CN112359064B - Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells - Google Patents
Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells Download PDFInfo
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Abstract
The application of methyl-beta-cyclodextrin (M beta CD) in improving transduction efficiency of baculovirus to mammalian cells comprises the steps of incubating normally cultured mammalian cells with a certain concentration of M beta CD, removing the incubation liquid after incubating for 30min, and then transducing the mammalian cells with BmNPV, so that the transduction efficiency of BmNPV to the mammalian cells can be obviously improved. Part of baculovirus can carry foreign gene into mammalian cell for gene transduction and gene therapy vector. However, the transduction efficiency of baculovirus into mammalian cells is generally low, which greatly limits its application as a vector for gene therapy. The method provided by the invention can effectively improve the transduction efficiency of the baculovirus to the mammalian cells, and is simple to operate and easy to understand.
Description
Technical Field
The invention belongs to the field of systems for transducing mammalian cells to express foreign proteins, and particularly relates to application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus on the mammalian cells.
Background
Baculoviruses are a class of double-stranded DNA viruses with a cyst membrane, the natural hosts in nature being lepidopteran, hymenopteran and dipteran insects. Since Smith et al successfully expressed human interferon-beta using Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector for the first time, there have been thousands of proteins that could be expressed using baculovirus (Acharya A, Sriram S & Saehragat S. Bombyx mori nuclear polyhedrosis virus molecular biology and biotechnology applications for large-scale synthesis [ J ]. Curr Sci 2002,83: 455. 465.). The currently applied insect baculovirus expression systems mainly include AcMNPV and Bombyx mori nuclear polyhedrosis virus (BmNPV), and the expressed foreign protein can be modified and processed well due to the fact that the baculovirus has strong polyhedrin and P10 promoters, and has good biological activity, and the genome of the baculovirus can be inserted into a large-fragment foreign DNA, so the insect baculovirus expression system is known as an excellent eukaryotic expression system.
The baculovirus host domain is very narrow, for example, BmNPV can only infect silkworm or silkworm cells with high efficiency, but part of baculovirus can transduce mammal cells but cannot replicate in mammals, so that the baculovirus host domain is harmless to human and livestock. Because part of baculovirus can transduce mammalian cells, it can be used as an excellent mammalian cell gene transfer vector, and has played a great role in gene therapy, vaccine development, drug screening, gene regulation and research, and the like. For example, recombinant BmNPV has been shown to be a DNA vaccine vector against Neocanicillium caninum (Kato, T., Itagaki, K., Yoshimoto, M., et. al. transfer of a Neospora caninum antigen cells using a modified Bomby x mole nuclear hydrophylicity for antibody production [ J ]. Biosci Bioeng,2017.124: 606-. Mammalian cells are not natural hosts for baculovirus, and there are limitations to using them for delivery and expression of foreign genes, the biggest challenge being the low efficiency of transduction of mammalian cells by baculovirus. In recent years, many researchers at home and abroad have devoted themselves to research on how to improve the transduction efficiency of mammalian cells by baculovirus, such as the fusion of cytoplasmic transduction peptide with baculovirus envelope protein GP64 to produce plasma membrane-permeable baculovirus, or the fusion of protein transduction domain of HIV TAT protein with baculovirus envelope protein VP39 to form nuclear membrane-permeable baculovirus, all of which can improve the transduction efficiency of mammalian cells (Chen, H.Z., Wu, C.P., Chao, Y.C.et al.Membrane transduction peptides in mammalian cells [ J ]. Biochem Biophys Res mu, 2011.405: 297-. In addition to the modification of the virus, Histone Deacetylase (HDAC) inhibitors such as butyrate, trichophyton A (Tri-chlorostatin A), valproic acid and the like are added to remarkably increase the gene expression mediated by the baculovirus. However, HDAC inhibitors at high concentrations are cytotoxic, and their effect on transduction efficiency varies from promoter to promoter and cell line (Spenger, A., Ernst, W., Condreay, JP, el. Influence of promoter choice and trichostatin A2 expression on expression of bacterial delayed genes in mammalian cells [ J ]. Protein Expr Punf,2004.38: 17-23). Transduction efficiency for mammalian cells can also be significantly improved by allowing baculovirus to stay on the cell membrane using low temperature (4 ℃) and then inducing the virus to fuse with the cell membrane surface using extrinsic low pH induction (Dong, S., Wang, M., Qiu, Z. autogra california multicatalytic complex infection Sf9cells and transmission mammalian cells direct fusion with the plasma membrane at low pH [ J ]. J Virol,2010.84: 5351-9.). There are also reports of increasing transduction efficiency by displaying foreign proteins on the surface of the virus, but this requires additional genetic manipulation of the virus. If the method with simple operation can be applied to improve the transduction efficiency and further improve the expression quantity of the exogenous gene, a great deal of manpower and material resources can be saved, and the effect of achieving twice the result with half the effort can be achieved.
