CN116178571B - Endoplasmic reticulum-targeted artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum-targeted vesicle, immune adjuvant and vaccine - Google Patents

Abstract

The invention provides endoplasmic reticulum targeting artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum targeting vesicle, immunoadjuvant and vaccine, and belongs to the technical field of biomedicine and drug delivery. The endoplasmic reticulum targeting artificial protein of the invention targets the endoplasmic reticulum of eukaryotic cells through Pardaxin peptide, is anchored on vesicles through Atg8 fragments and self-assembles, and forms endoplasmic reticulum targeting vesicles with Pardaxin targeting polypeptides displayed on the surfaces. The invention also provides an endoplasmic reticulum targeting magnetic vesicle, which is obtained by coating the endoplasmic reticulum targeting vesicle prepared by the preparation method according to the scheme with the magnetic mesoporous silicon nanoparticle. The invention provides a novel broad-spectrum vaccine adjuvant for different pathogenic infections and provides a new idea for the preparation and production of synthetic biological vaccines.

Description

An endoplasmic reticulum-targeted artificial protein, recombinant cerevisiae, endoplasmic reticulum-targeted vesicle, immune adjuvant and vaccine

Technical Field

The present invention belongs to the technical field of biomedicine and drug delivery, and specifically relates to an endoplasmic reticulum-targeted artificial protein, recombinant brewer's yeast, endoplasmic reticulum-targeted vesicles, immune adjuvants and vaccines.

Background technique

As one of the most important membranous organelles in eukaryotic cells, the endoplasmic reticulum (ER) is the main site for protein folding and transport, lipid synthesis, vesicle transport, and calcium ion storage, and is involved in the regulation of multiple signal transduction pathways. In addition, the ER also establishes connection sites with other membranous structures such as plasma membrane, mitochondria, endosomes, and lysosomes through its developed extended structure, playing an important role in the information, material, and energy exchange between intracellular organelles and plasma membranes. Given the extreme importance of the ER to the growth and metabolic process of eukaryotic cells, the disorder of its structure and function will have a significant impact on eukaryotic cells and biological organisms. A large number of studies have found that ER dysfunction is closely related to many major human diseases discovered so far, such as cancer, autoimmune diseases, pathogenic microbial infections, neurodegenerative diseases, and diabetes. With the continuous development of precision medicine theory and technology, the development of drugs with precise ER targeting has become an important trend in preventing and conquering related diseases.

The endoplasmic reticulum is inseparable from the synthesis, processing and transport of intracellular proteins. Based on the exploration of protein transport in cells, more signal peptides with endoplasmic reticulum tropism have been discovered. These endoplasmic reticulum signal peptides are mostly modified at the N-terminus of proteins, and perform different functions according to their sequences. Endoplasmic reticulum signal peptides include KDEL peptide, Eriss peptide and Pardaxin peptide. Among them, Pardaxin peptide (GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE, SEQ ID NO.1) is a natural antimicrobial peptide with endoplasmic reticulum localization ability. Its endoplasmic reticulum targeting mechanism may be related to the efficient phosphatidylcholine vesicle pore-forming ability (phosphatidylcholine is the main glycerophospholipid of the endoplasmic reticulum, accounting for 60%).

Currently, there is no record of using Pardaxin peptides to prepare ER-targeted vesicles.

Summary of the invention

In view of this, the object of the present invention is to provide an endoplasmic reticulum-targeted artificial protein, recombinant cerevisiae, endoplasmic reticulum-targeted vesicles, immune adjuvants and vaccines. The endoplasmic reticulum-targeted artificial protein of the present invention can be used to prepare endoplasmic reticulum-targeted vesicles.

The present invention provides an endoplasmic reticulum-targeted artificial protein, whose amino acid sequence from N-terminus to C-terminus is shown in SEQ ID NO.2.

The present invention also provides a gene encoding the endoplasmic reticulum-targeted artificial protein described in the above scheme, and the nucleotide sequence is shown in SEQ ID NO.3.

The present invention also provides a recombinant Saccharomyces cerevisiae, which uses ura3-deficient Saccharomyces cerevisiae as the original cell and contains a recombinant plasmid; the coding gene described in the above scheme is inserted into the recombinant plasmid.

The present invention also provides the use of the artificial protein or the encoding gene or the recombinant Saccharomyces cerevisiae described in the above scheme in preparing endoplasmic reticulum-targeted vesicles.

The present invention also provides a method for preparing an endoplasmic reticulum-targeted vesicle, comprising the following steps:

The recombinant Saccharomyces cerevisiae described in the above scheme was inoculated into SC-Gal medium for the first induction culture;

The recombinant Saccharomyces cerevisiae after the first induction culture is inoculated into a SC-Gal medium without a nitrogen source for a second induction culture;

Centrifuging the culture after the second induction culture, collecting precipitated yeast cells, extracting protoplasts of the yeast cells, and obtaining yeast protoplasts;

The yeast protoplasts are sequentially crushed and ultracentrifuged, and the precipitate is collected to obtain a crude product of endoplasmic reticulum-targeted vesicles;

Purifying the crude vesicles using a nickel column to obtain endoplasmic reticulum-targeted vesicles;

The SC-Gal medium uses water as a solvent and includes the following components in concentrations: 6.7 g/L of amino-free yeast nitrogen source, 20 g/L of galactose, and 2 g/L of mixed amino acids;

The SC-Gal medium without amino-free yeast nitrogen source uses water as solvent and includes the following components at the following concentrations: 20 g/L galactose and 2 g/L mixed amino acids;

The mixed amino acid comprises the following components in parts by mass: 2 parts of L-proline, 10 parts of L-leucine, 2 parts of L-valine, 2 parts of L-alanine, 2 parts of L-serine, 2 parts of L-lysine, 2 parts of L-glutamic acid, 2 parts of L-methionine, 2 parts of L-threonine, 2 parts of L-glutamine, 2 parts of L-glycine, 2 parts of L-isoleucine, 2 parts of L-tryptophan, 2 parts of L-aspartic acid, 2 parts of L-tyrosine, 2 parts of L-phenylalanine, 2 parts of inositol, 2 parts of L-cysteine, 2 parts of p-aminobenzoic acid, 2 parts of L-asparagine and 0.5 parts of adenine.

