CN106047932B - Methyl-B-cyclodextrin is improving the application in baculoviral exogenous protein expression amount - Google Patents
Methyl-B-cyclodextrin is improving the application in baculoviral exogenous protein expression amount Download PDFInfo
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Abstract
Application of the methyl-B-cyclodextrin in raising baculoviral exogenous protein expression amount, present invention a certain concentration M β CD are incubated for the cell for expressing foreign protein, can significantly improve insect baculovirus efficiency of infection and improve the expression quantity of foreign protein.The present invention can obviously improve the expression efficiency of baculovirus expression system, M β CD itself is soluble easily in water simultaneously and organic solvent, it has been widely used in drug and food service industry, there is good safety, therefore be equally applicable this method using baculovirus expression pharmaceutical protein.Method provided by the invention is readily appreciated that operation is simple, and effect is obvious.
Description
Technical field
The invention belongs to field of virology and protein expression system fields, are related to methyl-B-cyclodextrin (Methyl-
β-cyclodextrin, abbreviation M β CD are also expressed as MBCD, Me- β CD, MeBCD) insect baculovirus invasion efficiency is improved,
And then improve the concrete application method of exogenous protein expression amount.
Background technique
Baculoviral is a kind of double-stranded DNA virus with cyst membrane, main infection Lepidoptera in nature, Hymenoptera and double
Homopterous insect.Autographa california nuclear polyhedrosis virus (Autographa californica is utilized for the first time from Smith et al.
Nucleopolyhedrovirus, AcMNPV) make carrier successful expression humanβ-interferon since, used baculovirus expression so far
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 applied at present
System mainly has AcMNPV and bombyx mori nuclear polyhydrosis virus (Bombyx mori nucleopolyhedrovirus, BmNPV),
Since baculoviral has powerful polyhedron promoter and P10 promoter, the foreign protein of expression has preferable modification processing,
With good biological activity, and larger segment exogenous DNA etc. can be inserted in its genome, so insect baculovirus table
Excellent eukaryotic expression system has been acknowledged as up to system.In recent years, the drug and vaccine produced using recombinant baculovirus
It has been listed that, such as the Human-papilloma Vaccine (Cervarix produced using baculovirus expression systemTM, GlaxoSmithKline PLC)
It has been listed that, and obtain extraordinary immune effect.
In order to preferably utilize baculovirus expression foreign protein, many scholars at home and abroad are dedicated to how improving its expression
Measure the research of aspect: French scholar is by cathepsin gene, chitinase gene and p10 gene disruption in virus to increase table
Up to amount (patent: improved baculovirus expression system, publication number: 103748229A)；American scientist, which passes through, utilizes a variety of letters
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 quantity.A series of baculovirals by transformation require to pass through in the application
The breeding of recombinant virus, massive amplification link, just can apply to the expression of foreign protein, in recombinant virus preparation and amplification procedure
In, need to consume a large amount of manpower financial capacity, if can during virus infection, using certain means improve efficiency of infection,
And then its expression quantity is improved, it can use manpower and material resources sparingly, reduce cost, achieve the effect that get twice the result with half the effort.
M β CD is the alkyl derivative of beta-cyclodextrin (β-CD), and white powder is nontoxic, odorless, slightly sweet, soluble easily in water
And organic solvent, it is widely applied in food and drug.Xie Baitai etc. reviews M β CD in 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 sorbefacient, but it is noted that M β CD dosage, to guarantee it with preferable safety (Xie Baitai, Ma Xiaoming, Yao
The characteristic of Sun Xian, Xie Weimei methyl-β-cyclodextrin and its Chinese Journal of New Drugs 2009 volume 18 of application in drug
8th phase 705-709.).This seminar early period finds M β CD at higher concentrations when studying baculoviral phagocytic process, can be with
BmNPV is inhibited to infect BmN cell, exactly during inhibiting invasion with a series of continuous concentration gradient M β CD research,
It is found surprisingly that when using low concentration M β CD, not only the infection without inhibiting virus, instead the infectivity of enhanced virus, finally
Increase the expression quantity to foreign protein of baculoviral.Baculovirus infection rate and exogenous protein expression amount are improved using M β CD
Aspect is not yet reported that, so the present invention is for the first time to this method for improving rhabdovirus expression vector exogenous protein expression amount
Do specific report.
