CN105085639B - Truncated 8 albumen of rotavirus vp and application thereof - Google Patents

Abstract

The present invention relates to a kind of truncated 8 albumen of rotavirus vp, fusion protein comprising the truncated protein, conjugate comprising the truncated protein, the coded sequence and preparation method of the truncated protein and fusion protein, pharmaceutical composition and vaccine comprising the truncated protein, fusion protein or conjugate, the truncated protein, fusion protein, conjugate, pharmaceutical composition and vaccine can be used for preventing, mitigate or treat rotavirus infection and by rotavirus infection caused by disease, such as rotaviral gastroenteritis and diarrhea etc..The invention further relates to the purposes that above-mentioned truncated protein, fusion protein, conjugate are used to prepare pharmaceutical composition or vaccine, described pharmaceutical composition or vaccine be used to prevent, mitigate or treat rotavirus infection and by rotavirus infection caused by disease, such as rotaviral gastroenteritis and diarrhea etc..

Description

Truncated 8 albumen of rotavirus vp and application thereof

Technical field

The present invention relates to biochemistry, molecular biology, Molecular Virology and field of immunology.Specifically, the present invention relates to And a kind of 8 albumen of truncated rotavirus vp, the fusion protein comprising the truncated protein, the conjugation comprising the truncated protein The coded sequence and preparation method of object, the truncated protein and fusion protein include the truncated protein, fusion protein or conjugation The pharmaceutical composition and vaccine of object, the truncated protein, fusion protein, conjugate, pharmaceutical composition and vaccine can be used for preventing, Mitigate or treatment rotavirus infection and by rotavirus infection caused by disease, such as rotaviral gastroenteritis and Diarrhea etc..The invention further relates to the use that above-mentioned truncated protein, fusion protein, conjugate are used to prepare pharmaceutical composition or vaccine On the way, described pharmaceutical composition or vaccine are used to prevent, mitigate or treat the infection of rotavirus and the infection institute by rotavirus Caused disease, such as rotaviral gastroenteritis and diarrhea etc..

Background technique

Diarrhea Caused by Porcine rotavirus is the most important acute infectious intestinal disease of infant in worldwide, with diarrhea and is taken off Water is characterized, and the death rate is high, causes serious burden on society and financial burden.The whole world about 1.1 hundred million 5 years old or less every year Children suffer from rotaviral gastroenteritis, wherein the necessary hospitalization of 2,000,000 children, about 453000 children die of rotavirus every year Property diarrhea, wherein the death of developing country and less developed country account for sum 85% or more (LeBaron, C.W.et al.1990)。

After infecting rotavirus (Rotavirus, RV), because dehydration and electrolyte disturbance lead to severe diarrhea and death, mesh Before there is no specific medicament, clinical treatment is also symptomatic treatment, therefore, is badly in need of exploitation efficient vaccine and therapeutic strategy, to reduce The morbidity and mortality of the disease mitigate society caused by the disease and financial burden (Parashar, U.D.et al.2003).

However, the two kinds of Rotavirus Vaccines listed at present: RotaTeq (Merk) and Rotarix (GSK), in colyliform The place of viral threat most serious, vaccine immunity effect are barely satisfactory.Meanwhile it has been reported that can after oral Rota Teq vaccine The molecular recombination between severe diarrhea and vaccine strain can occur；In addition, the relevance of RotaTeq and Rotarix and entembole It is not excluded (Lepage, P.et al.2007).Therefore, exploitation a new generation substitution vaccine, the vaccine are badly in need of in this field It can cause significant cell response and humoral response simultaneously, the antibody that can neutralize viral infection be generated, so as to be used for Prevention, mitigate or treatment rotavirus infection and by rotavirus infection caused by disease.

In recent years, in the vaccine research and development of rotavirus, attention is gone to from inactivation and Attenuated Virus Vaccines Recombinant vaccine.

Rotavirus (Rotavirus, RV) belongs to arc reovirus virus section (Reoviridae), and genome is by 11 segments DsRNA composition, encodes 6 structural proteins (VP1-4, VP6, VP7) and 6 non-structural proteins (NSP1-6).Mature virion Son is three layers of capsid structure, and outermost layer forms (Bridger, J.C.et by furcella shape albumen VP4 and glycoprotein VP7 al.1976).VP7 is directly related with duplication of the virus in mature intestinal epithelial cell.VP4 albumen is directed not only to the viscous of RV Attached, intrusion, and with the physicochemical properties phase such as RV is coagulation, infectious enhancing after neutralization activity, virulence and protease cracking It closes.VP7 and VP4 can independently induce virucidin, and the antibody of anti-VP7 and VP4 can all prevent the intrusion of virus.Therefore VP7 and VP4 becomes the preferred object molecule (Arias, C.F.et al.2002) of vaccine development.

VP8 albumen is a part (AA1-231) of the outermost layer structural proteins VP4 (AA1-776) of RV virion.VP4 Can form VP8* and VP5* segment after pancreatin cracks, and the former contain VP4 specificity Main Antigenic (Clark, S.M.et al.1981).This prompt, can use VP8* albumen to develop novel RV subunit vaccine.

Although being classified as structural proteins, now clear VP4 the viropexis stage exercise critical function.Therefore, VP4 plays the role of extremely important (Arias, C.F.et not only in virus structure in RV reproduction process al.1996)。

VP4 played during viropexis effect depend on the albumen and target cell surface expression specificity by The interaction of body.In RV course of infection, SA is considered as first and is one of most important cell receptor, it passes through It plays a role in conjunction with VP8.This interaction is so that RV is adsorbed to cell surface, to start the of RV infection cell One step.It for the RV of NA responsive type, is found by the means of NMR spectroscopy, the core part and α-N- second of VP8* Acyl neuraminic acid combines, this binding site is highly conserved, it is no longer necessary to the participation of other glycosyl sequences；And it is non-for NA Whether the RV strain of responsive type, VP8* still can not be very clear (Blanchard H, et al.2007) using identical site.

The first step of VP8* and SA interacts so that virion is anchored on cell surface.VP8* and SA motif is mutually known After not combining, occurred conformation changes virus capsid protein therewith, in favor of searching subsequent more specific acceptor molecules, thus Mediate retroviral next step enters born of the same parents' process (Dormitzer PR, et al.2002).

It can inhibit viropexis for the neutralization immune response of VP8, to inhibit target cell infected, this makes the albumen It is considered as the outstanding target for establishing anti-RV vaccine.One it is important it is a prerequisite that VP8 evoke before viropexis it is immune Neutralizing antibody caused by response can interact with the VP8 of virus, prevent the VP8 of virus in conjunction with cell receptor, to hinder Only VP8 allosteric inhibits viropexis.

The purification expression of VP8 is based on escherichia expression system more at present, mainly has solubility expression to purify and wrap Contain body and purifies two kinds of forms.

Although solubility expression purifying is able to maintain the relatively natural conformation of VP8 and high immune neutralization activity, soluble table Low up to measuring, way of purification is relative complex, purifies that resulting protein conformation is inhomogenous, and there are polymers, and stability is poor, is easy hair Raw degradation (Favacho AR, et al.2002).Particularly, the present inventor it has been investigated that, purified VP8 is put at 4 DEG C It is significant to degrade (referring to Examples below 3 and Fig. 1) after setting three days.This brings greatly to large-scale industrial production VP8 It is difficult.

Although inclusion body purification mode technique is relatively simple, resulting albumen and native protein are purified in conformation It has differences, the ability that induction body generates neutralizing antibody is obviously reduced, and is not suitable for being used as vaccine.

Therefore, the destination protein of soluble expression how is obtained, and keeps its native conformation simultaneously, becomes researcher Research emphasis.

Chinese patent application CN 103319604A discloses a kind of * subunit recombinant protein of 8 albumen of rotavirus vp, △ VP8* is made of the AA 64-223 of VP8 full-length proteins.The albumen is expressed by purification it has been found that △ VP8* is in stabilization Property and homogeneity in terms of, hence it is evident that be better than VP8 full-length proteins.However, the present inventor has found in further research, △ VP8* is lured The ability for leading body generation neutralizing antibody is markedly less than VP8 full-length proteins (see below embodiment 6 and Fig. 5).This leads to △ VP8* is not suitable for being used as efficient anti-rotavirus vaccine.

Therefore, it there is an urgent need in the art to develop a kind of new VP8 protein variant, can be purified by solubility expression Method easily, high expression quantity generates, and is able to maintain the native conformation of VP8 albumen, possesses good homogeneity and stability, And body can be induced to generate the neutralizing antibody for rotavirus of high titre, in order to develop efficient anti-rotavirus Vaccine, and make it possible the efficient anti-rotavirus vaccine of large-scale industrial production.

Summary of the invention

The present inventor it has been unexpectedly found that: the VP8 albumen that N-terminal has truncated 21-60 amino acid can be With high expression quantity solubility expression in Escherichia coli, and purifying can be easy to carry out by chromatography, and thus obtained High-purity truncated protein (purity can reach at least 50% or higher, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) possess good homogeneity and stability (being not easy to degrade), and body can be induced to generate high titre The neutralizing antibody for rotavirus, to efficiently solve above-mentioned technical problem.

Therefore, in one aspect, truncated 21-60 amino acid the present invention relates to N-terminal, for example, 21,22,23, 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 A, 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54 A, 55,56,57,58,59 or 60 amino acid 8 albumen of rotavirus vp or its variant.

In one aspect, the present invention relates to a kind of truncated 8 albumen of rotavirus vp or its variants, with wild type colyliform Viral VP8 albumen is compared, and N-terminal has truncated 21-60 amino acid, such as 21,22,23,24,25,26,27 A, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42 A, 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57 A, 58,59 or 60 amino acid.

In certain preferred aspects, compared with 8 albumen of wild type rotavirus vp, the truncated rotavirus The N-terminal of VP8 albumen has truncated 21-60 amino acid, for example, 21,25,30,35,40,45,50,55 or 60 amino acid.

In certain preferred aspects, truncated 8 albumen of rotavirus vp (hereinafter also referred to as truncated protein) With SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, Amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

The present inventor is also further studied truncated protein obtained.The results show that truncation of the invention The combination of albumen and Inner adjuvant can further improve the immunogenicity of truncated protein.Particularly, when by truncation of the invention When albumen and Inner adjuvant amalgamation and expression, compared with individual VP8 albumen or truncated protein, fusion protein obtained has Higher immunogenicity, and higher immune protective can be provided for host.In addition, can also be by by truncation egg of the invention It is white to be conjugated with Inner adjuvant to further increase the immunogenicity of truncated protein.

Therefore, on the other hand, the present invention provides a kind of fusion protein, it includes the first polypeptide and the second polypeptide, Wherein, first polypeptide is truncated 8 albumen of rotavirus vp as described above or its variant；Also, second polypeptide For Inner adjuvant.

In certain embodiments, the fusion protein optionally also includes peptide linker, label, signal peptide, proteolytic cleavage Site is cut, or any combination thereof.

In certain preferred aspects, the Inner adjuvant be selected from diphtheria toxin non-toxic mutant CRM197 and its The A subunit of truncated protein such as CRM197, cholera toxin, b subunit of cholera toxin CTB, cholera toxin mutants such as CTA112/ KDEV and CTA1-DD, e. coli heat-labile toxin (LT) and its non-toxic mutant LTR192G, e. coli heat-labile toxin B Subunit LTB, tetanus toxin and any combination thereof.

In certain preferred aspects, first polypeptide by way of directly merging or pass through peptide linker and institute The second polypeptide is stated to be attached.In certain preferred aspects, first polypeptide is located at the N-terminal of second polypeptide Or C-terminal, and the two is connected optionally by peptide linker.In certain preferred aspects, the peptide linker is selected from GS (SEQID NO:32), GGS (SEQ ID NO:33), GGGS (SEQ ID NO:34), SGGGS (SEQ ID NO:35), GGGG (SEQ ID NO:36), GGSS (SEQ ID NO:37), GGGGS (SEQ ID NO:38), (GGGGS)₃(SEQ ID NO:39), And any combination thereof.

