CN118021957A - MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof - Google Patents
MRNA for encoding anti-avian influenza H7N9 virus antibody and preparation method and application thereof Download PDFInfo
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- CN118021957A CN118021957A CN202211410073.9A CN202211410073A CN118021957A CN 118021957 A CN118021957 A CN 118021957A CN 202211410073 A CN202211410073 A CN 202211410073A CN 118021957 A CN118021957 A CN 118021957A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention relates to mRNA for encoding an anti-avian influenza H7N9 virus antibody, a preparation method and application thereof. Specifically, an isolated mRNA composition is provided that includes an mRNA comprising an open reading frame encoding a monoclonal antibody heavy chain of H7N9 and an mRNA comprising an open reading frame encoding a monoclonal antibody light chain of H7N9, the mrnas further comprising: a 5' cap structure; a 5' UTR sequence; 3' UTR sequence and 4) polyadenylation sequence, the mass ratio of mRNA comprising the open reading frame encoding the H7N9 monoclonal antibody heavy chain to mRNA comprising the open reading frame encoding the H7N9 monoclonal antibody light chain in the composition is 1:1. The mRNA composition of the invention can be directly expressed as a monoclonal antibody in a subject, has quick response and can be directly used for treating H7N9 infection. And the effect is better than that of directly injecting monoclonal antibody.
Description
Technical Field
The invention belongs to the fields of immunology and molecular biology, and particularly relates to mRNA for encoding an anti-avian influenza H7N9 virus antibody, a preparation method and application thereof.
Background
The H7N9 virus is an avian influenza virus, has drug resistance to traditional antiviral drugs amantadine (amantadine) and rimantadine (rimantadine), and has no effective treatment means at present. In 2013, the H7N9 bird flu outbreak deprives lives of 40% of infected patients, and cities such as Shanghai shut down the live bird market, kill at least 17 ten thousand birds, and continue to prevail for years. The H7N9 virus needs to rely on specific molecules expressed by the virus itself to bind to receptors on human cells when invading the cells to infect the cells and further expand. The humanized antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocytes, and can be combined with antigens on the surface of the virus, so that the virus is prevented from adhering to target cell receptors, the virus is prevented from invading cells, and the H7N9 influenza can be effectively prevented and treated. However, the high development and production costs of antibody drugs and the complex technical requirements of the neck limit their worldwide use. Therapeutic monoclonal antibodies are typically produced by cell lines, such as chinese hamster ovary cells, and then the antibodies are purified from the cell culture supernatant in large quantities and pharmaceutical formulations are developed. Production of antibodies presents a number of challenges, including misfolding or incorrect post-translational modifications that can lead to adverse events. The purification of each monoclonal antibody is specific and requires the development of a new method for each antibody, and thus the production cost is high.
In contrast, mRNA production and purification is simple, fast and cost effective because it does not require complex and expensive laboratory infrastructure, and the same method can be used for all mRNA. The sudden burst of new crown epidemic makes mRNA technology free and gets unprecedented attention. Nucleic acid-encoded monoclonal antibodies, particularly mRNA-based monoclonal antibodies, offer great promise for improved therapeutic efficacy and wide-ranging applications. The therapeutic mRNA vaccine has short research and development period, high safety and effectiveness, simple production process and high productivity, and is the most advanced biomedical technology at present. mRNA vaccines encoding neutralizing antibodies are an important trend in the development of drugs for the treatment of infectious diseases: mRNA encoding a broad spectrum neutralizing HIV antibody VRC01 successfully produced VRC01 in vivo after injection into mice and protected human mice from HIV-1 virus infection; aiming at human RSV, tiwari develops the prior drug palivizumab into mRNA encoding membrane anchoring neutralizing antibody, which has higher efficiency than palivizumab and significantly inhibits RSV after 7 days of transfection; mRNA encoding the chikungunya virus neutralizing antibody was efficiently expressed in mice and protected from arthritis and musculoskeletal tissue infection 2 days after inoculation, and viremia was reduced to undetectable levels. Anti-avian influenza virus monoclonal antibodies can prevent and treat avian influenza virus infection. However, the antibody is subject to purification and post-translational modification during cell production, and is expensive to develop and produce, so that the use of mRNA technology encoding anti-avian influenza antibodies for more efficient, safe, and low-cost monoclonal antibody therapy is a new approach to preventing and treating avian influenza.
