NL2034221B1 - Anti-h1n1 subtype influenza virus nanoparticles based on self-assembled ferritin, preparation method and application thereof - Google Patents
Anti-h1n1 subtype influenza virus nanoparticles based on self-assembled ferritin, preparation method and application thereof Download PDFInfo
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- NL2034221B1 NL2034221B1 NL2034221A NL2034221A NL2034221B1 NL 2034221 B1 NL2034221 B1 NL 2034221B1 NL 2034221 A NL2034221 A NL 2034221A NL 2034221 A NL2034221 A NL 2034221A NL 2034221 B1 NL2034221 B1 NL 2034221B1
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- influenza virus
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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Abstract
Disclosed are an anti-H1 N1 subtype influenza virus nanoparticles based on self-assembled 5 ferritin, a preparation method and an application thereof, and relate to the technical field of genetic engineering. The nanoparticles are formed by connecting a signal peptide-ST-FE fusion protein and a signal peptide-SC-HA-VHH fusion protein in vitro; the signal peptide-ST-FE fusion protein is obtained by connecting a signal peptide-SpyTag shown in SEQ ID NO: 3 to an N-terminal of a ferritin monomer subunit; the signal peptide-SC-HA-VHH fusion protein is obtained by connecting 10 the N-terminal of nano antibody against hemagglutinin protein of influenza type A (H1 N1) subtype virus with a signal peptide-SpyCatcher shown in SEQ ID NO: 4. The anti-H1 N1 subtype influenza virus nanoparticles of the present invention are of great significance for monitoring, prevention and treatment of the H1 N1 subtype influenza virus.
Description
ANTI-H1N1 SUBTYPE INFLUENZA VIRUS NANOPARTICLES BASED ON SELF-
ASSEMBLED FERRITIN, PREPARATION METHOD AND APPLICATION THEREOF
The invention relates to the technical field of genetic engineering, and in particular to an anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin, a preparation method and an application thereof.
Influenza is very common in actual production and life. It is caused by human or animal infection with influenza virus. The typical clinical symptoms of influenza include acute high fever, physical pain, fatigue and respiratory symptoms. Influenza itself has obvious seasonality, and it has a high incidence in autumn and winter. Some highly contagious viruses may cause influenza pandemic in a large scale or even in the world, and there are very serious complications and even individual deaths caused by different individual constitutions. Influenza viruses may be roughly divided into three different types, which are type A, type B and type C in turn.
H1N1 virus, a kind of influenza type A virus, is also one of the most commonly infected influenza viruses for humans. Many studies have found that influenza type A (H1N1) virus is a new type of influenza type A virus after mutation, and there are four sources of recombination in its genome, which is a mixture of human influenza, swine influenza and avian influenza virus genes. The PBI gene of this virus is derived from human influenza H3N2 virus, HA, NA, NP, NS and M genes are derived from swine influenza H1N1 virus, and PB2 and PA genes are derived from avian influenza H1N1 virus. Human influenza is mainly caused by influenza type A virus.
Seasonal influenza may cause 250,000 to 500,000 deaths worldwide every year, and the number of people who die from seasonal influenza in the United States may reach 3,000 to 49,000 every year. Influenza type A virus has many subtypes, and most antibodies induced by different subtypes may not play a cross-protection role.
For a long time, genetic engineering antibody is one of the main means and research hotspots for preventing and treating influenza type A (H1N1) virus. It is of great practical significance to develop anti-H1N1 subtype influenza virus nanoparticles based on self- assembled ferritin for the prevention and treatment of influenza type A (H1N1) influenza.
The objective of the present invention is to provide an anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin, and a preparation method and application thereof, so as to solve the problems existing in the prior art. The method combines the advantages of iron nanoparticles and anti-H1N1 subtype influenza virus nanoparticles to prepare anti-H1N1 subtype influenza virus nanoparticles, and it is of great significance for monitoring, prevention and treatment of the H1N1 subtype influenza virus.
In order to achieve the above objective, the present invention provides the following schemes.
The invention provides an anti-H1N1 subtype influenza virus nanoparticles based on self- assembled ferritin, wherein the nanoparticles are formed by connecting a signal peptide-ST-FE fusion protein and a signal peptide-SC-HA-VHH fusion protein in vitro; the signal peptide-ST-FE fusion protein is obtained by connecting a signal peptide-SpyTag shown in SEQ ID NO: 3 to an
N-terminal of a ferritin monomer subunit; the signal peptide-SC-HA-VHH fusion protein is obtained by connecting the N-terminal of nano antibody against hemagglutinin protein of influenza type A (H1N1) subtype virus with a signal peptide-SpyCatcher shown in SEQ ID NO: 4.
Further, an amino acid sequence of the signal peptide-SpyTag and the N-terminal connecting peptide of the ferritin monomer subunit is shown in SEQ ID NO: 5; the amino acid sequence of the N-terminal of the nano antibody against the hemagglutinin protein of influenza type A (H1N1) subtype virus and the connecting peptide of the signal peptide-SpyCatcher is shown in SEQ ID NO: 5.