Summary of the invention
The technical issues of solution: the object of the present invention is to provide a kind of chemicals --- and the new application of M β CD is provided and is answered
The new method of baculoviral exogenous protein expression amount is improved with certain density methyl-B-cyclodextrin, this method is easy, at low cost
It is honest and clean, easily operated popularization.
Technical solution: methyl-B-cyclodextrin is improving answering in insect baculovirus expression system exogenous protein expression amount
With.
The insect baculovirus is AcMNPV virus or BmNPV virus.
The expression system is sf21, sf9 or BmN cell.
The expression system is the cell of adhere-wall culture and the culture that suspends.
The working concentration of the methyl-B-cyclodextrin is 0.125-2mM.
Application of the methyl-B-cyclodextrin in the efficiency for improving insect baculovirus invasion host cell.
The composition of insect baculovirus expression system exogenous protein expression amount is improved, effective component includes methyl-β-ring
Dextrin.
The preparation of 1.M β CD (being purchased from Sigma company) solution and baculoviral prepare
M β CD is dissolved with 1 × PBS, compound concentration is that the storing liquid of 50~100mM is spare.
By recombinate shape virus infection sensitive host cell, 60-96h harvest budding virus after infection (Budded virus,
BV), virus titer is measured with Endpoint Dilution Method, 4 DEG C are kept in dark place for follow-up study.
2. the incubation of cell
The insect cell of logarithmic growth phase is inoculated into culture dish/plate, different cell strains keep its optimal population, such as
Sf21 and sf9 is maintained at about 5 × 104A cell/cm2, BmN cell about 1 × 104A cell/cm2, after adherent 12-24h, with matching
The M β CD storing liquid made is added in adherent Insect cellculture liquid, and making M β CD final concentration is respectively 0.125~2mM, is incubated for
10-120min, incubated cell temperature are 24-29 DEG C, using 0mM M β CD (i.e. isometric 1 × PBS) Incubating Solution as control.It is incubated for
After time arrives, the M β CD Incubating Solution of (or not removing) 1 × PBS and various concentration are removed respectively, are trained with the TC-100 of serum-free
Feeding base gently rinses cell 2 times.
3. virus infection efficiency analysis and the analysis of exogenous protein expression amount
Then the baculovirus infection for being 0.01~50 with infection multiplicity (multiplicity of infection, MOI)
Corresponding host cell after infecting 60-120min, removes virus liquid, cell is gently washed with serum free medium twice (can also
Do not wash), normal culture medium is added, 6h is infected luciferase expression efficiency in cell using flow cytometry analysis after infection, really
Determine virus infection efficiency.24-120h cell sample after infection is then collected in the analysis of exogenous protein expression amount, is expressed in detection cell
Uciferase activity compares relative luciferase activity difference between different disposal.
The utility model has the advantages that present invention firstly discovers that a kind of new application of M β CD.It is incubated for a certain concentration M β CD outer for expressing
The cell of source protein can significantly improve insect baculovirus efficiency of infection and improve the expression quantity of foreign protein.When with
When 0.25mM M β CD is incubated for BmN cell, the cell quantity of BmNPV virus infection improves 35% or so, and the work of luciferase
Property than control improve 799%, the reinforcing effect of other concentration also reaches extremely significant level；AcMNPV invasion efficiency improves
51%.Purposes disclosed by the invention can obviously improve the expression efficiency of baculovirus expression system, while M β CD itself is soluble in
Water and organic solvent, have been widely used in drug and food service industry, have good safety, therefore apply baculoviral table
This method is equally applicable up to pharmaceutical protein.Method provided by the invention is readily appreciated that operation is simple, and effect is obvious.
Detailed description of the invention
Fig. 1 various concentration M β CD pre-processes sf21 cell to AcMNPV viral infection rate analysis chart.Respectively with final concentration of
The M β CD processing 30min of 0mM (1 isometric × PBS is as control, CTRL), 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM
Afterwards, then with AcBac-egfp (MOI=5) 1h is infected, is normally cultivated after removal infection liquid.Flow cytomery cell is used after 6h
Infection rate relatively, wherein CTRL infection rate is set as 100%, other each processing infection rates relatively are as shown, * is indicated and to photograph
Comparing difference is significant (P < 0.05), and it is extremely significant (P < 0.01) that * * expression is compared with a control difference.
