CN101418286A - Artificial pseudotype virus of human immunodeficiency virus I type as well as preparation and application thereof - Google Patents
Artificial pseudotype virus of human immunodeficiency virus I type as well as preparation and application thereof Download PDFInfo
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- CN101418286A CN101418286A CNA2008102041354A CN200810204135A CN101418286A CN 101418286 A CN101418286 A CN 101418286A CN A2008102041354 A CNA2008102041354 A CN A2008102041354A CN 200810204135 A CN200810204135 A CN 200810204135A CN 101418286 A CN101418286 A CN 101418286A
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Abstract
The invention discloses an artificial fake virus of I-type human immunodeficiency virus, preparation and application thereof. The artificial fake virus of the I-type human immunodeficiency virus is a retrovirus which is generated from PT67 cells transformed from pLXSN plasmid vectors, wherein the pLXSN plasmid vectors contain a SEQ ID NO: 1 polynucleotide sequence. The artificial fake virus is available and is safe and cheap, and can be used as PCR of a HIV-1 virus, a positive reference of RNA extraction and reverse transcription, or a standard substance of the HIV-1 virus.
Description
Technical field
The present invention relates to the artificial pseudovirus of human immunodeficiency virus I type.
Background technology
Acquired immune deficiency syndrome (AIDS) full name behaviour acquired immunodeficiency syndrome, its pathogenic agent is human immunodeficiency virus (Human ImmunodeficiencyVirus, HIV), be a kind ofly to survive among people's blood and can destroy the human immune system, and then make human body lose resistivity to other disease, infection that initiation can not be cured and tumour finally cause the virus of infected person's death.Wherein, HIV-1 is main popular and pathogenic hypotype.
When HIV detects, have following three key steps to need positive reference:
1. extract HIV RNA from sample, comprising: outer virionic membrane, nucleocapsid are opened/fragmentation, and nucleic acid discharges.
2.HIV being reversed record (also claiming reverse transcription), RNA is cDNA.
3.cDNA mix amplification and obtain signal in the PCR instrument with PCR reaction solution and DNA polymerase.
Existing positive reference mainly contains following several source: patient blood (/ blood product); Interior or the in-vitro transcription RNA of body; Plasmid or chemosynthesis.
In all positive references, the positive of accurate appropriateness is with reference to should being the natural sample (as patient blood, lymph) that contains this HIV virus, its homogeneity can fully guarantee its positive reference as aforementioned three steps, but its defective is that natural sample source is few, and, therefore has infectivity and hazardness owing to contain natural viral.
Overcome natural viral source less and have a communicable shortcoming, can solve by isolated culture in essence, promptly HIV (especially genome is through the HIV of innoxious transformation) is imported suitable artificial culture host cell system, the Recombinant HIV particle can produce justacrine to the substratum supernatant from cell.Yet the committed step of this technology is a confidential state, is only had by a few experiments chamber at present; On the other hand, this HIV production method may remain in some defectives, and is still lower as cost height, viral load, structure is not natural, poor repeatability etc.
The HIV RNA of in-vitro transcription because it is the same with index to be measured in the sample, is RNA, therefore can be used as the positive reference of reverse transcription; But,, therefore be not suitable as the positive reference of from sample, extracting the viral RNA operation because it does not possess virus coat.
As positive reference, its advantage is that the preparation purifying is easy with the plasmid that contains the HIV dna sequence dna, but because plasmid is a closed hoop superhelix double chain form, non-single linear, neither RNA, so it can only be as the PCR reference, and can't extract and the reference of reverse transcription as RNA.
And the positive reference of the HIV RNA that adopts the chemosynthesis preparation, technology is still immature at present, and the cost height, also can degrade easily because of the protection that does not have nucleocapsid, film, does not therefore up to the present also have the report of practical application.
Summary of the invention
The purpose of this invention is to provide a kind of being easy to and obtain, safety, the artificial pseudovirus of cheap human immunodeficiency virus I type (HIV-1), its preparation and application.
Pseudovirus promptly refers to retrovirus vector, is the retrovirus through genetic modification, has basic structure and the part life history of protovirus, and does not have most regulatory gene and the main composition that causes a disease.So-called basic structure, be the outer membrane protein of pseudovirus and lipid, capsid protein, replicative enzyme (playing the albumen of cutting, reverse transcription, integration), rna gene group, coded/representative that origin comes from env, gag, pol, the goal gene (sequence) of natural viral respectively.So-called part life history is that pseudovirus only can generation component and assembling in host cell, and target cell infection once, does not produce new virus.
