CN104830905B - Iba1 and the double gene coexpression recombinant adenoviral vectors of LAG 3 and its preparation method and application - Google Patents
Iba1 and the double gene coexpression recombinant adenoviral vectors of LAG 3 and its preparation method and application Download PDFInfo
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Abstract
The invention discloses Iba1 and the double gene coexpression recombinant adenoviral vectors of LAG 3 and its preparation method and application, recombinant adenoviral vector contains expression Iba1 and LAG 3 double gene expression box, the double gene expression box is successively by CMV promoter, the genes of LAG 3, internal ribosome entry site IRES, Iba1 genes and terminator composition, recombinant adenoviral vector Prepare restructuring adenovirus can be utilized, after the recombined adhenovirus transfection microglia of preparation, the inflammatory reaction after cerebral hemorrhage can be suppressed, effectively improve the nervous function of cerebral hemorrhage mouse, and extend the life cycle of cerebral hemorrhage mouse, available for the medicine for preparing anti-Activated Microglia, there is good development prospect in the therapy field of cerebrovascular disease.
Description
Technical field
The invention belongs to biological technical field, and in particular to Iba1 and LAG-3 double gene coexpression recombinant adenoviral vectors,
Further relate to the preparation method and application of the carrier.
Background technology
Cerebral hemorrhage (Intracerebral hemorrhage, ICH) is common ACVD, its incidence of disease, disease
Dead rate and disability rate are high, there is the trend risen year by year in China's incidence of disease.Intracranial hematoma formation is to cause neurotrosis most after ICH
Important pathology.The inflammatory reaction of hemotoncus main component and contractile effect of erythrocyte breakdown products induction, perihematoma metabolic disorder, local cerebral
CBF change etc. is the main cause for causing Secondary cases neurotrosis to aggravate.It is now recognized that the inflammatory reaction that hemotoncus composition is excited
Play an important roll in Secondary cases neurotrosis and receive much concern.The inflammatory cell such as perihematoma microglia swashs after ICH
It is living, the pro-inflammatory cytokine such as release TNF-α, IL-1, blood-brain barrier disruption in addition, the inflammatory cell such as peripheral blood mononuclear macrophage is to blood
Swollen peripheral region aggregation, thus induces inflammatory cascade iodine, ultimately results in secondary brain injury and neurologic impairment aggravation.
Therefore, suppress the inflammatory reaction of microglia after cerebral hemorrhage, can effectively control the nervous function damage after cerebral hemorrhage.
Calcium ion is as the first messenger in signal transduction passage, and the life for including axoneure in all cells is lived
Played an important role in dynamic.Calcium ion carries out cascade reaction after being combined from different calbindins.Iba1(ionized
Calcium binding adapter molecule 1) it can be specifically bound with macrophage and microglia
Calbindin, after these cell-stimulatings, Iba1 expression quantity up-regulation, therefore Iba1 is usually as identification microglia
Label is used.
Lymphocyte activation gene -3 (lymphocyte activation gene-3, LAG-3, CD223) is immune ball
One of member of superfamily protein, is the molecule inhibited to lymphocyte.LAG-3 is positioned at No. 12 chromosomes of people,
There is substantial connection with CD4.Research finds that the structure and expression pattern of pig LAG-3 molecules are shared in mammalian species
, and soluble pig LAG-3 has effect to control people-pig xenogenesis t cell immune response.LAG-3 molecules are mainly expressed in
NK cells, the T lymphocytic cell surfaces of activation, are combined with HLA-II high-affinities.Tumor-infiltrated CD8+T cell surfaces LAG-3
Up-regulated expression, LAG-3 inhibitory action plays an important role in HCC cellullar immunologic response, it is seen that block LAG-3 points
The expression of son is likely to become the new method for the treatment of tumour.In addition, thin using the targeted activation T of LAG-3 toxic antibodies selectivity
Born of the same parents can hinder the delayed allergy that T cell is participated in.
