WO2016184425A1 - 截短的轮状病毒vp4蛋白及其用途 - Google Patents

截短的轮状病毒vp4蛋白及其用途 Download PDF

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WO2016184425A1
WO2016184425A1 PCT/CN2016/082780 CN2016082780W WO2016184425A1 WO 2016184425 A1 WO2016184425 A1 WO 2016184425A1 CN 2016082780 W CN2016082780 W CN 2016082780W WO 2016184425 A1 WO2016184425 A1 WO 2016184425A1
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protein
rotavirus
truncated
seq
infection
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PCT/CN2016/082780
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French (fr)
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葛胜祥
李廷栋
贾连智
李毅坚
薛淼舸
曾渊君
张军
夏宁邵
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厦门大学
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Priority to HRP20211838TT priority Critical patent/HRP20211838T1/hr
Priority to PL16795912T priority patent/PL3299384T3/pl
Priority to EP21194967.2A priority patent/EP3981782A1/en
Priority to SI201631412T priority patent/SI3299384T1/sl
Priority to RS20211464A priority patent/RS62664B1/sr
Priority to US15/575,140 priority patent/US10723767B2/en
Application filed by 厦门大学 filed Critical 厦门大学
Priority to ES16795912T priority patent/ES2898701T3/es
Priority to LTEPPCT/CN2016/082780T priority patent/LT3299384T/lt
Priority to JP2017560565A priority patent/JP6808650B2/ja
Priority to EP16795912.1A priority patent/EP3299384B1/en
Publication of WO2016184425A1 publication Critical patent/WO2016184425A1/zh
Priority to US16/901,410 priority patent/US11339194B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12351—Methods of production or purification of viral material

Definitions

  • the present invention relates to the fields of biochemistry, molecular biology, molecular virology and immunology.
  • the present invention relates to a truncated rotavirus VP4 protein, a coding sequence thereof and a method of preparation, a pharmaceutical composition comprising the protein and a vaccine, the protein, pharmaceutical composition and vaccine being useful for prevention, alleviation or Treatment of rotavirus infections and diseases caused by rotavirus infections, such as rotavirus gastroenteritis and diarrhea.
  • the invention further relates to the use of a protein as described above for the preparation of a pharmaceutical composition or vaccine for the prevention, alleviation or treatment of a rotavirus infection and a disease caused by the infection of a rotavirus, such as a round Viral gastroenteritis and diarrhea.
  • Rotavirus belongs to the reoviridae family, a genus of rotavirus, which is the main pathogen causing diarrhea in infants and young children. It was found in the duodenum of patients with gastroenteritis in Bishop in 1973 (Bishop, Davidson). , Holmes, et al. Lancet, 2 (7841), 1281-1283, 1973). Studies have shown that more than 95% of children have had at least one rotavirus infection before the age of five. According to WHO statistics, the annual death toll from rotavirus infection is as high as 600,000, and the number of cases of diarrhea is as high as 200 million.
  • Rotavirus is a non-enveloped RNA virus whose genome consists of 11 double-stranded RNAs encoding six structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6) (Estes and Cohen) .Microbiol Rev, 53(4), 410-449, 1989).
  • the rotavirus is icosahedral symmetry, and its capsid is composed of three concentric layers, namely a core layer composed of VP1, VP2 and VP3, an undergarment shell composed of VP6, and an outer casing composed of VP4 and VP7.
  • the VP6 protein is the highest capsid protein in rotavirus. According to the antigenicity of VP6, rotavirus can be divided into 7 groups of A-G, and group A rotavirus is the main pathogen causing diarrhea in infants. VP4 and VP7 are mainly neutralizing antigens, and group A rotavirus can be divided into different P serotypes and G serotypes according to their antigenicity. G serotypes and P serotypes are independent of each other and interact with each other. Common combinations include G1P[8], G2P[4], G3P[8], and G4P[8]. In recent years, the prevalence of G9P [8] and G9P [6] has increased significantly (Li, Liu, Yu, et al. Vaccine, 27 F40-F45, 2009).
  • Non-replicating vaccines are the main direction of current rotavirus vaccine research, and genetic engineering vaccines have attracted much attention due to their low cost, safety and effectiveness.
  • VP4 and VP7 proteins can stimulate the body to produce neutralizing antibodies, thereby inhibiting rotavirus infection. Therefore, both VP4 and VP7 are the major candidate antigens for rotavirus subunit vaccines.
  • the VP7 protein is a glycosylated protein containing four pairs of intrachain disulfide bonds to form a Ca 2+ -dependent trimer. The recombinantly expressed VP7 protein stimulates the body to produce lower levels of neutralizing antibodies.
  • VP4 protein is aglycosylated, and recombinantly expressed VP4 protein can stimulate the body to produce a higher immune response and reduce the level of diarrhea in suckling mice (Mackow, Vo, Broome, et al. J Virol, 64 (4), 1698 -703, 1990).
  • VP4 protein is more suitable as a candidate antigen for rotavirus genetic engineering vaccine than VP7 protein.
  • the VP4 protein consists of 776 amino acids and can be digested by trypsin to form VP8* (aa1-231) and VP5* (aa248-776).
  • the VP4 protein consists of three parts: the head, the neck and the pedestal. It acts as a spike protein for rotavirus and plays an important role in the infection of rotavirus.
  • the head of the spike is composed of two molecules of VP8 protein, which binds to the sialic acid receptor on the cell surface to mediate the rotavirus adsorption process.
  • the neck consists of three molecules of the VP5 antigenic region, wherein the VP5 antigenic region of the two molecules forms a dimer and the other VP5 antigenic region exists as a monomer.
  • the structure of VP4 is rearranged, exposing the VP5 membrane fusion site and mediating rotavirus
  • the pedestal portion is composed of three molecules of the VP5 protein C-terminal domain, and the outer capsid and the undergarment are inserted through the domain.
  • the N-terminal 25 amino acids of the VP8 protein form an alpha helix, and the three alpha helices form a helical bundle inserted into the VP5 pedestal portion and are connected to the VP8 head through a flexible linking region.
  • the structure of the linking region has not yet been fully resolved (Settieri, Chen , Dormitzer, et al. EMBO J, 30 (2), 408-16, 2011).
  • the neutralizing epitope of VP4 protein is mainly located in the VP8 head and VP5 antigenic region.
  • the neutralizing antibody against VP8 can inhibit the rotavirus adsorption process, and the neutralizing antibody against VP5 has cross-neutralizing activity, which can inhibit the round. Entry into the bacterium (Dormitzer, Nason, Venkataram Prasad, et al. Nature, 430 (7003), 1053-1058, 2004; Abdelhakim, Salgado, Fu, et al. PLoS Pathog, 10(9), e1004355, 2014) .
  • VP4 protein can stimulate the body to produce a protective immune response (Dunn, Fiore, Werner, et al. Arch Virol, 140 (11), 1969-78, 1995; Gil, De Souza, Asensi, et al. Viral Immunology, 13(2), 187-200, 2000).
  • the expression of the VP4 protein by the eukaryotic system is long, costly, and low in expression; and, the full-length VP4 protein is mainly expressed in the form of inclusion bodies in the prokaryotic system, which is difficult to purify and cannot maintain its native conformation.
  • truncated VP8 protein ( ⁇ VP8*, aa65) which can be efficiently expressed in soluble form in E. coli. -223) (Wen, Cao, Jones, et al. Vaccine, 30 (43), 6121-6, 2012); however, the immunogenicity of the truncated VP8 protein is significantly lower than the full length VP8 protein.
  • our laboratory has conducted intensive research to obtain a new immunocompetent new VP8 truncated protein, which is immunized mice under Freund's adjuvant conditions.
  • the inventors of the present application have unexpectedly discovered after extensive research that the N-terminal truncated 1-64 amino acids (for example, 5-64 amino acids), and the VP4 protein whose C-terminus terminates between amino acid positions 276-497 can Expressed in soluble form in E. coli and can be easily purified by chromatography; and the high-purity truncated protein thus obtained (purity can be at least 50% or higher, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) have good homogeneity and immunogenicity, and can induce high titers of the body under the conditions of aluminum adjuvant.
  • the neutralizing antibody of the prion virus effectively solves the above technical problems.
  • the invention relates to a rotavirus VP4 protein or variant thereof having a N-terminal truncation of 1-64 amino acids (eg, 5-64 amino acids) and a C-terminus ending at amino acid position 276-497 .
  • the invention relates to a truncated rotavirus VP4 protein or variant thereof, which is N-terminally truncated from 1 to 64 amino acids (eg, 5-64) compared to wild-type rotavirus VP4 protein Amino acid), and the C-terminus terminates at the following position of the wild-type rotavirus VP4 protein: as opposed to any of amino acid positions 276-497 of SEQ ID NO:40 The location should be.
  • the N-terminus of the truncated rotavirus VP4 protein is truncated from 1 to 64 amino acids, such as 1, 2, 3, compared to the wild-type rotavirus VP4 protein. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 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, 60, 61, 62, 63 or 64 amino acids (eg, 1-5, 5-25, 5-21) , 21-25, 21-64, or 25-64 amino acids, such as 1, 5, 21, 25, or 64 amino acids); and the C-terminus ends in wild-type rotavirus VP4 protein Position of: amino acid position 276-497 of SEQ ID NO: 40 (eg, amino acid positions 281-497, 291-497
  • the N-terminus of the truncated rotavirus VP4 protein is truncated by 1, 5, 21, 25, or 64 compared to the wild-type rotavirus VP4 protein.
  • Amino acid, and the C-terminus terminates at the following position of the wild-type rotavirus VP4 protein: amino acid positions 276, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371 with SEQ ID NO: a position corresponding to 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 476, 482, 487, 492 or 497 (for example, amino acid positions 331, 351, 381 with SEQ ID NO: 40 , 411, 441, 461, 471, 476, 482, 487, 492 or 497 corresponding position).
  • the truncated rotavirus VP4 protein has a characteristic selected from the group consisting of:
  • the N-terminus is truncated by 25 amino acids and the C-terminus is terminated at the following positions: amino acid positions 276, 281, 291, 301, 311, 321 , 331 , 341 , 351 , 361 , 371 with SEQ ID NO: 40 a position corresponding to 381, 391, 401, 411, 421, 431, 441, 451, 461, 471, 476, 482, 487, 492 or 497 (preferably, amino acid position 331, 351 with SEQ ID NO: 40) , 381, 411, 441, 461, 471, 476, 482, 487, 492 or 497 corresponding position);
  • N-terminus is 21 amino acids truncated and the C-terminus terminates at a position corresponding to amino acid position 476 of SEQ ID NO: 40;
  • N-terminus is truncated by 64 amino acids and the C-terminus is terminated at a position corresponding to amino acid position 476 of SEQ ID NO:40.
  • the wild-type rotavirus VP4 protein is a VP4 protein derived from a rotavirus LLR strain, a SA11 strain, an EDIM strain, or is derived from P[4], P[6], Or the VP4 protein of the P[8] genotype rotavirus;
  • the wild-type rotavirus VP4 protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 87-91.
  • the truncated rotavirus VP4 protein (hereinafter also simply referred to as a truncated protein) has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5 and 10-39.
  • the invention relates to a polynucleotide encoding the above-described truncated protein or variant thereof, and a vector comprising the polynucleotide.
  • Vectors that can be used to insert polynucleotides of interest are well known in the art, including but not limited to Cloning vector and expression vector.
  • 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 host cell of the invention is a prokaryotic cell such as an E. coli cell.
  • the invention also relates to a multimer comprising or consisting of the truncated protein or variant thereof.
  • the multimer is a trimer.
  • the multimer is a multimer having a molecular weight of at least 600 kDa.
  • the multimer comprises a truncated rotavirus VP4 protein that is N-terminally truncated by 25 amino acids compared to the wild-type rotavirus VP4 protein, and the C-terminus ends in A position in the wild-type rotavirus VP4 protein that corresponds to amino acid position 476 of SEQ ID NO:40.
  • the multimer comprises a truncated rotavirus VP4 protein having the amino acid sequence set forth in SEQ ID NO:30.
  • the multimer is a trimer or a multimer of at least 600 kDa comprising a truncated rotavirus VP4 protein as set forth in SEQ ID NO:30.
  • the present invention also relates to a composition
  • a composition comprising the above-described truncated protein or variant thereof, or the above polynucleotide, or the above vector, or the above host cell, or the above multimer.
  • the composition comprises a truncated protein of the invention or a variant thereof.
  • the composition comprises a multimer of the invention.
  • the invention also relates to a pharmaceutical composition (eg, a vaccine) comprising a truncated protein of the invention, or a variant thereof, or a multimer of the invention, optionally further comprising a pharmaceutically acceptable carrier and / or excipients.
  • a pharmaceutical composition of the present invention (for example, a vaccine) can be used for preventing or treating a rotavirus infection or a disease caused by a rotavirus infection such as rotavirus gastroenteritis or diarrhea.
  • the truncated proteins of the invention, or variants thereof, or multimers of the invention are present in an amount effective to prevent or treat a disease caused by rotavirus infection or infection by rotavirus.
  • the pharmaceutical compositions (e.g., 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.
  • the pharmaceutical compositions (e.g., vaccines) of the invention further comprise an adjuvant, such as an aluminum adjuvant.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, stabilizer or capable of providing advantageous properties for administration (e.g., administration to a human subject) of the pharmaceutical composition.
  • Suitable pharmaceutical carriers include, for example, sterile water, saline, dextrose, a condensation product of castor oil and ethylene oxide, a liquid acid, a lower alcohol (e.g., a C 1-4 alcohol), an oil (e.g., corn oil, peanut oil, sesame oil; It optionally further comprises an emulsifier such as a mono- or di-glyceride of a fatty acid or a phospholipid such as lecithin, ethylene glycol, polyalkylene glycol, sodium alginate, poly(vinylpyrrolidone) and the like.
  • the carrier optionally may further comprise an adjuvant, a preservative, a stabilizer, a wetting agent, an emulsifier, a penetration enhancer, and the like.
  • the pharmaceutical composition is sterile.
  • the viscosity of the pharmaceutical combination can be controlled and maintained by the selection of a suitable solvent or excipient.
  • the pharmaceutical composition is formulated to have a pH of 4.5-9.0, 5.0-8.0, 6.5-7.5, or 6.5-7.0.
  • compositions of the invention can be administered by methods well known in the art, such as, but not limited to, by oral or injection.
  • the pharmaceutical compositions (e.g., vaccines) of the invention are administered in unit dosage form.
  • the amount of the pharmaceutical composition of the invention (e.g., vaccine) 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, and the like, which may be According to the actual situation, it is reasonable to determine.
  • the invention in another aspect, relates to a method of making the above-described truncated protein or variant thereof, comprising culturing a host cell of the invention under conditions permitting expression of the truncated protein or variant thereof; The expressed truncated protein or variant thereof is recovered.
  • the method comprises using E. coli to express a truncated protein of the invention or a variant thereof, then lysing the E. coli and purifying it from a lysate to obtain the truncated protein or Its variant.
  • the purification comprises chromatography.
  • the purification comprises two-step chromatography.
  • the two-step chromatography comprises: anion exchange chromatography (eg, anion exchange chromatography using Q-sepharose-HP); and, followed by hydrophobic affinity chromatography (eg, Hydrophobic affinity chromatography using Phenyl sepharose-HP).
  • the present invention is also a method of preparing a vaccine comprising mixing a truncated protein of the present invention or a variant thereof or a multimer of the present invention with a pharmaceutically acceptable carrier and/or excipient,
  • an adjuvant such as an aluminum adjuvant
  • an additional active ingredient such as an additional active ingredient capable of preventing or treating a disease caused by rotavirus infection or infection by rotavirus
  • the method of preparing a vaccine comprises the step of mixing a truncated protein of the invention, or a variant thereof, or a multimer of the invention, with an adjuvant, such as an aluminum adjuvant.
  • the obtained vaccine can be used for the prevention or treatment of rotavirus infection or diseases caused by rotavirus infection such as rotavirus gastroenteritis and diarrhea.
  • the present invention relates to a method of preventing or treating a disease caused by a rotavirus infection or a rotavirus infection in a subject, comprising a prophylactically or therapeutically effective amount of a truncation according to the present invention
  • the protein or variant thereof or a multimer of the invention or a pharmaceutical composition of the invention is administered to the 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 also relates to the use of a truncated protein of the invention, or a variant thereof, or a multimer of the invention, in the preparation of a pharmaceutical composition, such as a vaccine, for use in a pharmaceutical composition, such as a vaccine Prevention or treatment of 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 present invention relates to the above-described truncated protein or variant thereof or the above-described multimer for use in preventing or treating 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 expression "the N-terminal truncation of X amino acids” means that the methionine residue encoded by the initiation codon is substituted for the amino acid residue of the N-th position of the N-terminus of the protein.
  • a rotavirus VP4 protein having a N-terminal truncation of 25 amino acids means that the methionine residue encoded by the initiation codon replaces the amino acid residues 1 to 25 of the N-terminus of the wild-type rotavirus VP4 protein.
  • the protein obtained by the base is obtained by the base.
  • C-terminus at amino acid position X means that all amino acid residues after amino acid position X (i.e., starting from amino acid position X+1) are all deleted.
  • termination of the C-terminus at amino acid position 441 of the wild-type rotavirus VP4 protein means that all amino acid residues after the amino acid position 441 of the wild-type rotavirus VP4 protein (ie, starting from amino acid position 442) are all deleted.
  • the term "variant” refers to a protein having one or more amino acid sequences (for example, 1-10 or 1-) compared to the amino acid sequence of the truncated rotavirus VP4 protein of the present invention. 5 or 1-3) amino acids differ (eg conservative amino acid substitutions) or have at least 60%, 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 sequences CTGACT and CAGGTT share 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.).
  • the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAM120 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 PAM250 matrix and 16, 14, 12, 10, 8, A gap weight of 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 are used to determine the percent identity between the 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.
  • Load The bodies are well known to those skilled in the art and include, but are not limited to, plasmids; phage; cosmid and the like.
  • VP4 protein refers to a structural protein that together with VP7 constitutes the outer capsid of the RV viral particle.
  • the amino acid sequences of the VP4 proteins are known to those skilled in the art and are found in various public databases (e.g., the GenBank database).
  • exemplary amino acid sequences of VP4 proteins can be set forth in SEQ ID NOs: 40 and 87-91.
  • the term "truncated rotavirus VP4 protein” refers to a protein produced after the removal of one or more amino acids at the N-terminus and/or C-terminus of the wild-type rotavirus VP4 protein, wherein the wild-type round
  • GenBank database such as GenBank Accession Nos. AEV53329.1, BAA03850.1, AAB94758.2, AIS93087.1, ACJ06216.1, and AAA66953.1.
  • an exemplary amino acid sequence of the VP4 protein of wild-type rotavirus LLR is shown in SEQ ID NO:40. Therefore, in the present invention, when referring to the sequence of the VP4 protein, it is described using the sequence shown in SEQ ID NO:40.
  • amino acid position 276-497 of the VP4 protein refers to amino acid position 276-497 of SEQ ID NO:40.
  • mutations or variations may be naturally occurring or artificially introduced in SEQ ID NO: 40 without affecting the biological properties of the VP4 protein.
  • the VP4 proteins of different strains of rotavirus may naturally differ in amino acid sequence but have substantially the same biological properties.
  • the term "VP4 protein” is intended to include all such polypeptides and variants, including the polypeptide set forth in SEQ ID NO: 40, as well as natural or artificial variants thereof, which retain the VP4 protein. Biological characteristics.
  • sequence fragment and amino acid position of the VP4 protein it includes not only the sequence fragment and amino acid position of the polypeptide represented by SEQ ID NO: 40, but also the corresponding sequence fragment and amino acid in the natural or artificial variant of the polypeptide. position.
  • amino acid position 276-497 of VP4 protein is intended to include amino acid positions 276-497 of SEQ ID NO: 40, and the corresponding amino acids in variants (natural or artificial) of the polypeptide set forth in SEQ ID NO: 40. position.
  • exemplary amino acid sequences of the VP4 proteins of wild-type rotavirus SA11, EDIM, P[4], P[6], P[8] are shown in SEQ ID NOs: 87-91, respectively.
  • the expression “corresponding sequence fragment” or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • the expression “corresponding amino acid position” means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity, the amino acid sites at the equivalent positions in the compared sequences are/ Residues.
  • truncated rotavirus VP4 protein gene fragment refers to a gene fragment which lacks the coding at the 5' or 3' end compared to the wild type rotavirus VP4 protein gene.
  • the nucleotide sequence of the VP4 protein gene of wild-type rotavirus LLR can be as shown in SEQ ID NO:40.
  • the term "multimer” refers to a polymer composed of a polypeptide molecule (for example, a truncated protein of the invention) as a monomer, which may generally comprise at least two (eg, three, four, 5 or more) polypeptide monomers (eg, truncated proteins of the invention).
  • monomer molecules polymerize to form multimers by intermolecular interactions (eg, hydrogen bonding, van der Waals forces, hydrophobic interactions).
  • the multimer is a trimer comprising 3 monomers.
  • the multimer is a multimer comprising a plurality of monomers and having a molecular weight of at least 600 kDa (such polymers are also referred to herein as highmers).
  • 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, aluminum hydroxide), Freund's adjuvant (eg, complete Freund's adjuvant)
  • An ionic strength enhancer includes, but is not limited to, sodium chloride.
  • the term "adjuvant” refers to a non-specific immunopotentiator which, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials. In the present invention, it is particularly preferred that the adjuvant is an aluminum adjuvant.
  • an effective amount means an amount effective to achieve the intended purpose.
