CN116178571B

CN116178571B - Endoplasmic reticulum targeting artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum targeting vesicle, immunoadjuvant and vaccine

Info

Publication number: CN116178571B
Application number: CN202310144827.9A
Authority: CN
Inventors: 喻其林; 赵妍
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2023-02-21
Filing date: 2023-02-21
Publication date: 2024-05-28
Anticipated expiration: 2043-02-21
Also published as: CN116178571A

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

Endoplasmic reticulum targeting artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum targeting vesicle, immunoadjuvant and vaccine

Technical Field

The invention belongs to the technical field of biomedicine and drug delivery, and particularly relates to an endoplasmic reticulum targeting artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum targeting vesicle, an immunoadjuvant and a vaccine.

Background

Endoplasmic reticulum (Endoplasmic reticulum, ER), one of the most important membranous organelles in eukaryotic cells, is the primary site of protein folding and transport, lipid synthesis, vesicle transport, calcium ion storage, and is involved in the regulation of a variety of signal transduction pathways. Furthermore, the endoplasmic reticulum also establishes a connecting site with other membranous structures such as plasma membrane, mitochondria, endocytosis, lysosome and the like through a developed extension structure, and plays an important role in the information, substance and energy exchange process of intracellular organelles and plasma membranes. Given the extreme importance of the endoplasmic reticulum to the metabolic processes of eukaryotic cell growth, disorders in structure and function will have a significant impact on eukaryotic cells and organisms. Numerous studies have found that endoplasmic reticulum dysfunction is closely linked to numerous major human diseases found to date, such as cancer, autoimmune diseases, pathogenic microbial infections, neurodegenerative diseases, diabetes, etc. Along with the continuous development of accurate medical theory and technology, the development of drugs with accurate endoplasmic reticulum targeting becomes an important trend for preventing and overcoming related diseases.

The endoplasmic reticulum is inseparable from the synthesis, processing and transport of intracellular proteins. Based on the search for intracellular transport of proteins, more signal peptides with endoplasmic reticulum tropism were found. These endoplasmic reticulum signal peptides are modified at the N-terminus of the protein and perform different functions depending on their sequence. Endoplasmic reticulum signal peptides include KDEL peptides, eriss peptides, pardaxin peptides, and the like. Of these, pardaxin peptide (GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE, SEQ ID No. 1) is a natural antimicrobial peptide with endoplasmic reticulum localization capability, and its endoplasmic reticulum targeting mechanism may be related to efficient phosphatidylcholine vesicle pore-forming capability (phosphatidylcholine is the main glycerophospholipid of endoplasmic reticulum, 60% in ratio).

At present, no description has been made of the preparation of endoplasmic reticulum targeting vesicles using Pardaxin peptides.

Disclosure of Invention

In view of the above, the present invention aims to provide an endoplasmic reticulum targeting artificial protein, recombinant saccharomyces cerevisiae, endoplasmic reticulum targeting vesicle, immunoadjuvant and vaccine, and the endoplasmic reticulum targeting artificial protein can be used for preparing the endoplasmic reticulum targeting vesicle.

The invention provides endoplasmic reticulum targeting artificial protein, which has an amino acid sequence shown as SEQ ID NO.2 from N end to C end.

The invention also provides a coding gene of the endoplasmic reticulum targeting artificial protein, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.

The invention also provides a recombinant saccharomyces cerevisiae, which takes ura 3-defective saccharomyces cerevisiae as an original cell and comprises recombinant plasmids; the recombinant plasmid is inserted with the coding gene of the scheme.

The invention also provides application of the artificial protein or the coding gene or the recombinant saccharomyces cerevisiae in preparation of endoplasmic reticulum targeting vesicles.

The invention also provides a preparation method of the endoplasmic reticulum targeting vesicle, which comprises the following steps:

inoculating the recombinant saccharomyces cerevisiae in the scheme to an SC-Gal culture medium, and performing first induction culture;

Inoculating the recombinant saccharomyces cerevisiae subjected to the first induction culture to an SC-Gal culture medium without a 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.

The invention also provides an endoplasmic reticulum targeting magnetic vesicle, which comprises 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 according to the scheme.

