CN109566539A - A kind of method for building up of EBV virus tree shrew animal model - Google Patents
A kind of method for building up of EBV virus tree shrew animal model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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Abstract
The invention discloses a kind of method for building up of Epstein-Barr virus tree shrew animal model, periodically draw blood after animal selection, virus liquid preparation, inoculation, observation tree shrew state, inoculation tree shrew, and separate EBV copy number in PBMCs, qPCR measurement PBMCs, the expression of EBV related gene in RT-PCR detection PBMCs, pathological examination and establish.Present invention firstly discloses the method for building up of tree shrew Epstein-Barr virus animal model, lay the foundation for preferably research Epstein-Barr virus biological characteristics and its related disease；The tree shrew selected in the present invention is novel experimental animal, is more nearly primate than current existing experimental animal, and secondly tree shrew has many advantages, such as neurological susceptibility to a variety of viruses, have it is easy raise, be small in size, easy acquisition.
Description
Technical field
The present invention relates to animal model preparation fields, more particularly to a kind of foundation side of EBV virus tree shrew animal model
Method.
Background technique
Epstein-Barr virus (Epstein-Barrvirus, EBV) be Epstein and Barr in 1964 for the first time from African children
It is isolated in the cell of Burkitt lymthoma.Infection rate of the EBV in crowd is up to 90%, mostly in recessive i.e. latent
Infection Status is lied prostrate, the mankind are usually just infected in infant period, have no the lifelong carrying of clinical symptoms.EBV is passed by saliva
It broadcasts, can also be bone-marrow-derived lymphocyte by blood or sex track transmission, target cell, secondly be epithelial cell.Studies have shown that in body
Outer EBV can excite bone-marrow-derived lymphocyte to immortalize.The generation of EBV and Several Kinds of Malignancy is closely related, including nasopharyngeal carcinoma, Huo Qijin
Disease, Burkitt lymthoma, gastric cancer, International Cancer Research Center (IARC) are classified to carcinogen, are clearly located as
I class carcinogen.Furthermore EBV is also related to lymphoproliferative disease, infectious mononucleosis, virus after transplanting
The generation of the diseases such as property erythrophage increase disease is closely related.There is presently no prevent and treat EBV infection drug, for
Associated malignancies caused by EBV also cannot get effectively preventing or treatment at present.Therefore, EBV relevant animal models pair are constructed
In the research and development for studying especially its effect and vaccine and drug in Several Kinds of Malignancy occurrence and development of its biological characteristics
It is most important.
So far from discovery EBV, the EBV animal model that domestic and foreign scholars establish includes primate, Severe Combined Immune
Deficient mice, transgenic animals and rabbit and four major class of cavy.The above animal model is in research EBV infection, pathogenic mechanism
And prophylactic treatment etc. important role, but some shortcomings are also individually present.In terms of studying the distinctive disease of the mankind, such as
It can use more close to the animal model of primate, it will be with irreplaceable advantage in integral level.New World monkey conduct
Unique primate in most above-mentioned animal model, this has irreplaceable advantage, but New World monkey species are endangered,
Extremely precious and protective policy, ethics requirement limits its continuing to use in EBV animal model, causes from 20
The research stagnation of EBV New World monkey animal model after the nineties in century.Therefore, a kind of more practical, effective EBV is established
Animal model becomes an urgent demand of Recent study EBV and its related disease.
Tree shrew is a kind of meiofauna for being grown on the torrid zone and subtropical zone, belongs to tree shrew section, easily raises, the service life about 8 years.
In China, tree shrew is mainly distributed on the ground such as Yunnan, Guangxi, and tree shrew is just more and more led by scientist and biological medicine in recent years
Domain uses.In the recent period, Kunming Institute of Zoology, Chinese Academy of Sciences is by the parsing to tree shrew full-length genome, transcript profile and protein groups,
Confirm that the hereditary feature of tree shrew and primate is close, in terms of physiology dissection, neurodevelopment and psychological stress with
There are the similitudes of height between the primate even mankind.Have at present it is multinomial studies at home and abroad show that, many viruses of tree shrew
Infection characterization is similar to the mankind, and tree shrew can be by virus infections such as hepatitis A, hepatitis B, hepatitis, colyliform, gland and blebs.In addition, tree shrew
It has successfully been built as the animal models such as drug-resistant bacteria infection and septicemia, psychological stress and depression, myopia and metabolic disease
It is vertical.Thus it will be seen that tree shrew is just obtaining close concern as a kind of novel animal more close with primate.
Studies have shown that the natural reservoir (of bird flu viruses) range of EBV is only the mankind and including Cotton-Topped monkey, it is new including common marmoset and cat owl monkey etc.
World monkey.Since tree shrew and primate have the similar characteristic of height, we perhaps can see tree shrew for the neurological susceptibility of EBV
To establish the good prospect of tree shrew EBV animal model.
The disclosure of background above technology contents is only used for auxiliary and understands inventive concept and technical solution of the invention, not
The prior art for necessarily belonging to present patent application shows above content in the applying date of present patent application in no tangible proof
In the case where having disclosed, above-mentioned background technique should not be taken to the novelty and creativeness of evaluation the application.