  • an effective amount for preventing or treating a disease means effectively preventing, preventing or delaying the onset of a disease (eg, a rotavirus infection), or alleviating, alleviating or treating an existing disease (eg, by a round)
  • the amount of severity of the disease caused by a viral infection is well within the abilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • 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 term “immunogenicity” means the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • subject refers to an animal, such as a vertebrate.
  • the subject is a mammal, such as a human, bovine, equine, feline, canine, rodent or primate.
  • the subject is a human.
  • the term can be used interchangeably with "patient.”
  • the truncated rotavirus VP4 protein provided by the present invention and its preparation method effectively solve the technical problems existing in the art.
  • the truncated protein of the present invention or a variant thereof can be stably expressed in Escherichia coli, achieving high yield. This provides favorable conditions for large-scale industrial production.
  • the truncated protein of the present invention or a variant thereof is purified in a relatively simple manner and is easy to handle.
  • the E. coli can be cleaved and then subjected to chromatographic treatment of the lysate (for example, by anion exchange chromatography and hydrophobic affinity chromatography), thereby obtaining High purity truncated protein (purity can be at least 50% or higher, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%). This provides favorable conditions for large-scale industrial production.
  • the high-purity truncated protein obtained by the present invention has good uniformity and stability.
  • the purified truncated proteins of the invention are capable of being present in a uniform form and are not susceptible to degradation. This provides favorable conditions for mass production, avoids variability between batches, and facilitates accurate drug use.
  • the truncated protein of the present invention or a variant thereof is capable of inducing the body to produce a high titer neutralizing antibody against rotavirus under aluminum adjuvant conditions. Since aluminum adjuvants are commonly used in the clinic without the use of Freund's adjuvant, the truncated proteins of the invention or variants thereof can be advantageously used in clinical conditions for protecting a subject against rotavirus infection.
  • the truncated protein of the present invention or a variant thereof is not only easy to express, but also easy to purify, and has high uniformity and stability. More importantly, the cut of the present invention Short proteins or variants thereof are highly immunogenic and are capable of inducing the body to produce high titers of neutralizing antibodies under aluminum adjuvant conditions, thereby providing strong protection to the subject under clinical conditions. Thus, the truncated proteins of the present invention or variants thereof enable large-scale industrial production of highly efficient anti-rotavirus vaccines, thereby providing an effective solution to the technical problems existing in the art.
  • Figure 1A shows the results of SDS-PAGE of purified VP8-5 protein. The results showed that the purity of the purified VP8-5 protein obtained was over 90%.
  • Figure 1B shows the results of an indirect ELISA analysis of VP8-5 protein and immune sera obtained by immunizing Balb/c mice, wherein the abscissa indicates the experimental group (VP8-5), the positive control group (LLR), and the negative.
  • the ordinate indicates the maximum dilution factor (ie, antibody titer) of the immune serum reactive with the VP8-5 protein.
  • the results showed that VP8-5 induced antibody production in mice on day 42 after immunization under the conditions of using aluminum adjuvant, but the efficiency was lower than that of inactivated virus LLR.
  • This result indicates that the VP8-5 protein is immunogenic under the conditions of using an aluminum adjuvant, and can induce antibody production in mice, but its ability to induce antibody production in animals is lower than that of inactivated virus LLR.
  • Figure 1C shows the results of analysis of neutralizing antibody titers of immune serum induced by VP8-5 in Balb/c mice, wherein the abscissa indicates the experimental group (VP8-5), the positive control group (LLR), and the negative control. Group (NC), the ordinate indicates the log value (log 2 NT50, neutralizing antibody titer) of the maximum dilution factor of immune serum up to 50% inhibition of infection.
  • the results showed that VP8-5 was able to induce neutralizing antibodies in mice on day 42 after immunization (after three immunizations) under conditions of aluminum adjuvant, but the neutralizing antibody titer (NT50) of the immune serum was lower than that. Inactivate virus LLR.
  • the neutralizing antibody titer (NT50) of the immune serum induced by the VP8-5 protein was only about 64. This result indicates that VP8-5 is capable of inducing the production of neutralizing antibodies in the body under the conditions of using an aluminum adjuvant, but the neutralizing antibody titer of the induced immune serum is lower than that of the inactivated virus LLR.
  • Figures 2A-2C show the scoring criteria for diarrhea in protective experiments. According to the different degrees of diarrhea in suckling rats, the score is divided into 3 grades: 1 point for normal feces (Fig. 2C), 2 points for soft stools (Fig. 2B), and 3 points for unformed water samples (Fig. 2A) .
  • Figure 3 shows the diarrhea scores of the suckling mice in different immunization groups after challenge, in which the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after the mice were challenged.
  • the results showed that there was no significant difference in the diarrhea score between the experimental group (VP8-5) and the negative control group (NC). This indicates that under the conditions of using aluminum adjuvant, VP8-5 has a lower ability to protect against rotavirus infection (below inactivated virus) and cannot fully alleviate diarrhea caused by rotavirus infection.
  • Figures 4A-4D show SDS-PAGE results for individual truncated proteins expressed in E. coli. The results showed that other truncated proteins were highly expressed in E. coli except for the relatively low expression levels of 26-311, 26-331 and 26-381.
  • Figures 5A-5B show SDS-PAGE results of various truncated VP4 proteins purified.
  • the lanes from left to right are: Protein Molecular Markers (Marker), 1-476, 6-476, 22-476, 26-476, 65-476, 26-231, 26-271, 26- 331 and 26-351.
  • the lanes from left to right are: Protein Molecular Markers (Marker), 26-381, 26-411, 26-441, 26-461, 26-471, 26-482, 26-487, 26- 492 and 26-497.
  • the results of Figures 5A and 5B show that after the above purification step, the concentration of each truncated protein is above 0.2 mg/ml and the purity is greater than 80%.
  • Figure 6 shows G3000 PWXL for truncated proteins 1-476, 6-476, 22-476, 26-476, 26-271, 26-471, 26-482, 26-487, 26-492, 26-497
  • the results of molecular sieve analysis The results of molecular sieve analysis.
  • the abscissa axis represents the retention time, and the ordinate axis represents the absorbance value at 280 nm.
  • Figure 7 shows the results of analysis by G5000 PWXL molecular sieves of truncated proteins 26-331, 26-351, 26-381, 26-411, 26-441 and 26-461.
  • the abscissa axis represents the retention time, and the ordinate axis represents the absorbance value at 280 nm.
  • the results showed that under truncated conditions of TB8.0, these truncated proteins were present in the sample as high polymers (peak time was about 10-11 minutes).
  • Fig. 8A shows the results of molecular sieve analysis of the truncated protein 26-476 after standing at 37 ° C for 12 hours in 50 mM Tris-HCl (pH 8.0).
  • the abscissa axis represents the retention time, and the ordinate axis represents the absorbance value at 280 nm.
  • the results showed that the truncated protein 26-476 was able to form a uniform polymer (retention time of 12.4 minutes) after standing at 37 ° C for 12 hours in 50 mM Tris-HCl (pH 8.0), and the proportion of high polymer was as high as 97.2. %.
  • Fig. 8B shows the results of electron microscopic observation of the truncated protein 26-476 after standing at 37 ° C for 12 hours in 50 mM Tris-HCl (pH 8.0). The results indicate that the truncated protein 26-476 can be assembled into a uniform polymer in vitro.
  • Figure 9 shows the results of SDS-PAGE of the cleaved and uncut truncated proteins 26-476, 26-482, 26-487, 26-492, 26-497.
  • the number 1 on the lane indicates that the sample was not trypsinized; the number 2 indicates that the sample was treated with 0.1 mg/ml trypsin.
  • the results showed that the above truncated proteins were recognized by pancreatin and digested, and the restriction sites were exposed.
  • Figure 10 shows various truncated VP4 proteins (1-476, 6-476, 22-476, 65-476, 26-271, 26-441, 26-461, 26-471, 26-476, 26-) 482, 26-487, 26-492, 26-497) and neutralizing antibody A3 (Fig. 10A), B1 (Fig. 10B), B5 (Fig. 10C), Results of indirect ELISA analysis of B6 (Fig. 10D), D6 (Fig. 10E), E2 (Fig. 10F), E5 (Fig. 10G), and 8F6 (Fig.
  • Figures 11A-11D show truncated protein 26-476 and each sample to be tested (1-476, 6-476, 22-476, 65-476, 26-271, 26-331, 26-351, 26-381) , trimer of 26-411, 26-441, 26-461, 26-471, 26-476, 26-482, 26-487, 26-492, 26-497, 26-476, high of 26-476
  • the result of indirect ELISA analysis of immune sera obtained by immunizing Balb/c mice wherein the abscissa indicates a protein sample used to prepare immune sera; the ordinate indicates reactivity with 26-476 Maximum dilution factor of immune serum (ie, antibody titer); RV: inactivated rotavirus; NC: negative control (PBS); trimer: trimer of 26-476; polymer: 26-476 High polymer.
  • FIGS 11A, 11B, 11C and 11D show results from different immunization batches.
  • the results showed that under the condition of using aluminum adjuvant, on the 42nd day after immunization, each protein sample can induce antibodies in mice (the antibody titer (GMT) in immune serum can reach 10 2 -10 5 or Higher); and, except for 26-271, antibody titers induced by other protein samples were higher than RV-induced antibody titers (1-476, 6-476, 22-476, 26-331, 26-351) , trimers of 26-381, 26-411, 26-441, 26-461, 26-471, 26-476, 26-487, 26-492, 26-476 and high polymers of 26-476), Or at least comparable to RV-induced antibody titers (65-476, 26-482, and 26-497).
  • Figures 12A-12D show individual protein samples (1-476, 6-476, 22-476, 65-476, 26-271, 26-331, 26-351, 26-381, 26-411, 26-441, 26-461, 26-471, 26-476, 26-482, 26-487, 26-492, 26-497, 26-476 trimer, 26-476 polymer, inactivated virus) in Balb
  • the results of analysis of neutralizing antibody titers of immune sera induced in /c mice wherein the abscissa indicates the protein sample used to prepare the immune serum; the ordinate indicates the maximum dilution factor of the immune serum that reached the 50% infection inhibition rate (NT50) , neutralizing antibody titer); RV: inactivated rotavirus; NC: negative control (PBS); trimer: trimer of 26-476; polymer: polymer of 26-476.
  • Figures 12A, 12B, 12C and 12D show results from different immunization batches. The results showed that under the condition of using aluminum adjuvant, on the 42nd day after immunization (after three immunizations), each protein sample can induce neutralizing antibodies in mice, and the neutralizing antibody titer (NT 50 ) can reach 2 8 -2 14 or higher; and, except for 26-271, the neutralizing antibody titers induced by other protein samples are comparable to RV-induced neutralizing antibody titers (6-476, 22-476, Trimers of 26-476, 65-476, 26-471, 26-482, 26-487, 26-492, 26-497, and 26-476), or even higher than RV-induced neutralizing antibody titers ( High polymers of 1-476, 26-331, 26-351, 26-381, 26-411, 26-441, 26-461 and 26-476).
  • Figures 13A-13D show 1-7 days after challenge from different immunization groups (using 22-476, 26-476, 65-476, 26-271, 26-331, 26-351, 26-381, 26-411 , 26-441, 26-461, 26-471, 26-482, 26-487, 26-492, 26-497, 26-476 trimer, 26-476 polymer, inactivated wheel Diarrhea scores in suckling mice (RV, positive control) or PBS (NC, negative control), where the vertical axis represents the mean diarrhea score; the horizontal axis represents the number of days after challenge in mice; RV: inactivated Rotavirus; NC: negative control (PBS); trimer: trimer of 26-476; polymer: polymer of 26-476.
  • RV positive control
  • NC negative control
  • trimer trimer of 26-476
  • polymer polymer of 26-476.
  • Figures 14A-14D show different immunization groups (using 22-476, 26-476, 65-476, 26-271, 26-331, 26-351, 26-381, 26-411, 26-441, 26-461, Triad of 26-471, 26-482, 26-487, 26-492, 26-497, 26-476, high polymer of 26-476, inactivated rotavirus (RV, positive control) or PBS
  • RV inactivated rotavirus
  • PBS negative control
  • trimer 26 a trimer of -476
  • a polymer a polymer of 26-476.
  • Figure 15 shows the results of SDS-PAGE of 26-476 proteins derived from different strains, wherein the lanes from left to right are: truncated protein 26-476 derived from rotavirus LLR; derived from rotavirus SA11 Truncated protein 26-476-SA11; 26-476-EDIM derived from rotavirus EDIM; truncated protein 26-476-P [8] derived from rotavirus P[8]; derived from rotavirus P[6] truncated protein 26-476-P [6]; truncated protein 26-476-P [4] derived from rotavirus P [4]; and protein molecular weight marker (Marker).
  • the method of the present invention is applicable to different rotavirus strains; the truncated VP4 protein (26-476) derived from different virus strains can be effectively expressed in Escherichia coli, and the purity thereof is purified by chromatography. Can reach more than 80%.
  • Figure 16 shows the results of molecular sieve analysis of truncated VP4 protein 26-476 derived from different strains.
  • the abscissa axis represents the retention time, and the ordinate axis represents the absorbance value at 280 nm.
  • the results showed that under the condition of TB8.0, 26-476 proteins derived from different strains were mainly in the form of trimers (peak time was about 13-14 minutes), and their contents were all above 80%. This indicates that under the condition of TB8.0, 26-476 proteins derived from different strains have good homogeneity.
  • Figure 17 shows each truncated protein (26-476-P[4], 26-476-P[6], 26-476-P[8], 26-476-EDIM, 26-476-SA11, and derived from LLR 26-476) results of indirect ELISA analysis of immune sera obtained by immunizing Balb/c mice with corresponding truncated proteins, wherein the abscissa indicates the virus strain from which the truncated protein used to prepare the immune serum is derived, The ordinate indicates the maximum dilution factor (ie, antibody titer) of the immune serum reactive with the corresponding truncated protein; P[4]: 26-476-P[4]; P[6]: 26-476-P [ 6]; P[8]: 26-476-P [8]; SA11: 26-476-SA11; EDIM: 26-476-EDIM; LLR: 26-476 prepared in Example 4.
  • the abscissa indicates the virus strain from which the truncated protein used to
  • Figure 18 shows neutralizing antibodies of immune sera induced in Balb/c mice by 26-476 protein (26-476-SA11, 26-476-EDIM, 26-476 derived from LLR) derived from different strains
  • 26-476 protein (26-476-SA11, 26-476-EDIM, 26-476 derived from LLR) derived from different strains
  • the analysis result of the titer wherein the abscissa indicates the virus strain from which the protein sample for preparing the immune serum is derived; the ordinate indicates the maximum dilution factor (NT 50 , neutralizing antibody titer) of the immune serum which reaches 50% inhibition rate of infection.
  • mice The results showed that 26-476 of SA11, EDIM and LLR strains induced high titer neutralizing antibodies in mice on day 42 after immunization (after three immunizations) using aluminum adjuvant.
  • the neutralizing antibody titer (NT 50 ) can reach a level of 2 10 -2 14 or higher; and the neutralizing antibody titers induced by 26-476-SA11 and 26-476-EDIM are even higher than LLR 26-476.
  • Figure 19A shows diarrhea scores for 1-7 days after challenge with SA11 virus in different immunized groups (immunized with 26-476-SA11 or PBS (NC, negative control);
  • Figure 19B shows different immunization groups Diabetes scores 1-12 days after challenge with EDIM virus in mice (immunized with 26-476-EDIM or PBS (NC, negative control); where abscissa indicates days after challenge and ordinate indicates mean diarrhea score.
  • the results showed that similar to 26-476 derived from LLR, the 26-476 proteins derived from SA11 and EDIM were significantly protective and could help mice resist rotavirus infection and diarrhea caused by rotavirus infection.
  • Figure 19C shows the amount of virus in a stool suspension sample of mice immunized with 26-476-EDIM or PBS (NC, negative control) 1-7 days after challenge with EDIM, wherein the abscissa indicates the challenge The number of days after day, the ordinate indicates the copy number of the EDIM genome contained in each ml of the stool suspension sample.
  • the results showed that after the challenge, the feces of the mice immunized with PBS Significant detoxification was detected in the feces of mice immunized with 26-476-EDIM, and no detoxification was detected.
  • Sequence 7 (SEQ ID NO: 7):
  • Sequence 8 (SEQ ID NO: 8):
  • Sequence 9 (SEQ ID NO: 9):
  • Sequence 50 (SEQ ID NO: 50):
  • Sequence 57 (SEQ ID NO: 57):
  • Sequence 58 (SEQ ID NO: 58):
  • Sequence 62 (SEQ ID NO: 62):
  • Sequence 65 (SEQ ID NO: 65):
  • Sequence 68 (SEQ ID NO: 68):
  • Sequence 69 (SEQ ID NO: 69):
  • Sequence 70 (SEQ ID NO: 70):
  • Sequence 71 (SEQ ID NO: 71):
  • Sequence 73 (SEQ ID NO: 73):
  • Sequence 74 (SEQ ID NO: 74):
  • Sequence 76 (SEQ ID NO: 76):
  • Sequence 77 (SEQ ID NO: 77):
  • Sequence 78 (SEQ ID NO: 78):
  • Sequence 81 (SEQ ID NO: 81):
  • Sequence 82 (SEQ ID NO: 82):
  • Sequence 83 (SEQ ID NO: 83):
  • Sequence 84 (SEQ ID NO: 84):
  • Sequence 85 (SEQ ID NO: 85):
  • Sequence 87 (SEQ ID NO: 87):
  • Sequence 88 (SEQ ID NO: 88):
  • Sequence 90 (SEQ ID NO: 90):
  • 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, The second edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Guide to Editing Molecular Biology, Third Edition, John Wiley & Sons, Inc., 1995, or in accordance with product specifications get on.
  • 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 rotavirus SA11 strain was purchased from the China Veterinary Deposit Center; the rotavirus Wa and DS-1 strains were purchased from ATCC; the rotavirus EDIM strain was the pathogen The research institute gave a gift; the prokaryotic expression vector PTO-T7 was built independently by the laboratory; E. coli ER2566 and BL21 (DE3) were purchased from New England Biolabs; the primers used were all produced by bio-organisms (Shanghai Engineering Co., Ltd. synthesis.
  • Example 1 Study on immunogenicity and immunoprotection of truncated VP8 protein (VP8-5)
  • the truncated rotavirus VP8 protein VP8-5 (the amino acid sequence of which is shown in SEQ ID NO: 1) was expressed and purified according to the method described in Chinese patent application CN 201510165746.2. Briefly, rotavirus genomic RNA was extracted from cultures of rotavirus LLR strains and cDNA encoding VP4 protein was obtained by reverse transcription. Subsequently, the obtained cDNA was used as a template, and a gene fragment encoding VP8-5 was amplified by a PCR reaction. Then, the obtained gene fragment was used to construct a VP8-5 expression plasmid, and the expression plasmid was transformed into Escherichia coli.
  • the VP8-5 protein in the soluble fraction was collected by anion exchange chromatography, wherein the instrument system used was an AKTAexplorer 100 preparative liquid chromatography system manufactured by GE Healthcare; the chromatographic medium used was Q- sepharose-HP (GE Healthcare); the buffer used was 50 mM Tris-HCl pH 8.0 and 50 mM Tris-HCl pH 8.0, 2 M NaCl; the elution procedure was: 1000 mM NaCl eluted the heteroprotein, 50 mM NaCl eluted the protein of interest, collected The product was eluted with 50 mM NaCl. The obtained eluted product was identified by 13.5% SDS-PAGE, and the results are shown in Fig. 1. The results of Figure 1 show that the purity of the purified VP8-5 protein obtained is over 90%.
  • the Chinese patent application CN 201510165746.2 has confirmed using a mouse model that the purified VP8-5 protein has good immunogenicity and immunoprotection under the conditions of using Freund's adjuvant (see, Examples 5-8 of the application) And Figure 4-9).
  • the immunoprotective effect of the VP8-5 protein under conditions using an aluminum adjuvant was evaluated by a mouse model.
  • mice were randomly divided into 3 groups, 7 mice in each group, 2 of which were control group and 1 group was experimental group.
  • the purified VP8-5 protein, an equal dose of the inactivated virus LLR strain and PBS were mixed with the aluminum phosphate adjuvant in a ratio of 1:1, and then the mice were immunized by intramuscular injection at an immunization dose of 10 ⁇ g/mouse.
  • the experimental group mice were immunized with VP8-5, the positive control mice were immunized with the inactivated virus LLR, and the negative control mice were immunized with PBS.
  • Each group of mice was immunized three times, with each immunization separated by two weeks.
  • Mouse eye blood was collected on days 0, 14, 28 and 42 of the immunization procedure for detection of antibody titers and neutralizing antibody titers.
  • the immune serum was serially diluted and then subjected to indirect ELISA analysis with VP8-5 coated on the plate (the secondary antibody used was a goat anti-mouse antibody (Wanyumei)) to determine that it had VP8-5
  • the maximum dilution factor of reactive immune serum The greater the maximum dilution factor of the immune serum, the higher the titer of the anti-VP8-5 antibody in the immune serum, and the higher the immunogenicity of the protein used to produce the immune serum.
  • MA104 cells were plated in 96-well cell culture plates (1.9*10 4 cells/well). After 20 hours, the neutralizing antibody titer of the immune serum was detected by ELISPOT (Li, Lin, Yu, et al. J Virol Methods, 209 7-14, 2014).
  • the specific protocol is as follows: the immune serum sample to be tested (including the neutralizing antibody to be tested) is serially diluted by DMEM supplemented with trypsin; then 100 ⁇ L of each diluted sample is separately diluted with the rotavirus in DMEM.
