CN102492662A

CN102492662A - Method for marking live viral particles in multicolor

Info

Publication number: CN102492662A
Application number: CN2011103586887A
Authority: CN
Inventors: 何治柯; 王汉中; 周鹏
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2011-11-14
Filing date: 2011-11-14
Publication date: 2012-06-13
Anticipated expiration: 2031-11-14
Also published as: CN102492662B

Abstract

The invention discloses a method for marking live viral particles in multicolor, which comprises the steps of: A, with help of a virus showing system, executing fusion expression of green fluorescent protein and virus envelope protein GP64, and constructing recombinant viruses with the green fluorescent protein on the GP64; B, adding metal complex [Ru(phen)2(dppz)]2+ solution into Grace's culture medium of fetal calf serum to cultivate sf host cells; adding the constructed recombinant viruses Bac-EGFP into the cultivated host cells for virus infection; putting the recombinant viruses and the host cells in a cultivation box for cultivation, and harvesting progeny viruses; and C, collecting, purifying progeny virus particles marked in two colors, treating the obtained progeny viruses through a transmission electron microscope, expressing structures of the progeny virus particles through inductive coupling plasma-mass spectrometry and a laser con-focal scanning microscope; measuring content of the metal complex in each single virus particle; and showing dual fluorescent signals of the single virus particle. The method disclosed by the invention is simple and convenient in operation, and high in marking efficiency, and can obtain marked viruses with excellent fluorescent signals, and satisfy the demands of researches about tracing the live virus particles to infect the host cells in real time in a better way.

Description

A kind of method of multi-color marking live body virion

Technical field

The present invention relates to the interdisciplinary field of subjects such as biology, chemistry, material, medical science, virusology, more specifically relate to a kind of method of multi-color marking live body virion.

Background technology

The research that real-time spike live virus particle infects host cell causes the early treatment of disease all significant for deeply understanding virus infection mechanism and virus.In research in the past, the investigator mainly carries out mark and is used for tracer study virus through two kinds of means: 1) at one section GFP of fusion of the outer albumen zone of virus target; 2) directly fluorescent molecular probe and viral protein are carried out coupling through chemical reaction.These methods can provide many and get into host cell about viropexis, and the information of togavirus and host cell membrane fusion.Yet; The virus infection host cell is an extremely complicated process; Its main process comprises: virus envelope and host cell generation film merge; Viral nucleic acid moves in the intravital release of lyase and in intracytoplasmic orientation, viral nucleic acid duplicating and the expression of viral protein in host cell nuclear.Only will be difficult to spike to the outer proteic mark of virus moves to the orientation that virus and cytolemma merge the back viral nucleic acid.The virological development of having lost big limitations of this key message.Therefore, the mark to the polycomponent mark, particularly its viral nucleic acid of virion is a hot issue always.Though for many years, investigators are always at attempts mark's viral nucleic acid, and the work of its successful mark also rarely has report.Return its reason mainly to be because the unique imporosity of its virus.Complete virion is generally by cyst membrane, and nucleocapsid protein and nucleic acid three parts are formed, in case virus assembling in host cell is accomplished, its viral nucleic acid just is protected in nucleocapsid protein and the cyst membrane.Dyestuff is difficult to carry out with it coupling.If through external force with virus dissociate the back mark, its viral infection ability will reduce greatly, even completely lose.And the application is in the cell internal breeding process of virus; Adopt metal complexes that recombinant virus is carried out nucleic acid marking; Not only realized the same tense marker of virus envelope and viral nucleic acid, and do not influenced the infection ability of labeled virus, simultaneously; Labeling effciency is high, can satisfy the needs that real-time spike live virus particle infects host cell research better.

Summary of the invention

To the restriction of prior art, the objective of the invention is to be to provide a kind of method of multi-color marking live body virion.This method is mixed metal complexes based on viral self-assembly system with cell culture medium, mark viral nucleic acid in the cell internal breeding process of virus; In conjunction with genetic engineering technique, two different virus components (envelope protein, nucleic acid) of fluorescent mark in the cell have been realized.Labeling process environmental protection, step are simple; Only, just can be had the green fluorescence cyst membrane in a large number, the sub-virion of double-colored mark of red fluorescence nucleic acid through the self-reproduction of virus in host cell.Through characterizing the progeny virion of purifying, can confirm that the virus structure of mark is complete, every virus contains a large amount of metal complexess, and in the cell imaging experiment, excellent fluorescent signal can be provided.