Fig. 2 various concentration M β CD pre-processes sf9 cell to AcMNPV viral infection rate analysis chart.Respectively with final concentration of
The M of 0mM (1 isometric × PBS is as control, CTRL), 0.125mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.5mM, 2mM
After β CD handles sf9 cell 30min, (MOI=5) 1h is infected with AcBac-egfp, normally cultivates after removal infection liquid, is used after 6h
Flow cytomery cell infection rate.It is extremely significant (P < 0.01) that * expression is compared with a control difference.
The M β CD of Fig. 3 various concentration influences schematic diagram (96h after infection) to AcMNPV virus exogenous protein expression amount.Point
Final concentration of 0mM (1 isometric × PBS as compare, CTRL), 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM M β are not used
After CD handles sf21 30min, different disposal cell 1h is then infected respectively with AcBac-Luc (MOI=5), after liquid is infected in removal
Normal culture, 96h collects cell detection uciferase activity after infection.* expression be compared with a control difference it is extremely significant (P <
0.01)。
The M β CD of Fig. 4 .0.25mM handles cell, the phase analysis figure (MOI=5) of AcMNPV expressing luciferase.With
After 0.25mM M β CD is incubated for sf21 cell 30min, M β CD and control cell (MOI=5) are infected respectively with AcBac-Luc, 27 DEG C
After infecting 1h, remove virus liquid, after infection for 24 hours, 48h, 72h, 96h, 120h each time point collect sample, test and analyze fluorescence
Plain enzyme relative activity.* it indicates to be compared with a control significant difference (P < 0.05), * * expression is compared with a control the extremely significant (P of difference
<0.01)。
To BmNPV infection rate analysis chart after Fig. 5 various concentration M β CD processing BmN cell.It is respectively using M β CD final concentration
0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, control are added 1 × PBS of respective volume, remove and be incubated for after 27 DEG C of incubation 30min
Liquid uses BmBac-egfp (MOI=5) 27 DEG C of infection 1h after being rinsed 2 times with the TC-100 culture medium of serum-free, removes virus liquid,
Twice of cleaning, after being placed in 27 DEG C of incubator culture 6h, flow cytometer counts infection cell number.* expression is compared with a control
Difference is extremely significant (P < 0.01).
Fig. 6 .M β CD (0.25mM) incubated cell time influences schematic diagram to BmNPV infection rate.Final concentration of 0.25mM M β
27 DEG C of CD is incubated for 10,30,45,60,90,120min respectively, and 1 × PBS of respective volume is added in control.Then Incubating Solution is removed, is used
The TC-100 culture medium of serum-free gently rinses cell 2 times, then uses BmBac-egfp infection cell (MOI=5), 27 DEG C of infection
Remove virus liquid after 1h, is rinsed 2 times with the TC-100 culture medium of serum-free, after being placed in 27 DEG C of incubator culture 6h, streaming is thin
Born of the same parents' instrument counts fluorecyte quantity.* it indicates to be compared with a control significant difference (P < 0.05), * * expression is compared with a control difference
Extremely significant (P < 0.01).
Fig. 7 various concentration M β CD influences signal to the uciferase activity of BmNPV viral (different infective doses) expression
Figure.Using 27 DEG C of 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM M β CD final concentration incubation BmN cell 30min, phase is added in control
1 × PBS of volume is answered, the rear Incubating Solution that removes rinses cell 2 times, infects BmN with viral BmBac-Luc (MOI=1 and MOI=0.1)
Cell removes virus liquid after 27 DEG C of infection 1h, is rinsed 2 times with the TC-100 culture medium of serum-free, is placed in 27 DEG C of incubators trainings
It supports and measures uciferase activity in cell afterwards for 24 hours.Dark grey histogram is MOI=1 infective dose, and * * indicates each and handles and compare
Reach extremely significant horizontal (P < 0.01)；Light gray is MOI=0.1 infective dose,Indicate that processing reaches the level of signifiance (P with compareing
< 0.05),Indicate it is each processing with compare reach it is extremely significant level (P < 0.01).
