WO2015176586A1 - 截短的轮状病毒vp8蛋白及其用途 - Google Patents

截短的轮状病毒vp8蛋白及其用途 Download PDF

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WO2015176586A1
WO2015176586A1 PCT/CN2015/076259 CN2015076259W WO2015176586A1 WO 2015176586 A1 WO2015176586 A1 WO 2015176586A1 CN 2015076259 W CN2015076259 W CN 2015076259W WO 2015176586 A1 WO2015176586 A1 WO 2015176586A1
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protein
rotavirus
truncated
variant
fusion protein
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French (fr)
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葛胜祥
薛淼舸
俞林岐
车耀健
李廷栋
张军
夏宁邵
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厦门大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/15—Reoviridae, e.g. calf diarrhea virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the fields of biochemistry, molecular biology, molecular virology and immunology.
  • the present invention relates to a truncated rotavirus VP8 protein, a fusion protein comprising the truncated protein, a conjugate comprising the truncated protein, a coding sequence of the truncated protein and the fusion protein, and
  • a method of preparation comprising a pharmaceutical composition and vaccine of the truncated protein, fusion protein or conjugate, the truncated protein, fusion protein, conjugate, pharmaceutical composition and vaccine can be used for preventing, reducing or treating a wheel Infection with viruses and diseases caused by infection with rotavirus, such as rotavirus gastroenteritis and diarrhea.
  • the invention also relates to the use of the above-described truncated proteins, fusion proteins, conjugates for the preparation of a pharmaceutical composition or vaccine for the prevention, alleviation or treatment of rotavirus infection and by rotavirus Diseases caused by infections, such as rotavirus gastroenteritis and diarrhea.
  • Rotavirus diarrhea is the most important acute intestinal infectious disease in infants and young children worldwide. It is characterized by diarrhea and dehydration, and the mortality rate is high, causing serious social burden and economic burden. About 110 million children under the age of 5 suffer from rotavirus gastroenteritis every year in the world, of which 2 million children must be hospitalized. About 453,000 children die each year from rotavirus diarrhea, including deaths in developing and less developed countries. Cases accounted for more than 85% of the total (LeBaron, CW et al. 1990).
  • Rotavirus belongs to the family Reoviridae, whose genome consists of 11-segment dsRNA, encoding 6 structural proteins (VP1-4, VP6, VP7) and 6 non-structural proteins (NSP1). -6).
  • the mature virion is a three-layered capsid structure, and the outermost layer is composed of spiked protein VP4 and glycoprotein VP7 (Bridger, J.C. et al. 1976).
  • VP7 is directly involved in the replication of the virus in mature intestinal epithelial cells.
  • the VP4 protein is involved not only in the adhesion and invasion of RV, but also in the physical and chemical properties such as RV hemagglutination, neutralizing activity, virulence, and enhanced infectivity after protease cleavage. Both VP7 and VP4 independently induce virus-neutralizing antibodies, and antibodies against both VP7 and VP4 prevent viral invasion. Therefore, both VP7 and VP4 have become the preferred target molecules for vaccine development (Arias, C.F. et al. 2002).
  • the VP8 protein is part of the outermost structural protein VP4 (AA1-776) of the RV virion (AA1-231). VP4 is cleaved by trypsin to form VP8* and VP5* fragments, while the former contains a major epitope specific for VP4 (Clark, S.M. et al. 1981). This suggests that the VP8* protein can be used to develop a novel RV subunit vaccine.
  • VP4 plays an important role in the viral entry phase. Therefore, VP4 plays an extremely important role not only in the viral structure but also in the RV replication process (Arias, C.F. et al. 1996).
  • VP4 The role of VP4 in the process of viral entry depends on the interaction of the protein with specific receptors expressed on the surface of the target cell.
  • SA is considered to be one of the first and most important cellular receptors during RV infection, and it works by binding to VP8. This interaction causes RV to adsorb to the cell surface, thus starting the first step in RV-infected cells.
  • NA-sensitive RV it was found by nuclear magnetic resonance spectroscopy that the core of VP8* binds to ⁇ -N-acetylneuraminic acid, and this binding site is highly conserved, eliminating the need for other glycosylation. Participation; for NA non-sensitive RV strains, It is not clear whether VP8* can also utilize the same locus (Blanchard H, et al. 2007).
  • the first step of interaction of VP8* with SA allows the virions to anchor to the cell surface.
  • VP8* and SA motifs recognize each other, the viral coat protein undergoes a conformational change, which facilitates the search for more specific receptor molecules, which mediates the next entry process of the virus (Dormitzer PR, et al. 2002).
  • the neutralizing immune response against VP8 inhibits viral entry into cells, thereby inhibiting infection of target cells, making this protein an excellent target for establishing anti-RV vaccines.
  • An important prerequisite is that the neutralizing antibody produced by the immune response evoked by VP8 before the virus enters the cell can interact with the VP8 of the virus, preventing the VP8 of the virus from binding to the cell receptor, thereby preventing VP8 allosteric and inhibiting the virus. Enter the cell.
  • the in vitro purified expression of VP8 is based on the E. coli expression system, mainly in the form of soluble expression purification and inclusion body purification.
  • soluble expression and purification can maintain the relative natural conformation and high immunoneutralizing activity of VP8, the soluble expression is low and the purification method is relatively complicated.
  • the purified protein has a heterogeneous conformation, a multimer, and poor stability, which is prone to degradation. Favacho AR, et al. 2002).
  • the inventors have found through research that the purified VP8 degrades significantly after being left at 4 ° C for three days (see, Example 3 and Figure 1 below). This has brought great difficulties to large-scale industrial production of VP8.
  • the process of purification of inclusion bodies is relatively simple, the purified protein has a difference in conformation with the natural protein, and its ability to induce neutralizing antibodies is significantly weakened, which is not suitable for use as a vaccine.
  • Cisoka virus VP8 protein ⁇ VP8*
  • ⁇ VP8* subunit recombinant protein of rotavirus VP8 protein
  • Expression of this protein by in vitro purification has found that ⁇ VP8* is significantly superior to VP8 full-length protein in terms of stability and homogeneity.
  • the inventors found in further studies that the ability of ⁇ VP8* to induce the production of neutralizing antibodies was significantly weaker than that of VP8 full-length protein (see implementation below).
  • Example 6 and Figure 5 This results in ⁇ VP8* being unsuitable for use as a highly effective anti-rotavirus vaccine.
  • a VP8 protein having a N-terminally truncated 21-60 amino acid can be expressed in a high expression amount in Escherichia coli, and can be easily purified by chromatography, and thereby High purity truncated protein obtained (purity of at least 50% or higher, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) with good uniformity
  • the above technical problem is effectively solved by the nature and stability (not easily degradable) and the ability to induce the body to produce a high titer neutralizing antibody against rotavirus.
  • the invention relates to N-terminal truncation of 21-60 amino acids, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 Rotavirus VP8 protein, or 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 59 or 60 amino acids body.
  • the invention relates to a truncated rotavirus VP8 protein or variant thereof, which has a N-terminal truncation of 21-60 amino acids compared to the wild-type rotavirus VP8 protein, eg, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59 or 60 amino acids.
  • the N-terminus of the truncated rotavirus VP8 protein is truncated by 21-60 amino acids, such as 21, 25, 30, compared to the wild-type rotavirus VP8 protein. , 35, 40, 45, 50, 55 or 60 amino acids.
  • the truncated rotavirus VP8 protein (hereinafter also simply referred to as a truncated protein) has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.
  • SEQ ID NO: 5 The amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the inventors also conducted further studies on the obtained truncated proteins.
  • the results show that the combination of the truncated protein of the present invention and an intramolecular adjuvant further enhances the immunogenicity of the truncated protein.
  • the obtained fusion protein has higher immunogenicity and can provide a host with a VP8 protein or a truncated protein alone. Higher immune protection.
  • the immunogenicity of the truncated protein can be further enhanced by conjugating the truncated protein of the present invention to an intramolecular adjuvant.
  • the present invention provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a truncated rotavirus VP8 protein as described above or a variant thereof; and the second polypeptide is an intramolecular adjuvant.
  • the fusion protein optionally further comprises a peptide linker, a tag, a signal peptide, a protease cleavage site, or any combination thereof.
  • the intramolecular adjuvant is selected from the group consisting of diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as the A subunit of CRM197, cholera toxin, cholera toxin B subunit CTB, cholera toxin mutant
  • the intramolecular adjuvant is selected from the group consisting of diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as the A subunit of CRM197, cholera toxin, cholera toxin B subunit CTB, cholera toxin mutant
  • CTA112/KDEV and CTA1-DD Escherichia coli heat labile toxin (LT) and its avirulent mutant LTR192G
  • Escherichia coli heat labile toxin B subunit LTB tetanus toxin, and any combination thereof.
  • the first polypeptide is linked to the second polypeptide by direct fusion or by a peptide linker. In certain preferred embodiments, the first polypeptide is located at the N-terminus or C-terminus of the second polypeptide, and the two are optionally joined by a peptide linker.
  • the peptide linker is selected from the group consisting of GS (SEQ ID NO: 32), GGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), SGGGS (SEQ ID NO: 35) , GGGG (SEQ ID NO: 36), GGSS (SEQ ID NO: 37), GGGGS (SEQ ID NO: 38), (GGGGS) 3 (SEQ ID NO: 39), and any combination thereof.
  • the fusion protein further comprises a tag at its N-terminus and/or C-terminus.
  • X Key isomerase tag, DsbA tag, DsbC tag, SUMO tag, msyB tag, TF tag, trigger tag, ubiquitin tag, Myc tag, Flag tag, fluorescent protein (eg GFP) tag, biotin tag, avidin Label, and any combination thereof.
  • the fusion protein further comprises a signal peptide at its N-terminus.
  • the signal peptide is selected from the group consisting of OmpA, OmpT, pelB, CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and a signal peptide of IL-3.
  • the fusion protein further comprises a protease cleavage site.
  • the protease cleavage site is between adjacent two elements selected from the group consisting of the first polypeptide, the second polypeptide, a peptide linker, a tag, and a signal peptide.
  • the fusion protein has the amino acid sequence set forth in any one of SEQ ID NOs: 12-13 and 15-16.
  • the invention provides a conjugate comprising a truncated rotavirus VP8 protein or variant thereof as described above, and conjugated to the truncated VP8 protein or variant thereof Intramolecular adjuvant.
  • the intramolecular adjuvant is selected from the group consisting of diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as the A subunit of CRM197, cholera toxin, cholera toxin B subunit CTB, cholera toxin mutant
  • the intramolecular adjuvant is selected from the group consisting of diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as the A subunit of CRM197, cholera toxin, cholera toxin B subunit CTB, cholera toxin mutant
  • CTA112/KDEV and CTA1-DD Escherichia coli heat labile toxin (LT) and its avirulent mutant LTR192G
  • Escherichia coli heat labile toxin B subunit LTB tetanus toxin, and any combination thereof.
  • the intramolecular adjuvant is covalently (eg, chemically coupled) or non-covalently (eg, adsorbed) with the rotavirus VP8 protein or Variant conjugation.
  • the invention relates to a polynucleotide encoding a truncated protein of the invention, or a variant thereof, or a fusion protein of the invention, and a vector comprising the polynucleotide.
  • Vectors useful for insertion of a polynucleotide of interest include, but are not limited to, cloning vectors and expression vectors.
  • the vector is, for example, a plasmid, a cosmid, a phage, and the like.
  • the invention also relates to a host cell comprising the above polynucleotide or vector.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the invention in another aspect, relates to a composition
  • a composition comprising the above-described truncated protein or variant thereof, or the above-described fusion protein, or the above-described conjugate, or the above polynucleotide or vector or host cell.
  • the composition comprises a truncated protein of the invention, or a variant thereof, or a fusion protein of the invention, or a conjugate of the invention.
  • compositions of the invention comprise a N-terminal truncation of 21-60 amino acids, such as 21, 25, 30, 35, compared to the wild-type rotavirus VP8 protein. 40, 45, 50, 55 or 60 amino acid truncated rotavirus VP8 protein.
  • the compositions of the invention comprise a truncated rotavirus VP8 protein having a sequence selected from the group consisting of SEQ ID NOs: 1-8.
  • the compositions of the invention comprise a fusion protein having a sequence selected from the group consisting of SEQ ID NOS: 12-13 and 15-16.
  • the invention also relates to a pharmaceutical composition or vaccine comprising a truncated protein of the invention or a variant thereof, or a fusion protein of the invention, or a conjugate of the invention, and optionally further A pharmaceutically acceptable carrier and/or excipient is included.
  • the pharmaceutical composition or vaccine of the present invention can be used for preventing or treating rotavirus infection or caused by rotavirus infection
  • the disease is, for example, rotavirus gastroenteritis or diarrhea.
  • the pharmaceutical composition or vaccine further comprises an adjuvant, such as an aluminum adjuvant.
  • a truncated protein of the invention, or a variant thereof, or a fusion protein of the invention, or a conjugate of the invention is caused by the prevention or treatment of a rotavirus infection or by a rotavirus infection.
  • An effective amount of the disease exists.
  • the pharmaceutical compositions or vaccines of the invention further comprise additional active ingredients.
  • the additional active ingredient is capable of preventing or treating a rotavirus infection or a disease caused by a rotavirus infection.
  • compositions or vaccines of the invention may be administered by methods well known in the art such as, but not limited to, administration by oral or injection.
  • pharmaceutical compositions or vaccines of the invention are administered in unit dosage form.
  • the amount of the pharmaceutical composition or vaccine of the invention required to prevent or treat a particular condition will depend on the route of administration, the severity of the condition being treated, the sex, age, weight and general health of the patient, etc., which may be The situation is reasonably determined.
  • the invention in another aspect, relates to a method of obtaining a truncated protein of the invention, or a variant thereof, or a fusion protein of the invention, comprising allowing expression of said truncated protein or variant thereof or said fusion protein
  • the host cell of the present invention is cultured under the conditions; and the expressed truncated protein or a variant thereof or the fusion protein is recovered.
  • the method comprises using an E. coli expression system to express a truncated protein of the invention, or a variant thereof, or the fusion protein, and then lysing the E. coli from the lysate Purification results in the inclusion of the truncated protein or variant thereof or the fusion protein.
  • the purification comprises chromatography.
  • the invention in another aspect, relates to a method of preparing a vaccine comprising the use of a truncated protein of the invention, or a variant thereof, or a fusion protein of the invention, or a conjugate of the invention, and a pharmaceutically acceptable carrier And/or excipients, optionally together with adjuvants such as aluminum adjuvants, and/or additional active ingredients, for example additional agents capable of preventing or treating rotavirus infections or diseases caused by rotavirus infections Active ingredient.
  • adjuvants such as aluminum adjuvants
  • additional active ingredients for example additional agents capable of preventing or treating rotavirus infections or diseases caused by rotavirus infections Active ingredient.
  • the vaccine can be used to prevent or treat rotavirus infection or diseases caused by rotavirus infection such as rotavirus gastroenteritis and diarrhea.
  • the present invention relates to a method for preventing or treating a disease caused by rotavirus infection or infection by rotavirus, which comprises preventing or treating a therapeutically effective amount of a truncated protein according to the present invention or a variant thereof Or a fusion protein of the invention, or a conjugate of the invention, or a pharmaceutical composition or vaccine of the invention, is administered to a subject.
  • the disease caused by rotavirus infection includes, but is not limited to, rotavirus gastroenteritis and diarrhea.
  • the subject is a mammal, such as a mouse and a human.
  • the invention relates to a truncated protein according to the invention or a variant thereof, or a fusion protein of the invention, or the use of a conjugate of the invention in the preparation of a pharmaceutical composition or vaccine,
  • the vaccine is used to prevent or treat a rotavirus infection or a disease caused by a rotavirus infection in a subject.
  • the disease caused by rotavirus infection includes, but is not limited to, rotavirus gastroenteritis and diarrhea.
  • the subject is a mammal, such as a mouse and a human.
  • the invention relates to a truncated protein according to the invention or a variant thereof, or a fusion protein according to the invention, or a conjugate according to the invention for use in the prevention or treatment of rotavirus in a subject Infection or disease caused by rotavirus infection.
  • the disease caused by rotavirus infection includes, but is not limited to, rotavirus gastroenteritis and diarrhea.
  • the subject is a mammal, such as a mouse and a human.
  • the expression "protein having a N-terminally truncated X amino acid” refers to a protein obtained by substituting a methionine residue encoded by a start codon for the amino acid residue of the N-th position of the N-terminus of the protein.
  • a rotavirus VP8 protein having a 21-amino acid truncation at the N-terminus means that the amino acid residue at the N-terminus of the wild-type rotavirus VP8 protein is replaced by a methionine residue encoded by the initiation codon.
  • the term "variant” refers to the amino acid sequence of a protein having an amino acid sequence to a truncated rotavirus VP8 protein of the invention (such as the protein of any one of SEQ ID NOS: 1-8). Having one or more (eg, 1-10 or 1-5 or 1-3) amino acid differences (eg, conservative amino acid substitutions) or having at least 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identity, and it retains the necessary properties of the truncated protein.