In certain embodiments, the fusion protein also includes label in its N-terminal and/or C-terminal.In certain implementations In scheme, the label is selected from histidine tag, glutathione transferase (GST) label, maltose-binding protein (MBP) mark Label, thioredoxin (Trx) label, NusA label, disulfide bond isomerase label, DsbA label, DsbC label, SUMO label, MsyB label, TF label, triggering factor label, ubiquitin label, Myc label, Flag label, fluorescin (such as GFP) label, Biotin label, Avidin label and any combination thereof.

In certain embodiments, the fusion protein includes also signal peptide in its N-terminal.In certain embodiments, The signal peptide is selected from OmpA, OmpT, pelB, CSP, mschito, the signal peptide of MF- α, pho1, HBM, t-pA and IL-3.

In certain embodiments, the fusion protein also includes proteolytic cleavage site.In certain embodiments, institute Proteolytic cleavage site is stated between two adjacent elements, the element is selected from first polypeptide, the second polypeptide, and peptide connects Head, label and signal peptide.

In certain preferred aspects, the fusion protein has any one of SEQ ID NO:12-13 and 15-16 institute The amino acid sequence shown.

On the other hand, the present invention provides a kind of conjugates, and it includes truncated rotavirus vps 8 as described above Albumen or its variant, and the Inner adjuvant being conjugated with the truncated VP8 albumen or its variant.

In certain preferred aspects, the Inner adjuvant be selected from diphtheria toxin non-toxic mutant CRM197 and its The A subunit of truncated protein such as CRM197, cholera toxin, b subunit of cholera toxin CTB, cholera toxin mutants such as CTA112/ KDEV and CTA1-DD, e. coli heat-labile toxin (LT) and its non-toxic mutant LTR192G, e. coli heat-labile toxin B Subunit LTB, tetanus toxin and any combination thereof.

In certain preferred aspects, the Inner adjuvant passes through covalent manner (such as chemical coupling) or non-total Valence mode (such as absorption) is conjugated with 8 albumen of rotavirus vp or its variant.

On the other hand, the present invention relates to encode truncated protein or its variant of the invention or fusion egg of the invention White polynucleotides and containing the polynucleotide carrier.

The carrier that can be used for being inserted into polynucleotide of interest is it is known in the art that including but not limited to cloning vector and expression Carrier.In one embodiment, carrier is such as plasmid, clay, bacteriophage etc..

On the other hand, the invention further relates to the host cells comprising above-mentioned polynucleotides or carrier.Such host is thin Born of the same parents include but is not limited to prokaryotic cell such as Bacillus coli cells and eukaryocyte such as yeast cells, insect cell, are planted Object cell and zooblast (such as mammalian cell, such as mouse cell, people's cell etc.).Host cell of the invention can be with It is cell line, such as 293T cell.

On the other hand, the invention further relates to comprising above-mentioned truncated protein or its variant or above-mentioned fusion protein, or on State the composition of conjugate or above-mentioned polynucleotides or carrier or host cell.In certain preferred aspects, described group Closing object includes truncated protein or its variant of the invention or fusion protein of the invention or conjugate of the invention.

In certain preferred aspects, composition of the invention include compared with 8 albumen of wild type rotavirus vp, N-terminal has truncated 21-60 amino acid, such as 21,25,30,35,40,45,50,55 or 60 amino 8 albumen of truncated rotavirus vp of acid.In certain preferred aspects, composition of the invention includes and has to be selected from 8 albumen of truncated rotavirus vp of the sequence of SEQ ID NO:1-8.In certain preferred aspects, group of the invention Closing object includes the fusion protein with the sequence selected from SEQ ID NO:12-13 and 15-16.

On the other hand, the invention further relates to a kind of pharmaceutical composition or vaccines, and it includes truncated proteins of the invention Or its variant or fusion protein of the invention or conjugate of the invention, and optionally also include pharmaceutically acceptable carrier And/or excipient.Pharmaceutical composition or vaccine of the invention can be used for preventing or treating rotavirus infection or by colyliform disease Disease such as rotaviral gastroenteritis or diarrhea caused by poison infection etc..

In certain preferred aspects, described pharmaceutical composition or vaccine also include adjuvant, such as aluminium adjuvant.

In certain preferred aspects, truncated protein of the invention or its variant or fusion protein of the invention, or Conjugate of the invention is to prevent or treat rotavirus infection or be deposited by the effective quantity of the disease caused by rotavirus infection ?.In certain preferred aspects, pharmaceutical composition of the invention or vaccine also include other active constituent.It is preferred that Ground, the other active constituent can prevent or treat rotavirus infection or by the disease caused by rotavirus infection.

Pharmaceutical composition or vaccine of the invention can be administered by methods known in the art, such as, but not limited to logical Oral or injection is crossed to be administered.In certain preferred aspects, pharmaceutical composition of the invention or vaccine are with unit Dosage form is administered.

Pharmaceutical composition of the present invention needed for prevention or treatment particular condition or the amount of vaccine will depend on administration method, to Severity, the gender of patient, age, weight and general health of the patient's condition for the treatment of etc., can be by doctor according to reality Border situation rationally determines.

On the other hand, truncated protein or its variant of the invention or fusion of the invention are obtained the present invention relates to a kind of The method of albumen comprising, under conditions of allowing the truncated protein or its variant or the expressing fusion protein, culture is originally The host cell of invention；With recycle expressed truncated protein or its variant or the fusion protein.

In certain preferred aspects, the method includes expressing the present invention using escherichia expression system Truncated protein or its variant or the fusion protein, then the Escherichia coli are cracked, and purify and obtain from lysate Contain the truncated protein or its variant or the fusion protein.In certain preferred aspects, purifying includes chromatography layer Analysis.

On the other hand, the invention further relates to a kind of methods for preparing vaccine comprising by truncated protein of the invention Or its variant or fusion protein of the invention or conjugate of the invention and pharmaceutically acceptable carrier and/or excipient it is mixed It closes, optionally also mixes adjuvant such as aluminium adjuvant and/or other active constituent, such as can prevent or treat rotavirus Infection or other active constituent by the disease caused by rotavirus infection.As discussed above, vaccine obtained can For preventing or treating rotavirus infection or by the disease such as rotaviral gastroenteritis caused by rotavirus infection With diarrhea etc..

On the other hand, it is led the present invention relates to a kind of prevention or treatment rotavirus infection or by rotavirus infection The method of the disease of cause comprising by prevention or the truncated protein or its variant according to the present invention or this hair of therapeutically effective amount Bright fusion protein or conjugate of the invention or pharmaceutical composition of the invention or vaccine administration are to subject.Certain excellent In the embodiment of choosing, the disease by caused by rotavirus infection includes but is not limited to, rotaviral gastroenteritis and Diarrhea.In certain preferred aspects, the subject is mammal, such as mouse and people.

On the other hand, truncated protein according to the present invention or its variant or fusion protein of the invention are further related to, or The purposes of conjugate of the invention in preparation pharmaceutical composition or vaccine, described pharmaceutical composition or vaccine are used in subject Middle prevention or treatment rotavirus infection or by the disease caused by rotavirus infection.In certain preferred aspects, The disease by caused by rotavirus infection includes but is not limited to rotaviral gastroenteritis and diarrhea.Certain preferred Embodiment in, the subject is mammal, such as mouse and people.

On the other hand, truncated protein according to the present invention or its variant or fusion egg according to the present invention are further related to Conjugate white or according to the present invention is used to prevent in subject or treat rotavirus infection or by rotavirus sense Disease caused by dye.In certain preferred aspects, the disease by caused by rotavirus infection includes but not It is limited to, rotaviral gastroenteritis and diarrhea.In certain preferred aspects, the subject is mammal, such as Mouse and people.

The explanation and explanation of relational language in the present invention

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, cell culture used herein, molecular genetics, nucleic acid chemistry, immunological experiment Room operating procedure is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, it is provided below The definition and explanation of relational language.

According to the present invention, statement " protein that N-terminal has truncated X amino acid " refers to, the first encoded with initiation codon The 1-X amino acids residue protein obtained of methyllanthionine residue substitution protein N-terminal.Such as N-terminal has truncated 21 8 albumen of rotavirus vp of amino acid refers to, substitutes wild type rotavirus with the methionine residues that initiation codon encodes The 1-21 amino acids residue protein obtained of VP8 protein N terminal.

According to the present invention, term " variant " refers to such albumen, amino acid sequence and truncated colyliform of the invention The amino acid sequence of viral VP8 albumen (albumen as shown in SEQ ID any one of NO:1-8) has one or more (such as 1- 10 or 1-5 or 1-3) amino acid different (such as conservative amino acid replacements) or have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and its necessary characteristic for remaining the truncated protein.Art herein Language " necessary characteristic " can be one or more in following characteristic:

(i) its can in Escherichia coli with high expression quantity solubility expression, and can by chromatography easily into Row purifying；

(ii) it possesses good homogeneity and stability (being not easy to degrade)；

(iii) it can specifically bind anti-VP8 antibody, and the anti-VP8 antibody can inhibit virus to cell in vitro Infection；

(iv) it can inhibit infection of the virus to cell with rotavirus competitive binding cell receptor；

(v) it can induce the neutralizing antibody for rotavirus of body generation high titre；With

(vi) it can protect subject (such as people and mouse), resist the infection of rotavirus.

Preferably, " variant " of the invention remains all above-mentioned characteristics of truncated protein.

According to the present invention, term " identity " be used to refer between two polypeptides or between two nucleic acid sequence matching feelings Condition.(the example when some position in the sequence that two are compared all is occupied by identical base or amino acid monomer subunit Such as, some position in each of two DNA moleculars by adenine occupy or two polypeptides each in some position Set and all occupied by lysine), then each molecule is same on the position." percentage identity " between two sequences is The matching position number shared by the two sequences divided by position number × 100 being compared function.For example, if two There are 6 matchings in 10 positions of sequence, then the two sequences have 60% identity.For example, DNA sequence dna CTGACT and CAGGTT shares 50% identity (having 3 location matches in 6 positions in total).In general, by two sequence alignments to produce It is compared when raw maximum identity.Such comparison can be by using for example, can pass through computer program such as Align journey The method of Needleman that sequence (DNAstar, Inc.) easily carries out et al. (1970) J.Mol.Biol.48:443-453 is come It realizes.E.Meyers and the W.Miller (Comput.Appl for being integrated into ALIGN program (version 2 .0) also can be used Biosci., (1988) 4:11-17) algorithm, use PAM120 weight residue table (weight residue table), 12 Gap Length Penalty and 4 Gap Penalty measure the percentage identity between two amino acid sequences.In addition, can be used The Needleman and Wunsch (J MoI being integrated into the GAP program of GCG software package (can be obtained on www.gcg.com) Biol.48:444-453 (1970)) algorithm, use 62 matrix of Blossum or PAM250 matrix and 16,14,12,10,8,6 Or 4 Gap Weight (gap weight) and 1,2,3,4,5 or 6 Length Weight measure hundred between two amino acid sequences Score identity.

