CN111569064B - Efficient targeted antitumor drug nano-carrier system and application thereof - Google Patents
Efficient targeted antitumor drug nano-carrier system and application thereof Download PDFInfo
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The invention provides a high-efficiency targeted antitumor drug nano-carrier system, which is mainly prepared by coupling magnetosomes and recombinant SPA through SPDP; the efficient targeted antitumor drug nano-carrier system provided by the invention has stronger targeting property, and particularly has stronger performance of targeting breast cancer cells (SK-BR-3).
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a high-efficiency targeted antitumor drug nano-carrier system and application thereof.
Background
In recent years, the nano magnetic beads are gradually highlighted in the field of medical inspection and treatment, the nano materials have the characteristics of large specific surface area, low toxicity, targeting function and the like, and have been a research hotspot in tumor diagnosis and treatment, but because the biological environment is relatively complex, after the nano materials enter a human body, one or more layers of protein (protein corona) can be adsorbed on the surface of the nano materials, which is called protein corona, and the phenomenon can be completed when the nano particles enter blood for 30s, and research shows that the nano materials completely lose the tumor targeting capability after being connected with transferrin and entering the biological environment. The macrophage surface contains various receptors, which can recognize the nano material and phagocytose the nano material as foreign matter and activate NF-kB system, thereby generating a series of reactions. The targeting efficiency is reduced or lost, resulting in a large loss of therapeutic or diagnostic effect.
It has been reported that when the nanomaterials are incubated with plasma and plasma rich in immunoglobulin (IgG), phagocytosis of the nanomaterials by macrophages after the plasma rich in IgG is found to be far greater than that of the control group. In addition, studies have reported that nonspecific adsorption increases phagocytosis efficiency of chemically coupled nanomaterials by macrophages rather than chemical coupling, and that the exposure of Fab region decreases upon chemical coupling, the presence of primary amines in the side chains of lysine amino acid residues, and the N-terminus of each polypeptide chain. The lysine distribution of mouse IgG1 was plotted to show that lysine residues were distributed on the Fc and Fab regions (lysine distribution Fab:58% vs. Fc: 42%). This is because chemical conjugation mostly uses an amino group in the antibody, which not only reduces the potency of the antibody, but more importantly exposes the FC end of the antibody to more external recognition by macrophages.
Disclosure of Invention
In order to solve the technical problems, the FC end of the antibody is specifically combined by using the recombinant SPA, the antibody is positively coupled on the magnetosome, and the high-efficiency targeted antitumor drug nano-carrier system is mainly prepared by coupling the magnetosome and the recombinant SPA through SPDP.
Furthermore, the efficient targeted antitumor drug nano-carrier system is prepared by coupling magnetosomes and recombinant SPAs through SPDP to prepare BMP-SPDP-RA, and then coupling the BMP-SPDP-RA and antibodies.
Further, the nano-carrier system is prepared by the following method:
s1, dissolving SPDP in dimethyl sulfoxide to prepare a first solution, wherein the concentration of the first solution is 5-15mM;
s2, adding magnetosomes into PBS (phosphate buffer solution) and uniformly mixing to prepare a solution II, wherein the mass ratio of the magnetosome to the SPDP is (0.5-1.5);
s3, adding the solution II into the solution I, ultrasonically mixing uniformly and coupling to prepare a first mixed solution;
s4, magnetically adsorbing the first mixed solution, removing unbound SPDP after adsorption, and then washing with PBS to obtain BMP-SPDP;
s5, carrying out mixing, ultrasonic mixing and coupling on BMP-SPDP and the recombinant SPA in a mass ratio of 0.5-1.5;
s6, magnetically adsorbing the second mixed solution, washing away the unbound recombinant SPA, and washing for three times by using PBS to prepare the BMP-SPDP-RA.
Wherein, SPDP is bifunctional reagent 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester, BMP is magnetosome, and RA represents recombinant SPA coupled with BMP-SPDP.
Further, the ultrasound conditions of step S3 and step S5 are ultrasound for 1min, pause for 1min, and repeat for 30 times.