  • essential characteristic herein may be one or more of the following characteristics:
  • the "variant" of the invention retains all of the above properties of the truncated protein.
  • the term "identity" is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequence CTGACT and CAGGTT has a total of 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological activity of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein E. coli (strain) is derived from commercially available strains such as, but not limited to, GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3), etc.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of a protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phage; cosmid and the like.
  • VP8 protein refers to the outermost structural protein VP4 (AA 1-776) from RV virions.
  • AA 1-231 consists of a protein.
  • An exemplary amino acid sequence of the VP8 protein can be as set forth in SEQ ID NO:9.
  • the term "truncated rotavirus VP8 protein” refers to a protein after removal of one or more amino acids at the N-terminus and/or C-terminus of the wild-type rotavirus VP8 protein, wherein the wild-type rotavirus
  • the specific amino acid sequence of the VP8 protein can be readily obtained from public databases such as the GenBank database, such as GenBank Accession No. JQ013506.1.
  • the amino acid sequence of the wild-type rotavirus VP8 protein can be as shown in SEQ ID NO: 9.
  • truncated rotavirus VP8 protein gene fragment refers to a gene fragment which lacks the coding at the 5' or 3' end compared to the wild type rotavirus VP8 protein gene.
  • the nucleotide sequence of the wild-type rotavirus VP8 protein gene can be as shown in SEQ ID NO: 10.
  • intramolecular adjuvant refers to an adjuvant which forms a fusion protein with a protein of interest (ie, an antigen) such that it is present in the same molecule as the antigen (ie, comprises its fusion with the antigen) In the protein), and acts as an adjuvant to the antigen to enhance the immunogenicity of the antigen. That is, an intramolecular adjuvant is an adjuvant capable of enhancing the immunogenicity of a target protein (antigen) expressed by fusion therewith. Such intramolecular adjuvants are well known to those skilled in the art and have been described in detail in the prior art literature. Typically, the intramolecular adjuvant is a toxin-like polypeptide fragment.
  • intramolecular adjuvants include, but are not limited to, the diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as the A subunit of CRM197 (Chinese Patent Application CN102807621; Ilyina, N., S. Kharit, L. Namazova- Baranova, A. Asatryan, M. Benashvili, E. Tkhostova, C. Bhusal and AK Arora (2014).
  • Hum Vaccin Immunother 10 2471-2481
  • Cholera Toxin CT O'Neal, CM, JDClements, MKEstes and MEConner , 1998, J Virol 72(4): 3390-3393
  • cholera toxin B subunit CTB Gloudemans, AK, M. Plantinga, M. Guilliams, MAWillart, A. Ozir-Fazalalikhan, A. van der Ham, L. Boon , NL Harris, H. Hammad, HC Hoogsteden, M.
  • the term "peptide linker” refers to a short peptide used to join two molecules, such as proteins.
  • a fusion protein is obtained by introducing a polynucleotide sequence encoding the short peptide (for example, by PCR amplification or ligase) between two DNA fragments of two proteins of interest to be ligated, and performing protein expression.
  • the target protein 1-peptide linker - the target protein 2 the target protein 2.
  • peptide linkers include, but are not limited to, flexible linker peptides such as GGGG (SEQ ID NO: 36), GGSS (SEQ ID NO: 37), GGGGS (SEQ ID NO: 38) and (GGGGS) 3 (SEQ ID NO: 39) and the like.
  • GGGG SEQ ID NO: 36
  • GGSS SEQ ID NO: 37
  • GGGGS SEQ ID NO: 38
  • GGGGS 3
  • a detailed description of such peptide linkers can also be found, for example, in Robinson, CR and RTSauer (1998). Proc Natl Acad Sci 95(11): 5929-5934.
  • the term "tag” refers to a short peptide which is fused or linked to a protein of interest, such as a truncated protein or fusion protein of the invention, and thereby facilitates soluble expression, detection and/or purification of the recombinant protein.
  • the tag can be fused or ligated to the N-terminus and/or C-terminus of the protein of interest (optionally through a linker or protease cleavage site).
  • labels are well known to those skilled in the art and have been described in detail in the prior art literature.
  • such tags include, but are not limited to, histidine tags (Sockolosky, JTand FCSzoka (2013).
  • Protein Expr Purif 86 (1): 53-57 Protein Expr Purif 86 (1): 53-57
  • ubiquitin label (Sabin, EA) , Lee-Ng, Chun Ting, Shuster, Jeffrey R., Barr, Philip J. (1989). Nature Biotechnology 7 (7): 705-709), Myc tag, Flag tag, fluorescent protein (eg GFP) tag (Pedelacq, JD, S. Cabantous, T. Tran, TCTerwilliger and GSWaldo (2006). Nat Biotechnol 24(1): 79-88), biotin tag, and avidin tag.
  • GFP fluorescent protein
  • the term "signal peptide” refers to a short peptide which, when fused to a protein of interest (for example, a truncated protein or fusion protein of the present invention), is capable of promoting secretion of a protein of interest expressed by a cell to the outside of the cell.
  • the signal peptide is typically located at the N-terminus of the protein of interest, and various signal peptides are known to those skilled in the art.
  • Such signal peptides include, but are not limited to, OmpA (Sockolosky, JTand FCSzoka (2013). Protein Expr Purif 87(2): 129-135), OmpT (Pohlmann, C., M. Thomas, S. Forster, M.
  • Vet Immunol Immunopathol 132 (2-4): 314-317), t-pA (Wang, JY, WTSong, Y.Li, WJChen, D.Yang, G .C.Zhong, HZZhou, CYRen, HTYu and H.Ling (2011). Appl Microbiol Biotechnol 91 (3): 731-740), and signal peptides of IL-3 (Tessier, DC, DYThomas, HEKhouri , F. Laliberte and T. Vernet (1991). Gene 98 (2): 177-183).
  • protease cleavage site refers to a site that is capable of being specifically recognized and cleaved by a protease.
  • protease Various specific proteases and their recognition sites are well known to those skilled in the art and are found in many prior art documents.
  • One skilled in the art can use a suitable protease cleavage site in the fusion protein and cleave with the corresponding protease, depending on the actual situation.
  • the use of a protease cleavage site can be advantageous, for example, it can be used to excise a signal peptide and/or tag from a fusion protein to obtain a mature protein having the desired activity.
  • a recombinant protein comprising a tag, a first polypeptide and a second polypeptide is constructed and expressed, wherein a protease cleavage site is designed between the tag and the first polypeptide;
  • the expressed recombinant protein is purified; then, the tag is excised from the recombinant protein using the corresponding protease to obtain a purified fusion protein comprising the first polypeptide and the second polypeptide.
  • conjugation means that two or more molecules (eg, proteins and proteins, proteins and polysaccharides, proteins and labels, etc.) are covalently (eg, chemically coupled) or non-covalently (eg, Adsorption) to connect.
  • conjugate refers to two or more molecules joined together by covalent means (eg, chemical coupling) or non-covalent means (eg, adsorption).
  • a conjugate of the invention can refer to, for example, a truncated VP8 protein or a variant thereof linked to an intramolecular adjuvant by covalent or non-covalent means.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient which is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is in the art It is well known (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) Aluminum), Freund's adjuvant (eg complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) Aluminum), Freund's adjuvant (eg complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount means an amount effective to achieve the intended purpose.
  • an effective amount for preventing or treating a disease means that it can be effectively prevented.
  • the amount that prevents or delays the onset of a disease eg, a rotavirus infection
  • alleviates, alleviates, or treats the severity of an existing disease such as a disease caused by a rotavirus infection. Determination of such effective amounts is well within the abilities of those skilled in the art.
  • chromatography includes, but is not limited to, ion exchange chromatography (eg cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (eg hydroxyapatite chromatography), gel filtration (gel discharge) Resistance) chromatography, affinity chromatography.
  • lysis/crushing of host cells can be achieved by various methods well known to those skilled in the art including, but not limited to, homogenizer disruption, homogenizer disruption, sonication, milling, high pressure extrusion, lysozyme treatment. and many more.
  • the N-terminally truncated rotavirus VP8 protein provided by the present invention and a preparation method thereof effectively solve the technical problems existing in the art.
  • the truncated protein of the present invention or a variant thereof can be expressed in a high expression amount in Escherichia coli, achieving high yield.
  • the truncated protein of the present invention or a variant thereof is purified in a relatively simple manner and is easy to handle.
  • the Escherichia coli can be lysed, and then the lysate is subjected to a chromatographic treatment such as ion exchange chromatography to obtain a high-purity truncated protein (purity can be obtained).
  • a chromatographic treatment such as ion exchange chromatography
  • the truncated proteins of the present invention or variants thereof have good homogeneity and stability and are not susceptible to degradation.
  • the truncated proteins of the present invention or variants thereof are capable of inducing the body to produce high titers of neutralizing antibodies against rotavirus.
  • a fusion protein comprising a truncated protein of the invention and an intramolecular adjuvant (eg, CRM197-A or CTB) can stimulate a mouse to produce higher levels than a single truncated protein or a truncated protein + aluminum adjuvant.
  • an intramolecular adjuvant eg, CRM197-A or CTB
  • the immune response produces a stronger immune protection.
  • the truncated protein of the present invention or a variant thereof and the fusion protein of the present invention have the ability to induce neutralizing antibodies in a strong induction body of the full-length wild-type VP8 protein.
  • it has high expression, easy purification, high uniformity and stability. Therefore, the truncated protein of the present invention (or a variant thereof) and the fusion protein can be used as a highly efficient anti-rotavirus vaccine, and enable large-scale industrial production of a highly effective anti-rotavirus vaccine, thereby being present in the art.
  • the technical issues provide an effective solution.
  • Figure 1 shows the results of SDS-PAGE of freshly purified full length VP8 protein and its placement at 4 °C for three days.
  • Lane 1 freshly purified full length VP8 protein
  • Lane 2 full length VP8 protein after 3 days at 4 °C. The results showed that the full-length VP8 protein was significantly degraded after being placed at 4 ° C for three days, and its stability was poor.
  • Figures 2A-2B show various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) at The results of SDS polyacrylamide gel electrophoresis after purification was completed (Fig. 2A) and after 3 days at 4 °C (Fig. 2B).
  • Fig. 2A the lanes are from left to right: VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.
  • concentration of VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12 protein is about 2.0 mg. /ml, and the purity is greater than 98%, and its expression level is significantly better than VP-8 full-length protein.
  • the lanes from left to right are: VP8 protein, VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.
  • the results in Figure 2B show that the stability of VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12 proteins is significantly better than VP-8 full-length protein. After being placed at 4 ° C for 3 days, No significant degradation occurred.
  • Figures 3A-3D show various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) and Results of indirect ELISA analysis of antibodies 1B11 (Fig. 3A), 11C1 (Fig. 3B), 3C5 (Fig. 3C), and 1E1 (Fig. 3D), in which the abscissa indicates each truncated protein and the ordinate indicates reaction with each truncated protein. Maximum antibody dilution factor for the primary antibody (1E1, 1B11, 11C1 or 3C5).
  • each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) was recognized by four neutralizing antibodies. It has good antigenicity (ie, antibody reactivity), which is equivalent to or even stronger than the antigenicity of ⁇ VP8*.
  • Figure 4 shows various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) and used Results of an indirect ELISA assay of immune sera obtained by immunizing Balb/c mice, wherein the abscissa indicates each truncated protein and the ordinate indicates the maximum dilution factor of the immune serum reactive with the corresponding truncated protein (ie, antibody drop degree). The results showed that the truncated protein was able to efficiently induce antibody production in mice on the 42nd day after immunization.
  • the antibody titer (GMT) in the immune serum can reach 10 5 or higher. There were no significant differences between the experimental groups. These results indicate that each of the truncated proteins of the present invention (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) has a good immunogen. Sexually, it can effectively induce the production of antibodies in animals, and its efficacy is not significantly different from VP8 and ⁇ VP8*.
  • Figure 5 shows various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) in Balb/ Analysis results of neutralizing antibody titers of immune sera induced in c mice, wherein the abscissa indicates each truncated protein, and the ordinate indicates the maximum dilution factor of immune serum (NT50, neutralizing antibody titer) that achieves 50% infection inhibition rate. ).
  • NT50 neutralizing antibody titer
  • the neutralizing antibody titer (NT50) of the immune serum induced by the truncated protein of the present invention can reach a level of 10 3 or higher.
  • Figure 6 shows the results of analysis of neutralizing antibody titers of positive sera after incubation with each truncated protein, wherein the abscissa indicates each truncated protein incubated with positive serum, and the ordinate indicates that the infection inhibition rate was 50%.
  • Maximum dilution of positive serum after incubation (NT50, neutralizing antibody titer).
  • the results show that the truncated proteins of the invention (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) result in positive serum after incubation.
  • the titer of neutralizing antibody was significantly decreased: the neutralizing antibody titer of positive serum incubated with TB8.0 could exceed 6*10 3 ; the neutralizing antibody titer of positive serum incubated with ⁇ VP8* could exceed 5*10 3 ; while the positive serum incubated with the truncated protein of the present invention has a neutralizing antibody titer of less than 4*10 3 or even less than 3*10 3 .
  • Figures 7A-7C show the scoring criteria for diarrhea in a protective experiment. According to the different degrees of diarrhea in suckling rats, the score is divided into 3 grades: 1 point for normal feces (Fig. 7C), 2 points for soft stools (Fig. 7B), and 3 points for unformed water samples (Fig. 7A). .
  • Figure 8 shows the diarrhea score after immunization of Balb/c mice with each of the truncated proteins prepared in Example 3, wherein the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after challenge in mice.
  • Figure 9 shows the average number of days of diarrhea after immunization of Balb/c mice with each of the truncated proteins prepared in Example 3, wherein the vertical axis represents the average number of days of diarrhea; and the horizontal axis represents each truncated protein.
  • Figure 10 shows the results of SDS-PAGE of the fusion proteins (CTB-VP8-5 and VP8-5-CTB) containing the truncated protein VP8-5 and the intramolecular adjuvant CTB prepared in Example 9 after completion of purification.
  • the lanes from left to right are: CTB-VP8-5, VP8-5-CTB, VP8-5 and CTB.
  • Figure 11 shows the results of analysis of total antibody titer and neutralizing antibody titer in immune sera induced by fusion proteins (CTB-VP8-5 and VP8-5-CTB) in Balb/c mice, wherein the abscissa indicates The various proteins used, the left ordinate indicates the neutralizing antibody titer (NT50, ie, the maximum dilution factor of the immune serum that achieves 50% inhibition of infection), and the right ordinate indicates the total antibody titer (log); and, The antibody titer is represented by a bar graph and the total antibody titer is represented by a graph.
  • CTB-VP8-5 and VP8-5-CTB fusion proteins
  • the fusion proteins of the present invention can efficiently induce immune sera with high antibody titers in mice on day 42 after immunization (after three immunizations), and The total anti-titer and neutralizing antibody titers in the immunized serum were significantly higher than the total anti-titer and neutralizing antibody titers in the immune serum of the VP8-5 and CTB groups.
  • These results indicate that the immunogenicity of the truncated proteins of the present invention can be further enhanced by linking the truncated proteins of the present invention to intramolecular adjuvants (e.g., CTB) to achieve a better immune effect.
  • intramolecular adjuvants e.g., CTB
  • Figure 12 shows the diarrhea score after immunization of Balb/c mice with the fusion protein prepared in Example 9, in which the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after challenge in mice.
  • CTB-VP8-5 and VP8-5-CTB fusion proteins of the present invention
  • Figure 13 shows the fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) comprising the truncated protein VP8-5 and the intramolecular adjuvant CRM-A prepared in Example 10 after purification. SDS-PAGE results. The lanes from left to right are: CRM-A-VP8-5, VP8-5-CRM-A, VP8-5 and CTB.
  • Figure 14 shows the results of analysis of total antibody titers and neutralizing antibody titers in immune sera induced by fusion proteins (CRM-A-VP8-5 and VP8-5-CRM-A) in Balb/c mice, Wherein, the abscissa indicates various proteins used, the left ordinate indicates the neutralizing antibody titer (NT50, ie, the maximum dilution factor of the immune serum that achieves 50% infection inhibition rate), and the right ordinate indicates the total antibody titer (log) And, the neutralizing antibody titer is represented by a histogram, and the total antibody titer is represented by a graph.
  • NT50 neutralizing antibody titer
  • NT50 the neutralizing antibody titer
  • log total antibody titer
  • the fusion proteins of the present invention can effectively induce immunity with high antibody titers in mice on day 42 after immunization (after three immunizations). Serum, and the total anti-titer and neutralizing antibody titers in the immune serum were significantly higher than the total anti-titer and medium in the sera of the VP8-5 and CRM-A groups. And antibody titer.
  • These results indicate that the immunogenicity of the truncated proteins of the present invention can be further enhanced by linking the truncated proteins of the present invention to intramolecular adjuvants (e.g., CRM-A) to achieve a better immune effect.
  • intramolecular adjuvants e.g., CRM-A
  • Figure 15 shows the diarrhea score after immunization of Balb/c mice with the fusion protein prepared in Example 10, wherein the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after the mouse was challenged.
