CN104164442B - Pig SAMHD1 genes, albumen, monoclonal antibody and its application - Google Patents
Pig SAMHD1 genes, albumen, monoclonal antibody and its application Download PDFInfo
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- CN104164442B CN104164442B CN201410028266.7A CN201410028266A CN104164442B CN 104164442 B CN104164442 B CN 104164442B CN 201410028266 A CN201410028266 A CN 201410028266A CN 104164442 B CN104164442 B CN 104164442B
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Abstract
The invention discloses a boar SAMHD1 genes, which includes the nucleotide sequence of amino acid sequence shown in coding SEQIDNO.1.The invention also discloses pig SAMHD1 protein, which has amino acid sequence shown in SEQIDNO.1.The invention also discloses the monoclonal antibodies of anti-pig SAMHD1 albumen.Pig SAMHD1 genes, the protein of the present invention, has antiviral activity, suitable for developing antiviral drugs, has a extensive future.The monoclonal antibody of the present invention has good specific reaction with pig SAMHD1 albumen, and the biological function for further research pig SAMHD1 albumen has established important basis.
Description
Technical field
The present invention relates to technical field of bioengineering more particularly to a boar SAMHD1 genes, albumen, monoclonal antibody and
It is applied.
Background technology
Congenital immunity plays a significant role during body resists pathogen infection, can after cause of disease invades body
It quick response is made, controls the diffusion of cause of disease, and then inducing specific immunity response eliminates cause of disease.Body is in long-term evolution
During, it forms with the natural restriction factor for resisting cause of disease invasion.Have now been found that 4 kinds of natural restrictions because
Son, TRIM5 α, Tetherin, APOBEC3G and SAMHD1 (Neil et al., 2008；Sheehy et al.,2002；
Stremlau et al.,2004).Research confirms that SAMHD1 is as a kind of natural restriction factor, in marrow source cell recently
Expression quantity is higher, can prevent HIV-1 types virus in marrow source cell duplication (Chen et al., 2012；Goldstone
et al.,2011；Laguette et al.,2011).SAMHD1 albumen can regulate and control the congenital immunity of body, hence it is evident that up-regulation
Body disease-resistant poison immune response mediates the inflammatory reaction as caused by interferon, participates in defense system of the host to intrusion cause of disease.So
And the biological characteristics of SAMHD1 is also known little about it at present.Recent research indicate that SAMHD1 albumen has deoxynucleoside triphosphate
The function of hydrolase, can with the dNTP in hydrolyzed cellular, so as to inhibit the forming process of the reverse transcription of HIV-1 viruses and cDNA,
And then prevent virus infection and duplication (Franzolin et al., 2013；Goldstone et al.,2011).And HIV-2
With some monkey immunodeficiency virus (SIVsm/mac) then can by its coding Vpx protein degradations SAMHD1, Vpx albumen with
DCAF1 interacts, and then forms Cullin4A/DDB1 and E3 ubiquitin-connection combined enzyme agent, compound by ubiquitin-ligase
Body targeting degradation SAMHD1 albumen, so as to cause infection of the virus to such cell, this degradation can pass through protease
Inhibitor come release (Ahn et al., 2012；Hrecka et al.,2011；Srivastava et al.,2008).Except HIV
Outside, if having other viruses that can interact therewith unclear.In addition, SAMHD1 albumen also with Aicardi-
Goutieres (ASG) disease is related (Rice et al., 2009).
SAMHD1 albumen whether can antagonism other viral infection, it is not fully aware of at present.In recent years, hog cholera
The great epidemic disease virus of pigs such as malicious (CSFV), high-pathogenicity blue ear disease viral (HP-PRRSV), porcine pseudorabies virus (PRV) is endangered always
The health of evil China's pig breeding industry, sustainable development.How the generation of the great epidemic disease of prevention and control, reduce loss caused by it, improve livestock and poultry
Body disease-resistant poison reaction is that scientific research personnel endeavours the target pursued.SAMHD1 as the innate immunity limit sex factor, function and
Characteristic is known little about it at present.Therefore, carry out function and characteristic research to it, there is weight to the generation and development of the great epidemic disease of prevention and control
Want meaning.
