CN103695468A - Method for expressing EGF-TCSKDEL fusion protein by bombyx mori baculovirus Bac-to-Bac system - Google Patents
Method for expressing EGF-TCSKDEL fusion protein by bombyx mori baculovirus Bac-to-Bac system Download PDFInfo
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Abstract
The invention discloses a method for expressing EGF-TCSKDEL fusion protein by a bombyx mori baculovirus Bac-to-Bac system, which comprises the following steps: subcloning EGF-TCSKDEL gene into a multiple cloning site of donor plasmid pFastBacHTb of a Bac-toBac system, transforming into competent bacterial DH10BmBac cells with bombyx mori baculovirus Bacmid genome, performing blue-white selection to obtain a positive clone, extracting plasmid DNA from the positive clone, transfecting bombyx mori cells BmN with the extracted plasmid DNA to obtain a recombinant baculovirus with the EGF-TCSKDEL gene, and infecting a lot of BmN cells with the collected recombinant baculovirus to obtain greatly-expressed EGF-TCSKDEL fusion protein. The method of the invention has a high expression quantity and biological activity, and lays the foundation of creation of new targeting antitumor drugs with high activity, high tumor inhibition rate, low immunogenicity, and low molecular weight.
Description
Technical field
The present invention relates to a kind of method of expression EGF-TCSKDEL fusion rotein, relate in particular to a kind of method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein.
Background technology
At present, the protein drug that is applied to clinical cancer therapy mainly contains Interferon, rabbit, interleukin etc., although successfully treated clinically the tumour of some particular source, but clinical efficacy is general on the whole, also produced serious toxic side effect and dose-dependently etc., and only had part patient to treating sensitivity simultaneously.Therefore be necessary that research has the protein drug of better clinical efficacy and less side effect.
Human epidermal growth factor (human epidemal growth factor, EGF) be a kind of important somatomedin that works and stimulate cellular proliferation and break up by acceptor, a lot of malignant tumours overexpression EGF acceptor as equal in kidney, cancer of the stomach, prostate cancer, liver cancer, nonsmall-cell lung cancer, bladder cancer etc.EGF can compete in conjunction with cancer cells surface receptor, carries toxin and kills and wounds specifically cancer cells.Trichosanthin (TrichosantIlin, TCS) is a kind of plant strand toxin naturally existing, and has the effect that suppresses tumor growth.KDEL is one section high hydrophobic sequence, the end that is applied to TCS can improve the activity of TCS, EGF-TCSKDEL the fusion rotein that utilizes the targeting of EGF and the cytotoxicity of TCSKDEL albumen to build, can targeting killing tumor cell, is conducive to the treatment of clinical tumor.Experiment in vitro and transplanting solid tumor Integral animal experiment are studied and are shown, this fusion rotein has obvious targeting anti-tumor effect.
Silkworm baculovirus Bac-to-Bac system by donor plasmid, contain the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac and form, its principle that produces recombinant virus is: first external source goal gene is cloned in donor plasmid, then donor plasmid is transformed in DH10BmBac competent cell, by the fixed point transposition effect of bacterial transposon, promotor is proceeded to silkworm baculovirus genome together with goal gene and produce recombinant virus dna, then obtain by transfection bombyx mori cell the recombinant baculovirus that contains goal gene.We utilize the quick generation of this system can express the recombinant virus of EGF-TCSKDEL fusion rotein, and utilize Bombyx noriN cell to give expression to the activated EGF-TCSKDEL of tool fusion rotein, for utilizing silkworm biological reactor to produce this fusion rotein, lay the first stone.
Summary of the invention
Goal of the invention: in order to solve the existing problem of prior art, the invention provides and a kind ofly have compared with high expression level amount and the bioactive method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein.
