CN108893474A - The separation and identification of adhesion molecule gene between Grass Carp Cell - Google Patents
The separation and identification of adhesion molecule gene between Grass Carp Cell Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The present invention relates to the separation of adhesion molecule gene between Grass Carp Cell and identifications, include the following steps：Clone obtains grass carp ICAM-1 coded sequence；Extract the recombinant expression plasmid of grass carp ICAM-1 activated protein；Expression of the ICAM-1 albumen in COS-7 cell；The separation and dyeing of grass carp blood lymphocyte；The adhesion experiment of grass carp blood lymphocyte.Technical characterstic of the invention is：ICAM-1 effect across endothelial cell to lymphocyte be it is required, which can be used for fish immunology Mechanism Study；There is soluble ICAM-1 in serum（sICAM-1）, it cuts the ICAM-1 of form membrane from protease；Clinically, the sICAM-1 level detected in serum can be used for auxiliary diagnosis fish disease.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to obtain and reflect using molecular biology and cell biology method
Determine the gene order of Adhesion molecule1 (ICAM-1) between Grass Carp Cell.
Background technique
Cell adhesion molecule is distributed across the class protein of cell surface, they can mediated cell and cell, cell with
Interaction between medium.In immune system, cell adhesion molecule has and can not replace for the transport and differentiation of leucocyte
The effect in generation.They not only can direct active cell signal transduction pathway, and can be protected during other receptors signal transductions
Necessary cell is held to contact with cell.Because by cell be physically contacted based on cell communication the coordination of immune response is carried out
Play a significant role, therefore antibody recognition reaction, costimulation effect and the cell adherence that adhesion molecule occurs in immune system
In all play important role.
Intercellular adhesion molecule-1 (ICAM-1) is the single pass transmembrane glycoprotein of a kind of contactin.
The expression of ICAM-1 can by bacteria lipopolysaccharide (LPS), phorbol exters and some cell factors, such as:Interleukin-1, TNF- and IFN-
Inducing expression.ICAM-1 is played an important role in inflammation and immune associated process.Particularly, it can be as secondary stimulus point
Son plays a significant role in across the endodermis migration of leucocyte and t cell activation.Currently, the ligand of a variety of ICAM-1 is sent out
It is existing, including lymphocyte function-associated antigen-1 (LFA-1), macrophage 1, antigen rhinovirus, fibrinogen and malignant malaria
Protozoan infection red blood cell.So far, fish ICAM-1 has not been reported, and the ICAM-1 molecule of grass carp is not found also.
Summary of the invention
The purpose of the present invention is to provide the separation and identification of Adhesion molecule1 (ICAM-1) gene between a kind of Grass Carp Cell
Method.Present invention finds the genes that one in grass carp genome can encode mediation grass carp ICAM-1, and use cell biology
Method confirm that it can specifically mediate the adherency of blood lymphocyte (PBLs).To achieve the purpose of the present invention, the present invention provides
Including gene cloning, the building of adhesion molecule maturation protein expression plasmid, the conversion of adhesion molecule expression plasmid, grass carp hemolymph
The methods of cell adherence.