Further, an amino acid sequence of the signal peptide-ST-FE fusion protein is shown in
SEQ ID NO: 1; and an amino acid sequence of the signal peptide-SC-HA-VHH fusion protein is shown in SEQ ID NO: 2.
Further, a nucleotide sequence encoding the signal peptide-ST-FE fusion protein is shown in SEQ ID NO: 10.
Further, a nucleotide sequence encoding the signal peptide-SC-HA-VHH fusion protein is shown in SEQ ID NO: 11.
The invention also provides a preparation method of the anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin, including the following steps: (1) respectively constructing genes encoding fusion proteins shown in SEQ ID NO: 1 and SEQ
ID NO: 2, then transferring the constructed genes into vectors to construct expression plasmids, and then transfecting to induce the expression of fusion proteins to obtain two kinds of fusion proteins; (2) connecting the two fusion proteins obtained in the step (1) in vitro to obtain the anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin.
Further, in the step (2), conditions of in vitro connection are: 4°C, and 16 h.
The invention also provides an application of the anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin in preparing medicines for treating H1N1 subtype influenza.
The fibronectin binding protein FbaB of Mlicrococcus scarlatinae contains a domain with spontaneous peptide bond between Lys and Asp. This protein contains two fragments: one is called SpyTag, and the other is called SpyCatcher. The reaction is carried out under different pH, temperature and buffer conditions. The reaction is simple and the yield is high. SpyTag may fuse at the end or inside, and produce specific reaction on the surface of mammalian cells.
Peptide binding may not be reversed by boiling or competing peptides.
There is a kind of natural heavy chain antibody (HCAb) that only contains heavy chain but not light chain in Camelidae (dromedary, bactrian camel, llama, etc.). Heavy chain antibody (HCAb) contains no L-chain polypeptide, and it is unique in that it lacks a first constant domain (CH1). In its N-terminal region, an H chain of homodimer protein contains a special variable domain called VHH, and the VHH is used to bind to homologous antigen of the HCAb.
After the variable region of HCAb in camel is cloned, a single domain antibody consisting of only one variable region of heavy chain is obtained, which is called variable domain of heavy chain of heavy chain antibody (VHH), with a diameter of 2.5 nm and a length of 4 nm, and is a smallest known unit that may bind the target antigen. Its molecular weight is only 1/10 of that of monoclonal antibody, and it is a smallest antibody unit with stable structure and antigen binding activity, so itis also called nanoantibody (Nb). As a small genetic engineering antibody, nanoantibody has the advantages of high expression, water solubility, stability, tissue penetration and weak immunogenicity, and these advantages make the antibody have broad application prospects in basic research and drug development. However, it is difficult to achieve the ideal therapeutic effect because of the phenomenon of immune escape of a single targeted nanoantibody of the nanoantibody.
Nanomaterials formed by self-assembled proteins not only have characteristics of good biocompatibility, uniform particle size and stability, but also have broad application prospects in cell imaging, lesion detection and drug sustained release.
Ferritin is an important functional protein that participates in and regulates storage and release of iron, and it is a kind of protein with high iron content which widely exists in animals, plants and microbial cells. Naturally synthesized ferritin mostly presents a hollow spherical nano-cage structure, with an outer diameter of 12 nm and an inner diameter of 8 nm. Its spherical structure consists of an inner core and an outer shell. The inner core is mainly composed of minerals, and the outer shell is assembled from 24 identical trimer subunits composed of every three ferritin subunits. When ferritin is used as a nano-carrier, it may wrap the target molecule inside the cage structure to realize the function of slow release or targeted release, and it may also fix the target molecule on the outer surface of the cage structure to realize the functions of stabilizing the structure and exposing the target protein.
The target protein is connected to the N-terminal of ferritin monomer subunit for fusion expression, so that the target antigen protein is anchored on the outer surface of the self- assembled ferritin nano-cage. Due to the unique assembly mode of ferritin subunit, it has obvious advantages for antigen expression with a natural conformation of trimer. With the self- assembly of ferritin monomer subunit into trimer, the N-terminal fusion expression antigen is very close in space, and it is easy to form a natural trimer structure. Such trimer reduces the natural conformation of antigen protein to the greatest extent, it is stable at the same time, and the immunogenicity is greatly enhanced compared with the single expression. Ferritin is resistant to high temperature and various denaturants because of its stability, and does not affect its natural structure.
The invention discloses the following technical effects.
A first objective of the present invention is to provide a method for realizing multimerization of nanoantibody, and another objective of the present invention is to provide nanoparticles that may be directly and specifically combined with the influenza type A (H1N1) virus.
In the invention, the signal peptide-SpyTag is connected to the N-terminal of ferritin monomer subunit for fusion expression by utilizing the spontaneous connection characteristic of
SpyTag and SpyCatcher in CnaB2 domain of Streptococcus pyogenes, and then the N-terminal of the nanoantibody of anti-H1N1 subtype influenza virus hemagglutinin protein is connected to the signal peptide-SpyCatcher for fusion expression. Finally, the two fusion proteins are connected in vitro, and the nanoantibody of anti-H1N1 subtype influenza virus hemagglutinin protein is displayed on the surface of self-assembled ferritin cage structure, thus achieving the preparation of the nanoparticles of anti-H1N1 subtype influenza virus.