The present invention utilizes the (RetroX of Clontech company of retrovirus system
(R)System), to go into pLXSN (plasmid-LTR-X-SV40 promoter-Neomycin) plasmid vector through the target DNA fragment reorganization (molecular biology routine techniques) that screening obtains, import (method: virus production cell (PT67 of Clontech company) liposome transfection) then, the pseudovirus that carries aim sequence (with rna form) just can produce from produce cell, is secreted in the liquid substratum.Collect this substratum, 2000RPM removed most of cell debris and impurity in centrifugal 10 minutes, just obtained the pseudovirus raw product.
RetroX
(R)System retrovirus system comprises that PT67 produces cell, 2 kinds of cloning vector pLXSN and pLNCX2 is selective, carrier attaches forward and reverse sequencing primer, clones (AP gene) plasmid pLAPSN over against shining, be X=AP, Alkalinephosphatase (alkaline phosphatase reporter gene).It is NIH3T3 that the PT67 cell derives from common mouse embryo fibroblasts, stable transfection Moloney retrovirus (Mo-MLV, Moloney murine leukemia virus, Moloney plant type Muridae leukosis virus) gag-pol gene, 10A1 retrovirus (10A1-MLV, 10A1 plant type Muridae leukosis virus) env gene (also claiming 10A1), the assisting sifting gene that forms stable transfection (being integrated into cell chromosome) is respectively TKgene (thymidine kinase, the thymus gland kinases) and DHFR gene (dihydrofolate reductase, Tetrahydrofolate dehydrogenase).Adopt RetroX
(R)The step that system retrovirus system makes retrovirus is: the aim sequence into a certain carrier of recombinating, amplification recombinant plasmid, transfection PT67 cell, results supernatant.
First aspect present invention discloses a kind of artificial pseudovirus of human immunodeficiency virus I type, is retrovirus, and the PT67 cell that is transformed by the pLXSN plasmid vector produces, and wherein, the pLXSN plasmid vector contains the polynucleotide sequence of SEQID NO:1.
Preferable, SEQ ID NO:1 is between the EcoRI and XhoI site of pLXSN carrier.
The pLXSN plasmid vector is the retroviral vector of Clontech company exploitation, the pLXSN plasmid vector is the abbreviation of plasmid-LTR-X-SV40 promoter-Neomycin, LTR=Long terminal repeat, the distinctive long terminal repeat of retrovirus, promptly in the base of the 500nt left and right sides, two ends of 10000nt full-length gene group identical sequence, can guide the generation of intermediate DNA and be integrated in the genome/karyomit(e) of people's cell, also can guide from intermediate DNA and produce new viral RNA genome/long RNA chain, X=Sequence of Interest, interested sequence/aim sequence, it in this patent unique ~ 1300nt HIV virus sequence, SV40 promoter=Simian vacuolating virus 40 promoter, the promoter sequence of vacuolating virus of monkey, in people's cell, still has the very strong ability that downstream sequence produces RNA that starts, Neomycin=Neomycin resistant gene, neomycin resistance gene, cell is if contain this gene, and expression product albumen, then can resist the Xin Meisu toxicity in the environment, SV40-Neomycin closes with aim sequence to be put, and just can screen and choose cell strain---sieve stable transfection/production cell strain that (/ make survival) contains aim sequence by Xin Meisu.
Second aspect present invention discloses a kind of pLXSN plasmid vector, and it contains the polynucleotide sequence of SEQ ID NO:1.
Third aspect present invention discloses a kind of host cell, the PT67 cell that it is transformed by the pLXSN plasmid vector, and wherein, the pLXSN plasmid vector contains the polynucleotide sequence of SEQ ID NO:1.
Fourth aspect present invention discloses the preparation method of the artificial pseudovirus of above-mentioned human immunodeficiency virus I type, comprises the following steps:
3) cultivate above-mentioned host cell;
4) from culture, isolate the artificial pseudovirus of human immunodeficiency virus I type.
Above-mentioned steps 1 can adopt the conventional formulation of field of cell culture to cultivate, as:
DMEM, 90%; Calf serum, 10%; 4mM L-glutamine; The 100U/ml Benzylpenicillin sodium; 100 μ g/ml Vetstreps.; The 1mM Sodium.alpha.-ketopropionate.
Fifth aspect present invention discloses the artificial pseudovirus of above-mentioned human immunodeficiency virus I type in the positive reference material of preparation HIV-1 virus detection or the application in the viral standard substance that detect of HIV-1.
The positive reference material that above-mentioned HIV-1 virus detects comprises that the positive reference material of PCR, RNA that HIV-1 virus detects extract positive reference material and the positive reference material of reverse transcription.