The present invention also provides an endoplasmic reticulum-targeted magnetic vesicle, comprising magnetic mesoporous silicon nanoparticles and endoplasmic reticulum-targeted vesicles coated in the pores of the magnetic mesoporous silicon nanoparticles. The endoplasmic reticulum-targeted vesicles are prepared by the preparation method described in the above scheme.

Preferably, the core of the magnetic mesoporous silicon nanoparticles is MnFe ₂ O ₄ nanoparticles; the shell of the magnetic mesoporous silicon nanoparticles is mesoporous silicon dioxide; the preparation method of the magnetic mesoporous silicon nanoparticles comprises the following steps:

suspending MnFe ₂ O ₄ in an organic solvent to obtain a suspension;

The suspension, the pore-forming agent and water are first mixed, and the organic solvent is removed to obtain a first mixed solution;

The first mixed solution, water, methanol and ethyl acetate are mixed for a second time to obtain a second mixed solution;

The second mixed solution, ammonium hydroxide solution, tetraethoxysilane and 3-aminopropyltriethoxysilane are mixed for the third time to obtain a third mixed solution;

The third mixed solution is centrifuged to collect the precipitate, and the precipitate is washed to remove the pore-forming agent to obtain magnetic mesoporous silicon nanoparticles.

The present invention also provides the use of the endoplasmic reticulum-targeted magnetic vesicles described in the above scheme in the preparation of immune adjuvants.

The present invention also provides a vaccine, comprising magnetic mesoporous silicon nanoparticles, and endoplasmic reticulum targeting vesicles and antigens encapsulated in the pores of the magnetic mesoporous silicon nanoparticles, wherein the endoplasmic reticulum targeting vesicles are prepared by the preparation method described in the above scheme.

Preferably, the antigen includes a pathogen antigen or a tumor antigen.

The present invention provides an endoplasmic reticulum-targeted artificial protein, and the amino acid sequence from the N-terminus to the C-terminus is shown in SEQ ID NO.2. The endoplasmic reticulum-targeted artificial protein of the present invention contains an N-terminal 6×His tag, a Pardaxin peptide, a green fluorescent protein, and a saccharomyces cerevisiae Atg8 fragment. The endoplasmic reticulum-targeted artificial protein of the present invention targets the endoplasmic reticulum of eukaryotic cells through the Pardaxin peptide, is anchored on a vesicle through the Atg8 fragment, and self-assembles to form an endoplasmic reticulum-targeted vesicle displaying the Pardaxin targeting polypeptide on the surface.

The present invention also provides an endoplasmic reticulum-targeted magnetic vesicle, comprising magnetic mesoporous silicon nanoparticles and endoplasmic reticulum-targeted vesicles coated in the pores of the magnetic mesoporous silicon nanoparticles. The endoplasmic reticulum-targeted vesicles are prepared by the preparation method described in the above scheme.

The endoplasmic reticulum-targeted magnetic vesicle of the present invention is a broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvant, which can load antigens. The present invention provides a new broad-spectrum vaccine adjuvant for different pathogenic infections and provides a new idea for the preparation and production of synthetic biological vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the characterization of MnFe ₂ O ₄ nanoparticles; A is a transmission electron microscope image, and B is a magnetothermal curve;

Figure 2 shows the characterization of MagMSN and MagParV, where A is the energy spectrum analysis, B is the transmission electron microscopy image, C is the Zeta potential, D is the antigen loading capacity, and E is the antigen release capacity;

FIG3 shows the serum CaAg IgG levels of mice after immunization with free antigen or antigen-loaded adjuvant;

Fig. 4 is a mouse survival curve;

Figure 5 shows the serum SaAg IgG levels of mice after immunization with free antigen or antigen-loaded adjuvant;

FIG6 shows the inhibitory effect of free antigen and antigen-loaded adjuvant immunization on tumor growth in mice.

Detailed ways

The present invention provides an endoplasmic reticulum-targeted artificial protein ParGA protein, whose amino acid sequence from N-terminus to C-terminus is shown in SEQ ID NO.2, specifically:

MHHHHHHGFFALIPKIISSPLFKTLLSAVGSALSSSGGQEGGGGGGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGMKSTFKSEYPFEKRKAESERIADRFKNRIPVICEKAEKSDIPEIDKRKYLVPADLTVGQFVYVIRKRIMLPPEKAIFIFVNDTLPPTAALMSAIYQEHKDKDGFLYVTYSGENTFGR.

The endoplasmic reticulum targeting artificial protein of the present invention contains an N-terminal 6×His tag, a Pardaxin peptide, a green fluorescent protein and a saccharomyces cerevisiae Atg8 fragment. The artificial protein of the present invention targets the endoplasmic reticulum of eukaryotic cells through the Pardaxin peptide, is anchored on the vesicle through the Atg8 fragment and self-assembles to form an endoplasmic reticulum targeting vesicle with the Pardaxin targeting polypeptide displayed on the surface. The 6×His tag and the green fluorescent protein serve as a separation and purification tag and a marker, respectively.