Compared with prokaryotic expression, the expression product of CHO eukaryotic cell system expression is properly modified and distributed regionally, having weak immunogenicity and being not prone to rejection. In the process of protein expression, by introducing a signal peptide before the target protein, the target protein may be directly obtained in the cell culture solution, and this avoids cell fragmentation, improves the protein purification efficiency, is conducive to improving the purity of the protein, and further reduces the interference of non- target proteins, thus improving the connection efficiency between SpyTag and SpyCatcher.
Compared with traditional nanoantibodies, nanoparticles of anti-H1N1 subtype influenza virus hemagglutinin protein have higher sensitivity and therapeutic effect for the detection, diagnosis, prevention and treatment of the H1N1 subtype influenza virus. Anti-H1N1 subtype influenza virus nanoparticles may improve the accuracy and repeatability of detection and diagnosis of the H1N1 subtype influenza virus, being of great significance for monitoring the
H1N1 subtype influenza virus.
The anti-H1N1 subtype influenza virus nanoparticles based on self-assembled ferritin prepared by the invention are a kind of polymerized nanoantibody; compared with the H1N1 subtype influenza virus vaccine, the polymerized nanoantibody may directly treat diseases, thereby eliminating the immune process and shortening the treatment process; compared with the traditional nanoantibody, the polymerized nanoantibody has higher sensitivity and therapeutic effect on the detection, diagnosis, prevention and treatment of the H1N1 subtype influenza virus, especially the anti-H1N1 subtype influenza virus nanoparticles improve the accuracy and repeatability of the detection and diagnosis of the H1N1 subtype influenza virus, being of great significance for the monitoring of the H1N1 subtype influenza virus.
5 In order to explain the embodiment of the invention or the technical scheme in the prior art more clearly, the drawings used in the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the invention. For ordinary technicians in the field, other drawings may be obtained according to these drawings without making creative efforts.
Fig. 1 is a series structure diagram of gene signal peptide-ST-FE and signal peptide-SC-HA-
VHH.
Fig. 2 is an agar gel electrophoresis diagram of the target protein gene.
Fig. 3 is a linearized agar gel electrophoresis diagram of plasmid signal peptide-ST-FE and the signal peptide-SC-HA-VHH.
Fig. 4 is a protein Western Blot (WB) identification diagram of the target protein gene.
Fig. 5 is a cell expression identification diagram of signal peptide-ST-ferritin monomer subunit
RT-PCR.
Fig. 6 is a WB identification diagram of a connection between ferritin monomer subunit and
HA-VHH.
Fig. 7 is an electron microscope observation.
Fig. 8 is a cellular effect diagram of anti-H1N1 subtype influenza virus nanoparticles; PBS group is virus-free, PR8 is influenza virus-infected, and antivirus particles+PR8 are a combination incubation group of anti-virus particles and PR8 virus in vitro.
Fig. 9 is a trend diagram of weight change of mice.
Fig. 10 is a trend diagram of mouse survival rate.
Fig. 11 is an ELISA diagram of anti-H1N1 subtype influenza virus nanoparticle antigen recognition; Nbs-HA is a traditional nanoantibody against HA, and F-Nbs-HA is a nanoparticle against HA.
Fig. 12 is a comparison diagram of antigen recognition ability between anti-H1N1 subtype influenza virus nanoparticles and traditional nanoantibodies; Nbs-HA is a traditional nanoantibody against HA, and F-Nbs-HA is a nanoparticle against HA.
Various exemplary embodiments of the present invention will now be described in detail, which should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terms described in the present invention are only for describing specific embodiments, and are not intended to limit the present invention. In addition, as for the numerical range in the present invention, it should be understood that every intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range and every smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention relates. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.
Without departing from the scope or spirit of the invention, it is obvious to those skilled in the art that many modifications and changes may be made to the specific embodiments of the specification of the invention. Other embodiments derived from the description of the present invention will be apparent to the skilled person. The specification and examples of this application are only exemplary.
As used herein, the terms “including”, “comprising”, “having”, “containing”, etc. are all open terms, and they mean including but not limited to.
Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the experimental materials and reagents used are commercially available unless otherwise specified.
Embodiment 1
Construction of plasmid signal peptide-SpyTag-ferritin monomer subunit (signal peptide-
ST-FE) and signal peptide-SpyCatcher-nano-antibody (signal peptide -SC-HA-VHH) against hemagglutinin protein of H1N1 subtype influenza virus.
Firstly, the gene signal peptide-ST-FE and signal peptide-SC-HA-VHH are synthesized.
Their gene serial structure is shown in Fig. 1, optimized by codon of CHO eukaryotic expression system, and is synthesized by Kingsley Biotechnology Co., Ltd. The amino acid sequence of the signal peptide-ST-FE (SEQ ID NO: 1) is as follows:
MGWSCIILFLVATATGVHSAHIVMVDAYKPTKGGGSGGGSGGGSRMLKALNDQLNR
ELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIP
KPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEAS
VKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE.