Obtain to be suitable as the artificial pseudovirus of the human immunodeficiency virus I type of positive control or standard substance, it is crucial that suitable aim sequence is chosen, the factor of choosing main needs consideration the following aspects of aim sequence, at first, the sequence of aim sequence should be guarded in the human immunodeficiency virus I of various different genotype type, be most genotypic HIV-1, the entrained HIV-1 virus of most humans just, all has this section specific base sequence, secondly, for PCR and virus packing, the fugitive more rate of fragment is high more, and for purpose/function of simulating HIV-1, then representative more near the genome total length more.
Take all factors into consideration above-mentioned factor, the present invention has chosen the sequence of SEQ ID NO:1 as aim sequence, according to standard NCBIreference sequence (NC_001802), this base sequence is the sequence of 337 to 1641 in HIV-1 virus, the major part (coding capsid protein) that contains the HIV-1gag gene, but there be not (made ATG--〉TTG sudden change) in translation initiation codon.Choosing this sequence at first is because this sequence has comprised the most conservative sequence of genome sequence of HIV, with this detect parameters/index/object as the universal dosed detection kit of HIV-1, can improve recall rate, reduce loss, and at present main commercialization and laboratory PCR detection method are all at this zone.Secondly, this sequence PCR and viral packaging efficiency height are compared with other long segment, and relatively easy subclone advances pseudovirus carrier pLXSN, and its corresponding virus yield is up to 10
7~ 10
8, and titre, stability, sensitivity etc. all can meet the demands; Test is found, contain that the HIV-1 gag gene of HIV-1 gag initiator codon or HIV-1 gag-pol are dual-gene all can't to produce high titre pseudovirus, infer that HIV-1 gag protein expression may disturb the assembling of retrovirus, because the capsid protein MMLV gag of HIV-1 gag albumen and retrovirus is at character, molecular weight, similar nominally; In other words, HIV-1 is similar virus to MMLV.
Artificial pseudovirus of the present invention is easy to obtain and the safety cheapness, can be used as the positive reference material or the HIV-1 virus standard substance of HIV-1 virus PCR, RNA extraction and reverse transcription.
Description of drawings
Fig. 1: with the pSPAX2 plasmid is template, the HIV dna sequence dna electrophorogram that pcr amplification obtains
Swimming lane 1:DNA molecular weight marker is 2000,1000,750,500,250 from top to bottom, 100bp.
Swimming lane 2:DNA molecular weight marker is 10000,8000,7000,6000,5000,4000,3000,2000 from top to bottom, 1000bp.(obviously do not separate for top 7.)
Swimming lane 3:PCR product, about 4300bp, template is the pSPAX2 plasmid.
Fig. 2: cleavage map, PCR fragment and carrier segments produce sticking terminal through the restriction enzyme cutting.
Swimming lane 1:DNA molecular weight marker is 10000,8000,7000,6000,5000,4000,3000,2000 from top to bottom, 1000bp.(obviously do not separate for top 7.)
Swimming lane 2:PCR product is cut through EcoRI and BglII enzyme, about 3000bp of gained band and 1300bp; The 1300bp band is the purpose band.
Swimming lane 3: blank.
Swimming lane 4:pLXSN carrier is cut through EcoRI and BamH I enzyme, the about 5850bp of gained band.
Fig. 3: enzyme is cut evaluation figure.Plasmid is extracted in the microbial culture that the transforms gained back of spending the night, and enzyme is cut evaluation and whether is contained the purpose fragment.
Swimming lane 1:DNA molecular weight marker is 10000,8000,7000,6000,5000,4000,3000,2000 from top to bottom, 1000bp.(obviously do not separate for top 6.)
Swimming lane 2:1 bacterium colony plasmid is cut through Hind III enzyme and is obtained 627,731, and the band of 5828bp shows and contains the purpose fragment.
Swimming lane 3:2 bacterium colony plasmid is cut through Hind III enzyme and is obtained 627,731, and the band of 5828bp shows and contains the purpose fragment.
Swimming lane 4:3 bacterium colony plasmid is cut through Hind III enzyme and is obtained 627,731, and the band of 5828bp shows and contains the purpose fragment.
Fig. 4: DNA standard form realtime PCR detects figure
Fig. 5: the Ct value typical curve that DNA standard form real time PCR detects
Fig. 6: pseudovirus real time PCR detects (quantitatively) figure
Ct＝20.48
Be scaled titre 8.1 * 10
7Copy/mL.
Fig. 7: embodiment 2 pseudoviruss are as sun ginseng/standard substance, and gradient dilution real time PCR detects figure.Extension rate: former times, 10,100,1000,10000 times
Fig. 8: embodiment 2 pseudovirus realtime PCR detect Ct value dilution curve
Fig. 9: embodiment 2 national standard product real time PCR detect 00,1000,100 viruses of Figure 100.