The present invention also provides a gene encoding the endoplasmic reticulum-targeted artificial protein PARGA described in the above scheme, the nucleotide sequence of which is shown in SEQ ID NO.3, specifically:

atgcatcatc accaccatca cggtttcttt gctttgatcc caaagattatctcttctccattgtttaagacattgttgtc agctgtcggt tccgctttgt cctcttccggtggtcaagaaggtggcgggg gtggtggtgtgtccaaaggt gaagaattat tcactggtgttgttccaatcttggttgaat tggatggtga tgttaacggtcacaagttct ccgtttctggtgaaggtgaaggtgatgcta cctacggtaa gttgaccttg aaattcatctgtaccaccggtaagctgcccgtcccttggc caaccttggt caccactttg acctacggtgttcaatgtttctctagatacccagaccaca tgaagcaaca cgacttcttc aagtccgctatgccagaaggttacgtccaagaaagaacca ttttcttcaa ggatgacgga aactataagaccagagctgaagtcaagtttgaaggtgaca ctttggtcaa ccgtatcgaa ttgaagggtattgatttcaaggaagacggtaacatcttag gtcacaaatt ggaatacaac tacaactctcacaacgtctacattatggctgacaagcaaa agaacggtat caaggtcaat ttcaagatcagacacaacattgaggatggttctgtccaat tggctgatca ttaccaacaa aacactccaattggtgacggtccagtcttactacctgaca accactactt atccactcaa tctgctctctccaaggacccaaacgaaaagagagatcaca tggttttgtt ggaatttgtt actgctgctggtatcactttgggtatggacgaattgtaca agggtggtgg tatgaaatct actttcaagagtgaatatccattcgaaaagagaaaggccg aatctgaacg tattgctgac agattcaagaacagaatcccagttatctgtgaaaaggccg aaaagtctga cattccagaa attgacaaaagaaagtacttagttccagctgatttgactg ttggtcaatt cgtttacgtt attcgtaagagaatcatgttgccaccagaaaaagctatat tcatttttgt taatgacacc ctacctccaactgccgccttgatgtctgccatctaccaag aacacaagga caaggatggt ttcttgtacgttacttactctggtgaaaacaccttcggta gataa.

The coding gene of the present invention is obtained based on the optimization of the most suitable codon of Saccharomyces cerevisiae.

The present invention also provides a recombinant Saccharomyces cerevisiae, which uses ura3-deficient Saccharomyces cerevisiae as the original cell and contains a recombinant plasmid; the coding gene described in the above scheme is inserted into the recombinant plasmid.

In the present invention, the original plasmid of the recombinant plasmid is an expression plasmid of Saccharomyces cerevisiae, preferably a plasmid pESC-URA with an inducible promoter PGAL1. When pESC-URA is used as the original plasmid, the insertion site of the coding gene is BamH1/Xho1.

In the present invention, the ura3-deficient Saccharomyces cerevisiae is preferably chassis cell Saccharomyces cerevisiae Sc0, genotype: MATahis3Δ1leu2 trp1-289 ura3-52.

The present invention has no particular limitation on the method for constructing the recombinant plasmid and the recombinant Saccharomyces cerevisiae, and conventional methods in the art may be used.

The present invention also provides the use of the artificial protein or the encoding gene or the recombinant Saccharomyces cerevisiae described in the above scheme in preparing endoplasmic reticulum-targeted vesicles.

The present invention also provides a method for preparing an endoplasmic reticulum-targeted vesicle, comprising the following steps:

The recombinant Saccharomyces cerevisiae described in the above scheme was inoculated into SC-Gal medium for the first induction culture;

The recombinant Saccharomyces cerevisiae after the first induction culture is inoculated into a SC-Gal medium without an amino-free yeast nitrogen source for a second induction culture;

Centrifuging the culture after the second induction culture, collecting precipitated yeast cells, extracting protoplasts of the yeast cells, and obtaining yeast protoplasts;

The yeast protoplasts are sequentially crushed and ultracentrifuged, and the precipitate is collected to obtain a crude product of endoplasmic reticulum-targeted vesicles;

Purifying the crude vesicles using a nickel column to obtain endoplasmic reticulum-targeted vesicles;

In the present invention, the recombinant Saccharomyces cerevisiae described in the above scheme is firstly inoculated into SC-Gal culture medium for the first induction culture.

Before inoculating the recombinant Saccharomyces cerevisiae into the SC-Gal medium, it is preferred that the recombinant Saccharomyces cerevisiae strain is inoculated into the YPD liquid medium for expansion culture to obtain a large amount of recombinant Saccharomyces cerevisiae; the temperature of the expansion culture is preferably 30°C; the method of the expansion culture is preferably shaking culture; the time of the expansion culture is preferably 9 to 16 hours, more preferably 12 hours.

In the present invention, the SC-Gal medium uses water as a solvent and includes the following components in concentrations: 6.7 g/L of amino-free yeast nitrogen source, 20 g/L of galactose and 2 g/L of mixed amino acids; the water is preferably distilled water; and the pH value of the SC-Gal medium is preferably 6.0-6.5.

In the present invention, the amino-free yeast nitrogen source is preferably yeast nitrogen base (without aminoacids), purchased from Solarbio Company, with a specification of 500 grams and a model number of Y8040.

In the present invention, the temperature of the first induction culture is preferably 30°C; the mode of the first induction culture is preferably shaking culture; the time of the first induction culture is preferably 24h; the function of the first induction culture is to induce the expression of ParGA controlled by the GAL10 promoter.

After the first induction culture, the present invention inoculates the recombinant Saccharomyces cerevisiae after the first induction culture into a SC-Gal medium without a nitrogen source to perform a second induction culture.

In the present invention, the SC-Gal medium containing no amino yeast nitrogen source uses water as solvent and includes the following components at the following concentrations: 20 g/L galactose and 2 g/L mixed amino acids; the water is preferably distilled water; the pH value of the SC-Gal medium containing no amino yeast nitrogen source is preferably 6.0 to 6.5;

In the present invention, the mixed amino acid includes the following components in parts by mass: 2 parts of L-proline, 10 parts of L-leucine, 2 parts of L-valine, 2 parts of L-alanine, 2 parts of L-serine, 2 parts of L-lysine, 2 parts of L-glutamic acid, 2 parts of L-methionine, 2 parts of L-threonine, 2 parts of L-glutamine, 2 parts of L-glycine, 2 parts of L-isoleucine, 2 parts of L-tryptophan, 2 parts of L-aspartic acid, 2 parts of L-tyrosine, 2 parts of L-phenylalanine, 2 parts of inositol, 2 parts of L-cysteine, 2 parts of p-aminobenzoic acid, 2 parts of L-asparagine and 0.5 parts of adenine.

In the present invention, the temperature of the second induction culture is preferably 30°C; the method of the second induction culture is preferably shaker culture, and the shaker speed is preferably 100-140rpm; the time of the second induction culture is preferably 2h; the role of the second induction culture is to induce the ParGA protein containing the Atg8 fragment to rapidly self-assemble to form autophagosome vesicles through the autophagy pathway.