  • the results show that the fusion proteins of the invention (CRM-A-VP8-5 and VP8-5-CRM-A) have significant immunoprotection and are equivalent or superior to those not fused with the intramolecular adjuvant CRM-A. VP8-5.
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, And the method described in FMAusubel et al., Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995 or in accordance with product specifications.
  • the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
  • the invention is described by way of example, and is not intended to limit the scope of the invention. Modifications or substitutions of the methods, steps or conditions of the invention are intended to be included within the scope of the invention.
  • the rotavirus LLR strain was donated by Beijing Wantai Biopharmaceutical Co., Ltd.; the prokaryotic expression vector PTO-T7 was constructed independently by the laboratory; E. coli ER2566 was purchased from New England Biolab Company; the primers used were used. They are all synthesized by Shenggong Biological (Shanghai) Engineering Co., Ltd.
  • Example 1 Construction of an expression vector encoding a truncated rotavirus VP8 protein
  • the rotavirus LLR strain was cultured with a rhesus embryonic kidney cell line (MA-104) cell.
  • the medium used was DMEM supplemented with 2 ⁇ g/ml trypsin, 0.5 mg/ml ampicillin and 0.4 mg/ml streptomycin, 3.7 mg/ml sodium bicarbonate, and 0.34 mg/ml L-valley. Aminoamide.
  • the viral DNA/RNA extraction kit produced by Beijing Jinmaige Biotechnology Co., Ltd. was used to extract the genomic RNA of rotavirus and pass the counter.
  • the cDNA encoding the VP4 protein was obtained by transcription.
  • the obtained cDNA is used as a template, and a gene fragment encoding the truncated rotavirus VP8 protein is amplified by a PCR reaction.
  • the primers used are as follows:
  • the cleavage site is underlined, and the introduced stop codon is indicated in italics.
  • the PCR reaction system used is as follows:
  • the PCR reaction conditions were as follows: pre-denaturation at 95 ° C for 5 min, 35 cycles (95 ° C, 40 s; 55 ° C, 40 s; 72 ° C for 1 min), and a final extension of 72 ° C for 10 min.
  • the obtained amplification product was detected by 2% agarose gel electrophoresis.
  • Each PCR amplification product was ligated to a commercially available pMD18-T vector (TAKARA) and transferred into Escherichia coli DH5 ⁇ . Then, the positive colonies were screened, and the plasmid was extracted and identified by NdeI/HindIII digestion to obtain the positive clone plasmid pMD 18-T-VP8-5A, pMD 18-T-VP8-5, pMD 18-T-VP8 inserted into the target gene fragment. -6, pMD 18-T-VP8-8, pMD 18-T-VP8-9, pMD 18-T-VP8-10, pMD 18-T-VP8-11, and pMD 18-T-VP8-12.
  • TAKARA commercially available pMD18-T vector
  • the above positive clones were sequenced using the M13(+)/(-) primers at Shanghai Shenggong Bioengineering Co., Ltd.
  • the sequencing results showed that the nucleotide sequence of the target fragment inserted in the above positive clone plasmid was identical to the expected one, and the encoded amino acid sequence was as shown in SEQ ID NOs: 1-8, which correspond to the N-terminal truncation 21, respectively. 25, 30, 40, 45, 50, 55, 60 amino acids, C-terminal untruncated VP8 protein.
  • the above positive clone plasmid was digested with NdeI/HindIII to obtain a fragment of the gene encoding each truncated VP8 protein, and the non-fusion expression vector pTO-T7 digested with NdeI/HindIII (Luo Wenxin et al., Journal of Bioengineering, 2000, 16:53-57) was ligated into E. coli ER2566; then, the positive colonies were screened, and the plasmid was extracted and identified by NdeI/HindIII digestion to obtain the positive expression plasmid inserted into the target fragment: pTO-T7-VP8-5A.
  • an expression plasmid expressing the full-length VP8 protein, PTO-T7-VP8, was also constructed by a method similar to the above.
  • the PCR amplification primers used are as follows:
  • Upstream primer 5'- GGATCCCATATG GCTTCGCTCATT-3' (SEQ ID NO: 26);
  • Downstream primer 5'- AAGCTT A GGATCC GGTGTTTTGTATTGGTGG-3 '(SEQ ID NO: 28);
  • the cleavage site is underlined, and the introduced stop codon is indicated in italics.
  • Upstream primer 5'- GGATCCCATATG TTGAATGGACCA-3' (SEQ ID NO: 27);
  • Downstream primer 5'- AAGCTT ATCCATTATTTACGTATTCTGTG-3' (SEQ ID NO: 29).
  • Example 2 Expression of truncated rotavirus VP8 protein
  • the recombinant plasmids pTO-T7-VP8-5A, pTO-T7-VP8-5, pTO-T7-VP8-6, pTO-T7-VP8-8, pTO-T7-VP8 prepared in Example 1 were taken from -70 °C. -9, pTO-T7-VP8-10, pTO-T7-VP8-11 or pTO-T7-VP8-12 E. coli bacterial solution, inoculated into 50 ml of kanamycin-containing LB liquid medium, Incubate at 180 rpm for approximately 4 hours at 37 ° C; then transfer to 10 bottles of 500 ml LB medium containing kanamycin (500 ul of bacteria per vial). When the absorbance of the culture at 600 nm reached 0.5, IPTG was added to a final concentration of 1 mM, and incubation was continued at 180 rpm, 25 ° C for 6 hours.
  • Example 3 Purification and characterization of truncated rotavirus VP8 protein
  • the cell wall of E. coli cells was disrupted by using a sonicator (Thermo) at 4 min/1 g of wet bacteria under Tris-HCl 8.0 buffer at 4 ° C, and soluble fractions were collected and compared by the following protocol.
  • the recombinant protein expressed by E. coli cells was purified.
  • Chromatography medium Q-sepharose-HP (GE Healthcare).
  • the sample is a previously prepared E. coli lysate soluble fraction containing recombinantly expressed VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12.
  • the elution procedure was as follows: 1000 mM NaCl eluted the heteroprotein, 50 mM NaCl was used to elute the protein of interest, and 50 mM NaCl was eluted to obtain 30 mL of recombinantly expressed VP8-5A, VP8-5, VP8-6, VP8-8, VP8. A purified sample of -9, VP8-10, VP8-11 or VP8-12.
  • purified samples containing recombinantly expressed ⁇ VP8* were also obtained by a similar purification protocol; and purified samples containing recombinantly expressed VP8 full-length protein were obtained by DEAE-FF chromatography column (GE) purification.
  • Figure 1 shows the results of SDS-PAGE of freshly purified full length VP8 protein and its placement at 4 °C for three days.
  • Lane 1 freshly purified full length VP8 protein
  • Lane 2 full length VP8 protein after 3 days at 4 °C.
  • the results in Fig. 1 show that the full-length VP8 protein is significantly degraded after being left at 4 ° C for three days, and its stability is poor.
  • Figure 2A shows various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) that have just been purified.
  • SDS Results of polyacrylamide gel electrophoresis.
  • the lanes are from left to right: VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.
  • Figure 2B shows purified various truncated rotavirus VP8 proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, after 3 days of placement at 4 °C, Results of SDS polyacrylamide gel electrophoresis of VP8-11 or VP8-12).
  • the results in Figure 2B show that the stability of VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12 proteins is significantly better than VP-8 full-length protein. After 3 days at 4 ° C, no significant degradation occurred.
  • VP8 full-length protein is poor in homogeneity and is prone to form multimers; whereas the truncated proteins of the present invention (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8)
  • the homogeneity of -11 or VP8-12 was significantly better than that of the VP-8 full-length protein, and substantially no multimer was present in the purified sample.
  • the purified truncated rotavirus VP8 protein obtained in the above Example 3 was diluted to 0.5 ng/ul with a coating buffer (20 mM PBS, pH 7.4), and the plate was coated at room temperature for 2 hours. The plate was washed once with 0.1% PBS-Tween 20 (PBST), and the plate was blocked with a 1% BSA in PBST solution for 2 hours at room temperature, and patted dry.
  • PBST 0.1% PBS-Tween 20
  • Neutralizing antibodies 1E1, 1B11, 11C1, and 3C5 (the laboratory was prepared by hybridoma technology at a concentration of 1 mg/ml, and the neutralizing potency IC50 of each monoclonal antibody was 200 ng/ml, 200 ng/ml, 50 ng/ml, and 5 ng/ml) gradient dilution, and then indirect ELISA analysis with the coated truncated protein, respectively.
  • the secondary antibody used was goat anti-mouse antibody (Wan Meimei) to determine the reaction with each truncated protein.
  • the maximum antibody dilution factor of the primary antibody (1E1, 1B11, 11C1, 3C5). The greater the dilution factor of the antibody, the higher the reactivity of the truncated protein with the antibody.
  • the ⁇ VP8* and VP-8 full-length proteins were used as controls. .
  • Example 5 Analysis of immunogenicity of truncated rotavirus VP8 protein
  • the purified truncated rotavirus VP8 protein obtained in the above Example 3 was diluted to 0.5 ng/ul with a coating buffer (20 mM PBS, pH 7.4), and the plate was coated at room temperature for 2 hours. The plate was washed once with 0.1% PBS-Tween 20 (PBST), and the plate was blocked with a 1% BSA in PBST solution for 2 hours at room temperature, and patted dry.
  • PBST 0.1% PBS-Tween 20
  • the purified truncated rotavirus VP8 protein obtained in the above Example 3 was used to immunize Balb/c mice.
  • the immunization protocol was as follows: 70 4-5 week old female Balb/c mice were randomly divided into 10 groups, 7 mice in each group, 2 of which were the control group and the other 8 groups were the experimental group.
  • the experimental group was injected subcutaneously with each of the truncated proteins (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) prepared in Example 3.
  • Each mouse was injected at a dose of 10 ⁇ g of a recombinant protein mixed with 1:1 adjuvant.
  • immune sera were collected from the immunized mice.
  • the immune serum was diluted in a gradient and then subjected to indirect ELISA analysis with the corresponding truncated protein after coating (the secondary antibody used was a goat anti-mouse antibody (Wan Meimei) to determine the reaction with the corresponding truncated protein.
  • the maximum dilution factor of the sexual immune serum The larger the dilution factor of the immune serum, the higher the titer of the anti-truncated protein antibody in the immune serum, and the higher the immunogenicity of the truncated protein used to generate the immune serum.
  • the results of the indirect ELISA are shown in Figure 4, in which the abscissa indicates each truncated protein and the ordinate indicates the maximum dilution factor (i.e., antibody titer) of the immune serum reactive with each truncated protein.
  • the results show that truncation
  • the protein can effectively induce antibody production in mice on the 42nd day after immunization. After the immunization program is completed, the antibody titer (GMT) in the immune serum can reach 10 5 or higher. There is no significant difference between the experimental groups.
  • Example 6 Analysis of the ability of truncated rotavirus VP8 protein to induce neutralizing antibodies
  • mice per group The experimental group of Balb/c mice (7 mice per group) were immunized with the purified truncated rotavirus VP8 protein obtained in Example 3 above using the immunization protocol described in Example 5, and collected. Immune serum. In addition, using the same immunization protocol, ⁇ VP8*, VP-8 full-length protein, rotavirus RV and Freund's adjuvant were used as controls for the immunization control group Balb/c mice (7 mice per group) ) and collect immune serum.
  • Infection inhibition rate (virus dot count of wells not added to serum - virus dot count of wells added to serum) / virus dot count of wells not added to serum * 100%.
  • the neutralizing antibody titer in the immune serum is defined as the maximum dilution factor of the immune serum that achieves a 50% inhibition rate of infection. Immune sera that achieved 50% inhibition of infection after 50-fold dilution were considered to have neutralizing ability.
  • the results of the analysis of the neutralizing antibody titer of the immune serum are shown in Fig. 5, wherein the abscissa indicates each truncated protein, and the ordinate indicates the maximum dilution factor (NT50, neutralizing antibody titer) of the immune serum which reaches 50% inhibition rate of infection. .
  • the results show that the truncated protein of the present invention can effectively induce immune sera with high neutralizing antibody titers in mice on day 42 after immunization (after three immunizations). After the completion of the immunization procedure, the neutralizing antibody titer (NT50) of the immune serum induced by the truncated protein of the present invention can reach a level of 10 3 or higher.
  • each of the truncated proteins of the present invention has a strong induction body.
  • the ability to produce neutralizing antibodies enables the induction of immune sera with high neutralizing antibody titers in animals that are effective in inhibiting rotavirus infection.
  • the ability of the truncated proteins of the invention to induce neutralizing antibodies is significantly superior to ⁇ VP8* and is close to VP8 full-length protein and RV.
  • Example 7 Reactivity of truncated rotavirus VP8 protein with polyclonal antiserum
  • mice were immunized with rotavirus and immune sera were collected and serially diluted (from a dilution of 200 fold, gradient dilution to 25600 fold). Then, each diluted serum sample was separately labeled with each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) (1 mg/) Ml) was mixed and incubated for 1 hour at 37 °C. After the incubation, the mixture was added to a 96-well cell culture plate pre-plated with MA104 cells, and further cultured at 37 ° C for 14 h.
  • truncated protein VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12
  • the amount of viral infection was detected using Elispot, and the neutralizing antibody titer (NT50) of the positive serum after incubation with each truncated protein was calculated as described in Example 6.
  • Fig. 6 The results of the analysis of the neutralizing antibody titer of the positive serum after incubation with each truncated protein are shown in Fig. 6, wherein the abscissa indicates each truncated protein incubated with the positive serum, and the ordinate indicates that the infection inhibition rate was 50%. Maximum dilution of positive serum after incubation (NT50, neutralizing antibody titer).
  • NT50 neutralizing antibody titer
  • the titer of neutralizing antibody was significantly decreased: the neutralizing antibody titer of positive serum incubated with TB8.0 could exceed 6*10 3 ; the neutralizing antibody titer of positive serum incubated with ⁇ VP8* could exceed 5*10 3 ; while the positive serum incubated with the truncated protein of the present invention has a neutralizing antibody titer of less than 4*10 3 or even less than 3*10 3 .
  • Example 8 Protective evaluation of truncated rotavirus VP8 protein in animals
  • mice per group The experimental group of Balb/c mice (7 mice per group) were immunized with the purified truncated rotavirus VP8 protein obtained in the above Example 3 using the immunization protocol described in Example 5.
  • ⁇ VP8*, VP-8 full-length protein, rotavirus RV and Freund's adjuvant were used as controls for the immunization control group Balb/c mice (7 mice per group) ).
  • Mouse eye blood was collected before immunization and at 14 days, 28 days and 42 days after immunization, and serum was separated and stored at -20 °C.
  • mice After completion of the immunization procedure (42 days after immunization), each group of mice was mated with one male mouse per two female rats. About 20 days after mating, the mother rat gave birth to the suckling rat for 7 days. The 7-day-old suckling mice were intragastrically challenged with LLR strain, and the challenge dose was 5*10 6 TCID50/only.
  • FIGS 7A-7C The scoring criteria are shown in Figures 7A-7C. According to the different degrees of diarrhea in suckling rats, the score is divided into 3 grades: 1 point for normal feces (Fig. 7C) and 2 points for soft stools. (Fig. 7B), the water sample was not formed and counted as 3 points (Fig. 7A).
  • Figure 8-9 shows the diarrhea score after immunization of Balb/c mice with each of the truncated proteins prepared in Example 3, wherein the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after challenge in mice.
  • Figure 9 shows the average number of days of diarrhea after immunization of Balb/c mice with each of the truncated proteins prepared in Example 3, wherein the vertical axis represents the average number of days of diarrhea; and the horizontal axis represents each truncated protein.
  • Example 9 Expression and purification of fusion proteins comprising VP8-5 and intramolecular adjuvant CTB and their ability to induce neutralizing antibodies and their immunoprotective properties
  • the CTB gene of the N-terminal and C-terminal restriction sites BglII/NdeI and BamHI/HindIII was synthesized from Shenggong Biological (Shanghai) Engineering Co., Ltd.
  • the CTB gene was digested with Nde I/Hind III and cloned into the PTO-T7 vector to obtain PTO-T7-CTB (the cloning process was the same as in Example 1); subsequently, the VP8-5 gene was utilised with BamH I/Hind III. Double digestion and cloning into PTO-T7-CTB (cloning procedure as in Example 1), thereby obtaining recombinant plasmid PTO-T7-CTB-VP8-5.
  • primers (SEQ ID NO: 18 and SEQ ID NO: 28) were used to amplify the VP8-5 gene; subsequently, the amplified product was double digested with Nde I/Hind III and cloned into the PTO-T7 vector, thereby PTO-T7-VP8-5' was obtained; further, the CTB gene was digested with BglII/HindIII, and then cloned into PTO-T7-VP8-5' (the cloning process was the same as in Example 1) to obtain the recombinant plasmid PTO- T7-VP8-5-CTB.