The Ct value typical curve that Figure 10: embodiment 2 national standard product realtime PCR detect
Figure 11: embodiment 2 negative control real time PCR detect figure
Figure 12: 4 kinds of HIV pseudoviruss design pairing titer determination figure (real time PCR method)
Embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment only are used to illustrate the present invention, but not limit the scope of the invention.The reagent of the experimental technique of unreceipted actual conditions and undeclared prescription is according to people such as normal condition such as Sambrook in the following example, molecular cloning: the condition of the condition described in the test handbook or manufacturers's suggestion is carried out or is disposed.
The preparation of embodiment 1 human immunodeficiency virus I type artificial pseudovirus
1. contain the plasmid vector preparation of HIV-1 sequence, and cell is produced in transfection.
1.1 clone's purpose HIV-1DNA.
The pSPAX2 plasmid comes from Addgene company, contains the dual-gene zone of gag-pol of HIV-1 virus, and this tests required DNA just for this.As template, pcr amplification to enough HIV-1 dna sequence dnas (result as shown in Figure 1,4300bp) with the pSPAX2 plasmid.
Mix:
H
2O ——34μL，
10×PCR?buffer ——5μL，
dNTPs ——5μL，
Mg
2+ ——2μL，
HIV-1?gagTTG?sense ——1μL，
HIV-1?pol?antis ——1μL，
KOD+ ——1μL，
PSPAX2 template---1 μ L,
The PCR thermal cycle conditions:
The first step: 94 ℃ of 3min.
Second step: 94 ℃ of 25sec, 57 ℃ of 30sec, 68 ℃ of 4min 30sec; 30 circulations.
The 3rd step: 68 ℃ of 3min.
Electrophoresis, UV-light incision glue reclaims: reclaim the operation of test kit specification sheets according to glue.
Primer sequence:
gag?EcoR?I?sense：CTGGAATTCACTGGTGAGAG
TTGG
pol?XhoI?antis：GGTCCTCGAGTTAATCCTCATCCT
Annotate:
Archaeal dna polymerase: the KOD+DNA polymerase of TOYOBO company (Co., Ltd.).And 10 corresponding * PCR
Buffer, Mg
2+(25mM, 25 *) and dNTPs (2mM each of dATP, dTTP, dCTP, dGTP, 10 *).
PCR pipe: common thin-walled PCR pipe (0.2mL type)
PCR instrument: rich day qualitative PCR instrument.
Primer underscore place is the mutational site.
1.2HIV-1 DNA PCR product and plasmid vector enzyme are cut.
PCR product enzyme is cut:
PCR product (gagTTG-pol)---17 μ L
10×H?buffer ——2μL
EcoR?I ——0.5μL
Bgl?II ——0.5μL
Handle 10-20hr for 37 ℃.
The carrier enzyme is cut:
H
2O ——16μL
PLXSN carrier---1 μ L
10×H?buffer ——2μL
EcoR?I ——0.5μL
BamH?I ——0.5μL
Handle 10-20hr for 37 ℃.
Annotate: EcoR I, Bgl II, BamH I and supporting 10 * H buffer, 10 * K buffer is in precious biological (Dalian)
Co., Ltd.'s product.Bgl II and BamH I are isocaudarner.
1.3 HIV-1 DNA PCR product and plasmid vector after enzyme is cut carry out ligation.
Enzyme is cut back PCR product (gagTTG)---16 μ L
Enzyme is cut back pLXSN carrier---1 μ L
10×T4?ligation?buffer ——2μL
T4?DNA?ligase ——1μL
Handle 10-20hr for 16 ℃.
Annotate: T4DNA ligase, and supporting 10 * T4 ligation buffer are precious biological (Dalian) Co., Ltd. product.
1.4 connect product transformed competence colibacillus intestinal bacteria.
Take out pipe (100 a μ L) competent cell (DH-5 α intestinal bacteria) from-70 ℃ of refrigerators, thaw.
Add and connect product (step 1.3), mixing.
Ice bath 30min.42 ℃ of heat shock 90sec.Ice bath 2min.
Add 1mL LB, 150-200rpm shakes 40-60min.37 ℃.
Centrifugal, 3000rpm, 3min.
Siphon away the part supernatant, stay 100-300 μ L,, coat penbritin LB agar plate with cleer and peaceful precipitation on the pipettor mixing.
Cultivate 14-16hr for 37 ℃.
1.5 the recombinant plasmid (enzyme cutting method) of screening/evaluation expection from bacterium colony.
1.5.1 extraction plasmid.