Preferably, the inner core of the magnetic mesoporous silicon nanoparticle is MnFe ₂O₄ nanoparticle; the shell of the magnetic mesoporous silicon nanoparticle is mesoporous silicon dioxide; the preparation method of the magnetic mesoporous silicon nanoparticle comprises the following steps:

Suspending MnFe ₂O₄ in an organic solvent to obtain a suspension;

Firstly mixing the suspension, the pore-forming agent and water, and removing the organic solvent 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 the pore-forming agent to obtain the magnetic mesoporous silicon nanoparticle.

The invention also provides application of the endoplasmic reticulum targeting magnetic vesicle in preparation of an immune adjuvant.

The invention also provides a vaccine, which comprises magnetic mesoporous silicon nano particles, and endoplasmic reticulum targeting vesicles and antigens coated in pore channels of the magnetic mesoporous silicon nano particles, wherein the endoplasmic reticulum targeting vesicles are prepared by the preparation method according to the scheme.

Preferably, the antigen comprises a pathogenic antigen or a tumor antigen.

The invention provides endoplasmic reticulum targeting artificial protein, which has an amino acid sequence shown as SEQ ID NO.2 from N end to C end. The endoplasmic reticulum targeting artificial protein contains an N-terminal 6 XHis tag, pardaxin peptide, green fluorescent protein and Saccharomyces cerevisiae Atg8 fragment. 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 endoplasmic reticulum targeting magnetic vesicle is a broad-spectrum endoplasmic reticulum targeting magnetic vesicle adjuvant and can load antigens. 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.

Drawings

FIG. 1 is a representation of MnFe ₂O₄ nanoparticles; wherein A is a transmission electron microscope image, and B is a magnetocaloric curve;

FIG. 2 is a representation of MagMSN and MagParV, where A is energy spectrum analysis, B is transmission electron microscopy, C is Zeta potential, D is antigen loading capacity, and E is antigen release capacity;

FIG. 3 shows serum CaAg IgG levels of mice after immunization with free antigen or antigen-loaded adjuvant;

FIG. 4 is a mouse survival curve;

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

figure 6 shows the inhibition of tumor growth in mice by free antigen and antigen-loaded adjuvant immunization.

Detailed Description

The invention provides endoplasmic reticulum targeting artificial protein ParGA, which has an amino acid sequence shown in SEQ ID NO.2 from N end to C end, and specifically comprises the following components:

MHHHHHHGFFALIPKIISSPLFKTLLSAVGSALSSSGGQEGGGGGGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGMKSTFKSEYPFEKRKAESERIADRFKNRIPVICEKAEKSDIPEIDKRKYLVPADLTVGQFVYVIRKRIMLPPEKAIFIFVNDTLPPTAALMSAIYQEHKDKDGFLYVTYSGENTFGR.

The endoplasmic reticulum targeting artificial protein contains an N-terminal 6 XHis tag, pardaxin peptide, green fluorescent protein and Saccharomyces cerevisiae Atg8 fragment. The 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 to form endoplasmic reticulum targeting vesicles with Pardaxin targeting polypeptides displayed on the surfaces. The 6 XHis tag and the green fluorescent protein respectively play roles of separating and purifying the tag and identifying.

The invention also provides a coding gene PARGA of the endoplasmic reticulum targeting artificial protein, which has a nucleotide sequence shown as SEQ ID NO.3, and specifically comprises the following steps:

atgcatcatc accaccatca cggtttcttt gctttgatcc caaagattat ctcttctccattgtttaagacattgttgtc agctgtcggt tccgctttgt cctcttccgg tggtcaagaaggtggcgggg gtggtggtgtgtccaaaggt gaagaattat tcactggtgt tgttccaatcttggttgaat tggatggtga tgttaacggtcacaagttct ccgtttctgg tgaaggtgaaggtgatgcta cctacggtaa gttgaccttg aaattcatctgtaccaccgg taagctgcccgtcccttggc caaccttggt caccactttg acctacggtg ttcaatgtttctctagatacccagaccaca tgaagcaaca cgacttcttc aagtccgcta tgccagaaggttacgtccaagaaagaacca ttttcttcaa ggatgacgga aactataaga ccagagctgaagtcaagtttgaaggtgaca ctttggtcaa ccgtatcgaa ttgaagggta ttgatttcaaggaagacggtaacatcttag gtcacaaatt ggaatacaac tacaactctc acaacgtctacattatggctgacaagcaaa agaacggtat caaggtcaat ttcaagatca gacacaacattgaggatggttctgtccaat tggctgatca ttaccaacaa aacactccaa ttggtgacggtccagtcttactacctgaca accactactt atccactcaa tctgctctct ccaaggacccaaacgaaaagagagatcaca tggttttgtt ggaatttgtt actgctgctg gtatcactttgggtatggacgaattgtaca agggtggtgg tatgaaatct actttcaaga gtgaatatccattcgaaaagagaaaggccg aatctgaacg tattgctgac agattcaaga acagaatcccagttatctgtgaaaaggccg aaaagtctga cattccagaa attgacaaaa gaaagtacttagttccagctgatttgactg ttggtcaatt cgtttacgtt attcgtaaga gaatcatgttgccaccagaaaaagctatat tcatttttgt taatgacacc ctacctccaa ctgccgccttgatgtctgccatctaccaag aacacaagga caaggatggt ttcttgtacg ttacttactctggtgaaaacaccttcggta gataa.