The amino acid sequence of the signal peptide-SC-HA-VHH (SEQ ID NO: 2) is as follows:
MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKE
LAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE
QGAVTVNGKATKGDAHIGGGSGGGSGGGSQVOLVESGGGLVQSGGSLRLSCAASG
SMSRITMGWYRQAPGMERELVAVIGNNDNTVYGDSVQGRFTVSRDNAKNTAYLQM
NSLNAEDTAMYYCKISTLTPPHEYWGQGTQVTVSSHHHHHH.
The amino acid sequence (SEQ ID NO: 3) of the signal peptide-SpyTag is as follows:
MGWSCIILFLVATATGVHSAHIVMVDAYKPTK.
The amino acid sequence (SEQ ID NO: 4) of the signal peptide-SpyCatcher is as follows:
MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKE
LAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE
QGQVTVNGKATKGDAHI.
The amino acid sequence of the connecting peptide (SEQ ID NO: 5) is as follows:
GGGSGGGSGGGS.
The amino acid sequence of the signal peptide (SEQ ID NO: 6) is as follows:
MGWSCIILFLVATATGVHS.
After the synthesis of signal peptide-ST-FE and signal peptide-SC-HA-VHH genes, the target fragment is amplified by PCR using the synthesized genes as a template, and the amplification primers are as follows: signal peptide-ST-FE: upstream primer sequence(SEQ ID NO: 7):
AATCTCTAGAATGGGCTGGAGCTGCAT; downstream primer sequence (SEQ ID NO: 8):
AATCAAGCTTTTACTCGCCGCCCTGCAT; signal peptide-SC-HA-VHH: upstream primer sequence: AATCTCTAGAATGGGCTGGAGCTGCAT, downstream primer sequence (SEQ ID NO: 9):
AATCAAGCTTTTAGTGGTGGTGGTGGTG.
The nucleotide sequences of signal peptide -ST-FE and signal peptide -SC-HA-VHH genes are as follows: signal peptide -ST-FE (SEQ ID NO: 10):
GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCG
TGCACTCTGCCCACATCGTGATGGTGGACGCCTACAAGCCCACAAAGGGCGGAG
GCAGCGGCGGCGGCTCTGGCGGAGGATCTCGGATGCTGAAGGCCCTGAACGAC
CAGCTGAATCGGGAGCTGTACTCCGCCTACCTGTACTTTGCCATGGCCGCTTACT
TOGAGGACCTGGGCCTGGAGGGCTTCGCCAACTGGATGAAAGCTCAGGCCGAG
GAAGAGATCGGCCACGCCTTGAGATTCTACAACTACATCTACGACAGAAACGGCA
GAGTGGAACTGGATGAGATTCCTAAGCCTCCAAAAGAGTGGGAGAGCCCCCTGA
AGGCTTTCGAGGCTGCTTACGAGCATGAGAAGTTCATCTCCAAGTCCATCTACGA
GCTGGCTGCTCTGGCAGAGGAAGAAAAGGATTATTCCACCAGAGCCTTCCTGGAA
TGGTTCATCAACGAGCAAGTCGAAGAAGAGGCCTCCGTGAAGAAGATCCTGGACA
AGCTGAAGTTTGCCAAGGACTCCCCTCAGATCCTGTTCATGCTCGATAAAGAACT
GTCTGCTCGGGCCCCTAAGCTGCECTGGCCTGCTGATGCAGGGCGGCGAGTGAG
GATCC; signal peptide -SC-HA-VHH (SEQ ID NO: 11):
GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTGC
ACTCCTCCTACTACCACCACCACCACCACCACGACTACGACATTCCCACCACCGAGAACCT
GTACTTCCAGGGCTCCGCCACACACATCAAGTTCTCCAAGAGAGACGAGGATGGCAAAGA
GCTGGCTGGCGCTACAATGGAACTGAGAGATAGCTCTGGCAAAACAATCTCTACCTGGAT
CAGCGACGGCCAAGTGAAGGACTTCTACCTCTATCCTGGCAAGTACACCTTCGTGGAAAC
AGCTGCTCCTGATGGCTACGAGGTGGCTACCGCCATCACCTTTACCGTGAACGAGCAGGG
CCAGGTCACCGTGAACGGCAAGGCCACCAAGGGCGATGCCCACATCGGCGGAGGATCTG
GCGGAGGCTCCGGCGGAGGCTCTCAGGTGCAGCTGGTGGAATCTGGAGGTGGCCTGGT
GCAGTCCGGCGGCAGCCTGCGGCTGTCCTGTGCCGCTTCTGGCTCCATGAGCCGGATCA
TCACCATGGGCTGGTACAGACAGGCCCCAGGCATGGAACGCGAGCTGGTCGCCGTGATC
GGCAACAACGACAATACCGTTTACGGCGACTCCGTGCAAGGCAGATTCACCGTGTCTCGG
GACAATGCCAAGAACACCGCTTATCTGCAGATGAACTCCCTGAACGCCGAGGACACCGCC
ATGTACTACTGCAAGATCTCCACCCTGACACCTCCTCACGAGTACTGGGGCCAGGGCACC
CAGGTGACCGTGTCCTCTCACCATCACCACCATCATTGAGGATCC.