  • the fusion proteins CTB-VP8-5 and VP8-5-CTB were expressed in E. coli using the constructed recombinant plasmid according to the method described in Example 2. Specifically, when the absorbance of the culture at 600 nm reached 0.5, IPTG was added to the culture to a final concentration of 1 mM; subsequently, Escherichia coli was further cultured at 180 rpm, 25 ° C for 6 hours to induce Escherichia coli expression of the fusion protein. .
  • Escherichia coli expressing the fusion protein was sonicated according to the method described in Example 3. Collect insoluble fractions and use Buffer I (50 mM Tris-HCl pH 8.0 + 150 mM) Reconstituted with NaCl). Subsequently, the obtained solution was centrifuged at 25000 g for 10 mins, and the precipitate was collected. The precipitate was dissolved with 8 M urea and dialyzed at 4 ° C to remove urea and anneal the fusion protein in the precipitate. Subsequently, the fusion protein in the solution was purified by the following protocol.
  • Chromatography medium Q-sepharose-HP (GE Healthcare).
  • the sample is a previously prepared solution comprising the renatured fusion protein CTB-VP8-5 or VP8-5-CTB.
  • the elution procedure was: elution of the protein with 1000 mM NaCl, elution of the protein of interest with 300 mM NaCl, collection of 300 mM NaCl eluted product, and a total of 30 mL of purified samples containing recombinantly expressed CTB-VP8-5 and VP8-5-CTB were obtained.
  • Figure 10 shows the results of SDS-PAGE of the fusion protein (CTB-VP8-5 and VP8-5-CTB) containing the truncated protein VP8-5 and the intramolecular adjuvant CTB prepared above after purification.
  • the results showed that the constructed fusion protein was highly expressed in E. coli, and the above method efficiently enriched and purified the fusion protein expressed in E. coli.
  • mice were immunized with the purified fusion protein prepared above using the immunization protocol described in Example 5 (the Freund's adjuvant used therein was replaced with an aluminum adjuvant). Subsequently, total antibody titers and neutralizing antibody titers in the immune sera of the immunized mice were analyzed using the protocol described in Examples 5-6. In addition, the party described in Embodiment 8 is also used. To analyze the immunoprotective properties of fusion proteins in animals. The results are shown in Figures 11-12.
  • Figure 11 shows the results of analysis of total antibody titer and neutralizing antibody titer in immune sera induced by fusion proteins (CTB-VP8-5 and VP8-5-CTB) in Balb/c mice, wherein the abscissa indicates For each of the various proteins used, the left ordinate indicates the neutralizing antibody titer (NT50, i.e., the maximum dilution factor of the immune serum that achieves 50% inhibition of infection), and the right ordinate indicates the total antibody titer (log).
  • NT50 neutralizing antibody titer
  • log total antibody titer
  • the fusion proteins of the present invention can efficiently induce immune sera with high antibody titers in mice on day 42 after immunization (after three immunizations), and The total anti-titer and neutralizing antibody titers in the immunized serum were significantly higher than the total anti-titer and neutralizing antibody titers in the immune serum of the VP8-5 and CTB groups.
  • These results indicate that the immunogenicity of the truncated proteins of the present invention can be further enhanced by linking the truncated proteins of the present invention to intramolecular adjuvants (e.g., CTB) to achieve a better immune effect.
  • intramolecular adjuvants e.g., CTB
  • Figure 12 shows the diarrhea score after immunization of Balb/c mice with the fusion protein prepared above, wherein the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after the mouse was challenged.
  • CTB-VP8-5 and VP8-5-CTB have significant immunoprotective properties; among them, CTB-VP8-5 and VP8-5-CTB have significantly better immunoprotective properties.
  • the negative control group was superior to VP8-5 which was not fused with the intramolecular adjuvant CTB, and CTB-VP8-5 had the highest immunoprotectiveness.
  • Example 10 Expression and purification of fusion proteins comprising VP8-5 and intramolecular adjuvant CRM-A and their ability to induce neutralizing antibodies and their immunoprotective properties
  • the CRM-A gene of NdeI and BamHI/HindIII at the N-terminus and C-terminus was synthesized from Shenggong Biological (Shanghai) Engineering Co., Ltd., and the C-terminus of the gene was ligated with a nucleotide sequence encoding a peptide linker. .
  • the CRM-A gene was cloned into the PTO-T7 vector by NdeI/Hind III double digestion according to the method described in Example 9, thereby obtaining PTO-T7-CRM-A; subsequently, double digestion by BamH I/HindIII VP8-5 was ligated into PTO-T7-CRM-A to obtain recombinant plasmid PTO-T7-CRM-A-VP8-5.
  • the CRM-A gene was amplified with primers (SEQ ID NOS: 30 and 31).
  • the obtained amplification product contains a nucleotide sequence encoding a peptide linker at its N-terminus.
  • the amplified product was cloned into PTO-T7-VP8-5' by double digestion, thereby obtaining the recombinant plasmid PTO-T7-VP8-5-CRM-A.
  • the recombinant plasmid PTO-T7-CRM-A-VP8-5 encodes the fusion protein CRM-A-VP8-5, wherein CRM-A is linked to the N-terminus of VP8-5 via a peptide linker.
  • the recombinant plasmid PTO-T7-VP8-5-CRM-A encodes the fusion protein VP8-5-CRM-A, wherein VP8-5 is linked to the N-terminus of CRM-A via a peptide linker.
  • the fusion proteins CRM-A-VP8-5 and VP8-5-CRM-A were expressed in E. coli using the constructed recombinant plasmid according to the method described in Example 2. Specifically, when the absorbance of the culture at 600 nm reached 0.5, IPTG was added to the culture to a final concentration of 1 mM; subsequently, Escherichia coli was further cultured at 180 rpm, 25 ° C for 6 hours to induce Escherichia coli expression of the fusion protein. .
  • Escherichia coli expressing the fusion protein was sonicated according to the method described in Example 3. The soluble fraction was collected, and 25% ammonium sulfate was added thereto to precipitate the protein therein. Protein pellets were collected and reconstituted with 50 mM Tris-HCl pH 8.0. The reconstituted solution was centrifuged and the supernatant was collected. Subsequently, the fusion protein in the supernatant was purified by the following protocol.
  • Chromatography medium Q-sepharose-HP (GE Healthcare).
  • the sample was a previously prepared supernatant containing the fusion protein CRM-A-VP8-5 or VP8-5-CRM-A.
  • the elution procedure was as follows: 1000 mM NaCl eluted the heteroprotein, 300 mM NaCl was used to elute the protein of interest, 300 mM NaCl was eluted, and a total of 30 mL of recombinantly expressed CRM-A-VP8-5 and VP8-5-CRM-A were obtained. Purified sample.
  • Figure 13 shows the SDS-PAGE of the fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) containing the truncated protein VP8-5 and the intramolecular adjuvant CRM-A prepared above after purification. result.
  • the results showed that the constructed fusion protein was highly expressed in E. coli, and the above method efficiently enriched and purified the fusion protein expressed in E. coli.
  • mice were immunized with the purified fusion protein prepared above using the immunization protocol described in Example 5 (the Freund's adjuvant used therein was replaced with an aluminum adjuvant). Subsequently, total antibody titers and neutralizing antibody titers in the immune sera of the immunized mice were analyzed using the protocol described in Examples 5-6. In addition, the immunoprotective properties of the fusion protein in animals were also analyzed using the protocol described in Example 8. The results are shown in Figures 14-15.
  • Figure 14 shows the results of analysis of total antibody titers and neutralizing antibody titers in immune sera induced by fusion proteins (CRM-A-VP8-5 and VP8-5-CRM-A) in Balb/c mice, Wherein, the abscissa indicates various proteins used, the left ordinate indicates the neutralizing antibody titer (NT50, ie, the maximum dilution factor of the immune serum that achieves 50% infection inhibition rate), and the right ordinate indicates the total antibody titer (log) ).
  • NT50 neutralizing antibody titer
  • log total antibody titer
  • the fusion proteins of the present invention (CRM-A-VP8-5 and VP8-5-CRM-A) can effectively induce immunity with high antibody titers in mice on day 42 after immunization (after three immunizations). Serum, and the total anti-titer and neutralizing antibody titers in the immune sera were significantly higher than the total anti-titer and neutralizing antibody titers in the sera of the VP8-5 and CRM-A groups.
  • These results indicate that the immunogenicity of the truncated proteins of the present invention can be further enhanced by linking the truncated proteins of the present invention to intramolecular adjuvants (e.g., CRM-A) to achieve a better immune effect.
  • intramolecular adjuvants e.g., CRM-A
  • Figure 15 shows the diarrhea score after immunization of Balb/c mice with the fusion protein prepared above, wherein the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after the mouse was challenged.
  • the results show that the fusion proteins of the invention (CRM-A-VP8-5 and VP8-5-CRM-A) have significant immunoprotection; among them, CRM-A-VP8-5 and VP8-5-CRM-A The immunoprotection was significantly better than the negative control group, and VP8-5-CRM-A was the most immunoprotective.

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Abstract

本发明公开了一种截短的轮状病毒VP8蛋白，包含所述截短蛋白的融合蛋白，包含所述截短蛋白的缀合物，所述截短蛋白和融合蛋白的编码序列和制备方法，包含所述截短蛋白、融合蛋白或缀合物的药物组合物和疫苗，所述截短蛋白、融合蛋白、缀合物、药物组合物和疫苗可用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病，例如轮状病毒性胃肠炎和腹泻等。还公开了上述截短蛋白、融合蛋白、缀合物用于制备药物组合物或疫苗的用途。

Description

截短的轮状病毒VP8蛋白及其用途 技术领域