Research for SAMHD1 protein functions is, it is necessary to obtain its complete genome sequence, recombinant protein and with good anti-
The monoclonal antibody of answering property.At present, the SAMHD1 such as people, monkey, horse have obtained its complete genome sequence, but without accurate pig SAMHD1
Complete genome sequence, and monoclonal antibody or mostly anti-very poor with pig SAMHD1 albumen reactivity prepared by people source SAMHD1, almost without
Reactivity.Therefore, it is necessary to obtain the complete genome sequence of accurate pig SAMHD1, and prepare the monoclonal with good reactivity and resist
Body, can realize to SAMHD1 protein functions deeper into research.
The content of the invention
The technical issues of the invention solves currently without accurate pig SAMHD1 complete genome sequences, provide a boar
SAMHD1 genes, the complete genome sequence of the gene can be used for the drug or biological products of developing the great epidemic disease of prevention and control pig.
For this purpose, it is also required to provide a boar SAMHD1 albumen and monoclonal antibody, the prevention great epidemic disease of pig is used to prepare
Drug or biological products.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a boar SAMHD1 genes are provided, which includes coding SEQ ID
The nucleotide sequence of amino acid sequence shown in NO.1.
Preferably, the pig SAMHD1 gene orders are the nucleotide sequence shown in SEQ ID NO.2.
Preferably, pig SAMHD1 complete genome sequences of the invention contain 3951 in addition to poly (A) sequence at 3 ' ends
Nucleotide, wherein comprising an open reading frame, between the 68th and 1951 nucleotide, length is the open reading frame
1884 nucleotide encode 627 amino acid.
In another aspect of this invention, a boar SAMHD1 protein is additionally provided, which has SEQ
Amino acid sequence shown in ID NO.1.
In another aspect of this invention, a kind of recombinant vector is additionally provided, it includes amino shown in coding SEQ ID NO.1
The nucleotide sequence of acid sequence.
The empty carrier of the recombinant vector includes prokaryotic expression carrier or carrier for expression of eukaryon.
In the present invention, the preferred pcold TF DNA prokaryotic expression carriers of prokaryotic expression carrier, carrier for expression of eukaryon are preferred
P3 × FLAG CMV7.1 carrier for expression of eukaryon.
In another aspect of this invention, a kind of host cell is additionally provided, it includes above-mentioned recombinant vectors.
In another aspect of this invention, a kind of recombinant protein is additionally provided, which passed through by above-mentioned recombinant vector
It expresses and is made.
In another aspect of this invention, a kind of monoclonal antibody of anti-pig SAMHD1 albumen is additionally provided.
Preferably, the monoclonal antibody is resisted by the monoclonal of hybridoma cell line CCTCC NO.C201422 secretions
Body.
In another aspect of this invention, a kind of kit for detecting pig SAMHD1 albumen is additionally provided, which includes
The monoclonal antibody of above-mentioned anti-pig SAMHD1 albumen.
In another aspect of this invention, a kind of antiviral drugs is additionally provided, which contains pig SAMHD1 protein.
The pig SAMHD1 genes of the present invention have the complete genome sequence of pig SAMHD1 genes, the mistake in MARC-145 cells
Pig SAMHD1 albumen is expressed, can substantially inhibit highly pathogenic PRRSV multiplication, illustrate pig SAMHD1 eggs
There is antiviral activity in vain, suitable for developing antiviral drugs.The restructuring obtained using pig SAMHD1 gene prokaryotics of the present invention
Soluble pig SAMHD1 albumen prepares the monoclonal antibody for having specific reaction with pig SAMHD1 albumen after mouse is immunized,
The monoclonal antibody has established important basis for the characteristic of further research pig SAMHD1 albumen with function.
Description of the drawings
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is the electrophoresis result figure that the embodiment of the present invention 1 expands pig SAMHD1 gene orders；
Fig. 2 is the amplification of 2 pig SAMHD1 gene coded sequences of the embodiment of the present invention and the expression and purification result of recombinant protein
Figure；
Fig. 3 is the Western blot of 2 pig SAMHD1 antagonisms HP-PRRSV of embodiment of the present invention multiplication and titration of virus knot
Fruit is schemed；
Fig. 4 is 4 pig SAMHD1 MAb mediated ELISA potency qualification figures of the embodiment of the present invention；
Fig. 5 is 5 pig SAMHD1 monoclonal antibody specificity qualification figures of the embodiment of the present invention.