Technical scheme: for achieving the above object, a kind of method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein of the present invention, comprises the following steps:
(1) EGF-TCSKDEL gene is cloned into the multiple clone site of donor plasmid pFastBacHTb of Bac-to-Bac baculovirus expression system from plasmid pET28a-EGF-TCSKDEL Central Asia, makes goal gene be positioned at the downstream of baculovirus polyhedrin body promotor;
(2) this plasmid is transformed into and contains in the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac cell, incubated overnight on the culture plate that contains the similar thing X-Gal of microbiotic and galactoside;
(3) by the screening of blue hickie, obtain positive colony, extracting plasmid DNA from positive colony subsequently, this plasmid DNA is the genome of recombinant virus;
(4), with this DNA transfection bombyx mori cell BmN, isolation medium supernatant liquor after 72h, obtains the recombinant baculovirus that contains EGF-TCSKDEL gene;
(5) utilize Ni-NTA post separation and purification restructuring EGF-TCSKDEL fusion rotein from the bombyx mori cell of infection recombinant baculovirus: with the recombinant baculovirus of collecting, infect in a large number BmN cell, observing cell has while infecting sign, collecting cell, by cell centrifuging and taking supernatant after ultrasonication, utilize Ni-NTA column separating purification to obtain corresponding target protein matter supernatant liquor, and by SDS-PAGE, analyze the purity of protein of purifying.
More preferred, in described step (1), multiple clone site is BamHI and HindIII.
Further, the middle microbiotic of described step (2) is one or more in kantlex and gentamicin.
Further, the concentration of described kantlex is 50 μ g/mL.
Further, the concentration of described gentamicin is 30 μ g/mL.
Further, the concentration of the similar thing X-Gal of described galactoside is 40 μ g/mL.
Beneficial effect: a kind of method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein provided by the present invention, by EGF-TCSKDEL gene subclone to the multiple clone site of the donor plasmid pFastBacHTb of Bac-to-Bac system, be transformed into and contain in the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac cell, by blue hickie screening, obtain positive colony, extracting plasmid DNA from positive colony subsequently, plasmid DNA transfection bombyx mori cell BmN with extracting, the recombinant baculovirus that acquisition contains EGF-TCSKDEL gene, with the recombinant baculovirus of collecting, infect in a large number BmN cell, obtain great expression EGF-TCSKDEL fusion rotein, the present invention has higher expression amount and biological activity, for creating the targeting antineoplastic medicine thing of new high reactivity, high tumour inhibiting rate, reduced immunogenicity, small molecules amount, lays a good foundation.
Accompanying drawing explanation
Fig. 1 is the donor plasmid pFastBacHTb figure of Bac-to-Bac baculovirus expression system of the present invention;
Fig. 2 is that the present invention SDS-PAGE of EGF-TCSKDEL fusion rotein after purified that recombinate analyzes;
Fig. 3 is that mtt assay of the present invention detects the 95-D Growth of Cells situation of processing through EGF-TCSKDEL.
Embodiment
Embodiment 1:
Material source: recombinant plasmid pET28a-EGF-TCSKDEL is that our company preserves; DNA marker, restriction enzyme, T4 ligase enzyme etc. are all purchased from Takara; Baculovirus transfer vector pFastBacHTb, lipofectin and Ni-NTA resin are Invitrogen company product; Foetal calf serum FCS, Bombyx noriN cell substratum TC-100 are the product of GibcoBRL.
A method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein, comprises the following steps:
(1) EGF-TCSKDEL gene is cloned into from plasmid pET28a-EGF-TCSKDEL Central Asia between the multiple clone site BamHI and HindIIII of donor plasmid pFastBacHTb of Bac-to-Bac baculovirus expression system, makes goal gene be positioned at the downstream of baculovirus polyhedrin body promotor;
(2) donor plasmid of restructuring is transformed into and contains in the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac cell, coat containing incubated overnight on the culture plate of kantlex 50 μ g/mL, gentamicin 30 μ g/mL and the similar thing X-Gal40 of galactoside μ g/mL;
(3) by the screening of blue hickie, obtain positive colony, extracting plasmid DNA from positive colony subsequently, this plasmid DNA is the genome of recombinant virus;
(4), with this DNA transfection bombyx mori cell BmN, isolation medium supernatant liquor after 72h, obtains the recombinant baculovirus that contains EGF-TCSKDEL gene;
(5) utilize Ni-NTA post separation and purification restructuring EGF-TCSKDEL fusion rotein from the bombyx mori cell of infection recombinant baculovirus: with the recombinant baculovirus of collecting, infect in a large number BmN cell, observing cell has while infecting sign, collecting cell, by cell centrifuging and taking supernatant after ultrasonication, utilize Ni-NTA column separating purification to obtain corresponding target protein matter supernatant liquor, and by SDS-PAGE, analyze the purity of protein of purifying.Fig. 2 analyzes for the SDS-PAGE of restructuring EGF-TCSKDEL fusion rotein after purified, and result shows, after purifying, recombinant protein shows through SDS-PAGE, and purity surpasses 95%.