本发明涉及生物化学、分子生物学、分子病毒学和免疫学领域。具体地，本发明涉及一种截短的轮状病毒VP8蛋白，包含所述截短蛋白的融合蛋白，包含所述截短蛋白的缀合物，所述截短蛋白和融合蛋白的编码序列和制备方法，包含所述截短蛋白、融合蛋白或缀合物的药物组合物和疫苗，所述截短蛋白、融合蛋白、缀合物、药物组合物和疫苗可用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病，例如轮状病毒性胃肠炎和腹泻等。本发明还涉及上述截短蛋白、融合蛋白、缀合物用于制备药物组合物或疫苗的用途，所述药物组合物或疫苗用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病，例如轮状病毒性胃肠炎和腹泻等。
背景技术
轮状病毒性腹泻，是全世界范围内婴幼儿最主要的急性肠道传染病，以腹泻和脱水为特征，死亡率高，造成了严重的社会负担和经济负担。全世界每年大约有1.1亿5岁以下儿童患轮状病毒性胃肠炎，其中200万儿童必须住院治疗，每年约453000儿童死于轮状病毒性腹泻，其中发展中国家和欠发达国家的死亡病例占总数的85％以上(LeBaron，C.W.et al.1990)。
感染轮状病毒(Rotavirus，RV)后，因脱水和电解质紊乱导致重症腹泻和死亡，目前尚无特效药物，临床治疗也只是对症治疗，因此，急需开发高效疫苗和治疗策略，以降低该病的发病率和死亡率，减轻该病所造成的社会和经济负担(Parashar，U.D.et al.2003)。
然而，目前已上市的两种轮状病毒疫苗：RotaTeq(Merk)和Rotarix(GSK)，在轮状病毒威胁最严重的地方，疫苗免疫效力却差强人意。同时，已报道，口服Rota Teq疫苗后可能发生严重腹泻以及疫苗株之间的分子重组；此外，RotaTeq和Rotarix与肠套叠的关联性 并没有被排除(Lepage，P.et al.2007)。因此，本领域急需开发新一代替代疫苗，所述疫苗能够同时引起显著的细胞应答和体液应答，产生能够中和病毒感染性的抗体，从而可用于预防、减轻或治疗轮状病毒的感染及由轮状病毒的感染所导致的疾病。
在最近几年中，在轮状病毒的疫苗研发上，注意力已从灭活和减毒病毒疫苗转到基因工程疫苗。
轮状病毒(Rotavirus，RV)属于呼肠弧病毒科(Reoviridae)，其基因组由11节段的dsRNA组成，编码6个结构蛋白(VP1-4，VP6，VP7)和6个非结构蛋白(NSP1-6)。成熟的病毒粒子为三层衣壳结构，最外层由刺突状蛋白VP4和糖蛋白VP7组成(Bridger，J.C.et al.1976)。VP7与病毒在成熟的小肠上皮细胞中的复制直接相关。VP4蛋白不仅涉及RV的粘附、侵入，而且与RV血凝性、中和活性、毒力以及蛋白酶裂解后的感染性增强等理化特性相关。VP7和VP4都可独立地诱导病毒中和抗体，抗VP7和VP4的抗体都可阻止病毒的侵入。故VP7和VP4都成为疫苗开发的首选目标分子(Arias，C.F.et al.2002)。
VP8蛋白是RV病毒颗粒的最外层结构蛋白VP4(AA1-776)的一部分(AA1-231)。VP4经胰酶裂解后可形成VP8＊和VP5＊片段，而前者包含了VP4特异性的主要抗原表位(Clark，S.M.et al.1981)。这提示，可以利用VP8＊蛋白来开发新型RV亚单位疫苗。
尽管已被分类为结构蛋白，现在已明确VP4在病毒入胞阶段行使重要功能。因此，VP4不仅在病毒结构中，而且在RV复制过程中起到了极其重要的作用(Arias，C.F.et al.1996)。
VP4在病毒入胞过程中发挥的作用取决于所述蛋白与靶细胞表面表达的特异性受体的相互作用。在RV感染过程中，SA被认为是第一个而且是最重要的细胞受体之一，它通过与VP8结合而发挥作用。这种相互作用使得RV吸附至细胞表面，从而开始了RV感染细胞的第一步。对于NA敏感型的RV来说，通过核磁共振光谱学的手段发现，VP8＊的核心部位与α-N-乙酰神经氨酸相结合，这个结合位点是高度保守的，不再需要其他糖基序的参与；而对于NA非敏感型的RV株， VP8＊是否也能利用相同的位点还不是很明确(Blanchard H，et al.2007)。
VP8＊与SA的第一步相互作用使得病毒粒子锚定在细胞表面。VP8＊与SA基序相互识别结合后，病毒外壳蛋白随之发生构象改变，以利于搜寻后续更多的特异性受体分子，从而介导病毒下一步的入胞过程(Dormitzer PR，et al.2002)。
针对VP8的中和免疫应答能抑制病毒入胞，从而抑制靶细胞被感染，这使得该蛋白被认为是建立抗RV疫苗的优秀靶标。一个重要的先决条件是，VP8在病毒入胞前激起的免疫应答所产生的中和抗体能与病毒的VP8相互作用，阻止病毒的VP8与细胞受体结合，从而阻止VP8变构，抑制病毒入胞。
目前VP8的体外纯化表达多基于大肠杆菌表达系统，主要有可溶性表达纯化和包涵体纯化两种形式。
可溶性表达纯化虽然能保持VP8相对天然的构象和高免疫中和活性，但可溶性表达量低，纯化方式相对复杂，纯化所得的蛋白构象不均一，存在多聚体，且稳定性差，容易发生降解(Favacho AR，et al.2002)。特别地，本发明人经研究发现，经纯化后的VP8在4℃放置三天后，显著降解(参见，下文实施例3和图1)。这给大规模工业化生产VP8带来了极大的困难。
包涵体纯化方式虽然工艺相对比较简单，但纯化所得的蛋白与天然蛋白在构象上存在差异，其诱导机体产生中和抗体的能力显著减弱，不适宜用作疫苗。
因此，如何获得可溶性高表达的目的蛋白，并同时保持其天然构象，成为了研究者们的研究重点。
中国专利申请CN 103319604A公开了一种轮状病毒VP8蛋白的亚单位重组蛋白，ΔVP8＊，其由VP8全长蛋白的AA 64-223组成。通过体外纯化表达该蛋白已发现，ΔVP8＊在稳定性和均一性方面，明显优于VP8全长蛋白。然而，本发明人在进一步的研究中发现，ΔVP8＊诱导机体产生中和抗体的能力明显弱于VP8全长蛋白(参见下文实施 例6和图5)。这导致ΔVP8＊不适宜用作高效的抗轮状病毒疫苗。
因此，本领域迫切需要开发一种新的VP8蛋白变体，其能够通过可溶性表达纯化的方法方便地、高表达量地产生，能够保持VP8蛋白的天然构象，拥有良好的均一性和稳定性，并且能够诱导机体产生高滴度的针对轮状病毒的中和抗体，以便可开发高效的抗轮状病毒疫苗，并使得大规模工业化生产高效的抗轮状病毒疫苗成为可能。
发明内容
本发明的发明人已出人意料地发现：N端截短了21-60个氨基酸的VP8蛋白能够在大肠杆菌中以高表达量可溶性表达，且可通过色谱层析容易地进行纯化，并且由此所获得的高纯度截短蛋白(纯度可达到至少50％或更高，例如60％，70％，80％，90％，95％，96％，97％，98％，99％)拥有良好的均一性和稳定性(不易于降解)，且能够诱导机体产生高滴度的针对轮状病毒的中和抗体，从而有效地解决了上述技术问题。
因此，在一个方面，本发明涉及N端截短了21-60个氨基酸，例如21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、43个、44个、45个、46个、47个、48个、49个、50个、51个、52个、53个、54个、55个、56个、57个、58个、59个或60个氨基酸的轮状病毒VP8蛋白或其变体。
在一个方面，本发明涉及一种截短的轮状病毒VP8蛋白或其变体，其与野生型轮状病毒VP8蛋白相比，N端截短了21-60个氨基酸，例如21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、43个、44个、45个、46个、47个、48个、49个、50个、51个、52个、53个、54个、55个、56个、57个、58个、59个或60个氨基酸。
在某些优选的实施方案中，与野生型轮状病毒VP8蛋白相比，该截短的轮状病毒VP8蛋白的N端截短了21-60个氨基酸，例如21个、25个、30个、35个、40个、45个、50个、55个或60个氨基酸。
在某些优选的实施方案中，该截短的轮状病毒VP8蛋白(以下也简称为截短蛋白)具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7或者SEQ ID NO：8所示的氨基酸序列。
本发明人还对所获得的截短蛋白进行了进一步的研究。结果显示，本发明的截短蛋白与分子内佐剂的组合可进一步提高截短蛋白的免疫原性。特别地，当将本发明的截短蛋白与分子内佐剂融合表达时，与单独的VP8蛋白或截短蛋白相比，所获得的融合蛋白具有更高的免疫原性，且能够为宿主提供更高的免疫保护性。此外，还可通过将本发明的截短蛋白与分子内佐剂缀合来进一步提高截短蛋白的免疫原性。
因此，在另一个方面，本发明提供了一种融合蛋白，其包含第一多肽和第二多肽，其中，所述第一多肽为如上所述的截短的轮状病毒VP8蛋白或其变体；并且，所述第二多肽为分子内佐剂。
在某些实施方案中，所述融合蛋白任选地还包含肽接头，标签，信号肽，蛋白酶切割位点，或其任何组合。
在某些优选的实施方案中，所述分子内佐剂选自白喉毒素无毒突变体CRM197及其截短蛋白例如CRM197的A亚基，霍乱毒素，霍乱毒素B亚基CTB，霍乱毒素突变体例如CTA112/KDEV和CTA1-DD，大肠杆菌不耐热毒素(LT)及其无毒突变体LTR192G，大肠杆菌不耐热毒素B亚基LTB，破伤风毒素，及其任何组合。
在某些优选的实施方案中，所述第一多肽通过直接融合的方式或通过肽接头与所述第二多肽进行连接。在某些优选的实施方案中，所述第一多肽位于所述第二多肽的N末端或C末端，并且二者任选地通过肽接头连接。在某些优选的实施方案中，所述肽接头选自GS(SEQ ID NO：32)，GGS(SEQ ID NO：33)，GGGS(SEQ ID NO：34)，SGGGS (SEQ ID NO：35)，GGGG(SEQ ID NO：36)，GGSS(SEQ ID NO：37)，GGGGS(SEQ ID NO：38)，(GGGGS)3(SEQ ID NO：39)，及其任何组合。
在某些实施方案中，所述融合蛋白在其N末端和/或C末端还包含标签。在某些实施方案中，所述标签选自组氨酸标签、谷胱甘肽转移酶(GST)标签、麦芽糖结合蛋白(MBP)标签、硫氧还蛋白(Trx)标签、NusA标签、二硫键异构酶标签、DsbA标签、DsbC标签、SUMO标签、msyB标签、TF标签、引发因子标签、泛素标签、Myc标签、Flag标签、荧光蛋白(例如GFP)标签、生物素标签、亲和素标签、及其任何组合。
在某些实施方案中，所述融合蛋白还在其N末端包含信号肽。在某些实施方案中，所述信号肽选自OmpA，OmpT，pelB，CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。
在某些实施方案中，所述融合蛋白还包含蛋白酶切割位点。在某些实施方案中，所述蛋白酶切割位点位于相邻的两个元件之间，所述元件选自所述第一多肽，第二多肽，肽接头，标签和信号肽。
在某些优选的实施方案中，所述融合蛋白具有SEQ ID NO：12-13和15-16任一项所示的氨基酸序列。
在另一个方面，本发明提供了一种缀合物，其包含如上所述的截短的轮状病毒VP8蛋白或其变体，以及与所述截短的VP8蛋白或其变体缀合的分子内佐剂。
在某些优选的实施方案中，所述分子内佐剂选自白喉毒素无毒突变体CRM197及其截短蛋白例如CRM197的A亚基，霍乱毒素，霍乱毒素B亚基CTB，霍乱毒素突变体例如CTA112/KDEV和CTA1-DD，大肠杆菌不耐热毒素(LT)及其无毒突变体LTR192G，大肠杆菌不耐热毒素B亚基LTB，破伤风毒素，及其任何组合。
在某些优选的实施方案中，所述分子内佐剂通过共价方式(例如化学偶联)或非共价方式(例如吸附)与所述轮状病毒VP8蛋白或其 变体缀合。
在另一个方面，本发明涉及编码本发明的截短蛋白或其变体或者本发明的融合蛋白的多核苷酸以及含有该多核苷酸的载体。
可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个实施方案中，载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，本发明还涉及包含上述多核苷酸或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明还涉及包含上述截短蛋白或其变体，或上述融合蛋白，或上述缀合物，或上述多核苷酸或载体或宿主细胞的组合物。在某些优选的实施方案中，所述组合物包含本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物。
在某些优选的实施方案中，本发明的组合物包含与野生型轮状病毒VP8蛋白相比，N端截短了21-60个氨基酸，例如21个、25个、30个、35个、40个、45个、50个、55个或60个氨基酸的截短的轮状病毒VP8蛋白。在某些优选的实施方案中，本发明的组合物包含具有选自SEQ ID NO：1-8的序列的截短的轮状病毒VP8蛋白。在某些优选的实施方案中，本发明的组合物包含具有选自SEQ ID NO：12-13和15-16的序列的融合蛋白。
在另一个方面，本发明还涉及一种药物组合物或疫苗，其包含本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物，并且任选地还包含药学可接受的载体和/或赋形剂。本发明的药物组合物或疫苗可以用于预防或治疗轮状病毒感染或由轮状病毒感染所导致 的疾病例如轮状病毒性胃肠炎或腹泻等。
在某些优选的实施方案中，所述药物组合物或疫苗还包含佐剂，例如铝佐剂。
在某些优选的实施方案中，本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物以预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的有效量存在。在某些优选的实施方案中，本发明的药物组合物或疫苗还包含另外的活性成分。优选地，所述另外的活性成分能够预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病。
本发明的药物组合物或疫苗可通过本领域公知的方法进行施用，例如但不限于通过口服或者注射进行施用。在某些优选的实施方案中，本发明的药物组合物或疫苗以单位剂量形式进行施用。
预防或治疗特定病况所需的本发明药物组合物或疫苗的量将取决于施用途径、待治疗的病况的严重程度、患者的性别、年龄、体重和总体健康情况等等，其可由医生根据实际情况合理确定。
在另一个方面，本发明涉及一种获得本发明的截短蛋白或其变体或本发明的融合蛋白的方法，其包括，在允许所述截短蛋白或其变体或所述融合蛋白表达的条件下，培养本发明的宿主细胞；和，回收所表达的截短蛋白或其变体或所述融合蛋白。
在某些优选的实施方案中，所述方法包括，利用大肠杆菌表达系统来表达本发明的截短蛋白或其变体或所述融合蛋白，然后将所述大肠杆菌裂解，并从裂解液中纯化获得含有所述截短蛋白或其变体或所述融合蛋白。在某些优选的实施方案中，纯化包括色谱层析。
在另一个方面，本发明还涉及一种制备疫苗的方法，其包括将本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物与药学可接受的载体和/或赋形剂混合，任选地还混合佐剂例如铝佐剂，和/或另外的活性成分，例如能够预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的另外的活性成分。如上所论述的，所获得 的疫苗可以用于预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病例如轮状病毒性胃肠炎和腹泻等。
在另一个方面，本发明涉及一种预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的方法，其包括将预防或治疗有效量的根据本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物，或本发明的药物组合物或疫苗施用给受试者。在某些优选的实施方案中，所述由轮状病毒感染所导致的疾病包括但不限于，轮状病毒性胃肠炎和腹泻。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。
在另一个方面，还涉及根据本发明的截短蛋白或其变体，或本发明的融合蛋白，或本发明的缀合物在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病。在某些优选的实施方案中，所述由轮状病毒感染所导致的疾病包括但不限于，轮状病毒性胃肠炎和腹泻。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。
在另一个方面，还涉及根据本发明的截短蛋白或其变体，或根据本发明的融合蛋白，或根据本发明的缀合物，其用于在受试者中预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病。在某些优选的实施方案中，所述由轮状病毒感染所导致的疾病包括但不限于，轮状病毒性胃肠炎和腹泻。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。
本发明中相关术语的说明及解释
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
根据本发明，表述“N端截短了X个氨基酸的蛋白质”是指，用起始密码子编码的甲硫氨酸残基替代蛋白质N末端的第1-X位氨基酸残基所获得的蛋白质。例如N端截短了21个氨基酸的轮状病毒VP8蛋白是指，用起始密码子编码的甲硫氨酸残基替代野生型轮状病毒VP8蛋白N末端的第1-21位氨基酸残基所获得的蛋白质。
根据本发明，术语“变体”是指这样的蛋白，其氨基酸序列与本发明的截短的轮状病毒VP8蛋白(如SEQ ID NO：1-8任一项所示的蛋白)的氨基酸序列具有一个或多个(例如1-10个或1-5个或1-3个)氨基酸不同(例如保守氨基酸置换)或者具有至少80％，85％，90％，95％，96％，97％，98％，或99％的同一性，并且其保留了所述截短蛋白的必要特性。此处术语“必要特性”可以是如下特性中的一个或者多个：
(i)其能够在大肠杆菌中以高表达量可溶性表达，且能够通过色谱层析容易地进行纯化；
(ii)其拥有良好的均一性和稳定性(不易于降解)；
(iii)其能够特异性结合抗VP8抗体，所述抗VP8抗体能够体外抑制病毒对细胞的感染；
(iv)其能够与轮状病毒竞争性结合细胞受体，抑制病毒对细胞的感染；
(v)其能够诱导机体产生高滴度的针对轮状病毒的中和抗体；和
(vi)其能够保护受试者(例如人和小鼠)，抵抗轮状病毒的感染。
优选地，本发明的“变体”保留了截短蛋白的所有上述特性。
根据本发明，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和 CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学活性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)； Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
根据本发明，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698、ER2566、BL21(DE3)、B834(DE3)、BLR(DE3)等等。
根据本发明，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
根据本发明，术语“VP8蛋白”、“VP8全长蛋白”和“轮状病毒VP8蛋白”可互换使用，其是指，由RV病毒颗粒的最外层结构蛋白VP4(AA 1-776)的AA 1-231所组成的蛋白。VP8蛋白的示例性氨基酸序列可如SEQ ID NO：9所示。
根据本发明，术语“截短的轮状病毒VP8蛋白”是指在野生型轮状病毒VP8蛋白的N端和/或C端去掉一个或者多个氨基酸后的蛋白质，其中，野生型轮状病毒VP8蛋白的具体氨基酸序列可从公共数据库(例如GenBank数据库)容易地获得，例如GenBank登录号JQ013506.1。例如，野生型轮状病毒VP8蛋白的氨基酸序列可如SEQ ID NO：9所示。
在本发明中，术语“截短的轮状病毒VP8蛋白基因片段”是指这样的基因片段，其与野生型轮状病毒VP8蛋白基因相比，在5′端或3′端缺失编码一个或多个氨基酸的核苷酸，其中野生型轮状病毒VP8蛋白基因的全长序列从公共数据库(例如GenBank数据库)容易地获得，例如GenBank登录号JQ013506.1。例如，野生型轮状病毒VP8蛋白基因的核苷酸序列可如SEQ ID NO：10所示。
根据本发明，术语“分子内佐剂”是指这样的佐剂，其与目的蛋白(即，抗原)形成融合蛋白，从而其与抗原存在于一个相同的分子(即， 包含其与抗原的融合蛋白)中，并充当该抗原的佐剂以增强该抗原的免疫原性。即，分子内佐剂是能够增强与之融合表达的目的蛋白(抗原)的免疫原性的佐剂。此类分子内佐剂是本领域技术人员所熟知的，并且已在现有技术文献中进行了详细描述。通常，分子内佐剂为毒素类多肽片段。例如，此类分子内佐剂包括但不限于，白喉毒素无毒突变体CRM197及其截短蛋白例如CRM197的A亚基(中国专利申请CN102807621；Ilyina，N.，S.Kharit，L.Namazova-Baranova，A.Asatryan，M.Benashvili，E.Tkhostova，C.Bhusal and A.K.Arora(2014).Hum Vaccin Immunother10(8)：2471-2481)，霍乱毒素CT(O′Neal，C.M.，J.D.Clements，M.K.Estes and M.E.Conner，1998，J Virol72(4)：3390-3393)，霍乱毒素B亚基CTB(Gloudemans，A.K.，M.Plantinga，M.Guilliams，M.A.Willart，A.Ozir-Fazalalikhan，A.van der Ham，L.Boon，N.L.Harris，H.Hammad，H.C.Hoogsteden，M.Yazdanbakhsh，R.W.Hendriks，B.N.Lambrecht and H.H.Smits，2013，PLoS One8(3)：e59822)，霍乱毒素突变体例如CTA112/KDEV(Hagiwara，Y.，Y.I.Kawamura，K.Kataoka，B.Rahima，R.J.Jackson，K.Komase，T.Dohi，P.N.Boyaka，Y.Takeda，H.Kiyono，J.R.McGhee and K.Fujihashi 2006，J Immunol177(5)：3045-3054)和CTA1-DD(Agren，L.C.，L.Ekman，B.Lowenadler and N.Y.Lycke，1997，J Immunol158(8)：3936-3946)，大肠杆菌不耐热毒素(LT)及其无毒突变体LTR192G(Yuan，L.，A.Geyer，D.C.Hodgins，Z.Fan，Y.Qian，K.-O.Chang，S.E.Crawford，V.