As used in this article, term " conservative substitution " means to influence or change comprising amino acid sequence The amino acid replacement of the biological activity of protein/polypeptide.It is lured for example, can for example be pinpointed by standard technique known in the art Become and the mutagenesis of PCR mediation introduces conservative substitution.Conservative amino acid replacement includes being replaced with the amino acid residue with similar side chain For the displacement of amino acid residue, it is used for example in physically or functionally similar with corresponding amino acid residue (such as with phase Like size, shape, charge, chemical property, including forming covalent bond or the ability of hydrogen bond etc.) the displacement that carries out of residue.Exist The family of the amino acid residue with similar side chain is defined in the art.These families include having basic side chain (for example, relying Propylhomoserin, arginine and histidine), acid side-chain (such as aspartic acid, glutamic acid), uncharged polar side chain it is (such as sweet Propylhomoserin, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar sidechain (such as Alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β branched building block (for example, Soviet Union ammonia Acid, valine, isoleucine) and beta-branched side (for example, tyrosine, phenylalanine, tryptophan, histidine) amino acid. It is therefore preferable that substituting corresponding amino acid residue with another amino acid residue from same side chain family.Identify amino acid The method of conservative substitution is well known in the art (see, e.g., Brummell et al., Biochem.32:1180-1187 (1993)；Kobayashi et al. Protein Eng.12 (10): 879-884 (1999)；With Burks et al. Proc.Natl Acad.Set USA 94:412-417 (1997), is incorporated herein by reference).

According to the present invention, term " escherichia expression system " refers to the expression being made of Escherichia coli (bacterial strain) and carrier System, wherein Escherichia coli (bacterial strain) derive from bacterial strain available on the market, such as, but not limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3) etc..

According to the present invention, term " carrier (vector) " refers to, a kind of nucleic acid delivery that can be inserted polynucleotides Tool.When carrier can make the encoded albumen of polynucleotides of insertion obtain expression, carrier is known as expression vector.Carrier can be with By conversion, transduction or transfection import host cell, and the inhereditary material element for carrying it is expressed in host cell. Carrier is well known to those skilled in the art, including but not limited to: plasmid；Bacteriophage；Coemid etc..

According to the present invention, term " VP8 albumen ", " VP8 full-length proteins " and " 8 albumen of rotavirus vp " are used interchangeably, It refers to, the albumen as composed by the AA 1-231 of the outermost layer structural proteins VP4 (AA 1-776) of RV virion.VP8 egg White exemplary amino acid sequence can be as shown in SEQ ID NO:9.

According to the present invention, term " truncated 8 albumen of rotavirus vp " refers to the N in 8 albumen of wild type rotavirus vp End and/or C-terminal remove the protein after one or more amino acid, wherein the specific ammonia of 8 albumen of wild type rotavirus vp Base acid sequence can be readily available from public database (such as GenBank database), such as GenBank accession number JQ013506.1.For example, the amino acid sequence of 8 albumen of wild type rotavirus vp can be as shown in SEQ ID NO:9.

In the present invention, term " truncated 8 protein gene segment of rotavirus vp " refers to such genetic fragment, with 8 protein gene of wild type rotavirus vp is compared, and encodes the nucleotide of one or more amino acid at the end 5' or the end 3' missing, The full length sequence of middle 8 protein gene of wild type rotavirus vp is easily obtained from public database (such as GenBank database) , such as GenBank accession number JQ013506.1.For example, the nucleotide sequence of 8 protein gene of wild type rotavirus vp can be such as Shown in SEQ ID NO:10.

According to the present invention, term " Inner adjuvant " refers to such adjuvant, melts with destination protein (that is, antigen) formation Hop protein so that it is present in an identical molecule (that is, fusion protein comprising itself and antigen) with antigen, and serves as this The adjuvant of antigen is to enhance the immunogenicity of the antigen.That is, Inner adjuvant is the purpose egg that can enhance amalgamation and expression therewith The adjuvant of the immunogenicity of white (antigen).Such Inner adjuvant is well-known to those skilled in the art, and existing It is described in detail in technical literature.In general, Inner adjuvant is endotoxin polypeptide fragment.For example, such Inner adjuvant Including but not limited to, A subunit (the Chinese patent Shen of diphtheria toxin non-toxic mutant CRM197 and its truncated protein such as CRM197 It please CN102807621；Ilyina,N.,S.Kharit,L.Namazova-Baranova,A.Asatryan,M.Benashvili, E.Tkhostova,C.Bhusal and A.K.Arora (2014).Hum Vaccin Immunother10(8):2471- 2481), cholera toxin CT (O'Neal, C.M., J.D.Clements, M.K.Estes and M.E.Conner, 1998, J Virol72 (4): 3390-3393), b subunit of cholera toxin CTB (Gloudemans, A.K., M.Plantinga, M.Guilliams,M.A.Willart,A.Ozir-Fazalalikhan,A.van der Ham,L.Boon,N.L.Harris, H.Hammad,H.C.Hoogsteden,M.Yazdanbakhsh,R.W.Hendriks,B.N.Lambrecht and H.H.Smits, 2013, PLoS One8 (3): e59822), cholera toxin mutants such as CTA112/KDEV (Hagiwara, Y.,Y.I.Kawamura,K.Kataoka,B.Rahima,R.J.Jackson,K.Komase,T.Dohi,P.N.Boyaka, Y.Takeda, H.Kiyono, J.R.McGhee and K.Fujihashi 2006, J Immunol177 (5): 3045-3054) With CTA1-DD (Agren, L.C., L.Ekman, B.Lowenadler and N.Y.Lycke, 1997, J Immunol158 (8): 3936-3946), e. coli heat-labile toxin (LT) and its non-toxic mutant LTR192G (Yuan, L., A.Geyer, D.C.Hodgins,Z.Fan,Y.Qian,K.-O.Chang,S.E.Crawford,V.L.A.Ward and M.K.Estes, 2000, Journal of virology 74 (19): 8843-8853), e. coli heat-labile toxin B subunit LTB and tetanus toxin (Wen, X., K.Wen, D.Cao, G.Li, R.W.Jones, J.Li, S.Szu, Y.Hoshino and L.Yuan(2014).,Vaccine,Volume 32,Issue 35,Pages 4365-4598)。

According to the present invention, term " peptide linker " refers to the small peptide for connecting two molecules (such as albumen).In general, passing through The polynucleotide sequence for encoding the small peptide is introduced into (for example, by PCR amplification or ligase) and is separately encoded to be connected two Between two DNA fragmentations of kind destination protein, and protein expression is carried out to obtain fusion protein, such as destination protein 1- peptide connects Head-destination protein 2.As known to the skilled person, peptide linker includes but is not limited to flexible peptide linker, such as GGGG (SEQ ID NO:36), GGSS (SEQ ID NO:37), GGGGS (SEQ ID NO:38) and (GGGGS)₃(SEQ ID NO:39) etc.. The detailed description of such peptide linker is referring also to for example, Robinson, C.R.and R.T.Sauer (1998) .Proc Natl Acad Sci 95(11):5929-5934。

According to the present invention, term " label " refers to such small peptide, with destination protein (such as truncated protein of the invention Or fusion protein) merge or connect, and thus promote solubility expression, detection and/or the purifying of recombinant protein.Label can merge Or it is connected to the N-terminal and/or C-terminal of destination protein (optionally by connector or proteolytic cleavage site).Such label is ability Known to field technique personnel, and it has been described in detail in the prior art document.For example, such label includes but unlimited In histidine tag (Sockolosky, J.T.and F.C.Szoka (2013) .Protein Expr Purif87 (2): 129- 135), glutathione transferase (GST) label (Hayashi, K.and C.Kojima (2008) .Protein Expr Purif62 (1): 120-127), maltose-binding protein (MBP) label (Bataille, L., W.Dieryck, A.Hocquellet, C.Cabanne, K.Bathany, S.Lecommandoux, B.Garbay and E.Garanger, Protein Expression and Purification Volume 110, June 2015, Pages 165-171), sulphur oxygen Also albumen (Trx) label (Tomala, M., A.Lavrentieva, P.Moretti, U.Rinas, C.Kasper, F.Stahl, A.Schambach, E.Warlich, U.Martin, T.Cantz and T.Scheper, 2010, Protein Expr Purif73 (1): 51-57), NusA label (Li, K., T.Jiang, B.Yu, L.Wang, C.Gao, C.Ma, P.Xu and Y.Ma (2013) .Sci Rep3:2347), disulfide bond isomerase DsbA label (Zhang, Y., D.R.Olsen, K.B.Nguyen, P.S.Olson,E.T.Rhodes and D.Mascarenhas(1998).Protein Expr Purif12(2):159- 165), DsbC label (Kurokawa, Y., H.Yanagi and T.Yura (2001) .J Biol Chem276 (17): 14393- 14399), SUMO label (Marblestone, J.G., S.C.Edavettal, Y.Lim, P.Lim, X.Zuo and T.R.Butt (2006) (1) .Protein Sci15: 182-189), msyB label (Zou, Z., L.Cao, P.Zhou, Y.Su, Y.Sun and W.Li (2008) .J Biotechnol135 (4): 333-339), TF label, triggering factor label (Kim, E.K., J.C.Moon, J.M.Lee,M.S.Jeong,C.Oh,S.M.Ahn,Y.J.Yoo and H.H.Jang(2012).Protein Expr Purif86 (1): 53-57), ubiquitin label (Sabin, E.A., Lee-Ng, Chun Ting, Shuster, Jeffrey R., Barr, Philip J. (1989) .Nature Biotechnology7 (7): 705-709), Myc label, Flag label, fluorescence Albumen (such as GFP) label (Pedelacq, J.D., S.Cabantous, T.Tran, T.C.Terwilliger and G.S.Waldo (2006) .Nat Biotechnol24 (1): 79-88), biotin label and Avidin label.

According to the present invention, term " signal peptide " refers to such small peptide, when its (such as truncation of the invention with destination protein Albumen or fusion protein) fusion when, destination protein expressed by cell can be promoted to be secreted into extracellularly.The usual position of signal peptide In the N-terminal of destination protein, and various signal peptides are known to the skilled in the art.Such signal peptide includes but is not limited to, OmpA (Sockolosky, J.T.and F.C.Szoka (2013) .Protein Expr Purif87 (2): 129-135), OmpT (Pohlmann,C.,M.Thomas,S.Forster,M.Brandt,M.Hartmann,A.Bleich and F.Gunzer (2013) (3) .Bioengineered4: 172-179), pelB (Sockolosky, J.T.and F.C.Szoka (2013) .Protein (2) Expr Purif87: 129-135), CSP (Heggeset, T.M., V.Kucharova, I.Naerdal, S.Valla,H.Sletta,T.E.Ellingsen and T.Brautaset(2013).Appl Environ Microbiol79 (2): 559-568), mschito (Sun, Y., J.Zhang and S.Wang (2015) .Indian J Microbiol55 (2): 194-199), MF- α (Ghosalkar, A., V.Sahai and A.Srivastava (2008) .Protein Expr Purif60 (2): 103-109), pho1 (Braspenning, J., W.Meschede, A.Marchini, M.Muller, L.Gissmann and M.Tommasino (1998) .Biochem Biophys Res Commun245 (1): 166-171), HBM(Wang,Y.B.,Z.Y.Wang,H.Y.Chen,B.A.Cui,Y.B.Wang,H.Y.Zhang and R.Wang(2009) .Vet Immunol Immunopathol132 (2-4): 314-317), t-pA (Wang, J.Y., W.T.Song, Y.Li, W.J.Chen,D.Yang,G.C.Zhong,H.Z.Zhou,C.Y.Ren,H.T.Yu and H.Ling(2011).Appl Microbiol Biotechnol91 (3): 731-740) and the signal peptide of IL-3 (Tessier, D.C., D.Y.Thomas, H.E.Khouri,F.Laliberte and T.Vernet(1991).Gene98(2):177-183)。

According to the present invention, term " proteolytic cleavage site " refers to, can be by protease specificity identifies and cuts position Point.Various specific proteases and its recognition site are well-known to those skilled in the art, and see many prior art texts In offering.Those skilled in the art can use suitable proteolytic cleavage site, and use phase according to the actual situation in the fusion protein The protease answered is cut.The use of proteolytic cleavage site can be advantageous, for example, it can be used for from fusion protein Signal peptide and/or label are cut off, so that obtaining has the active maturation protein of purpose.For example, in one embodiment, building And expressing includes label, the recombinant protein of the first polypeptide and the second polypeptide, wherein design has egg between label and the first polypeptide White cleavage sites；Then, expressed recombinant protein is purified by label；Then, using corresponding protease, Label is cut off from recombinant protein, to obtain purified fusion protein comprising the first polypeptide and the second polypeptide.