To achieve these goals, the present invention adopts following technical measures:

A kind of method of multi-color marking live body virion, its step is following:

(1),, constructs the recombinant virus that has green fluorescent protein on the GP64 with green fluorescent protein (EGFP) and virus envelope Protein G P64 amalgamation and expression by viral display systems.

According to AcMNPV genome (GenBank:L22858.1) sequence among the GenBank with primer-design software Primer premier 5 (Premier company); Design EGFP/gp64 genetic expression primer (Fig. 6) adds EcoR I and two restriction enzyme sites of HindIII respectively at 5 ' and 3 ' of gp64.At first, as masterplate, FEG1 and REG-G are that primer carries out pcr amplification with the pEGFP-N1 plasmid (Clonetech company) of purifying; As template, is that primer once more carry out pcr amplification with FN2 and REG-G with the PCR product EGP0 that obtains, and obtains one section fragment that comprises GP64 signal peptide and EGFP sequence, with this sequence called after EGP1.Be template (the bac to bac rhabdovirus system of Invitrogen company) with the Bacmid DNA that purifies then, FG-EG and RG are that primer carries out pcr amplification, obtain one section fragment that includes the gp64 peptide section sequence, with this sequence called after EGP2.5 ' the terminal sequence of 27 EGP2 that the 3 ' end of EGP1 adds is introduced by primer REG-G; 3 ' the terminal sequence of 24 EGP1 that the 5 ' end of EGP2 adds is introduced by primers F G-EG; EGP1 and EGP2 have just had 24 complementary bases like this, for Overlap PCR at the back tests the condition that provides.Overlap PCR experiment is a template with EGP1 and EGP2, and FN2 and RG are primer, amplify at the gp64 signal peptide sequence to merge the fragment that the EGFP sequence is arranged at the back, with this sequence called after EGFP/gp64.The PCR reaction system is following:

(5’-GAATTCATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCG-3’)，

(5’-AAGCTTTTAATATTGTCTATTACGGTTTCTAATC-3’)

Ligation product electricity is transformed into preparation plasmid EGFP/gp64-T Easy Vector in intestinal bacteria (Escherichia.Coli) DH5 α (Wuhan Virology Institute,Chinan academy of Sciences) competent cell.Restriction enzyme EcoR I and HindIII double digestion donor plasmid EGFP/gp64-T Easy Vector and expression vector pFastBac1 (the bac to bac baculovirus expression system of Invitrogen company); Enzyme is cut product and is demonstrated the fragment that varies in size through gel electrophoresis; Under uv lamp, cut the adhesive tape that is consistent with pFastBac1 clip size after EGFP/gp64 fragment and enzyme are cut; DNA glue with Omega company reclaims test kit recovery dna fragmentation; Carry out ligation at 4 ℃ after the purifying and recovering, the EGFP/gp64 fragment is inserted in the corresponding MCS of expression vector pFastBac1.

Double digestion carries out 3h under 37 ℃ of conditions, reaction system is following:

The ligation body is 4 ℃ of reaction overnight, and two segmental add-ons are according to two segmental concentration and big or small to doing trickle adjustment, and reaction system is following:

Connecting the product electricity is transformed in intestinal bacteria (Escherichia.Coli) the DH5 α competent cell for preparing; On the flat board that contains 100 μ g/mL penbritins and 100 μ g/mL kantlex, carry out blue hickie screening; Extract the DNA of white colony; Carry out EcoR I-HindIII double digestion and identify, whether successful to confirm the clone.Identify correct clone's called after pFastBac1-EGFP, be stored in than 3: 7 in-80 ℃ of refrigerators so that 50% (glycerin/water volume ratio) glycerine/bacteria liquid is long-pending.