the coding gene is obtained based on optimization of the optimal codon of saccharomyces cerevisiae.

In the invention, the original plasmid of the recombinant plasmid is an expression plasmid of saccharomyces cerevisiae, preferably a plasmid pESC-URA of an inducible promoter PGAL1, and when the pESC-URA is taken as the original plasmid, the insertion site of the coding gene is BamH1/Xho1.

In the present invention, the ura 3-deficient Saccharomyces cerevisiae is preferably Saccharomyces cerevisiae Sc0, a chassis cell, genotype: MATahis.DELTA.1leu2 trp1-289 ura3-52.

The method for constructing the recombinant plasmid and the recombinant saccharomyces cerevisiae is not particularly limited, and conventional methods in the field can be adopted.

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;

the recombinant saccharomyces cerevisiae disclosed by the scheme is inoculated to an SC-Gal culture medium for first induction culture.

Before inoculating the recombinant saccharomyces cerevisiae into the SC-Gal medium, preferably further comprising inoculating a strain of the recombinant saccharomyces cerevisiae 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 ℃; the mode of the expansion culture is preferably shake culture; the time for the expansion culture is preferably 9 to 16 hours, more preferably 12 hours.

In the invention, 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 water is preferably distilled water; the pH value of the SC-Gal culture medium is preferably 6.0-6.5.

In the present invention, the amino-free yeast nitrogen source is preferably yeast nitrogen base (without amino acids), available from Solarbio under the specification of 500 g and model Y8040.

In the present invention, the temperature of the first induction culture is preferably 30 ℃; the first induction culture mode is preferably shake culture; the time of the first induction culture is preferably 24 hours; the first induction culture functions to induce ParGA expression under the control of the GAL10 promoter.

After the first induction culture, the recombinant saccharomyces cerevisiae after the first induction culture is inoculated into an SC-Gal culture medium without a nitrogen source for the second induction culture.

In the invention, the SC-Gal culture medium which does not contain an amino-free yeast nitrogen source takes water as a solvent and comprises the following components in concentration: galactose 20g/L and mixed amino acid 2g/L; the water is preferably distilled water; the pH value of the SC-Gal culture medium without the nitrogen source of the amino-free yeast is preferably 6.0-6.5;

In the invention, 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.

In the present invention, the temperature of the second induction culture is preferably 30 ℃; the second induction culture mode is preferably shake culture, and the rotation speed of the shake culture is preferably 100-140 rpm; the time of the second induction culture is preferably 2 hours; the second induction culture is used for inducing ParGA protein containing Atg8 fragment to form autophagosome vesicles through autophagy pathway in a rapid self-assembly mode.

After the second induction culture is carried out, the culture after the second induction culture is centrifuged, precipitated yeast cells are collected, protoplasts of the yeast cells are extracted, and the yeast protoplasts are obtained. In the present invention, the rotational speed of the centrifugation is preferably 4200rpm, and the time of the centrifugation is preferably 3min. The invention preferably further comprises washing the collected yeast cells prior to extracting the protoplasts; the reagent used for the washing is preferably a protoplast preparation buffer; the protoplast preparation buffer uses water as a solvent, and preferably comprises the following components in concentration: sorbitol 1M, trisodium citrate 0.02M, EDTANa ₂ 0.1.1: 0.1M and Na ₂HPO₄·12H₂ O0.02M.