The purpose of the present invention is achieved through the following technical solutions：Between Grass Carp Cell the separation of adhesion molecule gene and
Identification, which is characterized in that include the following steps：
S1, clone obtain grass carp ICAM-1 coded sequence：The sequence of ICAM-1 is found from grass carp genome, design is drawn
Object, clone obtains the coded sequence and amino acid sequence of grass carp ICAM-1 from grass carp head-kidney tissue；
S2, the recombinant expression plasmid for extracting grass carp ICAM-1 activated protein：Enzyme is carried out to the coded sequence of grass carp ICAM-1
Enzyme site scanning analysis, the restriction enzyme site in the multiple cloning sites of control vector pcDNA3.1/myc-His (-) select purpose to compile
Restriction enzyme site of the restriction enzyme site that code sequence is free of as building plasmid, primer of the design containing restriction enzyme site, PCR amplification,
Glue recovery product is carried out, double enzyme cutting digestion reactions is then carried out, is connected to required carrier with ligase, is turned in 42 DEG C of heat shocks
Change Escherichia coli, screening positive clone is simultaneously cultivated, and the recombinant expression plasmid of grass carp ICAM-1 activated protein is extracted；
Expression of S3, ICAM-1 albumen in COS-7 cell：Under set temperature, by the weight of grass carp ICAM-1 activated protein
Group expression plasmid is transfected into the culture medium of the cell containing COS-7, transfection expression；
The separation and dyeing of S4, grass carp blood lymphocyte：Blood of Ctenopharyngodon, which is collected, with disconnected cervical approach presses 1:1 addition culture medium simultaneously mixes
It is even, it handles to obtain grass carp blood lymphocyte by density gradient centrifugation, obtained grass carp blood lymphocyte is divided into two parts, wherein
After portion addition bacteria lipopolysaccharide is handled, fluorescent dyes are added to two parts of grass carp blood lymphocytes and be incubated at dyeing
Reason；
The adhesion experiment of S5, grass carp blood lymphocyte：Transfection, which is added, in the grass carp blood lymphocyte of fluorochrome label has
It is incubated in ICAM-1 expression plasmid or the COS-7 cell of zero load；Same number is added to without the cell of fluorescent dyeing
Transfection, which has in ICAM-1 expression plasmid or the COS-7 cell of zero load, to be incubated for as negative control, after incubation, with the buffering of preheating
Nonadherent cell is removed in liquid washout, carries out fluorometric investigation respectively to the cell of adherency.
Further, the primer in the step S1：5 ' TTTCTAAAGCCCAGTACTAGAG3 ' of gcICAM-1CDSF,
gcICAM-1CDSR 5’TGGCAGATGCAGCAAACCCAG 3’。
Further, the coded sequence of the grass carp ICAM-1 in the step S1 is as shown in SEQ ID No.1.
Further, the amino acid sequence of the grass carp ICAM-1 in the step S1 is as shown in SEQ ID No.2.
Further, the primer in the site of point containing digestion in the step S2：
5 ' ccgctcgagaagATGGCGAAGCAGTCGTTTTTTCT 3 ' of gcICAM-1EXF,
gcICAM-1EXR 5’cggggtaccAGCAAAGATATTGCTCTGAGAG 3’。
Further, the concrete operations of the step S3 are as follows：By 1 μ g pcDNA3.1/myc-His (-) or
The serum-free of pcDNA3.1/myc-His/gcICAM-1 plasmid and 1.5 μ l Lipofectamine, 2000 reagent in 500ul
It mixes in DMEM culture medium, is incubated for 15 minutes at room temperature；The COS-7 cell in 24 orifice plates is washed three times with PBS buffer solution, will be turned
Dye mixture is added thereto；After transfection 4 hours, changes transfection mixture into DMEM culture medium containing 10%FBS, cultivate 48 hours
It is used afterwards for cell adhesion experiments.
Further, the concrete operations of the step S4 are as follows：
(I) separation：Grass carp is put to death with disconnected cervical approach, syringe collecting blood is rapidly added heparin sodium containing 50mg and without FBS
RPM-1640 culture medium press 1:1 mixes；The Histopaque that 8ml density is 1.083kg/l is added in the centrifuge tube of 50ml
Solution, then isometric diluted Blood of Ctenopharyngodon is gently added on it；30 points are centrifuged on bucket centrifuge with revolving speed 1600g
Clock, the cell for collecting Histopaque solution and culture medium interface is grass carp blood lymphocyte；
(II) dyeing：Number of viable cells is checked with Trypan Blue, then cultivates base weight with the RPM-1640 containing 10%
Outstanding cell, and cell is transferred in culture medium after overnight incubation collects cell and is divided into two parts, and a copy of it is with 20 μ g/ml's
Bacteria lipopolysaccharide is handled 1 hour；It collects bacteria lipopolysaccharide processing or untreated cell is incubated at room temperature with 5 μM of calcein-AM
It educates 15 minutes；The calcein-AM dyestuff that not can enter cell is washed with grass carp blood lymphocyte to be removed three times；The staining cell
For following adhesion experiments.