The mode of gene association treatment mainly has two kinds：One is by a variety of recombinant expression carriers for carrying different genes respectively
While transfected target cells, this mode can freely adjust the ratio of each recombinant expression carrier, but need to build carrying respectively not
Isogenic recombinant expression carrier, construction work is cumbersome, time-consuming, and influence factor is more；Further, since transfection process have it is certain with
There is the inhomogenous problem of gene copy in machine, the cell after transfection, be both unfavorable for expression and the successive treatment of target gene, again
Experimental result is caused to be difficult to analysis and assessment；Disadvantages mentioned above limits further applying for such a mode.Two be by two or two
Gene above is carried by a carrier and expressed, and this mode can pass through multiple promoter carrier, polycistronic vector or fusion
The control measures such as gene improve efficiency gene transfection, increase expression intensity, and maintain relative expression ratio, so that before overcoming
A kind of deficiency of mode, is increasingly subject to the attention of researcher in recent years.
The content of the invention
In view of this, an object of the present invention is that providing Iba1 and LAG-3 double gene coexpressions recombined adhenovirus carries
Body；The second object of the present invention is the preparation method for providing above-mentioned recombinant adenoviral vector；The third object of the present invention is
Application of the recombination expression adenovirus vector in the medicine for preparing anti-Activated Microglia is provided；The fourth object of the present invention exists
In the recombined adhenovirus for providing exploitation right recombination expression adenovirus vector packaging；The fifth object of the present invention is to provide restructuring gland
Application of the virus in the medicine for preparing anti-Activated Microglia.
For achieving the above object, the present invention provides following technical scheme：
Iba1 and LAG-3 double gene coexpression recombinant adenoviral vectors, the recombinant adenoviral vector contains expression Iba1
With LAG-3 double gene expression box, the double gene expression box is entered by CMV promoter, LAG-3 genes, internal ribosome successively
Angle of striking IRES, Iba1 gene and terminator composition, the nucleotide sequence of the LAG-3 genes is as shown in SEQ ID NO.3, institute
The nucleotide sequence of Iba1 genes is stated as shown in SEQ ID NO.6.
It is preferred that, the recombinant adenoviral vector is by gene containing LAG-3, internal ribosome entry site IRES and Iba1
The fragment of gene by the restriction enzyme sites of Sal I and the restriction enzyme sites of Not I be connected into the restriction enzyme sites of pShuttle-CMV carrier Ss al I and
Between the restriction enzyme sites of Not I, then with the linearization for enzyme restriction of Pme I again with pAdEasy-1 carriers in Escherichia coli BJ902 it is homologous
Recombinate and obtain.
It is preferred that, comprise the following steps：
A. clone's LAG-3 genes and Iba1 gene coding regions, then insert pStar carriers by LAG-3 gene coding regions
The IRES downstreams of pStar carriers are inserted in IRES upstreams, then Iba1 gene coding regions, obtain recombinant vector pStar-LAG-3-IRES-
Iba1；
B. using recombinant vector pStar-LAG-3-IRES-Iba1 as template, shown in SEQ ID No.7 and SEQ ID No.8
Sequence is that primer enters performing PCR amplification, obtains and contains gene containing LAG-3, internal ribosome entry site IRES with restriction enzyme site
With the piece of Iba1 genes, the piece of gene containing LAG-3, internal ribosome entry site IRES and Iba1 gene is then inserted into adenopathy
Malicious shuttle vector pShuttle-CMV CMV promoter downstream, obtains shuttle vector of adenovirus pShuttle-CMV-LAG-3-
IRES-Iba1；
C. by shuttle vector of adenovirus pShuttle-CMV-LAG-3-IRES-Iba1 and adenoviral backbone carrier
PAdEasy-1 carries out intracellular homologous recombination in Escherichia coli BJ902, obtains recombinant adenoviral vector pAd-LAG-3/Iba1.
It is preferred that, the method that LAG-3 genes are cloned in step a is as follows：Human liver tissue total serum IgE is extracted, reverse transcription is obtained
CDNA, then using the cDNA as template, performing PCR expansion is entered by upstream and downstream primer of sequence shown in SEQ ID No.1 and SEQ ID No.2
Increase, obtain LAG-3 genes；The PCR conditions are 94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72
DEG C extension 2 minutes, totally 30 circulation；Last 72 DEG C extend 10 minutes.