After the second induction culture, the present invention centrifuges the culture after the second induction culture, collects the precipitated yeast cells, extracts the protoplasts of the yeast cells, and obtains yeast protoplasts. In the present invention, the rotation speed of the centrifugation is preferably 4200 rpm, and the time of the centrifugation is preferably 3 minutes. Before extracting the protoplasts, the present invention preferably further includes washing the collected yeast cells; the reagent used for the washing is preferably a protoplast preparation buffer; the protoplast preparation buffer uses water as a solvent, and preferably includes the following components in the following concentrations: sorbitol 1M, trisodium citrate 0.02M, EDTANa ₂ 0.1M and Na ₂ HPO ₄ ·12H ₂ O 0.02M.

In the present invention, the extraction of protoplasts preferably includes enzymolysis of the yeast cells and enzyme mixture after the second induction culture to obtain a protoplast suspension, centrifuging the protoplast suspension, and collecting precipitated yeast protoplasts; the enzyme preferably includes yeast lytic enzyme or snail enzyme; the working concentration of the yeast lytic enzyme is preferably 100U/mL; the working mass concentration of the snail enzyme is preferably 2% (W/V); the enzymolysis time is preferably 10 to 30 minutes, more preferably 20 minutes; the centrifugal speed is preferably 4200rpm; the centrifugal time is preferably 5 minutes.

After obtaining the yeast protoplasts, the present invention sequentially crushes and ultracentrifuges the yeast protoplasts, collects the precipitate, and obtains a crude endoplasmic reticulum-targeted vesicle product.

In the present invention, the crushing method is preferably homogenized crushing by a Dounce homogenizer. In the present invention, the speed of the ultracentrifugation is preferably 35000 rpm; the time of the ultracentrifugation is preferably 30 to 120 min, more preferably 60 to 90 min.

After obtaining the crude endoplasmic reticulum-targeted vesicles, the present invention uses a nickel column to purify the crude vesicles to obtain the endoplasmic reticulum-targeted vesicles.

In the present invention, the purification of the crude vesicles using a nickel column preferably includes: suspending the crude endoplasmic reticulum-targeted vesicles in 10 mM imidazole buffer, adding it to the nickel column for vesicle adsorption, then washing with 10 mM imidazole buffer, and finally eluting with 100 mM imidazole; the number of washing times is preferably 3 times.

The present invention also provides an endoplasmic reticulum-targeted magnetic vesicle, comprising magnetic mesoporous silicon nanoparticles and endoplasmic reticulum-targeted vesicles coated in the pores of the magnetic mesoporous silicon nanoparticles. The endoplasmic reticulum-targeted vesicles are prepared by the preparation method described in the above scheme.

In the present invention, the mass ratio of the endoplasmic reticulum targeting vesicles to the magnetic mesoporous silicon nanoparticles is preferably (0.1-1):(1-10), and more preferably 0.5:2.

In the present invention, the endoplasmic reticulum-targeted magnetic vesicle is round; the particle size of the endoplasmic reticulum-targeted magnetic vesicle is 100-200nm; the pore size of the endoplasmic reticulum-targeted magnetic vesicle is 10-20nm, and each MagMSN has multiple _{MnFe2O4 nanoparticle} clusters in the center; the Zeta potential of the MagMSN is positive (+26mV).

In the present invention, the core of the magnetic mesoporous silicon nanoparticles is MnFe ₂ O ₄ nanoparticles; the shell of the magnetic mesoporous silicon nanoparticles is mesoporous silicon dioxide; the magnetic mesoporous silicon nanoparticles are preferably prepared by the following method:

suspending MnFe ₂ O ₄ in an organic solvent to obtain a suspension;

The suspension, the pore-forming agent and water are first mixed, and the organic solvent is removed to obtain a first mixed solution;

The first mixed solution, water, methanol and ethyl acetate are mixed for a second time to obtain a second mixed solution;

The second mixed solution, ammonium hydroxide solution, tetraethoxysilane and 3-aminopropyltriethoxysilane are mixed for the third time to obtain a third mixed solution;

The third mixed solution is centrifuged to collect the precipitate, and the precipitate is washed to remove the pore-forming agent to obtain magnetic mesoporous silicon nanoparticles.

The present invention firstly suspends manganese iron oxide (MnFe ₂ O ₄ ) in an organic solvent to obtain a suspension.

In the present invention, the _MnFe2O4 is preferably obtained by thermal decomposition reaction using trivalent organic iron and divalent organic _manganese as iron source and manganese source respectively; the _MnFe2O4 is a magnetic nanoparticle, the diameter of the _MnFe2O4 nanoparticle is _9-11nm , and the _{MnFe2O4 nanoparticle} has good _{magnetothermal} conversion ability.

In the present invention, the iron (III) acetylacetonate is preferably iron (III) acetylacetonate; the divalent organic manganese is preferably manganese (II) acetylacetonate; the thermal decomposition reaction includes a first thermal decomposition reaction and a second thermal decomposition reaction performed sequentially; the temperature of the first thermal decomposition reaction is preferably 200°C; the time of the first thermal decomposition is preferably 1h; the temperature of the second thermal decomposition reaction is preferably 298°C; and the time of the second thermal decomposition reaction is preferably 1h.

In the present invention, the organic solvent is preferably chloroform, and the ratio of the mass of the MnFe ₂ O ₄ to the volume of the organic solvent is preferably 5 mg:2 mL.

After obtaining the suspension, the present invention first mixes the suspension, the pore-forming agent and water, removes the organic solvent, and obtains a first mixed solution. In the present invention, the pore-forming agent is preferably hexadecyltrimethylammonium bromide (CTAB); the water is preferably distilled water; based on the mass of _MnFe2O4 being _5mg , the amount of the pore-forming agent is preferably 200mg, and the amount of the water is preferably 10mL; the first mixing method is preferably ultrasonic mixing; the first mixing time is preferably 20min; the method of removing the organic solvent is preferably heating to 75°C to evaporate the organic solvent.