Antibody drugs are special-effect drugs for treating serious infectious diseases, but their high research, development and production costs and the complex technical requirements of the neck limit their worldwide application. The mRNA drug research and development period is short, the safety and the effectiveness are high, the production process is simple, the productivity is high, and the method is the most advanced biotechnology at present. Currently, no mRNA vaccine against H7N9 virus infection is seen, and there is a few direct uses of monoclonal antibody mRNA expressing neutralizing virus as a therapeutic mRNA vaccine for inhibiting H7N9 infection.
Disclosure of Invention
To solve the above problems, the inventors of the present invention used the sequence of a monoclonal antibody (bulletin No. CN 109810189B) against H7N9 avian influenza virus developed in the previous study. The invention transcribes this antibody gene into mRNA and wraps it in LNP to form an LNP-RNA vaccine, which is capable of treating the H7N9 virus after vaccination of a subject.
The first aspect of the invention provides an isolated mRNA composition comprising mRNA comprising a heavy chain of a monoclonal antibody encoding H7N9 and mRNA comprising a light chain of a monoclonal antibody encoding H7N9,
The amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the H7N9 monoclonal antibody are shown below, respectively:
heavy chain CDR1 region: GFTFSSYA SEQ ID No.1;
Heavy chain CDR2 region: ISGSGGST SEQ ID No.2;
heavy chain CDR3 region: AKNRRGSMIVSFLAKSRAGMDV SEQ ID No.3;
light chain CDR1 region: QSISSY SEQ ID No.4;
light chain CDR2 region: AAS;
light chain CDR3 region: QQSYSTPWT SEQ ID No.5;
The mRNAs also respectively comprise:
1) A 5' cap structure;
2) A 5' UTR sequence;
3) Sequence encoding a Signal peptide
4) Stop codon and enzyme cutting site sequence
5) 3' UTR sequences; and
6) A poly (A) sequence of a poly (A),
Wherein, the mRNA containing the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA containing the light chain of the monoclonal antibody encoding H7N9 respectively comprises the following elements in sequence along the 5 '. Fwdarw.3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, mRNA corresponding to the open reading frame of the heavy or light chain of a monoclonal antibody of H7N9, a stop codon and cleavage site sequence, a 3' UTR sequence and a polyadenylation sequence.
Further, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 6;
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSS SEQ ID NO.6; And/or
The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 7:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK SEQ ID NO.7.
further, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 8;
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.8; And/or
The light chain amino acid sequence of the antibody is shown as SEQ ID NO. 9:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.9.
further, the RNA sequence corresponding to the open reading frame of the heavy chain of the monoclonal antibody of H7N9 is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO. 10:
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG SEQ ID NO.10
The RNA sequence corresponding to the open reading frame of the H7N9 monoclonal antibody light chain is the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO. 11:
gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO.11
Further, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -ogppg, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1.
Further, the 5' UTR sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID NO. 12.
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC SEQ ID NO.12。
In other embodiments of the invention, the 3' UTR sequence is selected from the group consisting of the RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID NO. 13.
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC SEQ ID NO.13
Further, the polyadenylation sequence comprises a sequence of 25-400 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-400 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-300 adenylates.
Further, the polyadenylation sequence comprises a sequence of 50-250 adenylates.
Further, the polyadenylation sequence comprises a sequence of 80-120 adenylates.
Further, the sequence encoding the signal peptide is selected from the group consisting of RNA sequences corresponding to the sequence shown in SEQ ID NO. 16.
Further, the cleavage site sequence is selected from the group consisting of the RNA sequences corresponding to CTCGAG.
In a second aspect the invention provides a pharmaceutical composition comprising the mRNA composition of the first aspect, and optionally a delivery vehicle.
Further, the delivery vehicle is a nanoparticle.
Further, the delivery vehicle is a lipid nanoparticle.
In other embodiments of the present invention, the lipid nanoparticle comprises a cationic lipid and at least one selected from the group consisting of a non-cationic lipid, a sterol, a PEG-modified lipid.
In some embodiments of the invention, the lipid nanoparticle is a cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid.
In other embodiments of the invention, the cationic lipid is an ionizable cationic lipid selected from one or more of the following: 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane, di-lino-methyl-4-dimethylaminobutyrate and 9- ((4- (dimethylamino) butyryl) oxy) heptadecanedioic acid di ((Z) -non-2-en-1-yl) ester, preferably Di-lino-methyl-4-dimethylaminobutyrate. In some embodiments of the invention, the non-cationic lipid is a neutral lipid selected from at least one of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl lecithin (DOPC), and dioleoyl phosphatidylserine (DOPS).
