CN106047932A - Application of methyl-beta-cyclodextrins to increase of baculovirus foreign protein expression quantity - Google Patents

Application of methyl-beta-cyclodextrins to increase of baculovirus foreign protein expression quantity Download PDF

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CN106047932A
CN106047932A CN201610383426.9A CN201610383426A CN106047932A CN 106047932 A CN106047932 A CN 106047932A CN 201610383426 A CN201610383426 A CN 201610383426A CN 106047932 A CN106047932 A CN 106047932A
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cell
baculovirus
application
infection
virus
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CN106047932B (en
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黄金山
郝碧芳
南文斌
柳林
沈兴家
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Jiangsu University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention discloses application of methyl-beta-cyclodextrins (MbetaCD) to increase of the baculovirus foreign protein expression quantity. MbetaCD with a certain concentration is used for incubating cells for expressing foreign protein, insect baculovirus infection efficiency can be remarkably improved, and the foreign protein expression quantity can be remarkably increased. By means of the application, the expression efficiency of a baculovirus expression system can be remarkably improved; meanwhile, MbetaCD can be easily dissolved in water and organic solvent, has already been widely applied to medicine and food industries, and has high safety, and therefore the method can also be applied when baculovirus is used for expressing pharmaceutical protein. The method is easy to understand, easy to operate and remarkable in effect.

Description

Methyl-B-cyclodextrin application in improving baculovirus exogenous protein expression amount
Technical field
The invention belongs to field of virology and protein expression system field, relate to methyl-B-cyclodextrin (Methyl- β-cyclodextrin, is called for short M β CD, is also expressed as MBCD, Me-β CD, MeBCD) improve insect baculovirus invasion efficiency, And then improve the concrete application process of exogenous protein expression amount.
Background technology
Baculovirus is the double-stranded DNA virus that a class has cyst membrane, main infection Lepidoptera in nature, Hymenoptera and double Homopterous insect.Autographa california nuclear polyhedrosis virus (Autographa californica is utilized first from Smith et al. Nucleopolyhedrovirus, AcMNPV) make carrier successful expression humanβ-interferon since, the most used baculovirus expression Albumen have thousand kinds of (Acharya A, Sriram S&Saehrawat S.Bombyx mori nucleopolyhedrovirus molecular biology and biotechnological applications for large-scale synthesis Of recombinant proteins.Curr Sci 2002,83:455 465.), the baculovirus expression of application at present System mainly has AcMNPV and bombyx mori nuclear polyhydrosis virus (Bombyx mori nucleopolyhedrovirus, BmNPV), Owing to baculovirus has powerful polyhedron promoter and P10 promoter, the foreign protein of expression has preferably modifies processing, There is good biologic activity, and its genome may be inserted into larger piece section foreign DNA etc., so insect baculovirus table The system that reaches has been acknowledged as excellent eukaryotic expression system.In recent years, medicine and vaccine that recombinant baculovirus produces are utilized List, such as, utilized the Human-papilloma Vaccine (Cervarix that baculovirus expression system producesTM, GlaxoSmithKline PLC) List, and obtained extraordinary immune effect.
In order to preferably utilize baculovirus expression foreign protein, the most many scholars are devoted to how to improve it and express Research in terms of amount: France scholar by cathepsin gene, chitinase gene and p10 gene disruption in virus to increase table The amount of reaching (patent: the baculovirus expression system of improvement, publication number: 103748229A)；American scientist is by utilizing multiple letter Number peptide changes foreign protein processing and secretion (patent: Method to improve the efficiency of processing and secretion of foreign genes in insect systems.United States.Patent 5278050) to improve expression.A series of baculoviruss through transformation are required for passing through in the application The breeding of recombinant virus, expand link in a large number, just can apply to the expression of foreign protein, at recombinant virus preparation and amplification procedure In, need to consume substantial amounts of manpower financial capacity, if can virus infect during, apply certain means improve efficiency of infection, And then improve its expression, can use manpower and material resources sparingly, reduce cost, reach the effect got twice the result with half the effort.