Select 3 bacterium colonies from flat board, shaking table is cultivated in 3mL LB, extracts plasmid according to the Qiagen specification sheets after 15 hours.(alkaline lysis/silicon matrix absorption method.)
1.5.2 enzyme is cut evaluation.
H
2O ——14μL
Plasmid to be identified---4 μ L
10×M?buffer ——2μL
Hind?III ——0.5μL
Handle 10-20hr for 37 ℃.
Positive criteria: visible 627,731, the electrophoretic band that 5828bp is bright.
Annotate: Hind III, and supporting 10 * M buffer are precious biological (Dalian) Co., Ltd. product.
1.5.3 order-checking is identified.
Send a positive bacterium colony order-checking company to identify, sequencing primer incidental pLXSN5 ' primer of carrier and pLXSN3 ' primer.Measurement result meets expection.
1.6 cell is produced in transfection
Producing cell is the PT67 cell (clontech company) that is incubated at the 35mm ware, and cell degree of converging changes liquid when 50-80%, the beginning transfection.A liquid: DMEM250 μ L+ liposome 10 μ L, mixing leaves standstill 5min; B liquid: DMEM250 μ L+ plasmid (pLXSN-gagTTG) 4 μ g[20 μ L (0.2 μ g/ μ L)], mixing leaves standstill 5min; Mix A liquid and B liquid, mixing leaves standstill 20min; Join cell conditioned medium.Change liquid behind the 4-7hr once more.The thick viruses of results (cell culture medium supernatant) behind the transfection 48-72hr.
Annotate: liposome is Lipofectamine2000 (Invitrogen).
2. the acquisition of cell cultures and pseudovirus
2.1 the recovery of cell.The cell (in the frozen pipe) of liquid nitrogen cryopreservation is melted in 37 ℃ of water-baths, use the alcohol swab wiping immediately, move into culture dish (as the culture dish of 60mm diameter), add the perfect medium (being fit to the 60mm ware) of suitable volumes as 5mL.Change liquid behind the 12-24hr, remove not adherent cell.After this normally cultivate (perfect medium is used for the PT67 cell, conditioned medium has been used for stable transfection the PT67 cell of target plasmid).
2.2 the cultivation of cell
Adherent cell is cultivated with perfect medium (PT67 cell) or conditioned medium (stable transfection the PT67 cell of target plasmid), changes liquid in every 2-3 days, promptly removes old culture supernatant, adds the equal-volume fresh culture.Cell digest when 70-90% degree of converging, and goes down to posterity then or frozen.
Annotate 1: perfect medium:
DMEM (containing L-Glutamine 4mM and sodium pyruvate acid 1mM)+FBS (10%)+penicillin (100U/mL)+streptomycin (100 μ g/mL)
Annotate 2: conditioned medium:
DMEM (containing L-Glutamine 4mM and sodium pyruvate acid1mM)+FBS (10%)+G418 (0.6mg/mL).
Annotating 3:FBS (Fetus Bovine Serum, foetal calf serum) is the product of Invitrogen (Australia) company, and brand is Gibco; All the other are Sigma-Aldrich company product.
2.3 the digestion of cell.
Attached cell with the DMEM washing once adds pancreatin (0.25%) 1mL, behind the 1-2min, inhales and removes pancreatin.
Trysinization liquid: 0.25%Trypsin (bovine trypsin, Invitrogen company, Gibco brand).
2.4 going down to posterity of cell.
After the digestion, add complete substratum, blow afloat cell gently with pipettor, divide to required culture dish, general 1 part passes 3-10 part.
2.5 cell is frozen.
After the digestion, add the cell frozen storing liquid, be transferred to frozen pipe, be stored in-20 ℃ immediately, after this 1-2hr transfers to-70 ℃, and 5-24hr is after this frozen in liquid nitrogen.
Cells frozen storing liquid: DMEM (containing L-Glutamine 4mM and sodium pyruvate acid 1mM, 70% volume)+FBS (20% volume)+DMSO (Dimethyl sulphoxide, 10% volume)
Annotate: DMSO is a Sigma-Aldrich company product.
2.6 the screening of cell.
Working conditions culture medium culturing cell.G418 content is 0.6mg/mL, based on concentration gradient sensitive experiment (killingcurve).
2.7 the quantitative/titer determination of the acquisition of pseudovirus and purpose nucleic acid.
2.7.1
To the cell of transient transfection, the thick viruses of results (the cell culture medium supernatant behind the centrifugal 2000rpm10min, is got supernatant) behind the transfection 48-72hr.
To the cell of stable transfection, then work as cell density and reach 50-90%, the thick viruses of results (the cell culture medium supernatant behind the centrifugal 2000rpm 10min, is got supernatant) behind the cultivation 48-72hr.