In other embodiments of the invention, the sterol is cholesterol.
In some embodiments of the invention, the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG, and PEG-DMPE.
In other embodiments of the invention, the PEG-modified lipid has a PEG length of 0.5-200KDa.
In some embodiments of the invention, the pharmaceutical composition optionally contains an adjuvant.
The third aspect of the invention provides a kit comprising an mRNA composition according to the first aspect of the invention and/or a pharmaceutical composition according to the second aspect of the invention.
According to a fourth aspect of the invention, there is provided an mRNA composition according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, and the use of a kit according to the third aspect of the invention in the manufacture of a medicament for the prophylaxis and/or treatment of H7N9 virus infection diseases.
A fifth aspect of the present invention provides a method for producing the mRNA composition according to the first aspect of the present invention, comprising the steps of:
S1) respectively inserting heavy chain genes and light chain genes of the H7N9 monoclonal antibody into plasmid vectors to obtain plasmid vectors containing the heavy chain genes or the light chain genes of the H7N9 monoclonal antibody;
S2) respectively transferring the plasmid vectors containing the H7N9 monoclonal antibody heavy chain and light chain genes obtained in the step 1) into host bacteria for culture and sequencing;
s3) carrying out amplification culture on the monoclonal with correct sequencing in the step 2), and extracting plasmids;
s4) obtaining a linearization plasmid by enzyme digestion of the plasmid;
S5) transcribing the linearized plasmid into RNA;
S6) capping the RNA obtained in the step S5) to obtain mRNA,
S7) mRNA comprising the open reading frames of the H7N9 monoclonal antibody heavy and light chain genes were mixed at 1:1 to obtain mRNA compositions.
The invention has the beneficial effects that:
The H7N9 mRNA vaccine has a short research and development period, and is particularly suitable for developing new sudden infectious disease vaccines including H7N 9; the safety is high. The mRNA composition of the invention overcomes the problems possibly occurring in the prior art when antigen mRNA vaccines are used for treating virus infection diseases, such as different immune response degrees of different subjects after inoculation, not all subjects can produce corresponding antibodies, even if the antibodies are produced, the antibody production time is longer, and the problem that the yield is insufficient to inhibit virus infection can also occur. The mRNA composition can be directly expressed as a monoclonal antibody in a subject, has quick response, can be directly used for treating H7N9 infection, and has better effect than the direct injection of the monoclonal antibody.
Drawings
FIG. 1 is a photograph of plasmid linearized gel electrophoresis.
FIG. 2 is a photograph of transcribed mRNA gel electrophoresis.
FIG. 3 is an ELISA to verify antibody expression of LNP-mRNA in cells.
Detailed Description
The following detailed description of the present invention will be made in detail to make the above objects, features and advantages of the present invention more apparent, but should not be construed to limit the scope of the present invention.
In some embodiments of the invention, the mRNA is not chemically modified or is chemically modified, the chemical modification being a replacement of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA, replacement of uracil with pseudouridine, N1-methyl-pseudouridine, N1-ethyl-pseudouridine, 2-thiouridine, 4' -thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-1-methyl-uridine, and N-methyl-1-methyl-pseudouridine or further preferably N1-methyl-pseudouridine;
In some embodiments of the invention, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -OGpppG, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1.
According to the demands of different mRNAs, different 5 'cap structures can be flexibly added at the 5' end of the mRNAs.
"M7G" represents a 7-methylguanosine capped nucleoside, "ppp" represents a triphosphate bond between the 5 'carbon of the capped nucleoside and the first nucleotide of the primary RNA transcript, N1 is the most 5' nucleotide, "G" represents a guanosine, "m7" represents a methyl group at the 7-position of guanine, and "m2'-O" represents a methyl group at the 2' -O position of the nucleotide.
The nanoparticle provided by the invention can be used for efficiently delivering mRNA and has the following characteristics and advantages: for example, in the encapsulation of mRNA, acidic pH conditions cause the ionizable cationic lipid to carry a positive charge, compressing the negatively charged mRNA molecule, and thus achieving higher encapsulation efficiency; under the physiological pH condition, the ionizable lipid nanoparticle has neutrality, does not act with negatively charged cell membranes, and has high biocompatibility; after the ionizable lipid nanoparticle enters cells through endocytosis to form endosomes, the acidic condition in the endosomes enables the nanoparticle to be positively charged again and electrostatically interact with the endosomal membrane with negative charges, so that mRNA release is facilitated.