M beta CD is an alkylated derivative of beta-cyclodextrin (beta-CD) and is widely applied to food and medicines. The research of the subject group finds that before the BmNPV is used for transferring the mammalian cells, the normally cultured cells are firstly incubated with the M beta CD with a certain concentration, after the incubation, the incubation liquid is removed, and then the BmNPV is used for infecting the cells, so that the transfer efficiency of the BmNPV on the mammalian cells can be obviously improved, and the expression quantity of the BmNPV on the foreign proteins is finally increased. The method for improving the transduction efficiency of the BmNPV to the mammalian cells by utilizing the M beta CD has not been reported in research, so the method for improving the transduction efficiency of the baculovirus to the mammalian cells is specifically explained for the first time.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides an application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells, in particular to a novel method for improving the transduction efficiency of baculovirus to mammalian cells by using methyl-beta-cyclodextrin with a certain concentration so as to improve the expression quantity of exogenous genes.
The technical scheme is as follows: application of methyl-beta-cyclodextrin in preparing products for improving transduction efficiency of baculovirus on mammalian cells.
The concentration of the methyl-beta-cyclodextrin is 0.25mM-2 mM.
The concentration of the methyl-beta-cyclodextrin is 0.5 mM.
A product for improving transduction efficiency of baculovirus to mammalian cells contains methyl-beta-cyclodextrin as effective component.
A product for improving transduction efficiency of baculovirus to mammalian cells contains 0.5mM methyl-beta-cyclodextrin as effective component.
The specific contents are as follows:
preparation of MbetaCD (purchased from Sigma) solution and Virus preparation
M.beta.CD was dissolved in 1 XPBS (available from Biotechnology, Shanghai, Ltd.) to prepare a 50mM stock solution.
BmBacJS13 is a strain of Bacmid that has the same infection characteristics as BmNPV [ Huang JS et al. construction of the Bac-to-Bac System of Bombyx mori nucleolysis virus, virology sinica.2007, 22 (3): 218-225]. In order to facilitate observation and statistics of the transduction efficiency, the CMV promoter and the reporter gene egfp are simultaneously transposed to a Tn7 transposition insertion site of BmBacJS13, and the recombined bacmid is named BmBac-CMV-egfp. Extracting DNA of the recombinant bacmid, transfecting the bombyx mori BmN cells, harvesting a Budded Virus (BV) after 72h, continuously infecting the bombyx mori cells for Virus amplification, harvesting the Virus after 72h, determining the Virus titer by using an end point dilution method, and storing the Virus at 4 ℃ in a dark place, wherein the method is used in all methods disclosed by the invention.
2. Incubation of cells
A HeLa cell line (hereinafter abbreviated as HeLa cell) for human cervical cancer in logarithmic growth phase was inoculated into a 24-well plate to maintain the optimum density thereof at about 2X 105After 12h of each cell/well attaching, the prepared M beta CD storage solution is added into the culture solution of the adherent mammal cells to ensure that the final concentration of the M beta CD is 0.25mM, 0.5mM, 1mM and 2mM respectively, the cells are incubated for 30min, the temperature of the incubated cells is 37 ℃, and 1 XPBS with the same volume is used as a control. After the incubation time, 1 × PBS and various concentrations of M β CD incubation were removed (or not removed), respectively, and the cells were gently washed 2 times with serum-free DMEM medium.
3. Analysis of viral transduction efficiency and analysis of foreign protein expression level
Mammalian cells were then incubated with baculovirus with a multiplicity of infection (MOI) of 30 for 60min, virus fluid was removed, cells were gently washed twice with serum-free medium (and optionally with serum), and fresh medium was added. After 24h, the cells were harvested and the number of cells expressing egfp was measured by flow cytometry and compared between treatments.
Has the advantages that: the invention provides a new application of the M beta CD for the first time. The mammalian cells for expressing the exogenous protein are incubated by the M beta CD with a certain concentration, so that the transduction efficiency of the insect baculovirus to the cells can be obviously improved, and the expression quantity of the exogenous gene can be improved. When Hela cells were incubated with 0.5mM M.beta.CD, the transduction efficiency of BmNPV on the cells was increased by 8.71%, and the enhancement effect of the treatment at other concentrations was also significant. The application disclosed by the invention can obviously improve the efficiency of transduction of mammalian cells by baculovirus, and meanwhile, the M beta CD is easy to dissolve in water and organic solvents, is widely applied to the pharmaceutical and food industries and has good safety, so that the method can be applied to the expression of medicinal proteins by transduction of animal cells by baculovirus or gene therapy vectors. The method provided by the invention is easy to operate, low in cost and obvious in effect.
Drawings
FIG. 1 is a schematic diagram of a recombinant virus BmBac-CMV-egfp;
the following primers were designed and the promoter CMV was obtained from pcDNA6.0 using PCR:
the following primers were designed from pEGFP-N1The egfp gene was obtained by PCR:
cutting the CMV fragment by using EcoRI and SalI; double enzyme digestion of egfp gene with Sal I and Sal I; the pFastBacDual was digested simultaneously with EcoRI and XbaI. And connecting the enzyme-cut CMV and egfp with the enzyme-cut pFastBacDual vector through conventional molecular biology to construct a recombinant pFastBacDual-CMV-egfp plasmid. The recombinant virus BmBac-CMV-egfp (FIG. 1) was obtained by transposition according to the Bac-to-Bac system operation instructions.