Further, the concrete operations of the step S5 are as follows：By fluorescent dye, that is, calcein-AM label grass carp
Blood lymphocyte concentration is adjusted to 3.33 × 106A/ml, take 300ul cell suspension be added transfection have ICAM-1 expression plasmid or
In unloaded COS-7 cell, and in 27 DEG C of 2 hours of incubation；Same number is also added without the cell that calcein-AM is dyed
Have in ICAM-1 expression plasmid or the COS-7 cell of zero load to transfection as negative control；After incubation, nonadherent cell is used
The PBS of preheating gently washs removing；The fluorescence of adherent cell Nikon fluorescence microscope；The cell of adherency is with containing
The PBS of 0.1%Triton X-100 melts, and intracellular calcein is released, and measures fluorescence intensity with luminescent counter.
The beneficial effects of the invention are as follows：ICAM-1 effect across endothelial cell to lymphocyte is required, the Gene Isolation
Identification can be used for fish immunology Mechanism Study；There is soluble ICAM-1 (sICAM-1) in serum, it is from albumen
The ICAM-1 of form membrane is cut in digestion；Clinically, the sICAM-1 level detected in serum can be used for auxiliary diagnosis fish disease.
Detailed description of the invention
Fig. 1 is cell adhesion experiments；
A：Unloaded pcDNA3.1/myc-His (-) plasmid transfection COS-7 cell；B：ICAM-1 expression plasmid pcDNA3.1/
Myc-His/ICAM-1 transfects COS-7 cell, and PBLs is stimulated without LPS；C：ICAM-1 expression plasmid pcDNA3.1/myc-His/
ICAM-1 transfects COS-7 cell, and PBLs is stimulated through 20ug/ml LPS；D：Fluorescent strength determining result.
Fig. 2 is expression of the LFA-1 in each cellular component；
Specific embodiment
For a clearer understanding of the technical characteristics, objects and effects of the present invention, this hair of Detailed description of the invention is now compareed
Bright specific embodiment.
Technical solution of the present invention, specific work are described in further detail with attached drawing 1, attached drawing 2 combined with specific embodiments below
Steps are as follows for skill：
Embodiment one, clone obtain grass carp ICAM-1 coded sequence
Using bioinformatic analysis, the sequence of doubtful ICAM-1 is found from grass carp genome, and is set according to the sequence
Count primer：GcICAM-1CDSF 5 ' TTTCTAAAGCCCAGTACTAGAG 3 ', gcICAM-1CDSR 5 '
TGGCAGATGCAGCAAACCCAG 3 ', clone obtains the coding of grass carp ICAM-1 from the grass carp head-kidney tissue that phorbol exters are handled
Sequence.
After this step successfully carries out, the coded sequence of grass carp ICAM-1 is obtained, which is the SEQ in sequence table
ID No.1.(note：Sequence SEQ ID No.1 has been filed on GenBank ID and is：KY963996, but this can't be retrieved at present
Sequence.) translated to obtain grass carp ICAM-1 by the base sequence, which is the SEQ ID No.2 in sequence table.