Example 1 preparation of mRNA
(1) The neutralizing avian influenza virus antibody gene was inserted into pUC57-kana vector: the H7N9 antibody heavy chain gene, the H7N9 antibody light chain gene and the pUC57 vector (stored in this laboratory) were double digested with restriction enzymes EcoR1 and Hind3 (purchased from Thermo), respectively, and the antibody heavy chain gene and the antibody light chain gene were ligated by using T4 ligase (Thermo), respectively; obtaining a vector connected with a heavy chain gene of the antibody of the neutralizing avian influenza virus and a vector connected with the light chain gene of the antibody of the neutralizing avian influenza virus.
The H7N9 antibody heavy chain gene is shown as SEQ ID NO.10
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG SEQ ID NO.10.
The heavy chain gene sequence of the H7N9 antibody is encoded before the heavy chain gene sequence, and the heavy chain gene sequence also comprises a signal peptide sequence SEQ ID NO.16; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID No.16. The signal peptide can guide the newly synthesized protein to transfer to the secretion passage after being expressed, and the signal peptide used in the invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
Other gene sequences capable of encoding H7N9 antibody heavy chain SEQ ID NO.8 may also be used. The heavy chain sequence of the H7N9 antibody is shown in SEQ ID NO. 8:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRRGSMIVSFLAKSRAGMDVWGQGTTVTVSSRSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.8.
the H7N9 antibody light chain gene is shown in SEQ ID NO. 11:
gacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO.11.
The sequence of the light chain gene of the H7N9 antibody is encoded and also comprises a signal peptide sequence SEQ ID NO.16; ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC SEQ ID No.16. The signal peptide can guide the newly synthesized protein to transfer to the secretion passage after being expressed, and the signal peptide used in the invention is a signal peptide sequence known in the prior art, and can also be replaced by other signal peptide sequences with the same function.
Other gene sequences capable of encoding the H7N9 antibody light chain SEQ ID NO.9 may also be used. The H7N9 antibody light chain sequence is shown in SEQ ID NO. 9:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.9.
(2) Vector transformation XL1-blue strain: taking a tube of competent cells, adding 1 mu L (50 ng) of the plasmid containing the light chain gene or the plasmid containing the heavy chain gene obtained in the step (1), and uniformly mixing; ice bath for 5min, heat shock for 90 s, and ice placement for 5 min; 500. Mu.L of 2YT liquid medium was added at 37℃for 45 minutes at 200 rpm; plating by sucking 50 mu L of bacterial liquid and culturing overnight; selecting the monoclonal and sending to Jin Weizhi company for sequencing; the sequencing result is shown as SEQ ID NO.14 and SEQ ID NO.15 ：ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATCGTAGGGGGTCTATGATAGTGTCCTTCCTGGCGAAATCACGGGCGGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAcggtcgacCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO.14;
ATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCgacatccagATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAASEQ ID NO.15;
(3) Monoclonal expansion culture, plasmid extraction: the correctly sequenced monoclonal was cultured in 400ml of 2YT medium at 37℃overnight. Extracting plasmids by using a CWBIO company plasmid extraction kit to finally obtain 6.4mg of plasmids, and sequencing correctly;
(4) Plasmid linearization: plasmid was digested with Bsa I, and plasmid (2887. Mu.L), enzyme (333. Mu.L), buffer (1665. Mu.L) and DEPC water (11765. Mu.L) were added in this order to the centrifuge tube, followed by a water bath at 37℃overnight. Liquid is exchanged and recovered by a 100K ultrafiltration tube, and the concentration 387.5 ng/. Mu.L of the linearization plasmid is finally obtained;