Summary of the invention
Present invention aims at the method for building up for proposing a kind of Epstein-Barr virus tree shrew animal model, and specific technical solution is such as
Under:
A kind of method for building up of EBV virus tree shrew animal model is set by animal selection, virus liquid preparation, inoculation, observation
It periodically draws blood after Shrew state, inoculation tree shrew, and separates EBV copy number, RT-PCR detection in PBMCs, qPCR measurement PBMCs
The expression of EBV related gene in PBMCs, pathological examination and establish.
Preferably, adult, healthy tree shrew 13 (1-1.5) are chosen in animal selection, and male and female are unlimited, and weight is
120-160g。
Preferably, virus liquid preparation is cultivated cracking by B95-8 and is generated, this cell is purchased from Chinese Academy of Sciences Shanghai
Cell bank.
Preferably, the inoculation, 13 tree shrews are divided into experimental group (n=10) and control group (n=3), with Ts1-13 table
Show, experimental group via intranasal application collunarium 500 μ lEBV virus liquids of inoculation, about 3.9 × 108Copy；Control group connects in the same way
RPMI1640 culture medium 500 μ l of kind dual anti-containing 10%FBS and 1%.
Preferably, the observation tree shrew state, the state of close observation tree shrew after inoculation, when the poor (table of its life state
Now to be not active, often roll up one jiao of cage, feed less or do not feed and defecation is abnormal etc.) when, put to death in time to collect weight
It wants organ-tissue and analyzes the reason of present condition difference.
Preferably, it periodically draws blood after the inoculation tree shrew, and separates PBMCs
A) periodically blood drawing records the inoculation forward and backward blood drawing time in the mode in " 0,1,2 ... " week, blood drawing 1 time, inoculation before being inoculated with
It is periodically drawn blood afterwards by femoral artery/femoral vein into the anticoagulant blood-collecting pipe containing EDTA2Na.Blood drawing movement as far as possible is soft every time,
Protect animal；
B) separation of PBMCs is by the above-mentioned anticoagulation of 1ml (being drawn into disengaging time < 4h certainly), careful tiling to 3ml
PBMCs separating liquid ullage, room temperature 400g, centrifuge tube is from top to bottom divided into 4 layers after being centrifuged 20min, and second layer tunica albuginea layer is
For mononuclearcell layer, cellular layer is collected into centrifuge tube and PBS is added or cell washing solution mixes well, room temperature 250g, from
Washing is resuspended repeatedly and collects cell (being resuspended spare) after totally 3 times by heart 10min；
Preferably, the extraction of the DNA and RNA
A) DNA, which is extracted, is divided into 2 parts for the cell suspension of above-mentioned acquisition, is respectively intended to extract DNA and RNA.Add 1mlPBS weight
Outstanding cell, 1000rpm are centrifuged 7min, abandon supernatant；The piping and druming of 20 μ l protease is added to mix；200 μ l Buffer AL are added, gently
Shake 15sec, 56 DEG C, water-bath 10min；200 μ l dehydrated alcohols are added after gentle centrifugation, micro oscillator vibrates 15sec；It will mix
It closes liquid to move into adsorption column (adsorption column is placed in the collecting pipe of 2ml), 8000rpm is centrifuged 1min at room temperature；In adsorption column
500 μ l AW1 are added, 8000rpm is centrifuged 1min at room temperature；500 μ l AW2 are added in adsorption column, at room temperature 14000rpm from
Heart 3min；Adsorption column is placed in another new collecting pipe, 14000rpm is centrifuged 1min at room temperature；It is new that adsorption column is placed in one
In the EP pipe of 1.5ml, 50 μ l Buffer AE are added, are placed at room temperature for 2min, 8000rpm is centrifuged 1min at room temperature；Survey DNA concentration
And purity, sample are placed in -20 DEG C of preservations；
B) RNA, which is extracted, is centrifuged 7min in room temperature 1000rpm for another above-mentioned cell suspension, abandons supernatant, 1ml cracking is added
Liquid RZ, mixes gently；After placing 5min, 200 μ l chloroforms are added, acutely vibrates 15sec, is placed at room temperature for after 3min 4 DEG C,
12000rpm is centrifuged 10min, and sample can be divided into three layers, top layer's water phase is transferred in new EP pipe；It is anhydrous that about 250 μ l are added
Ethyl alcohol mixes；It will obtain solution and precipitating is transferred in adsorption column together, 4 DEG C, 12000rpm is centrifuged 30sec；Add into adsorption column
Enter 500 μ l protein liquid removal RD (using preceding addition ethyl alcohol), 4 DEG C, 12000rpm is centrifuged 30sec, abandons waste liquid；Into adsorption column CR3
500 μ l rinsing liquid RW (using preceding addition ethyl alcohol) is added, is stored at room temperature 2min, 4 DEG C, 12000rpm is centrifuged 30sec and repeats 1
It is secondary, abandon waste liquid；Adsorption column is put into 2ml collecting pipe, 4 DEG C, 12000rpm is centrifuged 2min to remove residual liquid；By adsorption column
It is transferred in a new 1.5ml centrifuge tube, adds 30 μ lRNase-Free ddH2O, is placed at room temperature for 2min, 4 DEG C, 12000rpm from
Heart 2min；RNA concentration and purity are surveyed, -80 DEG C of preservations are placed in.