In the invention, the extracting protoplast preferably comprises mixing yeast cells after the second induction culture with enzyme for enzymolysis to obtain protoplast suspension, centrifuging the protoplast suspension, and collecting precipitated yeast protoplast; the enzyme preferably comprises yeast muramidase or helicase; the working concentration of the yeast muramidase is preferably 100U/mL; the working mass concentration of the snailase is preferably 2% (W/V); the enzymolysis time is preferably 10-30 min, more preferably 20min; the rotational speed of the centrifugation is preferably 4200rpm; the time of the centrifugation is preferably 5min.

After the yeast protoplast is obtained, the yeast protoplast is crushed and ultracentrifuged in sequence, and sediment is collected to obtain a crude product of the endoplasmic reticulum targeting vesicle.

In the present invention, the crushing mode is preferably homogenizing and crushing by using a Dounce homogenizer. In the present invention, the rotational speed of the ultracentrifugation is preferably 35000rpm; the time of the ultracentrifugation is preferably 30 to 120min, more preferably 60 to 90min.

After obtaining the crude product of the endoplasmic reticulum targeting vesicle, the invention adopts a nickel column to purify the crude product of the vesicle to obtain the endoplasmic reticulum targeting vesicle.

In the present invention, the purification of the crude vesicle product using a nickel column preferably comprises: suspending the crude endoplasmic reticulum targeting vesicle in 10mM imidazole buffer, adding into a nickel column for vesicle adsorption, washing with 10mM imidazole buffer, and eluting with 100mM imidazole; the number of times of washing is preferably 3.

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

In the invention, the endoplasmic reticulum targeting magnetic vesicle is circular; the particle size of the endoplasmic reticulum targeting magnetic vesicle is 100-200 nm; the aperture of the endoplasmic reticulum targeting magnetic vesicle is 10-20 nm, and a plurality of MnFe ₂O₄ nanoparticle clusters are arranged in the center of each MagMSN; the Zeta potential of MagMSN was positive (+26 mV).

In the invention, the inner core of the magnetic mesoporous silicon nanoparticle is MnFe ₂O₄ nanoparticle; the shell of the magnetic mesoporous silicon nanoparticle is mesoporous silicon dioxide; the magnetic mesoporous silicon nanoparticle is preferably prepared by the following method:

Suspending MnFe ₂O₄ in an organic solvent to obtain a suspension;

Firstly, ferromanganese (MnFe ₂O₄) is suspended in an organic solvent to obtain a suspension.

In the invention, the MnFe ₂O₄ is preferably obtained by carrying out thermal decomposition reaction by taking trivalent organic iron and divalent organic manganese as an iron source and a manganese source respectively; the MnFe ₂O₄ is a magnetic nanoparticle, the diameter of the MnFe ₂O₄ nanoparticle is 9-11 nm, and the MnFe ₂O₄ nanoparticle has good magnetocaloric conversion capability.

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 comprises a first thermal decomposition reaction and a second thermal decomposition reaction which are sequentially carried out; the temperature of the first thermal decomposition reaction is preferably 200 ℃; the time of the first thermal decomposition is preferably 1h; the temperature of the second thermal decomposition reaction is preferably 298 ℃; 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 MnFe ₂O₄ to the volume of the organic solvent is preferably 5mg:2mL.

After the suspension is obtained, the suspension, the pore-forming agent and water are mixed for the first time, and the organic solvent is removed to obtain a first mixed solution. In the present invention, the pore-forming agent is preferably cetyl trimethylammonium bromide (CTAB); the water is preferably distilled water; the pore-forming agent is preferably used in an amount of 200mg and the water is preferably used in an amount of 10mL, based on the mass of MnFe ₂O₄ being 5 mg; the first mixing mode is preferably ultrasonic mixing; the time of the first mixing is preferably 20min; the organic solvent is preferably removed by evaporation of the organic solvent by heating to 75 ℃.

After the first mixed solution is obtained, the first mixed solution, water, methanol and ethyl acetate are mixed for the second time to obtain a second mixed solution. In the present invention, the water is preferably distilled water; the volume of the first mixed solution, water, methanol and ethyl acetate is preferably 10:95:5:20, a step of; the second mixing mode is preferably magnetic stirring mixing; the time of the second mixing is preferably 10 minutes.