Table 1: The target gene amplification system
Component Volume
DNA Template tag
Upstream primer 2 ul
Downstream primer 2 ul 10xEx Taq Buffer 5 HI dNTP Mixture 4 pl
Ex Taq 1 pl
Water, nuclease-free 36 ul
Total system 50 ul
Table 2: The PCR reaction procedure of the target gene
Steps = Procedure Time 1 Pre-denaturationat95°C ~~ 5min 2 Denaturing at 94°C 30 sec 3 Annealing at 58°C 30 sec 4 Extending at 72°C 30 sec
GOTO Step 2 33x 6 Final extension at 72°C 10 min 7 Storing at 12°C oo
The amplified product is electrophoresed with 1% agar gel, and the results are shown as
Fig. 2, which are the same as the expected fragment length. 5 The PCR products are recovered, and the two kinds of target fragments recovered from
PCR glue are digested with eukaryotic expression plasmid P3 using restriction endonucleases
Xba | and Hind III, respectively. T4 DNA Ligase is used to connect the above two Enzymatic fragments to construct two kinds of target gene vector plasmids, namely, plasmid signal peptide-ST-FE (p3-ST-FE) and signal peptide-SC-HA-VHH (p3-SC-HA-VHH), and they are stored at -20°C.
Embodiment 2
Linearization of plasmid signal peptide-ST-FE and signal peptide-SC-HA-VHH
The plasmid signal peptide-ST-FE and signal peptide-SC-HA-VHH stored at -20°C are taken out, and their concentrations are measured with NanoDrop one as 1200 ng/ul and 890 ng/k, respectively. The restriction endonuclease Pvu | is used for single digestion. The specific reaction systems are shown in Tables 3 and 4 below.
Table 3: Single enzyme digestion reaction system of plasmid signal peptide-ST-FE
Component Voume ~~ psST-FE s4u(oone)
Pvu | 10 pl 10xNEB Buffer 20 pl
Water, nuclease-free 86 ul
Total system 200 pl
Table 4 Single enzyme digestion reaction system of plasmid signal peptide-SC-HA-VHH -— Component elume ~~ p3SC-HAVHH 113ul(toopg
Pvu | 10 pl 10xNEB Buffer 20 pl
Water, nuclease-free 57 ul
Total system 200 pl
According to the single enzyme digestion reaction system table, the components are added and digested in a water bath at 37°C for 2 h.
During enzyme digestion, 1% agarose gel is prepared. First, the gel plate is prepared, the comb is selected and inserted according to the total system of enzyme digestion, then 0.5 g of agarose is weighed in a 100 ml beaker, 50 ml of 1 x TAE is weighed with a measuring cylinder, they are mixed well and put in a microwave oven, heated for 30 s, and taken out to observe the dissolution of agarose; then they are repeatedly heated to make them fully dissolved, then 2 u L of Gold View | nucleic acid dye is added, and is gently shaken evenly (at this time, the force is even to avoid bubbles), and the gel is poured; the gel is completely solidified by placing at room temperature for 40 min. Then, the products of single enzyme digestion are mixed well with a proper amount of 6xLoading Buffer and all of them are added to the gel wells. The plasmid without single enzyme digestion and DL 10000 DNA Marker are used as controls, and the electrophoresis conditions are constant pressure of 110 V, 600 mA and 30 min. After electrophoresis, the rubber block is gently taken out and placed in a blue light glue cutter for glue cutting. According to the instructions of San Prep Column DNA Gel Extraction Kit, the vector after single enzyme digestion is recovered and purified. The concentration and
OD260/280 of the recovered and purified products are determined with NanoDrop one, and the products are stored at -20°C. 1% agarose gel is shown in Fig. 3. The electrophoresis speed of linearized plasmid is slower than that of circular plasmid, which is in line with expectations.
Embodiment 3
Electroporation of linear signal peptide-ST-FE and signal peptide-SC-HA-VHH into GS gene- deficient CHO cells (1) Preparation of CHO suspension cells with GS gene deletion before electrotransformation: inoculating the cells in a 125 ml triangular shake flask with a living cell density of 5 x 10° cells/ml 24 h before electrotransformation, with a culture volume of 30 ml, and setting a rotating speed of the carbon dioxide shaking incubator at 110 rpm. (2) Collection of CHO suspension cells with GS gene deletion: on the day of electrotransformation, calculating the density and viability (98%) of living cells in suspension culture; calculating and placing the volume of cell suspension needed to collect 1x10 living cells in a sterile centrifuge tube at 1000 rpm for 5 min, and then resuspending in 400 HI of 302 medium (containing glutamine). (3) Cells and plasmids incubation: adding 40 ug of linear knockout plasmid (linear plasmid p3-ST-FE or linear plasmid p3-SC-HA-VHH) into the above-mentioned cell suspension, gently blowing and mixing, and incubating at room temperature for 10 min.