It is preferred that, the method that Iba1 genes are cloned in step a is as follows：Human Pancreas total serum IgE is extracted, reverse transcription is obtained
CDNA, then using the cDNA as template, performing PCR expansion is entered by upstream and downstream primer of sequence shown in SEQ ID No.4 and SEQ ID No.5
Increase, obtain Iba1 genes, the condition of the PCR amplifications is：94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C are denatured 50 seconds, 58 DEG C of annealing 50
Second, 72 DEG C extend 2 minutes, totally 30 circulations；Last 72 DEG C extend 10 minutes.
It is preferred that, in step b, amplification LAG-3-IRES-Iba1 condition is 94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C of changes
Property 50 seconds, 58 DEG C anneal 50 seconds, 72 DEG C extend 2 minutes, totally 30 circulation；Last 72 DEG C extend 10 minutes.
3rd, application of the recombination expression adenovirus vector in the medicine for preparing anti-Activated Microglia.
4th, the recombined adhenovirus of the recombination expression adenovirus vector packaging is utilized.
5th, application of the recombined adhenovirus in the medicine for preparing anti-Activated Microglia.
The beneficial effects of the present invention are：The invention discloses Iba1 and LAG-3 double gene coexpressions recombined adhenovirus load
Body, it contains the double gene expression box that recombinant adenoviral vector contains expression Iba1 and LAG-3, and the expression cassette can be simultaneously constant
Iba1 and LAG-3 albumen is given expression to, recombinant adenoviral vector is packaged as recombined adhenovirus, the recombined adhenovirus being made turns in vitro
Contaminate after microglia, the inflammatory reaction after cerebral hemorrhage can be suppressed, the nervous function of cerebral hemorrhage mouse is effectively improved, and extended
The life cycle of cerebral hemorrhage mouse, have available for the medicine for preparing anti-Activated Microglia, and in the therapy field of cerebrovascular disease
There is good development prospect.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：
Fig. 1 show LAG-3 full-length cDNA PCR primers agarose gel electrophoresis result (1 swimming lane be DNA molecular amount mark
Standard, 2 swimming lanes are PCR primer).
Fig. 2 show Iba1 full-length cDNA PCR primers agarose gel electrophoresis result (1 swimming lane be DNA molecular amount mark
Standard, 2 swimming lanes are PCR primer).
Fig. 3 show western blot detection LAG-3 and Iba1 expression of results (1 be untransfected Ad-LAG-3/Iba1
Virus transfection microglia；2 be Ad-LAG-3/Iba1 virus transfections microglia).
Fig. 4 shows the testing result of microglia multiplication capacity.
Fig. 5 shows the testing result of microglia secrete cytokines ability.
Fig. 6 shows the encephaledema situation of cerebral hemorrhage mouse.
Fig. 7 shows the Neuroscore of cerebral hemorrhage mouse.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific in embodiment
The experimental method of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. write)
Described in condition, or according to the condition proposed by manufacturer.
The preparation of embodiment 1, recombined adhenovirus Ad-LAG-3/Iba1
1st, LAG-3 full-length cDNAs are cloned
Extract human liver tissue total serum IgE by Trizol kit specifications, reverse transcription obtains total cDNA, then using total cDNA as
Template, with F1：5’-ggctcgagg(SEQ ID No.1, underscore part is Xho I to atgtgggaggctcagttcctg-3 '
Restriction enzyme site) and R1：5’-gggaattc(SEQ ID No.2, underscore part is EcoR I to tcagagctgctccggctc-3 '
Restriction enzyme site) it is upstream and downstream primer, PCR expands LAG-3 full-length cDNAs, and PCR reaction systems are 50 μ l, and cycling condition parameter is：
94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, totally 30 circulations；Last 72
DEG C extension 10 minutes.PCR primer identifies through agarose gel electrophoresis, as a result as shown in Figure 1.As a result show, PCR primer is about
It is in single specificity band at 1500bp, is consistent with expected results.Then by purpose band gel extraction, after purification withEasy carriers are connected, connection product conversion bacillus coli DH 5 alpha competent cell, with the LB containing ampicillin
Plate screening positive colony, extracts plasmid, positive colony plasmid is named as into pT-LAG-3.Then by positive colony plasmid order-checking
Identification, sequencing result shows the insertion gene order of positive colony plasmid as shown in SEQ ID No.3, the LAG-3 bases with report
Because of the consistent (GenBank of CDS sequences：NM_002286.5).