Specific embodiment
By taking AcMNPV as an example:
With 1 × PBS (being purchased from Shanghai Sheng Gong bioengineering limited liability company) dissolution M β CD, compound concentration is the storage of 50mM
Liquid storage.
For the ease of observing and counting the expression efficiency of the present invention for improving foreign protein, hsp70 is manipulated respectively
Under the control of reporter gene egfp and AcMNPV polyhedrosis gene promoter 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 after harvest budding virion (Budded Virus, BV), continues on for infecting sf21 cell, harvests virus after 72h,
Endpoint Dilution Method measures virus titer, and 4 DEG C are kept in dark place, for example involved in the present invention.
Embodiment 1: after being handled respectively with the M β CD of final concentration of 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, AcBac-
Egfp infection rate analyzes (MOI=5)
About 1 × 10 is respectively inoculated in 24 porocyte culture plates5After adherent, prepared M β CD is stored up for the sf21 cell in/hole
Liquid storage is added in the cell culture fluid of attached cell sf21, and making M β CD final concentration is respectively 0mM (isometric 1 × PBS, figure
1CTRL),0.125mM,0.25mM,0.5mM,1mM,2mM.Incubating Solution is removed after 27 DEG C of incubation 30min, with serum-free TC-100
Culture medium gently rinses cell 2 times (can not also wash), then uses the thin of the AcBac-egfp virus infection different disposal of MOI=5
Born of the same parents, 27 DEG C of infection 1h move back venom of preventing or cure a disease, and rinse 2 times (can not also wash) with the TC-100 culture medium of serum-free, add normal training
Base is supported, after being placed in 27 DEG C of incubator culture 6h, there is the cell quantity and total cell number of luciferase expression with flow cytometer statistics,
Handled using excel t test Analysis whether there is significant difference between control group.Experiment sets three repetitions, and repeats two
It is secondary.Fig. 1 is that MOI=5AcBac-egfp infection host insect cell sf21 is exemplary as a result, statistical result showed 0.125-2mM
The M β CD processing cell of concentration has significant increase with respect to infection rate relatively control.
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 handling sf9, AcBac-egfp infection rate analyzes (MOI=5)
About 1 × 10 is respectively inoculated in 24 porocyte culture plates5The sf9 in a/hole after adherent, is stored with prepared M β CD
Liquid is added in the cell culture fluid of attached cell sf9, and making M β CD final concentration is respectively 0mM (isometric 1 × PBS, figure
1CTRL), 0.125mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.5mM, 2mM remove Incubating Solution after 27 DEG C of incubation 30min, use
The TC-100 culture medium of serum-free gently rinses cell 2 times, then infects various concentration M β respectively with MOI=5AcBac-egfp
The cell that CD is incubated for after 27 DEG C of infection 1h, removes virus liquid, is rinsed 2 times with the TC-100 culture medium of serum-free, and addition is conventional to train
Base is supported, after being placed in 27 DEG C of incubator culture 6h, there is the cell quantity and total cell number of luciferase expression with flow cytometer statistics,
Handled using t test Analysis in excel whether there is significant difference between control group.Experiment sets three repetitions, and repeats two
It is secondary.Fig. 2 is that MOI=1AcBac-egfp infection host insect cell sf9 is exemplary as a result, statistical result shows to use 0.125-2mM
After concentration handles cell, the cell quantity for expressing fluorescin has extremely significant raising compared with control.