2.7.2
(adopt the TRIzol method to extract pseudovirus RNA.)
Add TRIzol (Invitrogen company) 0.5mL in the thick virus to 100 μ L HIV-1, fully shake up, leave standstill 5-15min and add 100 μ L trichloromethane (chloroform, CHCl
3), acutely shake up, leave standstill 10-20min.
Centrifugal, 12000rpm, 15min.
Draw centrifuge tube upper strata clear liquid carefully, volume is V (about common 400 μ L).
Add Virahol, volume 0.8V (about 300 μ L); Add glycogen (Glycogen) help heavy, about 1 μ L (20 μ g/ μ L mother liquor).
Centrifugal, 12000rpm, 15min.
Stay precipitation, add 70% ~ 80% washing with alcohol.
Centrifugal, 7000-8000rpm, 10-15min stays precipitation.
Centrifugal, 3000-5000rpm, 1min stays precipitation.
30 μ L DEPC water dissolution RNA precipitation.
2.7.3
After getting DNA standard substance (template) (HIV-1 linearizing DNA/PCR product is measured concentration through OD/ absorbance measuring and electrophoretic band densitometric scan) dilution certain multiple, obtain 10
2, 10
3, 10
4, 10
5, 10
6The template of quantity.The pseudovirus RNA that obtains with 2.7.2 carries out Real time PCR (adopting the HIV-1 RNA/DNA detection by quantitative test kit of the Zhijiang River, Shanghai company) simultaneously.The amplification curve of standard substance as shown in Figure 4, the quantitative curve of pseudovirus as shown in Figure 6, setting threshold is 0.02, obtains Ct value (Threshold Cycle) separately.The logarithm of the Ct of each order of magnitude standard substance and quantity (10 is the end) mapping, obtain typical curve, as Fig. 5.And obtain linear relationship equation, Y=-3.403X+41.34 (X is the logarithm of template DNA quantity, the Ct value that Y records for this template correspondence), coefficient R
2=0.99, illustrate linear very good.Through calculating (typical curve and extent of dilution), pseudovirus titre (pseudovirion quantity) about 8.1 * 10
7
3. the pseudovirus physicochemical property test that obtains, pathogenic evaluation test
3.1 physicochemical property test: protein blot experiment (Western Blot Assay).
Get 100 μ L pseudovirus stostes, handle with 20 μ L lysates (250mM Tris-HCl, 10%SDS, 0.5% tetrabromophenol sulfonphthalein, 50% glycerine, 5% beta-mercaptoethanol).Carry out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), then protein band is changeed film to nitrocellulose filter, use anti-p24 mAb (anti-retroviral virus nucleocapsid albumen capsid/p24 monoclonal antibody) and anti-10A1 mAb (anti-retroviral virus membrane antigen 10A1 monoclonal antibody) to hatch respectively, after 1-2 hour, add HRP-pAb (anti-mouse two resists the rabbit of horseradish peroxidase-labeled) and develop the color.
The result---obtain positive signal (single band appears in the position in expection), this test shows virus nucleocapsid albumen, membranin, and the common existence of viral RNA (seeing quantitative result), and this meets the feature of reverse transcription pseudovirus.
3.2 pathogenic test: cell culture experiments.
In the normal A549 (human pneumonocyte system) and HepG2 (human liver cell system) that cultivates, each drops into the 1mL pseudovirus, and the 1st, 3,5,10, use the microscope observing cell form in 15 days, and check cell viability (mtt assay).
The result---cellular form is normal, and vigor is also normal.Illustrate that the pseudovirus pair cell does not have overt toxicity.
4. the pseudovirus performance comparison test of various objectives gene:
With reference to preceding method, designed 4 kinds of sections of HIV-1, prepare pseudovirus respectively: [1] gag-pol is dual-gene, contains initiator codon and terminator codon, 4300bp; [2] gag-pol is dual-gene, and the initiator codon removal (ATG--〉TTG) that suddenlyd change contains terminator codon (TAA), 4300bp; [3] part gag gene contains initiator codon, does not contain terminator codon, 1300bp; [4] removal (ATG--〉TTG) that suddenlyd change of part gag gene (the present invention), initiator codon does not contain terminator codon (TAA), 1300bp.Measure titre simultaneously, relatively height.The result measures and finds as shown in figure 12, and first three titre of planting pseudovirus is very low, and the 4th kind of pseudovirus titre is higher.