Sequence SEQ ID No.1
cattgttttaaaatgaagaagcagtcgttttttctccccacgtcttttcggattttctgtatgcgactatttggatt
tggttttctgtacttcacactagttacagggacacatgctgatgaatgtcctcttcagctcaacccacagagagttg
ttgtggagtacgccggttctgtgtcagttaactgcagcacttctgtcacgcatcaagggatgggatgggaaaccagt
gagaaacagattggcaagagcagagacaatctgatcacatggagagtgtcaaacctgacacaatgggacatactgcc
atactgctttataaactataatggacagtgtcaattagagctcccaataactatttaccagactccagacagtgtgt
ccatcagcactgtgaatcacaccggaccaatgatggagggaaaccagtatgagcttcagtgtgacgttctcaatgtg
gcccctgttcagaatctcactgtcaaatggtacaaaggacagactctggtggatcaaaccaacttccctgacaccac
catcactccagtgaataaaacagtcacactcatgatccgtccaaacagagctgatgatggagttcagtacaggtgtg
aagcagagctggaactgggagaagaaggacctcaacctcctcctaaactcacatcagatcctctcagcattactgta
tattataaaccgaccatcaatgagaccaaactgccctccaatgtgtctgtgtttcgtggctatgaaatggtgctggt
ctgtgaagccgaaggaaacccaaaaccaacaatcagctggaatatcagcacaaataatccagtttacagtaaatctt
ttactgtaacagattcaacacctgaggatctgtactgcattgcaaacaattctgttgccatcaccatcagacatgta
aaagtgtccaaacaaggtcttccagatatttccatcagtgctggggatcataaaggaccaatgacaacgggccagca
gtataaactccagtgtgacgttctttatgtggcttctgttcggtatctcactgtcaaatggtacaaaggacagactc
tggtggatcaaaccaacttcactgacaccatcaagactccagtgaataaaacagtcacactcatgatccgtccagac
agatctgatgatggagctcagtacaggtgtgaagcagagctggaactgggagcagaaggacctcaacctcctcctaa
agtcacatcagatcctctcaacattactgtatattttaaaccgatcatcaatgagaccaaactgctttccaaagtgc
ctgtgtttcgtggctatgaaatggtgctggtctgtgaagccgaaggaaacccaaaaccaacaatcagctggaatatc
agcacaaataatccagtttatagtgaaacttttactataacagattcaacaccagaagaagtgtcctgcattgcaaa
caattctgttggcagcaccatcagacatgtaaaagtgattttaaaagaggattacctgcccctaatagcagggcttg
ttgccatcacagtggcgttcatctcagtcatcttcatcttcatctactctatctactacaaaacagccaagacgggc
cgttatagcctgaaggatgccaagcctagtgcacaaaatggaaacatagcacagaatggcaaagacaacactatccc
tatgaagaaactctctcagagcaatatctttgcttagacactatccaca
Sequence SEQ ID No.2
Embodiment two, the recombinant expression plasmid for extracting grass carp ICAM-1 activated protein
Since the protein sequence does not have signal peptide, resulting amino acid sequence is translated to the protein gene sequence first
Column carry out bioinformatic analysis and determine the mature peptide of the albumen by structure domain analysis.
Nucleic acid sequence corresponding to the protein maturation peptide is analyzed with NEB cutter software, is determined in nucleic acid sequence
The restriction enzyme site of contained restriction endonuclease.Selecting pcDNA3.1/myc-His (-) is carrier needed for expressing the protein maturation peptide,
The protein maturation peptide is corresponded to the restriction enzyme site in the multiple cloning sites on restriction enzyme site and the carrier contained in nucleic acid sequence
It is compared, selects to contain on expression vector and the Xhol I and the Kpn I site that do not have in purpose nucleic acid sequence is as building matter
The restriction enzyme site of grain, primer of the design containing restriction enzyme site are：gcICAM-1EXF 5'
CcgctcgagaagATGGCGAAGCAGTCGTTTTTTCT 3 ' and gcICAM-1EXR 5 '
cggggtaccAGCAAAGATATTGCTCTGAGAG 3'.PCR amplification is carried out using high fidelity enzyme phusion.