The monoclonal antibody hybridoma cell line of the present invention, is preserved in Chinese Typical Representative culture on January 14th, 2014
Collection (abbreviation CCTCC, address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection), preserving number CCTCC
NO.C201422, Classification And Nomenclature are hybridoma cell strain 5M1.
Specific embodiment
In the following example, the experimental method of actual conditions is not specified, usually routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(chief editors such as F.M. Ao Sibai, R.E. James Kingstons, J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates Beijing：Section
Learn publishing house, 2004) described in method carry out.
In order to develop the drug or biological products of the great epidemic disease of prevention and control pig, the present invention obtains pig using RACE methods
SAMHD1 complete genome sequences.On the basis of pig SAMHD1 complete genome sequences, expand pig SAMHD1 encoding genes, be inserted into p3 ×
FLAG CMV7.1 carrier for expression of eukaryon expresses pig SAMHD1 in MARC-145 cells, it is numerous can to significantly inhibit high-pathogenicity porcine
The infection with breath syndrome virus is grown, illustrating the pig SAMHD1 albumen of the present invention has antiviral activity.Pig SAMHD1 is compiled
Code gene insertion pcold TF DNA prokaryotic expression carriers, induced expression, purifying obtain soluble Recombinant Swine SAMHD1 albumen, exempt from
The monoclonal antibody of anti-pig SAMHD1 albumen is prepared after epidemic disease BALB/c mouse.The monoclonal antibody that the present invention obtains, with pig
SAMHD1 albumen has preferable specific reaction in source cell, poor with people source, monkey source SAMHD1 albumen atopics.
Embodiment 1 obtains pig SAMHD1 complete genome sequences using the amplification of RACE methods
By comparing pig SAMHD1 forecasting sequences (the Genbank ID being had been filed in GenBank：XM_003483952,XM_
003361435, AK236802) pair of primers amplification SAMHD1 partial nucleic acid sequences, are designed, this is s880- to primer sequence
forward：5 '-TCAAAGCAGACCCTTACAT-3 ' (SEQ ID NO.3), S880-reverse：5’-
TTGCCTAATTCCTGTTTCT-3’(SEQIDNO.4).As a result the pig SAMHD1 partial gene sequences of length about 880bp are amplified
(see Figure 1A).After sequence, comparison are correct, drawn according to 880bp sequence design a pair of the SAMHD1 gene specifics amplified
Object, GSP1-forward：5 '-AGATGCCTTCCTCAAAGCAGACCCT-3 ' (SEQ ID NO.5), GSP1-reverse：5’-
TATGCCCTAAATTGATTGGGGGGGC-3 ' (SEQ ID NO.6), and design a pair is about the internal sleeve of 220bp inside it
Formula primer, S220-forward：5 '-GCTGAACTGAAGGCTGAA GAT-3 ' (SEQ ID NO.7), S220-forward：
5 '-AAACAGACTCTTTTCATCCGT-3 ' (SEQ ID NO.8), for identifying the specificity of RACE PCR amplification sequences.It adopts
With Clontech companies SMARTerTMRACE cDNA amplification kits are complete using gene-specific primer GSP amplification pigs SAMHD1
Gene order.The results show that 5 ' PCR product length about 2000bp (Figure 1B), 3 ' PCR product length about 2700bp (Fig. 1 C), PCR
After product purification, pBlunt cloning vectors are connected, Shanghai life work is sequenced.It is compared by sequence assembly, obtains pig SAMHD1
Complete genome sequence (SEQ ID NO.2) and its coded sequence.Sequence analysis shows that pig SAMHD1 complete genome sequences contain 3981
Nucleotide, wherein including an open reading frame.Between the 68th and 1951 nucleotide, length is open reading frame
1884 nucleotide encode 627 amino acid (SEQ ID NO.1).