According to the present invention, term " conjugation " refers to, 2 or multiple molecules (such as albumen and albumen, albumen and polysaccharide, egg It is white with label etc.) be attached by covalent manner (such as chemical coupling) or non-covalent fashion (such as absorption).Therefore, at this In invention, term " conjugate " refers to be connected to by covalent manner (such as chemical coupling) or non-covalent fashion (such as absorption) 2 together or multiple molecules.For example, conjugate of the invention can refer to for example, being connected by covalent manner or non-covalent fashion The truncated VP8 albumen being connected together or its variant and Inner adjuvant.

According to the present invention, term " pharmaceutically acceptable carrier and/or excipient " refers in pharmacology and/or physiologically The carrier and/or excipient compatible with subject and active constituent is well known in the art (see, for example, Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company, 1995), and include but is not limited to: pH adjusting agent, surfactant, adjuvant, ionic strength increase Strong agent.For example, pH adjusting agent includes but is not limited to phosphate buffer；Surfactant include but is not limited to cation, yin from Son or nonionic surface active agent, such as Tween-80；Adjuvant includes but is not limited to aluminium adjuvant (such as aluminium hydroxide), not Family name's adjuvant (such as complete Freund's adjuvant)；Ionic strength reinforcing agent includes but is not limited to sodium chloride.

According to the present invention, term " effective quantity " is the amount for referring to effectively realize expected purpose.For example, prevention or treatment disease Sick (such as rotavirus infection) effective quantity refers to, can effectively prevent, prevents or postpone disease (such as rotavirus infection) Generation or alleviate, mitigate or treat the severity of existing disease (such as by disease caused by rotavirus infection) Amount.Such effective quantity is measured within the limit of power of those skilled in the art.

According to the present invention, term " chromatography " includes but is not limited to: ion-exchange chromatography (such as cation exchange color Spectrum), hydrophobic interaction chromatograph, adsorption chromatography (such as hydroxylapatite chromatography), gel filtration (gel exclusion) chromatography, parent And chromatography.

According to the present invention, the cracking of host cell/broken can carry out reality by various methods well known to those skilled in the art Existing, including but not limited to homogenizer is broken, homogeneous crusher machine, ultrasonication, grinding, high-pressure extrusion, bacteriolyze enzymatic treatment etc..

Advantageous effect of the invention

Truncated 8 albumen of rotavirus vp of N-terminal provided by the invention and preparation method efficiently solve in this field ?.

Firstly, truncated protein of the invention or its variant can be real in Escherichia coli with high expression quantity solubility expression High yield is showed.

Secondly, the way of purification of truncated protein of the invention or its variant is relatively easy, it is easily operated.Particularly, big After carrying out solubility expression in enterobacteria, the Escherichia coli can be cracked, chromatography processing, example then are carried out to lysate Such as ion-exchange chromatography processing, thus can get high-purity truncated protein (purity can reach at least 50% or higher, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%).

Third, truncated protein of the invention or its variant possess good homogeneity and stability, are not easy to degrade.

4th, truncated protein of the invention or its variant can induce body generate high titre in rotavirus And antibody.

5th, compared with individual truncated protein or truncated protein+aluminium adjuvant, comprising truncated protein of the invention and divide The fusion protein of adjuvant (such as CRM197-A or CTB) can stimulate mouse to generate higher immune response in son, generate stronger Immanoprotection action.

It can be seen that truncated protein of the invention or its variant and fusion protein of the invention are to remain overall length wild While the stronger induction body of type VP8 albumen generates neutralizing antibody ability, and there is high expression quantity, be easy to purify, Gao Jun The features such as one property and stability.Therefore, truncated protein of the invention (or its variant) and fusion protein can be used as efficiently Anti-rotavirus vaccine, and make it possible to the efficient anti-rotavirus vaccine of large-scale industrial production, to be this field Present in technical problem provide effective solution scheme.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but those skilled in the art Member it will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.With reference to the accompanying drawings With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art It will be apparent.

Detailed description of the invention

Fig. 1 shows the overall length VP8 albumen just purified and its SDS-PAGE result after placing three days at 4 DEG C.Swimming Road 1: the overall length VP8 albumen just purified；Swimming lane 2: the overall length VP8 albumen after being placed three days at 4 DEG C.The results show that overall length VP8 Albumen is significantly degraded after placing three days at 4 DEG C, and stability is poor.

Fig. 2A -2B shows various truncated rotavirus vp 8 albumen (VP8-5A, VP8-5, VP8-6, VP8-8, VP8- 9, VP8-10, VP8-11 or VP8-12) after the completion of purifying (Fig. 2A) and 4 DEG C place 3 days after (Fig. 2 B) SDS poly- third The result of acrylamide gel electrophoresis.

In fig. 2, swimming lane is from left to right successively are as follows: VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.Fig. 2A's the results show that after above-mentioned purification step, VP8-5A, VP8-5, VP8-6, VP8-8, VP8- The concentration of 9, VP8-10, VP8-11 or VP8-12 albumen is about 2.0mg/ml, and purity is greater than 98%, and expression quantity is significantly better than VP-8 full-length proteins.

In fig. 2b, swimming lane is from left to right successively are as follows: VP8 albumen, VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.Fig. 2 B's the results show that VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, The stability of VP8-11 or VP8-12 albumen is significantly better than VP-8 full-length proteins, after 4 DEG C are placed 3 days, does not occur significantly to drop Solution.

Fig. 3 A-3D shows various truncated rotavirus vp 8 albumen (VP8-5A, VP8-5, VP8-6, VP8-8, VP8- 9, VP8-10, VP8-11 or VP8-12) with antibody 1B11 (Fig. 3 A), 11C1 (Fig. 3 B), 3C5 (Fig. 3 C), 1E1 (Fig. 3 D) it is indirect Elisa assay as a result, wherein abscissa indicates that each truncated protein, ordinate indicate there is reactive one with each truncated protein The maximum antibody extension rate of anti-(1E1,1B11,11C1 or 3C5).The results show that each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) it can be identified have good anti-by four kinds of neutralizing antibodies Originality (that is, antibody response), is equal to or is even stronger than the antigenicity of △ VP8*.

Fig. 4 show various 8 albumen of truncated rotavirus vp (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) with Balb/c mouse is immunized with it and the knot of the indirect ELISA of the immune serum that obtains analysis Fruit, wherein abscissa indicates that each truncated protein, ordinate indicate there is reactive immune serum most with corresponding truncated protein Big extension rate (that is, antibody titer).The results show that truncated protein can effectively induce on the 42nd day after immune in Mice Body The generation of antibody.After the completion of immune programme, the antibody titer (GMT) in immune serum can reach 10⁵Or it is higher.Each experimental group Between without significant difference.These results indicate that each truncated protein of the invention (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) good immunogenicity is all had, the generation of antibody can be effectively induced in animal body, Its effect and VP8 and △ VP8* are without significant difference.

Fig. 5 show various 8 albumen of truncated rotavirus vp (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) analysis of the neutralizing antibody titers of immune serum that is induced in Balb/c mouse as a result, its Middle abscissa indicates that each truncated protein, ordinate indicate the immune serum maximum dilution multiple for reaching 50% infection inhibiting rate (NT50, neutralizing antibody titers).The results show that truncated protein of the invention can be (after being immunized three times) 42nd day after immune The immune serum with high neutralizing antibody titers is effectively induced in Mice Body.After the completion of immune programme, truncation of the invention is used The neutralizing antibody titers (NT50) for the immune serum that albumen induces can reach 10³Or higher level.These results indicate that this Invention each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) have compared with Strong induction body generates the ability of neutralizing antibody, can induce the immune blood with high neutralizing antibody titers in animal body Clearly, the immune serum can effectively inhibit the infection of rotavirus.During the induction body of each truncated protein of the invention generates △ VP8* is significantly better than with the ability of antibody, and close to VP8 full-length proteins and RV.

Fig. 6 shows the analysis of the neutralizing antibody titers of the positive serum after incubating with each truncated protein as a result, wherein horizontal Each truncated protein that coordinate representation and positive serum incubate, ordinate indicate to reach positive blood after the incubation of 50% infection inhibiting rate Clear maximum dilution multiple (NT50, neutralizing antibody titers).The results show that after incubation, truncated protein (VP8- of the invention 5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) cause the neutralizing antibody titers of positive serum aobvious Write decline: the neutralizing antibody titers with the TB8.0 positive serum incubated can be more than 6*10³；The positive serum incubated with △ VP8* Neutralizing antibody titers can be more than 5*10³；And it is low with the neutralizing antibody titers of the positive serum of truncated protein incubation of the invention In 4*10³, even lower than 3*10³.These results indicate that each truncated protein (VP8-5A, VP8-5, VP8-6, VP8- of the invention 8, VP8-9, VP8-10, VP8-11 or VP8-12) with positive serum high response is all had, and the reactivity is significantly stronger than △ VP8*；This shows the conformation of each truncated protein of the invention or closer with natural viral in turn, can be preferably by natural disease The immune antibody (positive polyvalent antibody) generated of poison is identified.

Fig. 7 A-7C shows the standards of grading of diarrhea in protective effect.According to the different degrees of of suckling mouse diarrhea, scoring point For 3 grades: normal fecal stools are calculated as 1 point (Fig. 7 C), and soft excrement is calculated as 2 points (Fig. 7 B), and shapeless watery stool is calculated as 3 points of (figures 7A)。

Fig. 8 shows the diarrhea scoring after Balb/c mouse is immunized with each truncated protein prepared in embodiment 3, wherein The longitudinal axis indicates diarrhea scoring；Horizontal axis indicates that mouse attacks number of days after poison.

Fig. 9 shows the average diarrhea number of days after Balb/c mouse is immunized with each truncated protein prepared in embodiment 3, In, the longitudinal axis indicates average diarrhea number of days；Horizontal axis indicates each truncated protein.

No matter Fig. 8's -9 the results show that respectively truncate egg from average diarrhea severity or from the point of view of average diarrhea number of days White (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) all has significant protectiveness, and It is more preferable than △ VP8* protectiveness.

Figure 10 shows the fusion protein comprising truncated protein VP8-5 and Inner adjuvant CTB prepared in embodiment 9 The SDS-PAGE result of (CTB-VP8-5 and VP8-5-CTB) after the completion of purifying.Wherein, each swimming lane is from left to right successively are as follows: CTB-VP8-5, VP8-5-CTB, VP8-5 and CTB.

Figure 11 shows the immune serum that fusion protein (CTB-VP8-5 and VP8-5-CTB) induces in Balb/c mouse In total antibody titer and neutralizing antibody titers analysis as a result, wherein abscissa indicate used in various albumen, a left side is vertical to be sat Mark indicates neutralizing antibody titers (NT50, that is, reach the immune serum maximum dilution multiple of 50% infection inhibiting rate), right ordinate Indicate total antibody titer (log)；Also, neutralizing antibody titers are indicated with column diagram, and total antibody titer is with graphical representation.As a result It has been shown that, fusion protein of the invention (CTB-VP8-5 and VP8-5-CTB) can be in mouse (after being immunized three times) 42nd day after immune The immune serum with antibody titers, also, total anti-titre and neutralizing antibody titers in immune serum are effectively induced in vivo Total anti-titre and the neutralizing antibody titers being significantly higher than in the immune serum of VP8-5 group and CTB group.These results indicate that can lead to It crosses and connect truncated protein of the invention to further increase the immune of truncated protein of the present invention with Inner adjuvant (such as CTB) Originality, to realize better immune effect.