  • 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 serum samples that achieved 50% inhibition of infection after 50-fold dilution were considered to have neutralizing ability.
  • Fig. 1C The results of the analysis of the neutralizing antibody titer of the immune serum are shown in Fig. 1C, wherein the ordinate indicates the maximum dilution factor (NT50, neutralizing antibody titer) of the immune serum which reached the 50% infection inhibition rate.
  • NT50 neutralizing antibody titer
  • the results showed that VP8-5 was able to induce neutralizing antibodies in mice on day 42 after immunization (after three immunizations) under conditions of aluminum adjuvant, but the neutralizing antibody titer (NT50) of the immune serum was lower than that. Inactivate virus LLR. After the immunization procedure was completed, the neutralizing antibody titer (NT50) of the immune serum induced by the VP8-5 protein was only about 64. The above results indicate that VP8-5 can induce the production of neutralizing antibodies in the body under the condition of using aluminum adjuvant, but the neutralizing antibody titer of the induced immune serum is lower than that of the inactivated virus LLR.
  • each group of mice was mated in proportion to one male mouse per male mouse. 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. After the challenge, the diarrhea of the suckling rats was observed and recorded every day for 7 days and scored according to the morphology of the feces. The scoring criteria are shown in Figure 2. According to the different degrees of diarrhea in suckling rats, the score is divided into 3 grades: 1 point for normal feces (Fig. 2C), 2 points for soft stools (Fig. 2B), and 3 points for unformed water samples (Fig. 2A) .
  • Figure 3 shows the diarrhea scores of the suckling mice in different immunization groups after challenge, in which the vertical axis represents the diarrhea score and the horizontal axis represents the number of days after the mice were challenged.
  • the results showed that there was no significant difference in the diarrhea score between the experimental group (VP8-5) and the negative control group (NC). This indicates that under the conditions of using aluminum adjuvant, VP8-5 has a lower ability to protect against rotavirus infection (below inactivated virus) and cannot fully alleviate diarrhea caused by rotavirus infection.
  • Example 2 Construction of an expression plasmid encoding a truncated VP4 protein
  • Rotavirus LLR and SA11 strains were cultured with Rhesus embryonic kidney cell line (MA-104) cells.
  • 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 rotavirus genomic RNA was extracted using the viral DNA/RNA extraction kit produced by Beijing Jinmaige Biotechnology Co., Ltd. according to the manufacturer's instructions, and the cDNA encoding the LLR strain VP4 protein was obtained by reverse transcription.
  • the obtained cDNA was used as a template, and a gene fragment encoding the truncated rotavirus LLR strain VP4 protein was amplified by a PCR reaction.
  • PCR primers used are as follows:
  • the cleavage site is underlined, and the introduced stop codon is indicated in italics.
  • the gene encoding the truncated VP4 protein was amplified by PCR using the above primers, and the PCR reaction system used was 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, 80 s; 72 ° C, 1 min), and a final extension of 72 ° C for 10 min.
  • the obtained amplification product was detected by 1.5% agarose gel electrophoresis.
  • Each PCR amplification product was ligated into the pMD18-T vector and transformed into E. coli DH5 ⁇ . Then, positive colonies were screened, and plasmids were extracted and identified by Nde I/Hind III digestion to obtain a positive clone plasmid into which the gene fragment of interest was inserted. Each positive clone plasmid obtained was sequenced. 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 NO: 2-34.
  • the above positive cloned plasmids were digested with Nde I/Hind III, respectively, to obtain gene fragments encoding the truncated VP4 proteins, 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 DH5 ⁇ . Then, the positive colonies were screened, and the plasmid was extracted, and the positive expression plasmid inserted into the gene fragment of interest was identified by NdeI/HindIII digestion.
  • Example 3 Expression of truncated VP4 protein
  • the Escherichia coli carrying the positive expression plasmid prepared in Example 2 was taken out from -70 ° C, inoculated into 50 ml of LB liquid medium containing kanamycin, and cultured at 180 rpm, 37 ° C for about 4 hours; Access to 10 bottles of 500 ml LB medium containing kanamycin (500 ul of bacteria per vial). When the absorbance of the culture at a wavelength of 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.
  • the E. coli bacterial solution obtained in Example 3 was centrifuged, and the bacterial cell pellet was collected. According to 15ml / g The proportion of wet bacteria was weighed with 50 mM TB8.0 expressing the truncated VP4 protein. Then, the E. coli cells were disrupted by sonication, and the ultrasonication conditions were as follows: ultrasonic 2 s, interval 4 s, and ultrasonic pulverization time per gram of bacteria was 4 min. After sonication, centrifugation was carried out at 25000 g, and the supernatant (i.e., the E. coli lysate soluble fraction containing the recombinantly expressed truncated VP4 protein) was collected.
  • the supernatant i.e., the E. coli lysate soluble fraction containing the recombinantly expressed truncated VP4 protein
  • the truncated VP4 protein in the soluble fraction of E. coli lysate can be purified by two-step chromatography.
  • the E. coli lysate soluble grade was treated with 40% ammonium sulphate prior to the two-step chromatography.
  • the fractions were then centrifuged and protein precipitates were collected; subsequently, the obtained protein precipitate was dissolved in 50 mM Tris-HCl pH 8.0 and applied to two-step chromatography.
  • the scheme for the two-step chromatography is as follows.
  • AKTA explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatography medium Q-sepharose-HP (GE Healthcare).
  • Buffer A pump: 50 mM Tris-HCl pH 8.0;
  • the sample was a previously prepared supernatant containing recombinantly expressed truncated VP4 protein (i.e., E. coli lysate soluble fraction or protein sample dissolved in 50 mM Tris-HCl pH 8.0).
  • recombinantly expressed truncated VP4 protein i.e., E. coli lysate soluble fraction or protein sample dissolved in 50 mM Tris-HCl pH 8.0.
  • the elution procedure was: elution of the protein of interest with 50 mM NaCl and elution of the heteroprotein with 1 M NaCl.
  • a fraction eluted with 50 mM NaCl was collected to obtain a total of 30 mL of a preliminary purified sample containing the recombinantly expressed truncated protein (Note: during the above preliminary purification, truncated proteins 1-476, 26-331, 26-351 , 26-381, 26-411, 26-441, and 26-461 are not combined with the column and are included in the flow-through. Therefore, the collection contains this The penetrating fraction of these truncated proteins was used as a preliminary purified sample).
  • Chromatography medium Phenyl sepharose-HP (GE Healthcare).
  • Buffer A pump: 50 mM Tris-HCl pH 8.0, 2 M NaCl;
  • the sample is a product purified by Q-HP column and dialyzed into 2M NaCl solution.
  • the elution procedure was as follows: 1.5 M NaCl eluted the heteroprotein, 1 M NaCl eluted the target protein, and 50 mM NaCl eluted the heteroprotein. A fraction eluted with 1 M NaCl was collected to obtain a total of 30 mL of purified recombinantly expressed truncated VP4 protein (Note: during the second purification described above, truncated protein 1-476, 26-331, 26-351 26-381, 26-411, 26-441, 26-461, and 26-471 were eluted with 50 mM TB8.0, and fractions eluted with 50 mM TB8.0 were collected).
  • Figures 5A-5B show SDS-PAGE results of various truncated VP4 proteins purified.
  • the lanes from left to right are: Protein Molecular Markers (Marker), 1-476, 6-476, 22-476, 26-476, 65-476, 26-231, 26-271, 26- 331 and 26-351.
  • the lanes from left to right are: Protein Molecular Markers (Marker), 26-381, 26-411, 26-441, 26-461, 26-471, 26-482, 26-487, 26- 492 and 26-497.
  • Figures 5A and 5B show truncated proteins 1-476, 6-476, 22-476, 26-476, 65-476, 26-231, 26-271, 26-331, 26 after the above purification steps. -351,26-381,26-411,26-441,26-461,26-471,26-482,26-487, The concentrations of both 26-492 and 26-497 were both above 0.2 mg/ml and the purity was greater than 80%.
  • the homogeneity of the above purified samples under different buffer conditions was also analyzed using HPLC.
  • the instrument used was an Agilent 1200 high performance liquid chromatograph with a G3000 PWXL or G5000 PWXL column with a column volume of 7.8*300 mm, a flow rate of 0.5 ml/min and a detection wavelength of 280 nm; 26-331,26-
  • the homogeneity of 351, 26-381, 26-411, 26-441 and 26-461 was tested with G5000 PWXL ; other proteins were tested using G3000 PWXL .
  • the results of the analysis by SEC-HPLC are shown in Fig. 6 and Fig. 7.
  • the retention time of the truncated protein 26-271 was about 16 min; this indicates that the protein is predominantly present as a monomer.
  • the remaining proteins (6-476, 22-476, 26-476, 26-471, 26-482, 26-487, 26-492, 26-497) have retention times of approximately 13-14 min, with IgG (150 kDa) The retention time is comparable; this indicates that these proteins are mainly present in the form of trimers.
  • Example 5 In vitro assembly and characterization of truncated VP4 protein
  • truncated protein 26-476 was dialyzed from TB8.0 buffer to the dialysis buffer specified in Table 4 at room temperature, and the dialysis buffer was replaced once after 6 hours. After the end of the dialysis, the protein solution was centrifuged at 12,000 rpm/min for 10 minutes, and the supernatant was taken. Subsequently, the supernatant was allowed to stand at the temperature specified in Table 4 for 30 minutes to 24 hours. After standing, the supernatant was rapidly ice-cooled and centrifuged at 12,000 rpm/min for 10 minutes. The supernatant after the second centrifugation (which contains in vitro assembled 26-476) was collected for further analysis.
  • the homogeneity of the polymer formed by in vitro assembly of the truncated protein 26-476 in the obtained supernatant was analyzed by HPLC.
  • the HPLC analysis instrument was a 1200 high performance liquid chromatograph produced by Agilent or an E2695 high performance liquid chromatograph produced by Waters.
  • the column was G5000 PWXL , the column volume was 7.8*300 mm, the flow rate was 0.5 ml/min, and the detection wavelength was It is 280 nm.
  • the results of the analysis by SEC-HPLC are shown in Table 4 and Figure 8A.
  • Fig. 8A shows the results of molecular sieve analysis of the truncated protein 26-476 after standing at 37 ° C for 12 hours in 50 mM Tris-HCl (pH 8.0). The results showed that the truncated protein 26-476 was able to form a uniform polymer (retention time of 12.4 minutes) after standing at 37 ° C for 12 hours in 50 mM Tris-HCl (pH 8.0), and the proportion of high polymer was as high as 97.2. %.
  • the truncated protein 26-476 in the range of pH 7.4-9.6, can be assembled to form a uniform polymer after standing at a temperature of 37 ° C to 50 ° C. And the proportion of high polymers can exceed 90%. Furthermore, the results in Table 4 also show that the presence of a salt ion (e.g., NaCl) inhibits the formation of high polymers by the truncated protein 26-476. The higher the concentration of salt ions (e.g., NaCl), the lower the proportion of polymer formed.
  • a salt ion e.g., NaCl
  • the purified truncated VP4 protein obtained in Example 4 was digested with trypsin for 1 hour at 37 °C. 100 ⁇ l of the digested fraction was added, 20 ⁇ L of 6X Loading Buffer was added, mixed and incubated for 10 min in a 100 ° C water bath. Subsequently, 10 ⁇ l was electrophoresed in a 13.5% SDS-polyacrylamide gel at 120 V for 120 min; then the electrophoresis band was visualized by Coomassie blue staining. The electrophoresis results are shown in Fig. 9.
  • Figure 9 shows the results of SDS-PAGE of the cleaved and uncut truncated proteins 26-476, 26-482, 26-487, 26-492, 26-497.
  • the number 1 on the lane indicates that the sample was not trypsinized; the number 2 indicates that the sample was treated with 0.1 mg/ml trypsin.
  • the sample used in the rightmost lane was VP8-5 and used as a control.
  • the results in Figure 9 show that the above truncated proteins are recognized by pancreatin and cleaved, and the cleavage sites are exposed.
  • Example 7 Antigenicity analysis of truncated VP4 protein
  • the purified truncated VP4 protein obtained in Example 4 was coated on a plate to obtain a coated plate.
  • Neutralizing antibodies A3, B1, B5, B6, D6, E2, E5, 8F6 (this laboratory was prepared by hybridoma technique, the concentration was 1 mg/ml), and then diluted in accordance with the indirect ELISA method described in Example 1. Test.
  • Example 8 Analysis of immunogenicity of truncated VP4 protein
  • the purified truncated protein 26-476 obtained in Example 4 was coated on a plate to obtain a coated plate.
  • each sample to be tested (various truncated VP4 protein obtained in Example 4, trimer of 26-476, obtained in Example 5) was obtained under the conditions of using an aluminum adjuvant.
  • Figures 11A-11D The results of the indirect ELISA are shown in Figures 11A-11D, wherein the abscissa indicates the protein sample used to prepare the immune serum, and the ordinate indicates the maximum dilution factor (i.e., antibody titer) of the immune serum reactive with 26-476. And, Figures 11A, 11B, 11C, and 11D show results from different immunization batches.
  • each protein sample can induce antibodies in mice (the antibody titer (GMT) in immune serum can reach 10 2 -10 5 or Higher); and, except for 26-271, antibody titers induced by other protein samples were higher than RV-induced antibody titers (1-476, 6-476, 22-476, 26-331, 26-351) , trimers of 26-381, 26-411, 26-441, 26-461, 26-471, 26-476, 26-487, 26-492, 26-476 and high polymers of 26-476), Or at least comparable to RV-induced antibody titers (65-476, 26-482, and 26-497).
  • each protein sample (except 26-271) has good immunogenicity and can stimulate mice to produce high titer antibodies;
  • Example 9 Analysis of immunological neutralizing activity of truncated VP4 protein
  • each sample to be tested (various truncated VP4 proteins obtained in Example 4, trimers of 26-476, highmers of 26-476 obtained in Example 5, Inactivated virus (RV, used as a positive control) and PBS (NC, negative control))
  • the experimental group Balb/c mice (7 mice per group) were immunized and immune sera were collected.
  • FIGS 12A-12D The results of the analysis of the neutralizing antibody titer of the immune serum are shown in Figures 12A-12D, wherein the abscissa indicates the protein sample used to prepare the immune serum, and the ordinate indicates the maximum dilution factor of the immune serum that achieves 50% infection inhibition rate (NT). 50 , neutralizing antibody titer).
  • Figures 12A, 12B, 12C and 12D show the results from different immunization batches.
  • each protein sample can induce neutralizing antibodies in mice, and the neutralizing antibody titer (NT 50 ) can reach 2 8 -2 14 or higher; and, except for 26-271, the neutralizing antibody titers induced by other protein samples are comparable to RV-induced neutralizing antibody titers (6-476, 22-476, Trimers of 26-476, 65-476, 26-471, 26-482, 26-487, 26-492, 26-497, and 26-476), or even higher than RV-induced neutralizing antibody titers ( High polymers of 1-476, 26-331, 26-351, 26-381, 26-411, 26-441, 26-461 and 26-476).
  • each protein sample except 26-271 has a strong ability to induce the production of neutralizing antibodies, and can induce high neutralizing antibodies in animals.
  • a titer of immune serum that is effective to inhibit rotavirus infection.
  • Each protein sample except 26-271) induced the body to produce neutralizing antibodies significantly better than VP8-5, thus having a stronger ability to resist/prevent RV infection.
  • the experimental results of Figure 12D also showed that the high polymer of truncated protein 26-476 induced the body to produce neutralizing antibodies significantly higher than the 26-476 trimer (p ⁇ 0.001), with significantly stronger resistance. / Ability to prevent RV infection.
  • Example 10 Evaluation of the protective properties of truncated VP4 protein in animals
  • each sample to be tested (the truncated VP4 protein obtained in Example 4, the trimer of 26-476, the high polymer of 26-476 obtained in Example 5, and the like) were used.
  • Figures 13A-13D show 1-7 days after challenge from different immunization groups (using 22-476, 26-476, 65-476, 26-271, 26-331, 26-351, 26-381, 26-411 , 26-441, 26-461, 26-471, 26-482, 26-487, 26-492, 26-497, 26-476 trimer, 26-476 polymer, inactivated wheel Diarrhea scores in suckling mice (RV, positive control) or PBS (NC, negative control), where the vertical axis represents the mean diarrhea score; the horizontal axis represents the number of days after challenge in mice; RV: inactivated Rotavirus; NC: negative control (PBS); trimer: trimer of 26-476; polymer: polymer of 26-476.
  • RV positive control
  • NC negative control
  • trimer trimer of 26-476
  • polymer polymer of 26-476.
  • Figures 14A-14D show different immunization groups (using 22-476, 26-476, 65-476, 26-271, 26-331, 26-351, 26-381, 26-411, 26-441, 26-461 a trimer of 26-471, 26-482, 26-487, 26-492, 26-497, 26-476, a polymer of 26-476, an inactivated rotavirus (RV, positive control) or Mean diarrhea duration after challenge and diarrhea score of 48 hours after challenge in PBS (NC, negative control), with mean diarrhea days in a bar graph and mean diarrhea score as a graph
  • the left vertical axis represents the average number of diarrhea days; the right vertical axis represents the diarrhea score; and the horizontal axis represents the immune group corresponding to each protein sample.
  • Example 11 Expression and purification of truncated VP4 protein derived from different virus strains And evaluation of its immunoprotective properties
  • VP4 gene sequence of EDIM strain provided in Gene bank (accession number: AF039219.2)
  • a gene fragment encoding 26-476 derived from the rotavirus EDIM strain was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.
  • 26-derived from rotavirus P[6] was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.
  • Gene fragment of 476 was amplified by PCR reaction.
  • the rotavirus SA11 strain was cultured with a rhesus embryonic kidney cell line (MA-104) to obtain a virus culture of rotavirus SA11.
  • Rotavirus P[4] and P[8] were obtained from diarrhea specimens collected by the Children's Hospital of Chongqing Medical University. The specimen numbers were 20131281 (P[4]) and No. 20131028 (P[8]).
  • RNA, and cDNA encoding VP4 protein derived from each strain was obtained by reverse transcription.
  • the obtained cDNA was used as a template, and a gene fragment encoding the truncated protein 26-476 derived from the strains SA11, P[4], and P[8] was amplified by a PCR reaction.
  • the cloning plasmid and the expression plasmid were constructed as described in Example 2 using the following PCR primers:
  • the cleavage site is underlined, and the introduced stop codon is indicated in italics.
  • amino acid sequences of the truncated proteins 26-476-P[4], 26-476-P[6], 26-476-P[8], 26-476-EDIM, 26-476-SA11 are respectively SEQ ID NO: 35-39.
  • the truncated protein 26-476 derived from different strains was expressed in E. coli according to the method described in Example 3-4 (ie, 26-476-P[4], 26-476-P[6], 26 -476-P[8], 26-476-EDIM, 26-476-SA11), and purification by two-step chromatography; then the purified protein was identified by SDS-PAGE.
  • the truncated VP4 protein (26-476) derived from different strains can be efficiently expressed in E. coli, and the purity can reach 80% or more after purification by chromatography.
  • the results of Fig. 16 also show that the ratio of the main absorption peaks of the obtained truncated proteins 26-476 is almost 80% or more; this indicates that these truncated proteins have high homogeneity and are suitable for industrial batches. Production, and is conducive to accurately determine the dosage.
  • truncated proteins 26-476 derived from different rotavirus strains were individually coated on a plate to obtain coated plates.
  • Purified truncated protein 26-476 obtained as above ie, 26-476-P[4], 26-476-P[6], 26-476-P[8] was used according to the protocol described in Example 1.
  • 26-476-EDIM, 26-476-SA11, 26-476 from LLR and PBS (negative control) to immunize Balb/c mice and collect mouse serum.
  • antibody titers in mouse sera were detected by indirect ELISA using coated plates according to the protocol described in Example 1.
  • Fig. 17 The results of the indirect ELISA are shown in Fig. 17, in which the abscissa indicates the virus strain from which the truncated protein used to prepare the immune serum is derived, and the ordinate indicates the maximum dilution factor of the immune serum reactive with the corresponding truncated protein ( Namely, antibody titer); P[4]: 26-476-P[4]; P[6]: 26-476-P[6]; P[8]: 26-476-P[8]; SA11 : 26-476-SA11; EDIM: 26-476-EDIM; LLR: 26-476 prepared in Example 4.
  • the experimental group Balb/c was immunized with 26-476 protein (26-476-SA11; 26-476-EDIM; 26-476 derived from LLR) derived from different strains. Mice (7 mice per group) and immune sera were collected. Subsequently, the neutralizing antibody titer in each of the collected immune serum samples was evaluated in accordance with the detection method described in Example 1. The results of the analysis of the neutralizing antibody titer of the immune serum are shown in Fig. 18, wherein the abscissa indicates the virus strain from which the protein sample for preparing the immune serum is derived; the ordinate indicates that the immune serum having the 50% infection inhibition rate is the largest. Dilution factor (NT 50 , neutralizing antibody titer); SA11: 26-476-SA11; EDIM: 26-476-EDIM; LLR: 26-476 prepared in Example 4.
  • NT 50 neutralizing antibody titer
  • Figure 19A shows diarrhea scores for 1-7 days after challenge with SA11 virus in different immunized groups (immunized with 26-476-SA11 or PBS (NC, negative control);
  • Figure 19B shows different immunization groups (Figure 19B) The diarrhea score of 1-12 days after challenge with the EDIM virus in the suckling mice immunized with 26-476-EDIM or PBS (NC, negative control);
  • the coordinates indicate the number of days after the attack and the ordinate indicates the average diarrhea score.