M β CD, is the alkyl derivative of beta-schardinger dextrin-(β-CD), and white powder is nontoxic, odorless, micro-sweet, soluble in water And organic solvent, it is widely applied in food and medicine.Xie Baitai etc. review M β CD at oral medicine, nasal spray Application prospect in terms of agent, suppository and cutaneous permeable agent, and point out that M β CD is the smaller drug stabilizing agent of toxicity, solubilising Agent and absorption enhancer, but it is noted that M β CD using dosage, to ensure that it has preferable safety (Xie Baitai, Ma Xiaoming, Yao Sun Xian, Xie Weimei. the characteristic of methyl-β-cyclodextrin and the application in medicine thereof. Chinese Journal of New Drugs 2009 volume 18 8th phase 705-709.).This seminar early stage finds M β CD at higher concentrations when studying baculovirus phagocytic process, permissible Suppression BmNPV infects BmN cell, just during using a series of continuous Concentraton gradient M β CD research suppression invasion, It is found surprisingly that when using low concentration M β CD, not only does not suppress the infection of virus, the infectivity of enhanced virus on the contrary, finally Add the expression to foreign protein of baculovirus.M β CD is utilized to improve baculovirus infection rate and exogenous protein expression amount Aspect it is not yet reported that, so the present invention is method to this raising rhabdovirus expression vector exogenous protein expression amount first Do concrete report.
Summary of the invention
Solve the technical problem that: it is an object of the invention to provide the new application of a kind of chemicals M β CD, it is provided that should Improve the new method of baculovirus exogenous protein expression amount with certain density methyl-B-cyclodextrin, the method is easy, low cost Honest and clean, it is easy to operation is promoted.
Technical scheme: methyl-B-cyclodextrin answering in improving insect baculovirus expression system exogenous protein expression amount With.
Described insect baculovirus is AcMNPV virus or BmNPV virus.
Described expression system is sf21, sf9 or BmN cell.
Described expression system is the cell of adhere-wall culture and suspension culture.
The working concentration of described methyl-B-cyclodextrin is 0.125-2mM.
Methyl-B-cyclodextrin is in the application of the efficiency improving insect baculovirus invasion host cell.
Improving the compositions of insect baculovirus expression system exogenous protein expression amount, effective ingredient includes methyl-β-ring Dextrin.
The preparation of 1.M β CD (purchased from Sigma company) solution and baculovirus prepare
With 1 × PBS dissolve M β CD, compound concentration be 50～100mM storage liquid standby.
By recombinate shape virus infection sensitive host cell, after infection 60-96h results sprout virus (Budded virus, BV), measuring virus titer with Endpoint Dilution Method, 4 DEG C keep in Dark Place for follow-up study.
2. the hatching of cell
Being inoculated in culture dish/plate by the insect cell of exponential phase, different cell strains keep its optimal population, such as Sf21 and sf9 is maintained at about 5 × 104Individual cell/cm2, BmN cell about 1 × 104Individual cell/cm2, after adherent 12-24h, with joining The M β CD made stores liquid and joins in adherent Insect cellculture liquid, makes M β CD final concentration be respectively 0.125～2mM, hatches 10-120min, incubated cell temperature is 24-29 DEG C, using 0mM M β CD (i.e. equal-volume 1 × PBS) Incubating Solution as comparison.Hatch After time arrives, remove (or not removing) 1 × PBS and the M β CD Incubating Solution of variable concentrations respectively, train with the TC-100 of serum-free Foster base rinses cell 2 times gently.
3. virus efficiency of infection analysis and exogenous protein expression component analysis
Then with the baculovirus infection that infection multiplicity (multiplicity of infection, MOI) is 0.01～50 Corresponding host cell, after infecting 60-120min, removes virus liquid, (also may be used for twice with serum-free medium washed cell gently Do not wash), add normal culture medium, luciferase expression efficiency during 6h utilizes the infected cell of flow cytometry analysis after infection, really Fixed virus efficiency of infection.24-120h cell sample after infection is then collected in exogenous protein expression component analysis, expresses in detection cell Uciferase activity, compares relative luciferase activity difference between different disposal.
Beneficial effect: present invention firstly discovers that a kind of new application of M β CD.Hatch outside expressing with finite concentration M β CD The cell of source protein, can significantly improve insect baculovirus efficiency of infection and improve the expression of foreign protein.When with When 0.25mM M β CD hatches BmN cell, the cell quantity that BmNPV virus infects improves about 35%, and the work of luciferase Property improves 799% than comparison, and the reinforced effects of other concentration also reaches pole significant level；AcMNPV invasion efficiency improves 51%.Purposes disclosed by the invention, can obviously improve the expression efficiency of baculovirus expression system, and M β CD is soluble in itself simultaneously Water and organic solvent, be widely used in medicine and food service industry, had good safety, therefore application baculovirus table Reach pharmaceutical protein and be equally applicable this method.The method that the present invention provides is easy to understand, and operation is simple, and effect is obvious.