Embodiment 2 recombinantly expresses pig SAMHD1 albumen
The pig SAMHD1 complete genome sequences amplified according to embodiment 1 design specific primer, expand SAMHD1 code sequences
Row, the specific primer of amplification coding gene is as follows, pcold-pig SAMHD1 (pcold-pig SAMHD1-forward：5’-
CCAAGCTTATGCAGAGTGCCGACTCC-3 ' (SEQ ID NO.9), pcold-pig SAMHD1-reverse：5’-
CGGGATCCTCACACCGAGTCCTTTGCA-3 ' (SEQ ID NO.10)), wherein upstream and downstream primer contains Hind III respectively
With Bam HI restriction endonuclease sites, the SAMHD1 coded sequences of pcold-pig SAMHD1 primer amplifications, for being inserted into
Pcold TF DNA prokaryotic expression carriers；pFLAG-pig SAMHD1(pFLAG-pig SAMHD1 forward：
CAAGCTTATGCAGAGTGCCGACTCCCAGCAG(SEQ ID NO.11)；pFLAG-pig SAMHD1 reverse：
GCTCTAGATCACACCGAGTCCTTTGCAAA (SEQ ID NO.12)), the SAMHD1 of pFLAG-pig SAMHD1 primer amplifications
Coded sequence, for being inserted into p3 × FLAG CMV7.1 carrier for expression of eukaryon.The pig SAMHD1 that pcr amplification product is 1884bp is compiled
(see Fig. 2A, Tu2AZhong, M are DNA molecular amount standard DL2000 to code sequence fragment；1 is negative control, and 2 be pcr amplification product).
The recovered purifying of PCR product, after enzymes double zyme cutting, respectively with pcold TF DNA prokaryotic expression carriers and p3 × FLAG
CMV7.1 carrier for expression of eukaryon connects, and structure pig SAMHD1 prokaryotic expression carriers and pig SAMHD1 carrier for expression of eukaryon are ordered respectively
Entitled pcold-pSAMHD1 and pFLAG-pSAMHD1, positive plasmid are identified correct through sequencing, digestion.
Pcold-pSAMHD1 converts Rosseta competent cells.Prokaryotic expression, purification Recombinant Swine SAMHD1 albumen program is such as
Under：Bacterium solution inoculation 2 × YT culture mediums containing positive plasmid, inoculative proportion 1：100, it shakes bacterium to bacterium solution OD values through 37 DEG C of vibrations and reaches
To after 0.6, IPTG to final concentration of 0.5mM is added in, 16 DEG C are continued to induce 5h.Bacterium solution is collected by centrifugation and with after PBS cleaning 2 times,
After PBS is resuspended, ultrasound to bacterium solution is clarified, centrifugation removal ultrasound precipitation, the 4 DEG C of effects of supernatant and nickel column overnight, to specifications into
Row protein purification.Purifying protein carries out Westernblot identifications with the anti-His tag monoclonal antibodies of mouse.SDA-PAGE with
Weatern blot are consistent with expected albumen size the results show that the recombinant protein size of Prokaryotic expression, purification is about 130kDa
(see Fig. 2 B, 2C).Fig. 2 B are the SDA-PAGE electrophoresis knots that pcoldTFDNA prokaryotic expression carriers express Recombinant Swine SAMHD1 albumen
Fruit is schemed, and wherein M is pre-dyed albumen Marker；1 is the full bacterium of Pcold TF DNA empty carriers；2 induce empty carrier for 0.5mM IPTG
Full bacterium；3 do not induce the full bacterium of recombinant plasmid for IPTG；4 induce the full bacterium of recombinant plasmid for 0.5mM IPTG；5 induce for 0.5mMIPTG
Express bacterium solution ultrasound supernatant；6 be 0.5mM IPTG induced expression bacterium solution ultrasound precipitations；7 be purifying Recombinant Swine SAMHD1 albumen.Figure
2C identifies recombinant protein result figure for the anti-His tag monoclonal antibodies Western blot of mouse, and wherein M is pre-dyed albumen
Marker；1 is the full bacterium of Pcold TF DNA empty carriers；2 induce the full bacterium of empty plasmid for 0.5mM IPTG；3 be purifying Recombinant Swine
SAMHD1 albumen.From these experimental results, IPTG induction pcold-pSAMHD1 expression obtains soluble Recombinant Swine
SAMHD1 albumen, the soluble recombinant protein can be as the immunogenes for preparing follow-up monoclonal antibody.