Preferably, EBV copy number in qPCR measurement PBMCs, the method is as follows:
With the DNA of above-mentioned acquirement using EBV copy number in qPCR method measurement PBMCs, the target fragment of amplification is EBV's
BamHIW segment is as follows: upstream primer: 5 '-GCA GCC GCC CAG TCT CT-3 '；Downstream primer: 5 '-ACA GAC AGT
GCA CAG GAG CCT-3 ', probe: 5 '-(6FAM) AAA AGC TGG CGC CCT TGC CTG (TAMRA) -3 '.Specific side
Method: being first arranged standard items (DNA of Namalwa cell line), according to Namalwa cell total DNA concentration and above-mentioned conversion method, uses
QPCR standard dilutions dilute 7 equal standard items than gradient out, specifically: first it is diluted to 1.2 × 105Copies/2 μ l,
Gradient dilution is at 1.2 × 10 again4copies/2μl、1.2×103copies/2μl、1.2×102copies/2μl、1.2×
101copies/2μl、1.2×100copies/2μl、1.2×10-1copies/2μl.The standard items of 7 proportional diluteds (are divided
She Zhi 2 repeating holes) on after machine testing, standard curve can be drawn out (referred to as according to the copy number of standard items and its Ct value
Mark is bent), the Ct value of EBV virus liquid and sample is substituted into, you can get it its copy number.Qualification mark song need to meet the following conditions: related
Coefficients R2> 0.98, -3.5 < slope < -3,90% < amplification efficiency E% < 120%.By TaKaRa company qPCR kit
(Premix Ex TaqTMProbe qPCR) specification operation, 3 repeating holes are set, its average value is taken to show that EBV virus liquid is copied
Shellfish number, is recorded with copies/ml.Reaction system: 10 μ l, PCR Forward of Premix Ex Taq (Probe qPCR) (2 ×)
0.4 μ l, PCR Reverse Primer (10 μM) of Primer (10 μM) 0.4 μ l, ROX Reference Dye (50 ×) 0.4 μ l,
0.8 μ l, DNA template of TaqMan Probe (5 μM) 2 μ l, dH2O supply 20 μ l.QPCR amplification condition: reaction is in Stepone
It is carried out on the automatic qPCR instrument of plus, reaction condition are as follows: 95 DEG C of 20sec；95 DEG C of 1sec, 60 DEG C of 20sec, 40 circulations.
Preferably, the RT-PCR detects the expression of EBV related gene in PBMCs, comprising the following steps:
A the reaction system of 20 μ l is established in) cDNA synthesis using Reverse Transcriptase kit, takes 500ngRNA, by following component plus
Enter no RNA, DNA enzymatic 200 μ l PCR pipe in (on ice operate), reaction system: total serum IgE 500ng, AnchoredOligo
(dT) 10 μ l, EasyScript@RT/RI Enzyme Mix of 18Primer (0.5 μ g/ μ l) 1 μ l, 2 × ES Reaction Mix
1 μ l, gDNA Remover, 1 μ l, RNase-free Water complements to 20 μ l.Reaction condition: the above mixture is gently mixed
It is even, after centrifugation, terminate reaction in 42 DEG C of heating 30min, 85 DEG C of heating 5sec, be placed in -20 DEG C it is spare；
B) RT-PCR expands EBV related gene using cDNA as template, carries out RT-PCR expansion using following 20 μ l reaction system
Increase, detect the related gene expressions situations such as BZLF1, LMP1, EBNA1, EBNA2, EA respectively, β-actin is internal reference.Reactant
System: 10 μ l of PCR Mix, 1 μ l of upstream primer (5 μM), 3 μ l, Deionized Free of downstream primer (5 μM) 1 μ l, template CDNA
H2O supplies 20 μ l.RT-PCR amplification condition: 95 DEG C of initial denaturations 3min, 95 DEG C of denaturation 30sec, anneal 30sec, 72 DEG C of extensions
1min, 35 circulations, 72 DEG C re-extend 10min.β-actin is internal reference, and the cDNA of B95-8 cell is positive control, and mould is not added
Plate is negative control.
A) each target gene primer sequence, primer size and annealing temperature are as follows: BZLF1 (primer size: 129bp, annealing
Temperature: 60 DEG C) F:GCG GAC AAA AAT CAG GCG TT, R:GAA AAT GCC GGG CCA AGT TT；LMP1 (product
Size: 211bp/134bp, annealing temperature: 60 DEG C) F:AAT TTG CAC GGA CAG GCA TT, R:AAG GCC AAA AGC
TGC CAG AT；EBNA2 (primer size: 253bp, annealing temperature: 63 DEG C) F:AAC TCC TGG CCC ATC CAA TG, R:
GGA GGG GCG AGG TCT TTT AC；EA (primer size: 126bp, annealing temperature: 60 DEG C) F:AAC GAG CAG ATG
ATT GGG CA, R:CGT GGT GAC GTA GAG ATC CG；EBNA1 (primer size: 270bp, annealing temperature: 60 DEG C) F:
GTC ATC ATC ATC CGG GTC TC, R:TTC GGG TTG GAA CCT CCT TG；β-actin (primer size:
113bp, annealing temperature: 60 DEG C) F:ATT TTG AAT GAT CAG CCA CC, R:AGG TAA GCC CTG GCT GCC
TC；
B) Ago-Gel of agarose gel electrophoresis analysis preparation 2%, is placed on equipped with 1 × TBE electrophoretic buffer
In electrophoresis tank, RT-PCR amplified production is added in gel pore respectively, is instruction in left side plus the hole DNA Marker, connects electricity
Gel taking-up is placed in ultraviolet gel imaging system when 2/3 under instruction to the pact of gel on gel by source, 100V, 250mA
Analyze result.