Embodiment 3: the M β CD of various concentration influences (MOI=5,96h after infection) to AcBac-Luc luciferase expression amount
About 1 × 10 is respectively inoculated in 24 porocyte culture plates5The sf21 in a/hole after adherent, is stored with prepared M β CD
Liquid is added in cell culture fluid, and M β CD final concentration is made to be respectively 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, and isometric 1
× PBS removes Incubating Solution as control (i.e. Fig. 3 CTRL), after 27 DEG C of incubation 30min, gently with the TC-100 culture medium of serum-free
It rinses cell 2 times, then infects different disposal cell respectively with AcBac-Luc (MOI=5), after 27 DEG C of infection 1h, remove virus
Liquid is rinsed 2 times with the TC-100 culture medium of serum-free, normal incubation medium is added, and 96h collects cell detection fluorescein after infection
Enzymatic activity, each processing are all provided with three repetitions.When sample collection, removal culture medium first is washed twice with 1 × PBS, then with 500 μ
Lysate cracking, collection M β CD processing and control cell in L luciferase kit (Promega), -80 DEG C of preservations, until
All samples are collected after 120h, referring to Promega product guide the method with 20/20n Luminometer(Promega
Company) detection uciferase activity.4 DEG C of centrifugation 5min of cell cracking mixture 12000rpm take supernatant to carry out 10 times of dilutions,
It takes 4 μ L dilutions to mix with 20 μ L substrates again, measures relative luciferase activity.Fig. 3 is AcBac-Luc infection various concentration medicine
Object processing sf21's as a result, statistical result showed the cell fluorescence element enzyme of 0.25~2mM drug-treated the extremely significant height of activity
In control.
After the M β CD processing of the final concentration of 0.25mM of embodiment 4., different time points luciferase after AcBac-Luc infection
Activity analysis (MOI=5)
About 1 × 10 is respectively inoculated in 24 porocyte culture plates5The sf21 in a/hole after adherent, is stored with prepared M β CD
Liquid is added in cell culture fluid, makes the M β final concentration of 0.25mM of CD, and 1 × PBS (i.e. Fig. 4 CTRL) of corresponding amount is added in control,
Incubating Solution is removed after 27 DEG C of incubation 30min, is gently rinsed cell 2 times with the TC-100 culture medium of serum-free, then uses AcBac-
Luc infects M β CD and control cell (MOI=5) respectively, after 27 DEG C of infection 1h, removes virus liquid, is trained with the TC-100 of serum-free
Support base to rinse 2 times, normal incubation medium be added, after infection for 24 hours, 48h, 72h, 96h, 120h each time point collect sample, it is each
Processing is all provided with three repetitions.The embodiment of the present invention 3 is shown in the measurement of uciferase activity.Fig. 4 is that AcMNPV infection sf21 is exemplary
As a result, the activity of the cell fluorescence element enzyme of statistical result showed drug-treated is significantly higher than control (P < 0.05).
By taking BmNPV as an example:
BmBacJS13 is one plant of Bacmid [Huang JS et with BmNPV with 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 counting
Efficiency of infection of the present invention, will be under the reporter gene egfp and BmNPV polyhedrosis gene promoter manipulation under hsp70 manipulation
Luciferase gene distinguish swivel base Tn7 swivel base insertion point into BmBacJS13, name BmBac-egfp and BmBac-Luc,
DNA is extracted, bombyx mori cell is transfected, after harvesting BV, continues on for infected silkworm cell, virus is harvested after 72h, Endpoint Dilution Method is surveyed
Determine virus titer, 4 DEG C are kept in dark place, for following instance in the present invention.
Embodiment 5: after various concentration M β CD handles BmN cell, the investigation of expressing green fluorescent protein cell infection rate ratio
About 5 × 10 are respectively inoculated in 24 porocyte culture plates4The BmN cell in a/hole after adherent, takes different amounts of M respectively
β CD storing liquid is added in cell culture fluid, and M β CD final concentration is made to be respectively 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, right
According to 1 × PBS of respective volume is added, Incubating Solution is removed after 27 DEG C of incubation 30min, is gently rinsed with the TC-100 culture medium of serum-free
It cell 2 times, then infects the cell of different disposal respectively with BmBac-egfp (MOI=5), after 27 DEG C of infection 1h, removes virus
Liquid is rinsed 2 times with the TC-100 culture medium of serum-free, adds conventional medium, after being placed in 27 DEG C of incubator culture 6h, streaming
Cell instrument counts infection cell number, and t test Analysis, which compares various concentration M β CD, influences virus infection efficiency.Fig. 5 is BmBac-
Egfp infection BmN cell it is exemplary as a result, statistical result showed 0.125,0.25,0.5mM M β CD processing group have significant increasing
Potent fruit, efficiency of infection are respectively increased 29%, 35%, 33% than control, reach extremely significant horizontal (P < 0.01), other each processing
Also there is certain reinforcing effect.
