CN102321661A - Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid - Google Patents
Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering, and particularly relates to an eukaryotic expression recombinant plasmid and a construction method thereof and a Vero cell line stably expressing the plasmid. The eukaryotic expression recombinant plasmid has the nucleotide sequence shown in SEQ ID NO:1. The Vero cell line stably expressing the plasmid can be used for high-efficiency separation of canine distemper virus virulent strains and the research of the pathogenic mechanism of canine distemper virus.
Description
Technical field
The present invention relates to technical field of bioengineering, the Vero clone of particularly a kind of eukaryotic expression recombinant plasmid and construction process thereof and this plasmid of stably express.
Background technology
(Canine distemper is that (Canine distemper virus, animals such as a kind of Canidae that CDV) causes, Mustelidae are prone to suffer from the height contagious disease by CDV CD) to canine distemper.This disease is one of transmissible disease of supporting dog industry, furbearer aquaculture harm maximum, is characteristic feature with apocleisis, diphasic fever, conjunctivitis, gastro-enteritis and nervous symptoms, can cause animals morbidities such as large quantities of dogs, fox, racoon dog, and death rate of the onset is 30～80%.In recent years, the scope of CDV infection host enlarges day by day, the macaque of Primates, and the wild dog in the medium-and-large-sized animal lion of sea dog, giant panda, the cat family of clasper order Phocidae, Africa etc. all has report infected dogs distemper virus.In China; Along with army dog, police dog, experimental dog and the numbers of animals raised such as furbearer fox, racoon dog in recent years increase considerably and the strange land is alternative increases; Sickness rate and the lethality rate of canine distemper in dog, fox, racoon dog all has trend of rising, so the canine distemper prevention and control seem particularly important.
The virus stripping technique is the important means to the research of CDV mechanism of causing a disease, but the CDV with cyst membrane is difficult in environment, surviving, so is difficult to separate successfully with usual way.At present; Separation to CDV street strain generally is total to cultured method through susceptible animal pulmonary macrophage and continuous cell lines such as Vero or MCDK; But there are complicated operation in these class methods, expend time in long with separate shortcomings such as obtaining the virus titer instability outside; And because above passage cell is the non-sensitive cell of CDV, for the CDV that is separated to the sudden change of virulence gene has taken place in adapting to the process of cell probably, cause a little less than the causing of virus strain.Because CDV (especially virulent strain) lacks cell or clone responsive and that be suitable for when vitro culture; Cause CDV to separate into power lower (generally being lower than 20%), seriously hindered research CDV etiology biology and mechanism of causing a disease aspect.
Calendar year 2001; Tatsuo etc. infect CDV street strain on the Chinese hamster ovary celI of manual work expression dog signal lymphocyte activator molecule (SLAM claims CD150 again); Test confirms that the SLAM of dog is the acceptor that CDV infects, and CDV H albumen is adsorbed onto the course of infection of the initial virus of cell surface through identification SLAM.2003, the Vero clone (Vero.DogSLAMtag) of the stably express dog SLAM that application such as Seki are set up was separated to CDV more fast and sensitively than Vero and B95a cell in doubtful hundstaupe pyreticosis dog body.Because the tag label of expressing among the Vero.DogSLAMtag is the proteic epitope of bird flu virus HA, therefore need carry out immunoblotting or indirect immunofluorescence method by monoclonal antibody to this epi-position to the evaluation of this positive Vero clone.Therefore, identify that from upright the reaching of the structure of this clone method therefor is comparatively loaded down with trivial details.In addition,, cause viral isolating susceptibility not high, directly influenced isolation of canine distemper virus efficient because dog SLAM expression amount is lower in this positive Vero clone.2010, Zhao Jianjun etc. successfully cloned the SLAM gene of fox, racoon dog and mink, and confirmed that it all can be as the acceptor of CDV infection.
Therefore; Make up a kind of efficiently expressing and the recombinant plasmid of the SLAM gene that virus tropism is responsive and importing in the Vero cell; Obtain the Vero clone of this SLAM gene of stably express, have realistic meaning for quick and responsive separation CDV and to its mechanism of causing a disease research.
Summary of the invention
In view of this, the present invention provides the Vero clone of a kind of eukaryotic expression recombinant plasmid and construction process and this plasmid of stably express.This eukaryotic expression recombinant plasmid has nucleotide sequence shown in the SEQ ID NO:1.Use this Vero clone, can carry out high efficiency separation and be applied in the research of CDV mechanism of causing a disease the CDV virulent strain.
In order to realize the foregoing invention purpose, the present invention provides following technical scheme:
The invention provides a kind of eukaryotic expression recombinant plasmid and have nucleotide sequence shown in the SEQ ID NO:1.
This eukaryotic expression recombinant plasmid; By the GeneBank accession number is racoon dog SLAM gene (rSLAM), coding 6 Histidines (fusion gene of 6 * his) nucleotide sequence and Kozak sequence, Pcmv, IRES of EU678639; EGFP and SV40pA form, and be shown in figure 15.Wherein, 5 ' to 3 ' end of said plasmid is followed successively by Pcmv, said fusion gene, and IRES, EGFP and SV40pA, shown in figure 13.The Kozak sequence for being present in one section sequence of eukaryote mRNA, has the effect that strengthens expressing quantity in translation initial.The optimal sequence of initiator codon upstream and downstream does in the eukaryotic gene, and-3 is purine bases; + 4 is G, the A among the initiator codon AUG is in+and 1.