PFLAG-pSAMHD1 transfects MARC-145 cells, obtains the Marc-145 of transient expression Recombinant Swine SAMHD1 albumen
Cell, highly pathogenic PRRSV multiplication is substantially inhibited in the cell.Specific experiment step is as follows：
MARC-145 cells 6 orifice plates of paving, 3 × 105/ hole.Next day, 2 μ g pFALG-pSAMHD1 transfectional cells.After transfecting 48h, 0.1MOI
HuN4 plants of infection cells of highly pathogenic PRRSV.Infection 24 after, collect respectively cells and supernatant with
Cell carries out titration of virus and is identified with Western blot respectively.The results show that pig SAMHD1 is overexpressed in MARC-145 cells
After albumen, tag antibody detects the expression of recombinant protein, while HP-PRRSV virus multiplications are subject to substantially to inhibit (Fig. 3 A).On
There is apparent decline in virus titer in clear, shows that pig SAMHD1 albumen has apparent antiviral activity (Fig. 3 B).Fig. 3 A are
Western blot detection pigs SAMHD1 inhibits the result figure of HP-PRRSV multiplication, and wherein Anti-FLAG is FLAG tag antibodies
Detect Recombinant Swine SAMHD1 protein expressions；Anti-N detects HP-PRRSV multiplication for PRRSV N proteins monoclonal antibody；β-
Actin compares for internal reference.Fig. 3 B are that virus titer detection pig SAMHD1 inhibits what HP-PRRSV was proliferated in MARC-145 cells
Result figure.
The preparation of 3 pig SAMHD1 mouse resource monoclonal antibodies of embodiment
Using the SAMHD1 albumen after 2 Prokaryotic expression, purification of embodiment as immunogene, mouse is immunized, is used to prepare monoclonal
Antibody.8 week old BABL/c female mices are selected, the pig SAMHD1 albumen of Prokaryotic expression, purification is immunized.Immunizing dose is for the first time
100 micrograms of protein, the second to four time immune 50 micrograms, total volume 100ul, subcutaneous multi-point injection are spaced two weeks respectively.First
Secondary be immunized is emulsified with Freund's complete adjuvant, SAMHD1 albumen and Freund's complete adjuvant 1:1 ratio mixes, second of immune Freund
Freund's incomplete adjuvant emulsifies, SAMHD1 albumen and incomplete Freund's adjuvant 1:1 ratio mixes, third and fourth time immune to be added without adjuvant,
Direct immunization recombinates SAMHD1 albumen.After four times are immune, eye socket blood sampling, ELISA identification antibody titers.Antibody titer reaches certain
After level, take immunized mice spleen cell with SP2/0 cells by 5:1 ratio fusion, carries out monoclonal antibody preparation.Fusion two weeks
Afterwards, indirect ELISA method screening positive hybridoma cell, specific method are as follows：
1st, it is coated with：Purifying Recombinant Swine SAMHD1 albumen is coated with respectively with Pcold TF DNA expression vector His label proteins
ELISA ELISA Plates are 1 μ g/100 μ L per hole package amount, and 4 DEG C of coatings are overnight.
2nd, wash：It is washed 3 times with the PBST containing 0.5 ‰ tween-20,200 μ L/ holes.5 minutes every time.
3rd, close：When 5% 37 DEG C of skimmed milk incubation 2 is small, 200 μ L/ holes.
4th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
5th, cells and supernatant is added：Cells and supernatant is separately added into coating Recombinant Swine SAMHD1 albumen and His label eggs
White ELISA Plate, per 50 μ L of hole, when 37 DEG C of effects 1 are small.
6th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
7th, secondary antibody is added in：1 is pressed with PBS：After 10000 times of dilutions, ELISA Plate is added in per 50 μ L of hole, 37 DEG C are incubated 40 minutes.
8th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
9th, develop the color：50 μ LTMB developing solutions are added in per hole, are protected from light incubation at room temperature 10 minutes.
10th, terminate：50 μ L 2M H are added in per hole2SO4。
11st, OD values detect：Microplate reader measures OD450Value.