After obtaining the first mixed solution, the present invention secondly mixes the first mixed solution, water, methanol and ethyl acetate to obtain a second mixed solution. In the present invention, the water is preferably distilled water; the volumes of the first mixed solution, water, methanol and ethyl acetate are preferably 10:95:5:20; the second mixing method is preferably magnetic stirring mixing; and the second mixing time is preferably 10 minutes.

After obtaining the second mixed solution, the present invention performs a third mixing of the second mixed solution, ammonium hydroxide solution, tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) to obtain a third mixed solution. In the present invention, the ammonium hydroxide solution is aqueous ammonia, and based on the volume of the first mixed solution being 10 mL, the amount of the ammonium hydroxide solution is preferably 3 mL; the amount of TEOS is preferably 300 μL; the amount of APTES is preferably 30 μL; the third mixing method is preferably magnetic stirring mixing, and during the magnetic stirring mixing process, mesoporous silicon grows on the surface of the magnetic nanoparticles; the third mixing time is preferably 12 hours.

After obtaining the third mixed solution, the present invention centrifuges the third mixed solution, collects the precipitate, washes the precipitate and removes the pore-forming agent to obtain magnetic mesoporous silicon nanoparticles. In the present invention, the centrifugal speed is preferably 8000-10000 rpm, and the centrifugal time is preferably 10 min; the reagent used for the washing is preferably ethanol; the removal of the pore-forming agent preferably includes heating with an ammonium nitrate solution with a concentration of 200 mg/100 mL to remove the pore-forming agent in the pores; the solvent of the ammonium nitrate solution is preferably ethanol.

In the present invention, _MnFe2O4 is the initial magnetic _nanoparticle . After adding ammonium hydroxide solution, tetraethoxysilane and 3-aminopropyltriethoxysilane, the surface oxygen atoms of the nanoparticle react with tetraethoxysilane, and mesoporous silica is derived on this basis to finally obtain magnetic mesoporous silicon nanoparticles with a core-shell structure, wherein the core is _MnFe2O4 nanoparticles and the shell is mesoporous _silica .

In the present invention, the magnetic mesoporous silicon nanoparticles are magnetic mesoporous silicon nanoparticles MagMSN in which large-pore MSN encapsulates MnFe ₂ O ₄ .

The present invention also provides the use of the endoplasmic reticulum-targeted magnetic vesicles described in the above scheme in the preparation of immune adjuvants and/or anti-tumor drugs.

The immune adjuvant of the present invention is a broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvant that can load antigens. The immune adjuvant of the present invention has a high antigen loading capacity, significantly induces the production of fungal, bacterial and viral antigen-specific antibodies under alternating magnetic field treatment, and can effectively protect the body from serious systemic infection by pathogenic bacteria. The present invention provides a new broad-spectrum vaccine adjuvant for different pathogenic infections, and provides new ideas for the preparation and production of synthetic biology vaccines.

In the present invention, the anti-tumor drug achieves the anti-tumor effect by inhibiting tumor growth.

The present invention also provides a vaccine, comprising magnetic mesoporous silicon nanoparticles, and endoplasmic reticulum targeting vesicles and antigens encapsulated in the pores of the magnetic mesoporous silicon nanoparticles, wherein the endoplasmic reticulum targeting vesicles are prepared by the preparation method described in the above scheme.

In the present invention, both the endoplasmic reticulum targeting vesicles and the antigens are adsorbed into the pores of the magnetic mesoporous silicon nanoparticles.

In the present invention, the preparation method of the magnetic mesoporous silicon nanoparticles is the same as the above scheme, which will not be repeated here.

In the present invention, the antigen preferably includes pathogen antigens or tumor antigens; the pathogen antigen preferably includes pathogen protein antigens and/or inactivated virus antigens, and more preferably includes pathogen protein; the pathogen protein preferably includes Candida albicans antigen (CaAg), Staphylococcus aureus antigen (SaAg); one or more of the avian influenza virus antigen (AIVAg) and SARS-CoV-2s protein receptor binding domain (SRBD); the tumor antigen preferably includes glioma antigen; the glioma antigen preferably includes glioma GL261 antigen.

In the present invention, based on 1 part by mass of the antigen, the mass of the endoplasmic reticulum targeting vesicle is preferably (0.1-1) part, more preferably 0.5 part; the mass of the magnetic mesoporous silica nanoparticles is preferably 1-10 parts, more preferably 2 parts.

The present invention also provides a method for preparing the vaccine described in the above scheme, comprising the following steps:

The magnetic mesoporous silicon nanoparticles, antigens and endoplasmic reticulum targeting vesicles are mixed to obtain a vaccine.

In the present invention, the mixing preferably involves mixing the antigen and the endoplasmic reticulum-targeted magnetic vesicles in PBS.

The present invention also provides a method for using the vaccine, comprising the following steps:

The vaccine is administered to the immunized subject.

In the present invention, the inoculation amount of the vaccine is calculated based on the total mass of the antigen and the endoplasmic reticulum-targeted magnetic vesicles, preferably 20 to 100 μg per mouse, more preferably 35 to 50 μg per mouse.

After vaccination, it is preferred that the vaccinated subject is placed in an alternating magnetic field for 8 to 12 minutes, preferably 10 minutes. In the present invention, the frequency of the alternating magnetic field is preferably 50 to 500,000 Hz, more preferably 400 to 10,000 Hz; the power of the alternating magnetic field is preferably 10 to 5,000 W, more preferably 100 to 1,000 W.

In the present invention, since the vaccine contains superparamagnetic _{MnFe2O4 nanoparticles} , it can generate heat under the condition of alternating magnetic field treatment, and the heat promotes the release of antigens, activates the immunogenicity of antigens, significantly induces the production of fungal, bacterial, viral and tumor antigen-specific antibodies, effectively prevents pathogenic bacteria infection, and inhibits tumor growth.