Preferably, the pathological examination specific method: simultaneously anatomical trees Shrew is put to death in the different observation stages of experiment, is taken out
Its liver, spleen, lymphonodi mesenterici, lung, pharynx nasalis tissue observe it and substantially tissue change and carry out HE dyeing and EBER-
ISH detection.
Inventor is mainly reflected in following several respects compared with prior art through furtheing investigate the great innovation made:
1, present invention firstly discloses the method for building up of tree shrew Epstein-Barr virus animal model, for preferably research Epstein-Barr virus biology
It learns characteristic and its related disease lays the foundation.
2, the tree shrew selected in the present invention is novel experimental animal, and it is long to be more nearly spirit than current existing experimental animal
Class.
3, the present invention described in tree shrew to it is a variety of virus have neurological susceptibility, have easily raising, it is small in size, it is easy obtain etc. it is excellent
Point.
Detailed description of the invention
Fig. 1: qPCR as a result, the trend of interruption rising or of short duration rising is presented in EBV copy number in tree shrew PBMCs；
Fig. 2: qPCR as a result, EBV copy number does not increase in tree shrew PBMCs；
Fig. 3: RT-PCR as a result, EBV related gene expression；
Fig. 4: RT-PCR as a result, EBV related gene expression；
Fig. 5: RT-PCR as a result, EBV related gene expression；
Fig. 6: HE result；
Fig. 7: EBER-ISH result；
Fig. 8: Western blot result.
Specific embodiment
One, specific embodiment
Embodiment 1
A kind of method for building up of EBV virus tree shrew animal model is set by animal selection, virus liquid preparation, inoculation, observation
It periodically draws blood after Shrew state, inoculation tree shrew, and separates EBV copy number, RT-PCR detection in PBMCs, qPCR measurement PBMCs
The expression of EBV related gene in PBMCs, pathological examination and establish.
Adult, healthy tree shrew 13 (1-1.5) are chosen in the animal selection, and male and female are unlimited, weight 120-
160g。
The virus liquid preparation, cultivates cracking by B95-8 and generates, this cell is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.
The inoculation, 13 tree shrews are divided into experimental group (n=10) and control group (n=3), are indicated with Ts1-13, experiment
Group via intranasal application collunarium 500 μ lEBV virus liquids of inoculation, about 3.9 × 108Copy；Control group is inoculated with 500 μ l in the same way
Containing the dual anti-RPMI1640 culture medium of 10%FBS and 1%.
The observation tree shrew state, the state of close observation tree shrew after inoculation (are shown as bad when its life state is poor
It is dynamic, often roll up one jiao of cage, feed less or do not feed and defecation is abnormal etc.) when, put to death in time to collect vitals group
Knit and analyze the reason of present condition difference.
It periodically draws blood after the inoculation tree shrew, and separates PBMCs
A) periodically blood drawing records the inoculation forward and backward blood drawing time in the mode in " 0,1,2 ... " week, blood drawing 1 time, inoculation before being inoculated with
It is periodically drawn blood afterwards by femoral artery/femoral vein into the anticoagulant blood-collecting pipe containing EDTA2Na.Blood drawing movement as far as possible is soft every time,
Protect animal；
B) separation of PBMCs is by the above-mentioned anticoagulation of 1ml (being drawn into disengaging time < 4h certainly), careful tiling to 3ml
PBMCs separating liquid ullage, room temperature 400g, centrifuge tube is from top to bottom divided into 4 layers after being centrifuged 20min, and second layer tunica albuginea layer is
For mononuclearcell layer, cellular layer is collected into centrifuge tube and PBS is added or cell washing solution mixes well, room temperature 250g, from
Washing is resuspended repeatedly and collects cell (being resuspended spare) after totally 3 times by heart 10min；
The extraction of the DNA and RNA
A) DNA, which is extracted, is divided into 2 parts for the cell suspension of above-mentioned acquisition, is respectively intended to extract DNA and RNA.Add 1mlPBS weight
