
Turbidity, take it is a certain amount of be equably added dropwise in advance on the mycin LB solid medium of benzyl containing ammonia of good plate, will with spreading rod
Bacterium solution is equably coated with and opens, and is placed in biochemical cultivation case and cultivates, and after cultivating 14-18h, bacterium colony growing state is checked, if bacterium
It falls density and reaches 60%-80%, terminate growth, the clean 15ml test tube of access branch is separately added into 4ml LB liquid into test tube
Culture medium picks them separately different monoclonal colonies and is inoculated in different test tubes, and in 37 DEG C of shaking tables, it is left that 180rpm cultivates 12-16h
The right side, mini-scale plasmid extracting；
(3) packaging of Ube3a adenovirus: experiment first passes through PCR amplification and obtains the Ube3a containing restriction enzyme digestion sites
Then target fragment is assembled by target gene template segments by viral vectors double digestion by restriction endonuclease by T4 ligase
In adenovirus vector, building recombination Ube3a is overexpressed adenoviral plasmid, by the cotransfection with skeleton plasmid, packs Ube3a mistake
Express adenopathy；
(4) transfect: transfection includes the transfection and adenovirus infection of siRNA, and the transfection procedure of siRNA is the siRNA powder of synthesis
The end processed sterile water dissolution of DEPC, is configured to 20 μM of solution, is then calculated according to the cell hole count of transfection and need to match
SiRNA (the 3 μ l RNAi#1 of about 7 μ l RNAiMAX and 6 μ l synthesis are added generally for the hole of 3.5cm diameter for the transfection liquid of system
With 3 μ l RNAi#2) solution, adenovirus infection takes the adenovirus solution of packaged 1010 titre of 5 μ l or more to be added directly into
In 2ml cell culture medium, adenovirus will voluntarily infected cell；
(5) data are analyzed: data are analyzed by using SPSS16.0, and the comparison between group is analyzed using independent samples t test, multiple groups
Between comparison using single factor test Dunnett ' s t examine (ANOVA Dunnett ' s posthoc), data result is with Mean ± SD
It indicates, p < 0.05 indicates statistically significant sex differernce.
2. a kind of research side of mechanism of action of the Ube3a gene according to claim 1 in the myocardial hypertrophy that Iso is induced
Method, which is characterized in that Preparatory work of experiment step is that suitable distilled water is added in triangular flask to prepare premix in the step (2)
LB liquid medium, LB solid medium, EP pipe and pipetting tip are put into autoclave sterilization by LB liquid and solid medium
Moist heat sterilization in pot, sterilized LB liquid and solid medium are put into super-clean bench, and after temperature reduction, it is mould that ammonia benzyl is added
Element blendes together the LB culture medium of one thousandth ammonia benzyl, then pours into solid medium in culture dish, and every ware 15ml or so is placed in
Make its solidification in super-clean bench, other sterilized articles are put into drying in thermostatic drying chamber, are sprayed after being dried overnight with 75% alcohol
Bacterium operation super-clean bench middle-ultraviolet lamp irradiation 30min is put into after spilling.
3. a kind of research side of mechanism of action of the Ube3a gene according to claim 1 in the myocardial hypertrophy that Iso is induced
Method, which is characterized in that adenovirus vector first carries out adenovirus vector construct in the step (3), then carries out adenovirus vector
Amplification.
4. a kind of research side of mechanism of action of the Ube3a gene according to claim 1 in the myocardial hypertrophy that Iso is induced
Method, which is characterized in that the transfection procedure of siRNA in the step (4), respectively to streaming Guan Zhongjia first in Biohazard Safety Equipment
Enter 250 μ l Opti-MEM serum free mediums, then half is separately added into 7 μ l RNAiMAX transfection reagents, the other half adds respectively
Enter 6 μ l siRNA solution, then the siRNA solution mixed is added in the Opti-MEM culture medium containing transfection reagent, mixes
After uniformly, 15min or so is placed in Biohazard Safety Equipment, is finally replaced the culture medium in 3.5cm diameter culture hole, is added about
1.5ml DMEM cell culture medium, the transfection reagent mixed is added in cell culture well, is placed in cell incubator and trains
It supports, siRNA can be transferred to when necessary again and carry out secondary knockout.
5. a kind of research side of mechanism of action of the Ube3a gene according to claim 1 in the myocardial hypertrophy that Iso is induced
Method, which is characterized in that adenovirus infection in the step (4) first carries out the packaging that Ube3a is overexpressed adenovirus, and step includes
A large amount of preparations of recombinant adenovirus plasmid, then carry out cell culture and plasmid transfection, receive malicious (P1), freeze thawing and virus amplification and
Secondary receipts are malicious (P2).
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| CN113755498A (en) * | 2021-09-27 | 2021-12-07 | 赛业（苏州）生物科技有限公司 | gRNA of targeted mouse Ube3a gene and method for constructing AS disease mouse model |
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