As preferably, said Kozak sequence has the nucleotide sequence shown in SEQ No.4.
The present invention adopts carrier for expression of eukaryon pIRES2-EGFP; Available from U.S. Clontech company; Carrier for expression of eukaryon pIRES2-EGFP is a kind of binary expression vector; At goal gene and the EGFP coexpression under the CMV promotor that MCS (MCS) is located to insert, can monitor (expression that reacts target protein through the expression of observing green fluorescent protein under the fluorescent microscope) to expression, can keep the biological characteristics of target protein own well again.
This expression vector contains:
(1) is used to clone the MCS MCS of foreign gene;
(2) allow two encoding soxs on the mRNA molecule to obtain the suitable people encephalomyocarditis virus ribosome internal entry site IRES of branch;
(3) use enhanced green fluorescence protein gene EGFP;
(4) make the mRNA molecule add the SV40polyA signaling zone of polyA tail;
(5) Pcmv expresses the foreign gene of back and the moulding of EGFP genome.Behind Xho I and EcoR I double digestion, obtain Pcmv, IRES, EGFP and SV40pA.
The sequence of carrier for expression of eukaryon pIRES2-EGFP has the nucleotide sequence shown in SEQ No.5, and the ring-type collection of illustrative plates is seen Figure 14.
Wherein, Pcmv has nucleotide sequence shown in the SEQ ID NO:6, and IRES has nucleotide sequence shown in the SEQ ID NO:7, and EGFP has nucleotide sequence shown in the SEQ ID NO:8, and SV40pA has nucleotide sequence shown in the SEQ ID NO:9.
The present invention also provides a kind of preparation method of eukaryotic expression recombinant plasmid, comprising:
Step 1: with the clone plasmid pMD18-T-rSLAM of rSLAM being arranged is template, and amplification obtains said fusion gene in the presence of upstream primer, downstream primer, and said upstream primer sequence is shown in SEQ No.2, and said downstream primer sequence is shown in SEQ No.3;
Step 2:pIRES2-EGFP carrier is cut through enzyme, obtains Pcmv, IRES, EGFP and SV40pA.
Step 3: get said fusion gene: after enzyme is cut, be connected with Pcmv described in the step 2, said IRES, said EGFP and said SV40pA, the transformation receptor bacterium is identified positive colony, obtains said plasmid.
With plasmid pMD18-T-rSLAM is template, with the P of sequence shown in SEQ No.2
1: 5 '-CCG
ATGGATTCCAGGGGCTTCCTC-3 ' is a upstream primer, with the P of sequence shown in SEQ No.3
2: 5 '-CCG
TCA
GTGATGGTGATGGTGATGGCTCTCTGGGAACGTCAC-3 ' be downstream primer (square frame is a restriction site, overstriking be the Kozak sequence, underscore is histidine-tagged gene); Carry out pcr amplification, to obtain goal gene.
Reaction system can be dna profiling 1 μ L (0.2ng/ μ L), each 1 μ L (20pmoL/L) of upstream and downstream primer, and Ex taq enzyme 0.3 μ L (5U/ μ L), dNTP (each 2.5mmol/L) 2 μ L, 10 * PCRbuffer, 2.5 μ L, ddH2O supplies 25 μ L.Amplification program is: 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 30s, 61.8 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ of total elongation 10min; Preserve 10min for 4 ℃.The PCR product reclaims through 1% sepharose, is connected into the pMD18-T-simple carrier then, and enzyme is cut evaluation.
As preferably, said enzyme is cut to Xho I and EcoR I double digestion.
Said racoon dog SLAM gene is obtained through Xho I and EcoR I double digestion respectively by said plasmid pMD18-T-rSLAM.
Said Pcmv, IRES, EGFP and SV40pA are obtained through Xho I and EcoR I double digestion by the pIRES2-EGFP carrier.
A kind of Vero clone, the clone has above-mentioned eukaryotic expression recombinant plasmid gene in the Vero cellular genome, and cell surface can have CDV acceptor racoon dog SLAM (rSLAM) by stably express.
The construction process of Vero clone can for: designing 1 pair of primer, with the clone pMD18-T-rSLAM plasmid of racoon dog SLAM gene to be arranged be template; Behind PCR method acquisition Kozak sequence+racoon dog SLAM+6 * his fusion gene, make up the pIRES2-EGFP-rSLAMhis plasmid; With said pIRES2-EGFP-rSLAMhis plasmid transfection Vero cell, through observing the Vero clone that EGFP expresses and employing G418 pressure is cultivated colony screening stably express SLAM.
The present invention also provide above-mentioned Vero clone the canine distemper virulent strain separate fast, sensitively and CDV research in application.
The present invention through make up and transfection eukaryotic expression recombinant plasmid pIRES2-EGFP-rSLAMhis (by the GeneBank accession number is the racoon dog SLAM gene of EU678639, the nucleotide sequence of 6 Histidines of coding and the fusion gene of Kozak sequence, Pcmv, IRES; EGFP and SV40pA form; Wherein, 5 ' to 3 ' of said plasmid end is followed successively by Pcmv, said fusion gene; IRES, EGFP and SV40pA.) to Vero clone; Obtain the Vero cell of the CDV acceptor rSLAM of surface-stable, high expression level amount through colony screening; And then inoculation contains the tissue sample isolated viral of CDV; Connect malicious test-results and show, the Vero cell behind the transfection rSLAM has strengthened the susceptibility to CDV, can effectively separate the canine distemper strain.