Figure 12 is shown to be scored with the diarrhea after the fusion protein immunization Balb/c mouse prepared in embodiment 9, wherein vertical Axis indicates diarrhea scoring；Horizontal axis indicates that mouse attacks number of days after poison.The results show that fusion protein of the invention (CTB-VP8-5 and VP8-5-CTB significant immune protective) is all had, and better than the VP8-5 not merged with Inner adjuvant CTB.

Figure 13 shows that is prepared in embodiment 10 merges egg comprising truncated protein VP8-5 and Inner adjuvant CRM-A The SDS-PAGE result of white (CRM-A-VP8-5 and VP8-5-CRM-A) after the completion of purifying.Each swimming lane is from left to right successively are as follows: CRM-A-VP8-5, VP8-5-CRM-A, VP8-5 and CTB.

Figure 14 shows that fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) induces immune in Balb/c mouse The analysis result of total antibody titer in serum and neutralizing antibody titers, wherein abscissa indicates used various albumen, left Ordinate indicates neutralizing antibody titers (NT50, that is, reach the immune serum maximum dilution multiple of 50% infection inhibiting rate), and the right side is vertical The total antibody titer of coordinate representation (log)；Also, neutralizing antibody titers are indicated with column diagram, and total antibody titer is with graphical representation. The results show that fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) of the invention is 42nd day after immune (after being immunized three times) The immune serum with antibody titers, also, total anti-titre and neutralization in immune serum can be effectively induced in Mice Body Antibody titer is significantly higher than total anti-titre and neutralizing antibody titers in the immune serum of VP8-5 group and CRM-A group.These results The present invention can be further increased by the way that truncated protein of the invention to be connect with Inner adjuvant (such as CRM-A) by, which showing, cuts The immunogenicity of short albumen, to realize better immune effect.

Figure 15 is shown to be scored with the diarrhea after the fusion protein immunization Balb/c mouse prepared in embodiment 10, wherein The longitudinal axis indicates diarrhea scoring；Horizontal axis indicates that mouse attacks number of days after poison.The results show that fusion protein (CRM-A-VP8-5 of the invention And VP8-5-CRM-A) significant immune protective is all had, and be equivalent to or better than not melting with Inner adjuvant CRM-A The VP8-5 of conjunction.

Sequence information

In the table 1 that the information of sequence of the present invention is provided below.

Table 1: the description of sequence

Sequence 1 (SEQ ID NO:1):

MIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSE GVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYG VMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 2 (SEQ ID NO:2):

MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVV EGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKY NGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 3 (SEQ ID NO:3):

MQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNG TDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLY TYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 4 (SEQ ID NO:4):

MAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILI EPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNAT TNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 5 (SEQ ID NO:5):

MAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVP ETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYS TTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 6 (SEQ ID NO:6):

MGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRN YTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYD SVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 7 (SEQ ID NO:7):

MSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFG ETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMT SYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 8 (SEQ ID NO:8):

MVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASI SVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDF YIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 9 (SEQ ID NO:9):

MASLIYRQLLTNSYTVNLSDEIQLIGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPY QPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKT TANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNG LPPIQNTR

Sequence 10 (SEQ ID NO:10):

ATGGCTTCGCTCATTTACAGACAATTACTTACGAATTCATATACAGTGAATCTTTCAGATGAAATACAG TTAATTGGATCAGAAAAAACGCAGAGAACTACAGTAAATCCAGGTCCATTTGCACAAACTGGTTATGCACCAGTGAA TTGGGGGCCTGGGGAAACGAGTGATTCCACTACTGTTGAGCCAGTGTTGAATGGACCATATCAGCCGACGACTTTCA ATCCACCAGTAGAATATTGGATGCTTCTAGCACCAACATCAGAAGGGGTAGTTGTTGAAGGTACTAATGGTACGGAT AGATGGCTAGCTACAATACTTATAGAACCAAATGTGCCTGAGACGACTAGAAATTACACATTATTTGGGGAAACAGC GAGTATATCAGTAGCAAACCCATCACAAAGTAAATGGCGTTTTGTTGACGTAGCTAAGACCACTGCAAATGGAACAT ATTCACAATATGGACCATTACTATCAGATACAAAACTGTATGGAGTAATGAAATACAACGGGAAGTTGTATACGTAT AATGGTGAAACTCCGAATGCTACAACAAATTATTATTCAACTACAAATTATGACTCAGTGAATATGACATCTTATTG CGATTTTTACATTATACCAAGAGCACAAGAATCAAAGTGCACAGAATACGTAAATAATGGATTACCACCAATACAAA ACACCAGA

Sequence 11 (SEQ ID NO:11):

MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERM KDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGS

Sequence 12 (SEQ ID NO:12):

MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERM KDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGSHMGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEP VLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRF VDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCT EYVNNGLPPIQNTR

Sequence 13 (SEQ ID NO:13):

MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVV EGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKY NGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRGSHMTPQNITDLCAEY HNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWN NKTPHAIAAISMANGS

Sequence 14 (SEQ ID NO:14):

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVD NENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSS SVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR

Sequence 15 (SEQ ID NO:15):

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVD NENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSS SVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGGGGSGGGGSHMGSEKTQRTTVNPGPF AQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVVEGTNGTDRWLATILIEPNVPETTR NYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKYNGKLYTYNGETPNATTNYYSTTNY DSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTR

Sequence 16 (SEQ ID NO:16):

MGSEKTQRTTVNPGPFAQTGYAPVNWGPGETSDSTTVEPVLNGPYQPTTFNPPVEYWMLLAPTSEGVVV EGTNGTDRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVAKTTANGTYSQYGPLLSDTKLYGVMKY NGKLYTYNGETPNATTNYYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGLPPIQNTRGSGGGGSGGGGGSGGG GSGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSG KAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINN WEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR

Sequence 17 (SEQ ID NO:17):

GGATCCCATATGATACAGTTAATTGGATCAGAAAA

Sequence 18 (SEQ ID NO:18):

GGATCCCATATGGGATCAGAAAAAACGCAG

Sequence 19 (SEQ ID NO:19):

GGATCCCATATGCAGAGAACTACAGTAAATCCAGG

Sequence 20 (SEQ ID NO:20):

GGATCCCATATGGCACAAACTGGTTATGCA

Sequence 21 (SEQ ID NO:21):

GGATCCCATATGGCACCAGTGAATTGGGG

Sequence 22 (SEQ ID NO:22):

GGATCCCATATGGGGCCTGGGGAAACG

Sequence 23 (SEQ ID NO:23):

GGATCCCATATGAGTGATTCCACTACTGTTGAGC

Sequence 24 (SEQ ID NO:24):

GGATCCCATATGGTTGAGCCAGTGTTGAATG

Sequence 25 (SEQ ID NO:25):

AAGCTTAGGTGTTTTGTATTGGTGG

Sequence 26 (SEQ ID NO:26):

GGATCCCATATGGCTTCGCTCATT

Sequence 27 (SEQ ID NO:27):

GGATCCCATATGTTGAATGGACCA

Sequence 28 (SEQ ID NO:28):

AAGCTTAGGATCCGGTGTTTTGTATTGGTGG

Sequence 29 (SEQ ID NO:29):

AAGCTTATCCATTATTTACGTATTCTGTG

Sequence 30 (SEQ ID NO:30):

GGATCCGGTGGCGGTGGCAGCGGTGGCGGTGGCAGCGGTGGCGGTGGAAGCGGCGCTGATGATGTTGTT

Sequence 31 (SEQ ID NO:31):

AAGCTTAACGATTTCCTGCACAGGCTTG

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.

Unless specifically stated otherwise, otherwise the present invention used in experimental methods of molecular biology and immunodetection, substantially Upper reference J.Sambrook et al., molecular cloning: laboratory manual, second edition, CSH Press, 1989, and F.M.Ausubel et al., fine works molecular biology experiment guide, the 3rd edition, described in John Wiley&Sons, Inc., 1995 Method carry out or carried out according to product description.Reagents or instruments used without specified manufacturer is that can pass through The conventional products of commercially available acquisition.As known to those skilled in the art, embodiment describes the present invention by way of example, and is not intended to limit Scope of the present invention.Without departing from the spirit and substance of the present invention, to the method for the present invention, step or Modifications or substitutions made by condition, all belong to the scope of the present invention.

The source of biomaterial and reagent used in embodiment:

LLR plants of rotavirus are presented by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.；Prokaryotic expression carrier PTO-T7 is Laboratory independently constructs；Escherichia coli (E.coli) ER2566 is purchased from New England Biolabs, Inc. (US) Massachusetts, United States of America；Used primer By raw work, biological (Shanghai) Engineering stock Co., Ltd is synthesized.

Embodiment 1: the building of the expression vector of truncated 8 albumen of rotavirus vp is encoded

LLR plants of rotavirus are cultivated with rhesus monkey embryonic kidney cells system (MA-104) cell.Used medium is DMEM, It is supplemented with the pancreatin of 2 μ g/ml, the ampicillin of 0.5mg/ml and the streptomysin of 0.4mg/ml, the sodium bicarbonate of 3.7mg/ml, The L-Glutamine of 0.34mg/ml.

According to the manufacturer's instructions, it is mentioned using viral DNA/RNA that Beijing Genmagbio Biotechnology Co., Ltd. produces Kit is taken, the geneome RNA of rotavirus is extracted, and obtains the cDNA of coding VP4 albumen by reverse transcription.With acquisition CDNA is template, is reacted by PCR, and amplification obtains the genetic fragment for encoding truncated 8 albumen of rotavirus vp.

Used primer is as follows:

Upstream primer:

5'-GGATCCCATATGATACAGTTAATTGGATCAGAAAA-3'(SEQ ID NO:17)

5'-GGATCCCATATGGGATCAGAAAAAACGCAG-3'(SEQ ID NO:18)

5'-GGATCCCATATGCAGAGAACTACAGTAAATCCAGG-3'(SEQ ID NO:19)

5'-GGATCCCATATGGCACAAACTGGTTATGCA-3'(SEQ ID NO:20)

5'-GGATCCCATATGGCACCAGTGAATTGGGG-3'(SEQ ID NO:21)

5'-GGATCCCATATGGGGCCTGGGGAAACG-3'(SEQ ID NO:22)

5'-GGATCCCATATGAGTGATTCCACTACTGTTGAGC-3'(SEQ ID NO:23)

5'-GGATCCCATATGGTTGAGCCAGTGTTGAATG-3'(SEQ ID NO:24)

Downstream primer:

5'-AAGCTTAGGTGTTTTGTATTGGTGG-3'(SEQ ID NO:25)

Wherein, restriction enzyme site is indicated with underscore, the terminator codon of introducing is indicated with italic.

Used PCR reaction system is as follows:

PCR reaction condition is as follows: 95 DEG C of initial denaturation 5min, 35 circulations (95 DEG C, 40s；55 DEG C, 40s；72℃ 1min), extend 10min eventually for 72 DEG C.Amplified production obtained is detected with 2% agarose gel electrophoresis.

Each pcr amplification product is connect with pMD 18-T carrier (TAKARA company) commercially, is transferred to bacillus coli DH 5 In α.Then, positive bacterium colony is screened, plasmid is extracted, is identified through NdeI/HindIII digestion, obtains insertion target gene fragment Positive colony plasmid pMD18-T-VP8-5A, pMD 18-T-VP8-5, pMD 18-T-VP8-6, pMD 18-T-VP8-8, pMD 18-T-VP8-9, pMD 18-T-VP8-10, pMD 18-T-VP8-11 and pMD18-T-VP8-12.