(5) Transcription: into the centrifuge tube were added 412. Mu.L of plasmid, 320. Mu.L of buffer, 64. Mu.L of ATP, 64. Mu.L of GTP, 64. Mu.L of pUTP, 160. Mu.L of RNase inhibitor, 3.2. Mu.L of inorganic pyrophosphatase, 160. Mu. L T7 RNA polymerase, 1888.8. Mu.L of DEPC water. The reaction was carried out at 37℃for 3 hours. Adding 160 mu L DNase I enzyme for digestion for 15 minutes; adding 4800 mu L of LiCl precipitation solution and 4800 mu L of DEPC water, uniformly mixing, and standing in a refrigerator at-20 ℃ for 30 minutes; centrifuging at 12000rpm for 15 minutes, cleaning with 70% ethanol for 2 times, and removing residual ethanol in the centrifuge tube as much as possible; 1500 μl of water was added to dissolve RNA. Finally, 5037 ng/. Mu.L RNA was obtained, together with 7.56mg;
(6) Capping m7Gppp (5') N1: 596. Mu.L of RNA, 3424. Mu.L of water, and denaturing at 65℃for 5 minutes are added to the centrifuge tube; 600. Mu.L buffer, 600. Mu.L GTP, 150. Mu.L SAM, 150. Mu.L RNase inhibitor, 240. Mu.L capping enzyme were added; 240. Mu.L of methyltransferase, and reacted at 37℃for 1 hour; adding 9mL of LiCl precipitation solution and 9mLDEPC of water, uniformly mixing, and standing in a refrigerator at-20 ℃ for 30min; centrifuging at 12000rpm for 15 minutes, cleaning with 70% ethanol for 2 times, and removing residual ethanol in the centrifuge tube as much as possible; the cover is opened and placed in an ultra clean bench for airing, and 3000 mu L of water is added for dissolving RNA. The Nanodrop detection concentration is 715ng/mL, the yield is 2.145mg, and the yield is 71.5%.
(7) Preparation of mRNA mixture: mRNA containing the heavy chain and light chain gene open reading frames of the H7N9 monoclonal antibody were mixed at a mass ratio of 1:1, respectively, to obtain mRNA compositions.
EXAMPLE 2 preparation of LNP Encapsulated mRNA
The experiment of LNP encapsulation of mRNA was completed by offshore technology Co., ltd, LNP-mRNA was milky white in appearance with an encapsulation rate >98%, dispersion coefficient of 0.103, and nanoparticle size of 90.35nm.
LNP-mRNA was transfected into 293T cells using LIPO3000 (Invitrogen), after 48 hours, the supernatant and cell lysate were collected, 100. Mu.L/well of the coated ELISA plates were diluted 20 times with PBS, incubated 2 hours at 37℃for 2 times, PBS washed 2 times, 5% nonfat dry milk blocked for 1 hour, PBS washed 2 times, 100. Mu.L of anti-his tag antibody was added to each well, incubated 1 hour at 37℃for 3 times, 100. Mu.L of chromogenic solution was added to each well for 10 minutes, and finally 100. Mu.L of stop solution was added, and OD450 data were recorded on a spectrophotometer.
The results are shown in FIG. 3: cells transfected with LNP-mRNA express the antibody of interest.
Example 3
Sodium pentobarbital solution with final concentration of 1% is prepared with physiological saline, filtered by 0.22 μm filter, and stored at low temperature of 4deg.C. The mice were weighed and the anesthetized mice were intraperitoneally dosed at 150 μl/20g and were nasally infected with 20 μl of 10×LD 50-containing H7N9 virus. After 12 hours, the mice were randomly divided into PBS group, LNP-mRNA experimental group and antibody experimental group, 6 mice each, and tail vein was inoculated with PBS, LNP-mRNA (1.4 mg/kg) and antibody (30 mg/kg), respectively. The day of nasal drip was day 0 of the mouse experiment. From the day of infection, the number of mice survived was recorded until 2 weeks after infection
The results show that: animals in PBS group all died, mice in LNP-mRNA group survived 83% and mice in antibody-treated group survived 66%. LNP-mRNA encoding the antibody at lower doses was able to protect mice more significantly from lethal challenge with H7N9 virus than in the antibody-treated group. LNP-mRNA encoding anti-avian influenza antibodies is capable of expressing antibodies directly in vivo and significantly protecting mice from lethal challenge with H7N9 virus.