The pFastBac1-EGFP swivel base is gone among the full genome Bacmid of wild-type baculovirus; Operation steps is following: frozen DH10Bac (the bac to bac baculovirus expression system of Invitrogen company) competent cell is taken out from refrigerator; In ice bath, add 1 μ L pFastBac1-EGFP DNA, both transfer in the conversion cup of precooling behind the mixing gently, immediately swivel base on electric conversion instrument; Operating voltage is 1800V; In transforming cup, add 0.8mL SOC substratum after electricity transforms successfully fast, the mixture of mixing transfer to EP manage in 37 ℃, 200rpm shaking table cultivation 4h; Get 5 μ L bacterium liquid and add the X-gal 40 μ L of 20mg/mL and the IPTG 4uL of 200mg/mL; Be applied on the solid culture flat board that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs, flat board is inverted in 37 ℃ of cultivations, carry out blue hickie screening after 16-24 hour.More than operation is aseptic technique.

With the white colony on the transfering loop picking flat board, on the flat board of 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs, carry out blue hickie screening once more.White colony on the picking flat board; Be inoculated in respectively in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs; 37 ℃ of shaking table overnight cultures; Pick bacterium liquid in each pipe, bacterium liquid is carried out the PCR evaluation with M13Forward and M13Reverse and M13Forward and RG primer or FN2 and M13Reverse primer.Identify correct clone's called after Bacmid-EGFP, be stored in than 3: 7 in-80 ℃ of refrigerators so that 50% (volume ratio) glycerine/bacteria liquid is long-pending.

The PCR system is:

(5’-GAATTCATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCG-3’)

(5’-CAGGAAACAGCTATGAC-3’)

The glucose of solution I: 50mmol/L, the Tris-Cl of 25mmol/L, the EDTA of 10mmol/L,

Whole pH 8.0

The NaOH of solution II: 0.2mol/L, 1%SDS, fresh

The potassium acetate 60mL of solution III: 5mol/L, glacial acetic acid 11.5mL, water 28.5mL

Extract the solution III of Bacmid: the potassium acetate of 3mol/L, whole pH5.5

Being accredited as correct clone is transferred to again in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs and cultivates; Extract homogeneous tube bacterium liquid (5mL) plasmid after 16-24 hour, transfection Spodoptera frugiperda (sf9) cell is won for virus.The step of extracting the Bacmid-EGFP plasmid is: bacterium liquid poured in the eppendorf pipe of 1.5mL, and 5, outwell supernatant behind the centrifugal 2min of 000rpm, continue to pour into bacterium liquid remaining in the test tube, repetitive operation precipitates until thalline for three times fully; Add the vibration of 300 μ L solution I and break up bacterial sediment; Add 300 μ L solution II, put upside down three to four times immediately gently, leave standstill to clarification to mixing; Add the solution III of 300 μ L extraction Bacmid, put upside down mixing immediately gently, place 5min on ice; 12, the centrifugal 15min of 000rpm carefully draws in the eppendorf pipe that supernatant forwards another 1.5mL to; Add 800 μ L Virahols, place behind the 25min 12, the centrifugal 15min of 000rpm for-20 ℃; Outwell supernatant, add the ethanol 1mL of 70% (volume ratio), 12, the centrifugal 5min of 000rpm; Thoroughly remove supernatant, 20-25 ℃ of drying adds 20 μ L ddH ₂The O dissolving.

The sf9 passage in the 35mm capsule, in Grace ' the s substratum that contains 10% (mass volume ratio) foetal calf serum in 28 ℃ of no CO ₂Be cultured to adherently under the condition, use serum-free Grace ' s substratum to wash twice before the transfection, treat transfection.Prepare transfection liquid: A solution (16 μ LBacmid-EGFP plasmids and 84 μ L serum-free Grace ' s substratum mix), B solution (8 μ L lipofectamine and 92 μ L serum-free Grace ' s substratum mix).B solution dropwise adds and produces lipid-DNA mixture in the A solution, and 20-25 ℃ leaves standstill 45min, whenever flicks even at a distance from 5min.Every mixture adds 800 μ L serum-free Grace ' s substratum, aspirates mixing gently, is added dropwise in the sf9 cell of treating transfection, places 23 ℃ of no CO ₂Cultivated in the incubator 6-7 hour.The sucking-off transfection mixture adds Grace ' the s substratum that 2mL contains 10% (mass volume ratio) foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, collect first-generation recombinant virus, this recombinant virus called after Bac-EGFP after 6 days.