Above-mentioned positive colony is sequenced using M13 (+)/(-) primer in Shanghai Sheng Gong bio-engineering corporation.Sequencing The results show that the nucleotide sequence for the target fragment being inserted into above-mentioned positive colony plasmid and expected consistent, the ammonia of coding As shown in SEQ ID NO:1-8, they correspond respectively to N-terminal and are truncated 21,25,30,40,45,50,55,60 base acid sequence The VP8 albumen that amino acid, C-terminal are not truncated.

Above-mentioned positive colony plasmid is subjected to NdeI/HindIII digestion, obtains the coding base of each truncated VP8 albumen Because of segment, and by itself and nonfusion expression vector pTO-T7 (Luo Wenxin etc., biotechnology through NdeI/HindIII digestion Report, 2000,16:53-57) it is connected, it is transferred to Escherichia coli ER2566；Then, positive bacterium colony is screened, plasmid is extracted, through NdeI/ HindIII digestion is identified to obtain the positive expression plasmid of insertion target fragment: pTO-T7-VP8-5A, pTO-T7-VP8-5, pTO- T7-VP8-6, pTO-T7-VP8-8, pTO-T7-VP8-9, pTO-T7-VP8-10, pTO-T7-VP8-11, pTO-T7-VP8-12.

Take the positive expression plasmid (0.15mg/ml) of 1 μ L, the competent E.coli that 40 μ L of conversion are prepared with Calcium Chloride Method ER2566 (is purchased from New England Biolabs, Inc. (US) Massachusetts, United States of America), is coated on containing kanamycins (final concentration 25mg/mL, similarly hereinafter) Solid LB media (LB medium component: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, similarly hereinafter), and 37 DEG C of standings Culture 10-12 hours clear and legible to single colonie.Picking single bacterium drops down onto LB liquid medium containing 4mL (containing kanamycins), so Afterwards at 37 DEG C, 200 revs/min lower shaken cultivation 10 hours.After culture, 1mL bacterium solution is taken to save in -70 DEG C.

In addition, also constructing the expression plasmid of expression overall length VP8 albumen, PTO-T7- by method similar to the above VP8.Used PCR amplification primer is as follows:

Upstream primer: 5'-GGATCCCATATGGCTTCGCTCATT-3'(SEQ ID NO:26)；

Downstream primer: 5'-AAGCTTAGGATCCGGTGTTTTGTATTGGTGG-3'(SEQ ID NO:28)；

Wherein, restriction enzyme site is indicated with underscore, the terminator codon of introducing is indicated with italic.

The expression plasmid of expression △ VP8*, PTO-T7- △ VP8* are also constructed by similar methods.Used PCR Amplimer is as follows:

Upstream primer: 5'-GGATCCCATATGTTGAATGGACCA-3'(SEQ ID NO:27)；

Downstream primer: 5'-AAGCTTATCCATTATTTACGTATTCTGTG-3'(SEQ ID NO:29)。

Embodiment 2: the expression of truncated 8 albumen of rotavirus vp

Carrying recombinant plasmid pTO-T7-VP8-5A, pTO-T7-VP8-5, pTO- prepared by embodiment 1 is taken out from -70 DEG C T7-VP8-6, pTO-T7-VP8-8, pTO-T7-VP8-9, pTO-T7-VP8-10, pTO-T7-VP8-11 or pTO-T7-VP8-12 Escherichia coli bacteria liquid, be inoculated in LB liquid medium of the 50ml containing kanamycins, cultivated at 180rpm, 37 DEG C big About 4 hours；Then it transfers (every bottle of access 500ul bacterium solution) in LB culture medium of 10 bottles of 500ml containing kanamycins.Work as culture When light absorption value at 600nm is up to 0.5, IPTG to final concentration of 1mM is added, continues culture 6 hours at 180rpm, 25 DEG C.

In addition, also carrying out recombinant protein PTO-T7-VP8, PTO- using Escherichia coli by method similar to the above The expression of T7- △ VP8*.

Embodiment 3: the purifying and characterization of truncated 8 albumen of rotavirus vp

Under 8.0 buffer conditions of Tris-HCl, at 4 DEG C, the item of the wet bacterium of 4min/1g is pressed with Ultrasound Instrument (Thermo) Part destroys the cell wall of E.coli cell, collects soluble fraction, and the weight expressed by following proposal E.coli cell Histone is purified.

Instrument system: GE Healthcare company (former Amershan Pharmacia company) production 100 type preparative liquid chromatography system of AKTAexplorer.

Chromatography media: Q-sepharose-HP (GE Healthcare company).

Column volume: 5.5cm*20cm.

Buffer: 50mM Tris-HCl pH 8.0

50mM Tris-HCl pH 8.0,2M NaCl

Flow velocity: 25mL/min.

Detector wavelength: 280nm.

VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, the VP8-10 containing recombinant expression that sample is prepared before being, The E. coli lysate soluble fraction of VP8-11 or VP8-12.

Elution program are as follows: 1000mM NaCl elutes foreign protein, and 50mM NaCl elutes destination protein, collects 50mMNaCl and washes It is temporarily released from one's regular work object, obtains the VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 containing recombinant expression of 30mL altogether Or the purified sample of VP8-12.

In addition, also obtaining the purified sample of the △ VP8* containing recombinant expression by similar purification schemes；Also, The purified sample of the VP8 full-length proteins containing recombinant expression is obtained by DEAE-FF chromatographic column (GE) purifying.

The 150 μ L of sample of above scheme of learning from else's experience purifying, is added 30 μ L 6X LoadingBuffer, mixes and in 100 DEG C of water Bathe 10min；Then take 10 μ l in 13.5%SDS- polyacrylamide gel with 120V electrophoresis 120min；Then by examining The bright blue dyeing of Maas is to show electrophoretic band.Electrophoresis result is shown in Fig. 1 (swimming lane 1) and Fig. 2A.

In addition, above-mentioned purified sample is placed 3 days at 4 DEG C, sds polyacrylamide gel electrophoresis is then carried out again Detection.Electrophoresis result is shown in Fig. 1 (swimming lane 2) and Fig. 2 B.

Fig. 1 shows the overall length VP8 albumen just purified and its SDS-PAGE result after placing three days at 4 DEG C.Swimming Road 1: the overall length VP8 albumen just purified；Swimming lane 2: the overall length VP8 albumen after being placed three days at 4 DEG C.Fig. 1's the results show that complete Long VP8 albumen is significantly degraded after placing three days at 4 DEG C, and stability is poor.

Fig. 2A shows various truncated rotavirus vp 8 albumen (VP8-5A, VP8-5, VP8-6, the VP8- just purified 8, VP8-9, VP8-10, VP8-11 or VP8-12) sds polyacrylamide gel electrophoresis result.In fig. 2, swimming lane is from a left side Successively to the right side are as follows: VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11, VP8-12.The result of Fig. 2A is aobvious Show, after above-mentioned purification step, VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12 egg White concentration is about 2.0mg/ml, and purity is greater than 98%, and expression quantity is significantly better than VP-8 full-length proteins.

Fig. 2 B show after being placed 3 days at 4 DEG C purified various truncated 8 albumen of rotavirus vp (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) sds polyacrylamide gel electrophoresis result. Fig. 2 B's the results show that VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12 albumen stabilization Property be significantly better than VP-8 full-length proteins, 4 DEG C place 3 days after, do not occur significantly to degrade.

In addition, also being analyzed using G3000SEC-HPLC the homogeneity of above-mentioned purified sample.SEC-HPLC Analysis result is shown in following table 2.

Table 2: the G3000SEC-HPLC analysis of purified protein sample

Albumen	VP8	VP8-5	VP8-6	VP8-8	VP8-9	VP8-10	VP8-11	VP8-12	△VP8*
										Appearance time (minute)	10.9	14.63	14.74	14.6	14.7	14.62	14.7	15.12	15.51
Peak area %	70.56	98.81	97.86	98.74	97.94	97.05	98.29	99.34	98.9

The results show that polymer (appearance time is about 10.9 minutes) content in VP-8 full-length proteins sample is about 70.56%, substantially monomer-free exists；And truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) monomer (appearance time is about 14-15 minutes) content in sample is more than 97%.This shows VP8 The homogeneity of full-length proteins is poor, is easily formed polymer；And truncated protein (VP8-5A, VP8-5, VP8-6, VP8- of the invention 8, VP8-9, VP8-10, VP8-11 or VP8-12) homogeneity be significantly better than VP-8 full-length proteins, it is basic in purified sample It is upper that polymer is not present.

Embodiment 4: the detection of the antibody response of truncated 8 albumen of rotavirus vp

Purified 8 albumen of truncated rotavirus vp that will be obtained in above-described embodiment 3, with coating buffer (20mM PBS, PH7.4) it is diluted to 0.5ng/ul, room temperature is coated with plate 2 hours.With 0.1%PBS-Tween20 (PBST) board-washing 1 time, with PBST solution room temperature containing 1%BSA blocking of plates 2 hours, pats dry.By (this laboratory neutralizing antibody 1E1,1B11,11C1,3C5 Made by oneself by hybridoma technology, concentration is 1mg/ml, each monoclonal antibody neutralization titer IC50 be respectively 200ng/ml, 200ng/ml, 50ng/ml and 5ng/ml) gradient dilution, then carrying out indirect ELISA analysis with the truncated protein after coating respectively (is wherein made Secondary antibody is goat anti-mouse antibody (ten thousand domain U.S. billows), with measurement with each truncated protein have reactive primary antibody (1E1, 1B11,11C1,3C5) maximum antibody extension rate.Antibody extension rate is bigger, then the reactivity of truncated protein and antibody is got over It is high.△ VP8* and VP-8 full-length proteins are used as control.

As shown in figs. 3 a-3d, wherein abscissa indicates each truncated protein to the result of indirect ELISA, ordinate indicate with it is each Truncated protein has the maximum antibody extension rate of reactive primary antibody (1E1,1B11,11C1 or 3C5).The results show that each section Short albumen (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) can be neutralized by four kinds Antibody identification has good antigenic (that is, antibody response), is equal to or is even stronger than the antigenicity of △ VP8*.

Embodiment 5: the analysis of the immunogenicity of truncated 8 albumen of rotavirus vp

Purified 8 albumen of truncated rotavirus vp that will be obtained in above-described embodiment 3, with coating buffer (20mM PBS, PH7.4) it is diluted to 0.5ng/ul, room temperature is coated with plate 2 hours.With 0.1%PBS-Tween20 (PBST) board-washing 1 time, with PBST solution room temperature containing 1%BSA blocking of plates 2 hours, pats dry.

Balb/c mouse is immunized using purified 8 albumen of truncated rotavirus vp obtained in above-described embodiment 3. Immunization protocol is as follows: the female Balb/c mouse of 70 4-5 week old being randomly divided into 10 groups, every group of 7 mouse, wherein 2 groups are Control group, another 8 groups are experimental group.Experimental group be subcutaneously injected respectively embodiment 3 preparation each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12).The injection dosage of every mouse is being mixed with 1:1 for 10 μ g The recombinant protein of adjuvant.It is immunized three times altogether, each immunization interval two weeks.It is immune for the first time to use Freund's complete adjuvant；The Secondary and third time is immune to use incomplete Freund's adjuvant.△ VP8* and VP-8 full-length proteins are used as control, are respectively used to pair It is immunized according to group, and using identical immunization protocol.