Figure PCTCN2015076259-appb-000001
L.A.Ward and M.K.Estes，2000，Journal of virology 74(19)：8843-8853)，大肠杆菌不耐热毒素B亚基LTB，以及破伤风毒素(Wen，X.，K.Wen，D.Cao，G.Li，R.W.Jones，J.Li，S.Szu，Y.Hoshino and L.Yuan(2014).，Vaccine，Volume 32，Issue 35，Pages 4365-4598)。
根据本发明，术语“肽接头”是指用于连接两个分子(例如蛋白)的短肽。通常，通过将编码该短肽的多核苷酸序列引入(例如，通过PCR扩增或连接酶)分别编码所要连接的两种目的蛋白的两个DNA 片段之间，并进行蛋白质表达来获得融合蛋白，例如目的蛋白1-肽接头-目的蛋白2。如本领域技术人员公知的，肽接头包括但不限于柔性连接肽，如GGGG(SEQ ID NO：36)，GGSS(SEQ ID NO：37)，GGGGS(SEQ ID NO：38)和(GGGGS)3(SEQ ID NO：39)等等。此类肽接头的详细描述还可参见例如，Robinson，C.R.and R.T.Sauer(1998).Proc Natl Acad Sci 95(11)：5929-5934。
根据本发明，术语“标签”是指这样的短肽，其与目的蛋白(例如本发明的截短蛋白或融合蛋白)融合或连接，并由此促进重组蛋白的可溶性表达、检测和/或纯化。标签可融合或连接至目的蛋白的N端和/或C端(任选地通过接头或蛋白酶切割位点)。此类标签是本领域技术人员熟知的，并且已在现有技术文献中进行了详细描述。例如，此类标签包括但不限于，组氨酸标签(Sockolosky，J.T.and F.C.Szoka(2013).Protein Expr Purif87(2)：129-135)、谷胱甘肽转移酶(GST)标签(Hayashi，K.and C.Kojima(2008).Protein Expr Purif62(1)：120-127)、麦芽糖结合蛋白(MBP)标签(Bataille，L.，W.Dieryck，A.Hocquellet，C.Cabanne，K.Bathany，S.Lecommandoux，B.Garbay and E.Garanger，Protein Expression and Purification Volume 110，June 2015，Pages 165-171)、硫氧还蛋白(Trx)标签(Tomala，M.，A.Lavrentieva，P.Moretti，U.Rinas，C.Kasper，F.Stahl，A.Schambach，E.Warlich，U.Martin，T.Cantz and T.Scheper，2010，Protein Expr Purif73(1)：51-57)、NusA标签(Li，K.，T.Jiang，B.Yu，L.Wang，C.Gao，C.Ma，P.Xu and Y.Ma(2013).Sci Rep3：2347)、二硫键异构酶DsbA标签(Zhang，Y.，D.R.Olsen，K.B.Nguyen，P.S.Olson，E.T.Rhodes and D.Mascarenhas(1998).Protein Expr Purif12(2)：159-165)、DsbC标签(Kurokawa，Y.，H.Yanagi and T.Yura(2001).J Biol Chem276(17)：14393-14399)、SUMO标签(Marblestone，J.G.，S.C.Edavettal，Y.Lim，P.Lim，X.Zuo and T.R.Butt(2006).Protein Sci15(1)：182-189)、msyB标签(Zou，Z.，L.Cao，P.Zhou，Y.Su，Y.Sun and W.Li(2008).J Biotechnol135(4)：333-339)、TF标签、引发因子标 签(Kim，E.K.，J.C.Moon，J.M.Lee，M.S.Jeong，C.Oh，S.M.Ahn，Y.J.Yoo and H.H.Jang(2012).Protein Expr Purif86(1)：53-57)、泛素标签(Sabin，E.A.，Lee-Ng，Chun Ting，Shuster，Jeffrey R.，Barr，Philip J.(1989).Nature Biotechnology7(7)：705-709)、Myc标签、Flag标签、荧光蛋白(例如GFP)标签(Pedelacq，J.D.，S.Cabantous，T.Tran，T.C.Terwilliger and G.S.Waldo(2006).Nat Biotechnol24(1)：79-88)、生物素标签、以及亲和素标签。
根据本发明，术语“信号肽”是指这样的短肽，当其与目的蛋白(例如本发明的截短蛋白或融合蛋白)融合时，其能够促进细胞所表达的目的蛋白分泌到细胞外。信号肽通常位于目的蛋白的N端，并且各种信号肽是本领域技术人员已知的。此类信号肽包括但不限于，OmpA(Sockolosky，J.T.and F.C.Szoka(2013).Protein Expr Purif87(2)：129-135)，OmpT(Pohlmann，C.，M.Thomas，S.Forster，M.Brandt，M.Hartmann，A.Bleich and F.Gunzer(2013).Bioengineered4(3)：172-179)，pelB(Sockolosky，J.T.and F.C.Szoka(2013).Protein Expr Purif87(2)：129-135)，CSP(Heggeset，T.M.，V.Kucharova，I.Naerdal，S.Valla，H.Sletta，T.E.Ellingsen and T.Brautaset(2013).Appl Environ Microbiol79(2)：559-568)，mschito(Sun，Y.，J.Zhang and S.Wang(2015).Indian J Microbiol55(2)：194-199)，MF-α(Ghosalkar，A.，V.Sahai and A.Srivastava(2008).Protein Expr Purif60(2)：103-109)，pho1(Braspenning，J.，W.Meschede，A.Marchini，M.Muller，L.Gissmann and M.Tommasino(1998).Biochem Biophys Res Commun245(1)：166-171)，HBM(Wang，Y.B.，Z.Y.Wang，H.Y.Chen，B.A.Cui，Y.B.Wang，H.Y.Zhang and R.Wang(2009).Vet Immunol Immunopathol132(2-4)：314-317)，t-pA(Wang，J.Y.，W.T.Song，Y.Li，W.J.Chen，D.Yang，G.C.Zhong，H.Z.Zhou，C.Y.Ren，H.T.Yu and H.Ling(2011).Appl Microbiol Biotechnol91(3)：731-740)，以及IL-3的信号肽(Tessier，D.C.，D.Y.Thomas，H.E.Khouri，F.Laliberte and T.Vernet(1991).Gene98(2)：177-183)。
根据本发明，术语“蛋白酶切割位点”是指，能够被蛋白酶特异性识别并切割的位点。各种特异性蛋白酶及其识别位点是本领域技术人员所熟知的，并见于许多现有技术文献中。本领域技术人员可根据实际情况，在融合蛋白中使用合适的蛋白酶切割位点，并用相应的蛋白酶进行切割。蛋白酶切割位点的使用可以是有利的，例如，其可用于从融合蛋白中切除信号肽和/或标签，从而获得具有目的活性的成熟蛋白。例如，在一个实施方案中，构建并表达包含标签，第一多肽和第二多肽的重组蛋白，其中在标签与第一多肽之间设计有蛋白酶切割位点；随后，通过标签来对所表达的重组蛋白进行纯化；然后，使用相应的蛋白酶，将标签从重组蛋白中切除，从而获得经纯化的、包含第一多肽和第二多肽的融合蛋白。
根据本发明，术语“缀合”是指，2个或多个分子(例如蛋白与蛋白，蛋白与多糖，蛋白与标记等)通过共价方式(例如化学偶联)或非共价方式(例如吸附)进行连接。因此，在本发明中，术语“缀合物”是指通过共价方式(例如化学偶联)或非共价方式(例如吸附)连接在一起的2个或多个分子。例如，本发明的缀合物可以指例如，通过共价方式或非共价方式连接在一起的截短的VP8蛋白或其变体与分子内佐剂。
根据本发明，术语“药学可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)；离子强度增强剂包括但不限于氯化钠。
根据本发明，术语“有效量”是指能够有效实现预期目的的量。例如，预防或治疗疾病(例如轮状病毒感染)有效量是指，能够有效预防、 阻止或延迟疾病(例如轮状病毒感染)的发生、或缓解、减轻或治疗已有的疾病(例如由轮状病毒感染所导致的疾病)的严重程度的量。测定这样的有效量在本领域技术人员的能力范围之内。
根据本发明，术语“色谱层析”包括但不限于：离子交换色谱(例如阳离子交换色谱)、疏水相互作用色谱、吸附层析法(例如羟基磷灰石色谱)、凝胶过滤(凝胶排阻)层析、亲和层析法。
根据本发明，宿主细胞的裂解/破碎可通过本领域技术人员熟知的各种方法来实现，包括但不限于匀浆器破碎、均质机破碎、超声波处理、研磨、高压挤压、溶菌酶处理等等。
发明的有益效果
本发明提供的N端截短的轮状病毒VP8蛋白和其制备方法有效地解决了本领域中存在的技术问题。
首先，本发明的截短蛋白或其变体能够在大肠杆菌中以高表达量可溶性表达，实现了高产率。
其次，本发明的截短蛋白或其变体的纯化方式相对简单，易于操作。特别地，在大肠杆菌中进行可溶性表达后，可将所述大肠杆菌裂解，然后对裂解液进行色谱层析处理，例如离子交换层析处理，由此可获得高纯度的截短蛋白(纯度可达到至少50％或更高，例如60％，70％，80％，90％，95％，96％，97％，98％，99％)。
第三，本发明的截短蛋白或其变体拥有良好的均一性和稳定性，不易于降解。
第四，本发明的截短蛋白或其变体能够诱导机体产生高滴度的针对轮状病毒的中和抗体。
第五，与单独的截短蛋白或者截短蛋白+铝佐剂相比，包含本发明的截短蛋白与分子内佐剂(例如CRM197-A或CTB)的融合蛋白能刺激小鼠产生更高的免疫应答，产生更强的免疫保护作用。
由此可见，本发明的截短蛋白或其变体以及本发明的融合蛋白在保留了全长野生型VP8蛋白的较强的诱导机体产生中和抗体能力的 同时，又具有高表达量、易于纯化、高均一性和稳定性等特点。因此，本发明的截短蛋白(或其变体)以及融合蛋白能够被用作高效的抗轮状病毒疫苗，并使得能够大规模工业化生产高效的抗轮状病毒疫苗，从而为本领域中存在的技术问题提供了有效的解决方案。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了刚刚纯化的全长VP8蛋白以及其在4℃放置三天后的SDS-PAGE结果。泳道1：刚刚纯化的全长VP8蛋白；泳道2：在4℃放置三天后的全长VP8蛋白。结果显示，全长VP8蛋白在4℃放置三天后显著降解，其稳定性差。
图2A-2B显示了各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)在纯化完成后(图2A)以及在4℃放置3天之后(图2B)的SDS聚丙烯酰胺凝胶电泳的结果。
在图2A中，泳道从左到右依次为：VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11，VP8-12。图2A的结果显示，经过上述纯化步骤后，VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12蛋白的浓度约为2.0mg/ml，且纯度大于98％，其表达量显著优于VP-8全长蛋白。
在图2B中，泳道从左到右依次为：VP8蛋白、VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11，VP8-12。图2B的结果显示，VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12蛋白的稳定性显著优于VP-8全长蛋白，其在4℃放置3天后， 未发生显著降解。
图3A-3D显示了各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)与抗体1B11(图3A)、11C1(图3B)、3C5(图3C)、1E1(图3D)的间接ELISA分析的结果，其中横坐标表示各截短蛋白，纵坐标表示与各截短蛋白具有反应性的一抗(1E1、1B11、11C1或3C5)的最大抗体稀释倍数。结果显示，各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均能够被四种中和抗体识别，具有良好的抗原性(即，抗体反应性)，其等同于或甚至强于ΔVP8＊的抗原性。
图4显示了各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)与用其免疫Balb/c小鼠而获得的免疫血清的间接ELISA分析的结果，其中横坐标表示各截短蛋白，纵坐标表示与相应截短蛋白具有反应性的免疫血清的最大稀释倍数(即，抗体滴度)。结果显示，截短蛋白在免疫后第42天均能在小鼠体内有效诱发抗体的产生。免疫程序完成后，免疫血清中的抗体滴度(GMT)均可达到105或更高。各实验组间无显著差异。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均具有良好的免疫原性，能够在动物体内有效诱发抗体的产生，其功效与VP8和ΔVP8＊无显著差异。
图5显示了各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)在Balb/c小鼠中诱发的免疫血清的中和抗体滴度的分析结果，其中横坐标表示各截短蛋白，纵坐标表示达到50％感染抑制率的免疫血清最大稀释倍数(NT50，中和抗体滴度)。结果显示，本发明的截短蛋白在免疫后第42天(三次免疫后)均能在小鼠体内有效诱发具有高中和抗体滴度的免疫血清。免疫程序完成后，使用本发明的截短蛋白诱发的免疫血清的中和抗体滴度(NT50)均可达到103或更高的水平。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)具有较强的诱导机体产生中和抗体的能力，能够在 动物体内诱发具有高中和抗体滴度的免疫血清，所述免疫血清能够有效抑制轮状病毒的感染。本发明的各截短蛋白的诱导机体产生中和抗体的能力显著优于ΔVP8＊，且接近于VP8全长蛋白和RV。
图6显示了与各截短蛋白温育后的阳性血清的中和抗体滴度的分析结果，其中横坐标表示与阳性血清温育的各截短蛋白，纵坐标表示达到50％感染抑制率的温育后阳性血清的最大稀释倍数(NT50，中和抗体滴度)。结果显示，在温育后，本发明的截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)导致阳性血清的中和抗体滴度显著下降：用TB8.0温育的阳性血清的中和抗体滴度可超过6＊103；用ΔVP8＊温育的阳性血清的中和抗体滴度可超过5＊103；而用本发明的截短蛋白温育的阳性血清的中和抗体滴度低于4＊103，甚至低于3＊103。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)与阳性血清均具有高反应性，且该反应性显著强于ΔVP8＊；这进而表明，本发明的各截短蛋白的构象或与天然病毒更接近，能够更好的被天然病毒免疫产生的抗体(阳性多抗血清)所识别。
图7A-7C显示了保护性实验中腹泻的评分标准。根据乳鼠腹泻的不同程度，评分分为3个等级：正常粪便计为1分(图7C)，软粪便计为2分(图7B)，不成形水样便计为3分(图7A)。
图8显示了用实施例3中制备的各截短蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。
图9显示了用实施例3中制备的各截短蛋白免疫Balb/c小鼠后的平均腹泻天数，其中，纵轴表示平均腹泻天数；横轴表示各截短蛋白。
图8-9的结果显示，无论从平均腹泻严重程度还是从平均腹泻天数来看，各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均具有显著的保护性，且均比ΔVP8＊保护性更好。
图10显示了实施例9中制备的包含截短蛋白VP8-5与分子内佐剂CTB的融合蛋白(CTB-VP8-5和VP8-5-CTB)在纯化完成后的SDS-PAGE结果。其中，各泳道从左到右依次为：CTB-VP8-5， VP8-5-CTB，VP8-5和CTB。
图11显示了融合蛋白(CTB-VP8-5和VP8-5-CTB)在Balb/c小鼠中诱发的免疫血清中的总抗体滴度和中和抗体滴度的分析结果，其中横坐标表示所使用的各种蛋白，左纵坐标表示中和抗体滴度(NT50，即，达到50％感染抑制率的免疫血清最大稀释倍数)，右纵坐标表示总抗体滴度(log)；并且，中和抗体滴度以柱形图表示，总抗体滴度以曲线图表示。结果显示，本发明的融合蛋白(CTB-VP8-5和VP8-5-CTB)在免疫后第42天(三次免疫后)能在小鼠体内有效诱发具有高抗体滴度的免疫血清，并且，免疫血清中的总抗滴度和中和抗体滴度显著高于VP8-5组和CTB组的免疫血清中的总抗滴度和中和抗体滴度。这些结果表明，可通过将本发明的截短蛋白与分子内佐剂(例如CTB)连接来进一步提高本发明截短蛋白的免疫原性，从而实现更好的免疫效果。
图12显示了用实施例9中制备的融合蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。结果显示，本发明的融合蛋白(CTB-VP8-5和VP8-5-CTB)均具有显著的免疫保护性，并且优于未与分子内佐剂CTB融合的VP8-5。
图13显示了实施例10中制备的包含截短蛋白VP8-5与分子内佐剂CRM-A的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在纯化完成后的SDS-PAGE结果。各泳道从左到右依次为：CRM-A-VP8-5，VP8-5-CRM-A，VP8-5和CTB。
图14显示了融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在Balb/c小鼠中诱发的免疫血清中的总抗体滴度和中和抗体滴度的分析结果，其中，横坐标表示所使用的各种蛋白，左纵坐标表示中和抗体滴度(NT50，即，达到50％感染抑制率的免疫血清最大稀释倍数)，右纵坐标表示总抗体滴度(log)；并且，中和抗体滴度以柱形图表示，总抗体滴度以曲线图表示。结果显示，本发明的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在免疫后第42天(三次免疫后)能在小鼠体内有效诱发具有高抗体滴度的免疫血清，并且，免疫血清中的总抗滴度和中和抗体滴度显著高于VP8-5组和CRM-A组的免疫血清中的总抗滴度和中 和抗体滴度。这些结果表明，可通过将本发明的截短蛋白与分子内佐剂(例如CRM-A)连接来进一步提高本发明截短蛋白的免疫原性，从而实现更好的免疫效果。