After Recombinant Swine SAMHD1 albumen and the dual ELISA screenings of Pcold TF DNA vector label proteins, choose and carry
Body label protein reaction negative, the clone for recombinating SAMHD1 albumen reacting positives carry out 5 subclones, every time after expanding culture
Dual ELISA screening and identifications positive colony is carried out after subclone.After 5 subclones, 3 plants of stably excreting monoclonal antibodies are obtained
Hybridoma cell strain 5M1,5M2,5M5.The hybridoma of most at last one preferred secrete monoclonal antibody lies in 2014
On January 14, in is preserved in China typical culture collection center (abbreviation CCTCC, address：Luojiashan, Wuchang, Wuhan City, Hubei Province is military
Chinese university collection), preserving number is CCTCC NO.C201422, and Classification And Nomenclature is hybridoma cell strain 5M1.The sun of screening
Property hybridoma through expand cultivate, for odd contradictive hydroperitoneum prepare.
Prepared by odd contradictive hydroperitoneum, be as follows：8 week old BABL/c female mices are chosen, norphytane, 200 μ are injected intraperitoneally
L/ is only.After a week, positive hybridoma cell counts immune norphytane, and basic DMEM culture mediums are resuspended, and 106/ Mice Inoculated abdomen
Chamber.It observes mouse web portion variation after a week, swells if there is abdomen, i.e. progress ascites acquisition, acquisition ascites, 4,000rpm/min
10min is centrifuged, supernatant dispenses -80 DEG C of preservations.
4 pig SAMHD1 MAb mediated ELISAs potency of embodiment is identified
Using odd contradictive hydroperitoneum prepared by embodiment 3 as primary antibody, with the ELISA potency of indirect ELISA method detection ascites, tool
Body method is as follows：
1st, it is coated with：Purifying Recombinant Swine SAMHD1 albumen is coated with respectively with Pcold TF DNA expression vector His label proteins
ELISA ELISA Plates are 1 μ g/100 μ L per hole package amount, and 4 DEG C of coatings are overnight.
2nd, wash：It is washed 3 times with the PBST containing 0.5 ‰ tween-20,200 μ L/ holes.5 minutes every time.
3rd, close：When 5% 37 DEG C of skimmed milk incubation 2 is small, 200 μ L/ holes.
4th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
5th, primary antibody is added in：Using PBS by the ascites of acquisition according to 1:After 2000 dilutions, coating Recombinant Swine is separately added into
The ELISA Plate of SAMHD1 albumen and His label proteins, per 50 μ L of hole, when 37 DEG C of effects 1 are small.
6th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
7th, secondary antibody is added in：1 is pressed with PBS：After 10000 times of dilutions, ELISA Plate is added in per 50 μ L of hole, 37 DEG C are incubated 40 minutes.
8th, wash：It is washed 3 times with the PBST containing 0.5 ‰ Tween-20,200 μ L/ holes.5 minutes every time.
9th, develop the color：50 μ L TMB developing solutions are added in per hole, are protected from light incubation at room temperature 10 minutes.
10th, terminate：50 μ L 2M H are added in per hole2SO4。
11st, OD values detect：Microplate reader measures OD450Value.
The results are shown in Figure 4, and it is up to 1 that the titer of ascites obtained after mouse, which is immunized, in hybridoma prepared by embodiment 3：
58.In Fig. 4,1, MAb be pig SAMHD1 monoclonal antibodies；After positive serum is immunized 4 weeks for Purification of Pig SAMHD1 recombinant proteins
BABL/c mice serums；Negative serum is not immune BABL/c mice serums；His label proteins induce pure for pcold empty carriers
The label protein obtained after change.
Embodiment 5
The odd contradictive hydroperitoneum that will be prepared in embodiment 3, as primary antibody, the idiosyncrasy of detection pig SAMHD1 monoclonal antibodies
Property.Concrete operations are as follows：
Collect cell sample：Pig alveolar primary macrophage-PAM cells, the passage of pig kidney are paved in 6 porocyte culture plates carefully
Born of the same parents system-PK-15 cells, African green monkey kidney passage cell-MARC-145 cells, baby hamster kidney passage cell-BHK-21 cells, people
Cervical cancer cell-HeLa cells, human lung carcinoma cell-A549 cells, human glioma cells-U251 cells, total number of cells for 1 ×
106.Supernatant is discarded, after precooling PBS is washed twice, adds in 200 μ L Thermo company's T hermo Scientific Pierce
After Cell Lysis buffer, ice bath 5min, cell is collected into 1.5mL centrifuge tubes.4 DEG C, 12000rpm/min, centrifugation
10min takes supernatant to be transferred to new pipe.It is detected with Coomassie Brilliant Blue and collects protein concentration.According to 4:1 volume ratio mixing 5 ×
Sample buffer, after boiling 10min, -20 DEG C save backup.