After the second mixed solution is obtained, the second mixed solution, ammonium hydroxide solution, tetraethoxysilane (TEOS) and 3-aminopropyl triethoxysilane (APTES) are mixed in a third mode to obtain a third mixed solution. In the invention, the ammonium hydroxide solution is ammonia water, and the dosage of the ammonium hydroxide solution is preferably 3mL according to the volume of the first mixed solution being 10 mL; the TEOS is preferably used in an amount of 300. Mu.L; the dosage of APTES is preferably 30 mu L; the third mixing mode is preferably magnetic stirring mixing, and mesoporous silicon grows on the surfaces of the magnetic nano particles in the magnetic stirring mixing process; the time of the third mixing is preferably 12 hours.

After the third mixed solution is obtained, the invention carries out centrifugation on the third mixed solution, collects sediment, washes the sediment and removes pore-forming agent to obtain the magnetic mesoporous silicon nano particles. In the invention, the rotation speed of the centrifugation is preferably 8000-10000 rpm, and the centrifugation time is preferably 10min; the reagent used for the washing is preferably ethanol; the removal of the pore-forming agent preferably comprises heating with an ammonium nitrate solution having a concentration of 200mg/100mL to remove the pore-forming agent from the pores; the solvent of the ammonium nitrate solution is preferably ethanol.

In the invention, mnFe ₂O₄ is an initial magnetic nanoparticle, after ammonium hydroxide solution, tetraethoxysilane and 3-aminopropyl triethoxysilane are added, the surface oxygen atoms of the nanoparticle react with tetraethoxysilane, mesoporous silica is derived on the basis, and finally the magnetic mesoporous silica nanoparticle with a core-shell structure is obtained, wherein the inner core of the magnetic mesoporous silica nanoparticle is MnFe ₂O₄ nanoparticle, and the outer shell of the magnetic mesoporous silica nanoparticle is mesoporous silica.

In the invention, the magnetic mesoporous silicon nanoparticle is a magnetic mesoporous silicon nanoparticle MagMSN with a large-aperture MSN wrapped MnFe ₂O₄.

The invention also provides application of the endoplasmic reticulum targeted magnetic vesicle in preparation of an immunoadjuvant and/or an anti-tumor drug.

The immune adjuvant is a broad-spectrum endoplasmic reticulum targeting magnetic vesicle adjuvant and can load antigen. The immunoadjuvant of the invention has higher antigen loading capacity, obviously induces the generation of antigen specific antibodies of fungi, bacteria and viruses under the treatment of alternating magnetic field, and can effectively protect organisms from serious pathogenic bacteria systemic infection. 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.

In the present invention, the antitumor drug achieves an antitumor effect by inhibiting tumor growth.

In the invention, both the endoplasmic reticulum targeting vesicle and the antigen are adsorbed into the pores of the magnetic mesoporous silicon nanoparticle.

In the present invention, the preparation method of the magnetic mesoporous silicon nanoparticle is the same as the above scheme, and will not be described here again.

In the present invention, the antigen preferably includes a pathogenic bacteria antigen or a tumor antigen; the pathogen antigen preferably comprises a pathogen protein antigen and or an inactivated virus antigen, more preferably comprises a pathogen protein; the pathogen proteins preferably include 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 comprises a brain glioma antigen; the glioma antigen preferably comprises a glioma GL261 antigen.

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

The invention also provides a preparation method of the vaccine according to the scheme, which comprises the following steps:

and mixing the magnetic mesoporous silicon nanoparticle, the antigen and the endoplasmic reticulum targeting vesicle to obtain the vaccine.

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

The invention also provides a using method of the vaccine, which comprises the following steps:

the vaccine is administered to the subject.

In the present invention, the amount of vaccination is preferably 20 to 100. Mu.g, more preferably 35 to 50. Mu.g, per mouse based on the total mass of antigen and endoplasmic reticulum targeting magnetic vesicles.

After inoculation, it is preferable to further include treating the vaccinated subject 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 500000Hz, more preferably 400 to 10000Hz; the power of the alternating magnetic field is preferably 10 to 5000W, more preferably 100 to 1000W.

In the invention, the vaccine contains superparamagnetism MnFe ₂O₄ nano particles, so that heat can be generated under the alternating magnetic field treatment condition, the heat can promote the release of antigen, activate the immunogenicity of antigen, obviously induce the generation of specific antibodies of fungi, bacteria, viruses and tumor antigens, effectively prevent pathogenic bacteria infection and inhibit tumor growth.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.