Accompanying drawing explanation
Fig. 1. variable concentrations M β CD pretreatment sf21 cell is to AcMNPV viral infection rate analysis chart.Respectively with final concentration of 0mM (isopyknic 1 × PBS is as comparison, CTRL), the M β CD of 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM process 30min After, then infect (MOI=5) 1h with AcBac-egfp, remove and normally cultivate after infecting liquid.Flow cytomery cell is used after 6h Relatively infection rate, wherein CTRL infection rate is set to 100%, other respectively process relative infection rate as it can be seen, * represent with to photograph Comparing difference notable (P < 0.05), * * represents that to be compared with a control difference extremely notable (P < 0.01).
Fig. 2. variable concentrations M β CD pretreatment sf9 cell is to AcMNPV viral infection rate analysis chart.Respectively with final concentration of 0mM (isopyknic 1 × PBS is as comparison, CTRL), the M of 0.125mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.5mM, 2mM After β CD processes sf9 cell 30min, infect (MOI=5) 1h with AcBac-egfp, remove and normally cultivate after infecting liquid, use after 6h Flow cytomery cell infection rate.* represents that to be compared with a control difference extremely notable (P < 0.01).
Fig. 3. the M β CD of variable concentrations affects schematic diagram (96h after infection) to AcMNPV virus exogenous protein expression amount.Point Not with final concentration of 0mM (isopyknic 1 × PBS is as comparison, CTRL), 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM M β After CD processes sf21 30min, then infect different disposal cell 1h respectively with AcBac-Luc (MOI=5), remove after infecting liquid Normal cultivation, 96h collects cell detection uciferase activity after infection.* represent be compared with a control difference extremely notable (P < 0.01)。
The M β CD of Fig. 4 .0.25mM processes cell, the phase analysis figure (MOI=5) of AcMNPV expressing luciferase.With After 0.25mM M β CD hatches sf21 cell 30min, infect M β CD and compared with control cells (MOI=5) respectively with AcBac-Luc, 27 DEG C After infecting 1h, removing virus liquid, each time point of 24h, 48h, 72h, 96h, 120h collects sample after infection, detects analysis of fluorescence Element enzyme relative activity.* representing and be compared with a control significant difference (P < 0.05), * * represents and is compared with a control the extremely notable (P of difference <0.01)。
Fig. 5. to BmNPV infection rate analysis chart after variable concentrations M β CD process BmN cell.M β CD final concentration is used to be respectively 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, comparison adds respective volume 1 × PBS, 27 DEG C hatch 30min after remove and hatch Liquid, infects 1h with BmBac-egfp (MOI=5) 27 DEG C after rinsing 2 times by the TC-100 culture medium of serum-free, removes virus liquid, Clean twice, after being positioned over 27 DEG C of incubators cultivation 6h, flow cytometer statistics infection cell number.* represents and is compared with a control Difference is extremely notable (P < 0.01).
Fig. 6 .M β CD (0.25mM) incubated cell time affects schematic diagram to BmNPV infection rate.Final concentration of 0.25mM M β CD 27 DEG C hatches 10 respectively, 30,45,60,90,120min, comparison adds respective volume 1 × PBS.Then remove Incubating Solution, use The TC-100 culture medium of serum-free rinses cell 2 times gently, then with BmBac-egfp infection cell (MOI=5), and 27 DEG C of infection Removing virus liquid after 1h, rinse 2 times by the TC-100 culture medium of serum-free, after being positioned over 27 DEG C of incubators cultivation 6h, streaming is thin Born of the same parents' instrument statistics fluorecyte quantity.* representing and be compared with a control significant difference (P < 0.05), * * represents and is compared with a control difference Extremely notable (P < 0.01).
Fig. 7. the uciferase activity impact signal that BmNPV virus (different infective dose) is expressed by variable concentrations M β CD Figure.Using 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM M β CD final concentration 27 DEG C to hatch BmN cell 30min, comparison adds phase Answering volume 1 × PBS, rear Incubating Solution of removing rinses cell 2 times, infects BmN by virus BmBac-Luc (MOI=1 and MOI=0.1) Cell, 27 DEG C are infected removal virus liquid after 1h, rinse 2 times by the TC-100 culture medium of serum-free, are positioned over 27 DEG C of incubators trainings Uciferase activity in cell is measured after supporting 24h.Dark grey block diagram is MOI=1 infective dose, and * * represents each process and compares Reach pole significant level (P < 0.01)；Light gray is MOI=0.1 infective dose,Expression process reaches significant level (P with compareing < 0.05),Represent each process and reach pole significant level (P < 0.01) with compareing.