Further, the preparation method of the nanocarrier system further comprises a step S7 of coupling the BMP-SPDP-RA to an antibody, and the step S7 of coupling the BMP-SPDP-RA to an antibody specifically comprises the following steps:
s71, putting BMP-SPDP-RA and the antibody with the mass ratio of 0.5-1.5 in a shaker at 37 ℃ to mix for 1.5-2.5h to prepare a third mixed solution;
s72, magnetically adsorbing the third mixed solution, and then washing away unbound antibodies by PBS to prepare BMP-SPDP-RA-TZ, thus obtaining the nano-carrier system.
Wherein, TZ represents the antibody trastuzumab coupled with BMP-SPDP-RA.
Further, magnetosomes (wild-type) are purified prior to use, said purification comprising the following steps:
(1) Taking bacterial sludge and PBS to dissolve according to the mass volume ratio of 1;
(2) Ultrasonically treating the solution obtained in the step (1) by using an ultrasonic cell crusher, adsorbing magnetosomes in the ultrasonically treated mixed solution by using a magnet, discarding supernatant, adding PBS again for ultrasonic treatment and magnet adsorption, and repeating the steps until the albumin concentration is not reduced; obtaining the BMP-WT.
Wherein BMP-WT represents a purified magnetosome.
Further, the magnetosome is placed in distilled water for ultrasonic cleaning, the magnet is adsorbed, the adsorbed magnetosome is suspended by distilled water, and the magnetosome is frozen by liquid nitrogen and then freeze-dried by a freeze dryer for later use at-20 ℃.
Further, the concentration of PBS was 10mM, and the pH of PBS was 7.4.
Further, the step (2) comprises the following specific steps:
(21) Placing the mixed solution obtained in the step (1) in a container, and carrying out ultrasonic treatment by using an ultrasonic cell crusher under the ultrasonic condition of a No. 6 horn, 60% power, 3s of ultrasonic treatment and 5s of intermittent treatment for 30min;
(22) Placing a magnet at the bottom of the container, and adsorbing magnetosomes overnight at 4 ℃;
(23) Discarding the supernatant, adding PBS, performing ultrasound with an ultrasonic cell crusher under 50% power for 3s and 5s at intervals for 20min, and performing magnetic adsorption for 6h;
discarding the supernatant, adding PBS, and performing ultrasound with an ultrasonic cell crusher under the ultrasound conditions of 40% power, 3s ultrasound, 5s pause, and 20min totally, repeating the above steps until the albumin concentration is not reduced, and reducing the power and the ultrasound time with the increase of the ultrasound times.
Further, the purified magnetosome is further treated with proteinase K-tris, specifically comprising the following steps:
(3) Suspending magnetosome with protease K-tris solution with concentration of 0.5-1.5mg/ml, and treating in water bath (56 deg.C constant temperature water bath) for 2-4 hr;
(4) Taking out, and collecting magnetosomes by magnetic adsorption.
Further, the sequence of the recombinant SPA protein is as follows:
CGSGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPKG SGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPK。
further, the SPA recombinant protein is carried out on the basis of modifying the structure of the SPA gene from Staphylococcus aureus, and the synthetic method of the recombinant protein comprises the following steps:
(a) Adding a cysteine to the N end of B domain of the natural SPA gene to introduce a sulfhydryl;
(b) Adding enzyme cutting sites of BamH I and HindIII at two ends of the SPA gene introduced with sulfydryl;
(c) Cloning the SPA gene in the step (b) into a PET28 (a +) vector, and converting the SPA gene into E.coli BL 21;
(d) Carrying out induction expression by using IPTG;
(e) Separating and purifying with affinity chromatography column to obtain pure protein, and obtaining recombinant protein.
The invention also provides application of the nano drug delivery system in preparation of a product of a targeted antitumor drug.
The invention also provides application of the nano drug delivery system in preparation of SK-BR-3 targeted products.