After immune programme, immune serum is collected from immunized mouse.By immune serum gradient dilution, then divide Not carrying out indirect ELISA analysis to the corresponding truncated protein after coating, (secondary antibody used in wherein is goat anti-mouse antibody (ten thousand Domain U.S. billows), to measure the maximum dilution multiple that there is reactive immune serum with corresponding truncated protein.The dilution of immune serum Multiple is bigger, the titre of the anti-truncated protein antibody in immune serum is higher, for generating the truncated protein of immune serum Immunogenicity is also higher.The result of indirect ELISA as shown in figure 4, wherein abscissa indicates each truncated protein, ordinate indicate with Each truncated protein has the maximum dilution multiple (that is, antibody titer) of reactive immune serum.The results show that truncated protein exists The generation of antibody can be effectively induced within 42nd day after immune in Mice Body.Antibody after the completion of immune programme, in immune serum Titre (GMT) can reach 10⁵Or it is higher.Without significant difference between each experimental group.These results indicate that each truncation egg of the invention White (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) all has good immunogenicity, The generation of antibody can be effectively induced in animal body, and effect and VP8 and △ VP8* are without significant difference.

Embodiment 6: truncated 8 albumen of rotavirus vp induces the analysis of the ability of neutralizing antibody

Using immunization protocol described in embodiment 5, with the purified truncated colyliform disease obtained in above-described embodiment 3 Malicious VP8 albumen comes immunization experiment group Balb/c mouse (every group of 7 mouse), and collects immune serum.In addition, being exempted from using identical △ VP8*, VP-8 full-length proteins, rotavirus RV and Freund's adjuvant are used as control, are respectively used to immunized controls group by epidemic disease scheme Balb/c mouse (every group of 7 mouse), and collect immune serum.

MA104 cell is laid on (1.9*10 in 96 porocyte culture plates⁴A cells/well).In being carried out as follows after 20 hours And experiment: it is dilute that immune serum sample to be measured (containing neutralizing antibody to be measured) is subjected to continuous multiple proportions with the DMEM that pancreatin is added respectively It releases；Then each diluted sample of 100 μ L is taken to mix (TCID50=1.5* with the rotavirus liquid being diluted in DMEM respectively 10⁵)；After being incubated for 1h at 37 DEG C, mixture is separately added into pre- 96 porocyte culture plates for being covered with MA104 cell, and 37 DEG C of culture 14h；Then, each immune serum is calculated as follows to the infection inhibiting rate of virus.

Infection inhibiting rate=(the virus point that virus point counting-addition serum hole in the hole of serum is not added counts)/not The virus point that the hole of serum is added counts * 100%.

Neutralizing antibody titers in immune serum is defined as: reach the immune serum maximum dilution times of 50% infection inhibiting rate Number.The immune serum for remaining to reach 50% or more infection inhibiting rate after 50 times of dilutions is considered as with neutralising capacity.

The analysis results of the neutralizing antibody titers of immune serum is indulged as shown in figure 5, wherein abscissa indicates each truncated protein Coordinate representation reaches the immune serum maximum dilution multiple (NT50, neutralizing antibody titers) of 50% infection inhibiting rate.The results show that Truncated protein of the invention (immune rear three times) 42nd day after immune can effectively induce in Mice Body to be had senior middle school and resists The immune serum of body titre.After the completion of immune programme, the neutralizing antibody for the immune serum that truncated protein of the invention induces is used Titre (NT50) can reach 10³Or higher level.These results indicate that each truncated protein (VP8-5A, VP8- of the invention 5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) energy with stronger induction body generation neutralizing antibody Power, can induce the immune serum with high neutralizing antibody titers in animal body, and the immune serum can effectively inhibit to take turns The infection of shape virus.The ability that the induction body of each truncated protein of the invention generates neutralizing antibody is significantly better than △ VP8*, and Close to VP8 full-length proteins and RV.

Embodiment 7: the reactivity of truncated rotavirus vp 8 albumen and polyvalent antibody

Balb/c mouse is immunized with rotavirus, and collects immune serum, and its gradient dilution (is opened for 200 times from dilution Begin, gradient dilution is to 25600 times).Then, by each diluted blood serum sample respectively with each truncated protein (VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) (1mg/ml) mixing, and incubated 1 hour at 37 DEG C.It incubates Afterwards, it feeds the mixture into pre- 96 porocyte culture plates for being covered with MA104 cell, and further cultivates 14h at 37 DEG C.So Afterwards, virus infection amount is detected using Elispot, and as described in example 6 above, calculated after being incubated with each truncated protein, positive blood Clear neutralizing antibody titers (NT50).The neutralizing antibody titers (NT50) of positive serum after incubation are lower, and truncated protein is tied Neutralizing antibody in the positive serum of conjunction is more, and the reactivity of truncated protein and positive serum is higher.△ VP8* is eased up Fliud flushing TB8.0 is used as control.

The analysis result of the neutralizing antibody titers of positive serum after incubating with each truncated protein is as shown in fig. 6, wherein horizontal Each truncated protein that coordinate representation and positive serum incubate, ordinate indicate to reach positive blood after the incubation of 50% infection inhibiting rate Clear maximum dilution multiple (NT50, neutralizing antibody titers).The results show that after incubation, truncated protein (VP8- of the invention 5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) cause the neutralizing antibody titers of positive serum aobvious Write decline: the neutralizing antibody titers with the TB8.0 positive serum incubated can be more than 6*10³；The positive serum incubated with △ VP8* Neutralizing antibody titers can be more than 5*10³；And it is low with the neutralizing antibody titers of the positive serum of truncated protein incubation of the invention In 4*10³, even lower than 3*10³.These results indicate that each truncated protein (VP8-5A, VP8-5, VP8-6, VP8- of the invention 8, VP8-9, VP8-10, VP8-11 or VP8-12) with positive serum high response is all had, and the reactivity is significantly stronger than △ VP8*；This shows the conformation of each truncated protein of the invention or closer with natural viral in turn, can be preferably by natural disease The immune antibody (positive polyvalent antibody) generated of poison is identified.

Embodiment 8: protectiveness evaluation of truncated 8 albumen of rotavirus vp in animal

Using immunization protocol described in embodiment 5, with the purified truncated colyliform disease obtained in above-described embodiment 3 Malicious VP8 albumen comes immunization experiment group Balb/c mouse (every group of 7 mouse).In addition, using identical immunization protocol, by △ VP8*, VP-8 full-length proteins, rotavirus RV and Freund's adjuvant are used as control, are respectively used to immunized controls group Balb/c mouse (every group of 7 mouse).14d, 28d and 42d acquire eyeball of mouse blood before immune and after immune, serum are separated, by it It is saved in -20 DEG C.

After the completion of immune programme (after 42 days immune), each group mouse is handed over by every two female rats with a public mouse Match.Post-coitum 20 days or so, female rat gave birth to suckling mouse, raised 7.Stomach-filling is carried out to 7 age in days suckling mouses with LLR Strain and attacks poison, is attacked Toxic dose is 5*10⁶TCID50/ is only.

It attacks observation suckling mouse after poison time of diarrhea, duration occurs and scores to diarrhea serious conditions, record. Standards of grading are as shown in Figure 7 A-7C.According to the different degrees of of suckling mouse diarrhea, be divided into 3 grades: normal fecal stools are calculated as 1 point (Fig. 7 C), soft excrement are calculated as 2 points (Fig. 7 B), and shapeless watery stool is calculated as 3 points (Fig. 7 A).

Experimental result is as Figure 8-9.Fig. 8 shows small with the immune Balb/c of each truncated protein prepared in embodiment 3 Diarrhea scoring after mouse, wherein the longitudinal axis indicates diarrhea scoring；Horizontal axis indicates that mouse attacks number of days after poison.Fig. 9, which is shown, uses embodiment The average diarrhea number of days after Balb/c mouse is immunized in each truncated protein prepared in 3, wherein the longitudinal axis indicates average diarrhea number of days； Horizontal axis indicates each truncated protein.

The results show that no matter from average diarrhea severity or from the point of view of average diarrhea number of days, each truncated protein (VP8- 5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12) significant protectiveness is all had, and compare △ VP8* protectiveness is more preferable.

Embodiment 9: the expression and purifying of the fusion protein comprising VP8-5 and Inner adjuvant CTB and its induction neutralize The ability of antibody and the analysis of its immune protective

From biological (Shanghai) the Engineering stock Co., Ltd synthesis N-terminal of raw work and C-terminal respectively band restriction enzyme site BglII/NdeI and The CTB gene of BamHI/HindIII.With Nde I/Hind III by CTB gene double digestion, and it is cloned into PTO-T7 carrier, from And obtain PTO-T7-CTB (cloning procedure is with embodiment 1)；Then, with BamH I/Hind III by VP8-5 gene double digestion, And it is cloned into PTO-T7-CTB (cloning procedure is with embodiment 1), to obtain recombinant plasmid PTO-T7-CTB-VP8-5.In addition, VP8-5 gene is expanded using primer (SEQ ID NO:18 and SEQ ID NO:28)；Then, will be expanded with Nde I/Hind III Increase production object double digestion, and be cloned into PTO-T7 carrier, to obtain PTO-T7-VP8-5 '；Further, BglII/HindIII is used By CTB gene double digestion, (cloning procedure with embodiment 1) is then cloned into PTO-T7-VP8-5 ', to obtain recombinant plasmid PTO-T7-VP8-5-CTB。

According to method described in embodiment 2, with the recombinant plasmid of building in expression in escherichia coli fusion protein CTB- VP8-5 and VP8-5-CTB.Particularly, when light absorption value of the culture at 600nm reaches 0.5, IPTG is added into culture To final concentration of 1mM；Then, continue culture Escherichia coli 6 hours, at 180rpm, 25 DEG C to induce Bacillus coli expression to melt Hop protein.

According to method described in embodiment 3, the Escherichia coli of ultrasonication expressed fusion protein.Collect insolubility grade Point, and redissolved using Buffer I (50mM Tris-HCl pH 8.0+150mMNaCl).Then, by solution obtained It is centrifuged 10mins with 25000g, and collects precipitating.It is dissolved and is precipitated with 8M urea, and dialysed at 4 DEG C, to remove urea, And make precipitating in fusion protein renaturation.Then, the fusion protein in solution is purified by following proposal.

Instrument system: GE Healthcare company (former Amershan Pharmacia company) production 100 type preparative liquid chromatography system of AKTAexplorer.

Chromatography media: Q-sepharose-HP (GE Healthcare company).

Column volume: 5.5cm*20cm.

Buffer: 50mM Tris-HCl pH 8.0

50mM Tris-HCl pH 8.0,2M NaCl

Flow velocity: 25mL/min.

Detector wavelength: 280nm.

The solution for the fusion protein CTB-VP8-5 or VP8-5-CTB comprising renaturation that sample is prepared before being.

Elution program are as follows: 1000mM NaCl elutes foreign protein, and 300mM NaCl elutes destination protein, collects 300mMNaCl Eluted product obtains the purified sample of the CTB-VP8-5 containing recombinant expression and VP8-5-CTB of 30mL altogether.

The 150 μ L of sample of above scheme of learning from else's experience purifying, is added 30 μ L 6X Loading Buffer, mixes and in 100 DEG C Water-bath 10min；Then take 10 μ l in 13.5%SDS- polyacrylamide gel with 120V electrophoresis 120min；Then pass through Coomassie brilliant blue dyes to show electrophoretic band.Electrophoresis result is shown in shown in Figure 10.

Figure 10 shows the fusion protein (CTB- comprising truncated protein VP8-5 and Inner adjuvant CTB produced above VP8-5 and VP8-5-CTB) SDS-PAGE result after the completion of purifying.The results show that constructed fusion protein is in large intestine bar High expression in bacterium, and the above method is effectively enriched with and has purified the fusion protein of expression in escherichia coli.

Further, using immunization protocol described in embodiment 5 (Freund's adjuvant used in it is replaced with aluminium adjuvant), With purified fusion protein immunization Balb/c mouse produced above.Then, using scheme described in embodiment 5-6, divide Analyse total antibody titer and the neutralizing antibody titers in the immune serum of immunized mouse.It is described in embodiment 8 in addition, also using Scheme, immune protective of the analysis fusioning protein in animal.As a result it is shown in Figure 11-12.