PCR reaction condition is as follows：
PCR reaction condition：L circulation：98℃30s；35 circulations：98℃10s,59℃30s,72℃60s；L circulation：
72 DEG C of 10min, 4 DEG C of preservations.
Product is analyzed with agarose gel electrophoresis after amplification, and target stripe is cut and carries out glue recycling, Zhi Houyong
XhoI and KpnI carries out double digestion.
Double enzyme cutting digestion conditions are as follows：
Above-mentioned reaction system is mixed, is incubated for 3 hours in 37 DEG C.Digestion products carry out electrophoresis with agarose, by purpose piece
Duan Huishou, and target fragment is connected in required carrier with T4 ligase.Product is transformed into after 4 DEG C of connections overnight
In JM109 competent E.coli, filters out positive colony and expanded with LB liquid medium and cultivated, tried with Tiangeng plasmid extraction
Agent box extracts plasmid as the recombinant expression plasmid with grass carp ICAM-1 activated protein.
The expression of embodiment three, ICAM-1 albumen in COS-7 cell
By COS-7 (African green monkey kidney fibroblast,CRL-1651TM) containing 10% fetal calf serum and double
Anti- DMEM (is purchased from U.S. Thermo company, article No.：In 11885092 culture mediums, and containing 5%CO2The training that temperature is 37 DEG C
It supports and is cultivated in case.By 1 × 105The COS-7 cell kind in a/hole is in 24 orifice plates.Transfection experiment is carried out after overnight incubation.Firstly,
By 1 μ g pcDNA3.1/myc-His (-) or pcDNA3.1/myc-His/gcICAM-1 plasmid and 1.5 μ l
2000 reagent of Lipofectamine mixes in the plasma-free DMEM medium of 500ul, is incubated for 15 minutes at room temperature.Meanwhile
The COS-7 cell in 24 orifice plates is washed three times with PBS buffer solution, and transfection mixture is added thereto.After transfection 4 hours, it will turn
Dye mixture changes the DMEM culture medium containing 10%FBS into, uses after culture 48 hours for cell adhesion experiments.
The separation and dyeing of example IV, grass carp blood lymphocyte
Grass carp is put to death with disconnected cervical approach, with syringe collecting blood, is rapidly added heparin sodium containing 50mg and is free of the RPM- of FBS
1640 culture mediums (1：1) it and mixes.8ml Histopaque solution (density is added in the centrifuge tube of 50ml：1.083kg/l),
Isometric diluted Blood of Ctenopharyngodon is gently added on it again.With revolving speed 1600g centrifugation 30 minutes on bucket centrifuge, receive
The cell for collecting Histopaque solution and culture medium interface is grass carp blood lymphocyte (PBLs).It is contaminated after collection with trypan blue
Color checks number of viable cells, cell then is resuspended with the RPM-1640 culture medium containing 10%, and cell is transferred to the training of 24 orifice plates
Support (1 × 106A cells/well/ml).After overnight incubation, collects cell and be divided into two parts.A copy of it is with the bacterium of 20 μ g/ml
Lipopolysaccharides is handled 1 hour.It collects LPS processing or untreated cell and 5 μM of calcein-AM is incubated at room temperature 15 minutes.Not
The calcein-AM dyestuff that can enter cell is washed with PBLs to be removed three times.The staining cell can be used for adhesion experiment below.