The present invention provides simultaneously by above-mentioned Vero clone and has separated canine distemper virulent strain LN (10) the f1 strain that obtains.
The present invention provides a kind of eukaryotic expression recombinant plasmid and construction process thereof, and the Vero clone of this plasmid of stably express.In order to strengthen the translation efficiency of target protein gene (rSLAM), be convenient to the detection of protein expression, when the present invention designs primer, introduced the Kozak sequence respectively at the upstream and downstream of rSLAM gene.The present invention is histidine-tagged in 6 of carboxyl terminal introducings wanting expressing protein; Be convenient to use histidine-tagged monoclonal antibody (this monoclonal antibody is commercialization at home and abroad) with Western blot and cellular immunization groupization to SLAM proteic expression carry out semi-quantitative assessment, have vital role for the evaluation that makes up stable expression cell line.The Kozak sequence can significantly improve the proteic expression amount of SLAM when plasmid expression, help improving CDV and its joint efficiency, and the separation efficiency of express cell system virus is improved.The present invention adopts carrier for expression of eukaryon pIRES2-EGFP, is a kind of binary expression vector, and the goal gene of insertion and EGFP be coexpression under the CMV promotor; Can monitor expression; Can keep the attribute of target protein own well again, can the cell screening initial stage through green fluorescent protein location positive cell, and the mono-clonalization of carrying out the expressing protein cell is selected; In conjunction with the G418 resistance screening, improve the success ratio of screening.The pIRES2-EGFP-rSLAMh plasmid transfection in the Vero cell, is treated transfectional cell growth 48h, and behind the discovery pIRES2-EGFP-rSLAMh plasmid transfection, the expression rate of EGFP is higher.After 10 generations of step sizing, EGFP still expresses.
The present invention detects SLAM Expression of Fusion Protein after the transfection through change into merit to 6 histidine-tagged cellular immunization groups; The poison that connects through transient transfection Vero cell is tested; Really strengthened the susceptibility of cell to CDV behind the proof transfection SLAM, the C ' end of explaining at SLAM too merges 6 His label proteins does not make SLAM albumen lose and proteic identification of CDV H and binding ability.For the feasibility of the foundation of stable transfected cells provides reliable experiment basis.After 10 generations of transfection group cell step sizing,, also can detect the proteic expression of EGFP simultaneously with detecting the SLAM Expression of Fusion Protein in the cellular immunization group cell.By the transfectional cell of drawing (V-p-rSLAMh cell) and the growth curve of untransfected Vero cell; Can calculate the cell doubling time of Vero cell and V-p-rSLAMh cell; The doubling time of Vero cell is the shortest to be 16.69h; The doubling time of V-p-rSLAMh cell is greater than 16.69h, explains that the speed of growth of expressing the cell behind the foreign protein is slack-off, expresses the metabolism burden that foreign protein increases cell; It is slack-off that the speed of growth becomes nature, and the V-p-rSLAMh cell is big than the Vero cell on size on the external form in addition.The present invention successfully sets up the Vero clone of stably express racoon dog SLAM, and is significant for setting up of the sharp separation of CDV and research platform.
Inoculate 3 parts of dead foxes in source or the doubtful tissue sample of racoon dog canine distemper respectively with V-p-rSLAMh cell and Vero cell; The V-p-rSLAMh cell can form visible typical CDV CPE at the inoculation first-generation 36～48h, and 6 generations of the continuous blind passage of Vero cell are not seen the formation of CPE yet.Confirm that the V-p-rSLAMh cell is responsive to CDV than the Vero cell.The 3 strain poison that are separated to through the RT-PCR checking are CDV, and to isolating 3 strain CDV H gene clones order-checking back comparison, the result shows that 3 strain CDV are Asia-1 genotype street strain; With wherein 1 strain [called after LN (10) f1 strain] the V-p-rSLAMh cell connect pass 4 generations laggard action thing to attack its virulence of malicious experimental verification strong and weak, with 4 * 10
2.39TCID
50/ only the toxic agent amount of attacking can make all death of the racoon dog of attacking the poison group and fox (every kind animal each 3).Confirmation keeps its strong virulence through the CDV of V-p-rSLAMh cellular segregation, can reach 100% to the lethality rate of inoculating animal.
The present invention has set up the Vero clone of stably express racoon dog SLAM acceptor; And to the Vero clone stability of expressing racoon dog SLAM acceptor and CDV susceptibility has been carried out the evaluation of system; Confirm that this clone destination gene expression is stable, responsive to CDV virus, it is stronger that separation obtains the CDV virulence.The foundation of the Vero clone of stably express racoon dog CDV SLAM is for separation and the research of CDV provides a platform.In addition, in the vaccine development process of prevention CD, the CDV that virulence is stable in the vaccine potency evaluation is strong, and poison is indispensable, and the acquisition of the strong poison of LN (10) f1 strain CDV cell with the appraisal tool that efficient vaccine is provided, is significant to the epidemic prevention of CD.
Description of drawings
Glue reclaimed rSLAM purpose fragment after Fig. 1 showed PCR; Wherein, swimming lane 1 is 100bp DNA Ladder Marker, and swimming lane 2 is for being the purpose fragment of template behind pcr amplification with plasmid pMD18-T-rSLAM, and size is about 1100bp.
Fig. 2 shows that the recombinant expression vector enzyme cuts qualification result; Wherein, swimming lane 1 is 100bp DNALadder Marker, and swimming lane 2 is DL15000DNA Ladder Marker, and swimming lane 3 is about 1100bp and 5300bp two bands for pIRES2-EGFP-rSLAMh generates size through double digestion.