2nd, Iba1 full-length cDNAs are cloned
Human Pancreas total serum IgE is extracted by Trizol kit specifications, reverse transcription obtains total cDNA, then with total cDNA
For template, with F2：5’-gtctaga(SEQ ID No.4, underscore part is Xba I to atgagccaaaccagggatttac-3 '
Restriction enzyme site) and R2：5’-gggtcgac(SEQ ID No.5, underscore part is Sal to tcagggcaactcagagatag-3 '
I restriction enzyme site) it is upstream and downstream primer, PCR expands Iba1 full-length cDNAs, and PCR reaction systems are 50 μ l, and cycling condition parameter is：
94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, totally 30 circulations；Last 72
DEG C extension 10 minutes.PCR primer identifies through agarose gel electrophoresis, as a result as shown in Figure 2.As shown in Figure 2, Ago-Gel electricity
Result of swimming shows that PCR primer is in single specificity band at about 500bp, is consistent with expected results.Then purpose band is cut
Glue reclaim, after purification withEasy carriers connect, connection product conversion bacillus coli DH 5 alpha competent cell, with containing
There are the LB plate screening positive colonies of ampicillin, extract plasmid, positive colony plasmid is named as pT-Iba1.Then will
Positive colony plasmid order-checking identifies that sequencing result shows the insertion gene order of positive colony plasmid as shown in SEQ ID No.6,
(GenBank consistent with the Iba1 gene C DS sequences of report：NM_001623.3).
3rd, recombinant vector pStar-LAG-3/Iba1 structure
Restriction enzyme site according to designed by LAG-3 and Iba1 full-length cDNAs two ends, first inserts LAG-3 full-length cDNAs
PStar carriers (research of the DNA vaccination and adenovirus carrier vaccine of Guo Jianqiang, H5N1 subtype influenza virus double gene coexpression,
Thesis for the doctorate, 2009) IRES upstreams, then by Iba1 full-length cDNAs insert pStar carriers IRES downstreams.Specific method is：
First by pT-LAG-3 carriers Xho I and the double digestions of EcoR I, double digestion product is purified through gel reclaims kit gel extraction
Afterwards, it is connected with the pStar carriers equally through Xho I and the double digestions of EcoR I, connection product conversion bacillus coli DH 5 alpha competence
Cell, with the LB plate screening positive colonies containing ampicillin, extracts plasmid, Xho I and the identification of the double digestions of EcoR I, will
Positive colony plasmid is named as pStar-LAG-3；Again by pT-Iba1 carriers Xba I and the double digestions of Sal I, double digestion product
Through gel reclaims kit gel extraction after purification, connect with the pStar-LAG-3 carriers equally through Xba I and the double digestions of Sal I
Connect, connection product conversion bacillus coli DH 5 alpha competent cell, with the LB plate screening positive colonies containing ampicillin, is carried
Plasmid, Xba I and the identification of the double digestions of Sal I are taken, positive colony plasmid is named as pStar-LAG-3-IRES-Iba1.
4th, recombinant vector pShuttle-CMV-LAG-3-IRES-Iba1 structure
Using pStar-LAG-3-IRES-Iba1 carriers as template, with F3：5’-
gggtcgacAtgtgggaggctcagttcctg-3 ' (SEQ ID No.7, underscore part is the restriction enzyme sites of Sal I) and R3：
5’-ggcggccgcTcagggcaactcagagatag-3 ' (SEQ ID No.8, underscore part is the restriction enzyme sites of Not I) is
The restriction enzyme site at the fragment two ends is simultaneously replaced by the digestions of Sal I by upstream and downstream primer, PCR amplification LAG-3-IRES-Iba1 fragments
Site and the restriction enzyme sites of Not I, PCR reaction systems and cycling condition parameter are the same.PCR primer is reflected through agarose gel electrophoresis
Fixed, gel reclaims kit gel extraction after purification, withEasy carriers are connected, connection product conversion Escherichia coli
DH5 α competent cells, with the LB plate screening positive colonies containing ampicillin, extract plasmid, positive colony plasmid are ordered
Entitled pT-LAG-3-IRES-Iba1, most afterwards through sequencing identification.