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

Example 1 Preparation of recombinant Saccharomyces cerevisiae cells

The endoplasmic reticulum-targeted artificial protein ParGA (amino acid sequence as shown in SEQ ID NO.2) was designed, which contained an N-terminal 6×His tag, pardaxin, green fluorescent protein and a fragment of Saccharomyces cerevisiae Atg8. Based on the optimal codon of Saccharomyces cerevisiae, an artificial gene PARGA encoding the ParGA protein was designed (nucleotide sequence as shown in SEQ ID NO.3). The artificial gene ParGA was cloned into the plasmid pESC-URA with the inducible promoter PGAL1 to obtain the plasmid pESC-ParGA, which was then transformed into the chassis cell Saccharomyces cerevisiae Sc0 (MATa his3Δ1leu2 trp1-289 ura3-52) to obtain the recombinant Saccharomyces cerevisiae cell Sc0+ParV.

Example 2 Preparation of biosynthetic vesicles

Reagents:

SC-Gal medium formula: 0.67 g of amino-free yeast nitrogen source, 2 g of galactose, 0.2 g of mixed amino acid powder and 100 mL of sterile water;

The culture formula of SC-Gal medium without nitrogen source: 2g galactose, 0.2g mixed amino acid powder and 100mL sterile water;

The formula of the protoplast preparation buffer is as follows: water as solvent, sorbitol 1M, trisodium citrate 0.02M, EDTANa ₂ 0.1M and Na ₂ HPO ₄ ·12H ₂ O 0.02M.

method:

The synthetic yeast cells Sc0+ParV were cultured in YPD medium at 30°C overnight with shaking, and then transferred to SC-Gal medium and cultured at 30°C with shaking for 24h. They were further transferred to SC-Gal medium without nitrogen source and cultured on a shaking platform for 2h to form autophagosome vesicles. The cells containing ParV were collected, washed with protoplast preparation buffer, and then treated with yeast lytic enzyme (100U/mL, 10min) to obtain yeast protoplasts.

Yeast protoplasts were broken up using a Dounce homogenizer, and ParV vesicles were separated by ultracentrifugation at 35,000 rpm for 30 min. Finally, the ParV vesicles were suspended in 10 mM imidazole buffer and added to a nickel column for vesicle adsorption, followed by washing three times with 10 mM imidazole buffer; finally, elution was performed with 100 mM imidazole to obtain purified ParV vesicles.

Example 3 Preparation of biosynthetic vesicles

The same procedures as in Example 2 were followed except that the yeast lytic enzyme (100 U/mL, 10 min) treatment was replaced by snail enzyme (2%, 10 min) treatment.

Example 4 Preparation of anti-Candida albicans vaccine based on endoplasmic reticulum-targeted magnetic vesicles

1. Preparation process

(1) Preparation of magnetic mesoporous silica nanoparticles

MnFe ₂ O ₄ magnetic nanoparticles were synthesized by thermal decomposition method (200°C, 1h, then 298°C, 1h) using iron (III) acetylacetonate and manganese (II) acetylacetonate as iron and manganese sources.

5 mg of MnFe ₂ O ₄ nanoparticles were suspended in 2 mL of chloroform, added to 10 mL of distilled water containing 200 mg of CTAB, ultrasonicated for 20 min, and then heated to 75 °C to evaporate the chloroform. This solution was added to a mixture of 95 mL of distilled water, 5 mL of methanol, and 20 mL of ethyl acetate, magnetically stirred for 10 min, and then 3 mL of ammonium hydroxide, 300 μL of TEOS, and 30 μL of APTES were added. The mixture was magnetically stirred for another 12 h, centrifuged, and the precipitate was washed with ethanol. CTAB in the pores was removed by heating with 100 mL of ethanol containing 200 mg of ammonium nitrate. Finally, magnetic mesoporous silicon nanoparticles MagMSNs with MnFe ₂ O ₄ wrapped by large-pore MSNs were obtained.

(2) Preparation and characterization of anti-Candida albicans vaccine

The Candida albicans cells cultured overnight at 37°C were centrifuged and resuspended in sterile water to a bacterial concentration of 5×10 ⁸ cells/mL. Ultrasonic disruption was performed under the following conditions: ice bath of the sample, 100W, ultrasonication for 3s, stop for 3s, and repeat ultrasonication 100 times. Centrifugation was performed at 12000rpm for 5min, and the supernatant was the Candida albicans antigen CaAg.

CaAg, MagMSN and ParV vesicles prepared in Example 2 were fully mixed in PBS at a mass ratio of 1:2:0.5 to obtain an anti-Candida albicans vaccine CaAg+MagParV. At the same time, controls with mass ratios of 1:1:1 and 1:2:1 were set.

Transmission electron microscopy, Zeta potential analysis, and antigen release analysis were used to characterize the anti-Candida albicans vaccine.

(3) Performance evaluation of anti-Candida albicans vaccines

Balb/c female mice were inoculated with 35 μg CaAg+MagParV on day -14 (14 days before the inoculation of Candida albicans) and day -7 (7 days before the inoculation of Candida albicans) and treated with an alternating magnetic field (100-400 Hz, 100 W) for 10 min to complete the immunization. Candida albicans SC5314 cells were cultured in YPD medium with shaking at 30° overnight, and the bacterial solution concentration was adjusted to 5×10 ⁷ cells/mL in physiological saline. On day 0, the Candida albicans suspension was injected intravenously into non-immunized mice and immunized mice (100 μL/mouse), and the survival rate was recorded. Mouse sera were collected on days 0, 2, 4, 8, and 20, and the anti-CaAg IgG levels of infected mice were detected by ELISA.

2. Experimental results

(1) Characterization of broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvants

Transmission electron microscopy observations show that the diameter of the MnFe ₂ O ₄ nanoparticles is about 9 to 11 nm. Magnetothermal experiments show that the magnetic core has good magnetothermal conversion capabilities. After 10 minutes of alternating magnetic field treatment, the solution temperature can reach 25 to 48°C (Figure 1).