(2) metal complexes [Ru (phen) 2 (dppz)] ²⁺Cultivate the sf host cell in Grace ' the s substratum of solution adding 10% (mass volume ratio) foetal calf serum; Under 25 ℃ of conditions, carry out virus infection, propagation in the host cell that the recombinant virus Bac-EGFP adding that step (1) is constructed is cultivated.Concrete operations are: the sf9 cell is used for amplicon virus when in Grace ' the s substratum that contains 10% (mass volume ratio) foetal calf serum, growing to 70% big ware (diameter 9cm) floorage.Elder generation's sucking-off part substratum makes remaining substratum just flood cell, adds 500mL first-generation recombinant virus, places 23 ℃ of no CO ₂Infect 1.5h in the incubator, the every rolling gently at a distance from half hour makes a movement.Metainfective cell adds Grace ' the s substratum that 6mL contains 10% (mass volume ratio) foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, gather in the crops progeny virus after 4 days.

(3) collect; The progeny virion of the double-colored mark of purifying; The progeny virus that obtains is through transmission electron microscope (TEM); (laser confocal scanning microscope LSCM) characterizes its structure, measures its single viral metal complexes content and shows two fluorescent signals of its single virus for inductivity coupled plasma mass spectrometry (ICP-MS) and laser scanning co-focusing microscope.

The labeled virus particle that present method obtains can be easy to observe virion and cell and carries out after film merges realizing that spike live virus particle infects in the host cell experiment, the green fluorescence envelope protein moves to the phenomenon that host cell is examined in the enrichment of Cytolysosome and the viral nucleic acid orientation of red fluorescence.This crucial virus infection process is displayed with the form of fluorescence for the first time, for deeply understanding virus infection mechanism more technical support is provided.

The present invention compared with prior art has the following advantages and effect:

Compare with monochromatic virus signature, the multicolor fluorescence labeled virus can be realized the spike of virus infection cell whole process.This method utilization virus self-assembly system labeled virus nucleic acid can be good at solving the external difficult problem that can't carry out the viral nucleic acid mark; And can not cause the change of virus structure and the forfeiture of infection ability; The viral nucleic acid of the good metal complexes of mark has been given play to many advantages in cell imaging: for example hang down the color development light of background, anti-long-time photobleaching etc.In addition, this labelling strategies only just can prepare two fluorescently-labeled virions through virus in intracellular increment in a large number, and step is simple, environmental protection.Infect in the host cell experiment at real-time spike double-tagging virion; Can very clearly observe two kinds of fluorescence at first in intracellular altogether location with take place that viromembrane merges separating of back red fluorescence and orientation moves to the nucleus process, can the virus infection cell processes intactly be shown through the spike means.The present invention has easy and simple to handle, and labeling effciency is high, does not influence advantages such as labeled virus infection ability, and the labeled virus that obtains has excellent fluorescent signal, can satisfy the needs that real-time spike live virus particle infects host cell research better.

Description of drawings

Fig. 1 is the synoptic diagram of two fluorescent mark virions in a kind of cell; (embodiment)

Fig. 2 is a kind of metal complexes [Ru (phen) ₂(dppz)] ²⁺Picked-up in host cell and distribution; (embodiment)

Wherein A figure is that metal complexes is by the picked-up naturally of host cell; B figure is [Ru (phen) ₂(dppz)] ²⁺Structure iron; C figure is [Ru (phen) in the substratum ₂(dppz)] ²⁺Change level.

Fig. 3 is that a kind of virus infection is to [Ru (phen) ₂(dppz)] ²⁺The influence of in host cell, absorbing.(embodiment) wherein A figure monitors generation virus infection host cell process in real time.B figure be cell in virus-free infecting (circle), the variation of metal complexes concentration in the substratum under virus infection (triangle) situation is arranged.C figure is the fluorescence contrast figure of viral extracting solution through enzymolysis, no enzymolysis.D figure adds the influence of metal complexes to virus titer.E figure is the influence of shelf time to labeled virus (dark-grey) and wild-type (light gray).