From left to right, four curves are represented respectively:
1, gag-TTG, calculate titre 8.1 * 107;
2, gag-TTG pol, calculate titre 3.1 * 106;
3, gag pol, calculate titre 1.3 * 106;
4, gag, calculate titre 7.7 * 105;
Clearly, (removal (ATG--〉TTG) that suddenlyd change of part gag gene, initiator codon does not contain terminator codon (TAA) to the gag-TTG pseudovirus, and 1300bp) titre exceeds remaining design more than 20 times (25-100 doubly).
The application of the artificial pseudovirus of embodiment 2 human immunodeficiency virus I types
(1) contrast of extracting as RNA.
Take off and state sample:
A, 100 μ L pseudovirus stostes (mode by embodiment 1 prepares).Quantity is about 1 * 10
7
B carries out 10 times of dilutions with 10 μ L pseudovirus stostes+90 μ L bovine serums, and the pseudovirus total amount is equivalent to 10 μ L stostes.Quantity is about 1 * 10
6
C carries out 10 times of dilutions with 10 μ L higher levels virus liquid+90 μ L bovine serums, and the pseudovirus total amount is equivalent to 1 μ L stoste.Quantity is about 1 * 10
5
D carries out 10 times of dilutions with 10 μ L higher levels virus liquid+90 μ L bovine serums, and the pseudovirus total amount is equivalent to 0.1 μ L stoste.Quantity is about 1 * 10
4
E carries out 10 times of dilutions with 10 μ L higher levels virus liquid+90 μ L bovine serums, and the pseudovirus total amount is equivalent to 0.01 μ L stoste.Quantity is about 1 * 10
3
F, 100 μ L bovine serums.As negative control.
G, the quantitative criterion product B1 of 100 μ LHIV-1 RNA country, HIV-1 RNA theoretical concentration is 10
5IU/mL, content are 10
4IU.
H, the quantitative criterion product B2 of 100 μ LHIV-1 RNA country, HIV-1 RNA theoretical concentration is 10
4IU/mL, content are 10
3IU.
I, the quantitative criterion product B3 of 100 μ LHIV-1 RNA country, HIV-1 RNA theoretical concentration is 10
3IU/mL, content are 10
2IU.
J, the negative reference material N1 of 100 μ LHIV-1 RNA country, HIV-1 RNA theoretical concentration is 0IU/mL, content is 0IU.
Above-mentioned 10 kinds of samples extract RNA simultaneously, and are identical among method and " the embodiment 1-2.7.2 ".The reverse transcription of extraction gained RNA and detected result are referring to the part of following (2).
Annotate 1:HIV-1 RNA national standard product and comprise 6 quantitative criterion product, 5 linear standard substance, 8 negative controls, 8 positive controls), be present unique legal HIV-1 RNA diagnostic reagent reference material.By Nat'l Pharmaceutical ﹠ Biological Products Control Institute's preparation, all be human plasma.Quantitative criterion product concentration is 10
5, 10
4, 10
3Three levels of IU/mL can be used as positive reference material simultaneously; Negative reference material N1 is not for containing the serum of HIV-1; This experiment is got these four parts of crucial reference materials and is compared.
Annotate 2: bovine serum is the product of Invitrogen (Australia) company, and brand is Gibco.
(2) as the contrast of reverse transcription reaction.
Get the RNA extract of 10 duplicate samples described in (1), carry out reverse transcription reaction, method is as follows:
Mixed liquor A: RNA 15 μ L, 5 * RT buffer, 5 μ L, random primer hexamer 3 μ L, mixing is handled 5min, ice bath 5min immediately for 70 ℃.
Mixed liquid B: mixed liquor A 23 μ L, dNTPs (10mM each) 1 μ L, RTase (MMLV of Promega company reversed transcriptive enzyme) 1 μ L, mixing, 25 ℃ of 10-20min, 42 ℃ of 30-80min, 70 ℃ of 10-20min.Be reverse transcription product---cDNA.
Detected result is referring to following (3).
(3) as contrast and the standard substance of realtime PCR.
Get the RNA reverse transcription product (being cDNA) of 10 duplicate samples described in (2), it is quantitative to carry out real time PCR.Method is referring to instrument (the Applied Biosystems ABI7000 of company quantitative PCR instrument, Shanghai grand stone SLAN quantitative PCR instrument) and test kit specification sheets (the HIV-1 RNA/DNA of Zhijiang River company quantification kit).
Reaction solution:
PCR?mix ——30uL
Taq?DNA?polymerase ——0.4uL
CDNA sample---10uL
Reaction conditions:
The first step, 95 ' C---4min,
Second step, 95 ' C---15sec, 60 ' C---1min, fluoroscopic examination; Circulate 40 times.