Detailed description of the invention
As a example by AcMNPV:
Dissolving M β CD with 1 × PBS (purchased from Shanghai Sheng Gong biological engineering limited company), compound concentration is the storage of 50mM Liquid storage.
For the ease of observing and add up the expression efficiency of raising foreign protein of the present invention, respectively hsp70 is handled Under reporter gene egfp and AcMNPV polyhedrosis gene promoter control under luciferase gene swivel base to AcMNPV Bac- The Tn7 swivel base insertion point of to-Bac (Invitrogen), names AcBac-egfp and AcBac-Luc, extracts DNA, transfection Sf21, results are sprouted after virion (Budded Virus, BV), continue on for infecting sf21 cell, results virus after 72h, Endpoint Dilution Method measures virus titer, and 4 DEG C keep in Dark Place, the example related in the present invention.
Embodiment 1: after processing with the M β CD of final concentration of 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM respectively, AcBac- Egfp infection rate analyzes (MOI=5)
Each inoculation about 1 × 10 in 24 porocyte culture plates5The sf21 cell in/hole, adherent after, will prepare M β CD storage Liquid storage joins in the cell culture fluid of attached cell sf21, makes M β CD final concentration be respectively 0mM (equal-volume 1 × PBS, figure 1CTRL)、0.125mM、0.25mM、0.5mM、1mM、2mM.Hatch removal Incubating Solution after 30min, use serum-free TC-100 for 27 DEG C Culture medium rinses cell 2 times (also can not wash) gently, then infects the thin of different disposal by the AcBac-egfp virus of MOI=5 Born of the same parents, remove virus liquid after 27 DEG C of infection 1h, rinse 2 times (also can not wash) by the TC-100 culture medium of serum-free, add normal training Support base, after being positioned over 27 DEG C of incubators cultivation 6h, have the cell quantity of luciferase expression and total cell number by flow cytometer statistics, Utilize excel t test Analysis to process and whether there is significant difference between matched group.Experiment sets three repetitions, and repeats two Secondary.Fig. 1 is the result of MOI=5AcBac-egfp infection host insect cell sf21 example, statistical result showed 0.125-2mM The M β CD process cell of concentration relatively compares relative to infection rate all significantly increase.
Embodiment 2: respectively with the M β CD of final concentration of 0.125mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.5mM, 2mM After processing sf9, AcBac-egfp infection rate analyzes (MOI=5)
Each inoculation about 1 × 10 in 24 porocyte culture plates5The sf9 in individual/hole, adherent after, store with the M β CD for preparing Liquid joins in the cell culture fluid of attached cell sf9, makes M β CD final concentration be respectively 0mM (equal-volume 1 × PBS, figure 1CTRL), 0.125mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.5mM, 2mM, hatch for 27 DEG C and remove Incubating Solution after 30min, use The TC-100 culture medium of serum-free rinses cell 2 times gently, then infects variable concentrations M β respectively with MOI=5AcBac-egfp The cell that CD is hatched, after 27 DEG C are infected 1h, removes virus liquid, rinses 2 times by the TC-100 culture medium of serum-free, adds routine training Support base, after being positioned over 27 DEG C of incubators cultivation 6h, have the cell quantity of luciferase expression and total cell number by flow cytometer statistics, T test Analysis in excel is utilized to process and whether there is significant difference between matched group.Experiment sets three repetitions, and repeats two Secondary.Fig. 2 is the result of MOI=1AcBac-egfp infection host sf9 insect cell example, and statistical result shows to use 0.125-2mM After concentration processes cell, the cell quantity of express fluorescent protein relatively compares all to have and the most significantly improves.