By pT-LAG-3-IRES-Iba1 carriers Sal I and the double digestions of Not I, double digestion product is through gel reclaim reagent
Box gel extraction after purification, is connected with the pShuttle-CMV carriers equally through Sal I and the double digestions of Not I, and connection product turns
Change bacillus coli DH 5 alpha competent cell, with the LB plate screening positive colonies containing ampicillin, extract plasmid, Sal I
With the identification of the double digestions of Not I, positive colony plasmid is named as pShuttle-CMV-LAG-3-IRES-Iba1.
5th, recombinant adenoviral vector pAd-LAG-3/Iba1 structure
By the pShuttle-CMV-LAG-3-IRES-Iba1 carriers linearization for enzyme restriction of Pme I, then with pAdEasy-1 carriers
The Escherichia coli BJ902 competent cells of electricity conversion simultaneously, carry out bacterium In vivo homologous recombination, are trained with the LB flat boards containing kanamycins
Support, picking with the LB fluid nutrient medium overnight incubations containing kanamycins, extracts plasmid, the digestions of Pac I are identified compared with petite,
Positive plasmid is named as pAd-LAG-3/Iba1.
6th, recombinant adenoviral vector pAd-LAG-3/Iba1 packaging
Human embryonic kidney 293 cell is seeded in T-25 blake bottles with 40%~60% cell density, cell growth is treated
To nutrient solution is discarded during 30%~50% fusion, pAd-LAG-3/Iba1 carriers are transfected with the reagents of Lipofectamine 2000
293 cells, are changed by micro- sem observation cell pathology, cell, cell are collected by centrifugation during obvious cytopathic effect to appear
After precipitation is resuspended with PBS, multigelation cracks cell 4 times, and centrifugation removes cell fragment, collects the supernatant containing virus, i.e.,
Obtain Ad-LAG-3/Iba1 virus stock solution useds；Virus stock solution used is pressed 1:10 ratio re-infects 293 cells, collects containing the upper of virus
Clear liquid, repeats aforesaid operations 3 times, obtains forth generation recombined adhenovirus Ad-LAG-3/Iba1.
Embodiment 2, recombined adhenovirus Ad-LAG-3/Iba1 transfect the preparation of microglia
1st, the sorting of microglia
Microglia culture is by literature method (Lehnart et al, J Neurosci, 2002；22(7):2478-
2486, this method is classical microglia cultivation, is widely used in research).It is specific as follows：Take 1-2 days reproduction age
C57BL/6 mouse forebrain tissues, with trypsin digestion, grinding 37 DEG C, 20min, cell is transferred to the DMEM trainings containing calf serum
Base culture is supported, after 1 week, 180rpm vibrations 30min, cell suspension (contains>90% small colloid spongiocyte) be transferred to it is not coated
Non- adherent cell suspension is removed after culture plate, 15min, is washed with PBS 3 times, with immunostaining identification of cell purity (90% small glue
Matter spongiocyte).
2nd, recombined adhenovirus Ad-LAG-3/Iba1 transfects microglia
By microglia using cell concentration as 1 × 106Individual/ml is inoculated in 6 orifice plates, adds by infection multiplicity (MOI) for 100
Enter forth generation recombined adhenovirus Ad-LAG-3/Iba1, collect cell within 48 hours after infection, washed and be resuspended with PBS, produce Ad-
LAG-3/Iba1 virus transfection microglias.
Western Blot are detected：Ultrasonic degradation Ad-LAG-3/Iba1 virus transfections microglia and sky are viral respectively
Microglia is transfected, total protein of cell is collected and carries out SDS-PAGE, electricity transfer pvdf membrane, washes film after electrophoresis is finished, and closes, plus
Enter rabbit-anti people's LAG-3 or Iba1 polyclonal antibody, 37 DEG C are incubated 1 hour, wash film, add goat anti-rabbit igg, 37 DEG C of incubations 1 are small
When, film is washed, is developed the color, as a result as shown in Figure 3.As a result show, Ad-LAG-3/Iba1 viruses can be expressed in microglia
LAG-3 and Iba1.
Embodiment 3, recombined adhenovirus Ad-LAG-3/Iba1 transfection microglia anti-proliferative capacity detections
Microglia is taken to be randomly divided into two groups：Experimental group and control group, experimental group microglia transfection Ad-LAG-3/
Iba1 viruses, control group is normally cultivated.