图15显示了用实施例10中制备的融合蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。结果显示，本发明的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)均具有显著的免疫保护性，并且相当于或者优于未与分子内佐剂CRM-A融合的VP8-5。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2015076259-appb-000002
Figure PCTCN2015076259-appb-000003
Figure PCTCN2015076259-appb-000004
Figure PCTCN2015076259-appb-000005
Figure PCTCN2015076259-appb-000006
Figure PCTCN2015076259-appb-000007
Figure PCTCN2015076259-appb-000008
Figure PCTCN2015076259-appb-000009
Figure PCTCN2015076259-appb-000010
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。
除非特别指明，否则本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。在不背离本发明的精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
实施例中使用的生物材料和试剂的来源：
轮状病毒LLR株由北京万泰生物药业股份有限公司馈赠；原核表达载体PTO-T7为实验室自主构建；大肠杆菌(E.coli)ER2566购自新英格兰生物实验室公司；所使用的引物均由生工生物(上海)工程股份有限公司合成。
实施例1：编码截短的轮状病毒VP8蛋白的表达载体的构建
轮状病毒LLR株用罗猴胚肾细胞系(MA-104)细胞进行培养。所用培养基为DMEM，其补充有2μg/ml的胰酶，0.5mg/ml的氨苄青霉素和0.4mg/ml的链霉素，3.7mg/ml的碳酸氢钠，0.34mg/ml的L-谷氨酰胺。
根据制造商的说明书，采用北京金麦格生物技术有限公司生产的病毒DNA/RNA提取试剂盒，提取轮状病毒的基因组RNA，并通过反 转录获得编码VP4蛋白的cDNA。以获得的cDNA为模板，通过PCR反应，扩增获得编码截短的轮状病毒VP8蛋白的基因片段。
所使用的引物如下：
上游引物：
5′-GGATCCCATATGATACAGTTAATTGGATCAGAAAA-3′(SEQ ID NO：17)
5′-GGATCCCATATGGGATCAGAAAAAACGCAG-3′(SEQ ID NO：18)
5′-GGATCCCATATGCAGAGAACTACAGTAAATCCAGG-3′(SEQ ID NO：19)
5′-GGATCCCATATGGCACAAACTGGTTATGCA-3′(SEQ ID NO：20)
5′-GGATCCCATATGGCACCAGTGAATTGGGG-3′(SEQ ID NO：21)
5′-GGATCCCATATGGGGCCTGGGGAAACG-3′(SEQ ID NO：22)
5′-GGATCCCATATGAGTGATTCCACTACTGTTGAGC-3′(SEQ ID NO：23)
5′-GGATCCCATATGGTTGAGCCAGTGTTGAATG-3′(SEQ ID NO：24)
下游引物：
5′-AAGCTTAGGTGTTTTGTATTGGTGG-3′(SEQ ID NO：25)
其中，以下划线标示出酶切位点，以斜体标示出引入的终止密码子。
所使用的PCR反应体系如下：
Figure PCTCN2015076259-appb-000011
PCR反应条件如下：95℃预变性5min，35个循环的(95℃，40s；55℃，40s；72℃1min)，72℃终延伸10min。所获得的扩增产物用2％琼脂糖凝胶电泳进行检测。
将各个PCR扩增产物与商售的pMD 18-T载体(TAKARA公司)连接，转入大肠杆菌DH5α中。然后，筛选阳性菌落，提取质粒，经NdeI/HindIII酶切鉴定，得到插入目的基因片段的阳性克隆质粒pMD 18-T-VP8-5A，pMD 18-T-VP8-5，pMD 18-T-VP8-6，pMD 18-T-VP8-8，pMD 18-T-VP8-9，pMD 18-T-VP8-10，pMD 18-T-VP8-11，和pMD 18-T-VP8-12。
在上海生工生物工程公司，利用M13(+)/(-)引物，对上述阳性克隆进行测序。测序结果显示，上述阳性克隆质粒中插入的目的片段的核苷酸序列与预期的一致，其编码的氨基酸序列如SEQ ID NO：1-8所示，它们分别对应于N端被截短21、25、30、40、45、50、55、60个氨基酸、C端未被截短的VP8蛋白。
将上述阳性克隆质粒进行NdeI/HindIII酶切，获得各个截短的VP8蛋白的编码基因片段，并将其与经NdeI/HindIII酶切的非融合表达载体pTO-T7(罗文新等，生物工程学报，2000，16：53-57)相连接，转入大肠杆菌ER2566；然后，筛选阳性菌落，提取质粒，经NdeI/HindIII酶切鉴定得到插入目的片段的阳性表达质粒：pTO-T7-VP8-5A，pTO-T7-VP8-5，pTO-T7-VP8-6，pTO-T7-VP8-8，pTO-T7-VP8-9，pTO-T7-VP8-10，pTO-T7-VP8-11，pTO-T7-VP8-12。
取1μL的阳性表达质粒(0.15mg/ml)，转化40μL以氯化钙法制备的感受态大肠杆菌ER2566(购自新英格兰生物实验室公司)，将其涂布于含卡那霉素(终浓度25mg/mL，下同)的固体LB培养基(LB培养基成分：10g/L蛋白胨，5g/L酵母粉，10g/L氯化钠，下同)，并37℃静置培养10-12小时至单菌落清晰可辨。挑取单菌落至含4mL液体LB培养基(含卡那霉素)中，然后在37℃，200转/分钟下振荡培养10小时。培养后，取1mL菌液于-70℃保存。
另外，还通过与上述类似的方法来构建表达全长VP8蛋白的表达质粒，PTO-T7-VP8。所使用的PCR扩增引物如下：
上游引物：5′-GGATCCCATATGGCTTCGCTCATT-3′(SEQ ID NO：26)；
下游引物：5′-AAGCTTAGGATCCGGTGTTTTGTATTGGTGG-3′(SEQ ID NO：28)；
其中，以下划线标示出酶切位点，以斜体标示出引入的终止密码子。
还通过类似的方法来构建表达ΔVP8＊的表达质粒，PTO-T7-ΔVP8＊。所使用的PCR扩增引物如下：
上游引物：5′-GGATCCCATATGTTGAATGGACCA-3′(SEQ ID NO：27)；
下游引物：5′-AAGCTTATCCATTATTTACGTATTCTGTG-3′(SEQ ID NO：29)。
实施例2：截短的轮状病毒VP8蛋白的表达
从-70℃取出实施例1制备的携带重组质粒pTO-T7-VP8-5A，pTO-T7-VP8-5，pTO-T7-VP8-6，pTO-T7-VP8-8，pTO-T7-VP8-9，pTO-T7-VP8-10，pTO-T7-VP8-11或pTO-T7-VP8-12的大肠杆菌菌液，将其接种入50ml含卡那霉素的LB液体培养基中，在180rpm，37℃下培养大约4小时；然后转接入10瓶500ml含卡那霉素的LB培养基中(每瓶接入500ul菌液)。当培养物在600nm下的吸光值达0.5时，加入IPTG至终浓度为1mM，在180rpm，25℃下继续培养6小时。
另外，还通过与上述类似的方法，使用大肠杆菌来进行重组蛋白PTO-T7-VP8，PTO-T7-ΔVP8＊的表达。
实施例3：截短的轮状病毒VP8蛋白的纯化和表征
在Tris-HCl 8.0缓冲液条件下，在4℃下，用超声仪(Thermo)按4min/1g湿菌的条件，破坏E.coli细胞的细胞壁，收集可溶性级分，并通过下述方案来对E.coli细胞表达的重组蛋白进行纯化。
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTAexplorer 100型制备型液相色谱系统。
层析介质：Q-sepharose-HP(GE Healthcare公司)。
柱体积：5.5cm*20cm。
缓冲液：50mM Tris-HCl pH 8.0
50mM Tris-HCl pH 8.0，2M NaCl
流速：25mL/min。
检测器波长：280nm。
样品为之前制备的含有重组表达的VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12的大肠杆菌裂解液可溶性级分。
洗脱程序为：1000mM NaCl洗脱杂蛋白，50mM NaCl洗脱目的蛋白，收集50mMNaCl洗脱产物，共获得30mL的含有重组表达的VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12的经纯化的样品。
另外，还通过类似的纯化方案获得含有重组表达的ΔVP8＊的经纯化的样品；并且，通过DEAE-FF层析柱(GE)纯化获得含有重组表达的VP8全长蛋白的经纯化的样品。
取经上述方案纯化的样品150μL，加入30μL 6X LoadingBuffer，混匀并于100℃水浴10min；然后取10μl于13.5％SDS-聚丙烯酰胺凝胶中以120V电压电泳120min；然后通过考马斯亮兰染色来显示电泳条带。电泳结果示于图1(泳道1)和图2A中。
另外，将上述经纯化的样品在4℃放置3天，然后再次进行SDS聚丙烯酰胺凝胶电泳检测。电泳结果示于图1(泳道2)和图2B中。
图1显示了刚刚纯化的全长VP8蛋白以及其在4℃放置三天后的SDS-PAGE结果。泳道1：刚刚纯化的全长VP8蛋白；泳道2：在4℃放置三天后的全长VP8蛋白。图1的结果显示，全长VP8蛋白在4℃放置三天后显著降解，其稳定性差。
图2A显示了刚刚纯化的各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)的SDS 聚丙烯酰胺凝胶电泳的结果。在图2A中，泳道从左到右依次为：VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11，VP8-12。图2A的结果显示，经过上述纯化步骤后，VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12蛋白的浓度约为2.0mg/ml，且纯度大于98％，其表达量显著优于VP-8全长蛋白。
图2B显示了在4℃放置3天之后的经纯化的各种截短的轮状病毒VP8蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)的SDS聚丙烯酰胺凝胶电泳的结果。图2B的结果显示，VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12蛋白的稳定性显著优于VP-8全长蛋白，其在4℃放置3天后，未发生显著降解。
另外，还使用G3000SEC-HPLC对上述经纯化的样品的均一性进行了分析。SEC-HPLC分析结果示于下面的表2中。
表2：经纯化的蛋白样品的G3000SEC-HPLC分析
蛋白 VP8 VP8-5 VP8-6 VP8-8 VP8-9 VP8-10 VP8-11 VP8-12 ΔVP8＊
出峰时间(分钟) 10.9 14.63 14.74 14.6 14.7 14.62 14.7 15.12 15.51
峰面积％ 70.56 98.81 97.86 98.74 97.94 97.05 98.29 99.34 98.9
结果显示，VP-8全长蛋白样品中的多聚体(出峰时间为约10.9分钟)含量约为70.56％，基本上无单体存在；而截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)样品中的的单体(出峰时间为约14-15分钟)含量均超过97％。这表明，VP8全长蛋白的均一性差，易于形成多聚体；而本发明的截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)的均一性显著优于VP-8全长蛋白，经纯化的样品中基本上不存在多聚体。
实施例4：截短的轮状病毒VP8蛋白的抗体反应性的检测
将上述实施例3中获得的经纯化的截短的轮状病毒VP8蛋白，用包被缓冲液(20mM PBS，PH7.4)稀释到0.5ng/ul，室温包被平板2小时。以0.1％PBS-Tween20(PBST)洗板1次，以含1％BSA的PBST溶液室温封闭平板2小时，拍干。将中和抗体1E1、1B11、11C1、3C5(本实验室通过杂交瘤技术自制，浓度均为1mg/ml，各单抗中和效价IC50分别为200ng/ml、200ng/ml、50ng/ml和5ng/ml)梯度稀释，然后分别与包被后的截短蛋白进行间接ELISA分析(其中所使用的二抗为山羊抗小鼠抗体(万域美澜)，以测定与各截短蛋白具有反应性的一抗(1E1、1B11、11C1、3C5)的最大抗体稀释倍数。抗体稀释倍数越大，则截短蛋白与抗体的反应性越高。将ΔVP8＊和VP-8全长蛋白用作对照。
间接ELISA的结果如图3A-3D所示，其中横坐标表示各截短蛋白，纵坐标表示与各截短蛋白具有反应性的一抗(1E1、1B11、11C1或3C5)的最大抗体稀释倍数。结果显示，各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均能够被四种中和抗体识别，具有良好的抗原性(即，抗体反应性)，其等同于或甚至强于ΔVP8＊的抗原性。
实施例5：截短的轮状病毒VP8蛋白的免疫原性的分析
将上述实施例3中获得的经纯化的截短的轮状病毒VP8蛋白，用包被缓冲液(20mM PBS，PH7.4)稀释到0.5ng/ul，室温包被平板2小时。以0.1％PBS-Tween20(PBST)洗板1次，以含1％BSA的PBST溶液室温封闭平板2小时，拍干。
使用上述实施例3中获得的经纯化的截短的轮状病毒VP8蛋白来免疫Balb/c小鼠。免疫方案如下：将70只4-5周龄的雌性Balb/c小鼠随机分为10组，每组7只小鼠，其中2组为对照组，另8组为实验组。实验组分别皮下注射实施例3制备的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)。每只小鼠的注射剂量为10μg的以1∶1混合佐剂的重组蛋白。共进行三次免疫，每次 免疫间隔两周。第一次免疫使用弗氏完全佐剂；第二次和第三次免疫使用弗氏不完全佐剂。将ΔVP8＊和VP-8全长蛋白用作对照，分别用于对照组，并使用相同的免疫方案进行免疫。
在免疫程序结束后，从经免疫的小鼠收集免疫血清。将免疫血清梯度稀释，然后分别与包被后的相应截短蛋白进行间接ELISA分析(其中所使用的二抗为山羊抗小鼠抗体(万域美澜)，以测定与相应截短蛋白具有反应性的免疫血清的最大稀释倍数。免疫血清的稀释倍数越大，免疫血清中的抗截短蛋白抗体的滴度就越高，用于产生免疫血清的截短蛋白的免疫原性也越高。间接ELISA的结果如图4所示，其中横坐标表示各截短蛋白，纵坐标表示与各截短蛋白具有反应性的免疫血清的最大稀释倍数(即，抗体滴度)。结果显示，截短蛋白在免疫后第42天均能在小鼠体内有效诱发抗体的产生。免疫程序完成后，免疫血清中的抗体滴度(GMT)均可达到105或更高。各实验组间无显著差异。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均具有良好的免疫原性，能够在动物体内有效诱发抗体的产生，其功效与VP8和ΔVP8＊无显著差异。
实施例6：截短的轮状病毒VP8蛋白诱发中和抗体的能力的分析
使用实施例5中所述的免疫方案，用上述实施例3中获得的经纯化的截短的轮状病毒VP8蛋白来免疫实验组Balb/c小鼠(每组7只小鼠)，并收集免疫血清。另外，使用相同的免疫方案，将ΔVP8＊、VP-8全长蛋白、轮状病毒RV和弗氏佐剂用作对照，分别用于免疫对照组Balb/c小鼠(每组7只小鼠)，并收集免疫血清。
将MA104细胞铺于96孔细胞培养板中(1.9＊104个细胞/孔)。20小时后如下进行中和实验：将待测的免疫血清样品(含待测中和抗体)分别用加入胰酶的DMEM进行连续倍比稀释；然后取100μL各个经稀释的样品分别与稀释于DMEM中的轮状病毒液混合(TCID50＝1.5＊105)；在37℃下孵育1h后，将混合物分别加入预铺有MA104细 胞的96孔细胞培养板中，并在37℃培养14h；然后，如下计算各个免疫血清对病毒的感染抑制率。
感染抑制率＝(未加入血清的孔的病毒点计数-加入血清的孔的病毒点计数)/未加入血清的孔的病毒点计数＊100％。
免疫血清中的中和抗体滴度定义为：达到50％感染抑制率的免疫血清最大稀释倍数。经50倍稀释后仍能达到50％以上感染抑制率的免疫血清被视为具有中和能力。
免疫血清的中和抗体滴度的分析结果如图5所示，其中横坐标表示各截短蛋白，纵坐标表示达到50％感染抑制率的免疫血清最大稀释倍数(NT50，中和抗体滴度)。结果显示，本发明的截短蛋白在免疫后第42天(三次免疫后)均能在小鼠体内有效诱发具有高中和抗体滴度的免疫血清。免疫程序完成后，使用本发明的截短蛋白诱发的免疫血清的中和抗体滴度(NT50)均可达到103或更高的水平。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)具有较强的诱导机体产生中和抗体的能力，能够在动物体内诱发具有高中和抗体滴度的免疫血清，所述免疫血清能够有效抑制轮状病毒的感染。本发明的各截短蛋白的诱导机体产生中和抗体的能力显著优于ΔVP8＊，且接近于VP8全长蛋白和RV。
实施例7：截短的轮状病毒VP8蛋白与多抗血清的反应性
用轮状病毒免疫Balb/c小鼠，并收集免疫血清，且将其梯度稀释(从稀释200倍开始，梯度稀释至25600倍)。然后，将各个稀释的血清样品分别与各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)(1mg/ml)混合，并且在37℃温育1小时。温育后，将混合物加入预铺有MA104细胞的96孔细胞培养板中，并在37℃下进一步培养14h。然后，使用Elispot检测病毒感染量，并如实施例6中所述，计算与各截短蛋白温育后，阳性血清的中和抗体滴度(NT50)。温育后的阳性血清的中和抗体滴度(NT50)越低，截短蛋白所结合的阳性血清中的中和抗体就越多，截短蛋白与阳性血清的反应 性就越高。将ΔVP8＊和缓冲液TB8.0用作对照。
与各截短蛋白温育后的阳性血清的中和抗体滴度的分析结果如图6所示，其中横坐标表示与阳性血清温育的各截短蛋白，纵坐标表示达到50％感染抑制率的温育后阳性血清的最大稀释倍数(NT50，中和抗体滴度)。结果显示，在温育后，本发明的截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)导致阳性血清的中和抗体滴度显著下降：用TB8.0温育的阳性血清的中和抗体滴度可超过6＊103；用ΔVP8＊温育的阳性血清的中和抗体滴度可超过5＊103；而用本发明的截短蛋白温育的阳性血清的中和抗体滴度低于4＊103，甚至低于3＊103。这些结果表明，本发明的各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)与阳性血清均具有高反应性，且该反应性显著强于ΔVP8＊；这进而表明，本发明的各截短蛋白的构象或与天然病毒更接近，能够更好的被天然病毒免疫产生的抗体(阳性多抗血清)所识别。