Fig. 3 shows confirming of G418 working concentration; Wherein, Fig. 3 (a) is the Vero cell of no G418; Fig. 3 (b) is for containing the Vero cell of 200 μ g/mL G418; Fig. 3 (c) is for containing the Vero cell of 600 μ g/mL G418; Fig. 3 (d) is for containing the Vero cell of 800 μ g/mL G418.
Fig. 4 shows the expression of EGFP behind the recombinant plasmid transient transfection Vero cell 48h; Wherein, Fig. 4 (a) is the Vero cell of untransfected; Fig. 4 (b) is the Vero cell of pIRES2-EGFP transfection; Fig. 4 (c) is the Vero cell of pIRES2-EGFP-rSLAMh transfection.
CDV connect malicious 60h cytopathy (CPE) after Fig. 5 showed recombinant plasmid transient transfection Vero cell; Wherein, Fig. 5 (a) is the Vero cell of untransfected; Fig. 5 (b) is the Vero cell of pIRES2-EGFP transfection; Fig. 5 (c) is the Vero (arrow indicator cells lesion locations) of pIRES2-EGFP-rSLAMh transfection.
Fig. 6 shows the Vero cell with transfection 48h; Change the screening culture medium that G418 content is the 10%FBS of 800 μ g/mL; About screening and culturing 10 days, with behind the trypsin digestion cell by the Tissue Culture Dish that reaches 100mm at 1: 40, continue to add the Vero cell cluster that the screening culture medium screening obtains transfection.
Fig. 7 shows cellular immunization group detected result; Wherein, Fig. 7 (a) is the Vero cell, and Fig. 7 (b) is the Vero cell of transfection empty carrier, and Fig. 7 (c) is a transfection recombinant plasmid Vero cell (showing the SLAMh protein expression).
Fig. 8 shows the growth curve of transfectional cell and non-transfected cells; Wherein,
shows the Vero cell,
show the Vero cell (V-p-rSLAMh cell) after the pIRES2-EGFP-rSLAMh transfection.
Fig. 9 shows the cytopathy that connects behind the poison; Wherein, Fig. 9 (a) is for connecing the Vero cell behind the poison; Fig. 9 (b) is the V-p-rSLAMh cell of inoculation JL (10) r1 strain CDV 48h, and Fig. 9 (c) is the V-p-rSLAMh cell of inoculation HeB (10) f1 strain CDV 48h, and Fig. 9 (d) is the V-p-rSLAMh cell of inoculation LN (10) f1 strain CDV 24h.
Figure 10 shows the RT-PCR amplification of V-p-rSLAMh cellular segregation CDV.Wherein, swimming lane 1 is 100bp DNALadder Marker, and swimming lane 2 is JL (10) r1 strain, and swimming lane 3 is HeB (10) f1 strain, and swimming lane 4 is LN (10) f1 strain, swimming lane 5 positive contrasts.
Figure 11 shows the body temperature of attacking poison and contrast racoon dog; Wherein,
shows the body temperature of No. 1 racoon dog;
shows the body temperature of No. 2 racoon dogs;
shows the body temperature of No. 3 racoon dogs, and
shows the body temperature that contrasts racoon dog.
Figure 12 shows the body temperature of attacking poison and contrast fox; Wherein,
shows the body temperature of No. 1 fox;
shows the body temperature of No. 2 foxes;
shows the body temperature of No. 3 foxes, and
shows the body temperature that contrasts fox.
Figure 13 shows carrier for expression of eukaryon pIRES2-EGFP collection of illustrative plates.
Figure 14 shows carrier for expression of eukaryon pIRES2-EGFP ring-type collection of illustrative plates.
Figure 15 shows eukaryotic expression recombinant plasmid collection of illustrative plates provided by the invention.
Embodiment
The invention discloses a kind of eukaryotic expression recombinant plasmid and construction process thereof, and the Vero clone of this plasmid of stably express, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
Carrier for expression of eukaryon pIRES2-EGFP is available from U.S. Clontech company; Restriction enzyme XhoI, EcoR I carrier are available from precious biological (Dalian) ltd; The Vero cell is that Gao Yun prevention animal doctor research department preserves; Midiprep High-pure plasmid extraction kit is purchased the company in Invitrogen; The DMEM substratum is available from Gibco BRL company; G418 is available from Promega company; Transfection reagent
HD Transfection Reagent is available from Roche company;
Monoclonal Antibody is available from Novagen company; Cellular immunization group test kit SP kit (Mouse) is available from Beijing Bo Aosen Bioisystech Co., Ltd; Trizol is available from American I nvitrogen company; Inverted fluorescence microscope is purchased (DMI3000B) from Leca company; Doubtful canine distemper fox tissue sample is that Gao Yun prevention animal doctor research department preserves.
Below in conjunction with embodiment, further set forth the present invention:
Pcr amplification and gene clone: with plasmid pMD18-T-rSLAM is template, with
P
15 '-CCG
ATGGATTCCAGGGGCTTCCTC-3 ' is a upstream primer, with P
25 '-CCG
TCA
GTGATGGTGATGGTGATGGCTCTCTGGGAACGTCAC-3 ' be downstream primer (square frame is a restriction site, overstriking be the Kozak sequence, underscore is histidine-tagged gene); Carry out pcr amplification, to obtain goal gene, reaction system is: dna profiling 1 μ L (0.2ng/ μ L); Each 1 μ L (20pmol/L) of upstream and downstream primer, Ex taq enzyme 0.3 μ L (5U/ μ L), dNTP (each 2.5mmol/L) 2 μ L; 10 * PCR Buffer, 2.5 μ L, ddH
2O supplies 25 μ L.