  • the results showed that similar to 26-476 derived from LLR, the 26-476 proteins derived from SA11 and EDIM were significantly protective and could help mice resist rotavirus infection and diarrhea caused by rotavirus infection.
  • mice were also immunized (co-immunized three times) with 26-476-EDIM or PBS (NC, negative control) according to the protocol described in Example 1.
  • the mice were challenged with 500 ⁇ L of EDIM virus (2*10 7 copies/ml), and the stool samples of the mice were collected daily 1-7 days after challenge and resuspended in PBS to 1%. Fecal suspension. Subsequently, the virus in each stool suspension sample was quantitatively detected by real-time PCR. The experimental results are shown in Figure 19C.
  • Figure 19C shows the amount of virus in a stool suspension sample from mice immunized with 26-476-EDIM or PBS 1-7 days after challenge, wherein the abscissa indicates the number of days after challenge and the ordinate indicates each The copy number of the EDIM genome contained in the ml stool suspension sample. Since the detection limit of the PCR assay kit is 10 4 copies/ml, the negative test result is defined as 10 3 copies/ml. The results showed that after the challenge, significant detoxification was detected in the feces of mice immunized with PBS, whereas in the feces of mice immunized with 26-476-EDIM, no detoxification was detected. The results of Figures 19A-19C show that 26-476-EDIM not only enables mice to resist rotavirus infection and diarrhea caused by rotavirus infection, but also inhibits the release of virus in mouse feces (i.e., detoxification).

Abstract

一种截短的轮状病毒VP4蛋白，其编码序列和制备方法，包含所述蛋白的药物组合物和疫苗，所述蛋白、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。

Description

截短的轮状病毒VP4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VP4蛋白，其编码序列和制备方法，包含所述蛋白的药物组合物和疫苗，所述蛋白、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。本发明还涉及上述蛋白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(rotavirus,RV)属于呼肠孤病毒科，轮状病毒属，是引起婴幼儿腹泻的主要病原体，在1973年由Bishop在胃肠炎病人的十二指肠中发现(Bishop,Davidson,Holmes,et al.Lancet,2(7841),1281-1283,1973)。研究表明，95％以上的儿童在5岁前至少感染过一次轮状病毒。据WHO统计，每年因轮状病毒感染而导致的死亡病例高达60万，而腹泻病例则高达2亿；仅在美国，每年因轮状病毒感染而造成的经济损失就高达10亿美元(Hsu,Staat,Roberts,et al.Pediatrics,115(1),78-82,2005；Tate,Burton,Boschi-Pinto,et al.The Lancet Infectious Diseases,12(2),136-141,2011)，这造成了严重的经济负担和社会负担。
轮状病毒为无包膜RNA病毒，其基因组由11条双链RNA组成，分别编码6种结构蛋白(VP1-VP4，VP6和VP7)和6种非结构蛋白(NSP1-NSP6)(Estes and Cohen.Microbiol Rev,53(4),410-449,1989)。轮状病毒为二十面体对称，其衣壳由三个同心层构成，即由VP1、VP2和VP3构成的核心层，由VP6构成的内衣壳，以及由VP4和VP7构成的外衣壳。
VP6蛋白是轮状病毒中含量最高的衣壳蛋白。根据VP6的抗原性，可以将轮状病毒分为A-G 7个组，其中A组轮状病毒是引起婴幼儿腹泻的主要病原体。VP4和VP7为主要中和抗原，根据二者抗原性的不同可将A组轮状病毒分为不同的P血清型和G血清型。G血清型和P血清型相互独立又相互影响，常见的组合包括G1P[8]，G2P[4]，G3P[8]和G4P[8]。近年来，G9P[8]和G9P[6]的流行显著增加(Li,Liu,Yu,et al.Vaccine,27 F40-F45,2009)。
目前尚无针对轮状病毒的特效药。安全有效的疫苗是控制轮状病毒感染的重要手段。经过多年的研究，疫苗开发历经单价减毒疫苗，多价基因重配疫苗和基因工程疫苗三个阶段。目前已有五种轮状病毒疫苗相继上市，包括惠氏的四价人-猿基因重配疫苗，兰州所的单价减毒疫苗、默克的五价人-牛基因重配疫苗、葛兰素史克的单价减毒疫苗和印度巴拉特的单价减毒疫苗。然而，这些疫苗均为减毒活疫苗，存在较大的安全隐患，其中：惠氏的疫苗由于肠套叠问题在上市半年后即被撤回(Murphy,Gargiullo,Massoudi,et al.344(8),564-572,2001)；默克和葛兰素史克的疫苗尽管经过大量的临床实验证实其安全性和有效性(Bernstein,Sack,Rothstein,et al.Lancet(British edition),354(9175),287-290,1999；Vesikari,Matson,Dennehy,et al.New England Journal of Medicine,354(1),23-33,2006；Linhares,Velázquez,Pérez-Schael,et al.Lancet,371(9619),1181-1189,2008；Vesikari,Itzler,Karvonen,et al.Vaccine,28(2),345-351,2009；Snelling,Andrews,Kirkwood,et al.Clinical Infectious Diseases,52(2),191-200,2011)，但其在亚洲和非洲等轮状病毒死亡率较高的国家和地区的保护效率远低于欧美等发达国家(Armah,Sow,Breiman,et al.Lancet,376 606-614,2010；Zaman,Anh,Victor,et al.Lancet,376 568-570,2010；Madhi,Cunliffe,Steele,et al.N Engl J Med,362(4),289-298,2011)，并且越来越多的证据表明，二者接种后均产生排毒并可导致病毒的水平传播(Anderson.Lancet Infect Dis,8(10),642-9,2008；Rivera,Pena,Stainier,et al.Vaccine,29(51),9508-9513,2011；Yen, Jakob,Esona,et al.Vaccine,29(24),4151-5,2011)，并且还有研究表明，免疫缺陷儿童接种疫苗后可产生严重的胃肠炎(Steele,Cunliffe,Tumbo,et al.J Infect Dis,200 Suppl 1 S57-62,2009；Patel,Hertel,Estes,et al.N Engl J Med,362(4),314-9,2010)；兰州所的疫苗上市十多年，至今尚未发现严重问题，但仅能预防严重腹泻，而不能预防轮状病毒的感染(Fu,Wang,Liang,et al.Vaccine,25(52),8756-61,2007)。因此，尽管轮状病毒减毒疫苗的研究取得了可喜的结果，但也还存在一定的问题，其安全性和有效性还有待进一步提高；发展更加安全、有效的疫苗势在必行。
非复制型疫苗是目前轮状病毒疫苗研究的主要方向，其中基因工程疫苗由于成本低，安全、有效等特点而备受关注。
VP4和VP7蛋白作为轮状病毒的中和抗原，均可刺激机体产生中和抗体，从而抑制轮状病毒的感染。因此，VP4和VP7均是轮状病毒亚单位疫苗的主要候选抗原。VP7蛋白为糖基化蛋白，含有4对链内二硫键，形成Ca2+依赖性的三聚体，重组表达的VP7蛋白刺激机体产生的中和抗体水平较低。VP4蛋白无糖基化修饰，重组表达的VP4蛋白则可以刺激机体产生较高的免疫应答，并降低乳鼠腹泻的水平(Mackow,Vo,Broome,et al.J Virol,64(4),1698-703,1990)。另一方面，常见的P血清型仅2种，而常见的G血清型有5种。因此，相比于VP7蛋白，VP4蛋白更适合作为轮状病毒基因工程疫苗的候选抗原。
VP4蛋白由776个氨基酸组成，可以被胰酶酶切形成VP8*(aa1-231)和VP5*(aa248-776)两部分。VP4蛋白由头部、颈部和基座三个部分构成，其作为轮状病毒的刺突蛋白，在轮状病毒的感染中发挥着重要的作用。刺突的头部由两个分子的VP8蛋白组成，VP8蛋白可以与细胞表面的唾液酸受体结合，从而介导轮状病毒的吸附过程。颈部由三个分子的VP5抗原区组成，其中两个分子的VP5抗原区形成二聚体，另一个VP5抗原区以单体形式存在。在轮状病毒感染过程中，VP4的结构发生重排，暴露出VP5膜融合位点并介导轮状病 毒的入胞过程；在此过程中，VP5抗原区由二聚体转变为三聚体。基座部分由三个分子的VP5蛋白C端结构域组成，并且通过该结构域插入外衣壳和内衣壳。VP8蛋白的N端25个氨基酸形成α螺旋，三个α螺旋形成螺旋束插入VP5基座部分，并通过一段柔性连接区与VP8头部相连，该连接区的结构目前尚未完全解析(Settembre,Chen,Dormitzer,et al.EMBO J,30(2),408-16,2011)。
研究表明，VP4蛋白的中和表位主要位于VP8头部和VP5抗原区，针对VP8的中和抗体可以抑制轮状病毒的吸附过程，针对VP5的中和抗体具有交叉中和活性，可以抑制轮状病毒的入胞(Dormitzer,Nason,Venkataram Prasad,et al.Nature,430(7003),1053-1058,2004；Abdelhakim,Salgado,Fu,et al.PLoS Pathog,10(9),e1004355,2014)。同时，合成肽扫描的结果表明，VP8蛋白的N端α螺旋区和连接区也存在中和表位(Kovacs-Nolan,Yoo and Mine.Biochem J,376(Pt 1),269-75,2003)。
大量研究结果表明，VP4蛋白可以刺激机体产生保护性免疫应答(Dunn,Fiore,Werner,et al.Arch Virol,140(11),1969-78,1995；Gil,De Souza,Asensi,et al.Viral Immunology,13(2),187-200,2000)。然而，通过真核系统来表达VP4蛋白的周期长、成本高而且表达量低；并且，全长的VP4蛋白在原核系统中主要以包涵体形式表达，难以纯化且无法维持其天然构象。闻小波等人通过截短表达的方式解决了VP8蛋白在原核系统中以包涵体形式表达的问题，获得了在大肠杆菌中能够以可溶形式高效表达的截短VP8蛋白(△VP8*，aa65-223)(Wen,Cao,Jones,et al.Vaccine,30(43),6121-6,2012)；但是，该截短VP8蛋白的免疫原性显著低于全长的VP8蛋白。为了解决截短VP8蛋白免疫原性较低的问题，我们实验室在进行了深入研究后，获得了免疫原性更高的新的VP8截短蛋白，其在弗氏佐剂条件下免疫小鼠后，可以刺激小鼠产生高滴度的中和抗体并可以降低子代乳鼠的腹泻水平；并且，其可以与分子内佐剂进行融合表达，产生免疫原性和免疫保护性更高的融合蛋白(CN201510165746.2)。尽管该新的VP8 截短蛋白及其融合蛋白已实现了显著有利的技术效果，但其也仍然存在着不足。例如，针对VP8蛋白的抗体主要是血清型特异性抗体，因此，该新的VP8截短蛋白用作疫苗时对异型毒株的中和活性显著低于同型毒株(Wen,Cao,Jones,et al.Vaccine,30(43),6121-6,2012)。此外，后续的研究还发现，该新的VP8截短蛋白在铝佐剂条件下，免疫保护性不够理想，有待进一步提高。
因此，本领域仍然需要开发新的针对轮状病毒的疫苗，其能够方便地、高表达量地产生(例如通过可溶性表达和纯化的方法)，能够比现有的疫苗(例如上述VP8截短蛋白)具有更强的免疫原性，能够诱导机体产生高滴度的针对轮状病毒的中和抗体(特别是在铝佐剂条件下)，从而使得能够大规模工业化生产高效的抗轮状病毒疫苗。
发明内容
本申请的发明人经过大量研究后出人意料地发现：N端截短了1-64个氨基酸(例如，5-64个氨基酸)，且C端终止于氨基酸位置276-497之间的VP4蛋白能够在大肠杆菌中以可溶形式表达，且能够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％)具有良好的均一性和免疫原性，且能够在铝佐剂的条件下，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1-64个氨基酸(例如5-64个氨基酸)，且C端终止于氨基酸位置276-497之间的轮状病毒VP4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VP4蛋白或其变体，其与野生型轮状病毒VP4蛋白相比，N端截短了1-64个氨基酸(例如5-64个氨基酸)，并且C端终止于野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置276-497中的任一位置相对 应的位置。
在某些优选的实施方案中，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白的N端截短了1-64个氨基酸，例如1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、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、60个、61个、62个、63个或64个氨基酸(例如，1-5个，5-25个，5-21个，21-25个，21-64个，或25-64个氨基酸，例如1个、5个、21个、25个、或64个氨基酸)；并且C端终止于野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置276-497(例如氨基酸位置281-497、291-497、301-497、311-497、321-497、331-497、341-497、351-497、361-497、371-497、381-497、391-497、401-497、411-497、421-497、431-497、441-497、451-497、461-497、471-497、476-497、482-497、487-497、或492-497)中的任一位置相对应的位置，例如C端终止于下述位置：与SEQ ID NO:40的氨基酸位置276、281、291、301、311、321、331、341、351、361、371、381、391、401、411、421、431、441、451、461、471、476、482、487、492或497相对应的位置(例如，与SEQ ID NO:40的氨基酸位置331、351、381、411、441、461、471、476、482、487、492或497相对应的位置)。
在某些优选的实施方案中，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白的N端截短了1个、5个、21个、25个、或64个氨基酸，并且C端终止于野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置276、281、291、301、311、321、331、341、351、361、371、381、391、401、411、421、431、441、451、461、471、476、482、487、492或497相对应的位置(例如，与SEQ ID NO:40的氨基酸位置331、351、381、411、441、461、471、 476、482、487、492或497相对应的位置)。
在某些优选的实施方案中，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白具有选自下列的一个特征：
(1)N端截短了25个氨基酸且C端终止于下述位置：与SEQ ID NO:40的氨基酸位置276、281、291、301、311、321、331、341、351、361、371、381、391、401、411、421、431、441、451、461、471、476、482、487、492或497相对应的位置(优选地，与SEQ ID NO:40的氨基酸位置331、351、381、411、441、461、471、476、482、487、492或497相对应的位置)；
(2)N端截短了1个氨基酸且C端终止于与SEQ ID NO:40的氨基酸位置476相对应的位置；
(3)N端截短了5个氨基酸且C端终止于与SEQ ID NO:40的氨基酸位置476相对应的位置；
(4)N端截短了21个氨基酸且C端终止于与SEQ ID NO:40的氨基酸位置476相对应的位置；和
(5)N端截短了64个氨基酸且C端终止于与SEQ ID NO:40的氨基酸位置476相对应的位置。
在某些优选的实施方案中，所述野生型轮状病毒VP4蛋白是来源于轮状病毒LLR株、SA11株、EDIM株的VP4蛋白，或者是来源于P[4]、P[6]、或P[8]基因型轮状病毒的VP4蛋白；
在某些优选的实施方案中，所述野生型轮状病毒VP4蛋白具有选自下列的氨基酸序列：SEQ ID NO:40和87-91。
在某些优选的实施方案中，所述截短的轮状病毒VP4蛋白(以下也简称为截短蛋白)具有选自下列的氨基酸序列：SEQ ID NO:2-5和10-39。
在另一个方面，本发明涉及编码上述截短蛋白或其变体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。然而，特别优选地，本发明的宿主细胞是原核细胞例如大肠杆菌细胞。
在另一个方面，本发明还涉及一种多聚体，其包含上述截短蛋白或其变体或者由上述截短蛋白或其变体组成。在某些优选的实施方案中，所述多聚体为三聚体。在某些优选的实施方案中，所述多聚体为分子量至少600kDa的多聚体。在某些优选的实施方案中，所述多聚体包含截短的轮状病毒VP4蛋白，其与野生型轮状病毒VP4蛋白相比，N端截短了25个氨基酸，并且C端终止于野生型轮状病毒VP4蛋白中的、与SEQ ID NO:40的氨基酸位置476相对应的位置。在某些优选的实施方案中，所述多聚体包含截短的轮状病毒VP4蛋白，其氨基酸序列如SEQ ID NO:30所示。在某些优选的实施方案中，所述多聚体为三聚体或者分子量至少600kDa的多聚体，其包含如SEQ ID NO:30所示的截短的轮状病毒VP4蛋白。
在另一个方面，本发明还涉及包含上述截短蛋白或其变体，或上述多核苷酸，或上述载体，或上述宿主细胞，或上述多聚体的组合物。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。在某些优选的实施方案中，所述组合物包含本发明的多聚体。
在另一个方面，本发明还涉及一种药物组合物(例如疫苗)，其包含本发明的截短蛋白或其变体或者本发明的多聚体，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(例如疫苗)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，本发明的截短蛋白或其变体或者本发明的多聚体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(例如疫苗)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。在某些优选的实施方案中，本发明的药物组合物(例如疫苗)还包含佐剂，例如铝佐剂。
在某些优选的实施方案中，所述药物组合物还包含药学可接受的载体，赋形剂，稳定剂或能够为所述药物组合物的施用(例如施用给人受试者)提供有利性质的其他试剂。合适的药物载体包括例如，无菌水，盐水，葡萄糖，蓖麻油和环氧乙烷的缩合产物，液体酸，低级醇(例如C1-4醇)，油(例如玉米油，花生油，芝麻油；其任选还包含乳化剂例如脂肪酸的单-或二-甘油酯或磷脂例如卵磷脂)，乙二醇，聚亚烷基二醇，藻酸钠，聚(乙烯基吡咯烷酮)等等。所述载体任选地还可包含佐剂，防腐剂，稳定剂，润湿剂，乳化剂，渗透增强剂等。在某些优选的实施方案中，所述药物组合物是无菌的。此外，所述药物组合的粘度可通过选择合适的溶剂或赋形剂来控制和维持。在某些优选的实施方案中，所述药物组合物经配制具有4.5-9.0，5.0-8.0，6.5-7.5，或6.5-7.0的pH。
本发明的药物组合物(例如疫苗)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(例如疫苗)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(例如疫苗)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，本发明涉及一种制备上述截短蛋白或其变体的方法，其包括，在允许所述截短蛋白或其变体表达的条件下，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。
在某些优选的实施方案中，所述方法包括，利用大肠杆菌来表达本发明的截短蛋白或其变体，然后将所述大肠杆菌裂解，并从裂解液中纯化获得该截短蛋白或其变体。在某些优选的实施方案中，纯化包括层析。在某些优选的实施方案中，所述纯化包括，两步层析法。在某些优选的实施方案中，所述两步层析法包括：阴离子交换层析(例如，使用Q-sepharose-HP的阴离子交换层析)；和，随后的疏水亲和层析(例如，使用Phenyl sepharose-HP的疏水亲和层析)。
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者本发明的多聚体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。在某些优选的实施方案中，所述制备疫苗的方法包括下述步骤：将本发明的截短蛋白或其变体或者本发明的多聚体与佐剂(例如铝佐剂)混合。
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者本发明的多聚体或者本发明的药物组合物施用给所述受试者。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，本发明还涉及本发明的截短蛋白或其变体或者本发明的多聚体在制备药物组合物(例如疫苗)中的用途，所述药物组合物(例如疫苗)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，本发明还涉及上述的截短蛋白或其变体或者上述的多聚体，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”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。例如，N端截短了25个氨基酸的轮状病毒VP4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VP4蛋白N末端的第1-25位氨基酸残基所获得的蛋白质。
根据本发明，表述“C端终止于氨基酸位置X”是指，氨基酸位置X之后(即，自氨基酸位置X+1开始)的氨基酸残基全部被缺失。例如，C端终止于野生型轮状病毒VP4蛋白的氨基酸位置441是指，在野生型轮状病毒VP4蛋白的氨基酸位置441之后(即，自氨基酸位置442开始)的氨基酸残基全部被缺失。
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VP4蛋白的氨基酸序列相比，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60％，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可溶性表达；