EXAMPLE 1 preparation of recombinant Saccharomyces cerevisiae cells

Endoplasmic reticulum targeting artificial protein ParGA (the amino acid sequence is shown as SEQ ID NO. 2) is designed, and contains an N-terminal 6×His tag, pardaxin, green fluorescent protein and Saccharomyces cerevisiae Atg8 fragment. Based on the optimal codon of Saccharomyces cerevisiae, an artificial gene PARGA (the nucleotide sequence of which is shown as SEQ ID NO. 3) for encoding ParGA protein is designed. The artificial gene ParGA was cloned on plasmid pESC-URA from inducible promoter PGAL1 to obtain plasmid pESC-ParGA, and transformed into Saccharomyces cerevisiae Sc0 (MATA his 3.DELTA.1leu2 trp1-289 URA 3-52) as a chassis cell to obtain recombinant Saccharomyces cerevisiae cell Sc0+ ParV.

Example 2 preparation of biosynthetic vesicles

Reagent:

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

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

Protoplast preparation buffer formulation: with water as solvent, sorbitol 1M, trisodium citrate 0.02M, EDTANa ₂ 0.1.1: 0.1M and Na ₂HPO₄·12H₂ O0.02M.

The method comprises the following steps:

The synthetic yeast cells Sc0+ ParV were shake-cultured in YPD medium at 30℃overnight, transferred to SC-Gal medium and shake-cultured at 30℃for 24h. And transferring to SC-Gal culture medium without nitrogen source, and shake culturing for 2 hr to form autophagosome vesicle. Cells containing ParV were collected, washed with protoplast preparation buffer and then treated with yeast muramidase (100U/mL, 10 min) to obtain yeast protoplasts.

Breaking up yeast protoplasts with a Dounce homogenizer, ultracentrifugating at 35000rpm for 30min to separate ParV vesicles, finally suspending ParV vesicles in 10mM imidazole buffer, adding to a nickel column for vesicle adsorption, and then washing with 10mM imidazole buffer for 3 times; finally, 100mM imidazole was used to obtain purified ParV vesicles.

Example 3 preparation of biosynthetic vesicles

Example 2 was repeated except that the yeast muramidase (100U/mL, 10 min) treatment was replaced with the snailase (2%, 10 min) treatment.

Example 4 preparation of endoplasmic reticulum targeting magnetic vesicle-based anti-candida albicans vaccine

1. Preparation process

(1) Preparation of magnetic mesoporous silicon nano particles

Iron (III) acetylacetonate and manganese (II) acetylacetonate are used as an iron source and a manganese source, and a thermal decomposition method (200 ℃ C., 1h, then 298 ℃ C., 1 h) is adopted to synthesize the MnFe ₂O₄ magnetic nano-particles.

5Mg of MnFe ₂O₄ nanoparticles were suspended in 2mL of chloroform, added to 10mL of distilled water containing 200mg of CTAB, sonicated for 20min, and heated to 75℃to evaporate the chloroform. This solution was added to a mixture of 95mL of distilled water, 5mL of methanol and 20mL of ethyl acetate, magnetically stirred for 10min, and then 3mL of ammonium hydroxide, 300. Mu. LTEOS and 30. Mu. LAPTES were added. And magnetically stirring for 12h, centrifuging, and washing the precipitate with ethanol. CTAB in the pores was removed by heating with 100mL ethanol containing 200mg ammonium nitrate. Finally, the magnetic mesoporous silicon nanoparticle MagMSN with the MnFe ₂O₄ wrapped by the large-aperture MSN is obtained.

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

Candida albicans cells cultured overnight at 37℃were centrifuged and resuspended in sterile water at a concentration of 5X 10 ⁸ cells/mL. Ultrasonic crushing, wherein ultrasonic conditions are as follows: the sample was ice-bathed, 100W, sonicated for 3s, stopped for 3s, and sonicated 100 times. Centrifuging at 12000rpm for 5min, and collecting supernatant as Candida albicans antigen CaAg.