Western blotting：20 μ g equivalent total proteins loadings carry out SDS-PAGE electrophoresis, after electrophoresis, using primary
Happy half-dried transferring film instrument, voltage 15V transfer 1h, albumen are carried out to nitrocellulose membrane (NC films).It is carried out after transfer follow-up
Experiment, concrete operations are as follows：
Closing：With 5% skimmed milk room temperature closing NC films 1 it is small when.
Washing：NC films are washed with the TBST containing 0.5 ‰ Tween-20 3 times, 5 minutes every time.
Add in primary antibody：The odd contradictive hydroperitoneum prepared in embodiment 3, according to 1:The dilution proportion of 500-1000 is to containing 5%BSA
TBST buffer solutions in, room temperature effect 1 it is small when.
Washing：NC films are washed with the TBST containing 0.5 ‰ Tween-20 3 times, 5 minutes every time.
Add in secondary antibody：Company of Zhong Shan Golden Bridge HRP marks goat anti-mouse IgG antibody, and 1 is pressed with TBST：After 5000 times of dilutions
It adds in, room temperature effect 1h.
Washing：NC films are washed with the TBST containing 0.5 ‰ Tween-20 3 times, 5 minutes every time.
Development：Using Thermo companies ECL luminescence reagent box (article No.s：34080) it is exposed development.
The results are shown in Figure 5, and the specificity of pig SAMHD1 monoclonal antibodies prepared by embodiment 3 is good.In Fig. 5,1, PAM cells be
Pig alveolar primary macrophage；PK cells are porcine kidney cell；MARC-145 cells are African green monkey kidney passage cell；BHK-
21 cells are baby hamster kidney passage cell；HeLa cells are human cervical carcinoma cell；A549 cells are human lung carcinoma cell；U251 cells
For human glioma cells.
Embodiment described above only expresses embodiments of the present invention, and description is more specific and detailed, but can not
Therefore it is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection model of the present invention
It encloses.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
- A 1. boar SAMHD1 genes, which is characterized in that the sequence of the pig SAMHD1 genes is shown in coding SEQ ID NO.1 The nucleotide sequence of amino acid sequence.
- 2. pig SAMHD1 genes according to claim 1, which is characterized in that the pig SAMHD1 gene orders are SEQ ID Nucleotide sequence shown in NO.2.
- A 3. boar SAMHD1 protein, which is characterized in that the amino acid sequence of the pig SAMHD1 protein such as SEQ ID Shown in NO.1.
- 4. a kind of recombinant vector, which is characterized in that the nucleotide sequence of amino acid sequence shown in coding SEQ ID NO.1 is included, The sequence be placed in 7.1 carrier for expression of eukaryon of pcold TF DNA prokaryotic expression carriers or p3 × FLAG CMV Hind III and Between two restriction enzyme sites of Bam HI.
- 5. a kind of host cell, which is characterized in that include the recombinant vector described in claim 4.
- 6. a kind of recombinant protein, which is characterized in that the recombinant protein is that recombinant vector is made through expression as described in claim 4 .
- 7. a kind of monoclonal antibody of anti-pig SAMHD1 albumen, which divided by hybridoma cell line CCTCC NO.C201422 The monoclonal antibody secreted.
- 8. a kind of kit for detecting pig SAMHD1 albumen, which is characterized in that include the monoclonal antibody described in claim 7.
- 9. a kind of antiviral drugs, which is characterized in that the drug contains the pig SAMHD1 protein described in claim 3.
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| PREDICTED: Sus scrofa SAM domain and HD domain 1 (SAMHD1), partial mRNA;NCBI;《NCBI Reference Sequence: XM_003361435.2》;20130926;序列 * |
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