(ii)其能够通过色谱层析容易地进行纯化；
(iii)其在纯化后拥有良好的均一性和稳定性(不易于降解)；
(iv)其能够特异性结合抗VP4抗体，所述抗VP4抗体能够体外抑制病毒对细胞的感染；
(v)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；
(vi)其能够诱导机体产生高滴度的针对轮状病毒的中和抗体；
(vii)其能够保护受试者(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“VP4蛋白”、“VP4全长蛋白”和“轮状病毒VP4蛋白”可互换使用，其是指与VP7一起共同构成RV病毒颗粒外衣壳的结构蛋白。VP4蛋白的氨基酸序列是本领域技术人员已知的，并且见于各种公共数据库(例如GenBank数据库)。例如，VP4蛋白的示例性氨基酸序列可如SEQ ID NO：40和87-91所示。
根据本发明，术语“截短的轮状病毒VP4蛋白”是指，在野生型轮状病毒VP4蛋白的N端和/或C端去掉一个或者多个氨基酸后产生的蛋白质，其中，野生型轮状病毒VP4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AEV53329.1、BAA03850.1、AAB94758.2、AIS93087.1、ACJ06216.1和AAA66953.1。
在本发明中，野生型轮状病毒LLR的VP4蛋白的示例性氨基酸序列如SEQ ID NO：40所示。因此，在本发明中，当涉及VP4蛋白的序列时，其使用SEQ ID NO:40所示的序列来进行描述。例如，表述“VP4蛋白的氨基酸位置276-497”是指，SEQ ID NO:40的氨基酸位置276-497。然而，本领域技术人员理解，可在SEQ ID NO:40中天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加)，而不影响VP4蛋白的生物学特性。例如，轮状病毒的不同病毒株的VP4蛋白可在氨基酸序列上天然存在差异，但具有实质上相同的生物学特性。因此，在本发明中，术语“VP4蛋白”意欲包括所有此类多肽和变体，包括SEQ ID NO:40所示的多肽以及其天然或人工的变体，所述变体保留了VP4蛋白的生物学特性。并且，当描述VP4蛋白的序列片段和氨基酸位置时，其不仅包括SEQ ID NO:40所示的多肽的序列片段和氨基酸位置，还包括该多肽的天然或人工变体中的相应序列片段和氨基酸位置。例如，表述“VP4蛋白的氨基酸位置276-497”意欲包括，SEQ ID NO:40的氨基酸位置276-497，以及SEQ ID NO:40所示的多肽的变体(天然或人工)中的相应氨基酸位置。
在本发明中，野生型轮状病毒SA11、EDIM、P[4]、P[6]、P[8]的VP4蛋白的示例性氨基酸序列分别如SEQ ID NO：87-91所示。
根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。根据本发明，表述“相应氨基酸位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的氨基酸位点/残基。
在本发明中，术语“截短的轮状病毒VP4蛋白基因片段”是指这样的基因片段，其与野生型轮状病毒VP4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VP4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、D16346.1、AF039219.2、KJ940075.1、FJ183356.1和L34161.1。例如，野生型轮状病毒LLR的VP4蛋白基因的核苷酸序列可如SEQ ID NO：40所示。
根据本发明，术语“多聚体”是指，以多肽分子(例如，本发明的截短蛋白)为单体构成的聚合体，其通常可包含至少2个(例如，3个，4个，5个或更多个)多肽单体(例如，本发明的截短蛋白)。在此类多聚体中，单体分子通过分子间相互作用(例如氢键、范德华力、疏水相互作用)而聚合形成多聚体。在本发明的某些实施方案中，多聚体是包含3个单体的三聚体。在本发明的某些实施方案中，多聚体是包含多个单体、且分子量为至少600kDa的多聚体(此类多聚体在本文中也被称为高聚体)。
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“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。在本发明中，特别优选地，佐剂为铝佐剂。
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。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
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。
根据本发明，术语“免疫原性(immunogenicity)”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫应答。
在本发明中，术语“多肽”和“蛋白质”具有相同的含义，可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中使用的，“受试者”是指动物，例如脊椎动物。优选地，受试者为哺乳动物，例如人，牛科动物，马科动物，猫科动物，犬科动物，啮齿类动物或灵长类动物。特别优选地，受试者为人。在本文中，该术语可以与“患者”互换使用。
发明的有益效果
本发明提供的截短的轮状病毒VP4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可溶性表达，实现了高产率。这为大规模工业化生产提供了有利条件。
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(例如通过阴离子交换层析和疏水亲和层析进行处理)，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％)。这为大规模的工业化生产提供了有利条件。
第三，本发明所获得的高纯度截短蛋白具有良好的均一性和稳定性。特别地，本发明的经纯化的截短蛋白能够以均一的形式存在，且不易于降解。这为批量化生产提供了有利条件，避免了不同批次间的变异性，有利于精确用药。
第四，本发明的截短蛋白或其变体能够在铝佐剂条件下，诱导机体产生高滴度的针对轮状病毒的中和抗体。由于临床中通常使用铝佐剂，而不使用弗氏佐剂，因此，本发明的截短蛋白或其变体可有利地用于临床条件，用于保护受试者，抵抗轮状病毒感染。
综合来说，本发明的截短蛋白或其变体不仅具有易于表达、易于纯化等特点，而且具有高均一性和稳定性。更重要的是，本发明的截 短蛋白或其变体具有强免疫原性，能够在铝佐剂条件下诱导机体产生高滴度的中和抗体，从而在临床条件下为受试者提供强的保护作用。因此，本发明的截短蛋白或其变体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1A显示了经纯化的VP8-5蛋白的SDS-PAGE结果。结果显示，所获得的经纯化的VP8-5蛋白的纯度达到90％以上。
图1B显示了VP8-5蛋白与用其免疫Balb/c小鼠而获得的免疫血清的间接ELISA分析的结果，其中，横坐标表示实验组(VP8-5)、阳性对照组(LLR)和阴性对照组(NC)，纵坐标表示与VP8-5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，在使用铝佐剂的条件下，在免疫后第42天，VP8-5能在小鼠体内诱发抗体的产生，但是效率低于灭活病毒LLR。这个结果表明，在使用铝佐剂的条件下，VP8-5蛋白具有免疫原性，能在小鼠体内诱发抗体的产生，但是其在动物体内诱发抗体产生的能力低于灭活病毒LLR。
图1C显示了VP8-5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，横坐标表示实验组(VP8-5)、阳性对照组(LLR)和阴性对照组(NC)，纵坐标表示达到50％感染抑制率的免疫血清最大稀释倍数的log值(log2NT50，中和抗体滴度)。结果显示，在使用铝佐剂的条件下，VP8-5在免疫后第42天(三次免疫后)能够在小鼠体内诱发中和抗体，但是免疫血清的中和抗体滴度(NT50) 低于灭活病毒LLR。免疫程序完成后，使用VP8-5蛋白诱发的免疫血清的中和抗体滴度(NT50)仅达到64左右。这个结果表明，在使用铝佐剂的条件下，VP8-5能够诱导机体产生中和抗体，但是其所诱发的免疫血清的中和抗体滴度低于灭活病毒LLR。
图2A-2C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2C)，软粪便计为2分(图2B)，不成形水样便计为3分(图2A)。
图3显示了攻毒后不同免疫组的乳鼠的腹泻评分情况，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，实验组(VP8-5)乳鼠的腹泻评分与阴性对照组(NC)无显著差异。这说明，在使用铝佐剂的条件下，VP8-5保护机体、抵抗轮状病毒感染的能力较低(低于灭活病毒)，不能充分缓解轮状病毒感染导致的腹泻。
图4A-4D显示了在大肠杆菌中表达的各个截短蛋白的SDS-PAGE结果。结果显示，除了26-311，26-331和26-381的表达量相对较低以外，其他的截短蛋白均可在大肠杆菌中高表达。
图5A-5B显示了经纯化的各种截短VP4蛋白的SDS-PAGE结果。在图5A中，泳道从左到右依次为：蛋白质分子量标记(Marker)，1-476，6-476，22-476，26-476，65-476，26-231，26-271，26-331和26-351。在图5B中，泳道从左到右依次为：蛋白质分子量标记(Marker)，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492和26-497。图5A和5B的结果显示，经过上述纯化步骤后，各截短蛋白的浓度均达到0.2mg/ml以上，且纯度大于80％。
图6显示了截短蛋白1-476、6-476、22-476、26-476、26-271、26-471、26-482、26-487、26-492、26-497的经G3000PWXL分子筛分析的结果。横坐标轴表示保留时间，纵坐标轴表示在280nm处的吸光度值。结果显示，在TB8.0条件下，截短蛋白1-476以高聚体的形式存在于样品中(出峰时间为约10-11分钟)；26-271呈单体状态(出峰时间为约16分钟)，含量超过90％；其余的截短蛋白(6-476、22-476、26-476、26-471、26-482、26-487、26-492、26-497)均为三聚体状态(出峰时 间为约13-14分钟)，含量几乎都在90％以上。在盐存在的条件下(TB8.0+1M NaCl)，截短蛋白26-476、26-482、26-487、26-492和26-497转变为单体状态(出峰时间由约13-14分钟改变为约15-16分钟)，含量均超过90％。这表明，在盐存在的条件下，26-476、26-482、26-487、26-492和26-497的构象受到盐离子影响，导致三聚体发生解聚，形成单体。
图7显示了截短蛋白26-331，26-351，26-381，26-411，26-441和26-461的经G5000PWXL分子筛分析的结果。横坐标轴表示保留时间，纵坐标轴表示在280nm处的吸光度值。结果显示，在TB8.0条件下，这些截短蛋白均以高聚体的形式存在于样品中(出峰时间为约10-11分钟)。
图6和图7的结果还显示，所获得的截短VP4蛋白的主要吸收峰所占比例几乎都在80％或甚至90％以上；这说明，这些截短蛋白具有良好的均一性。
图8A显示了在50mM Tris-HCl(pH8.0)中37℃静置12小时后的截短蛋白26-476的分子筛分析结果。横坐标轴表示保留时间，纵坐标轴表示在280nm处的吸光度值。结果显示，截短蛋白26-476在50mM Tris-HCl(pH8.0)中37℃静置12小时后能够形成均一的高聚体(保留时间为12.4分钟)，并且高聚体的比例高达97.2％。
图8B显示了在50mM Tris-HCl(pH8.0)中37℃静置12小时后的截短蛋白26-476的电镜观察的结果。结果表明，截短蛋白26-476能够在体外组装成均一的高聚体。
图9显示了经酶切和未经酶切的截短蛋白26-476，26-482，26-487，26-492，26-497的SDS-PAGE结果。泳道上的数字1表示，样品未经胰酶处理；数字2表示，样品经0.1mg/ml胰酶处理。结果显示，上述截短蛋白均能被胰酶识别并发生酶切，其酶切位点是暴露的。
图10显示了各种截短的VP4蛋白(1-476、6-476、22-476、65-476、26-271、26-441、26-461、26-471、26-476，26-482，26-487，26-492，26-497)与中和抗体A3(图10A)、B1(图10B)、B5(图10C)、 B6(图10D)、D6(图10E)、E2(图10F)、E5(图10G)、8F6(图10H)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(A3、B1、B5、B6、D6、E2、E5、8F6)的最低抗体浓度。结果显示，各截短蛋白均具有良好的抗原性(即，抗体反应性)。
图11A-11D显示了截短蛋白26-476与用各待测样品(1-476、6-476、22-476、65-476、26-271、26-331、26-351、26-381、26-411、26-441、26-461、26-471、26-476、26-482、26-487、26-492、26-497、26-476的三聚体、26-476的高聚体、灭活病毒)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，横坐标表示用于制备免疫血清的蛋白样品；纵坐标表示与26-476具有反应性的免疫血清的最大稀释倍数(即，抗体滴度)；RV：灭活的轮状病毒；NC：阴性对照(PBS)；三聚体：26-476的三聚体；高聚体：26-476的高聚体。图11A、11B、11C和11D显示了来自不同免疫批次的结果。结果显示，在使用铝佐剂的条件下，在免疫后第42天，各蛋白样品均能在小鼠体内诱发抗体(免疫血清中的抗体滴度(GMT)均可达到102-105或更高)；并且，除了26-271外，其他蛋白样品所诱发的抗体滴度均高于RV诱发的抗体滴度(1-476、6-476、22-476、26-331、26-351、26-381、26-411、26-441、26-461、26-471、26-476、26-487、26-492、26-476的三聚体和26-476的高聚体)，或至少与RV诱发的抗体滴度相当(65-476、26-482和26-497)。
图12A-12D显示了各蛋白样品(1-476、6-476、22-476、65-476、26-271、26-331、26-351、26-381、26-411、26-441、26-461、26-471、26-476、26-482、26-487、26-492、26-497、26-476的三聚体、26-476的高聚体、灭活病毒)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，横坐标表示用于制备免疫血清的蛋白样品；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)；RV：灭活的轮状病毒；NC：阴性对照(PBS)；三聚体：26-476的三聚体；高聚体：26-476的高聚体。图12A、12B、 12C和12D显示了来自不同免疫批次的结果。结果显示，在使用铝佐剂的条件下，在免疫后第42天(三次免疫后)，各蛋白样品均能在小鼠体内诱发中和抗体，中和抗体滴度(NT50)均可达到28-214或更高的水平；并且，除了26-271外，其他蛋白样品所诱发的中和抗体滴度均与RV诱发的中和抗体滴度相当(6-476、22-476、26-476、65-476、26-471、26-482、26-487、26-492、26-497和26-476的三聚体)，或甚至高于RV诱发的中和抗体滴度(1-476、26-331、26-351、26-381、26-411、26-441、26-461和26-476的高聚体)。
图13A-13D显示了攻毒后1-7天来自不同免疫组(用22-476，26-476，65-476，26-271，26-331，26-351，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492，26-497，26-476的三聚体，26-476的高聚体，灭活的轮状病毒(RV，阳性对照)或PBS(NC，阴性对照)进行免疫)的乳鼠的腹泻评分情况，其中，纵轴表示平均腹泻评分；横轴表示小鼠攻毒后天数；RV：灭活的轮状病毒；NC：阴性对照(PBS)；三聚体：26-476的三聚体；高聚体：26-476的高聚体。
图14A-14D显示不同免疫组(用22-476，26-476，65-476，26-271，26-331，26-351，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492，26-497，26-476的三聚体，26-476的高聚体，灭活的轮状病毒(RV，阳性对照)或PBS(NC，阴性对照)进行免疫)的乳鼠在攻毒后的平均腹泻持续时间和攻毒后48小时的平均腹泻评分，其中，平均腹泻天数以柱形图表示，平均腹泻评分以曲线图表示，左纵轴表示平均腹泻天数；右纵轴表示腹泻评分；横轴表示各蛋白样品所对应的免疫组；RV：灭活的轮状病毒；NC：阴性对照(PBS)；三聚体：26-476的三聚体；高聚体：26-476的高聚体。
图13-14的结果显示，在平均腹泻评分和平均腹泻天数方面，各蛋白样品所对应的免疫组均显著优于NC组。这表明，各蛋白样品均具有显著的保护性，能够帮助小鼠抵抗轮状病毒感染和由轮状病毒感染导致的腹泻。此外，图13-14的结果还显示，26-331，26-351，26-381， 26-411，26-441，26-461，26-476，26-476的三聚体，以及26-476的高聚体的保护性与RV相当，或甚至优于RV。结合实施例1的实验结果可知，在使用铝佐剂的条件下，这些蛋白样品在动物中的保护性均显著优于VP8-5。此外，图13D和图14D的实验结果还表明，截短蛋白26-476的高聚体在动物中的保护能力显著高于26-476的三聚体，可用于制备具有更高效力的疫苗。
图15显示了来源于不同病毒株的26-476蛋白的SDS-PAGE结果，其中，泳道从左到右依次为：来源于轮状病毒LLR的截短蛋白26-476；来源于轮状病毒SA11的截短蛋白26-476-SA11；来源于轮状病毒EDIM的26-476-EDIM；来源于轮状病毒P[8]的截短蛋白26-476-P[8]；来源于轮状病毒P[6]的截短蛋白26-476-P[6]；来源于轮状病毒P[4]的截短蛋白26-476-P[4]；和，蛋白质分子量标记(Marker)。结果表明，本发明的方法适用于不同的轮状病毒株；来源于不同病毒株的截短VP4蛋白(26-476)均可在大肠杆菌中有效表达，且经色谱法纯化后，其纯度均可达到80％以上。
图16显示了来源于不同毒株的的截短VP4蛋白26-476的分子筛分析结果。横坐标轴表示保留时间，纵坐标轴表示在280nm处的吸光度值。结果显示，在TB8.0条件下，来源于不同毒株的26-476蛋白均主要以三聚体的形式存在(出峰时间为约13-14分钟)，其含量均在80％以上。这表明，在TB8.0条件下，来源于不同毒株的26-476蛋白都具有良好的均一性。
图17显示各截短蛋白(26-476-P[4]、26-476-P[6]、26-476-P[8]、26-476-EDIM、26-476-SA11和来源于LLR的26-476)与用相应截短蛋白免疫Balb/c小鼠而获得的免疫血清的间接ELISA分析的结果，其中，横坐标表示用于制备免疫血清的截短蛋白所源自的病毒株，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)；P[4]：26-476-P[4]；P[6]：26-476-P[6]；P[8]：26-476-P[8]；SA11：26-476-SA11；EDIM：26-476-EDIM；LLR：实施例4中制备的26-476。结果显示，在使用铝佐剂的条件下，在免疫后第42天， 来源于不同病毒株的26-476蛋白均能在小鼠体内有效诱发抗体的产生，且所诱发的免疫血清中的抗体滴度(GMT)基本上相当(抗体滴度均可达到104-105或更高，显著高于阴性对照组)。这些结果表明，在使用铝佐剂的条件下，来源于不同病毒株的26-476蛋白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；并且，来源于不同病毒株的26-476蛋白的免疫原性基本上相当，且均显著高于VP8-5。
图18显示了来源于不同病毒株的26-476蛋白(26-476-SA11、26-476-EDIM、来源于LLR的26-476)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，横坐标表示用于制备免疫血清的蛋白样品所源自的病毒株；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)；SA11：26-476-SA11；EDIM：26-476-EDIM；LLR：实施例4中制备的26-476。结果显示，在使用铝佐剂的条件下，在免疫后第42天(三次免疫后)，SA11、EDIM和LLR毒株的26-476蛋白均能在小鼠体内诱发高滴度的中和抗体，中和抗体滴度(NT50)均可达到210-214或更高的水平；并且，26-476-SA11和26-476-EDIM所诱发的中和抗体滴度甚至高于来源于LLR的26-476。
图19A显示了不同免疫组(用26-476-SA11或PBS(NC，阴性对照)进行免疫)的乳鼠在用SA11病毒攻毒后1-7天的腹泻评分；图19B显示了不同免疫组(用26-476-EDIM或PBS(NC，阴性对照)进行免疫)的乳鼠用EDIM病毒攻毒后1-12天的腹泻评分；其中，横坐标表示攻毒后天数，纵坐标表示平均腹泻评分。结果显示，与来源于LLR的26-476类似，来源于SA11和EDIM的26-476蛋白均具有显著的保护性，能够帮助小鼠抵抗轮状病毒感染和由轮状病毒感染导致的腹泻。
图19C显示了在用EDIM攻毒后1-7天，经26-476-EDIM或PBS(NC，阴性对照)免疫的小鼠的粪便悬液样本中的病毒量，其中，横坐标表示攻毒后天数，纵坐标表示每ml粪便悬液样品中所含的EDIM基因组的拷贝数。结果显示，在攻毒后，在用PBS免疫的小鼠的粪便 中检测到明显的排毒，而在用26-476-EDIM免疫的小鼠的粪便中，则未检测到排毒。
图19A-19C的结果表明，26-476-EDIM不仅能够使小鼠抵抗轮状病毒感染以及由轮状病毒感染导致的腹泻，而且能够抑制病毒在小鼠粪便中的排放(即，排毒)。
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 PCTCN2016082780-appb-000001
Figure PCTCN2016082780-appb-000002
Figure PCTCN2016082780-appb-000003
Figure PCTCN2016082780-appb-000004
序列1(SEQ ID NO:1)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR
序列2(SEQ ID NO:2)：
MASLIYRQLLTNSYTVNLSDEIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPS
序列3(SEQ ID NO:3)：
MYRQLLTNSYTVNLSDEIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPS
序列4(SEQ ID NO:4)：
MIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPS
序列5(SEQ ID NO:5)：
MLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPS
序列6(SEQ ID NO:6)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNV PETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARR
序列7(SEQ ID NO:7)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVN
序列8(SEQ ID NO:8)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSL
序列9(SEQ ID NO:9)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDI
序列10(SEQ ID NO:10)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSI
序列11(SEQ ID NO:11)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKW
序列12(SEQ ID NO:12)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANY
序列13(SEQ ID NO:13)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEE
序列14(SEQ ID NO:14)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVN
序列15(SEQ ID NO:15)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGG
序列16(SEQ ID NO:16)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISR
序列17(SEQ ID NO:17)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYV
序列18(SEQ ID NO:18)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQA
序列19(SEQ ID NO:19)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSL
序列20(SEQ ID NO:20)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCA
序列21(SEQ ID NO:21)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPV
序列22(SEQ ID NO:22)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGS
序列23(SEQ ID NO:23)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTL
序列24(SEQ ID NO:24)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSL
序列25(SEQ ID NO:25)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLA
序列26(SEQ ID NO:26)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISR
序列27(SEQ ID NO:27)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLP
序列28(SEQ ID NO:28)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDF
序列29(SEQ ID NO:29)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLI
序列30(SEQ ID NO:30)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPS
序列31(SEQ ID NO:31)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPSNDDYQT
序列32(SEQ ID NO:32)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPSNDDYQTPIMNS
序列33(SEQ ID NO:33)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPSNDDYQTPIMNSVTVRQ
序列34(SEQ ID NO:34)：
MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPSNDDYQTPIMNSVTVRQDLERQ
序列35(SEQ ID NO:35)：
MGSEKTQNVTVNPGPFAQTRYAPVNWGHGEINDSTTVEPVLDGPYQPTTFKPPNDYWLLISSNTDGVVYESTNNSDFWTAVIAVEPHVSQTNRQYVLFGENKQFNIENSSDKWKFLEMFRGSGQSDFSNRRTLTSNNRLVGMLKYGGRVWTFHGETPRATTDSSNTADLNNISIIIHSEFYIIPRSQESKCNEYINNGLPPIQNTRNVVPLSLSSRSIQYRRAQVNEDITISKTSLWKEMQYNRDIIIRFKFGNSVIKLGGLGYKWSEISYKAANYQYSYSRDGEQVTAHTTCSVNGVNNFSYNGGSLPTDFSISRYEVIKENSYVYIDYWDDSKAFRNMVYVRSLAANLNSVKCVGGSYDFRLPVGEWPIMNGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVDEPSFSIIRTRTMNLYGLPAANPNNGNEYYEVSGRFSLISLVPTN
序列36(SEQ ID NO：36)
MGSEKSQNVTINPGPFAQTNYAPVTWSHGEVNDSTTIEPVLDGPYQPTNFKPPNDYWILLNPTNQQVVLEGTNKTDIWVALLLVEPNVTNQSRQYTLFGETKQITVENNTNKWKFFEMFRSNVSAEFQHKRTLTSDTKLAGFMKFYNSVWTFHGETPHATTDYSSTSNLSEVETVIHVEFYIIPRSQESKCSEYINTGLPPMQNTRNIVPVALSSRSVTYQRAQVNEDIIISKTSLWKEMQYNRDIIIRFKFNNSIVKLGGLGYKWSEISFKAANYQYSYLRDGEQVTAHTTCSVNGVNNFSYNGGSLPTDFSVSRYEVIKENSYVYVDYWDDSQAFRNMVYVRSLAANLNSVKCSGGNYNFQIPVGAWPVMSGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVEEPPFSILRTRVSGLYGLPAFNPNNGHEYYEIAGRFSLISLVPSN
序列37(SEQ ID NO:37)：
MGSEKTQNVTINPSPFAQTRYAPVNWGHGEINDSTTVEPMLDGPYQPTTFTPPNDYWILINSNTNGVVYESTNNSDFWTAVVAIEPHVNPVDRQYTIFGESKQFNVSNDSNKWKFLEMFRSSSQNEFYNRRTLTSDTRFVGILKYGGRVWTFHGETPRATTDSSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGLPPIQNTRNVVPLPLSSRSIQYKRAQVNEDIIVSKTSLWKEMQYNRDIIIRFKFGNSIVKMGGLGYKWSEISYKAANYQYNYLRDGEQVTAHTTCSVNGVNNFSYNGGFLPTDFGISRYEVIKENSYVYVDYWDDSKAFRNMVYVRSLAANLNSVKCTGGSYNFSIPVGAWPVMNGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVDEPPFSILRTRTVNLYGLPAANPNNGNEYYEIS GRFSLIYLVPTN
序列38(SEQ ID NO:38)：
MGAEKTQNVTVNPGPFAQTGYAPANWGPGETNDSTTVEPVLDGPYQPIAFSPPPEYYILLSPTAPGVIAECTNTVNRWIAIIAIEPNVSPTNRTYTLFGITEQLTVENSSVDKWKFIDFMKTPTTGSYVRYNILLSSTKLCAVAKHTDNLYSYVGETPTAGQAYYSSFNIFNLTAHCDFYIIPWSQQSLCTQYVNNGLPPIQNTRNVVPRHLSARSIITQRAQANEDIVVSKTSLWKEMQFNRDITIRFKFANAIIKSGGLGYKWSEISFKPANYQYTYTRDGEEVTAHTTCSVNGVNNFDFFGGSLPTDFGISRYEVIKENSFVYIDYWDDSQAFRNMVYVRSLAADLNTVECTGGAYSFSLPVGQWPVMTGGAVSLRAAGVTLSTQFTDFVSLNSLRFRFRLSVEEPSFSITRTRVSGLYGLPAADPNNGREYYEIAGRFSLISLVPSND
序列39(SEQ ID NO:39)：
MGSTKSQNVTINPGPFAQTGYAPVNWGPGEINDSTTVEPLLDGPYQPTTFNPPVDYWMLLAPTTPGVIVEGTNNTDRWLATILIEPNVQSENRTYTIFGIQEQLTVSNTSQDQWKFIDVVKTTANGSIGQYGSLLSSPKLYAVMKHNEKLYTYEGQTPNARTGHYSTTNYDSVNMTAFCDFYIIPRSEESKCTEYINNGLPPIQNTRNVVPLSLTARDVIHYRAQANEDIVISKTSLWKEMQYNRDITIRFKFANTIIKSGGLGYKWSEISFKPANYQYTYTRDGEEVTAHTTCSVNGVNDFSFNGGSLPTDFVVSKFEVIKENSYVYIDYWDDSQAFRNVVYVRSLAANLNSVMCTGGSYNFSLPVGQWPVLTGGAVSLHSAGVTLSTQFTDFVSLNSLRFRFRLAVEEPHFKLTRTRLDRLYGLPAADPNNGKEYYEIAGRFSLISLVPS
序列40(SEQ ID NO:40)：
MASLIYRQLLTNSYTVNLSDEIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRNVVPLALSSRSIVARRAAVNEDIVISKTSLWKEMQYNRDIIIRFKFANSIIKSGGLGYKWSEISFKPANYQYTYIRDGEEVTAHTTCSVNGVNDFNYNGGSLPTDFVISRYEVIKENSYVYIDYWDDSQAFRNMVYVRSLAADLNEVTCAGGTYNFQLPVGQWPVMSGGSVSLRSAGVTLSTQFTDFVSLNSLRFRFSLAVEEPPFSISRTRISGLYGLPAANPNNGRDFYEIAGRFSLILLVPSNDDYQTPIMNSVTVRQDLERQLGELREEFNALSQEIAMSQLIDLALLPLDMFSMFSGIKTTIDAAKSMATNVMKKFKSSGLATSVSTLTDSLSDAASAVSRNSSIRSIGSTASAWTDISSQIVDTQASVNTLATQTSTISKRLRLKEIATQTEGMNFDDISAAVLKTKIDKSSQIGPSTLPDIVTEASEKFIPNRTYRVIDDDTVFEAGTDGRFYAYRVETFEEVPFDVQKFADLVTDSPVISAIIDFKTLKNLNDNYGITRSQALNLIRSDPRVLREFINQDNPIIRNRIEQLILQCRL
序列41(SEQ ID NO:41)：
ATGGCTTCGCTCATTTACAGACAATTACTTACGAATTCATATACAGTGAATCTTTCAGATGAAATACAGTTAATTGGATCAGAAAAAACGCAGAGAACTACAGTAAATCCAGGTCCATTTGCACAAACTGGTTATGCACCAGTGAATTGGGGGCCTGGGGAAACGAGTGATTCCACTACTGTTGAGCCAGTGTTGAATGGACCATATCAGCCGACGACTTTCAATCCACCAGTAGAATATTGGATGCTTCTAGCACCAACATCAGAAGGGGTAGTTGTTGAAGGTACTAATGGTACGGATAGATGGCTAGCTACAATACTTATAGAACCAAATGTGCCTGAGACGACTAGAAATTACACATTATTTGGGGAAACAGCGAGTATATCAGTAGCAAACCCATCACAAAGTAAATGGCGTTTTGTTGACGTAGCTAAGACCACTGCAAATGGAACATATTCACAATATGGACCATTACTATCAGATACAAAACTGTATGGAGTAATGAAATACAACGGGAAGTTGTATACGTATAATGGTGAAACTCCGAATGCTACAACAAATTATTATTCAACTACAAATTATGACTCAGTGAATATGACATCTTATTGCGATTTTTACATTATACCAAGAGCACAAGAATCAAAGTGCACAGAATACGTAAATAATGGATTACCACCAATACAAAACACCAGAAATGTCGTACCATTAGCTTTATCTTCACGATCAATAGTTGCTAGAAGAGCTGCAGTGAACGAAGACATAGTTATATCGAAAACGTCATTGTGGAAAGAAATGCAATATAATCGAGATATCATAATAAGATTTAAGTTTGCAAACTCAATTATTAAATCAGGTGGACTAGGGTATAAATGGTCAGAGATTTCATTCAAACCAGCAAACTATCAATATACATATATACGTGATGGAGAGGAAGTAACTGCACATACAACATGTTCAGTGAATGGAGTGAACGACTTCAACTATAACGGAGGATCATTACCAACTGACTTTGTAATATCACGTTATGAAGTTATAAAAGAGAACTCTTATGTATATATAGATTATTGGGATGATTCACAAGCATTCAGAAACATGGTATATGTGAGATCATTAGCTGCGGACTTAAATGAAGTGACATGTGCAGGGGGTACTTATAATTTCCAACTACCAGTTGGACAATGGCCTGTGATGAGTGGTGGCTCAGTATCATTGCGTTCAGCTGGAGTAACGTTATCAACTCAATTTACAGACTTTGTGTCATTAAATTCGTTAAGATTTAGGTTCAGTTTAGCAGTAGAAGAACCGCCATTCTCTATTTCAAGGACACGGATATCAGGGTTATATGGGTTACCGGCAGCCAATCCAAATAATGGAAGAGACTTCTATGAAATTGCGGGTAGATTTTCATTAATTTTATTAGTACCATCAAATGATGATTATCAAACTCCTATAATGAACTCAGTGACGGTGAGACAGGACTTAGAGAGGCAGTTAGGAGAATTGAGAGAAGAATTTAACGCATTATCACAAGAGATAGCTATGTCACAATTGATAGATCTAGCTTTACTACCATTGGACATGTTCTCAATGTTTTCAGGAATTAAAACAACGATAGATGCAGCTAAATCAATGGCCACTAATGTAATGAAGAAGTTTAAAAGCTCAGGCTTGGCCACGTCTGTATCCACGTTGACAGACTCATTATCTGACGCCGCATCAGCGGTATCAAGGAACAGCTCAATAAGATCAATTGGATCAACAGCATCAGCTTGGACAGACATTTCTTCACAAATAGTGGATACGCAAGCATCAGTCAATACGTTGGCAACTCAAACGTCAACTATCAGCAAGAGATTAAGGTTAAAAGAAATTGCGACTCAAACAGAGGGAATGAATTTCGACGACATATCAGCAGCTGTGTTAAAAACTAAAATTGACAAATCATCACAAATAGGACCAAGTACTTTACCAGATATTGTTACTGAAGCGTCGGAGAAGTTTATACCAAATAGAACGTATAGAGTAATTGACGATGATACTGTGTTTGAAGCAGGAACAGATGGGAGATTTTACGCATATAGAGTCGAGACGTTTGAGGAAGTTCCATTTGAT GTGCAAAAATTCGCAGATTTAGTAACTGACTCTCCAGTAATCTCGGCCATTATAGACTTTAAAACGCTTAAAAACTTGAATGATAACTATGGAATTACTCGTTCGCAAGCATTAAATCTAATTAGATCAGATCCAAGGGTTCTGCGAGAATTTATCAATCAAGATAATCCAATAATAAGAAACAGGATAGAGCAGTTAATTCTGCAATGTAGATTGTAA
序列42(SEQ ID NO:42)：
GGATCCCATATGATGGCTTCGCTCATTTAC
序列43(SEQ ID NO:43)：
GGATCCCATATGTACAGACAATTACTTACGAATTC
序列44(SEQ ID NO:44)：
GGATCCCATATGATACAGTTAATTGGATCAGAAAA
序列45(SEQ ID NO:45)：
GGATCCCATATGGGATCAGAAAAAACGCAG
序列46(SEQ ID NO:46)：
GGATCCCATATGTTGAATGGACCA
序列47(SEQ ID NO:47)：
AAGCTTAGGTGTTTTGTATTGGTGG
序列48(SEQ ID NO:48)：
AAGCTTATCTTCTAGCAACTATTGATCGT
序列49(SEQ ID NO:49)：
AAGCTTAGTTCACTGCAGCTCTTCTAGC
序列50(SEQ ID NO:50)：
AAGCTTACAATGACGTTTTCGATATAACTA
序列51(SEQ ID NO:51)：
AAGCTTAGATATCTCGATTATATTGCATTTC
序列52(SEQ ID NO:52)：
AAGCTTAAATTGAGTTTGCAAACTTAAAT
序列53(SEQ ID NO:53)：
AAGCTTACCATTTATACCCTAGTCCACC
序列54(SEQ ID NO:54)：
AAGCTTAATAGTTTGCTGGTTTGAATGA
序列55(SEQ ID NO:55)：
AAGCTTATTCCTCTCCATCACGTATATATG
序列56(SEQ ID NO:56)：
AAGCTTAATTCACTGAACATGTTGTATGTG
序列57(SEQ ID NO:57)：
AAGCTTATCCTCCGTTATAGTTGAAGTC
序列58(SEQ ID NO:58)：
AAGCTTAACGTGATATTACAAAGTCAGTTG
序列59(SEQ ID NO:59)：
AAGCTTATACATAAGAGTTCTCTTTTATAACTTC
序列60(SEQ ID NO:60)：
AAGCTTATGCTTGTGAATCATCCCAA
序列61(SEQ ID NO:61)：
AAGCTTATAATGATCTCACATATACCATGTTT
序列62(SEQ ID NO:62)：
AAGCTTATGCACATGTCACTTCATTTAAG
序列63(SEQ ID NO:63)：
AAGCTTAAACTGGTAGTTGGAAATTATAAGTA
序列64(SEQ ID NO:64)：
AAGCTTATGAGCCACCACTCATCACA
序列65(SEQ ID NO:65)：
AAGCTTATAACGTTACTCCAGCTGAAC
序列66(SEQ ID NO:66)：
AAGCTTATAATGACACAAAGTCTGTAAATTG
序列67(SEQ ID NO:67)：
AAGCTTATGCTAAACTGAACCTAAATCTTA
序列68(SEQ ID NO:68)：
AAGCTTACCTTGAAATAGAGAATGGCG
序列69(SEQ ID NO:69)：
AAGCTTACGGTAACCCATATAACCCT
序列70(SEQ ID NO:70)：
AAGCTTAGAAGTCTCTTCCATTATTTGGA
序列71(SEQ ID NO:71)：
AAGCTTAAATTAATGAAAATCTACCCGC
序列72(SEQ ID NO:72)：
AGATCTAAGCTTATGATGGTACTAATAAAATTAATGAAAATC
序列73(SEQ ID NO:73)：
AGATCTAAGCTTAAGTTTGATAATCATCATTTGATGGTACTA
序列74(SEQ ID NO:74)：
AGATCTAAGCTTATGAGTTCATTATAGGAGTTTGATAATCAT
序列75(SEQ ID NO:75)：
AGATCTAAGCTTATGTCTCACCGTCACTGAGTTCA
序列76(SEQ ID NO:76)：
AGATCTAAGCTTACTGCCTCTCTAAGTCCTGTCTCA
序列77(SEQ ID NO:77)：
GGATCCCATATGGGATCGGAGAAAACTCAA
序列78(SEQ ID NO:78)：
AAGCTTAATTAGTTGGAACTAAAGAAATAAGT
序列79(SEQ ID NO:79)
GGATCCCATATGGGATCAGAGAAAAGTCAAAAT
序列80(SEQ ID NO:80)
AAGCTTAATTAGACGGTACTAATGAAA
序列81(SEQ ID NO:81)：
GGATCCCATATGGGATCAGAAAAAACTCAAAATG
序列82(SEQ ID NO:82)：
AAGCTTAGTTGGTTGGAACTAAAGAAA
序列83(SEQ ID NO:83)：
GGATCCCATATGGGAGCAGAGAAGACACA
序列84(SEQ ID NO:84)：
AAGCTTAATCGTTGGACGGCAC
序列85(SEQ ID NO:85)：
GGATCCCATATGGGATCAACTAAATCACAAAATG
序列86(SEQ ID NO:86)：
AAGCTTATGATGGCACTAATGATATAAGT
序列87(SEQ ID NO:87)：
MASLIYRQLLTNSYTVDLSDEIQEIGSTKSQNVTINPGPFAQTGYAPVNWGPGEINDSTTVEPLLDGPYQPMTFNPPVDYWMLLAPTTPGVIVEGTNNTDRWLATILIEPNVQSENRTYTIFGIQEQLTVSNTSQDQWKFIDVVKTTANGSIGQYGSLLSSPKLYAVMKHNEKLYTYEGQTPNARTGHYSTTNYDSVNMTAFCDFYIIPRSEESKCTEYINNGLPPIQNTRNVVPLSLTARDVIHYRAQANEDIVISKTSLWKEMQYNRDITIRFKFANTIIKSGGLGYKWSEISFKPANYQYTYTRDGEEVTAHTTCSVNGVNDFSFNGGSLPTDFVVSKFEVIKENSYVYIDYWDDSQAFRNVMYVRSLAANLNSVMCTGGSYNFSLPVGQWPVLTGGAVSLHSAGVTLSTQFTDFVSLNSLRFRFRLAVEEPHFKLTRTRLDRLYGLPAADPNNGKEYYEIAGRFSLISLVPSNDDYQTPIANSVTVRQDLERQLGELREEFNALSQEIAMSQLIDLALLPLDMFSMFSGIKSTIDAAKSMATNVMKKFKKSGLANSVSTLTDSLSDAASSISRGSSIRSIGSSASAWTDVSTQITDISSSVSSVSTQTSTISRRLRLKEMATQTEGMNFDDISAAVLKTKIDKSTQISPNTIPDIVTEASEKFIPNRAYRVINNDDVFEAGIDGKFFAYKVDTFEEIPFDVQKFADLVTDSPVISAIIDFKTLKNLNDNYGITKQQAFNLLRSDPRVLREFINQDNPIIRNRIEQLIMQCRL
序列88(SEQ ID NO:88)：
MASLIYRQLLTNSFTVDISDEIETIGAEKTQNVTVNPGPFAQTGYAPANWGPGETNDSTTVEPVLDGPYQPIAFSPPPEYYILLSPTAPGVIAECTNTVNRWIAIIAIEPNVSPTNRTYTLFGITEQLTVENSSVDKWKFIDFMKTPTTGSYVRYNILLSSTKLCAVAKHTDNLYS YVGETPTAGQAYYSSFNIFNLTAHCDFYIIPWSQQSLCTQYVNNGLPPIQNTRNVVPRHLSARSIITQRAQANEDIVVSKTSLWKEMQFNRDITIRFKFANAIIKSGGLGYKWSEISFKPANYQYTYTRDGEEVTAHTTCSVNGVNNFDFFGGSLPTDFGISRYEVIKENSFVYIDYWDDSQAFRNMVYVRSLAADLNTVECTGGAYSFSLPVGQWPVMTGGAVSLRAAGVTLSTQFTDFVSLNSLRFRFRLSVEEPSFSITRTRVSGLYGLPAADPNNGREYYEIAGRFSLISLVPSNDNYQTPIMNSVTVRQDLERQLGELREEFNALSQEIALSQLVDLALLPLDMFSMFSGIKATLDVAKSMATNVMKKFKKSGLATSISAMTESLSDAASSVSRSGAIRSVSSTSSAWTDVSSRVANVENAASTVSTQTATISRRLRLKEITTQTEGMNFDDISAAVLKTKLDKSVRIAPNTLPDIVTEASEKFIPNRSYRVINNNEAFETGTDGRFFAYRVDTLEELPFDVQKFADLVAESPVISAIIDFKTLKNLNDNYGISKEQAFSLLRSDPRVLREFINQGNPIIRNRIEQLIMQCRL
序列89(SEQ ID NO:89)：