The invention provides a nano material coupled antibody and can effectively avoid the phenomenon of low targeting efficiency caused by the massive phagocytosis of macrophagocyte in blood; meanwhile, when the nano material is exposed in a biological environment, such as plasma, the nano material still has good targeting performance.
Drawings
FIG. 1 shows an affinity chromatogram of the recombinant protein SPA;
FIG. 2 shows an electron micrograph of magnetosomes after proteinase K-tris treatment;
FIG. 3 shows an electrophoretogram of magnetosomes before and after proteinase K-tris treatment;
FIG. 4 shows BMP-SPDP-RA-TZ coupled trastuzumab amounts;
FIG. 5 shows a light microscope photograph of the induction of human monocytes to macrophages;
FIG. 6 shows macrophages with BMP-SPDP-RA-IgG; BMP-WT; prussian blue staining pattern after BMP-GA-IgG co-incubation;
FIG. 7 shows macrophages with BMP-SPDP-RA-IgG; BMP-GA-IgG; BMP-WT mean fluorescence intensity plot;
FIG. 8 shows macrophages with BMP-SPDP-RA-TZ; BMP-SPDP-RA-TZ plasma; BMP-SPDP-RA-TZ IgG plasma; BMP-GA-TZ; BMP-GA-TZ plasma; prussian blue staining pattern after co-incubation of BMP-GA-TZ IgG plasma;
FIG. 9 shows immunofluorescence images of HER2 expression on the surface of breast cancer SK-BR-3/MDA-MB-468 cells;
FIG. 10 shows macrophages with BMP-SPDP-RA-TZ; BMP-SPDP-RA-TZ plasma; BMP-SPDP-RA-TZ IgG plasma; BMP-GA-TZ; BMP-GA-TZ plasma; a graph of the mean fluorescence intensity of cells after co-incubation with BMP-GA-TZ IgG plasma;
wherein, A in FIG. 5 is a normal THP-1 cell (10X); b is normal THP-1 cells (20X); c is 100ng/mL PMA induction for 72h (10X); d was 100ng/mLPMA induced for 72h (20X).
Detailed Description
Example 1
The embodiment provides a high-efficiency targeted antitumor drug nano-carrier system, which is prepared by coupling a magnetosome and a recombinant SPA through SPDP to prepare BMP-SPDP-RA, and coupling the BMP-SPDP-RA and an antibody, and is specifically prepared by the following method:
s1, dissolving 1mg of SPDP in an EP tube containing 100 mu L of dimethyl sulfoxide and having a volume of 1.5ml to prepare a solution I;
s2, taking 1mg of magnetosome, adding 900 mu L of PBS with the concentration of 10mM and the pH value of 7.4, and uniformly mixing to prepare a solution II;
s3, adding the solution II into an EP tube containing the solution I, ultrasonically mixing uniformly and coupling, ultrasonically treating for 1min, intermitting for 1min, and repeating for 30 times to prepare a first mixed solution;
s4, placing the EP tube with the first mixed solution in the step S3 on a magnetic frame, carrying out magnetic adsorption for 1min, removing supernatant after adsorption, and washing for three times by PBS with pH of 7.4 to obtain BMP-SPDP;
s5, mixing 1mg of BMP-SPDP and 1mg of recombinant SPA, performing ultrasonic mixing and coupling for 1min, performing intermittent 1min, and repeating for 30 times to prepare a second mixed solution;
s6, placing the EP tube with the second mixed solution in the step S5 on a magnetic frame for magnetic adsorption for 1min, washing away unbound recombinant SPA, and washing for three times by PBS with pH of 7.4 to obtain BMP-SPDP-RA;
wherein, the sequence of the recombinant SPA protein is as follows:
CGSGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPKG SGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPK；
the recombinant protein is carried out on the basis of modifying the structure of a SPA gene from Staphylococcus aureus, and the synthetic method of the recombinant protein comprises the following steps:
(a) Adding cysteine to the N end of Bdomain of a natural SPA gene to introduce sulfydryl;
(b) Adding enzyme cutting sites of BamH I and HindIII at two ends of the SPA gene introduced with sulfydryl;
(c) Cloning the SPA gene in the step (b) into a PET28 (a +) vector, and converting the SPA gene into E.coli BL 21;
(d) Adding IPTG with the final concentration of 1mM for induction expression;