CaAg, magMSN and ParV vesicles prepared in example 2 were mixed in PBS at a mass ratio of 1:2: mixing thoroughly at a ratio of 0.5 to obtain anti-Candida albicans vaccine CaAg + MagParV. Simultaneously, the mass ratio is set to be 1:1: 1. 1:2:1 ratio control.

The candida albicans vaccine is characterized by adopting transmission electron microscope observation, zeta potential analysis, antigen release analysis and other methods.

(3) Performance evaluation of anti-candida albicans vaccine

Balb/c female mice were vaccinated with 35 μ g CaAg + MagParV on day-14 (14 days before the date of vaccination with Candida albicans) and day-7 (7 days before the date of vaccination with Candida albicans), treated with alternating magnetic fields (100-400 Hz, 100W) for 10min to complete immunization. Candida albicans SC5314 cells were cultured overnight in YPD medium with shaking at 30℃and the concentration of the bacterial solution was adjusted to 5X 10 ⁷ cells/mL in physiological saline. The candida albicans suspension was injected intravenously into non-immunized and immunized mice (100 μl/mouse) on day 0 and survival was recorded initially. Mouse serum was collected on day 0, day 2, day 4, day 8 and day 20, and infected mice were assayed for anti-CaAg IgG levels by ELISA.

2. Experimental results

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

The transmission electron microscope observation result shows that the diameter of MnFe ₂O₄ nano particles is about 9-11 nm, and the magnetocaloric experimental result shows that the magnetic core has good magnetocaloric conversion capability, and the solution temperature can reach 25-48 ℃ after the alternating magnetic field is treated for 10min (figure 1).

Transmission electron microscope observation and energy spectrum analysis show that MagMSN is round, the grain diameter is 100-200 nm, the aperture is 10-20 nm, and a plurality of MnFe ₂O₄ nanoparticle clusters are arranged in the center of each MagMSN; magParV are round, the surface has a distinct coating, and the particle size is slightly increased compared with MagMSN, and is 110-220 nm (figure 2).

The Zeta potential analysis results showed that MagMSN had a positive (+ 26 mV) Zeta potential and MagParV had a negative (-10 mV) potential (FIG. 2).

The mass ratio of the vesicle to CaAg, magMSN and ParV vesicles is 1:2: vaccine group 0.5, 1:1: 1. 1:2: in the control group with the ratio of 1, the nanoparticles have obvious antigen or vesicle distribution, which indicates that the antigen or vesicle in the two control groups is excessive, thereby determining that the optimal mass ratio of the three components is 1:2:0.5.

To investigate the antigen loading capacity of the broad spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvant, magMSN and MagParV were incubated in different antigen solutions for 24h, 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), and as a result, magParV was shown to be loaded with higher levels of antigen than MagMSN (fig. 2), indicating that MagParV has higher antigen loading capacity. To investigate the antigen release capacity of the broad-spectrum endoplasmic reticulum-targeted magnetic vesicle adjuvant, the antigen release rate of MagParV reached 45% after 30min using alternating magnetic field (400 hz,100 w) to stimulate antigen-loaded MagParV, whereas MagMSN was only 15% (fig. 2), indicating that MagParV has a higher alternating magnetic field response antigen release capacity. Since MagMSN and MagParV both contain superparamagnetic MnFe ₂O₄ nano particles, heat can be generated under the alternating magnetic field treatment condition, and then the heat promotes the release of antigens.

(2) Immunoprotection of infection by anti-candida albicans vaccine

ELISA method detection of serum specific antibody shows that the inoculation of free CaAg antigen only produces lower level IgG, and the dilution titer is 50000; the level of IgG increases after CaAg antigen binds to CFA or MagMSN, the dilution titer is 100000 ～ 200000, the level of IgG produced by binding to MagParV is higher, the dilution titer is >200000, the IgG level is significantly up-regulated after alternating magnetic field treatment (50-500000 Hz, 10-5000W, 1-60 min; the optimal parameters are 400Hz,100W,10 min), and the dilution titer is >800000 (FIG. 3). The above results indicate that MagParV under alternating magnetic field treatment can cause the highest level CaAg IgG to be produced.

After systemic infection with candida albicans, all unvaccinated mice died within 4 days, mice vaccinated with CaAg, caag+cfa, caag+magmsn, caag+ MagMSN + alternating magnetic fields died within 7 days, and mice vaccinated with CaAg + MagParV died within 3-10 days, in contrast to the CaAg + MagParV + alternating magnetic fields which had a strong inhibitory effect on the death of mice, with 70% of mice still surviving on day 20 post-infection (fig. 4). The above results indicate that: the anti-candida albicans vaccine has remarkable protection effect on candida albicans infection and can effectively protect mice from serious candida albicans systemic infection.