Embodiment 3: the M β CD of variable concentrations affects (MOI=5,96h after infection) to AcBac-Luc luciferase expression amount
Each inoculation about 1 × 10 in 24 porocyte culture plates5The sf21 in individual/hole, adherent after, store with the M β CD for preparing Liquid joins in cell culture fluid, makes M β CD final concentration be respectively 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, and isopyknic 1 × PBS, as comparison (i.e. Fig. 3 CTRL), hatches removal Incubating Solution after 30min, by the TC-100 culture medium of serum-free gently for 27 DEG C Rinse cell 2 times, then infect different disposal cell respectively with AcBac-Luc (MOI=5), after 27 DEG C are infected 1h, remove virus Liquid, rinses 2 times by the TC-100 culture medium of serum-free, adds normal incubation medium, and 96h collects cell detection fluorescein after infection Enzymatic activity, each process is all provided with three repetitions.During sample collection, first remove culture medium, wash twice with 1 × PBS, then use 500 μ Lysate cracking in L luciferase kit (Promega), collection M β CD process and compared with control cells ,-80 DEG C of preservations, until All samples has been collected, with reference to method described in Promega product guide with 20/20 after 120hn Luminometer(Promega Company) detection uciferase activity.4 DEG C of centrifugal 5min of cell cleavage mixture 12000rpm, take supernatant and carry out 10 times of dilutions, Take 4 μ L diluents and the mixing of 20 μ L substrates again, measure relative luciferase activity.Fig. 3 is that AcBac-Luc infects variable concentrations medicine Thing processes the result of sf21, and statistical result showed is the most significantly high in the activity of the cell fluorescence element enzyme of 0.25～2mM drug treating In comparison.
After the M β CD of the final concentration of 0.25mM of embodiment 4. processes, different time points luciferase after AcBac-Luc infection Activity analysis (MOI=5)
Each inoculation about 1 × 10 in 24 porocyte culture plates5The sf21 in individual/hole, adherent after, store with the M β CD for preparing Liquid joins in cell culture fluid, makes the M final concentration of 0.25mM of β CD, comparison add the 1 × PBS (i.e. Fig. 4 CTRL) of respective amount, Hatch removal Incubating Solution after 30min, rinse cell gently 2 times by the TC-100 culture medium of serum-free, then use AcBac-for 27 DEG C Luc infects M β CD and compared with control cells (MOI=5) respectively, after 27 DEG C are infected 1h, removes virus liquid, trains with the TC-100 of serum-free Supporting base to rinse 2 times, add normal incubation medium, each time point of 24h, 48h, 72h, 96h, 120h collects sample after infection, each Process is all provided with three repetitions.The mensuration of uciferase activity is shown in the embodiment of the present invention 3.Fig. 4 is that AcMNPV infects sf21 example As a result, the activity of the cell fluorescence element enzyme of statistical result showed drug treating is significantly higher than comparison (P < 0.05).
As a example by BmNPV:
BmBacJS13 is Bacmid [the Huang JS et that a strain and BmNPV have identical infection characterization al.Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru.Virologica Sinica.2007,22 (3): 218-225].For the ease of observing and statistics Efficiency of infection of the present invention, under handling the reporter gene egfp under hsp70 manipulation and BmNPV polyhedrosis gene promoter Luciferase gene respectively swivel base to Tn7 swivel base insertion point in BmBacJS13, name BmBac-egfp and BmBac-Luc, Extracting DNA, transfect bombyx mori cell, after results BV, continue on for infected silkworm cell, results virus after 72h, Endpoint Dilution Method is surveyed Determining virus titer, 4 DEG C keep in Dark Place, following instance in the present invention.
Embodiment 5: after variable concentrations M β CD processes BmN cell, expressing green fluorescent protein cell infection rate ratio is investigated
Each inoculation about 5 × 10 in 24 porocyte culture plates4The BmN cell in individual/hole, adherent after, take different amounts of M respectively β CD stores liquid and joins in cell culture fluid, makes M β CD final concentration be respectively 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, right According to adding respective volume 1 × PBS, hatch removal Incubating Solution after 30min, rinse gently by the TC-100 culture medium of serum-free for 27 DEG C Cell 2 times, then infects the cell of different disposal respectively, after 27 DEG C are infected 1h, removes virus with BmBac-egfp (MOI=5) Liquid, rinses 2 times by the TC-100 culture medium of serum-free, adds conventional medium, after being positioned over 27 DEG C of incubators cultivation 6h, and streaming Cell instrument statistics infection cell number, t test Analysis compares variable concentrations M β CD to virus efficiency of infection impact.Fig. 5 is BmBac- Egfp infects the result of BmN cell example, statistical result showed 0.125,0.25,0.5mM M β CD process group have significantly increasing Potent fruit, efficiency of infection is respectively increased 29%, 35%, 33% than comparison, reaches pole significant level (P < 0.01), and other respectively processes Also there is certain reinforced effects.