(e) Separating and purifying with affinity chromatography column to obtain pure protein, and obtaining recombinant protein, wherein the affinity chromatography chart of the recombinant protein SPA is shown in figure 1;
magnetosomes are purified prior to use, said purification comprising the steps of:
(1) Taking 10g of bacterial sludge, adding 100g of PBS with the concentration of 10mM and the pH value of 7.4, and stirring with a glass rod to fully dissolve the bacterial sludge;
(2) Performing ultrasonic treatment on the solution obtained in the step (1) by using an ultrasonic cell crusher, adsorbing magnetosomes in the ultrasonic mixed solution by using a magnet, discarding the supernatant, adding PBS again for ultrasonic treatment and magnet adsorption, and repeating the steps until the albumin concentration is not reduced any more, thus obtaining BMP-WT; the step (2) specifically comprises the following steps:
(21) Putting the solution in the step (1) into a beaker, performing ultrasonic treatment by using an ultrasonic cell crusher, selecting a No. 6 amplitude transformer, performing ultrasonic treatment at 60% power for 3 seconds and 5 seconds at intervals for 30 minutes, (22) putting a magnet at the bottom of the beaker, adsorbing magnetosomes overnight at 4 ℃ (23) discarding supernatant, adding PBS with the concentration of 10mM and the pH of 7.4 again for ultrasonic treatment at 50% power for 3 seconds and 5 seconds at intervals for 20 minutes at intervals, performing magnetic adsorption for 6 hours, and (24) discarding supernatant, adding PBS with the concentration of 10mM and the pH of 7.4 again for ultrasonic treatment at 40% power for 3 seconds and 5 seconds at intervals for 20 minutes at intervals, repeating the steps until the concentration of albumin is not reduced any more, and reducing power and ultrasonic time along with the increase of ultrasonic times to obtain BMP-WT;
detecting OD in the supernatant obtained in the step (2) 260 、OD 290 And calculating the protein content in the supernatant, wherein the calculation formula is as follows: supernatant protein concentration C (mg/ml) =1.45OD 290 -0.74OD 260 (ii) a The calculation result is as follows: the protein content in the supernatant is 0.05mg/ml;
and (3) magnetic corpuscle preservation: placing the magnetosome prepared in the step (2) in distilled water for ultrasonic cleaning twice, adsorbing by a magnet, suspending the adsorbed magnetosome by distilled water, quickly freezing by liquid nitrogen, and freeze-drying by a freeze dryer for later use at-20 ℃;
the purified magnetosome needs to be treated by proteinase K-tris, and the method specifically comprises the following steps:
(3) Subpackaging the stored magnetosome into 10ml centrifuge tubes, suspending the magnetosome with 1mg/ml protease K-tris solution, placing in a water bath, and treating at 56 deg.C for 3h;
(4) Taking out the centrifugal tube from the water bath, and collecting magnetosomes by magnetic adsorption;
wherein proteinase K-tris is available from Shanghai Roche pharmaceuticals, inc.; the goods number is: 3115879001;
performing electron microscope detection on the magnetosome treated by the proteinase K-tris, wherein the result is shown in figure 2, and detecting an electrophoretogram of the magnetosome before and after the proteinase K-tris treatment, and the result is shown in figure 3;
example 2
The present embodiment provides a high-efficiency targeting antitumor drug nanocarrier system, which is prepared by coupling BMP-SPDP-RA to an antibody, wherein the antibody in the present embodiment is trastuzumab (trastuzumab), purchased from pharmaceutical limited, rochon, shanghai, through all steps of example 1 and step S7; coupling BMP-SPDP-RA with Trastuzole Monoantibody specifically comprises the following steps:
s71 coupling 1mg of BMP-SPDP-RA and 1mg of trastuzumab, and mixing in a shaking table at 37 ℃ for 2h;
s72, uniformly mixing, placing on a magnetic frame, carrying out magnetic adsorption for 1min, washing for three times by using PBS (phosphate buffer solution) with the pH value of 7.4, and washing away redundant unconjugated trastuzumab to obtain the BMP-SPDP-RA-TZ.