Fig. 4 is a kind of sign of purifying double-tagging progeny virus.(embodiment)

Wherein A-C figure is the imaging of double-tagging progeny virus under laser confocal microscope, and D figure is that transmission electron microscope characterizes virus particle structure, and E figure is the progeny virus band that sucrose gradient centrifugation is collected.

Fig. 5 is a kind of real-time spike of double-tagging virus infection host cell.(embodiment)

Fig. 6 is a kind of schematic flow sheet of green fluorescence labeled virus envelope protein.(embodiment)

Embodiment

Below in conjunction with concrete embodiment the inventive method is done further to describe, so that those skilled in the art further understands the present invention, but following examples do not limit the present invention in any form.

Embodiment 1:

A kind of based on viral self-assembly system, Two Colour Fluorescence mark live virus particulate method in the cell of metal complexes and genetic engineering technique, its step is following:

(1),, constructs the recombinant baculovirus that has green fluorescent protein on the GP64 with green fluorescent protein (EGFP) and baculovirus envelope protein GP64 amalgamation and expression by viral display systems.According to AcMNPV genome (GenBank:L22858.1) sequence among the GenBank with primer-design software Primerpremier 5 (Premier company); Design EGFP/gp64 genetic expression primer (Fig. 6) adds EcoR I and two restriction enzyme sites of HindIII respectively at 5 ' and 3 ' of gp64.At first; With the pEGFP-N1 plasmid (Clonetech company) of purifying as masterplate; FEG1 and REG-G are that primer carries out pcr amplification, as template, are that primer once more carry out pcr amplification with FN2 and REG-G with the PCR product EGP0 that obtains; Obtain one section fragment that comprises GP64 signal peptide and EGFP sequence, with this sequence called after EGP1.Be template (the bac to bac rhabdovirus system of Invitrogen company) with the Bacmid DNA that purifies then, FG-EG and RG are that primer carries out pcr amplification, obtain one section fragment that includes the gp64 peptide section sequence, with this sequence called after EGP2.5 ' the terminal sequence of 27 EGP2 that the 3 ' end of EGP1 adds is introduced by primer REG-G; 3 ' the terminal sequence of 24 EGP1 that the 5 ' end of EGP2 adds is introduced by primers F G-EG; EGP1 and EGP2 have just had 24 complementary bases like this, for ensuing Overlap PCR experiment provides condition.The OverlapPCR experiment is a template with EGP1 and EGP2, and FN2 and RG are primer, amplify at the gp64 signal peptide sequence to merge the fragment that the EGFP sequence is arranged at the back, with this sequence called after EGFP/gp64.The PCR reaction system is following:

(5’-GAATTCATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCG-3’)，

(5’-AAGCTTTTAATATTGTCTATTACGGTTTCTAATC-3’)

With the white colony on the transfering loop picking flat board, on the flat board of 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs, carry out blue hickie screening once more.White colony on the picking flat board; Be inoculated in respectively in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs; 37 ℃ of shaking table overnight cultures; Pick bacterium liquid in each pipe, bacterium liquid is carried out the PCR evaluation with M13Forward and M13Reverse primer and M13Forward and RG primer or FN2 and M13Reverse primer.Identify correct clone's called after Bacmid-EGFP, be stored in than 3: 7 in-80 ℃ of refrigerators so that 50% (glycerin/water volume ratio) glycerine/bacteria liquid is long-pending.

The PCR system is:

(5’-GAATTCATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCG-3’)

(5’-CAGGAAACAGCTATGAC-3’)

Whole pH 8.0

The NaOH of solution II: 0.2mol/L, 1%SDS, fresh

Extract the solution III of Bacmid: the potassium acetate of 3mol/L, whole pH5.5.