Result (shown in Fig. 7-11):
Form 1:
Conclusion: as can be known by quantitative result, the pseudovirus of our company (Zhijiang River company) is in nucleic acid extraction, reverse transcription reaction, real time PCR process, the sensitivity and the linear lag and national standard product (representative clinical sample) are close, point out it to can be used as reference material or standard substance use.But copy number is converted to international unit (IU) is about 10:1.That is, the pseudovirus original liquid concentration 8.1 * 10
7Copy/mL (according to the company standard product) is equivalent to international concentration 8.1 * 10
7IU/mL.
Sequence table
＜110〉Shanghai Zhijiang Biological Science Co., Ltd
＜120〉artificial pseudovirus of human immunodeficiency virus I type and preparation thereof and application
<130>PCNZY081214
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1306
<212>DNA
<213>Human?immunodeficiency?virus?type?1
<400>1
<210>2
<211>5874
<212>DNA
<213>artificial?sequence
<220>
＜223〉pLXSN carrier
<400>2
Claims (8)
1. the artificial pseudovirus of a human immunodeficiency virus I type is retrovirus, and the PT67 cell that is transformed by the pLXSN plasmid vector produces, and wherein, the pLXSN plasmid vector contains the polynucleotide sequence of SEQ ID NO:1.
2. the artificial pseudovirus of human immunodeficiency virus I type according to claim 1 is characterized in that described SEQ IDNO:1 is between the EcoRI and XhoI site of pLXSN carrier.
3. pLXSN plasmid vector, it contains the polynucleotide sequence of SEQ ID NO:1.
4. as pLXSN plasmid vector as described in the claim 3, it is characterized in that described SEQ ID NO:1 is between the EcoRI and XhoI site of pLXSN carrier.
5. host cell, the PT67 cell that it is transformed by claim 3 or 4 described pLXSN plasmid vectors.
6. the preparation method of the artificial pseudovirus of a human immunodeficiency virus I type comprises the following steps:
1) cultivates the described host cell of claim 5;
2) from culture, isolate the artificial pseudovirus of human immunodeficiency virus I type.
7. the application of the artificial pseudovirus of human immunodeficiency virus I type in the standard substance of viral positive reference material that detects of preparation HIV-1 or the detection of HIV-1 virus as claimed in claim 1 or 2.
8. as the application of the artificial pseudovirus of human immunodeficiency virus I type as described in the claim 7, it is characterized in that the positive reference material that described HIV-1 virus detects comprises that the positive reference material of PCR, RNA that HIV-1 virus detects extract positive reference material and the positive reference material of reverse transcription.
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Cited By (6)
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| CN101892326A (en) * | 2010-07-19 | 2010-11-24 | 复旦大学 | Method for preparing standard substance suitable for virus PCR detection |
| CN102199674A (en) * | 2011-03-17 | 2011-09-28 | 中国疾病预防控制中心病毒病预防控制所 | Enveloped ribonucleic acid (RNA) virus nucleic acid detection reference/standard platform and application method thereof |
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| CN105132583A (en) * | 2015-07-17 | 2015-12-09 | 上海市临床检验中心 | Influenza virus carried HCV nucleic acid test quality control product and preparation method thereof |
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| CN101892326A (en) * | 2010-07-19 | 2010-11-24 | 复旦大学 | Method for preparing standard substance suitable for virus PCR detection |
| CN101892326B (en) * | 2010-07-19 | 2012-08-22 | 复旦大学 | Method for preparing standard substance suitable for virus PCR detection |
| CN102199674A (en) * | 2011-03-17 | 2011-09-28 | 中国疾病预防控制中心病毒病预防控制所 | Enveloped ribonucleic acid (RNA) virus nucleic acid detection reference/standard platform and application method thereof |
| CN103789277A (en) * | 2014-01-16 | 2014-05-14 | 东北制药集团辽宁生物医药有限公司 | Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments |
| CN105132583A (en) * | 2015-07-17 | 2015-12-09 | 上海市临床检验中心 | Influenza virus carried HCV nucleic acid test quality control product and preparation method thereof |
| CN105648037A (en) * | 2015-12-30 | 2016-06-08 | 南方医科大学 | Application of recombinant lentivirus to HIV (human immunodeficiency virus) phenotypic drug resistance detection |
| CN105648037B (en) * | 2015-12-30 | 2020-06-05 | 南方医科大学 | Application of recombinant lentivirus in HIV (human immunodeficiency virus) phenotypic drug resistance detection |
| CN108728492A (en) * | 2017-04-19 | 2018-11-02 | 辽宁琦润生物科技有限公司 | A kind of preparation method of gene mutation and fusion positive reference product |
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| CN101418286B (en) | 2011-06-15 |
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