Test example 1 assay of BMP-SPDP-RA coupled trastuzumab content
The BCA assay kit was used to detect the amount of BMP-SPDP-RA coupled trastuzumab antibody of example 2, while glutaraldehyde was used to couple magnetosomes with trastuzumab as a control, as follows: taking 1mg of BMP-SPDP-RA, adding 1mg/ml trastuzumab (the trastuzumab is dissolved by PBS), carrying out sample retention detection, carrying out coupling according to the coupling step, carrying out sample retention on the supernatant after magnetic adsorption, carrying out supernatant protein content detection, wherein the amount of the trastuzumab before coupling and the amount of the trastuzumab after coupling are the amount of the trastuzumab coupled per milligram of magnetosome, and the test result is shown in figure 4.
Test example 2 interaction of magnetosome and macrophage
2.1 Monocyte (THP-1) induced macrophage
Human monocyte (THP-1) is taken and purchased from Beijing Beinana Chuanglian Biotechnology research institute, phorbol Myristate Acetate (PMA) with the final concentration of 100ng/mL is used for inducing the human monocyte (THP-1) to become macrophage, the induction result is shown in figure 5, and as can be seen from figure 5, the human monocyte (THP-1) is changed into adherent long fusiform and irregular type from a suspended oval shape, which indicates that the monocyte is successfully induced to be the macrophage cell.
2.2 Macrophage and magnetosome co-incubation
Preparation of BMP-GA-IgG: 1. diluting glutaraldehyde by 5 times by PBS to prepare glutaraldehyde solution with the final concentration of 5%; placing 1mgBMP-WT in 1.5mL EP tube, adding 1mL5% glutaraldehyde solution, performing ultrasound in an ultrasonic cleaner for 1min, intermittently repeating for 1min for 30 times, and washing away unbound glutaraldehyde solution with PBS to obtain BMP-GA; adding 1mg/ml IgG solution into an EP tube, performing ultrasonic treatment in an ultrasonic cleaner for 1min, intermittently performing ultrasonic treatment for 1min, repeating the ultrasonic treatment for 30 times, and washing away unbound IgG solution with PBS to obtain BMP-GA-IgG; GA represents glutaraldehyde;
preparation of BMP-SPDP-RA-IgG: coupling 1mg of BMP-SPDP-RA and 1mg of IgG, and mixing in a shaking table at 37 ℃ for 2 hours; uniformly mixing, placing on a magnetic frame, magnetically adsorbing for 1min, washing with PBS (phosphate buffer solution) with pH of 7.4 for three times, and washing off excessive unbound IgG to obtain BMP-SPDP-RA-IgG;
IgG was purchased from Saimer Feishell technologies, inc. (Thermo Fisher cat # 4506);
blank cells (human monocytes without any treatment), BMP-GA-IgG, BMP-WT, and BMP-SPDP-RA-IgG were co-incubated with macrophages, respectively, at a final concentration of 10. Mu.g/mL for 4h, and individual phagocytes were used as controls, and the intracellular magnetosome content was observed by Prussian blue staining, as shown in FIG. 6,
as can be seen from FIG. 6, when immunoglobulin (IgG) was coupled in the forward direction, the phagocytosis efficiency of macrophages was reduced, and when glutaraldehyde was selected as the bifunctional reagent, the phagocytosis efficiency of macrophages was significantly higher than that of BMP-SPDP-RA-IgG group.
2.3 flow cytometry detection of phagocytosis efficiency of magnetosomes by macrophages
Blank cells (untreated human monocytes), BMP-SPDP-RA-IgG and BMP-GA-IgG are stained with Fluorescein Isothiocyanate (FITC), incubated with macrophages at a final concentration of 10 mu g/mL for 4 hours respectively, the cells are collected after being washed with PBS three times, and the fluorescence intensity is detected by flow cytometry, and the result is shown in FIG. 7, and as can be seen from FIG. 7, BMP-SPDP-RA-IgG has significantly reduced phagocytosis efficiency of macrophages and significant difference compared with BMP-GA-IgG, which indicates that the coupling direction of the IgG influences the recognition and phagocytosis capacity of the macrophages.