Utilize mtt assay to detect the method for anti-tumor activity of restructuring EGF-TCSKDEL: by the high-transfer human lung adenocarcinoma cell 95-D(2 * 105cell/ml of logarithmic phase) be inoculated in 96 well culture plates, EGF-TCSKDEL the sample that adds respectively different concns, blank group adds isopyknic nutrient solution, put 37 ℃, in 5%CO2 incubator, cultivate 24h, draw containing EGF-TCSKDEL serum, every hole adds MTT working fluid 100 μ L, making EGF-TCSKDEL final concentration is 0.5g/L, under cell culture condition, is incubated 72h; Take out culture plate, supernatant 100 μ L are drawn in every hole, add 100 μ L DMSO, in microplate reader 570nm place, survey absorbance A value, calculate cell survival rate: cell survival rate=[experimental group A average-background group A average]/[blank group A average-background group A average] * 100%, and map with different concns and the cell survival rate of EGF-TCSKDEL, determine the IC50 of EGF-TCSKDEL to tumour cell; Experiment repeats 3 times, and Fig. 3 is mtt assay and detects the 95-D Growth of Cells situation of processing through EGF-TCSKDEL, has shown that restructuring EGF-TCSKDEL has obvious anti-tumor activity effect compared with control group.
Should be understood that, above embodiment is only not used in and limits the scope of the invention for the present invention is described, after having read the present invention, those skilled in the art all fall within the application's claims limited range to the modification of the various equivalent form of values of the present invention.
Claims (6)
1. by a method for silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein, it is characterized in that comprising the following steps:
(1) EGF-TCSKDEL gene is cloned into the multiple clone site of donor plasmid pFastBacHTb of Bac-to-Bac baculovirus expression system from plasmid pET28a-EGF-TCSKDEL Central Asia, makes goal gene be positioned at the downstream of baculovirus polyhedrin body promotor;
(2) this plasmid is transformed into and contains in the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac cell, incubated overnight on the culture plate that contains the similar thing X-Gal of microbiotic and galactoside;
(3) by the screening of blue hickie, obtain positive colony, extracting plasmid DNA from positive colony subsequently, this plasmid DNA is the genome of recombinant virus;
(4), with this DNA transfection bombyx mori cell BmN, isolation medium supernatant liquor after 72h, obtains the recombinant baculovirus that contains EGF-TCSKDEL gene;
(5) utilize Ni-NTA post separation and purification restructuring EGF-TCSKDEL fusion rotein from the bombyx mori cell of infection recombinant baculovirus: with the recombinant baculovirus of collecting, infect in a large number BmN cell, observing cell has while infecting sign, collecting cell, by cell centrifuging and taking supernatant after ultrasonication, utilize Ni-NTA column separating purification to obtain corresponding target protein matter supernatant liquor, and by SDS-PAGE, analyze the purity of protein of purifying.
2. the method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein according to claim 1, is characterized in that: in described step (1), multiple clone site is BamHI and HindIII.
3. the method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein according to claim 1, is characterized in that: in described step (2), microbiotic is one or more in kantlex and gentamicin.
4. the method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein according to claim 2, is characterized in that: the concentration of described kantlex is 50 μ g/mL.
5. the method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein according to claim 2, is characterized in that: the concentration of described gentamicin is 30 μ g/mL.
6. the method with silkworm baculovirus Bac-to-Bac system expression EGF-TCSKDEL fusion rotein according to claim 1, is characterized in that: the concentration of the similar thing X-Gal of described galactoside is 40 μ g/mL.
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| CN106636208A (en) * | 2016-11-10 | 2017-05-10 | 西南大学 | Method of utilizing bombyx mori nuclear polyhedrosis virus vector system to excessively express microRNA |
| CN110713532A (en) * | 2019-11-06 | 2020-01-21 | 吉林省蚕业科学研究院 | Truncated protein AP-EGF of tussah epidermal growth factor and preparation method and application thereof |
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| CN110713532B (en) * | 2019-11-06 | 2021-04-13 | 吉林省蚕业科学研究院 | Truncated protein AP-EGF of tussah epidermal growth factor and preparation method and application thereof |
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Application publication date: 20140402 |