Claims (10)
1. An isolated mRNA composition comprising mRNA comprising a heavy chain of a monoclonal antibody encoding H7N9 and mRNA comprising a light chain of a monoclonal antibody encoding H7N9,
The heavy and light chains of the H7N9 monoclonal antibody have the CDR1, CDR2 and CDR3 regions as shown below:
heavy chain CDR1 region: GFTFSSYA SEQ ID No.1;
Heavy chain CDR2 region: ISGSGGST SEQ ID No.2;
heavy chain CDR3 region: AKNRRGSMIVSFLAKSRAGMDV SEQ ID No.3;
light chain CDR1 region: QSISSY SEQ ID No.4;
light chain CDR2 region: AAS;
light chain CDR3 region: QQSYSTPWT SEQ ID No.5;
The mRNA comprising the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA comprising the light chain of the monoclonal antibody encoding H7N9, respectively, further comprises:
1) A 5' cap structure;
2) A 5' UTR sequence;
3) Sequence encoding a Signal peptide
4) Stop codon and enzyme cutting site sequence
5) 3' UTR sequences; and
6) A poly (A) sequence of a poly (A),
Wherein, the mRNA containing the heavy chain of the monoclonal antibody encoding H7N9 or the mRNA containing the light chain of the monoclonal antibody encoding H7N9 sequentially comprises the following elements in the 5 '. Fwdarw.3' direction: a 5' cap structure, a 5' UTR sequence, a sequence encoding a signal peptide, an RNA sequence corresponding to the open reading frame of the heavy or light chain of a monoclonal antibody encoding H7N9, a stop codon and cleavage site sequence, a 3' UTR sequence and a polyadenylation sequence;
Preferably, the 5 'cap structure is selected from at least one of m7GpppG, m27,3' -ogppg, m7Gppp (5 ') N1 or m7Gppp (m 2' -O) N1;
Preferably, the polyadenylation sequence comprises a sequence of 25-400 adenylates;
Preferably, the mass ratio of the mRNA comprising the heavy chain of the monoclonal antibody encoding H7N9 to the mRNA comprising the light chain of the monoclonal antibody encoding H7N9 in the composition is 1:1.
2. The isolated mRNA composition of claim 1, wherein the heavy chain variable region amino acid sequence of the monoclonal antibody to H7N9 is set forth in SEQ ID No. 6; and/or
The amino acid sequence of the light chain variable region of the monoclonal antibody of H7N9 is shown in SEQ ID NO. 7:
Preferably, the heavy chain amino acid sequence of the H7N9 monoclonal antibody is shown as SEQ ID NO. 8; and/or
The light chain amino acid sequence of the H7N9 monoclonal antibody is shown as SEQ ID NO. 9.
3. The isolated mRNA composition of any one of claims 1-2, wherein the open reading frame nucleic acid sequence encoding the heavy chain of the monoclonal antibody of H7N9 is set forth in SEQ ID No. 10;
The open reading frame nucleic acid sequence of the monoclonal antibody light chain of the coding H7N9 is shown in SEQ ID NO. 11.
4. The isolated mRNA composition of any one of claims 1-2, wherein the 5' utr sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID No. 12.
5. The isolated mRNA composition of any one of claims 1-3, wherein the 3' utr sequence is selected from the group consisting of RNA sequences corresponding to the nucleic acid sequence set forth in SEQ ID No. 13.
6. A pharmaceutical composition, characterized in that it comprises the mRNA composition of any one of claims 1 to 5, and optionally a delivery vehicle;
preferably, the delivery vehicle is a nanoparticle;
Preferably, the delivery vehicle is a lipid nanoparticle.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition optionally contains an adjuvant.
8. Kit, characterized in that it comprises an mRNA composition according to any one of claims 1 to 5 and/or a pharmaceutical composition according to claim 6 or 7.
9. Use of an mRNA composition according to any one of claims 1 to 5, an mRNA composition according to claim 6 or 7, a pharmaceutical composition according to claim 8, or a kit according to claim 8 for the preparation of a medicament for the prophylaxis and/or treatment of H7N9 virus infection diseases.
10. The method for preparing an mRNA composition according to any one of claims 1 to 5, wherein the method comprises the steps of:
S1) respectively inserting heavy chain genes and light chain genes of the H7N9 monoclonal antibody into plasmid vectors to obtain plasmid vectors containing the heavy chain genes or the light chain genes of the H7N9 monoclonal antibody;
S2) respectively transferring the plasmid vectors containing the H7N9 monoclonal antibody heavy chain and light chain genes obtained in the step 1) into host bacteria for culture and sequencing;
s3) carrying out amplification culture on the monoclonal with correct sequencing in the step 2), and extracting plasmids;
s4) obtaining a linearization plasmid by enzyme digestion of the plasmid;
S5) transcribing the linearized plasmid into RNA;
S6) capping the RNA obtained in the step S5) to obtain mRNA;
S7) mRNA comprising the open reading frames of the H7N9 monoclonal antibody heavy and light chain genes were mixed at 1:1 to obtain mRNA compositions.
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