Experimental example 3 interaction with Breast cancer cells (SK-BR-3)
3.1 Detection of SK-BR-3 cell surface protein HER2
Cell surface HER2 identification was performed by rabbit anti-HER 2 primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody, both antibodies were purchased from Thermo Fisher, SK-BR-3 cells expressed high-expression human epidermal growth factor receptor-2 (HER 2), and MDA-MB-468 did not express HER2, and the results are shown in FIG. 8, and it can be seen from FIG. 8 that SK-BR-3 cell lines correctly and highly express HER2.
3.2SK-BR-3 cell interaction
BMP-GA-TZ and BMP-SPDP-RA-TZ of example 2 were incubated with SK-BR-3 cells for two hours after incubation with Plasma (Plasma) and IgG-enriched Plasma (IgG-Plasma), respectively, which was obtained from Sigma under Catalogue No. 4506, in which IgG was additionally added to normal Plasma at an amount of 6 mg/ml; the SK-BR-3 cells in this test example were donated by the national center of Nano science; observed under an optical microscope after Prussian staining, the result is shown in figure 9, CK is blank, and it can be known from figure 9 that after BMP-SPDP-RA-TZ and BMP-GA-TZ are incubated with plasma and IgG-rich plasma, SK-BR-3 does not show a very obvious difference in BMP-SPDP-RA-TZ phagocytosis efficiency, but the BMP-GA-TZ phagocytosis efficiency is remarkably reduced, which indicates that the existence of the protein crown makes SK-BR-3 not have a very obvious influence on BMP SK-SPDP-RA-TZ phagocytosis, but seriously influences the SK-BR-3 phagocytosis on BMP-GA-TZ, and indicates that the existence of the protein crown makes BMP-GA-TZ reduce the ability of targeting SK-BR-3.
3.3 Detection of phagocytosis efficiency of SK-BR-3 to BMP-SPDP-RA-TZ and BMP-GA-TZ by flow cytometry
BMP-GA-TZ and BMP-SPDP-RA-TZ of example 2 were stained with Fluorescein Isothiocyanate (FITC) for two hours, incubated with SK-BR-3 at a final concentration of 10. Mu.g/mL for 4 hours, and cells were collected for fluorescence intensity detection; the results are shown in fig. 10, and it can be seen from fig. 10 that, after BMP-SPDP-RA-TZ and BMP-GA-TZ are incubated with plasma and IgG-rich plasma, the efficiency of targeting SK-BR-3 is reduced to different degrees, after the BMP-SPDP-RA-TZ and BMP-GA-TZ are remarkably different in the fluorescence intensity of SK-BR-3 after the BMP-SPDP-RA-TZ and the IgG-rich plasma are incubated, and except for the same trend, the BMP-SPDP-RA-TZ still has strong performance of targeting SK-BR-3 after the plasma is incubated.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered by the claims of the present invention.
Claims (8)
1. The efficient targeting antitumor drug nano-carrier system is characterized in that the nano-carrier system is prepared by coupling magnetosomes and recombinant SPAs through SPDP;
the sequence of the recombinant SPA is as follows:
CGSGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPKG
SGSGSADNKFNKEQQNAFYEILHLPNLTEEQRNAAIQSLKDDPSQSANLLAEAKKLNDAQAPK；
the recombinant SPA is carried out on the basis of modifying the structure of the SPA gene from Staphylococcus aureus, and the synthetic method of the recombinant protein comprises the following steps:
(a) Adding a cysteine to the N end of B domain of the natural SPA gene to introduce a sulfhydryl;
(b) Adding BamH I and HindIII enzyme cutting sites at two ends of the SPA gene with the introduced sulfhydryl respectively;
(c) Cloning the SPA gene in the step (b) into a PET28 (a +) vector, and converting the SPA gene into E.coli BL 21;
(d) Carrying out induction expression by using IPTG;
separating and purifying with affinity chromatography column to obtain pure protein, and obtaining recombinant protein.