Amplification program is: 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 30s, 61.8 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ of total elongation 10min; Preserve 10min for 4 ℃.The PCR product reclaims through 1% sepharose, is connected into the pMD18-T carrier then, and enzyme is cut evaluation.
To dilute back plasmid pMD18-T-rSLAM is template; Pcr amplification purpose fragment, the purpose fragment of gained reclaims purifying through 1% agarose gel, the agarose through 1%; Electrophoresis 45min under the 100V voltage; Under ultraviolet transilluminator, can see the band that the expection size is about 1100bp, ImageQuant 3000 gel imaging systems (GE company) are gathered image, and are as shown in Figure 1.
Construction of eukaryotic: plasmid pMD18-T-rSLAMh is with XhoI and EcoRI double digestion, and glue reclaims the purpose fragment and under the effect of T4 ligase enzyme, is inserted into the carrier segments through the pIRES2-EGFP glue recovery of XhoI and EcoRI double digestion.The carrier that connects is transformed among the clone strain DH5 α, is coated in card and receives on the LB flat board of penicillium mould (30 μ g/mL) resistance; After cultivating 24h, the aseptic mono-clonal bacterial strain of choosing has card to receive in the test tube of liquid LB of penicillium mould (30 μ g/mL) resistance in 3～4mL, and test tube is put into constant-temperature shaking culture case (37 ℃ 200r/min) are spent the night; Extract plasmid with plasmid extraction kit (rich day of Hangzhou) by specification operation.
Enzyme is cut evaluation: the pIRES2-EGFP-rSLAMh recombinant plasmid generates size through double digestion and is about 1100bp and 5300bp two bands.The result is as shown in Figure 2, shows successfully to make up eukaryon expression plasmid, and pIRES2-EGFP-rSLAMh is connected into the purpose fragment.
Send biotech firm's order-checking with the eukaryon expression plasmid that makes up, sequence has the nucleotide sequence shown in SEQ No.1.
Confirming of G418 optimal concentration: the Vero cell of recovery the cell tryptase enzymic digestion that will go down to posterity, is pressed every hole 1 * 10 after passing for 2～3 generations with the DMEM of 10%FBS
4The amount in individual/hole is spread 96 orifice plates, absorbs substratum after being paved with plate on 2nd, and PBS washes cell once, adds screening culture medium.The concentration of G418 is followed successively by 0,200,300,400,500,600,700,800,900,1000 μ g/mL; Each gradient is established 3 repetitions; Per color of looking substratum in about 3 days is changed screening culture medium, cultured continuously 10 days; Every day, the observation of cell death condition was the righttest screening concentration with the 10th day all dead minimum G418 concentration of cell.The Vero cell that adds different concns G418, the screening group all had small amounts of cells dead in the 2nd day; When screening 8 days, 800 μ g/mL group has 95% necrocytosis, and 900 μ g/mL group has 99% necrocytosis, 100% necrocytosis of 1000 μ g/mL; Screen (see figure 3) on the 10th, 200 μ g/mL group has 40% necrocytosis, and 600 μ g/mL group also has 90% necrocytosis, and the above group of 800 μ g/mL cell is all dead, confirms that the righttest screening concentration of Vero cell G418 is 800 μ g/mL.
Transfection is with the preparation of high purity plasmid: the bacterial classification of the pIRES2-EGFP-rSLAMh that pIRES2-EGFP and embodiment 1 are made, be connected to respectively in the LB nutrient solution of 100mL Kan resistance, and 37 ℃, 200r/min spends the night.Press process specifications with Midiprep High-pure plasmid extraction kit, purifying extracts dual-expression vector.Extract plasmid according to the by specification operation steps.
The cell transient transfection: with the growth conditions good cell with every hole 1 * 10
5The density of individual cell is spread 6 orifice plates, and (6 holes are set to untransfected plasmid group, transfection pIRES2-EGFP empty carrier group, transfection pIRES2-EGFP-rSLAMh group respectively; The Vero cell of transfection empty carrier and recombinant plasmid is called after V-p and V-p-rSLAMh respectively), at 37 ℃, 5%CO
2After the overnight cultures,, cell degree of converging carries out cell transfecting when being about 80% in the incubator: be placed on room temperature environment to the DMEM of serum-free, plasmid purification and transfection reagent before the transfection according to following operation steps; In the EP of the aseptic no silication of 1.5mL pipe, add 100 μ L serum-frees and antibiotic DMEM; The abundant mixing of plasmid that adds 2 μ g then; Use the pipettor of no silication liquid-transfering gun head, the unsettled HD Transfaction Reagen (adding the preceding mixing of transfection reagent) that splashes into 9 μ L puts upside down mixing; Incubated at room 15min is so that form transfection composite; Transfection composite in the EP pipe is dropwise joined in the 6 corresponding orifice plates, in the dropping process, rotate 6 orifice plates gently and be distributed in cell surface uniformly to guarantee transfection composite; 37 ℃, 5%CO
2After cultivating 48h in the incubator, fluorescent microscope is gathered image down.To Fig. 4 (c), behind the discovery pIRES2-EGFP-rSLAMh plasmid transfection, the expression rate of EGFP is higher like Fig. 4 (a).60h can see tangible CPE behind the inoculation CDV of this recombinant plasmid group of transfection; Visible CPE does not appear in the Vero of untransfected group and transfection empty plasmid group; See Fig. 5 (a) to Fig. 5 (c), this shows that the expression of rSLAM after the transfection can significantly improve the susceptibility of CDV pair cell.