Transmission electron microscopy observation and energy spectrum analysis showed that MagMSN was round in shape, with a particle size of 100-200nm and a pore size of 10-20nm. There were multiple _{MnFe2O4 nanoparticle} clusters in the center of each MagMSN. MagParV was round in shape, with an obvious coating layer on the surface. Compared with MagMSN, its particle size was slightly increased to 110-220nm (Figure 2).

Zeta potential analysis results showed that the Zeta potential of MagMSN was positive (+26 mV), while that of MagParV was negative (-10 mV) ( Figure 2 ).

Compared with the vaccine group with a CaAg, MagMSN, and ParV vesicle mass ratio of 1:2:0.5, the control groups with a ratio of 1:1:1 and 1:2:1 had obvious antigen or vesicle distribution outside the nanoparticles, indicating that there was an excess of antigens or vesicles in the two control groups, thus determining that the optimal mass ratio of the three components was 1:2:0.5.

To study the antigen loading capacity of the broad-spectrum ER-targeted magnetic vesicle adjuvant, MagMSN and MagParV were incubated in different antigen solutions for 24 h, including ovalbumin (OVA), bovine serum albumin (BSA), Candida albicans antigen (CaAg), Staphylococcus aureus antigen (SaAg), avian influenza virus antigen (AIVAg) and SARS-CoV-2s protein receptor binding domain (SRBD). The results showed that MagParV loaded a higher level of antigen than MagMSN (Figure 2), indicating that MagParV has a higher antigen loading capacity. To study the antigen release capacity of the broad-spectrum ER-targeted magnetic vesicle adjuvant, the antigen-loaded MagParV was stimulated with an alternating magnetic field (400 Hz, 100 W). After 30 min, the antigen release rate of MagParV reached 45%, while that of MagMSN was only 15% (Figure 2), indicating that MagParV has a higher ability to release antigens in response to alternating magnetic fields. Since both MagMSN and MagParV contain superparamagnetic MnFe ₂ O ₄ nanoparticles, they can generate heat under alternating magnetic field treatment conditions, and the heat then promotes the release of antigens.

(2) Immune protection against infection by anti-Candida albicans vaccines

The results of serum specific antibody detection by ELISA showed that only a low level of IgG was produced after vaccination with free CaAg antigen, with a dilution titer of 50,000; the IgG level increased after CaAg antigen combined with CFA or MagMSN, with a dilution titer of 100,000 to 200,000, and the IgG level produced by combining with MagParV was even higher, with a dilution titer of >200,000. After alternating magnetic field treatment (50 to 500,000 Hz, 10 to 5000 W, 1 to 60 min; the optimal parameters were 400 Hz, 100 W, 10 min), the IgG level was significantly increased, with a dilution titer of >800,000 (Figure 3). The above results show that MagParV under alternating magnetic field treatment can induce the highest level of CaAg IgG production.

After systemic infection with Candida albicans, all unvaccinated mice died within 4 days, mice vaccinated with CaAg, CaAg+CFA, CaAg+MagMSN, and CaAg+MagMSN+alternating magnetic field died within 7 days, and mice vaccinated with CaAg+MagParV died within 3 to 10 days. In comparison, CaAg+MagParV+alternating magnetic field had a strong inhibitory effect on mouse death, and 70% of mice were still alive on the 20th day after infection (Figure 4). The above results show that the anti-Candida albicans vaccine has a significant protective effect against Candida albicans infection and can effectively protect mice from severe systemic infection with Candida albicans.

Example 5 Preparation of anti-Staphylococcus aureus vaccine based on endoplasmic reticulum-targeted magnetic vesicles

1 Test method

(1) Synthesis and characterization of anti-Staphylococcus aureus vaccine:

Centrifuge the Staphylococcus aureus cells cultured overnight at 37°C and resuspend in sterile water to a bacterial concentration of 2×10 ⁸ cells/mL. Ultrasonic disruption, ultrasonic conditions: ice bath sample, 100W, ultrasonic for 3s, stop for 3s, repeat ultrasonication 100 times. Centrifuge at 12000rpm for 5min, the supernatant is the Staphylococcus aureus antigen SaAg.

SaAg, MagMSN prepared by the method described in Example 4, and ParV vesicles prepared in Example 2 were fully mixed in PBS at a mass ratio of 1:2:1 to obtain an anti-Staphylococcus aureus vaccine SaAg+MagParV. The anti-Staphylococcus aureus vaccine was characterized by transmission electron microscopy, Zeta potential analysis, antigen release analysis, and other methods.

(2) Evaluation of the anti-infection performance of anti-Staphylococcus aureus vaccines:

Balb/c female mice were inoculated with SaAg+MagParV+alternating magnetic field (10μg+25μg, alternating magnetic field treatment for 10 min) on days -14 and -7 to complete immunization. Mouse sera were collected on days 0, 2, 4, 8, and 20, and the anti-SaAg IgG levels of infected mice were detected by ELISA.

2 Test results

(1) Characterization of broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvants

Same as Example 4.

(2) Immunoprotective effect of anti-Staphylococcus aureus vaccine on infection

The results of ELISA for detecting serum specific antibodies showed that vaccination with free SaAg antigen only produced a low level of serum IgG, with a dilution titer of <100,000; the IgG level increased after SaAg combined with CFA or MagMSN, with a dilution titer of 100,000 to 200,000, and the IgG level produced by combining with MagParV was even higher, with a dilution titer of >200,000. After treatment with an alternating magnetic field, the IgG level was significantly increased, with a dilution titer of >650,000 (Figure 5). The above results show that MagParV under alternating magnetic field treatment can induce the highest level of SaAg IgG production.

Example 6 Preparation of endoplasmic reticulum-targeted magnetic vesicle anti-tumor vaccine

1 Test method

(1) Synthesis and characterization of anti-tumor vaccines:

The GL261 glioma cells cultured at 37°C for 48 hours were centrifuged, resuspended in sterile water, and ultrasonically disrupted. Centrifuged at 5000rpm for 1 minute, and the supernatant was the GL261 glioma antigen GLAg. GLAg, MagMSN, and ParV vesicles were fully mixed in PBS at a mass ratio of 1:2:1 to obtain the glioma vaccine GLAg+MagParV. Transmission electron microscopy, Zeta potential analysis, antigen release analysis and other methods were used to characterize the anti-tumor vaccine.