2. The system of claim 1, wherein the nanocarrier system is prepared by a method comprising:
s1, dissolving SPDP in dimethyl sulfoxide to prepare a first solution, wherein the concentration of the first solution is 5-15mM;
s2, adding a magnetosome into PBS (phosphate buffer solution) and uniformly mixing to prepare a solution II, wherein the mass ratio of the magnetosome to the SPDP is 0.5-1.5, and the concentration of the magnetosome in the PBS is 1mg/0.8-1ml;
s3, adding the solution II into the solution I, ultrasonically mixing uniformly and coupling to prepare a first mixed solution;
s4, magnetically adsorbing the first mixed solution, removing the supernatant after adsorption, and then washing with PBS to prepare BMP-SPDP;
s5, carrying out mixing ultrasonic uniform mixing coupling on BMP-SPDP and the recombinant SPA in a mass ratio of 0.5-1.5;
s6, magnetically adsorbing the second mixed solution, washing away unbound recombinant SPA, and washing with PBS to obtain BMP-SPDP-RA.
3. The system of claim 2, wherein the method for preparing the nanopharmaceutical system further comprises the steps of S7 coupling the BMP-SPDP-RA to an antibody;
the step S7 of coupling the BMP-SPDP-RA with the antibody specifically comprises the following steps:
s71, taking BMP-SPDP-RA and the antibody with the mass ratio of 0.5-1.5, placing the BMP-SPDP-RA and the antibody in a shaking table at 37 ℃, and mixing for 1.5-2.5h to prepare a third mixed solution;
s72, magnetically adsorbing the third mixed solution, and then washing away the unbound antibody by PBS to prepare BMP-SPDP-RA-TZ, namely the nano-carrier system.
4. A system according to any of claims 1-3, wherein the magnetosomes are purified prior to use, the purification comprising the steps of:
(1) Taking the bacterial sludge and PBS to dissolve according to the mass volume ratio of 1;
(2) And (2) carrying out ultrasonic treatment on the solution obtained in the step (1) by using an ultrasonic cell crusher, adsorbing magnetosomes in the ultrasonic mixed solution by using a magnet, discarding the supernatant, adding PBS again for ultrasonic treatment and magnet adsorption, and repeating the steps until the concentration of albumin is not reduced any more, thus obtaining the BMP-WT.
5. The system of claim 4, wherein the PBS has a concentration of 10mM and the pH of 7.4.
6. The system of claim 4, wherein the step (2) comprises the following steps:
(21) Placing the mixed solution obtained in the step (1) in a container, and carrying out ultrasonic treatment for 30min by using an ultrasonic cell crusher under the ultrasonic condition of a No. 6 horn with 60% power for 3s and 5s of pause;
(22) Placing a magnet at the bottom of the container, and adsorbing the magnetosome overnight at 4 ℃;
(23) Discarding the supernatant, adding PBS, performing ultrasonic treatment with an ultrasonic cell crusher under 50% power for 3s and 5s at intervals for 20min, and performing magnetic adsorption for 6h;
(24) Discarding the supernatant, adding PBS, and performing ultrasound with an ultrasonic cell crusher under the ultrasound conditions of 40% power, 3s ultrasound, 5s pause, and 20min totally, repeating the above steps until the albumin concentration is not reduced, and reducing the power and the ultrasound time with the increase of the ultrasound times.
7. The system of claim 4, wherein the purified magnetosomes are further treated with proteinase K-tris, comprising the steps of:
(3) Suspending magnetosome with protease K-tris solution with concentration of 0.5-1.5mg/ml, and treating in water bath for 2-4 hr;
(4) Taking out, and collecting magnetosome by magnetic adsorption.
8. The application of the nano drug delivery system of any one of claims 1 to 7 in preparation of a targeted antitumor drug product.
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