Embodiment 6:M β CD (0.25mM) processes cell stage and viral infection rate relation
Each inoculation about 5 × 10 in 24 porocyte culture plates4The BmN cell in individual/hole, adherent after, add M β CD store liquid Make final concentration of 0.25mM, compare addition 1 × PBS, 27 DEG C hatch 10 respectively, 30,45,60,90,120min.Then remove and incubate Educate liquid, rinse cell gently 2 times by the TC-100 culture medium of serum-free, then with BmBac-egfp infection cell (MOI=5), Remove virus liquid after 27 DEG C of infection 1h, rinse 2 times by the TC-100 culture medium of serum-free, be positioned over 27 DEG C of incubators and cultivate 6h After, flow cytometer statistics fluorecyte quantity, the impact on virus efficiency of infection of the t test Analysis M β CD incubation time.Experiment If three repetitions.Fig. 6 is the result that BmBac-egfp infection M β CD hatches BmN different time example.Statistics display M β CD process More than cell 10min i.e. has preferable reinforced effects, process in 10-120 minute to be all remarkably higher than comparison.
Embodiment 7: the variable concentrations M β CD luciferase table activity to expressing in different infective dose virus infected cells Impact
Each inoculation about 5 × 10 in 24 porocyte culture plates4The BmN cell in individual/hole, adherent after, take the M prepared respectively β CD stores liquid and joins in cell culture fluid, makes M β CD final concentration 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM respectively, comparison Add respective volume 1 × PBS, hatch removal Incubating Solution after 30min, rinse gently carefully by the TC-100 culture medium of serum-free for 27 DEG C Born of the same parents 2 times, then with virus BmBac-Luc (MOI=1 and MOI=0.1) infection cell, 27 DEG C are infected removal virus liquid after 1h, use The TC-100 culture medium of serum-free rinses 2 times, is positioned over after 27 DEG C of incubators cultivate 24h and measures uciferase activity in cell, Detection method is shown in the embodiment of the present invention 3.Experiment sets three repetitions.Fig. 7 is the result that BmBac-Luc infects BmN cell example, system Meter result shows that variable concentrations M β CD processes cell and all can improve luciferase expression amount, the brightest with 0.25mM treatment effect Aobvious, in infecting with MOI=1 dosage, process and improve 799% than the activity of comparison, reach pole significant level, other processes also All reach pole significant level (P < 0.01)；532% is improve with the activity processed than compareing in infecting with MOI=0.1 dosage, Reaching pole significant level (P < 0.01), other various dose virus infects and all reaches notable (0.125mM, P < 0.05) with the most notable Effect (P < 0.01).
Finally should be noted that: M β CD concentration described in embodiment described above and embodiment and the time of process are only It is unrestricted for explanatory purposes, it should be appreciated by those of ordinary skill in the art that at cell state, training method is (outstanding Floating cultivation and adhere-wall culture), cell culture medium kind (TC100, Grace's, SF900II SFM, ExpressSFM Deng), the small difference of culture medium physicochemical property etc. (such as acid-base value etc.), and cell type (such as Hi5 etc.), can be in form In upper and details, it is made various change, the spirit of the present invention limited without departing from appended claims with Scope, and within being included in spirit and scope；Also comprise other baculovirus and depend in poisoning intrusion simultaneously Rely other togavirus in cholesterol, within being also included in spirit and scope.

Claims (7)

1. methyl-B-cyclodextrin application in improving insect baculovirus expression system exogenous protein expression amount.
Application the most according to claim 1, it is characterised in that described insect baculovirus is AcMNPV virus or BmNPV disease Poison.
Application the most according to claim 1, it is characterised in that described expression system is sf21, sf9, High five (Hi5) Or BmN cell.
Application the most according to claim 3, it is characterised in that described expression system is the thin of adhere-wall culture and suspension culture Born of the same parents.
Application the most according to claim 1, it is characterised in that the working concentration of described methyl-B-cyclodextrin is 0.125- 2mM。
6. methyl-B-cyclodextrin is in the application of the efficiency improving insect baculovirus invasion host cell.
7. the compositions improving insect baculovirus expression system exogenous protein expression amount, it is characterised in that effective ingredient bag Include methyl-B-cyclodextrin.
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CN112359064A (en) * 2020-11-10 2021-02-12 江苏科技大学 Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells

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CN112359064A (en) * 2020-11-10 2021-02-12 江苏科技大学 Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells
CN112359064B (en) * 2020-11-10 2022-01-04 江苏科技大学 Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells

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