(4) Electric shock: setting the parameters of the electrometer in advance, wherein the electric shock conditions are: voltage 280 V, electric shock time 20 ms, and electric shock once; incubating at room temperature for 10 min, then quickly adding the cell plasmid mixture into a precooled 4 mm electric shock cup for electric shock once. (5) Cell treatment after electric shock: after the end of electric shock, quickly transferring the cells after electric shock to 20 ml of 302 preheated medium {containing glutamine), and culturing T75 cells in a flask for 24 h. (6) Calculating and recording cell viability after 24 h of electric shock: the cell viability of linear plasmid p3-ST-FE is 48% after 24 h of electroporation, and that of linear plasmid p3-SC-
HA-VHH is 52% after 24 h of electroporation. (7) Continuing to culture with a medium containing no glutamine until the cell viability is above 95%.
Embodiment 4
Screening of cell lines after electrotransformation of plasmid signal peptide-ST-F and plasmid signal peptide-SC-HA-VHH
After electrotransformation for 24 h, the static culture cell pool is a mixed group of stably transfected cells, which contains the cell lines that it is needed to stably express the target protein. Because the cells contain GS gene after successful electrotransformation of plasmid, glutamine-free culture medium is used for screening, and monoclonal cell lines are selected by limited dilution method. The specific steps are as follows: (1) preparation of subclone plate-laying conditioned medium: 24 h before subclone plating, inoculating the suspended cells after electroporation in 125 ml triangular shake flask according to 1 x 10° cells/ml, with a rotation speed of 110 rpm, and a culture volume of 30 ml. (2) On the day of subcloning, placing the cells inoculated and cultured in the step 1 in a 50 ml sterile centrifuge tube at 1000 rpm for 5 min, and filtering the cell debris in the supernatant of the cell culture solution by a 0.2 um sterile disposable filter and using as conditioned medium for later use. (3) Layout method of paving medium: 60% fresh 302 medium {without glutamine)+30% conditioned medium+10% foetal bovine serum (FBS)+streptomycin (PS), and the above ingredients are mixed evenly and preheated at 37°C for later use. (4) Plating: the cell density and viability are calculated according to the steps of cell counting: screening monoclonal cell lines by using 96-well cell culture plates; considering the possibility of death during the growth of monoclonal cells, the cells are continuously diluted in the plating medium for 24 h to the final density of 1 cell per 100 pl, and the diluted cell suspension is added to the 96-well cell culture plates according to 100 pl/well with a multi- channel pipette; five 96-well cell culture plates are all paved and cultured in a 5% CO: incubator at 37°C.
(5) On the 7" day of subclone plating, the growth of the clone is observed under the microscope, and the monoclonal cells radiated outward from the central point like round cells, and both the monoclonal and polyclonal holes are marked. According to the need, the culture medium is changed in the monoclonal hole once every 5-7 days. (6) Cell strains with good growth condition are selected for later use.
Embodiment 5 Protein Western Blot (WB) identification of target protein. 5 x 105 cells/ml of the selected cell lines is inoculated into a 125 ml triangular shake flask for shaking culture, the culture volume is 30 ml, and the rotating speed of the carbon dioxide shaking incubator is set at 110 rpm. The viability of cell lines is determined every 12 h. When the cell viability reaches 70%, the cell supernatant is taken for WB identification of the target protein, as shown in Fig. 4, which meets the expected stripe size. Signal peptide-ST-FE has no
HIS tag and no band, and signal peptide-SC-HA-VHH has signal peptide display band. (1) Glue mixing: the lower layer glue is 10% separation glue, and the upper glue is 5% concentrated glue.
Washing the comb and putting it in a natural air-drying place; brushing the glass plate clean and leaning against the glue rack to dry the water. (Ultra-pure water washing)
Using ultra-pure water to conduct leak detection, then preparing the separation glue according to the formula; pouring out the leaked water when it is added to 10%SDS, blotting it with filter paper, and then continuing to add the remaining 10%APS and TEMED and mixing well.
Adding the prepared separation glue along one part of the glass, with each piece of about 4.5 ml, and immediately sealing it with ultra-pure water (making the glue surface smooth); waiting until an obvious refraction surface is formed between the separation glue and water, and starting to prepare according to the formula of concentrated glue; pouring off the water sealed when it is added to 10%SDS, drying it with filter paper, and then continuing to add and mix the remaining 10%APS and TEMED.
Pouring the prepared concentrated glue into the plate, quickly inserting the comb, pulling out the comb after the upper glue is completely solidified, and putting the glue and the glass plate in an electrophoresis tank for electrophoresis, or putting it in an electrophoresis buffer and putting it in a refrigerator at 4°Cfor later use. (2) Sampling
Mixing the samples with appropriate amount of loading buffer and boiling for 5 min, then adding 10 - 15 pl to each empty space; replacing the blank hole with 1xSDS buffer. (3) Running electrophoresis
Setting the voltage to 100V; generally, electrophoresis lasts for 90-120 min, depending on the electrophoresis progress.
During running electrophoresis, it is necessary to prepare things for membrane transfer and prepare membrane transfer buffer (stored and reserved at 4°C).
Film cutting: slightly cutting the film by one more hole according to the number of samples.
Soaking film: firstly, soaking the film in methanol for ten more seconds, then rinsing it with ultra-pure water (just once), then soaking it in the buffer solution of the rotary film and putting it in the refrigerator for later use.