实施例8：截短的轮状病毒VP8蛋白在动物中的保护性评价
使用实施例5中所述的免疫方案，用上述实施例3中获得的经纯化的截短的轮状病毒VP8蛋白来免疫实验组Balb/c小鼠(每组7只小鼠)。另外，使用相同的免疫方案，将ΔVP8＊、VP-8全长蛋白、轮状病毒RV和弗氏佐剂用作对照，分别用于免疫对照组Balb/c小鼠(每组7只小鼠)。分别于免疫前以及免疫后14d、28d和42d采集小鼠眼球血，分离血清，将其于-20℃保存。
在免疫程序完成后(免疫42天以后)，将各组小鼠按每两只母鼠配一只公鼠进行交配。交配后20日左右，母鼠产下乳鼠，饲养7日。用LLR病毒株对7日龄乳鼠进行灌胃攻毒，攻毒剂量为5*106TCID50/只。
攻毒后观察乳鼠发生腹泻的时间、持续时间、并对腹泻严重情况进行评分，记录。评分标准如图7A-7C所示。根据乳鼠腹泻的不同程度，评分分为3个等级：正常粪便计为1分(图7C)，软粪便计为2分 (图7B)，不成形水样便计为3分(图7A)。
实验结果如图8-9所示。图8显示了用实施例3中制备的各截短蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。图9显示了用实施例3中制备的各截短蛋白免疫Balb/c小鼠后的平均腹泻天数，其中，纵轴表示平均腹泻天数；横轴表示各截短蛋白。
结果显示，无论从平均腹泻严重程度还是从平均腹泻天数来看，各截短蛋白(VP8-5A，VP8-5，VP8-6，VP8-8，VP8-9，VP8-10，VP8-11或VP8-12)均具有显著的保护性，且均比ΔVP8＊保护性更好。
实施例9：包含VP8-5和分子内佐剂CTB的融合蛋白的表达和纯化以及其诱发中和抗体的能力和其免疫保护性的分析
从生工生物(上海)工程股份有限公司合成N端和C端各带酶切位点BglII/NdeI和BamHI/HindIII的CTB基因。用Nde I/Hind III将CTB基因双酶切，并克隆入PTO-T7载体，从而获得PTO-T7-CTB(克隆过程同实施例1)；随后，用BamH I/Hind III将VP8-5基因双酶切，并克隆入PTO-T7-CTB(克隆过程同实施例1)，从而获得重组质粒PTO-T7-CTB-VP8-5。另外，使用引物(SEQ ID NO：18和SEQ ID NO：28)来扩增VP8-5基因；随后，用Nde I/Hind III将扩增产物双酶切，并克隆入PTO-T7载体，从而获得PTO-T7-VP8-5’；进一步，用BglII/HindIII将CTB基因双酶切，然后克隆入PTO-T7-VP8-5’中(克隆过程同实施例1)，从而获得重组质粒PTO-T7-VP8-5-CTB。
按照实施例2中描述的方法，用构建的重组质粒在大肠杆菌中表达融合蛋白CTB-VP8-5和VP8-5-CTB。特别地，当培养物在600nm下的吸光值达到0.5时，向培养物中加入IPTG至终浓度为1mM；随后，在180rpm，25℃下继续培养大肠杆菌6小时，以诱导大肠杆菌表达融合蛋白。
按照实施例3中描述的方法，超声破碎表达融合蛋白的大肠杆菌。收集不可溶性级分，并使用Buffer I(50mM Tris-HCl pH 8.0+150mM  NaCl)进行复溶。随后，将所获得的溶液以25000g离心10mins，并收集沉淀。用8M尿素溶解沉淀，并在4℃下进行透析，以除去尿素，并使沉淀中的融合蛋白复性。随后，通过下述方案来对溶液中的融合蛋白进行纯化。
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTAexplorer 100型制备型液相色谱系统。
层析介质：Q-sepharose-HP(GE Healthcare公司)。
柱体积：5.5cm＊20cm。
缓冲液：50mM Tris-HCl pH 8.0
50mM Tris-HCl pH 8.0，2M NaCl
流速：25mL/min。
检测器波长：280nm。
样品为之前制备的包含复性的融合蛋白CTB-VP8-5或VP8-5-CTB的溶液。
洗脱程序为：1000mM NaCl洗脱杂蛋白，300mM NaCl洗脱目的蛋白，收集300mMNaCl洗脱产物，共获得30mL的含有重组表达的CTB-VP8-5和VP8-5-CTB的经纯化的样品。
取经上述方案纯化的样品150μL，加入30μL 6X Loading Buffer，混匀并于100℃水浴10min；然后取10μl于13.5％SDS-聚丙烯酰胺凝胶中以120V电压电泳120min；然后通过考马斯亮兰染色来显示电泳条带。电泳结果示于图10所示。
图10显示了如上制备的包含截短蛋白VP8-5与分子内佐剂CTB的融合蛋白(CTB-VP8-5和VP8-5-CTB)在纯化完成后的SDS-PAGE结果。结果显示，所构建的融合蛋白在大肠杆菌中高表达，并且上述方法有效地富集和纯化了大肠杆菌中表达的融合蛋白。
进一步，使用实施例5中所述的免疫方案(将其中使用的弗氏佐剂替换为铝佐剂)，用如上制备的经纯化的融合蛋白免疫Balb/c小鼠。随后，使用实施例5-6中描述的方案，分析经免疫的小鼠的免疫血清中的总抗体滴度和中和抗体滴度。另外，还使用实施例8中描述的方 案，分析融合蛋白在动物中的免疫保护性。结果示于图11-12中。
图11显示了融合蛋白(CTB-VP8-5和VP8-5-CTB)在Balb/c小鼠中诱发的免疫血清中的总抗体滴度和中和抗体滴度的分析结果，其中横坐标表示所使用的各种蛋白，左纵坐标表示中和抗体滴度(NT50，即，达到50％感染抑制率的免疫血清最大稀释倍数)，右纵坐标表示总抗体滴度(log)。结果显示，本发明的融合蛋白(CTB-VP8-5和VP8-5-CTB)在免疫后第42天(三次免疫后)能在小鼠体内有效诱发具有高抗体滴度的免疫血清，并且，免疫血清中的总抗滴度和中和抗体滴度显著高于VP8-5组和CTB组的免疫血清中的总抗滴度和中和抗体滴度。这些结果表明，可通过将本发明的截短蛋白与分子内佐剂(例如CTB)连接来进一步提高本发明截短蛋白的免疫原性，从而实现更好的免疫效果。
图12显示了用如上制备的融合蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。结果显示，本发明的融合蛋白(CTB-VP8-5和VP8-5-CTB)均具有显著的免疫保护性；其中，CTB-VP8-5和VP8-5-CTB的免疫保护性均显著优于阴性对照组，且优于未与分子内佐剂CTB融合的VP8-5，并且CTB-VP8-5的免疫保护性最高。
实施例10：包含VP8-5和分子内佐剂CRM-A的融合蛋白的表达和纯化以及其诱发中和抗体的能力和其免疫保护性的分析
从生工生物(上海)工程股份有限公司合成N端和C端各带酶切位点NdeI和BamHI/HindIII的CRM-A基因，并且该基因的C端连接有编码肽接头的核苷酸序列。按照实施例9中描述的方法，通过NdeI/Hind III双酶切将CRM-A基因克隆入PTO-T7载体，从而获得PTO-T7-CRM-A；随后，通过BamH I/HindIII双酶切将VP8-5连入PTO-T7-CRM-A，从而获得重组质粒PTO-T7-CRM-A-VP8-5。另外，用引物(SEQ ID NO：30和31)扩增CRM-A基因。所获得的扩增产物在其N末端包含有编码肽接头的核苷酸序列。随后，通过BamH I/Hind III双酶切将扩增产物克隆入PTO-T7-VP8-5’，从而获得重组质粒PTO-T7-VP8-5-CRM-A。重组质粒PTO-T7-CRM-A-VP8-5编码融合蛋白CRM-A-VP8-5，其中CRM-A通过肽接头连接至VP8-5的N端。重组质粒PTO-T7-VP8-5-CRM-A编码融合蛋白VP8-5-CRM-A，其中VP8-5通过肽接头连接至CRM-A的N端。
按照实施例2中描述的方法，用构建的重组质粒在大肠杆菌中表达融合蛋白CRM-A-VP8-5和VP8-5-CRM-A。特别地，当培养物在600nm下的吸光值达到0.5时，向培养物中加入IPTG至终浓度为1mM；随后，在180rpm，25℃下继续培养大肠杆菌6小时，以诱导大肠杆菌表达融合蛋白。
按照实施例3中描述的方法，超声破碎表达融合蛋白的大肠杆菌。收集可溶性级分，并向其中添加25％硫酸铵以沉淀其中的蛋白质。收集蛋白质沉淀，并用50mM Tris-HCl pH 8.0进行复溶。对复溶后的溶液进行离心，并收集上清。随后，通过下述方案来对上清中的融合蛋白进行纯化。
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTAexplorer 100型制备型液相色谱系统。
层析介质：Q-sepharose-HP(GE Healthcare公司)。
柱体积：5.5cm＊20cm。
缓冲液：50mM Tris-HCl pH 8.0
50mM Tris-HCl pH 8.0，2M NaCl
流速：25mL/min。
检测器波长：280nm。
样品为之前制备的包含融合蛋白CRM-A-VP8-5或VP8-5-CRM-A的上清。
洗脱程序为：1000mM NaCl洗脱杂蛋白，300mM NaCl洗脱目的蛋白，收集300mMNaCl洗脱产物，共获得30mL的含有重组表达的CRM-A-VP8-5和VP8-5-CRM-A的经纯化的样品。
取经上述方案纯化的样品150μL，加入30μL 6X LoadingBuffer， 混匀并于100℃水浴10min；然后取10μl于13.5％SDS-聚丙烯酰胺凝胶中以120V电压电泳120min；然后通过考马斯亮兰染色来显示电泳条带。电泳结果如图13所示。
图13显示了如上制备的包含截短蛋白VP8-5与分子内佐剂CRM-A的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在纯化完成后的SDS-PAGE结果。结果显示，所构建的融合蛋白在大肠杆菌中高表达，并且上述方法有效地富集和纯化了大肠杆菌中表达的融合蛋白。
进一步，使用实施例5中所述的免疫方案(将其中使用的弗氏佐剂替换为铝佐剂)，用如上制备的经纯化的融合蛋白免疫Balb/c小鼠。随后，使用实施例5-6中描述的方案，分析经免疫的小鼠的免疫血清中的总抗体滴度和中和抗体滴度。另外，还使用实施例8中描述的方案，分析融合蛋白在动物中的免疫保护性。结果示于图14-15中。
图14显示了融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在Balb/c小鼠中诱发的免疫血清中的总抗体滴度和中和抗体滴度的分析结果，其中，横坐标表示所使用的各种蛋白，左纵坐标表示中和抗体滴度(NT50，即，达到50％感染抑制率的免疫血清最大稀释倍数)，右纵坐标表示总抗体滴度(log)。结果显示，本发明的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)在免疫后第42天(三次免疫后)能在小鼠体内有效诱发具有高抗体滴度的免疫血清，并且，免疫血清中的总抗滴度和中和抗体滴度显著高于VP8-5组和CRM-A组的免疫血清中的总抗滴度和中和抗体滴度。这些结果表明，可通过将本发明的截短蛋白与分子内佐剂(例如CRM-A)连接来进一步提高本发明截短蛋白的免疫原性，从而实现更好的免疫效果。
图15显示了用如上制备的融合蛋白免疫Balb/c小鼠后的腹泻评分，其中，纵轴表示腹泻评分；横轴表示小鼠攻毒后天数。结果显示，本发明的融合蛋白(CRM-A-VP8-5和VP8-5-CRM-A)均具有显著的免疫保护性；其中，CRM-A-VP8-5和VP8-5-CRM-A的免疫保护性均显著优于阴性对照组，并且VP8-5-CRM-A的免疫保护性最高。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (13)

  1. 一种截短的轮状病毒VP8蛋白或其变体，其与野生型轮状病毒VP8蛋白相比，N端截短了21-60个氨基酸，例如21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、43个、44个、45个、46个、47个、48个、49个、50个、51个、52个、53个、54个、55个、56个、57个、58个、59个或60个氨基酸；
    例如，与野生型轮状病毒VP8蛋白相比，该截短的轮状病毒VP8蛋白N端截短了21-60个氨基酸，例如21个、25个、30个、35个、40个、45个、50个、55个或60个氨基酸；
    例如，该截短的轮状病毒VP8蛋白具有SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ 1D NO：7或者SEQ ID NO：8所示的氨基酸序列。
  2. 一种融合蛋白，其包含第一多肽和第二多肽，其中，所述第一多肽为权利要求1的截短的轮状病毒VP8蛋白或其变体；并且，所述第二多肽为分子内佐剂；
    任选地，所述融合蛋白还包含肽接头，标签，信号肽，蛋白酶切割位点，或其任何组合；
    优选地，所述分子内佐剂选自白喉毒素无毒突变体CRM197及其截短蛋白例如CRM197的A亚基，霍乱毒素，霍乱毒素B亚基CTB，霍乱毒素突变体例如CTA112/KDEV和CTA1-DD，大肠杆菌不耐热毒素(LT)及其无毒突变体LTR192G，大肠杆菌不耐热毒素B亚基LTB，破伤风毒素，及其任何组合；
    优选地，所述第一多肽通过直接融合的方式或通过肽接头与所述第二多肽进行连接；
    优选地，所述第一多肽位于所述第二多肽的N末端或C末端，并 且二者任选地通过肽接头连接；优选地，所述肽接头选自GS(SEQ ID NO：32)，GGS(SEQ ID NO：33)，GGGS(SEQ ID NO：34)，SGGGS(SEQ ID NO：35)，GGGG(SEQ ID NO：36)，GGSS(SEQ ID NO：37)，GGGGS(SEQ ID NO：38)，(GGGGS)3(SEQ ID NO：39)，及其任何组合；
    任选地，所述融合蛋白在其N末端和/或C末端包含标签；例如，所述标签选自组氨酸标签、谷胱甘肽转移酶(GST)标签、麦芽糖结合蛋白(MBP)标签、硫氧还蛋白(Trx)标签、NusA标签、二硫键异构酶标签、DsbA标签、DsbC标签、SUMO标签、msyB标签、TF标签、引发因子标签、泛素标签、Myc标签、Flag标签、荧光蛋白(例如GFP)标签、生物素标签、亲和素标签、及其任何组合；
    任选地，所述融合蛋白在其N末端包含信号肽；例如，所述信号肽选自OmpA，OmpT，pelB，CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽；
    任选地，所述融合蛋白包含蛋白酶切割位点；例如，所述蛋白酶切割位点位于相邻的两个元件之间，所述元件选自所述第一多肽，第二多肽，肽接头，标签和信号肽；
    优选地，所述融合蛋白具有SEQ ID NO：12-13和15-16任一项所示的氨基酸序列。
  3. 一种缀合物，其包含权利要求1的截短的轮状病毒VP8蛋白或其变体，以及与所述截短的VP8蛋白或其变体缀合的分子内佐剂；
    优选地，所述分子内佐剂选自白喉毒素无毒突变体CRM197及其截短蛋白例如CRM197的A亚基，霍乱毒素，霍乱毒素B亚基CTB，霍乱毒素突变体例如CTA112/KDEV和CTA1-DD，大肠杆菌不耐热毒素(LT)及其无毒突变体LTR192G，大肠杆菌不耐热毒素B亚基LTB，破伤风毒素，及其任何组合；
    优选地，所述分子内佐剂通过共价方式(例如化学偶联)或非共价方式(例如吸附)与所述轮状病毒VP8蛋白或其变体缀合。
  4. 一种分离的核酸，其编码权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白。
  5. 包含权利要求4的分离的核酸的载体。
  6. 包含权利要求4的分离的核酸和/或权利要求5的载体的宿主细胞。
  7. 一种组合物，其包含权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，或权利要求4的分离的核酸，或权利要求5的载体，或权利要求6的宿主细胞。
  8. 一种药物组合物或疫苗，其包含权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，任选地还包含药学可接受的载体和/或赋形剂，
    优选地，所述药物组合物或疫苗还包含佐剂，例如铝佐剂；
    优选地，所述截短蛋白或其变体，或所述融合蛋白，或所述缀合物以预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的有效量存在；
    优选地，所述药物组合物或疫苗还包含另外的活性成分；
    优选地，所述另外的活性成分能够预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病。
  9. 获得权利要求1的截短的轮状病毒VP8蛋白或其变体或权利要求2的融合蛋白的方法，其包括，在允许所述截短蛋白或其变体或所述融合蛋白表达的条件下，培养权利要求6的宿主细胞；和，回收所表达的截短蛋白或其变体或所述融合蛋白，
    优选地，所述方法包括步骤：利用大肠杆菌表达系统来表达所述截短蛋白或其变体或所述融合蛋白，然后将大肠杆菌裂解，并从裂解液中纯化获得含有所述截短蛋白或其变体或所述融合蛋白，优选地，所述纯化包括色谱层析。
  10. 一种制备疫苗的方法，其包括将权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，或者通过权利要求9的方法获得的截短的轮状病毒VP8蛋白或其变体或融合蛋白与药学可接受的载体和/或赋形剂混合，任选地还混合佐剂例如铝佐剂，和/或另外的活性成分，例如能够预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的另外的活性成分。
  11. 一种预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病的方法，其包括将预防或治疗有效量的权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，或权利要求8的药物组合物或疫苗，或者通过权利要求9的方法获得的截短的轮状病毒VP8蛋白或其变体或融合蛋白，或通过权利要求10的方法获得的疫苗施用给受试者，
    优选地，所述由轮状病毒感染所导致的疾病是轮状病毒性胃肠炎和腹泻；
    优选地，所述受试者是哺乳动物，例如小鼠和人。
  12. 权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，或者通过权利要求9的方法获得的截短的轮状病毒VP8蛋白或其变体或融合蛋白在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病，
    优选地，所述由轮状病毒感染所导致的疾病是轮状病毒性胃肠炎和腹泻；
    优选地，所述受试者是哺乳动物，例如小鼠和人。
  13. 权利要求1的截短的轮状病毒VP8蛋白或其变体，或权利要求2的融合蛋白，或权利要求3的缀合物，或者通过权利要求9的方法获得的截短的轮状病毒VP8蛋白或其变体或融合蛋白，其用于在受试者中预防或治疗轮状病毒感染或由轮状病毒感染所导致的疾病，
    优选地，所述由轮状病毒感染所导致的疾病是轮状病毒性胃肠炎和腹泻；
    优选地，所述受试者是哺乳动物，例如小鼠和人。
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