Embodiment 6:M β CD (0.25mM) handles cell stage and viral infection rate relationship
About 5 × 10 are respectively inoculated in 24 porocyte culture plates4After adherent, M β CD storing liquid is added in the BmN cell in a/hole
Make final concentration of 0.25mM, 1 × PBS is added in control, and 27 DEG C are incubated for 10,30,45,60,90,120min respectively.Then removal is incubated
Liquid is educated, is gently rinsed cell 2 times with the TC-100 culture medium of serum-free, then uses BmBac-egfp infection cell (MOI=5),
Remove virus liquid after 27 DEG C of infection 1h, is rinsed 2 times with the TC-100 culture medium of serum-free, be placed in 27 DEG C of incubator culture 6h
Afterwards, flow cytometer counts fluorecyte quantity, influence of the t test Analysis M β CD incubation time to virus infection efficiency.Experiment
If three repetitions.Fig. 6 is that BmBac-egfp infection M β CD is incubated for the exemplary result of BmN different time.Statistics display M β CD processing
Cell 10min or more has preferable reinforcing effect, and processing in 10-120 minutes is all remarkably higher than control.
Embodiment 7: various concentration M β CD is to the luciferase table activity expressed in different infective dose virus infected cells
It influences
About 5 × 10 are respectively inoculated in 24 porocyte culture plates4The BmN cell in a/hole after adherent, takes prepared M respectively
β CD storing liquid is added in cell culture fluid, and M β CD final concentration is made to distinguish 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, control
1 × PBS of respective volume is added, removes Incubating Solution after 27 DEG C of incubation 30min, is gently rinsed carefully with the TC-100 culture medium of serum-free
Born of the same parents 2 times, virus BmBac-Luc (MOI=1 and MOI=0.1) infection cell is then used, removes virus liquid after 27 DEG C of infection 1h, is used
The TC-100 culture medium of serum-free rinses 2 times, is placed in 27 DEG C of incubator cultures and measures uciferase activity in cell afterwards for 24 hours,
Detection method is shown in the embodiment of the present invention 3.Experiment sets three repetitions.Fig. 7 is that BmBac-Luc infection BmN cell is exemplary as a result, system
Luciferase expression amount can be improved the result shows that various concentration M β CD handles cell in meter, the brightest with 0.25mM treatment effect
Aobvious, in the infection of MOI=1 dosage, the activity handled than control improves 799%, reaches extremely significant level, other processing
All reach extremely significant horizontal (P < 0.01)；532% is being improved than the activity compareed with processing in the infection of MOI=0.1 dosage,
Reach extremely significant horizontal (P < 0.01), other various dose virus infections all reach significant (0.125mM, P < 0.05) with it is extremely significant
Effect (P < 0.01).
Finally it should be noted that: M β CD concentration described in embodiment described above and embodiment and processing the time only
It for explanatory purposes rather than limits, it should be appreciated by those of ordinary skill in the art that training method is (outstanding in cell state
Floating culture and adhere-wall culture), cell culture medium type (TC100, Grace's, SF900II SFM, ExpressSFM
Deng), (such as pH value etc.) small difference and the cell type (such as Hi5) such as culture medium physicochemical property, can be in form
Various changes are made to it in upper and details, without departing from spirit of the invention defined by the appended claims with
Range, and be included within spirit and scope；Simultaneously also comprising other baculovirals and in poisoning intrusion according to
Rely other togavirus in cholesterol, is also included within spirit and scope.
Claims (4)
1. the methyl-B-cyclodextrin that concentration is 0.125-2mM is improving outside insect baculovirus AcMNPV or BmNPV expression system
Application in source protein expression quantity.
2. application according to claim 1, it is characterised in that the expression system is sf21, sf9, High five (Hi5)
Or BmN cell.
3. application according to claim 2, it is characterised in that the expression system is adhere-wall culture and the thin of culture that suspend
Born of the same parents.
4. the methyl-B-cyclodextrin that concentration is 0.125-2mM is thin in raising insect baculovirus AcMNPV or BmNPV invasion host
Application in the efficiency of born of the same parents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610383426.9A CN106047932B (en) | 2016-06-01 | 2016-06-01 | Methyl-B-cyclodextrin is improving the application in baculoviral exogenous protein expression amount |
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