Soaking the filter paper: putting the filter paper in the tray, soaking it in the rotary film buffer lotion, and putting it in the refrigerator for use; getting the ice when there are more than ten minutes from the rotary film. (4) Film transfer
Putting the tank for film transfer into a box filled with ice, taking the film out of the refrigerator at -20°C and putting it into the tank, pouring the film transfer buffer into the tank, and taking out all the film transfer articles that are originally put into the refrigerator; the order of placing during film transfer: because protein electrophoreses from the negative electrode to the positive electrode, they are placed from the positive electrode to the negative electrode respectively: sponge—two layers of filter paper—PVDF film—glue—two layers of filter paper— sponge (preventing air bubbles between glue and filter paper, between glue and film, and between glue and filter paper).
Stacking several layers and putting them into the groove, setting the current to 300 mA, turning the key to the time shift, and conducting film transfer for 120 min; preparing 5% BSA sealing solution (sealing solution: dissolving 2 g of BSA in 40 ml of TBST) when there are ten more minutes left after film transfer. (5) Sealing
Taking out the film, turning it over, soaking it in 5% BSA, and shaking it in a refrigerator at 4°C for 1 h; or sealing at room temperature for 1 h (depending on the room temperature at that time). (6) Primary resistance
The diluent of primary antibody is 1% BSA; taking a suitable volume of HIS primary antibody (mouse source) and diluting it according to 1 : 800 - 1000; incubating overnight in the shaking bed of 4°C refrigerator. (7) Washing
Washing with TBST for three times, 5 min each time, and preparing the second antibody at that time, wherein the second antibody is rabbit anti-mouse antibody. (8) Secondary antibody: incubating in a light-proof room temperature shaking table for 1 h. (9) Washing: washing with TBST for three times, with 5 min each time. (10) Exposure.
Embodiment 6 RT-PCR detection of signal peptide -ST-FE
Since the signal peptide-ST-FE protein does not contain HIS tag, its RNA is extracted to detect whether it is transcribed. The result is shown in Fig. 5, which is in line with the expected size.
(1) Centrifuging cells (107 cells) to discard the culture medium and washing twice with PBS; discarding PBS and sucking it clean. (2) Adding 1 ml of TOIZOL (total RNA extraction reagent) and grinding. (3) Adding 250 um of chloroform, shaking violently for 30 s, and placing on ice for 12 min. (4) Centrifuging at 4°C and 13500 rpm/min for 15 min. (5) Taking 400 pl of supernatant and the same amount of isopropanol, and fully mixing them with the new EP tube, and refrigerating at 4°C for 35 min; then centrifuging at 4°C and 13500 rpm/min for 10 min. (6) Discarding the supernatant, adding 1 ml of 75% ethanol and shaking gently; centrifuging at 4°C and 10600 rpm/min for 5 min. (7) Discarding the supernatant, and placing it or blotting it with filter paper; adding 10 pl of deionized water to dissolve for later use or store at -80°C. (8) Reversing the extracted RNA transcribed according to Table 5, with the result of electrophoresis with 1% agar gel shown in Fig. 5, which is the same as the expected fragment length.
Table 5: RNA reverse transcription ~~ Component ~~ Volume
Total RNA extraction solutions ~~ spl
Upstream primer (same as Table 1) 1 HI
Downstream primer (same as Table 1) 1 HI
RNase Inhibitor 1 HI dNTP Mixture 2 ul
Reverse Transcoriptase M-MLV 1 HI 5xM-MLV Buffer 4 ul
Water, nuclease-free 5 HI
Total system 20 pul
Embodiment 7
Purification of Target Protein
ST-FE protein is purified by size exclusion HPLC. SC-HA-VHH protein is purified by HIS protein purification column, and imidazole is removed by 3KD dialysis membrane. Finally, 0.5 mg/ml of ST-FE protein and 0.45 mg/ml of SC-HA-VHH protein are obtained.
Embodiment 8 In vitro connection and electron microscopic observation of the target protein
The protein of ST-FE monomer and the protein of SC-HA-VHH are mixed in the same molar amount, and connected continuously at 4°C for 16 h to obtain anti-H1N1 subtype influenza virus nanoparticles. According to the characteristics of HIS tags, WB is used to detect the connection between them. As shown in Fig. 6, the connection is displayed, which meets the expected size. ST-FE protein and FE-HA-VHH linker are observed by transmission electron microscope. As shown in Fig. 7, under the same magnification, the diameter of FE-HA-VHH connector particles is obviously larger than that of FE protein, which is in line with expectations.
Embodiment 9 Cell-level antiviral identification of anti-H1N1 subtype influenza virus nanoparticles
Before the experiment, 1 x 108 MDCK cells are prepared and transmitted to 6 empty plates.
The obtained anti-H1N1 subtype influenza virus nanoparticles are mixed with H1N1 subtype influenza virus (PR8) in equal amount into a mixed solution of 0.5 ml, and incubated at 37°C for 15 min, with the virus amount of 10° PFU. The results are observed after 72 h, as shown in Fig. 8. The results show that the anti-H1N1 subtype influenza virus nanoparticles may effectively bind to the H1N1 subtype influenza virus (PR8) and inhibit the infection of the H1N1 subtype influenza virus (PR8) to MDCK cells.