The checking that SLAM expresses behind the transient transfection:
With the cell plate after the transfection at 37 ℃, 5%CO
2After cultivating 48h in the incubator; Behind the fluorescence microscope; Select the every hole of a plate cell to inoculate canine distemper fox internal organs poison LN (10) the f1 strain (changing the DMEM cell maintenance medium of 2%FBS behind the absorption 1h) of 200 μ L processing, whether observation every day of inoculation back the cytopathy situation that typical CDV infects occurs.The treating processes of internal organs virus is following:
Get the serious lung tissue of pathology and put into mortar, will organize rubbing, add a small amount of silica sand and PBS and grinding rapidly on ice with little scissors; Pathological material of disease is about 1: 5 (g/mL) with the ratio of PBS, puts into-80 ℃ of refrigerator freeze thawing once; 4 ℃, behind the centrifugal 30min of 3000r/min, get the filter filtration sterilization of supernatant with 0.22 μ m; Connect before the poison nutrient solution of cell sopped up after, with PBS washing 3 times, after every hole meets malicious 200 μ L absorption 1h, add the cell maintenance medium of the DMEM that contains 2%FBS.
The colony screening of stably express SLAM cell: adopt G418 pressure to select and the cell clone ring combines carries out the stable transfected cells colony screening.With cell plate after the transfection at 37 ℃, 5%CO
2Incubator is cultivated the DMEM screening culture medium of changing the 10%FBS that contains 800 μ g/mL G418 after 3 days; Every day observation of cell growth conditions; Renewed in per 3 days screening culture medium; All die more than 99% until the control wells cell, the trypsin digestion cell with 0.25% passes the big plate of 100mm by 1: 30 extent of dilution.Screening culture medium with the 10%DMEM that contains 800 μ g/mL G418 is cultivated; Renewed in per 3 days substratum, cultured continuously is about 10 days, fluorescent microscope is observed the protein expression situation down; Mark green fluorescence and be the position of cell cluster with marking pen; Digest the big plate that this position cell changes 100mm over to clone's ring, so repeatable operation is 3～4 times, can obtain the cell of purer stably express.Every interval 5 generation freeze-stored cell in the process that goes down to posterity of cell.
Vero cell with transfection 48h; Change the screening culture medium that G418 content is the 10%FBS of 800 μ g/mL; Changed one time screening culture medium in per 3 days, screening and culturing is about 10 days, and the normal control cell is all died; By with behind the trypsin digestion cell by the Tissue Culture Dish that reaches diameter 100mm at 1: 40, continue to add the Vero cell that screening culture medium is screened transfection.Can see cell cluster at microscopically and form (as shown in Figure 6).Fluorescence microscope, after will having cell cluster to form and have mark that a large amount of EGFP express with marking pen with the digestion of clone's ring, screening and culturing in 6 orifice plates, the cloned cell line that cultured continuously gets final product for 3-4 time purelyr.The good generation of the clone mark that obtains is frozen, and V-p and V-p-rSLAMh have organized step sizing after 10 generations, and EGFP is stably express still.
The checking of stable expression cell line SLAM protein expression: the cell of isoacceptor and the cell of normal cell and empty carrier are not spread 6 orifice plates with the expression of stable transfection.Verify the proteic expression of stable cell lines with cellular immunization group technology.Behind cell cultures 3～4d, PBS is hatched 30min for 37 ℃; With 4 ℃ of 2mL methyl alcohol, 4h is fixing; PBS flushing 3min * 3 time, washed with methanol 3min * 3 time; After the drying at room temperature, with the PBS of 1%Tween-20 flushing 3min * 3 time; 3%H
2O
2Deionized water hatch 10～15min, to eliminate endogenous Peroxidase activity; With confining liquid incubated at room 10～15min, incline and do not wash; The Histidine one that drips dilution in 1: 1200 is anti-, hatches 2～3h or 4 ℃ for 37 ℃ and spends the night; PBS flushing, 3min * 3 time; Drip biotin labeling goat anti-mouse IgG room temperature or 37 ℃ and hatch 10～15min; PBS flushing, 3min * 3 time; Drip the avidin of horseradish enzyme labelling, room temperature or 37 ℃ are hatched 10～15min; PBS flushing, 3min * 3 time; DAB chromogenic reagent 5～15min, PBS fully washes; Haematoxylin redyeing; PBS rinses well, microscopic examination.
Draw cell growth curve: the cell and normal Vero cell of the expression CDV acceptor in 2 generations of the frozen stable screening of recovering, passed for 2 generations after, spread 24 orifice plates, every 24h or 12h cell counting are once drawn cell growth curve according to count results, see Fig. 8.It is vigorous between 72～108h, respectively to organize the cell growth; The Vero cell begins to reduce after crossing 108h.