Example 5 preparation of endoplasmic reticulum-targeting magnetic vesicle anti-Staphylococcus aureus vaccine

1 Test method

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

Staphylococcus aureus cells cultured overnight at 37℃were centrifuged and resuspended in sterile water at a concentration of 2X 10 ⁸ cells/mL. Ultrasonic crushing, wherein ultrasonic conditions are as follows: the sample was ice-bathed, 100W, sonicated for 3s, stopped for 3s, and sonicated 100 times. Centrifuging at 12000rpm for 5min, and collecting supernatant as staphylococcus aureus antigen SaAg.

SaAg, magMSN prepared as described in example 4, parV vesicles prepared as in example 2 were combined in PBS according to 1:2:1 to obtain the anti-staphylococcus aureus vaccine SaAg + MagParV. The method adopts transmission electron microscope observation, zeta potential analysis, antigen release analysis and other methods to characterize the anti-staphylococcus aureus vaccine.

(2) Anti-infective performance evaluation of anti-staphylococcus aureus vaccine:

balb/c female mice were vaccinated with SaAg + MagParV + alternating magnetic field (10 μg+25 μg, alternating magnetic field treatment for 10 min) on day-14 and day-7 to complete immunization. Mouse serum was collected on day 0, day 2, day 4, day 8 and day 20, and infected mice were assayed for anti-SaAg IgG levels by ELISA.

2 Test results

Same as in example 4.

(2) Immunoprotection of infection by anti-staphylococcus aureus vaccine

ELISA method detection of serum specific antibody shows that the inoculation of free SaAg antigen only produces lower level serum IgG, and the dilution titer is less than 100000; saAg increased IgG levels after binding to CFA or MagMSN, the dilution titer was 100000 ～ 200000, the IgG levels generated by binding to MagParV were higher, the dilution titer was >200000, the IgG levels were significantly up-regulated after alternating magnetic field treatment, and the dilution titer was >650000 (fig. 5). The above results indicate that MagParV under alternating magnetic field treatment can cause the highest level SaAg IgG to be produced.

Example 6 preparation of endoplasmic reticulum-targeting magnetic vesicle anti-tumor vaccine

1 Test method

(1) Synthesis and characterization of antitumor vaccine:

Brain glioma GL261 cells cultured at 37 ℃ for 48 hours were centrifuged, resuspended in sterile water and sonicated. Centrifuging at 5000rpm for 1min, and collecting supernatant as brain glioma GL261 antigen GLAg. GLAg, magMSN, parV vesicles were taken up in PBS according to 1:2:1 to obtain the glioma vaccine GLAg + MagParV. The anti-tumor vaccine is characterized by adopting methods such as transmission electron microscope observation, zeta potential analysis, antigen release analysis and the like.

(2) Anti-infective performance evaluation of anti-tumor vaccine:

C57 female mice were vaccinated with GLAg + MagParV + alternating magnetic field (10 μg+25 μg, alternating magnetic field treatment for 10 min) on day-14 and day-7 to complete immunization. Mouse serum was collected on day 0, day 10 and day 20, and infected mice were tested for anti GLAg IgG levels by ELISA. On day 30, GL261 cells were inoculated in an amount of 1X 10 ⁶ cells/mouse on the right armpit of immunized mice. On day 50, tumors were removed and weighed and analyzed for inhibition of tumorigenesis by the anti-tumor vaccine.

2 Test results

As in the first embodiment.

(2) Inhibition of tumorigenesis by anti-tumor vaccine

After 20 days of tumor inoculation of each immunized mouse, tumors were weighed, and the weights of free GLAg group and GLAg +cfa group tumors were 0.89 times and 0.51 times that of the non-immunized control group, respectively, while the weights of GLAg + MagParV + alternating magnetic field treatment group tumors were only 0.18 times that of the non-immunized control group (fig. 6), indicating that the anti-tumor vaccine GLAg + MagParV had a significant inhibition effect on tumor generation after being combined with an alternating magnetic field.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Claims

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;

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;

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;

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