MASLIYRQLLTNSYSVDLHDEIEQIGSEKTQNVTVNPGPFAQTRYAPVNWGHGEINDSTTVEPVLDGPYQPTTFKPPNDYWLLISSNTDGVVYESTNNSDFWTAVIAVEPHVSQTNRQYVLFGENKQFNIENSSDKWKFLEMFRGSGQSDFSNRRTLTSNNRLVGMLKYGGRVWTFHGETPRATTDSSNTADLNNISIIIHSEFYIIPRSQESKCNEYINNGLPPIQNTRNVVPLSLSSRSIQYRRAQVNEDITISKTSLWKEMQYNRDIIIRFKFGNSVIKLGGLGYKWSEISYKAANYQYSYSRDGEQVTAHTTCSVNGVNNFSYNGGSLPTDFSISRYEVIKENSYVYIDYWDDSKAFRNMVYVRSLAANLNSVKCVGGSYDFRLPVGEWPIMNGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVDEPSFSIIRTRTMNLYGLPAANPNNGNEYYEVSGRFSLISLVPTNDDYQTPIMNSVTVRQDLERQLNDLREEFNSLSQEIAMSQLIDLALLPLDMFSMFSGIKSTIDLTKSMATSVMKKFRKSKLATSISEMTNSLSDAASSASRSASIRSNLSTISNWSDASKSVLNVTDSVNDVSTQTSTISKKLRLREMITQTEGISFDDISAAVLKTKIDMSTQIGKNTLPDIVTEASEKFIPKRSYRVLKDDEVMEVNTEGKFFAYKVDTLNEIPFDINKFAELVTDSPVISAIIDFKTLKNLNDNYGITRIEALNLIKSNPNVLRNFINQNNPIIRNRIEQLILQCKL
序列90(SEQ ID NO:90)：
MASLIYRQLLTNSYTVELSDEINTIGSEKSQNVTINPGPFAQTNYAPVTWSHGEVNDSTTIEPVLDGPYQPTNFKPPNDYWILLNPTNQQVVLEGTNKTDIWVALLLVEPNVTNQSRQYTLFGETKQITVENNTNKWKFFEMFRSNVNAEFQHKRTLTSDTKLAGFMKFYNSVWTFHGETPHATTDYSSTSNLSEVETVIHVEFYIIPRSQESKCSEYINTGLPPMQNTRNIVPVALSSRSVTYQRAQVNEDIIISKTSLWKEMQYNRDIIIRFKFNNSIVKLGGLGYKWSEISFKAANYQYSYLRDGEQVTAHTTCSVNGVNNFSYNGGSLPTDFSVSRYEVIKENSYVYVDYWDDSQAFRNMVYVRSLAANLNSVKCSGGNYNFQIPVGAWPVMSGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVEEPPFSILRTRVSGLYGLPAFNPNNGHEYYEIAGRFSLISLVPSNDDYQTPIMNSVTVRQDLERQLGDLREEFNSLSQEIAMTQLIDLALLPLDMFSMFSGIKSTIDVAKSMVTKVMKKFKKSGLATSISELTGSLSNAASSVSRSSSIRSNISSISVWTDVSEQIAGSSDSVRNISTQTSAISKRLRLREITTQTEGMNFDDISAAVLKTKIDRSTHISPDTLPDIITESSEKFIPKRAYRVLKDDEVMEADVDGKFFAYKVGTFEEVPFDVDKFVDLVTDSPVISAIIDFKTLKNLNDNYGITRSQALDLIRSDPRVLRDFINQNNPIIKNRIEQLILQCRL
序列91(SEQ ID NO:91)：
MASLIYRQLLTNSYSVDLHDEIEQIGSEKTQNVTINPSPFAQTRYAPVNWGHGEINDSTTVEPMLDGPYQPTTFTPPNDYWILINSNTNGVVYESTNNSDFWTAVVAIEPHVNPVDRQYTIFGESKQFNVSNDSNKWKFLEMFRSSSQNEFYNRRTLTSDTRFVGILKYGGRVWTFHGETPRATTDSSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGLPPIQNTRNVVPLPLSSRSIQYKRAQVNEDIIVSKTSLWKEMQYNRDIIIRFKFGNSIVKMGGLGYKWSEISYKAANYQYNYLRDGEQVTAHTTCSVNGVNNFSYNGGFLPTDFGISRYEVIKENSYVYVDYWDDSKAFRNMVYVRSLAANLNSVKCTGGSYNFSIPVGAWPVMNGGAVSLHFAGVTLSTQFTDFVSLNSLRFRFSLTVDEPPFSILRTRTVNLYGLPAANPNNGNEYYEISGRFSLIYLVPTNDDYQTPIMNSVTVRQDLERQLTDLREEFNSLSQEIAMAQLIDLALLPLDMFSMFSGIKSTIDLTKSMATSVMKKFRKSKLATSISEMTNSLSDAASSASRNVSIRSNLSAISNWTNVSNDVSNVTNSLNDISTQTSTISKKFRLKEMITQTEGMSFDDISAAVLKTKIDMSTQIGKNTLPDIVTEASEKFIPKRSYRILKDDEVMEINTEGKFFAYKINTFDEVPFDVNKFAELVTDSPVISAIIDFKTLKNLNDNYGITRTEALNLIKSNPNMLRNFINQNNPIIRNRIEQLILQCKL
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；轮状病毒SA11株购自中国兽医保藏中心；轮状病毒Wa和DS-1株购自ATCC；轮状病毒EDIM株为病原生物学研究所馈赠；原核表达载体PTO-T7为实验室自主构建；大肠杆菌(E.coli)ER2566和BL21(DE3)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：截短的VP8蛋白(VP8-5)的免疫原性和免疫保护性的研究
按照中国专利申请CN 201510165746.2中描述的方法表达并纯化截短的轮状病毒VP8蛋白VP8-5(其氨基酸序列如SEQ ID NO:1所示)。简言之，从轮状病毒LLR株的培养物中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。随后，以获得的cDNA为模板，通过PCR反应，扩增获得编码VP8-5的基因片段。然后，使用获得的基因片段构建VP8-5表达质粒，并将该表达质粒转化入大肠杆菌中。在37℃下培养含有VP8-5表达质粒的大肠杆菌至OD600＝0.6左右，然后将温度降低至25℃，并加入终浓度为0.8mM的IPTG，继续培养6h。培养结束后，通过离心收集菌体，并进行超声破碎，收集可溶性级分。随后，通过阴离子交换层析来收集可溶性级分中的VP8-5蛋白，其中所使用的仪器系统为GE Healthcare公司生产的AKTAexplorer 100型制 备型液相色谱系统；所使用的层析介质为Q-sepharose-HP(GEHealthcare公司)；所使用的缓冲液为50mM Tris-HCl pH 8.0和50mMTris-HCl pH 8.0，2M NaCl；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。通过13.5％的SDS-PAGE来鉴定所获得的洗脱产物，结果示于图1中。图1的结果显示，所获得的经纯化的VP8-5蛋白的纯度达到90％以上。
中国专利申请CN 201510165746.2已利用小鼠模型证实，在使用弗氏佐剂的条件下，经纯化的VP8-5蛋白具有良好的免疫原性和免疫保护性(参见，该申请的实施例5-8和图4-9)。为了进一步考察VP8-5蛋白在使用铝佐剂的条件下的免疫保护效果，通过小鼠模型来评价经纯化的VP8-5蛋白+铝佐剂的免疫原性和免疫保护性。
具体方案如下：将5-6周龄的雌性Balb/c小鼠随机分为3组，每组7只小鼠，其中2组为对照组，1组为实验组。将纯化的VP8-5蛋白、等剂量的灭活病毒LLR株和PBS分别与磷酸铝佐剂按照1:1的比例混合，然后通过肌肉注射的方式免疫小鼠，免疫剂量为10μg/只，其中，实验组小鼠用VP8-5进行免疫，阳性对照组小鼠用灭活病毒LLR进行免疫，阴性对照组小鼠用PBS进行免疫。各组小鼠分别进行三次免疫，每次免疫间隔两周。在免疫程序的第0、14、28和42天分别采集小鼠的眼球血，用于抗体滴度和中和抗体滴度的检测。
抗体滴度的检测
将免疫血清梯度稀释，然后分别与包被于平板上的VP8-5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VP8-5具有反应性的免疫血清的最大稀释倍数。免疫血清的最大稀释倍数越大，免疫血清中的抗VP8-5抗体的滴度就越高，用于产生免疫血清的蛋白的免疫原性也越高。
间接ELISA的结果如图1B所示，其中，纵坐标表示与VP8-5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，在使用铝佐剂的条件下，在免疫后第42天，VP8-5能在小鼠体内诱发抗体的产生，但是效率低于灭活病毒LLR。上述结果表明，在使用铝佐剂的 条件下，VP8-5蛋白具有免疫原性，能在小鼠体内诱发抗体的产生，但是其在动物体内诱发抗体产生的能力低于灭活病毒LLR。
中和抗体滴度的检测
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，通过ELISPOT检测免疫血清的中和抗体滴度(Li,Lin,Yu,et al.J Virol Methods,209 7-14,2014)。具体方案如下：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样品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样品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1C所示，其中，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，在使用铝佐剂的条件下，VP8-5在免疫后第42天(三次免疫后)能够在小鼠体内诱发中和抗体，但是免疫血清的中和抗体滴度(NT50)低于灭活病毒LLR。免疫程序完成后，使用VP8-5蛋白诱发的免疫血清的中和抗体滴度(NT50)仅达到64左右。上述结果表明，在使用铝佐剂的条件下，VP8-5能够诱导机体产生中和抗体，但是其所诱发的免疫血清的中和抗体滴度低于灭活病毒LLR。
在动物中的保护性的分析
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的比例进行交配。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，每天观察并记录乳鼠的腹泻情况，连续观察7天， 并根据粪便的形态进行评分。评分标准如图2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2C)，软粪便计为2分(图2B)，不成形水样便计为3分(图2A)。
图3显示了攻毒后不同免疫组的乳鼠的腹泻评分情况，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，实验组(VP8-5)乳鼠的腹泻评分与阴性对照组(NC)无显著差异。这说明，在使用铝佐剂的条件下，VP8-5保护机体、抵抗轮状病毒感染的能力较低(低于灭活病毒)，不能充分缓解轮状病毒感染导致的腹泻。
实施例2：编码截短的VP4蛋白的表达质粒的构建
轮状病毒LLR和SA11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LLR株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LLR株VP4蛋白的基因片段。
所使用的PCR引物如下：
上游引物：
5'-GGATCCCATATGATGGCTTCGCTCATTTAC-3'(SEQ ID NO:42)
5'-GGATCCCATATGTACAGACAATTACTTACGAATTC-3'(SEQ ID NO:43)
5'-GGATCCCATATGATACAGTTAATTGGATCAGAAAA-3'(SEQ ID NO:44)
5'-GGATCCCATATGGGATCAGAAAAAACGCAG-3'(SEQ ID NO:45)
5'-GGATCCCATATGTTGAATGGACCA-3'(SEQ ID NO:46)
下游引物：
5'-AAGCTTAGGTGTTTTGTATTGGTGG-3'(SEQ ID NO:47)
5'-AAGCTTATCTTCTAGCAACTATTGATCGT-3'(SEQ ID NO:48)
5'-AAGCTTAGTTCACTGCAGCTCTTCTAGC-3'(SEQ ID NO:49)
5'-AAGCTTACAATGACGTTTTCGATATAACTA-3'(SEQ ID NO:50)
5'-AAGCTTAGATATCTCGATTATATTGCATTTC-3'(SEQ ID NO:51)
5'-AAGCTTAAATTGAGTTTGCAAACTTAAAT-3'(SEQ ID NO:52)
5'-AAGCTTACCATTTATACCCTAGTCCACC-3'(SEQ ID NO:53)
5'-AAGCTTAATAGTTTGCTGGTTTGAATGA-3'(SEQ ID NO:54)
5'-AAGCTTATTCCTCTCCATCACGTATATATG-3'(SEQ ID NO:55)
5'-AAGCTTAATTCACTGAACATGTTGTATGTG-3'(SEQ ID NO:56)
5'-AAGCTTATCCTCCGTTATAGTTGAAGTC-3'(SEQ ID NO:57)
5'-AAGCTTAACGTGATATTACAAAGTCAGTTG-3'(SEQ ID NO:58)
5'-AAGCTTATACATAAGAGTTCTCTTTTATAACTTC-3'(SEQ ID NO:59)
5'-AAGCTTATGCTTGTGAATCATCCCAA-3'(SEQ ID NO:60)
5'-AAGCTTATAATGATCTCACATATACCATGTTT-3'(SEQ ID NO:61)
5'-AAGCTTATGCACATGTCACTTCATTTAAG-3'(SEQ ID NO:62)
5'-AAGCTTAAACTGGTAGTTGGAAATTATAAGTA-3'(SEQ ID NO:63)
5'-AAGCTTATGAGCCACCACTCATCACA-3'(SEQ ID NO:64)
5'-AAGCTTATAACGTTACTCCAGCTGAAC-3'(SEQ ID NO:65)
5'-AAGCTTATAATGACACAAAGTCTGTAAATTG-3'(SEQ ID NO:66)
5'-AAGCTTATGCTAAACTGAACCTAAATCTTA-3'(SEQ ID NO:67)
5'-AAGCTTACCTTGAAATAGAGAATGGCG-3'(SEQ ID NO:68)
5'-AAGCTTACGGTAACCCATATAACCCT(SEQ ID NO:69)
5'-AAGCTTAGAAGTCTCTTCCATTATTTGGA-3'(SEQ ID NO:70)
5'-AAGCTTAAATTAATGAAAATCTACCCGC-3'(SEQ ID NO:71)
5'-AGATCTAAGCTTATGATGGTACTAATAAAATTAATGAAAATC-3'(SEQ ID NO:72)
5'-AGATCTAAGCTTAAGTTTGATAATCATCATTTGATGGTACTA-3'(SEQ ID NO:73)
5'-AGATCTAAGCTTATGAGTTCATTATAGGAGTTTGATAATCAT-3'(SEQ ID NO:74)
5'-AGATCTAAGCTTATGTCTCACCGTCACTGAGTTCA-3'(SEQ ID NO:75)
5'-AGATCTAAGCTTACTGCCTCTCTAAGTCCTGTCTCA-3'(SEQ ID NO:76)
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。
利用上述引物，通过PCR扩增编码截短的VP4蛋白的基因，所使用的PCR反应体系如下：
Figure PCTCN2016082780-appb-000005
用于扩增编码截短的VP4蛋白的基因的引物对如表2所示：
表2：VP4截短蛋白编码基因扩增引物
Figure PCTCN2016082780-appb-000006
Figure PCTCN2016082780-appb-000007
PCR反应条件如下：95℃预变性5min，35个循环的(95℃，40s；55℃，80s；72℃，1min)，72℃终延伸10min。所获得的扩增产物用1.5％的琼脂糖凝胶电泳进行检测。
将各个PCR扩增产物连接入pMD18-T载体，并转化入大肠杆菌DH5α。然后，筛选阳性菌落，提取质粒，经Nde I/Hind III酶切鉴定，得到插入目的基因片段的阳性克隆质粒。对获得的各个阳性克隆质粒进行测序。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，其所编码的氨基酸序列如SEQ ID NO：2-34所示。
将上述阳性克隆质粒分别通过Nde I/Hind III进行酶切，获得各个截短的VP4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，转化入大肠杆菌DH5α。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表达质粒。
取1μL阳性表达质粒，转化40μL感受态大肠杆菌Bl21(DE3)(购自NEB公司)。将转化后的大肠杆菌涂布于含卡那霉素(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，加入终浓度为10％的甘油，于-70℃保存。
实施例3：截短的VP4蛋白的表达
从-70℃取出实施例2制备的携带阳性表达质粒的大肠杆菌菌液，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波长条件下的吸光值达0.5时，加入IPTG至终浓度为1mM，在180rpm，25℃下继续培养6小时。
取1ml上述菌液，进行离心，并收集菌体沉淀。向菌体沉淀中加入100μL去离子水，将菌体重悬。然后，加入20μL 6*上样缓冲液，混匀，并在沸水浴中温育10min，以裂解细胞。取10μL样品，通过12％的SDS-PAGE进行分析。SDS-PAGE结果如图4A-4D所示。结果显示，除了26-311，26-331和26-381的表达量相对较低以外，其他的截短蛋白均可在大肠杆菌中高表达。
实施例4：截短的VP4蛋白的纯化和表征
离心实施例3获得的大肠杆菌菌液，收集菌体沉淀。按照15ml/g 湿菌的比例，用50mM TB8.0将表达截短的VP4蛋白的菌体重悬。然后，通过超声破碎大肠杆菌细胞，超声破碎条件为：超声2s，间隔4s，每克菌体超声破碎时间为4min。超声破碎后，以25000g进行离心，收集上清(即，含有重组表达的截短VP4蛋白的大肠杆菌裂解液可溶性级分)。
可通过两步层析法来纯化大肠杆菌裂解液可溶性级分中的截短VP4蛋白。对于26-331，26-351，26-381，26-411，26-441和26-461而言，在进行两步层析法之前，先用40％的硫酸铵处理大肠杆菌裂解液可溶性级分，然后离心并收集蛋白质沉淀；随后，将获得的蛋白质沉淀溶解于50mM Tris-HCl pH 8.0中，并应用于两步层析法。两步层析法的方案如下。
首先，通过Q-HP阴离子交换层析进行初步纯化，获得纯度约60％的截短的VP4蛋白，其具体纯化条件如下：
仪器系统：GE Healthcare公司(原Amershan Pharmacia公司)生产的AKTA explorer 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：A泵：50mM Tris-HCl pH 8.0；
        B泵：50mM Tris-HCl pH 8.0，2M NaCl
流速：6mL/min。
检测器波长：280nm。
样品为之前制备的含有重组表达的截短的VP4蛋白的上清(即，大肠杆菌裂解液可溶性级分或溶解于50mM Tris-HCl pH 8.0中的蛋白质样品)。
洗脱程序为：50mM NaCl洗脱目的蛋白，1M NaCl洗脱杂蛋白。收集用50mMNaCl洗脱的级份，共获得30mL的含有重组表达的截短蛋白的经初步纯化的样品(注意：在上述初步纯化过程中，截短蛋白1-476，26-331，26-351，26-381，26-411，26-441和26-461不与层析柱结合，包含在穿透级份(flow-through)中。因此，收集含有这 些截短蛋白的穿透级份作为经初步纯化的样品)。
将上述经阴离子交换层析初步纯化的样品分别透析至含有2MNaCl的TB8.0缓冲液中，然后使用Phenyl sepharose-HP疏水亲和层析进行第二次纯化。
层析介质：Phenyl sepharose-HP(GE Healthcare公司)。
柱体积：5.5cm*20cm。
缓冲液：A泵：50mM Tris-HCl pH 8.0，2M NaCl；
        B泵：50mM Tris-HCl pH 8.0
流速：6mL/min。
检测器波长：280nm。
样品为经Q-HP层析柱纯化且透析至2M NaCl溶液中的产物
洗脱程序为：1.5M NaCl洗脱杂蛋白，1M NaCl洗脱目的蛋白，50mM NaCl洗脱杂蛋白。收集用1M NaCl洗脱的级份，共获得30mL经纯化的重组表达的截短的VP4蛋白(注意：在上述第二次纯化过程中，截短蛋白1-476，26-331，26-351，26-381，26-411，26-441，26-461和26-471用50mM TB8.0进行洗脱，收集用50mM TB8.0洗脱的级份)。
取经上述方案纯化的样品150μL，加入30μL 6X Loading Buffer，混匀并于100℃水浴中温育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5中。
图5A-5B显示了经纯化的各种截短VP4蛋白的SDS-PAGE结果。在图5A中，泳道从左到右依次为：蛋白质分子量标记(Marker)，1-476，6-476，22-476，26-476，65-476，26-231，26-271，26-331和26-351。在图5B中，泳道从左到右依次为：蛋白质分子量标记(Marker)，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492和26-497。图5A和5B的结果显示，经过上述纯化步骤后，截短蛋白1-476，6-476，22-476，26-476，65-476，26-231，26-271，26-331，26-351，26-381，26-411，26-441，26-461，26-471，26-482，26-487， 26-492和26-497的浓度均达到0.2mg/ml以上，且纯度大于80％。
另外，还使用HPLC对上述经纯化的样品在不同缓冲条件下的均一性进行了分析。所使用的仪器为安捷伦1200高效液相色谱仪，层析柱为G3000PWXL或G5000PWXL，柱体积为7.8*300mm，流速为0.5ml/min，检测波长为280nm；其中，26-331，26-351，26-381，26-411，26-441和26-461的均一性用G5000PWXL来进行检测；其他蛋白使用G3000PWXL来进行检测。SEC-HPLC的分析结果示于图6和图7中。
结果显示：在TB8.0的条件下，截短蛋白1-476，26-331，26-351，26-381，26-411，26-441和26-461的保留时间均在11min左右，其分子量超过600kDa；这说明，这些截短蛋白主要以高聚体的形式存在。截短蛋白26-271的保留时间为约16min；这说明，该蛋白主要以单体形式存在。其余蛋白(6-476、22-476、26-476、26-471、26-482、26-487、26-492、26-497)的保留时间均为约13-14min，与IgG(150kDa)的保留时间相当；这说明，这些蛋白主要以三聚体的形式存在。
另外，在TB8.0+1M NaCl的条件下，截短的VP4蛋白26-476，26-482，26-487，26-492和26-497的保留时间均为约15min，与VP8二聚体(40kDa)相当；这说明，这些截短蛋白在TB8.0+1M NaCl条件下主要以单体形式存在。这进而表明，在盐存在的条件下，26-476，26-482，26-487，26-492，26-497的构象受到盐离子影响，导致三聚体发生解聚，形成单体。
此外，图6和图7的结果还显示，所获得的截短VP4蛋白的主要吸收峰所占比例几乎都在80％或甚至90％以上；这说明，这些截短蛋白具有良好的均一性，适合于工业化批量生产，且有利于准确确定用药剂量。
图6和图7的实验结果还概述于下面的表3中。
表3
Figure PCTCN2016082780-appb-000008
Figure PCTCN2016082780-appb-000009
实施例5：截短的VP4蛋白的体外组装及表征
通过下述方案来进行截短蛋白26-476的体外组装。在室温条件下，将截短蛋白26-476由TB8.0缓冲液透析至表4指定的透析缓冲液中，6小时后更换透析缓冲液一次。透析结束后，以12000rpm/min离心蛋白溶液10分钟，留取上清液。随后，将上清液在表4指定的温度下静置30分钟到24小时。静置后，将上清液迅速冰浴，并以12000rpm/min离心10分钟。收集第二次离心后的上清液(其含有体外组装的26-476)，用于下一步分析。
通过HPLC来分析所获得的上清液中由截短蛋白26-476体外组装所形成的高聚体的均一性。HPLC分析所用仪器为安捷伦公司生产的1200高效液相色谱仪或Waters公司生产的E2695高效液相色谱仪，层析柱为G5000PWXL，柱体积为7.8*300mm，流速为0.5ml/min，检测波长为280nm。SEC-HPLC的分析结果示于表4和图8A中。