FIG. 2 shows that the MOI-30 BmBac-CMV-egfp transduced Hela cells incubated with different concentrations of M beta CD, and the fluorescence expression efficiency in the transduced cells was analyzed 24h later by flow cytometry. When Hela cells were incubated with 0.5mM M.beta.CD, the transduction efficiency of BmNPV on the cells was increased by 8.71%, and the enhancement effect of the treatment at other concentrations was also significant.
Detailed Description
Taking BmNPV as an example:
M.beta.CD was dissolved in 1 XPBS (available from Shanghai Biotechnology Ltd.) to prepare a 50mM stock solution.
In order to facilitate observation and statistics of the invention for improving the transduction efficiency of mammalian cells, a CMV promoter and a reporter gene egfp are simultaneously transposed to a Tn7 transposition insertion site of BmBacJS13, and the recombined bacmid is named BmBac-CMV-egfp (a schematic diagram is shown in figure 1). Extracting DNA of the recombinant bacmid, transfecting the bombyx mori BmN cells for 72h, harvesting a Budded Virus (BV), continuously infecting the bombyx mori cells for Virus amplification, harvesting the Virus after 72h, determining the Virus titer by using an end point dilution method, and storing the Virus at 4 ℃ in a dark place, wherein the method is used in all methods disclosed by the invention.
Example 1:
each of the 24-well cell culture plates was seeded at about 2X 105Hela attached to the wall for 24h in each well, prepared M beta CD stock solutions of 2.5. mu.L, 5. mu.L, 10. mu.L and 20. mu.L were added to the cell culture medium (total volume of cell culture medium is 500. mu.L/well) so that the final M beta CD concentrations were 0.25, 0.5, 1 and 2mM, respectively, 20. mu.L of 1 XPBS was added to healthy Hela cells as a control, and incubation was carried out at 37 ℃ for 30 min. After the incubation time is up, removing 1 XPBS and different concentrations of M beta CD incubation liquid, gently washing cells for 2 times by serum-free DMEM medium, then transducing the cells incubated by 0.25, 0.5, 1 and 2mM M beta CD and PBS by BmBac-CMV-egfp virus with MOI (equal to 30) carrying green fluorescent protein gene, incubating for 1h at 37 ℃, washing for 2 times by serum-free DMEM medium, placing in an incubator at 37 ℃ for 24h, removing the cell medium, suspending the cells by PBS, counting the number of cells with fluorescent expression by a flow cytometer, and determining the efficiency of transduction of mammalian cells by BmNPV after the cells incubated by different concentrations of M beta CD.
FIG. 2 shows the results of BmNPV transducing mammalian cells Hela, and statistics show that the efficiency of BmNPV transducing cells was 8.71% higher than that of the control group when 0.5mM M.beta.CD was treated, and the transduction efficiencies after 0.25, 1, and 2mM M.beta.CD treatments were 3.72%, 5.28%, and 3.59%, respectively, than that of the control group.
Finally, it should be noted that: the above examples and embodiments described M β CD concentration and treatment time are for illustrative purposes only and not intended to be limiting, but it will be understood by those of ordinary skill in the art that minor differences in cell status or physicochemical properties of the cell culture medium, etc., may be varied in form and detail without departing from the spirit and scope of the present invention as defined by the appended claims.
Sequence listing
<110> university of Jiangsu science and technology
Application of <120> methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells
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Claims (3)
1. Application of methyl-beta-cyclodextrin in preparing products for improving transduction efficiency of baculovirus BmNPV on mammalian Hela cells.
2. Use according to claim 1, characterized in that the concentration of methyl- β -cyclodextrin is between 0.25mM and 2 mM.
3. Use according to claim 2, characterized in that the concentration of methyl- β -cyclodextrin is 0.5 mM.
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| CN106047822A (en) * | 2016-06-01 | 2016-10-26 | 江苏科技大学 | Application of methyl-beta-cyclodextrins to expansion of silkworm bombyx mori nuclear polyhedrosis virus host range |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2337794A1 (en) * | 1998-07-02 | 2000-01-13 | Jennifer D. Tousignant | Transgene expression in polarized cells |
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| CN102199629A (en) * | 2011-03-29 | 2011-09-28 | 中国科学院武汉病毒研究所 | Method for improving transduction efficiency of rhabdovirus on mammalian cells |
| CN102994550A (en) * | 2012-10-24 | 2013-03-27 | 中国农业科学院生物技术研究所 | Method for expressing exogenous gene in animal cell or animal tissue |
| CN104027347A (en) * | 2014-05-26 | 2014-09-10 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells |
| CN106047822A (en) * | 2016-06-01 | 2016-10-26 | 江苏科技大学 | Application of methyl-beta-cyclodextrins to expansion of silkworm bombyx mori nuclear polyhedrosis virus host range |
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