Be accredited as correct clone and be transferred to again in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs and cultivate, extract homogeneous tube bacterium liquid (5mL) plasmid after 16-24 hour, the transfection sf9 cell generation virus of winning.The step of extracting the Bacmid-EGFP plasmid is: bacterium liquid poured in the eppendorf pipe of 1.5mL, and 5, outwell supernatant behind the centrifugal 2min of 000rpm, continue to pour into bacterium liquid remaining in the test tube, repetitive operation precipitates until thalline for three times fully; Add the vibration of 300 μ L solution I and break up bacterial sediment; Add 300 μ L solution II, put upside down three to four times immediately repeatedly gently, leave standstill to clarification to mixing; Add the solution III of 300 μ L extraction Bacmid, put upside down mixing immediately gently, place 5min on ice; 12, the centrifugal 15min of 000rpm carefully draws in the eppendorf pipe that supernatant forwards another 1.5mL to; Add 800 μ L Virahols, place behind the 25min 12, the centrifugal 15min of 000rpm for-20 ℃; Outwell supernatant, add the ethanol 1mL of 70% (volume ratio), 12, the centrifugal 5min of 000rmp; Thoroughly remove supernatant, drying at room temperature adds 20 μ L ddH ₂The O dissolving.

The sf9 passage in the 35mm capsule, in Grace ' the s substratum that contains 10% (mass volume ratio) foetal calf serum in 28 ℃ of no CO ₂Be cultured to adherently under the condition, use serum-free Grace ' s substratum to wash twice before the transfection, treat transfection.Prepare transfection liquid: A solution: 16 μ L Bacmid-EGFP plasmids and 84 μ L serum-free Grace ' s substratum mix; B solution: 8 μ L lipofectamine and 92 μ L serum-free Grace ' s substratum mix.B solution dropwise adds and produces lipid-DNA mixture in the A solution, and 25 ℃ leave standstill 45min, whenever flicks even at a distance from 5min.Every mixture adds 800 μ L serum-free Grace ' s substratum, aspirates mixing gently, is added dropwise in the sf9 cell of treating transfection, places 23 ℃ of no CO ₂Cultivated in the incubator 6-7 hour.The sucking-off transfection mixture adds Grace ' the s substratum that 2mL contains 10% foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, collect first-generation recombinant virus, this recombinant virus called after Bac-EGFP after 6 days.

(2) metal complexes [Ru (phen) ₂(dppz)] ²⁺Cultivate the sf host cell in Grace ' the s substratum of solution adding 10% (mass volume ratio) foetal calf serum; Under 25 ℃ of conditions, carry out virus infection, propagation in the host cell that the recombinant virus Bac-EGFP adding that step (1) is constructed is cultivated.Concrete operations are: the sf9 cell is used for amplicon virus when in Grace ' the s substratum that contains 10% (mass volume ratio) foetal calf serum, growing to 70% big ware (diameter 9cm) floorage.Elder generation's sucking-off part substratum makes remaining substratum just flood cell, adds 500mL first-generation recombinant virus, places 23 ℃ of no CO ₂Infect 1.5h in the incubator, the every rolling gently at a distance from half hour makes a movement.Metainfective cell adds Grace ' the s substratum that 6mL contains 10% (mass volume ratio) foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, gather in the crops progeny virus after 4 days.

(4) progeny virion of the double-colored mark of purifying is used for the tracer study of infecting of host cell SF9.

Through the elements are contained and the virus titer statistics of inductivity coupled plasma mass spectrometry (ICP-MS), the progeny virion of every mark contains 2.1 * 10 ^-7[the Ru (phen) of nmol ₂(dppz)] ²⁺In the virus except with [the Ru (phen) of viral nucleic acid mark ₂(dppz)] ²⁺Also contain excessive [Ru (phen) outward ₂(dppz)] ²⁺Molecule, these excessive metal complexess can guarantee that virion keeps higher labeling effciency and stable fluorescence signal, for real-time spike branch virus infection host cell provides reliable guidance.