The adhesion experiment of embodiment five, grass carp blood lymphocyte
Fluorescent dye (calcein-AM) PBLs concentration marked is adjusted to 3.33 × 106A/ml takes 300ul cell
Transfection, which is added, in suspension has in ICAM-1 expression plasmid or the COS-7 cell of zero load, and in 27 DEG C of 2 hours of incubation.Same number
Cell without calcein-AM dyeing, which is also added to transfection, to be had in ICAM-1 expression plasmid or the COS-7 cell of zero load as negative
Control.After incubation, the PBS of nonadherent cell preheating gently washs removing.The fluorescence of adherent cell Nikon fluorescence
Micro- sem observation and preservation of taking a picture.PBS of the cell of adherency containing 0.1%Triton X-100 melts, intracellular
Calcein is released, and fluorescence intensity measures (excitation and launch wavelength are respectively 485nm and 538nm) with luminescent counter.Knot
Fruit sees Fig. 1
To further confirm that the adhesion molecule is ICAM-1, the PBLs on COS-7 cell is adhered to under PBS heavy duty wash
Come.The total serum IgE of LPS processing or the untreated PBLs without adhesion experiment and the PBLs of adherency elution are mentioned with Tripure reagent
It takes, the 2ug total serum IgE of each sample is cDNA by reverse transcription.Detect the mRNA table of the ICAM-1 receptor (LFA-1) in these cells
Up to level, judge the adhesion molecule for ICAM-1 indirectly.As a result such as Fig. 2.
The ICAM-1 obtained through the foregoing embodiment has following application prospect：
1.ICAM-1 effect across endothelial cell to lymphocyte be it is required, which can be used for fish immunity
Learn Mechanism Study.
2. there is soluble ICAM-1 (sICAM-1) in serum, it cuts the ICAM- of form membrane from protease
1.Clinically, the sICAM-1 level detected in serum can be used for auxiliary diagnosis.Thus, it is found that and identify fish ICAM-1, make
It is possibly realized by the horizontal auxiliary diagnosis fish disease of sICAM-1 in detection fish serum.
It should be noted that for simple description, therefore, it is stated as a series of movements for embodiment above-mentioned
Combination, but those skilled in the art should understand that, the application is not limited by the described action sequence, because according to this
Application, certain some step can be performed in other orders or simultaneously.Secondly, those skilled in the art should also know that, it says
Embodiment described in bright book belongs to preferred embodiment, and related movement and unit not necessarily the application institute are necessary
's.
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.
Claims (8)
1. the separation and identification of adhesion molecule gene between Grass Carp Cell, which is characterized in that include the following steps：
S1, clone obtain grass carp ICAM-1 coded sequence：Find the sequence of ICAM-1 from grass carp genome, design primer, from
Clone obtains the coded sequence and amino acid sequence of grass carp ICAM-1 in grass carp head-kidney tissue；
S2, the recombinant expression plasmid for extracting grass carp ICAM-1 activated protein：Digestion position is carried out to the coded sequence of grass carp ICAM-1
Spot scan is analyzed, and the restriction enzyme site in the multiple cloning sites of control vector pcDNA3.1/myc-His (-) selects target gene to compile
Restriction enzyme site of the restriction enzyme site that code sequence is free of as building plasmid, primer of the design containing restriction enzyme site, PCR amplification,
Glue recovery product is carried out, double enzyme cutting digestion reactions is then carried out, is connected to required carrier with ligase, is turned in 42 DEG C of heat shocks
Change Escherichia coli, screening positive clone is simultaneously cultivated, and the recombinant expression plasmid of grass carp ICAM-1 activated protein is extracted；
Expression of S3, ICAM-1 albumen in COS-7 cell：Under set temperature, by the recombination table of grass carp ICAM-1 activated protein
Up to plasmid transfection into the culture medium of the cell containing COS-7, transfection expression；
The separation and dyeing of S4, grass carp blood lymphocyte：Blood of Ctenopharyngodon, which is collected, with disconnected cervical approach presses 1:1 addition culture medium simultaneously mixes,
It handles to obtain grass carp blood lymphocyte by density gradient centrifugation, obtained grass carp blood lymphocyte is divided into two parts, wherein one
After part addition bacteria lipopolysaccharide is handled, fluorescent dyes are added to two parts of grass carp blood lymphocytes and carry out being incubated for dyeing processing；
The adhesion experiment of S5, grass carp blood lymphocyte：Transfection, which is added, in the grass carp blood lymphocyte of fluorochrome label ICAM-
It is incubated in 1 expression plasmid or the COS-7 cell of zero load；Same number, which is added to transfection without the cell of fluorescent dyeing, to be had
It is incubated in ICAM-1 expression plasmid or the COS-7 cell of zero load and is used as negative control, after incubation, removed with the buffer of preheating
Nonadherent cell is removed, fluorometric investigation is carried out respectively to the cell of adherency.
2. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that the step
Primer in rapid S1：GcICAM-1CDSF 5 ' TTTCTAAAGCCCAGTACTAGAG 3 ', gcICAM-1CDSR 5 '
TGGCAGATGCAGCAAACCCAG 3’。
3. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that the step
The coded sequence of grass carp ICAM-1 in rapid S1 is as shown in SEQ ID No.1.
4. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that described
The amino acid sequence of grass carp ICAM-1 is as shown in SEQ ID No.2 in step S1.
5. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that described
The primer in the site of point containing digestion in step S2：
5 ' ccgctcgagaagATGGCGAAGCAGTCGTTTTTTCT 3 ' of gcICAM-1EXF,
gcICAM-1EXR 5’cggggtaccAGCAAAGATATTGCTCTGAGAG 3’。
6. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that described
The concrete operations of step S3 are as follows：By 1 μ g pcDNA3.1/myc-His (-) or pcDNA3.1/myc-His/gcICAM-1 matter
Grain and 1.5 μ l Lipofectamine, 2000 reagent mix in the plasma-free DMEM medium of 500ul, are incubated for 15 at room temperature
Minute；The COS-7 cell in 24 orifice plates is washed three times with PBS buffer solution, and transfection mixture is added thereto；After transfection 4 hours,
It changes transfection mixture into DMEM culture medium containing 10%FBS, is used after culture 48 hours for cell adhesion experiments.
7. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that described
The concrete operations of step S4 are as follows：
(I) separation：Grass carp is put to death with disconnected cervical approach, syringe collecting blood is rapidly added heparin sodium containing 50mg and is free of FBS's
RPM-1640 culture medium presses 1:1 mixes；It is molten that the Histopaque that 8ml density is 1.083kg/l is added in the centrifuge tube of 50ml
Liquid, then isometric diluted Blood of Ctenopharyngodon is gently added on it；30 points are centrifuged on bucket centrifuge with revolving speed 1600g
Clock, the cell for collecting Histopaque solution and culture medium interface is grass carp blood lymphocyte；
(II) dyeing：Number of viable cells is checked with Trypan Blue, is then resuspended with the RPM-1640 culture medium containing 10% thin
Born of the same parents, and cell is transferred in culture medium after overnight incubation collect cell and are divided into two parts, and a copy of it is with the bacterium of 20 μ g/ml
Lipopolysaccharides is handled 1 hour；It collects bacteria lipopolysaccharide processing or untreated cell and 5 μM of calcein-AM is incubated at room temperature 15
Minute；The calcein-AM dyestuff that not can enter cell is washed with grass carp blood lymphocyte to be removed three times；The staining cell is used for
Following adhesion experiments.
8. the separation and identification of adhesion molecule gene between Grass Carp Cell according to claim 1, which is characterized in that described
The concrete operations of step S5 are as follows：Fluorescent dye, that is, calcein-AM label grass carp blood lymphocyte concentration is adjusted to 3.33
×106A/ml, taking 300ul cell suspension that transfection is added has in ICAM-1 expression plasmid or the COS-7 cell of zero load, and 27
DEG C be incubated for 2 hours；Same number without the calcein-AM cell dyed be also added to transfection have ICAM-1 expression plasmid or
Negative control is used as in unloaded COS-7 cell；After incubation, the PBS of nonadherent cell preheating gently washs removing；It is viscous
The fluorescence of attached cell Nikon fluorescence microscope；PBS of the cell of adherency containing 0.1%Triton X-100 melts,
Intracellular calcein is released, and measures fluorescence intensity with luminescent counter.
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