表4：截短蛋白的体外组装的条件及结果
Figure PCTCN2016082780-appb-000010
图8A显示了在50mM Tris-HCl(pH8.0)中37℃静置12小时后的截短蛋白26-476的分子筛分析结果。结果显示，截短蛋白26-476在50mM Tris-HCl(pH8.0)中37℃静置12小时后能够形成均一的高聚体(保留时间为12.4分钟)，并且高聚体的比例高达97.2％。
从表4和图8A的结果可以看出，在pH7.4-9.6的范围内，截短蛋白26-476在37℃-50℃的温度下静置后，能够组装形成均一的高聚体，并且高聚体的比例可超过90％。此外，表4的结果还显示，盐离子(例如NaCl)的存在可抑制截短蛋白26-476形成高聚体。盐离子(例如NaCl)的浓度越高，所形成的高聚体的比例就越低。
此外，还使用电镜来观察由截短蛋白26-476体外组装所形成的高聚体，所使用的仪器为FEI公司生产的G2 Spirit电镜。简言之，将样品固定于喷炭的铜网上，并用2％的磷钨酸(pH7.4)负染30分钟，随后使用电镜进行观察。结果示于图8B，其中可见大量不对称结构的颗粒，半径为约10nm。这些结果表明，截短蛋白26-476能够在体外组装成均一的高聚体。
实施例6：截短的VP4蛋白的酶切验证
将实施例4中获得的经纯化的截短的VP4蛋白在37℃下用胰蛋白酶酶切1小时。取酶切后的组分100ul，加入20μL 6X Loading Buffer，混匀并于100℃水浴中温育10min。随后取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9。
图9显示了经酶切和未经酶切的截短蛋白26-476，26-482，26-487，26-492，26-497的SDS-PAGE结果。泳道上的数字1表示，样品未经胰酶处理；数字2表示，样品经0.1mg/ml胰酶处理。最右侧的泳道所使用的样品为VP8-5，用作对照。图9的结果显示，上述截短蛋白均能被胰酶识别并发生酶切，其酶切位点是暴露的。
实施例7：截短的VP4蛋白的抗原性分析
将实施例4中获得的经纯化的截短VP4蛋白包被于平板上，获得经包被的平板。将中和抗体A3、B1、B5、B6、D6、E2、E5、8F6(本实验室通过杂交瘤技术自制，浓度均为1mg/ml)梯度稀释，然后按照实施例1中描述的间接ELISA方法进行检测。
检测结果示于图10中，其中横坐标表示各截短蛋白，纵坐标表示与各截短蛋白发生反应(OD450/620高于0.2)的最低抗体浓度。结果显示，各截短的VP4蛋白均具有良好的抗原性(即，抗体反应性)。
实施例8：截短的VP4蛋白的免疫原性的分析
将实施例4中获得的经纯化的截短蛋白26-476包被于平板上，获得经包被的平板。按照实施例1中描述的方案，在使用铝佐剂的条件下，用各待测样品(实施例4中获得的各种截短VP4蛋白、26-476的三聚体、实施例5中获得的26-476的高聚体、灭活病毒(RV，用作阳性对照)和PBS(NC，阴性对照))来分别免疫Balb/c小鼠，并收集小鼠血清。随后，按照实施例1中描述的方案，使用包被了26-476的平板通过间接ELISA来检测小鼠血清中的抗体滴度。
间接ELISA的结果如图11A-11D所示，其中，横坐标表示用于制备免疫血清的蛋白样品，纵坐标表示与26-476具有反应性的免疫血清的最大稀释倍数(即，抗体滴度)；并且，图11A、11B、11C和11D显示了来自不同免疫批次的结果。结果显示，在使用铝佐剂的条件下，在免疫后第42天，各蛋白样品均能在小鼠体内诱发抗体(免疫血清中的抗体滴度(GMT)均可达到102-105或更高)；并且，除了26-271外，其他蛋白样品所诱发的抗体滴度均高于RV诱发的抗体滴度(1-476、6-476、22-476、26-331、26-351、26-381、26-411、26-441、26-461、26-471、26-476、26-487、26-492、26-476的三聚体和26-476的高聚体)，或至少与RV诱发的抗体滴度相当(65-476、26-482和26-497)。
结合实施例1的实验结果可知，在使用铝佐剂的条件下，各蛋白样品(除了26-271外)均具有良好的免疫原性，能够刺激小鼠产生高滴度的抗体；其免疫原性显著强于VP8-5，免疫血清中的抗体滴度显著高于来自用VP8-5免疫的小鼠的血清。此外，图11D的实验结果还表明，截短蛋白26-476的高聚体的免疫原性显著高于26-476的三聚体(p＝0.005)。
实施例9：截短的VP4蛋白的免疫中和活性的分析
使用实施例1中描述的方案，用各待测样品(实施例4中获得的各种截短VP4蛋白、26-476的三聚体、实施例5中获得的26-476的高聚体、灭活病毒(RV，用作阳性对照)和PBS(NC，阴性对照)) 来免疫实验组Balb/c小鼠(每组7只小鼠)，并收集免疫血清。
随后，按照实施例1中描述的检测方法，评价所收集的各个免疫血清样品中的中和抗体滴度。免疫血清的中和抗体滴度的分析结果如图12A-12D所示，其中，横坐标表示用于制备免疫血清的蛋白样品，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)。图12A、12B、12C和12D显示了来自不同免疫批次的结果。结果显示，在使用铝佐剂的条件下，在免疫后第42天(三次免疫后)，各蛋白样品均能在小鼠体内诱发中和抗体，中和抗体滴度(NT50)均可达到28-214或更高的水平；并且，除了26-271外，其他蛋白样品所诱发的中和抗体滴度均与RV诱发的中和抗体滴度相当(6-476、22-476、26-476、65-476、26-471、26-482、26-487、26-492、26-497和26-476的三聚体)，或甚至高于RV诱发的中和抗体滴度(1-476、26-331、26-351、26-381、26-411、26-441、26-461和26-476的高聚体)。
结合实施例1的实验结果可知，在使用铝佐剂的条件下，各蛋白样品(除了26-271外)均具有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。各蛋白样品(除了26-271外)诱导机体产生中和抗体的能力均显著优于VP8-5，从而具有更强的抵抗/预防RV感染的能力。此外，图12D的实验结果还表明，截短蛋白26-476的高聚体诱导机体产生中和抗体的能力显著高于26-476的三聚体(p<0.001)，具有显著更强的抵抗/预防RV感染的能力。
实施例10：截短的VP4蛋白在动物中的保护性的评价
使用实施例1中所述的方案，用各待测样品(实施例4中获得的截短VP4蛋白、26-476的三聚体、实施例5中获得的26-476的高聚体、灭活病毒(RV，用作阳性对照)和PBS(NC，阴性对照))免疫Balb/c小鼠(每组7只小鼠)，并收集血清。
按照实施例1中所述的方案来评价各蛋白样品在动物中的保护性。 除1-476和6-476免疫组(这两组的动物未交配成功)之外，其余免疫组的实验结果如图13-14所示。
图13A-13D显示了攻毒后1-7天来自不同免疫组(用22-476，26-476，65-476，26-271，26-331，26-351，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492，26-497，26-476的三聚体，26-476的高聚体，灭活的轮状病毒(RV，阳性对照)或PBS(NC，阴性对照)进行免疫)的乳鼠的腹泻评分情况，其中，纵轴表示平均腹泻评分；横轴表示小鼠攻毒后天数；RV：灭活的轮状病毒；NC：阴性对照(PBS)；三聚体：26-476的三聚体；高聚体：26-476的高聚体。图14A-14D显示了不同免疫组(用22-476，26-476，65-476，26-271，26-331，26-351，26-381，26-411，26-441，26-461，26-471，26-482，26-487，26-492，26-497，26-476的三聚体，26-476的高聚体，灭活的轮状病毒(RV，阳性对照)或PBS(NC，阴性对照)进行免疫)的乳鼠在攻毒后的平均腹泻持续时间和攻毒后48小时的平均腹泻评分，其中，平均腹泻天数以柱形图表示，平均腹泻评分以曲线图表示，左纵轴表示平均腹泻天数；右纵轴表示腹泻评分；横轴表示各蛋白样品所对应的免疫组。
结果显示，在平均腹泻评分和平均腹泻天数方面，各蛋白样品所对应的免疫组均显著优于NC组。这表明，各蛋白样品均具有显著的保护性，能够帮助小鼠抵抗轮状病毒感染和由轮状病毒感染导致的腹泻。此外，结果还显示，26-331，26-351，26-381，26-411，26-441，26-461，26-476，26-476的三聚体，以及26-476的高聚体的保护性与RV相当，或甚至优于RV。结合实施例1的实验结果可知，在使用铝佐剂的条件下，这些蛋白样品在动物中的保护性均显著优于VP8-5。此外，图13D和图14D的实验结果还表明，截短蛋白26-476的高聚体在动物中的保护能力显著高于26-476的三聚体，可用于制备具有更高效力的疫苗。
实施例11：来源于不同病毒株的截短VP4蛋白的表达、纯化以 及其免疫保护性的评价
根据Gene bank中提供的EDIM毒株的VP4基因序列(登录号：AF039219.2)，由上海生工生物技术有限公司合成编码来源于轮状病毒EDIM株的26-476的基因片段。另外，根据Gene bank中提供的轮状病毒P[6]的VP4基因序列(登录号：FJ183356.1)，由上海生工生物技术有限公司合成编码来源于轮状病毒P[6]的26-476的基因片段。随后，以合成的基因片段为模板，通过PCR反应，扩增获得编码来源于轮状病毒P[6]和EDIM的截短蛋白26-476的基因片段。
另外，如实施例2中所述，用罗猴胚胎肾细胞系(MA-104)细胞培养轮状病毒SA11株，获得轮状病毒SA11的病毒培养物。轮状病毒P[4]和P[8]来源于重庆医科大学附属儿童医院收集的腹泻标本，标本编号为20131281(P[4])和编号20131028(P[8])。
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，从病毒培养物或病毒标本中提取轮状病毒SA11、P[4]、P[8]的基因组RNA，并通过反转录获得编码来源于各个毒株的VP4蛋白的cDNA。以获得的cDNA为模板，通过PCR反应，扩增获得编码来源于毒株SA11、P[4]、P[8]的截短蛋白26-476的基因片段。
按照实施例2中描述的方法构建克隆质粒和表达质粒，所使用的PCR引物如下：
上游引物：
5'-GGATCCCATATGGGATCGGAGAAAACTCAA-3'(SEQ ID NO：77)
5'-GGATCCCATATGGGATCAGAGAAAAGTCAAAAT-3'(SEQ ID NO:79)
5'-GGATCCCATATGGGATCAGAAAAAACTCAAAATG-3'(SEQ ID NO：81)
5'-GGATCCCATATGGGAGCAGAGAAGACACA-3'(SEQ ID NO：83)
5'-GGATCCCATATGGGATCAACTAAATCACAAAATG-3'(SEQ ID NO：85)
下游引物：
5'-AAGCTTAATTAGTTGGAACTAAAGAAATAAGT-3'(SEQ ID NO:78)
5'-AAGCTTAATTAGACGGTACTAATGAAA-3'(SEQ ID NO:80)
5'-AAGCTTAGTTGGTTGGAACTAAAGAAA-3'(SEQ ID NO:82)
5'-AAGCTTAATCGTTGGACGGCAC-3'(SEQ ID NO:84)
5'-AAGCTTATGATGGCACTAATGATATAAGT-3'(SEQ ID NO:86)
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。
用于扩增各基因片段的引物对如表5所示：
表5：用于扩增来源于不同毒株的26-476的编码基因的引物对
Figure PCTCN2016082780-appb-000011
截短蛋白26-476-P[4]、26-476-P[6]、26-476-P[8]、26-476-EDIM、26-476-SA11的氨基酸序列分别如SEQ ID NO:35-39所示。
按照实施例3-4中描述的方法，在大肠杆菌中表达来源于不同病毒株的截短蛋白26-476(即，26-476-P[4]、26-476-P[6]、26-476-P[8]、26-476-EDIM、26-476-SA11)，并通过两步层析法进行纯化；然后通过SDS-PAGE鉴定所纯化的蛋白。
SDS-PAGE结果如图15所示，其中，泳道从左到右依次为：来源于轮状病毒LLR的截短蛋白26-476；来源于轮状病毒SA11的截短蛋白26-476-SA11；来源于轮状病毒EDIM的26-476-EDIM；来源于轮状病毒P[8]的截短蛋白26-476-P[8]；来源于轮状病毒P[6]的截短蛋白26-476-P[6]；来源于轮状病毒P[4]的截短蛋白26-476-P[4]；和，蛋白质分子量标记(Marker)。
结果表明，本发明的方法适用于不同的病毒株。来源于不同病毒株的截短VP4蛋白(26-476)均可在大肠杆菌中有效表达，且经色谱法纯化后，其纯度均可达到80％以上。
另外，还按照实施例4描述的方法，使用HPLC对上述经纯化的 截短蛋白26-476在50mM TB8.0条件下的均一性进行了分析。SEC-HPLC的分析结果示于图16中。
结果显示：在TB8.0的条件下，来源于不同毒株的截短VP4蛋白26-476的保留时间均在13-14min左右，与IgG(150kDa)的保留时间相当；这说明这些蛋白主要以三聚体的形式存在。此外，图16的结果还显示，所获得的截短蛋白26-476的主要吸收峰所占比例几乎都在80％以上；这说明这些截短蛋白均具有较高的均一性，适合于工业化批量生产，且有利于准确确定用药剂量。
进一步，将来源于不同轮状病毒毒株的截短蛋白26-476分别包被于平板上，获得经包被的平板。按照实施例1中描述的方案，使用如上获得的经纯化的截短蛋白26-476(即，26-476-P[4]、26-476-P[6]、26-476-P[8]、26-476-EDIM、26-476-SA11，来源于LLR的26-476和PBS(阴性对照))来免疫Balb/c小鼠，并收集小鼠血清。随后，按照实施例1中描述的方案，使用经包被的平板通过间接ELISA来检测小鼠血清中的抗体滴度。
间接ELISA的结果如图17所示，其中，横坐标表示用于制备免疫血清的截短蛋白所源自的病毒株，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)；P[4]：26-476-P[4]；P[6]：26-476-P[6]；P[8]：26-476-P[8]；SA11：26-476-SA11；EDIM：26-476-EDIM；LLR：实施例4中制备的26-476。
结果显示，在使用铝佐剂的条件下，在免疫后第42天，来源于不同病毒株的26-476蛋白均能在小鼠体内有效诱发抗体的产生，且所诱发的免疫血清中的抗体滴度(GMT)基本上相当(抗体滴度均可达到104-105或更高，显著高于阴性对照组)。这些结果表明，在使用铝佐剂的条件下，来源于不同病毒株的26-476蛋白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；并且，来源于不同病毒株的26-476蛋白的免疫原性基本上相当，且均显著强于VP8-5。
进一步地，使用实施例1中描述的方案，用来源于不同病毒株的26-476蛋白(26-476-SA11；26-476-EDIM；来源于LLR的26-476) 免疫实验组Balb/c小鼠(每组7只小鼠)，并收集免疫血清。随后，按照实施例1中描述的检测方法，评价所收集的各个免疫血清样品中的中和抗体滴度。免疫血清的中和抗体滴度的分析结果如图18所示，其中，横坐标表示用于制备免疫血清的蛋白样品所源自的病毒株；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)；SA11：26-476-SA11；EDIM：26-476-EDIM；LLR：实施例4中制备的26-476。
结果显示，在使用铝佐剂的条件下，在免疫后第42天(三次免疫后)，SA11、EDIM和LLR毒株的26-476蛋白均能在小鼠体内诱发高滴度的中和抗体，中和抗体滴度(NT50)均可达到210-214或更高的水平；并且，26-476-SA11和26-476-EDIM所诱发的中和抗体滴度甚至高于来源于LLR的26-476。由此可见，来源于SA11毒株和EDIM毒株的26-476蛋白的免疫中和活性甚至优于来源于LLR的26-476蛋白。
此外，通过类似的方法还可证实，来源于轮状病毒P[4]、P[6]和P[8]的26-476蛋白具有良好的免疫中和活性，能够在小鼠体内诱发高滴度的中和抗体。
这些结果表明，在使用铝佐剂的条件下，来源于不同病毒株的26-476蛋白均具有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。
进一步地，使用实施例1中所述的方案，用来源于不同病毒株的26-476蛋白(26-476-SA11，26-476-EDIM和PBS(NC，阴性对照))免疫Balb/c小鼠(每组7只小鼠)，并收集血清。随后，按照实施例1中所述的方案来评价各蛋白样品在动物中的保护性。实验结果如图19A-19B所示。
图19A显示了不同免疫组(用26-476-SA11或PBS(NC，阴性对照)进行免疫)的乳鼠用SA11病毒攻毒后1-7天的腹泻评分；图19B显示了不同免疫组(用26-476-EDIM或PBS(NC，阴性对照)进行免疫)的乳鼠用EDIM病毒攻毒后1-12天的腹泻评分；其中，横 坐标表示攻毒后天数，纵坐标表示平均腹泻评分。结果显示，与来源于LLR的26-476类似，来源于SA11和EDIM的26-476蛋白均具有显著的保护性，能够帮助小鼠抵抗轮状病毒感染和由轮状病毒感染导致的腹泻。
此外，还按照实施例1中描述的方案，用26-476-EDIM或PBS(NC，阴性对照)来对成年小鼠进行免疫(共免疫三次)。免疫程序完成后，用500μL的EDIM病毒(2*107copies/ml)来攻击这些小鼠，并在攻毒后1-7天，每天收集小鼠的粪便标本，并用PBS重悬为1％的粪便悬液。随后，通过荧光定量PCR法对各个粪便悬液样本中的病毒进行定量检测。实验结果如图19C所示。
图19C显示了在攻毒后1-7天，来自经26-476-EDIM或PBS免疫的小鼠的粪便悬液样本中的病毒量，其中，横坐标表示攻毒后天数，纵坐标表示每ml粪便悬液样品中所含的EDIM基因组的拷贝数。由于荧光定量PCR检测试剂盒的检测下限为104copies/ml，因此，将阴性检测结果定义为103copies/ml。结果显示，在攻毒后，在用PBS免疫的小鼠的粪便中检测到明显的排毒，而在用26-476-EDIM免疫的小鼠的粪便中，则未检测到排毒。图19A-19C的结果表明，26-476-EDIM不仅能够使小鼠抵抗轮状病毒感染以及由轮状病毒感染导致的腹泻，而且能够抑制病毒在小鼠粪便中的排放(即，排毒)。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且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 (12)

  1. 一种截短的轮状病毒VP4蛋白或其变体，其与野生型轮状病毒VP4蛋白相比，N端截短了1-64个氨基酸(例如5-64个氨基酸)，并且C端终止于野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置276-497中的任一位置相对应的位置；
    优选地，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白的N端截短了1-5个，5-21个，21-25个或25-64个氨基酸；并且C端终止于所述野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置276-497(例如氨基酸位置281-497、291-497、301-497、311-497、321-497、331-497、341-497、351-497、361-497、371-497、381-497、391-497、401-497、411-497、421-497、431-497、441-497、451-497、461-497、471-497、476-497、482-497、487-497、或492-497)中的任一位置相对应的位置；
    优选地，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白的N端截短了1个、5个、21个、25个或64个氨基酸，并且C端终止于野生型轮状病毒VP4蛋白的下述位置：与SEQ ID NO:40的氨基酸位置281、291、301、311、321、331、341、351、361、371、381、391、401、411、421、431、441、451、461、471、476、482、487、492或497相对应的位置；
    优选地，与野生型轮状病毒VP4蛋白相比，该截短的轮状病毒VP4蛋白的N端截短了25个氨基酸且C端终止于下述位置：与SEQ ID NO:40的氨基酸位置331、351、381、411、441、461、471、476、482、487、492或497相对应的位置；或者，该截短的轮状病毒VP4蛋白的N端截短了1个、5个、21个、25个或64个氨基酸且C端终止于下述位置：与SEQ ID NO:40的氨基酸位置476相对应的位置；
    优选地，所述野生型轮状病毒VP4蛋白是来源于轮状病毒LLR株、SA11株、EDIM株的VP4蛋白，或者是来源于P[4]、P[6]、或P[8]基因型轮状病毒的VP4蛋白；
    优选地，所述野生型轮状病毒VP4蛋白具有选自下列的氨基酸序列：SEQ ID NO:40和87-91；
    优选地，所述截短的轮状病毒VP4蛋白具有选自下列的氨基酸序列：SEQ ID NO:2-5和10-39。
  2. 一种分离的核酸，其编码权利要求1的截短的轮状病毒VP4蛋白或其变体。
  3. 包含权利要求2的分离的核酸的载体。
  4. 包含权利要求2的分离的核酸或权利要求3的载体的宿主细胞。
  5. 一种多聚体，其包含或者由权利要求1的截短的轮状病毒VP4蛋白或其变体组成。
  6. 一种组合物，其包含权利要求1的截短的轮状病毒VP4蛋白或其变体，或权利要求2的分离的核酸，或权利要求3的载体，或权利要求4的宿主细胞，或权利要求5的多聚体。
  7. 一种药物组合物(例如疫苗)，其包含权利要求1的截短的轮状病毒VP4蛋白或其变体或者权利要求5的多聚体，任选地还包含药学可接受的载体和/或赋形剂，
    优选地，所述药物组合物还包含佐剂，例如铝佐剂；
    优选地，所述截短蛋白或其变体或者所述多聚体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。
  8. 制备权利要求1的截短的轮状病毒VP4蛋白或其变体的方法，其包括，在允许所述截短蛋白或其变体表达的条件下，培养权利要求4的宿主细胞；和，回收所表达的截短蛋白或其变体，
    优选地，所述方法包括步骤：利用大肠杆菌来表达所述截短蛋白或其变体，然后将大肠杆菌裂解，并从裂解液中纯化获得所述截短蛋白或其变体，优选地，所述纯化包括层析(例如两步层析法)。
  9. 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VP4蛋白或其变体或者权利要求5的多聚体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。
  10. 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VP4蛋白或其变体，或权利要求5的多聚体，或权利要求7的药物组合物施用给受试者，
    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。
  11. 权利要求1的截短的轮状病毒VP4蛋白或其变体或者权利要求5的多聚体在制备药物组合物(例如疫苗)中的用途，所述药物组合物(例如疫苗)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。
  12. 权利要求1的截短的轮状病毒VP4蛋白或其变体或者权利要求5的多聚体，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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RS62664B1 (sr) 2021-12-31
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CN106167518A (zh) 2016-11-30
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JP6808650B2 (ja) 2021-01-06
CN112724207A (zh) 2021-04-30
PT3299384T (pt) 2021-11-23
EP3299384A1 (en) 2018-03-28
US11339194B2 (en) 2022-05-24
EP3299384B1 (en) 2021-09-08
US20180141978A1 (en) 2018-05-24
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US20200308233A1 (en) 2020-10-01
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