Claims

1. the method for a multi-color marking live body virion, its step is following:

(1),, constructs the recombinant virus that has green fluorescent protein on the GP64 with green fluorescent protein and virus envelope Protein G P64 amalgamation and expression by viral display systems;

According to AcMNPV genome sequence among the GenBank with primer-design software Primer premier 5; Design EGFP/gp64 genetic expression primer; Add EcoR I and two restriction enzyme sites of HindIII respectively at 5 ' and 3 ' of gp64; At first, as masterplate, FEG1 and REG-G are that primer carries out pcr amplification with the pEGFP-N1 plasmid of purifying; As template, is that primer once more carry out pcr amplification with FN2 and REG-G with the PCR product EGP0 that obtains, and obtains one section fragment that comprises GP64 signal peptide and EGFP sequence; Called after EGP1 is a template with the Bacmid DNA that purifies then, and FG-EG and RG are that primer carries out pcr amplification; Obtain one section fragment that includes the gp64 peptide section sequence, called after EGP2, the 5 ' terminal sequence of 27 EGP2 that the 3 ' end of EGP1 adds is introduced by primer REG-G; 3 ' the terminal sequence of 24 EGP1 that the 5 ' end of EGP2 adds is introduced by primers F G-EG, and EGP1 and EGP2 have just had 24 complementary bases like this, and the OverlapPCR experiment is a template with EGP1 and EGP2; FN2 and RG are primer; Amplify at the gp64 signal peptide sequence and merge the fragment that the EGFP sequence is arranged at the back, called after EGFP/gp64, the PCR reaction system is following:

Ligation product electricity is transformed into preparation plasmid EGFP/gp64-T Easy Vector in the bacillus coli DH 5 alpha competent cell; Restriction enzyme EcoR I and HindIII double digestion donor plasmid EGFP/gp64-T Easy Vector and expression vector pFastBac1; Enzyme is cut product and is demonstrated the fragment that varies in size through gel electrophoresis; Under uv lamp, cut the adhesive tape that is consistent with pFastBac1 clip size after EGFP/gp64 fragment and enzyme are cut; Reclaim dna fragmentation, carry out ligation at 4 ℃ after the purifying and recovering, the EGFP/gp64 fragment is inserted in the corresponding MCS of expression vector pFastBac1; Double digestion carries out 3h under 37 ℃ of conditions, reaction system is following:

The ligation body is 4 ℃ of reaction overnight, and two segmental add-ons are according to two segmental concentration and big or small to adjusting, and reaction system is following:

Connecting the product electricity is transformed in the bacillus coli DH 5 alpha competent cell for preparing; On the flat board that contains 100 μ g/mL penbritins and 100 μ g/mL kantlex, carry out blue hickie screening; Extract the DNA of white colony; Carry out EcoR I-HindIII double digestion and identify, called after pFastBac1-EGFP was stored in than 3: 7 in-80 ℃ of refrigerators so that 50% glycerine/bacteria liquid is long-pending;

The pFastBac1-EGFP swivel base is gone among the full genome Bacmid of wild-type baculovirus, and operation steps is following: frozen DH10Bac competent cell is taken out from refrigerator, in ice bath, add 1 μ LpFastBac1-EGFP DNA; Both transfer in the conversion cup of precooling behind the mixing gently; Swivel base on electric conversion instrument, operating voltage are 1800V, in transforming cup, add 0.8mL SOC substratum after electricity transforms successfully fast; The mixture of mixing is transferred in the EP pipe 37 ℃; The 200rpm shaking table is cultivated 4h, gets 5 μ L bacterium liquid and adds the X-gal 40 μ L of 20mg/mL and the IPTG 4uL of 200mg/mL, is applied on the solid culture flat board that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs; Flat board is inverted in 37 ℃ of cultivations, carries out blue hickie screening after 16-24 hour; With the white colony on the transfering loop picking flat board; On the flat board of 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs, carry out blue hickie screening once more; White colony on the picking flat board is inoculated in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs 37 ℃ of shaking table overnight cultures respectively; Pick bacterium liquid in each pipe; With M13Forward and M13Reverse primer and M13Forward and RG primer or FN2 and M13Reverse primer bacterium liquid is carried out PCR and identify that called after Bacmid-EGFP was stored in than 3: 7 in-80 ℃ of refrigerators so that 50% glycerine/bacteria liquid is long-pending;

The PCR system is:

Being accredited as correct clone is transferred to again in the LB liquid nutrient medium that contains 100 μ g/mL kantlex and 100 μ g/mL qingfengmeisu qiongs and cultivates; Extract homogeneous tube bacterium liquid plasmid after 16-24 hour, transfection Spodopterafrugiperda cell is won for virus, and the step of extracting the Bacmid-EGFP plasmid is: bacterium liquid is poured in the eppendorf pipe of 1.5mL; 5; Outwell supernatant behind the centrifugal 2min of 000rpm, continue to pour into bacterium liquid remaining in the test tube, repetitive operation precipitates until thalline for three times fully; Add the vibration of 300 μ L solution I and break up bacterial sediment; Add 300 μ L solution II, put upside down three to four times repeatedly, leave standstill to clarification to mixing; Add the solution III of 300 μ L extraction Bacmid, put upside down mixing, place 5min on ice; 12, the centrifugal 15min of 000rpm, absorption supernatant forward in the eppendorf pipe of another 1.5mL; Add 800 μ L Virahols, place behind the 25min 12, the centrifugal 15min of 000rpm for-20 ℃; Outwell supernatant, add the ethanol 1mL of 70% volume ratio, 12, the centrifugal 5min of 000rpm; Thoroughly remove supernatant, drying at room temperature adds 20 μ L ddH ₂The O dissolving;

The sf9 passage in the 35mm capsule, in containing Grace ' the s substratum of 10% mass volume ratio foetal calf serum in 28 ℃ of no CO ₂Be cultured to adherently under the condition, use serum-free Grace ' s substratum to wash twice before the transfection, preparation transfection liquid: A solution: 16 μ LBacmid-EGFP plasmids and 84 μ L serum-free Grace ' s substratum mix; B solution: 8 μ Llipofectamine and 92 μ L serum-free Grace ' s substratum mix; B solution dropwise adds and produces lipid-DNA mixture in the A solution, and 20-25 ℃ leaves standstill 45min, whenever flicks even at a distance from 5min; Every mixture adds 800 μ L serum-free Grace ' s substratum; The suction mixing is added dropwise in the sf9 cell of treating transfection, places 23 ℃ of no CO ₂Cultivated in the incubator 6-7 hour, the sucking-off transfection mixture adds Grace ' the s substratum that 2mL contains 10% mass volume ratio foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, collect first-generation recombinant virus, called after Bac-EGFP after 6 days;

(2) metal complexes [Ru (phen) ₂(dppz)] ²⁺Solution adds cultivates the sf host cell in Grace ' the s substratum of 10% mass volume ratio foetal calf serum; Under 25 ℃ of conditions, carry out virus infection in the host cell that the recombinant virus Bac-EGFP adding that step 1 is constructed is cultivated; Propagation; Be operating as: the sf9 cell is used for amplicon virus when in containing Grace ' the s substratum of 10% mass volume ratio foetal calf serum, growing to 70% big ware floorage, and first sucking-off part substratum makes remaining substratum just flood cell; Add 500mL first-generation recombinant virus, place 23 ℃ of no CO ₂Infect 1.5h in the incubator, every rolling makes a movement at a distance from half hour, and metainfective cell adds Grace ' the s substratum that 6mL contains 10% mass volume ratio foetal calf serum, places 23 ℃ of no CO ₂Cultivate in the incubator, gather in the crops progeny virus after 4 days;

(3) collect; The progeny virion of the double-colored mark of purifying; The progeny virus that obtains is through transmission electron microscope, and inductivity coupled plasma mass spectrometry and laser scanning co-focusing microscope characterize its structure, measure its single viral metal complexes content and show two fluorescent signals of its single virus.

2. the method for a kind of multi-color marking live body virion according to claim 1 is characterized in that: the glucose of described solution I: 50mmol/L, the Tris-Cl of 25mmol/L, the EDTA of 10mmol/L, whole pH 8.0; The NaOH of solution II: 0.2mol/L, 1%SDS, fresh; The potassium acetate 60mL of solution III: 5mol/L, glacial acetic acid 11.5mL, water 28.5mL; Extract the solution III of Bacmid: the potassium acetate of 3mol/L, whole pH5.5.