Outstanding cell, 1000rpm are centrifuged 7min, abandon supernatant；The piping and druming of 20 μ l protease is added to mix；200 μ l Buffer AL are added, gently
Shake 15sec, 56 DEG C, water-bath 10min；200 μ l dehydrated alcohols are added after gentle centrifugation, micro oscillator vibrates 15sec；It will mix
It closes liquid to move into adsorption column (adsorption column is placed in the collecting pipe of 2ml), 8000rpm is centrifuged 1min at room temperature；In adsorption column
500 μ l AW1 are added, 8000rpm is centrifuged 1min at room temperature；500 μ l AW2 are added in adsorption column, at room temperature 14000rpm from
Heart 3min；Adsorption column is placed in another new collecting pipe, 14000rpm is centrifuged 1min at room temperature；It is new that adsorption column is placed in one
In the EP pipe of 1.5ml, 50 μ l Buffer AE are added, are placed at room temperature for 2min, 8000rpm is centrifuged 1min at room temperature；Survey DNA concentration
And purity, sample are placed in -20 DEG C of preservations；
B) RNA, which is extracted, is centrifuged 7min in room temperature 1000rpm for another above-mentioned cell suspension, abandons supernatant, 1ml cracking is added
Liquid RZ, mixes gently；After placing 5min, 200 μ l chloroforms are added, acutely vibrates 15sec, is placed at room temperature for after 3min 4 DEG C,
12000rpm is centrifuged 10min, and sample can be divided into three layers, top layer's water phase is transferred in new EP pipe；It is anhydrous that about 250 μ l are added
Ethyl alcohol mixes；It will obtain solution and precipitating is transferred in adsorption column together, 4 DEG C, 12000rpm is centrifuged 30sec；Add into adsorption column
Enter 500 μ l protein liquid removal RD (using preceding addition ethyl alcohol), 4 DEG C, 12000rpm is centrifuged 30sec, abandons waste liquid；Into adsorption column CR3
500 μ l rinsing liquid RW (using preceding addition ethyl alcohol) is added, is stored at room temperature 2min, 4 DEG C, 12000rpm is centrifuged 30sec and repeats 1
It is secondary, abandon waste liquid；Adsorption column is put into 2ml collecting pipe, 4 DEG C, 12000rpm is centrifuged 2min to remove residual liquid；By adsorption column
It is transferred in a new 1.5ml centrifuge tube, adds 30 μ lRNase-Free ddH2O, is placed at room temperature for 2min, 4 DEG C, 12000rpm from
Heart 2min；RNA concentration and purity are surveyed, -80 DEG C of preservations are placed in.
EBV copy number in the qPCR measurement PBMCs, the method is as follows:
With the DNA of above-mentioned acquirement using EBV copy number in qPCR method measurement PBMCs, the target fragment of amplification is EBV's
BamHIW segment is as follows: upstream primer: 5 '-GCA GCC GCC CAG TCT CT-3 '；Downstream primer: 5 '-ACA GAC AGT
GCA CAG GAG CCT-3 ', probe: 5 '-(6FAM) AAA AGC TGG CGC CCT TGC CTG (TAMRA) -3 '.Specific side
Method: being first arranged standard items (DNA of Namalwa cell line), according to Namalwa cell total DNA concentration and above-mentioned conversion method, uses
QPCR standard dilutions dilute 7 equal standard items than gradient out, specifically: first it is diluted to 1.2 × 105Copies/2 μ l,
Gradient dilution is at 1.2 × 10 again4copies/2μl、1.2×103copies/2μl、1.2×102copies/2μl、1.2×
101copies/2μl、1.2×100copies/2μl、1.2×10-1copies/2μl.The standard items of 7 proportional diluteds (are divided
She Zhi 2 repeating holes) on after machine testing, standard curve can be drawn out (referred to as according to the copy number of standard items and its Ct value
Mark is bent), the Ct value of EBV virus liquid and sample is substituted into, you can get it its copy number.Qualification mark song need to meet the following conditions: related
Coefficients R2> 0.98, -3.5 < slope < -3,90% < amplification efficiency E% < 120%.By TaKaRa company qPCR kit
(Premix Ex TaqTMProbe qPCR) specification operation, 3 repeating holes are set, its average value is taken to show that EBV virus liquid is copied
Shellfish number, is recorded with copies/ml.Reaction system: 10 μ l, PCR Forward of Premix Ex Taq (Probe qPCR) (2 ×)
0.4 μ l, PCR Reverse Primer (10 μM) of Primer (10 μM) 0.4 μ l, ROX Reference Dye (50 ×) 0.4 μ l,
0.8 μ l, DNA template of TaqMan Probe (5 μM) 2 μ l, dH2O supply 20 μ l.QPCR amplification condition: reaction is in Stepone
It is carried out on the automatic qPCR instrument of plus, reaction condition are as follows: 95 DEG C of 20sec；95 DEG C of 1sec, 60 DEG C of 20sec, 40 circulations.