Clone is to the susceptibility of CDV relatively: when Vero and V-p-rSLAMh cell growth state are good; Spread each 3 hole in six orifice plates (Denmark Nunc company) with two kinds of cells; The Vero cell is with the DMEM cell growth medium of 10%FBS; The G418 that has also added 800 μ g/mL in the V-p-rSLAMh cell growth medium is at 37 ℃, 5%CO
2Incubator in cultivate 48h after, adsorb respectively and connect poison, the V-p-rSLAMh cell keep the G418 that also contains 800 μ g/mL in the liquid, the pathology situation of every 12h observation of cell.3 kinds of street strains of inoculation are respectively Hebei fox internal organs poison [HeB (10) f1] in 2010; Jiutai racoon dog internal organs poison [JL (10) r1] in 2010; 2010 Lian Ning Jinzhou fox internal organs poison [LN (10) f1)].More than 3 parts of canine distemper samples detect through RT-PCR that to be CDV nucleic acid positive.Cell to pathology not occurring carries out blind passage, and the feminine gender that is regarded as of CPE does not appear in blind passage 6 generations yet.
Two kinds of cells are to the separating resulting of 3 strain street strains:
The V-p-rSLAMh cell: meet the malicious first-generation 36～48h, tangible cytogamy CPE all appears in 3 strain street strains.LN (10) f1 tangible CPE promptly occurs at 36h, and all the other 2 strains all tangible CPE occurs at 48h, see Fig. 9 (b), Fig. 9 (c), Fig. 9 (d).
The Vero cell: visible CPE appears in 3 strain street strains in the first-generation, and after this 6 generations of cell blind passage are not seen CPE yet, are regarded as virus and separate negative.See Fig. 9 (a)
The RT-PCR checking and the H gene sequencing of isolated viral:
By the V-p-rSLAMh cellular segregation to 3 strain CDV, divide you can well imagine viral RNA after the freeze thawing, reverse transcription cDNA, the RT-PCR checking confirms that 3 strain virus that are separated to all are CDV, and is shown in figure 10.
Primer design is with synthetic: according to the CDV N that delivers on the Genbank and H gene order respectively at a pair of detection primer of its conserved regions design; Amplification sheet degree is 430bp; Upstream primer (P1): 5 '-ACCAGACAAAGTTGGCTAWGGAT-3 ', downstream primer (P2): 5 '-ATGCTTGGTATTACCTCTACTAACTTG-3 '; A pair of amplimer, amplification length are 1879bp, upstream primer (P3): 5 '-TTAGGGCTCAGGTAGTCCA-3 ', and downstream primer (P4): 5 '-CTAAGKCCAATTGARATGTGT-3 ', designed primer is synthetic by the precious biotechnology in Dalian ltd.
Synthetic and the pcr amplification of the extraction of viral RNA, cDNA: to extracting the CDV viral RNA, then cDNA is synthesized in reverse transcription with Trizol method by specification, and the reverse transcription system is following:
With cDNA is template applications pcr amplification specific fragment; The PCR reaction system is: dna profiling 1 μ L, each 1 μ L (20pmol/L) of upstream and downstream primer, Ex Taq archaeal dna polymerase 0.3 μ L, 10 * PCR Buffer2.5 μ L; DNTP (each 2.5mmol/L) 2 μ L supply 25 μ L with ddH2O.The pcr amplification program: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 52 ℃ of annealing 40s (2min), 72 ℃ are extended 40s (2min), 35 circulations; Last 72 ℃ are extended 10min.1% agarose is identified the PCR product.After the recovery of pcr amplification product glue, respectively it is cloned into the pMD18-T carrier.Enzyme is cut and is identified that 3 positive colonies of each fragment picking of back send the order-checking of the precious biotech firm in Dalian, to confirm concensus sequence.
CDV H sequential analysis: the H gene that will check order correct; Other CDV strain gene orders with DNAStar software MegAlign/Clustal W and GenBank login compare analysis and constructing system evolutionary tree, and use the potential N-connection of the H albumen glycosylation site that NetNGlyc 1.0Server software is predicted.
The V-p-rSLAMh cellular segregation to H gene order and other strain sequence alignments of LN (10) f1, [JL (10) r1] and [HeB (10) f1] strain CDV after show that 3 strain virus are CDV, and all belong to Asia-1 genotype street strain.The glycosylation site number that H gene potential N-connects, street strain generally has 8～9, and vaccine strain has 4 (Onderstepoort) or 7 (CDV
3), it is the distinctive glycosylation site of street strain that 309～311 N-connects glycosylation site, the 3 strain CDV H genes that increased contain and comprise that 309～311 have 9 glycosylation sites, meet the characteristic of pathogenic street strain (virulent strain).Wherein the homology of LN (10) f1 strain H gene nucleotide series and vaccine strain Onderstepoort (AF305419) and CDV3 is merely 91.2%, and with the homology of the genotypic HBF-1 of Asia-1 street strain (EU325721) be 98.4%.Confirm that LN (10) the f1 strain CDV that is separated to is the genotypic street strain of Asia-1.