1st, microglia multiplication capacity is detected
It is 1 × 10 with the culture mediums of the RPMI 1640 adjustment cell concentration for the hyclone for being 10% containing volume fraction6
Individual/ml, is seeded to 96 orifice plates, and per the μ l of hole 100, every group sets 3 multiple holes, puts temperature for 37 DEG C, CO2Gas volume fraction is 5%
Incubator in culture 96 hours, add the μ l of splitting erythrocyte suspension 100, cultivate 96 hours, then 0.1ml concentration is added per hole and be
1×103μ Ci/L 3H- thymidines (3H-TdR) solution, continues to cultivate 12 hours, rinses cell 3 times with PBS, formaldehyde
10 minutes are fixed, then is rinsed 3 times for 5% trichloroacetic acid solution with the volume fraction of 4 DEG C of precoolings of temperature, 0.5ml is added per hole
Concentration is 0.3mol/L NaOH solution, and 60 DEG C of water-baths 30 minutes are cooled to room temperature, are transferred in scintillation vial, add 5ml
Scintillation solution, flashing times (cpm) per minute are counted with liquid scintillation counter, determine DPM values (reflection cell DNA synthesis speed
Rate), replication 3 times, as a result as shown in Figure 4.As a result show, the cpm values of experimental group microglia are 32000 ± 3100,
And the cpm values of control group microglia are 59000 ± 6500, it was demonstrated that Ad-LAG-3/Iba1 virus transfection microglia energy
The multiplication capacity of enough effectively reduction microglias.
2nd, microglia secrete cytokines ability is detected
It is 1 × 10 with the culture mediums of the RPMI 1640 adjustment cell concentration for the hyclone for being 10% containing volume fraction6
Individual/ml, is seeded to 96 orifice plates, and per the μ l of hole 100, every group sets 3 multiple holes, puts temperature for 37 DEG C, CO2Gas volume fraction is 5%
Incubator in culture 96 hours, add the μ l of splitting erythrocyte suspension 100, cultivate 96 hours, with ELISA method detection IL-1 β and
The content of TNF-α, as a result as shown in Figure 5.As shown in Figure 5, the IL-1 β and TNF-α content of experimental group microglia are respectively
55.3 ± 5.8pg/ml and 49.7 ± 4.9pg/ml, and the IL-1 β of control group mice lymphocyte and TNF-α content are respectively
104.8 ± 12.3pg/ml and 95.3 ± 11.2pg/ml, it was demonstrated that Ad-LAG-3/Iba1 virus transfections microglia can be effective
Reduce the ability of microglia secrete cytokines.
Embodiment 3, recombined adhenovirus Ad-LAG-3/Iba1 transfection microglias mitigate cerebral hemorrhage nervous function damage energy
Power
With 4% chloral hydrate anesthesia mouse, it is fixed on afterwards on mouse stereotaxic apparatus.Regioselective coordinate is bregma
0.2mm forward, 2.3mm, deep 3.5mm are opened in side.Experiment point sham groups, physiological saline group (blank control) and erythrocytolysis composition
6 groups.Collection mouse vein blood is crushed into the μ l of liquid 30 autologous blood is injected into sprocket bit with 2 μ l/min speed with syringe pump immediately
Suspend 7min after point, 5 μ l of injection, then remaining 25 μ l blood syringes are finished with 2 μ l/min speed again, stopped afterwards
10min flows back to prevent Hemostatic Oral Liquid.After pin is extracted, mouse skull is closed with bone wax, skin suture, treat that mouse recovers meaning
Continue normal raise after knowledge.Ensure that mouse temperature maintains 37 ± 0.5 degree or so during operation, in 1h after 1h before bleeding and bleeding
Recombined adhenovirus Ad-LAG-3/Iba1 and control PBS are injected respectively respectively at brain parenchym same location, then in after bleeding 72h,
Dry and wet weight method, the water content of neurological deficits score method brain tissue, the change of neurologic impairment are utilized respectively, figure is as a result seen
Shown in 6 and Fig. 7.As a result show, the brain water content of experimental mice is (78.3 ± 6.4) %, the brain group of control group mice
Knitting water content is
(82.1 ± 7.4) %；The Neuroscore of experimental mice is (18 ± 2) point, the neural work(of control group mice
It can score as (12 ± 1) point.Experiment confirms that Ad-LAG-3/Iba1 viruses can effectively improve the nervous function damage of cerebral hemorrhage mouse
Wound.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment the present invention is described in detail, it is to be understood by those skilled in the art that can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (9)
1.Iba1 and LAG-3 double gene coexpression recombinant adenoviral vectors, it is characterised in that：The recombinant adenoviral vector contains
Iba1 and LAG-3 double gene expression box is expressed, the double gene expression box is successively by CMV promoter, LAG-3 genes, inside
Ribosome entry site IRES, Iba1 gene and terminator composition, the nucleotide sequence such as SEQ ID of the LAG-3 genes
Shown in NO.3, the nucleotide sequence of the Iba1 genes is as shown in SEQ ID NO.6.