(2) Evaluation of anti-infection performance of anti-tumor vaccines:

C57 female mice were inoculated with GLAg+MagParV+alternating magnetic field (10μg+25μg, alternating magnetic field treatment for 10min) on days -14 and -7 to complete immunization. Mouse sera were collected on days 0, 10, and 20, and the anti-GLAg IgG levels of infected mice were detected by ELISA. On day 30, GL261 cells were inoculated in the right axilla of immunized mice at a number of 1×10 ⁶ cells/mouse. On day 50, the tumors were removed and weighed to analyze the inhibitory effect of the anti-tumor vaccine on tumorigenesis.

2 Test results

(1) Characterization of broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvants

Same as Example 1.

(2) The inhibitory effect of anti-tumor vaccines on tumorigenesis

20 days after the inoculation of the tumor in each immunized mouse, the tumor was weighed. The tumor weights of the free GLAg group and the GLAg+CFA group were 0.89 times and 0.51 times that of the non-immunized control group, respectively, while the tumor weight of the GLAg+MagParV+alternating magnetic field treatment group was only 0.18 times that of the non-immunized control group (Figure 6), indicating that the anti-tumor vaccine GLAg+MagParV combined with the alternating magnetic field has a significant inhibitory effect on tumor occurrence.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (10)

1. An endoplasmic reticulum targeting artificial protein is characterized in that the amino acid sequence is shown as SEQ ID NO.2 from the N end to the C end.

2. The endoplasmic reticulum targeting artificial protein coding gene of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 3.

3. A recombinant saccharomyces cerevisiae, which is characterized by taking ura 3-defective saccharomyces cerevisiae as an original cell and comprising a recombinant plasmid; the recombinant plasmid has the coding gene of claim 2 inserted therein.

4. Use of the artificial protein of claim 1 or the coding gene of claim 2 or the recombinant saccharomyces cerevisiae of claim 3 for the preparation of endoplasmic reticulum targeting vesicles.

5. The preparation method of the endoplasmic reticulum targeting vesicle is characterized by comprising the following steps of:

Inoculating the recombinant saccharomyces cerevisiae of claim 3 into an SC-Gal medium for a first induction culture;

inoculating the recombinant saccharomyces cerevisiae subjected to the first induction culture to an SC-Gal culture medium without an amino-free yeast nitrogen source, and performing a second induction culture;

Centrifuging the culture after the second induction culture, collecting precipitated yeast cells, and extracting protoplasts of the yeast cells to obtain yeast protoplasts;

Crushing and ultracentrifugation are sequentially carried out on the yeast protoplast, and sediment is collected to obtain a crude product of the endoplasmic reticulum targeting vesicle;

Purifying the crude vesicle product by adopting a nickel column to obtain an endoplasmic reticulum targeting vesicle;

The SC-Gal culture medium takes water as a solvent and comprises the following components in concentration: 6.7g/L of nitrogen source of the yeast without amino group, 20g/L of galactose and 2g/L of mixed amino acid;

the SC-Gal culture medium without the nitrogen source of the amino-free yeast takes water as a solvent and comprises the following components in concentration: galactose 20g/L and mixed amino acid 2g/L;

the mixed amino acid comprises the following components in parts by mass: 2 parts of L-proline, 10 parts of L-leucine, 2 parts of L-valine, 2 parts of L-alanine, 2 parts of L-serine, 2 parts of L-lysine, 2 parts of L-glutamic acid, 2 parts of L-methionine, 2 parts of L-threonine, 2 parts of L-glutamine, 2 parts of L-glycine, 2 parts of L-isoleucine, 2 parts of L-tryptophan, 2 parts of L-aspartic acid, 2 parts of L-tyrosine, 2 parts of L-phenylalanine, 2 parts of inositol, 2 parts of L-cysteine, 2 parts of p-aminobenzoic acid, 2 parts of L-asparagine and 0.5 part of adenine.

6. An endoplasmic reticulum targeting magnetic vesicle, which is characterized by comprising magnetic mesoporous silicon nano-particles and endoplasmic reticulum targeting vesicles coated in pore channels of the magnetic mesoporous silicon nano-particles, wherein the endoplasmic reticulum targeting vesicles are prepared by the preparation method of claim 5;

The magnetic mesoporous silicon nanoparticle is a magnetic mesoporous silicon nanoparticle MagMSN with a large-aperture MSN coated with MnFe ₂O₄.

7. The endoplasmic reticulum targeting magnetic vesicle as claimed in claim 6, wherein the preparation method of the magnetic mesoporous silicon nanoparticle comprises the following steps:

suspending MnFe ₂O₄ in chloroform to obtain MnFe ₂O₄ suspension;

Firstly mixing the MnFe ₂O₄ suspension, cetyltrimethylammonium bromide and water, and removing the chloroform to obtain a first mixed solution;

Mixing the first mixed solution, water, methanol and ethyl acetate for the second time to obtain a second mixed solution;

Thirdly mixing the second mixed solution, an ammonium hydroxide solution, tetraethoxysilane and 3-aminopropyl triethoxysilane to obtain a third mixed solution;

and centrifuging the third mixed solution, collecting precipitate, washing the precipitate, and removing cetyl trimethyl ammonium bromide to obtain the magnetic mesoporous silicon nanoparticle.

8. Use of an endoplasmic reticulum-targeted magnetic vesicle according to claim 6 or 7 for the preparation of an immunoadjuvant.

9. A vaccine comprising magnetic mesoporous silicon nanoparticles, and endoplasmic reticulum targeting vesicles and antigens coated in the pores of the magnetic mesoporous silicon nanoparticles, wherein the endoplasmic reticulum targeting vesicles are prepared by the preparation method of claim 5;

The magnetic mesoporous silicon nanoparticle is a magnetic mesoporous silicon nanoparticle MagMSN with a large-aperture MSN coated with MnFe ₂O₄.

10. The vaccine of claim 9, wherein the antigen comprises a pathogenic antigen or a tumor antigen.