The mensuration of CDV viral level: select the good V-p-rSLAMh cell of growth conditions, with 0.25% trysinization after, the DMEM growth media (G418 that contains 800 μ g/mL) that usefulness contains 10% FBS is 3 * 10 with cell concn
5/ mL.Be added in 96 orifice plates with every hole 100 μ L with the volley of rifle fire.Culture plate is placed 5%CO
2In the incubator, cultivate 12-18h, make cell grow up to individual layer for 37 ℃.Virus to be measured is kept 10 times of serial dilutions of liquid with the DMEM that contains 2%FBS, make the liquid concentration of virus be respectively 10
-1～10
-6Discard the culture plate growth media, each porocyte is with PBS liquid washing 2 times, from viral liquid from 10
-2Extent of dilution begins, and every hole adds different dilution viral liquid 100 μ L, each repetition in extent of dilution 8 holes; Surplus two arrange and do positive control; One row does and does not add viral liquid normal control, and behind 37 ℃ of absorption 1h, each hole adds again keeps liquid (G418 that contains 800 μ g/mL) 100 μ L; After shaking up gently, 37 ℃, 5%CO
2The static cultivation of incubator.Day by day observe each porocyte pathology, observed continuously 5 days, calculate TCID with the Reed-Munch method
50
LN (10) f1 strain animal is attacked poison experiment: the LN that is separated to (10) f1 strain was passed for 4 generations continuously at the V-p-rSLAMh cell, the cell freeze thawing is used for fox (racoon dog) for 2 times attacks poison, measure viral level before attacking poison.Select the fox (racoon dog) of 5 CDV neutralizing antibodies negative (less than 1: 4), 3 experimental group, 2 as control group.Adopt the mode of subcutaneous multi-point injection, to the poison of attacking of the every fox of experimental group (racoon dog), attacking the toxic agent amount is 4 * 10
2.39TCID
50/ only, the DMEM nutritive medium of the same dosage of the subcutaneous multi-point injection of control group.Measure the body temperature of every fox (racoon dog) morning every day, and the state of every fox of observed and recorded (racoon dog).
Attack malicious racoon dog clinical symptom:
Attack the poison group since 4 days, body temperature begins to be elevated to more than 39.5 ℃, reaches the highest (more than 41 ℃) at 5～9 days body temperature, and 2,3 racoon dog body temperature descend subsequently, and No. 2 racoon dogs opened and make body temperature begin again to rise in 11 days, and are shown in figure 11, attack malicious racoon dog death in 11～13 days.The control group racoon dog is tested omnidistance body temperature normal (below 39.5 ℃).
Attack malicious fox clinical symptom:
The course of disease that fox is attacked poison is longer, attacks the poison group since 4 days, and body temperature begins to be elevated to more than 40 ℃, and No. 3 fox is positive at the 10th day anal swab CDV; The 12nd～13 day, attack the poison group and all begin to occur appetite decline, conjunctivitis is arranged, draw bloody stool, nasal cavity dryness, foot to be lined with and seriously to thicken inflammation; Attack the death in the 18th～22 day of malicious fox for 3, the control group appetite and the mental status are good, and body temperature normal (below 39.5 ℃) is shown in figure 12.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (5)
1. an eukaryotic expression recombinant plasmid is characterized in that, has nucleotide sequence shown in the SEQ ID NO:1.
2. the preparation method of plasmid according to claim 1 is characterized in that, comprising:
Step 1: with the pMD18-T-rSLAM plasmid is template, and amplification obtains said fusion gene in the presence of upstream primer, downstream primer, and said upstream primer sequence is shown in SEQ No.2, and said downstream primer sequence is shown in SEQ No.3;
Step 2:pIRES2-EGFP carrier is cut through enzyme, obtains Pcmv, IRES, EGFP and SV40pA;
Step 3: after getting said fusion gene enzyme and cutting, be connected with Pcmv described in the step 2, said IRES, said EGFP and said SV40pA, the transformation receptor bacterium is identified positive colony, obtains said plasmid.
3. preparation method as claimed in claim 2 is characterized in that, said enzyme is cut to Xho I and EcoR I double digestion.
4. a Vero clone is characterized in that, after the Vero transit cell dyes eukaryotic expression recombinant plasmid as claimed in claim 1, through clone and screening, obtains the clone that the cell surface stably express has CDV acceptor racoon dog SLAM.
5. Vero clone as claimed in claim 4 is in the separation and the application in CDV research thereof of canine distemper virulent strain.
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| CN104805107A (en) * | 2014-01-26 | 2015-07-29 | 中国人民解放军军事医学科学院生物工程研究所 | GS screening system-based animal cell high efficiency expression vector and application thereof |
| CN106755110A (en) * | 2016-12-23 | 2017-05-31 | 中国农业科学院兰州兽医研究所 | A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications |
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| CN109750006A (en) * | 2019-01-14 | 2019-05-14 | 青岛农业大学 | A kind of canine distemper virus replication defective strain and its construction method |
| CN110872597A (en) * | 2019-11-22 | 2020-03-10 | 浙江省农业科学院 | Construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector |
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| CN104805107B (en) * | 2014-01-26 | 2018-03-23 | 中国人民解放军军事医学科学院生物工程研究所 | It is a kind of based on the zooblast efficient expression vector of GS screening systems and application |
| CN106755110A (en) * | 2016-12-23 | 2017-05-31 | 中国农业科学院兰州兽医研究所 | A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications |
| CN106755110B (en) * | 2016-12-23 | 2020-09-01 | 中国农业科学院兰州兽医研究所 | Preparation method of sensitive cell subcloned Vero/Slam/V for enhancing PPRV replication |
| CN107760653A (en) * | 2017-11-08 | 2018-03-06 | 扬州大学 | A kind of the NF κ B Dual-Luciferases reporter cells and construction method of stable expression pig source TLR5 acceptor genes |
| CN109750006A (en) * | 2019-01-14 | 2019-05-14 | 青岛农业大学 | A kind of canine distemper virus replication defective strain and its construction method |
| CN110872597A (en) * | 2019-11-22 | 2020-03-10 | 浙江省农业科学院 | Construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector |
| CN114214268A (en) * | 2021-12-13 | 2022-03-22 | 广东省农业科学院动物卫生研究所 | African green monkey kidney cell line for stably expressing SLAM protein and construction method and application thereof |
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