2. recombinant adenoviral vector according to claim 1, it is characterised in that：The recombinant adenoviral vector be by containing
LAG-3 genes, the fragment of internal ribosome entry site IRES and Iba1 gene pass through the restriction enzyme sites of Sal I and the digestions of Not I position
Point be connected between the restriction enzyme sites of pShuttle-CMV carrier Ss al I and the restriction enzyme sites of Not I, then with the linearization for enzyme restriction of Pme I again with
PAdEasy-1 carriers in Escherichia coli BJ902 homologous recombination and obtain.
3. the preparation method of recombinant adenoviral vector described in claim 1 or 2, it is characterised in that comprise the following steps：
A. clone's LAG-3 genes and Iba1 gene coding regions, then insert LAG-3 gene coding regions the IRES of pStar carriers
Upstream, then by the IRES downstreams of Iba1 gene coding regions insertion pStar carriers, obtain recombinant vector pStar-LAG-3- IRES-
Iba1；
B. using recombinant vector pStar-LAG-3- IRES-Iba1 as template, sequence shown in SEQ ID No.7 and SEQ ID No.8
Enter performing PCR amplification for primer, obtain and contain LAG-3 genes, internal ribosome entry site IRES and Iba1 with restriction enzyme site
The fragment of gene, then inserts adenovirus by the fragment of gene containing LAG-3, internal ribosome entry site IRES and Iba1 gene
Shuttle vector pShuttle-CMV CMV promoter downstream, obtains shuttle vector of adenovirus pShuttle-CMV-LAG-3-
IRES- Iba1；
C. by shuttle vector of adenovirus pShuttle-CMV-LAG-3-IRES- Iba1 and adenoviral backbone carrier
PAdEasy-1 carries out intracellular homologous recombination in Escherichia coli BJ902, obtains recombinant adenoviral vector pAd-LAG-3/Iba1.
4. preparation method according to claim 3, it is characterised in that the method that LAG-3 genes are cloned in step a is as follows：
Human liver tissue total serum IgE is extracted, reverse transcription obtains cDNA, then using the cDNA as template, with SEQ ID No.1 and SEQ ID No.2
Shown sequence is that upstream and downstream primer enters performing PCR amplification, obtains LAG-3 genes；The PCR conditions are 94 DEG C of pre-degenerations 5 minutes；Then
94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, totally 30 circulations；Last 72 DEG C extend 10 minutes.
5. preparation method according to claim 3, it is characterised in that the method that Iba1 genes are cloned in step a is as follows：Carry
Human Pancreas total serum IgE is taken, reverse transcription obtains cDNA, then using the cDNA as template, with SEQ ID No.4 and SEQ ID No.5
Shown sequence is that upstream and downstream primer enters performing PCR amplification, obtains Iba1 genes, and the condition of the PCR amplifications is：94 DEG C of 5 points of pre-degenerations
Clock；Then 94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, totally 30 circulations；Last 72 DEG C extend 10 minutes.
6. preparation method according to claim 3, it is characterised in that：In step b, LAG-3-IRES-Iba1 bar is expanded
Part is 94 DEG C of pre-degenerations 5 minutes；Then 94 DEG C are denatured 50 seconds, and 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, totally 30 circulations；Most
72 DEG C extend 10 minutes afterwards.
7. application of the recombinant adenoviral vector described in claim 1 or 2 in the medicine for preparing anti-Activated Microglia.
8. utilize the recombined adhenovirus of recombinant adenoviral vector packaging described in claim 1 or 2.
9. application of the recombined adhenovirus described in claim 8 in the medicine for preparing anti-Activated Microglia.
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