The expression of EBV related gene in the RT-PCR detection PBMCs, comprising the following steps:
A the reaction system of 20 μ l is established in) cDNA synthesis using Reverse Transcriptase kit, takes 500ngRNA, by following component plus
Enter no RNA, DNA enzymatic 200 μ l PCR pipe in (on ice operate), reaction system: total serum IgE 500ng, AnchoredOligo
(dT) 10 μ l, EasyScript@RT/RI Enzyme Mix of 18Primer (0.5 μ g/ μ l) 1 μ l, 2 × ES Reaction Mix
1 μ l, gDNA Remover, 1 μ l, RNase-free Water complements to 20 μ l.Reaction condition: the above mixture is gently mixed
It is even, after centrifugation, terminate reaction in 42 DEG C of heating 30min, 85 DEG C of heating 5sec, be placed in -20 DEG C it is spare；
B) RT-PCR expands EBV related gene using cDNA as template, carries out RT-PCR expansion using following 20 μ l reaction system
Increase, detect the related gene expressions situations such as BZLF1, LMP1, EBNA1, EBNA2, EA respectively, β-actin is internal reference.Reactant
System: 10 μ l of PCR Mix, 1 μ l of upstream primer (5 μM), 3 μ l, Deionized Free of downstream primer (5 μM) 1 μ l, template CDNA
H2O supplies 20 μ l.RT-PCR amplification condition: 95 DEG C of initial denaturations 3min, 95 DEG C of denaturation 30sec, anneal 30sec, 72 DEG C of extensions
1min, 35 circulations, 72 DEG C re-extend 10min.β-actin is internal reference, and the cDNA of B95-8 cell is positive control, and mould is not added
Plate is negative control.
A) each target gene primer sequence, primer size and annealing temperature are as follows: BZLF1 (primer size: 129bp, annealing
Temperature: 60 DEG C) F:GCG GAC AAA AAT CAG GCG TT, R:GAA AAT GCC GGG CCA AGT TT；LMP1 (product
Size: 211bp/134bp, annealing temperature: 60 DEG C) F:AAT TTG CAC GGA CAG GCA TT, R:AAG GCC AAA AGC
TGC CAG AT；EBNA2 (primer size: 253bp, annealing temperature: 63 DEG C) F:AAC TCC TGG CCC ATC CAA TG, R:
GGA GGG GCG AGG TCT TTT AC；EA (primer size: 126bp, annealing temperature: 60 DEG C) F:AAC GAG CAG ATG
ATT GGG CA, R:CGT GGT GAC GTA GAG ATC CG；EBNA1 (primer size: 270bp, annealing temperature: 60 DEG C) F:
GTC ATC ATC ATC CGG GTC TC, R:TTC GGG TTG GAA CCT CCT TG；β-actin (primer size:
113bp, annealing temperature: 60 DEG C) F:ATT TTG AAT GAT CAG CCA CC, R:AGG TAA GCC CTG GCT GCC
TC；
B) Ago-Gel of agarose gel electrophoresis analysis preparation 2%, is placed on equipped with 1 × TBE electrophoretic buffer
In electrophoresis tank, RT-PCR amplified production is added in gel pore respectively, is instruction in left side plus the hole DNA Marker, connects electricity
Gel taking-up is placed in ultraviolet gel imaging system when 2/3 under instruction to the pact of gel on gel by source, 100V, 250mA
Analyze result.
The pathological examination, the method is as follows: put to death simultaneously anatomical trees Shrew in the different observation stages of experiment, take out its liver
Dirty, spleen, lymphonodi mesenterici, lung, pharynx nasalis tissue, observe its substantially tissue change and carry out HE dyeing and EBER-ISH inspection
It surveys.
Two, testing result:
1, qPCR result: in 7 tree shrew PBMCs in 15 EBV copy number present interruption rise, becoming of fluctuating up and down
Gesture, in addition of short duration rising is presented in 3 EBV copy numbers, is typically raised to the 2nd week or the 4th week to declining after reach to peak value, and after decline
The trend no longer risen (see Fig. 1).And remaining 5 tree shrew EBV copy number has no raising.(see Fig. 2).
2, RT-PCR is as the result is shown: in 10 tree shrew PBMCs different degrees of appearance EBV related gene expression (see
Fig. 3,4).And the expression of remaining 5 tree shrew EBV related gene.(see Fig. 5).
3, HE result: compared with the control group, the spleen enlargement of 7 tree shrews (Ts1,2,3,4,8,12,14), HE in experimental group
Coloration result shows acini lienalis hyperplasia, and HE coloration result shows massive inflammatory cells infiltrated (see Fig. 6)
4, EBER-ISH is as the result is shown: the cell of the EBERs positive is detected in the spleen of 4 tree shrews (Ts2,3,4,14)
(see Fig. 7).
5, Western blot is as the result is shown: detecting EBNA1 egg in 7 tree shrew (Ts1,2,3,4,8,12,14) spleens
White expression (see Fig. 8).
Invention is further described in detail With reference to embodiment.It is emphasized that following the description is only
It is merely exemplary, the range and its application being not intended to be limiting of the invention.It is specific/preferred real the above content is combining
Applying mode, further detailed description of the invention, and it cannot be said that specific implementation of the invention is only limited to these instructions.
It for those of ordinary skill in the art to which the present invention belongs, without departing from the inventive concept of the premise, can be with
Some replacements or modifications are made to the embodiment that these have been described, and these substitutions or variant all shall be regarded as belonging to this hair
Bright protection scope.
Claims (9)
1. a kind of method for building up of Epstein-Barr virus tree shrew animal model, which is characterized in that by animal selection, virus liquid preparation, connect
It periodically draws blood after kind, observation tree shrew state, inoculation tree shrew, and separates EBV copy number, RT- in PBMCs, qPCR measurement PBMCs
The expression of EBV related gene in PCR detection PBMCs, pathological examination and establish.
2. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, it is characterised in that: the animal choosing
It selects, chooses the healthy tree shrew 13 of adult, 1-1.5, male and female are unlimited, weight 120-160g.
3. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, it is characterised in that: the virus liquid system
It is standby, cracking is cultivated by B95-8 and is generated.
4. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, it is characterised in that: the inoculation, 13
Tree shrew is divided into experimental group n=10 and control group n=3, is indicated with Ts1-13,500 μ lEBV disease of experimental group via intranasal application collunarium inoculation
Venom is 3.9 × 108Copy；Control group is inoculated with the 500 μ l RPMI1640 dual anti-containing 10%FBS and 1% in the same way
Culture medium.
5. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, it is characterised in that: the observation tree shrew
State, the state of close observation tree shrew after inoculation are put to death in time when its life state difference to collect organs in adult and divide
Be precipitated the reason of present condition difference, the life state difference be show as it is not active, often roll up one jiao of cage, feed less or not into
Food and defecation are abnormal etc..
6. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, it is characterised in that: the inoculation tree shrew
Periodically blood drawing afterwards, and separate PBMCs；
A) periodically blood drawing records the inoculation forward and backward blood drawing time in the mode in " 0,1,2 ... " week, and blood drawing 1 time, fixed after inoculation before being inoculated with
Phase is drawn blood by femoral artery/femoral vein into the anticoagulant blood-collecting pipe containing EDTA2Na, and blood drawing movement as far as possible is soft every time, protection
Animal；
B) the anticoagulation that 1ml step A) is extracted in the separation of PBMCs, careful tiling to 3mlPBMCs separating liquid ullage,
Room temperature 400g, centrifuge tube is from top to bottom divided into 4 layers after being centrifuged 20min, and second layer tunica albuginea layer is mononuclearcell layer, collects thin
Born of the same parents' layer is into centrifuge tube and PBS is added or cell washing solution mixes well, room temperature 250g, is centrifuged 10min, and washing is resuspended repeatedly altogether
Cell is collected after 3 times, it is spare；
C) the step B) cell suspension obtained is divided into 2 parts by the extraction of DNA and RNA, is respectively intended to extract DNA and RNA；
A) DNA is extracted plus cell is resuspended in 1mlPBS, and 1000rpm is centrifuged 7min, abandons supernatant；The piping and druming of 20 μ l protease is added to mix；
200 μ l Buffer AL are added, gently shake 15sec, 56 DEG C, water-bath 10min；200 μ l dehydrated alcohols are added after gentle centrifugation,
Micro oscillator vibrates 15sec；Mixed liquor is moved into adsorption column, 8000rpm is centrifuged 1min at room temperature；It is added in adsorption column
500 μ l AW1,8000rpm is centrifuged 1min at room temperature；500 μ l AW2 are added in adsorption column, 14000rpm is centrifuged at room temperature
3min；Adsorption column is placed in another new collecting pipe, 14000rpm is centrifuged 1min at room temperature；It is new that adsorption column is placed in one
In the EP pipe of 1.5ml, 50 μ l Buffer AE are added, are placed at room temperature for 2min, 8000rpm is centrifuged 1min at room temperature；Survey DNA concentration
And purity, sample are placed in -20 DEG C of preservations；
B) RNA, which is extracted, is centrifuged 7min in room temperature 1000rpm for cell suspension, abandons supernatant, 1ml lysate RZ is added, mixes gently；
After placing 5min, 200 μ l chloroforms are added, acutely vibrate 15sec, is placed at room temperature for after 3min 4 DEG C, 12000rpm is centrifuged 10min, sample
Product can be divided into three layers, top layer's water phase is transferred in new EP pipe；About 250 μ l dehydrated alcohols are added, mix；It will obtain solution
It is transferred in adsorption column together with precipitating, 4 DEG C, 12000rpm is centrifuged 30sec；It is added 500 μ l protein liquid removal RD into adsorption column, 4
DEG C, 12000rpm is centrifuged 30sec, abandons waste liquid；500 μ l rinsing liquid RW are added into adsorption column CR3, are stored at room temperature 2min, 4 DEG C,
12000rpm centrifugation 30sec is simultaneously repeated 1 times, and abandons waste liquid；Adsorption column is put into 2ml collecting pipe, 4 DEG C, 12000rpm centrifugation
2min is to remove residual liquid；Adsorption column is transferred in a new 1.5ml centrifuge tube, 30 μ lRNase-Free dd H are added2O,
It is placed at room temperature for 2min, 4 DEG C, 12000rpm is centrifuged 2min；RNA concentration and purity are surveyed, -80 DEG C of preservations are placed in.