Embodiment 10 In vivo antiviral identification of anti-H1N1 subtype influenza virus nanoparticles
Female SPF mice of 6-8 weeks old are divided into 2 groups, 8 in each group. One group is PBS group, and the other group is anti-H1N1 subtype influenza virus nanoparticle treatment group. Four hours before the infection of the H1N1 subtype influenza virus (PR8), 200 ul of PBS is instilled into the nasal cavity of each mouse in PBS group, and 200 pl of anti-H1N1 subtype influenza virus nanoparticles (50 ug) are instilled into the nasal cavity of each mouse in anti-
H1N1 subtype influenza virus nanoparticles treatment group. The virus infection amount of each mouse is 2 x LD50. 24 hours after infection, 200 pl of PBS is instilled into the nasal cavity of each mouse in PBS group, and 200 pl of anti-H1N1 subtype influenza virus nanoparticles (50
Hg) are instilled into the nasal cavity of each mouse in anti-H1N1 subtype influenza virus nanoparticles treatment group. The weight and survival rate of mice are recorded. The experimental results are shown in Fig. 9 and Fig. 10, which show the weight change and survival rate of mice. The weight of mice in the anti-H1N1 subtype influenza virus nanoparticles treatment group has no obvious change, no abnormality and no death. The clinical symptoms of mice in PBS control group are obvious, such as rough hair, shortness of breath, hunchback, slow activity and clustering, and their weight decreases obviously, and all mice die within 8 days.
Embodiment 11
Detection of binding ability of anti-H1N1 subtype influenza virus nanoparticles to virus
Firstly, the binding ability of anti-H1N1 subtype influenza virus nanoparticles with the H1N1 subtype influenza virus (PR8) is tested: diluting the H1N1 subtype influenza virus (PR8) to 10°
PFU, adding 50 ul each well to the ELISA plate and placing at 37°C for 2 h or 4°C for 24 h; discarding the liquid in the hole (to avoid evaporation, the plate should be covered or placed flat in a metal wet box with wet gauze at the bottom); sealing the enzyme-labelled reaction well: adding 2% BSA to 200 pl/well, and sealing at 37°C for 60 min or 4°C for 24 h; removing bubbles in each hole, washing the hole with washing liquid for 3 times after sealing, each time for 3 min; washing method: sucking up the reaction solution in the hole, filling the plate hole with the washing solution, and then adding the washing solution again after suction, and repeating for 3 times; pouring off the liquid and patting it to dry on absorbent paper with washing times of 3. The anti-
H1N1 subtype influenza virus nanoparticles are 50 pl per hole, and three gradients are set, placed at 37°C for 40-60 min. The holes are washed with washing liquid for 3 times, 3 min each time;
HIS antibody is added at 4°C overnight, and the washing is as before. Enzyme-labelled antibody (secondary antibody) is added, and dilution is performed according to the reference work provided by the enzyme conjugate provider, at 37°C, for 30-60 min, added 50 pl to each hole; the washing is the same as before; the substrate solution (currently prepared) is added, TMB-urea hydrogen peroxide solution is the first choice, followed by OPD-hydrogen peroxide substrate solution system. Substrate dosage: 50 pul of the substrate is added per well, placed at 37°C in the dark for 3-5 min, and stop liquid is added to develop colour. Termination of the reaction: 50ul of termination solution is added to each well to terminate the reaction, and the experimental results are determined within 20 min. The results show that the wavelength of 492 nm is used after OPD colour development, and 450 nm is required for TMB reaction product detection. At the same time, the positive group and the negative group are set with 3 replicates in each group. The traditional nanoantibody treatment is the same as above. The results show that the anti-H1N1 subtype influenza virus nanoparticles may combine with the H1N1 subtype influenza virus (PR8), and the sensitivity is superior to that of traditional nanoantibodies, as shown in Fig. 11.
Then, the H1N1 subtype influenza virus (PR8) is set with six gradients, which are 10° PFU, 5x 105 PFU, 2 x 105 PFU, 1 x 105 PFU, 5 x 10% PFU, 103PFU and PBS as control, and the same amount of anti-H1N1 subtype influenza virus nanoparticles are combined. The steps are the same as above. Traditional nanoantibody treatment is the same as above. The results show that the maximum specific binding of anti-H1N1 subtype influenza virus nanoparticles is better than that of traditional nanoantibody, and the binding sensitivity of anti-H1N1 subtype influenza virus nanoparticles is 7.18 times higher than that of traditional nanoantibody, as shown in Fig. 12.
The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention.
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Claims (8)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210548788.4A CN114805608B (en) | 2022-05-20 | 2022-05-20 | Self-assembled ferritin-based nanoparticle for resisting H1N1 subtype influenza A virus as well as preparation method and application thereof |
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| CN112010984B (en) * | 2020-08-04 | 2021-10-12 | 广州千扬生物医药技术有限公司 | Novel coronavirus S protein polymer nano vaccine based on helicobacter pylori ferritin |
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