7. the method for building up of Epstein-Barr virus tree shrew animal model as claimed in claim 6, it is characterised in that: the qPCR measurement
EBV copy number in PBMCs, for the DNA of acquirement using EBV copy number in qPCR method measurement PBMCs, the target fragment of amplification is EBV
BamHIW segment, it is as follows: upstream primer: 5 '-GCA GCC GCC CAG TCT CT-3 '；Downstream primer: 5 '-ACA GAC
AGT GCA CAG GAG CCT-3 ', probe: 5 '-(6FAM) AAA AGC TGG CGC CCT TGC CTG (TAMRA) -3 ', tool
Body method: being first arranged standard items, according to Namalwa cell total DNA concentration and above-mentioned conversion method, with qPCR standard dilutions
7 equal standard items than gradient out are diluted, specifically: first it is diluted to 1.2 × 105Copies/2 μ l, then gradient dilution at 1.2 ×
104copies/2μl、1.2×103copies/2μl、1.2×102copies/2μl、1.2×101copies/2μl、1.2×
100copies/2μl、1.2×10-1copies/2μl；After machine testing on the standard items of 7 proportional diluteds, according to standard items
Copy number and its Ct value can draw out standard curve, substitute into the Ct value of EBV virus liquid and sample, you can get it its copy number；
Qualification mark song need to meet the following conditions: coefficient R2> 0.98, -3.5 < slope < -3,90% < amplification efficiency E% <
120%.
8. the method for building up of Epstein-Barr virus tree shrew animal model as described in claim 1, which is characterized in that the RT-PCR inspection
Survey PBMCs in EBV related gene expression the following steps are included:
A) reaction system of 20 μ l is established in cDNA synthesis using Reverse Transcriptase kit, 500ngRNA is taken, on ice by following component
Be added without RNA, 200 μ l of DNA enzymatic PCR pipe in, reaction system: total serum IgE 500ng, AnchoredOligo 18Primer 1
10 1 μ l, gDNA Remover of μ l, EasyScript@RT/RI Enzyme Mix of μ l, 2 × ES Reaction Mix, 1 μ l,
RNase-free Water complements to 20 μ l, reaction condition: the above mixture being mixed gently, after centrifugation, is heated at 42 DEG C
30min, 85 DEG C of heating 5sec terminate reaction, be placed in -20 DEG C it is spare；
B) RT-PCR expands EBV related gene using cDNA as template, carries out RT-PCR amplification using following 20 μ l reaction system, point
Not Jian Ce the related gene expressions situation such as BZLF1, LMP1, EBNA1, EBNA2, EA, β-acti internal reference, reaction system: PCR Mix
10 μ l, 1 μ l of upstream primer, 3 μ l, Deionized Free H2O of downstream primer 1 μ l, template CDNA supply 20 μ l, RT-PCR expansions
Increasing condition: 95 DEG C of initial denaturations 3min, 95 DEG C of denaturation 30sec, anneal 30sec, and 72 DEG C of extension 1min, 35 circulations, 72 DEG C are prolonged again
10min is stretched, β-actin is internal reference, and the cDNA of B95-8 cell is positive control, and it is negative control that template, which is not added,；
A) each target gene primer sequence, primer size and annealing temperature are as follows: BZLF1 primer size: 129bp, annealing temperature:
60 DEG C, F:GCG GAC AAA AAT CAG GCG TT, R:GAA AAT GCC GGG CCA AGT TT；LMP1 primer size:
211bp/134bp, annealing temperature: 60 DEG C, F:AAT TTG CAC GGA CAG GCA TT, R:AAG GCC AAA AGC TGC
CAG AT；EBNA2 primer size: 253bp, annealing temperature: 63 DEG C, F:AAC TCC TGG CCC ATC CAA TG, R:GGA
GGG GCG AGG TCT TTT AC；EA primer size: 126bp, annealing temperature: 60 DEG C, F:AAC GAG CAG ATG ATT
GGG CA, R:CGT GGT GAC GTA GAG ATC CG；EBNA1 primer size: 270bp, annealing temperature: 60 DEG C, F:GTC
ATC ATC ATC CGG GTC TC, R:TTC GGG TTG GAA CCT CCT TG；β-actin primer size: 113bp is moved back
Fiery temperature: 60 DEG C, F:ATT TTG AAT GAT CAG CCA CC, R:AGG TAA GCC CTG GCT GCC TC；
B) Ago-Gel of agarose gel electrophoresis analysis preparation 2%, is placed on the electrophoresis equipped with 1 × TBE electrophoretic buffer
In slot, RT-PCR amplified production is added in gel pore respectively, is instruction in left side plus the hole DNA Marker, powers on,
Gel taking-up is placed in ultraviolet gel imaging system and analyzed by 100V, 250mA when 2/3 under instruction to the pact of gel on gel
As a result.
9. the method for building up of EBV virus tree shrew animal model as described in claim 1, it is characterised in that: the pathology inspection
It surveys and puts to death simultaneously anatomical trees Shrew in the different observation stages of experiment, take out its liver, spleen, lymphonodi mesenterici, lung, pharynx nasalis group
Knit, observe its substantially tissue change and carry out HE dyeing and EBER-ISH detection.
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| CN113604431A (en) * | 2021-09-02 | 2021-11-05 | 广西医科大学第一附属医院 | Separation method of tree shrew peripheral blood mononuclear cells |
| CN114731988A (en) * | 2022-05-23 | 2022-07-12 | 昆明市第一人民医院 | Tree shrew model construction method infected by HBV and tree shrew model |
| CN114731988B (en) * | 2022-05-23 | 2023-10-20 | 昆明市第一人民医院 | Method for constructing HBV-infected tree shrew model and tree shrew model |
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