Figure 11 shows the immune serum that fusion protein (CTB-VP8-5 and VP8-5-CTB) induces in Balb/c mouse In total antibody titer and neutralizing antibody titers analysis as a result, wherein abscissa indicate used in various albumen, a left side is vertical to be sat Mark indicates neutralizing antibody titers (NT50, that is, reach the immune serum maximum dilution multiple of 50% infection inhibiting rate), right ordinate Indicate total antibody titer (log).The results show that fusion protein (CTB-VP8-5 and VP8-5-CTB) of the invention is the after immune (after being immunized three times) can effectively induce the immune serum with antibody titers in Mice Body within 42 days, also, in immune serum Total anti-titre and neutralizing antibody titers be significantly higher than total anti-titre in the immune serum of VP8-5 group and CTB group and neutralize anti- Body titre.These results indicate that can be by truncated protein of the invention being connect with Inner adjuvant (such as CTB) come further The immunogenicity for improving truncated protein of the present invention, to realize better immune effect.

Figure 12 is shown to be scored with the diarrhea after fusion protein immunization Balb/c mouse produced above, wherein longitudinal axis table Show that diarrhea scores；Horizontal axis indicates that mouse attacks number of days after poison.The results show that fusion protein (CTB-VP8-5 and VP8-5- of the invention CTB significant immune protective) is all had；Wherein, the immune protective of CTB-VP8-5 and VP8-5-CTB is significantly better than yin Property control group, and better than the VP8-5 that is not merged with Inner adjuvant CTB, and the immune protective highest of CTB-VP8-5.

Embodiment 10: in the expression and purifying of the fusion protein comprising VP8-5 and Inner adjuvant CRM-A and its induction With the ability of antibody and the analysis of its immune protective

From biological (Shanghai) the Engineering stock Co., Ltd synthesis N-terminal of raw work and C-terminal respectively with restriction enzyme site NdeI and BamHI/ The CRM-A gene of HindIII, and the C-terminal of the gene is connected with the nucleotide sequence of coding peptide linker.According in embodiment 9 CRM-A gene cloning is entered PTO-T7 carrier by NdeI/Hind III double digestion, to obtain PTO-T7- by the method for description CRM-A；Then, VP8-5 is connected by PTO-T7-CRM-A by BamH I/HindIII double digestion, to obtain recombinant plasmid PTO-T7-CRM-A-VP8-5.In addition, expanding CRM-A gene with primer (SEQ ID NO:30 and 31).Amplification obtained produces Object includes the nucleotide sequence for encoding peptide linker in its N-terminal.Then, it will be expanded by BamH I/Hind III double digestion Product cloning enters PTO-T7-VP8-5 ', to obtain recombinant plasmid PTO-T7-VP8-5-CRM-A.Recombinant plasmid PTO-T7- CRM-A-VP8-5 encoding fusion protein CRM-A-VP8-5, wherein CRM-A is connected to the N-terminal of VP8-5 by peptide linker.Recombinate matter Grain PTO-T7-VP8-5-CRM-A encoding fusion protein VP8-5-CRM-A, wherein VP8-5 is connected to the N of CRM-A by peptide linker End.

According to method described in embodiment 2, with the recombinant plasmid of building in expression in escherichia coli fusion protein CRM- A-VP8-5 and VP8-5-CRM-A.Particularly, it when light absorption value of the culture at 600nm reaches 0.5, is added into culture IPTG to final concentration of 1mM；Then, continue culture Escherichia coli 6 hours, at 180rpm, 25 DEG C to induce Escherichia coli table Up to fusion protein.

According to method described in embodiment 3, the Escherichia coli of ultrasonication expressed fusion protein.Collect soluble fraction Point, and add 25% ammonium sulfate thereto to precipitate protein therein.Protein precipitation is collected, and with 50mM Tris-HCl PH 8.0 is redissolved.Solution after redissolution is centrifuged, and collects supernatant.Then, by following proposal come in supernatant Fusion protein purified.

Instrument system: GE Healthcare company (former Amershan Pharmacia company) production 100 type preparative liquid chromatography system of AKTAexplorer.

Chromatography media: Q-sepharose-HP (GE Healthcare company).

Column volume: 5.5cm*20cm.

Buffer: 50mM Tris-HCl pH 8.0

50mM Tris-HCl pH 8.0,2M NaCl

Flow velocity: 25mL/min.

Detector wavelength: 280nm.

The supernatant comprising fusion protein CRM-A-VP8-5 or VP8-5-CRM-A that sample is prepared before being.

Elution program are as follows: 1000mM NaCl elutes foreign protein, and 300mM NaCl elutes destination protein, collects 300mMNaCl Eluted product obtains the purified sample of the CRM-A-VP8-5 containing recombinant expression and VP8-5-CRM-A of 30mL altogether.

The 150 μ L of sample of above scheme of learning from else's experience purifying, is added 30 μ L 6X LoadingBuffer, mixes and in 100 DEG C of water Bathe 10min；Then take 10 μ l in 13.5%SDS- polyacrylamide gel with 120V electrophoresis 120min；Then by examining The bright blue dyeing of Maas is to show electrophoretic band.Electrophoresis result is as shown in figure 13.

Figure 13 shows the fusion protein (CRM- comprising truncated protein VP8-5 and Inner adjuvant CRM-A produced above A-VP8-5 and VP8-5-CRM-A) SDS-PAGE result after the completion of purifying.The results show that constructed fusion protein is big High expression in enterobacteria, and the above method is effectively enriched with and has purified the fusion protein of expression in escherichia coli.

Further, using immunization protocol described in embodiment 5 (Freund's adjuvant used in it is replaced with aluminium adjuvant), With purified fusion protein immunization Balb/c mouse produced above.Then, using scheme described in embodiment 5-6, divide Analyse total antibody titer and the neutralizing antibody titers in the immune serum of immunized mouse.It is described in embodiment 8 in addition, also using Scheme, immune protective of the analysis fusioning protein in animal.As a result it is shown in Figure 14-15.

Figure 14 shows that fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) induces immune in Balb/c mouse The analysis result of total antibody titer in serum and neutralizing antibody titers, wherein abscissa indicates used various albumen, left Ordinate indicates neutralizing antibody titers (NT50, that is, reach the immune serum maximum dilution multiple of 50% infection inhibiting rate), and the right side is vertical The total antibody titer of coordinate representation (log).The results show that fusion protein (CRM-A-VP8-5 and VP8-5-CRM-A) of the invention exists The immune serum with antibody titers can effectively be induced 42nd day after immune (after being immunized three times) in Mice Body, also, exempted from Total anti-titre and neutralizing antibody titers in epidemic disease serum are significantly higher than total anti-drop in the immune serum of VP8-5 group and CRM-A group Degree and neutralizing antibody titers.These results indicate that can be by by truncated protein of the invention and Inner adjuvant (such as CRM-A) Connection is to further increase the immunogenicity of truncated protein of the present invention, to realize better immune effect.

Figure 15 is shown to be scored with the diarrhea after fusion protein immunization Balb/c mouse produced above, wherein longitudinal axis table Show that diarrhea scores；Horizontal axis indicates that mouse attacks number of days after poison.The results show that fusion protein (CRM-A-VP8-5 and VP8- of the invention 5-CRM-A) all have significant immune protective；Wherein, the immune protective of CRM-A-VP8-5 and VP8-5-CRM-A is aobvious It writes and is better than negative control group, and the immune protective highest of VP8-5-CRM-A.

Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (43)

1. a kind of 8 albumen of truncated rotavirus vp, compared with 8 albumen of wild type rotavirus vp, N-terminal has truncated 21-60 A amino acid, wherein the amino acid sequence of 8 albumen of wild type rotavirus vp is as shown in SEQ ID NO:9.

2. 8 albumen of truncated rotavirus vp of claim 1, compared with 8 albumen of wild type rotavirus vp, N-terminal is truncated 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 A, 52,53,54,55,56,57,58,59 or 60 amino acid.

3. 8 albumen of truncated rotavirus vp of claim 1, amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 institute Show.

4. a kind of fusion protein, it includes the first polypeptides and the second polypeptide, wherein first polypeptide is appointed for claim 1-3 One 8 albumen of truncated rotavirus vp；Also, second polypeptide is Inner adjuvant.

5. the fusion protein of claim 4, wherein the fusion protein also includes peptide linker, label, signal peptide, proteolytic cleavage Site is cut, or any combination thereof.

6. the fusion protein of claim 4 or 5, wherein the Inner adjuvant is selected from diphtheria toxin non-toxic mutant CRM197 And its truncated protein, cholera toxin, b subunit of cholera toxin CTB, cholera toxin mutants, e. coli heat-labile toxin (LT) And its non-toxic mutant LTR192G, e. coli heat-labile toxin B subunit LTB, tetanus toxin and any combination thereof.

7. the fusion protein of claim 6, wherein the truncated protein of the CRM197 is the A subunit of CRM197；Alternatively, described Cholera toxin mutants are selected from CTA112/KDEV and CTA1-DD.

8. the fusion protein of claim 4, wherein first polypeptide by way of directly merging or pass through peptide linker and institute The second polypeptide is stated to be attached.

9. the fusion protein of claim 4, wherein first polypeptide is located at the N-terminal or C-terminal of second polypeptide.

10. the fusion protein of claim 9, wherein first polypeptide is connect with second polypeptide by peptide linker.

11. the fusion protein of claim 4 or 5, wherein the fusion protein includes label in its N-terminal and/or C-terminal.

12. the fusion protein of claim 11, wherein the label is selected from histidine tag, glutathione transferase (GST) is marked Label, maltose-binding protein (MBP) label, thioredoxin (Trx) label, NusA label, disulfide bond isomerase label, DsbA Label, DsbC label, SUMO label, msyB label, TF label, triggering factor label, ubiquitin label, Myc label, Flag mark Label, fluorescent protein tag, biotin label, Avidin label and any combination thereof.

13. the fusion protein of claim 4 or 5, wherein the fusion protein includes signal peptide in its N-terminal.

14. the fusion protein of claim 13, wherein the signal peptide is selected from OmpA, OmpT, pelB, CSP, mschito, MF- The signal peptide of α, pho1, HBM, t-pA and IL-3.

15. the fusion protein of claim 5, wherein the fusion protein includes proteolytic cleavage site.

16. the fusion protein of claim 15, wherein the proteolytic cleavage site is between two adjacent elements, institute It states element and is selected from first polypeptide, the second polypeptide, peptide linker, label and signal peptide.

17. the fusion protein of claim 4, wherein the amino acid sequence of the fusion protein such as SEQ ID NO:12-13 and Shown in any one of 15-16.

18. a kind of conjugate, it includes 8 albumen of truncated rotavirus vp of any one of claim 1-3, and with described section The Inner adjuvant of short VP8 albumen conjugation.

19. the conjugate of claim 18, wherein the Inner adjuvant be selected from diphtheria toxin non-toxic mutant CRM197 and its Truncated protein, cholera toxin, b subunit of cholera toxin CTB, cholera toxin mutants, e. coli heat-labile toxin (LT) and its Non-toxic mutant LTR192G, e. coli heat-labile toxin B subunit LTB, tetanus toxin and any combination thereof.

20. the conjugate of claim 19, wherein the truncated protein of the CRM197 is the A subunit of CRM197；Alternatively, described Cholera toxin mutants are selected from CTA112/KDEV and CTA1-DD.

21. the conjugate of any one of claim 18-20, wherein the Inner adjuvant passes through covalent manner or non-covalent side Formula and 8 albumen of rotavirus vp are conjugated.

22. the conjugate of claim 21, wherein the covalent manner is chemical coupling；Alternatively, the non-covalent fashion is to inhale It is attached.

23. a kind of isolated nucleic acid, 8 albumen of truncated rotavirus vp or right of any one of coding claim 1-3 is wanted Seek the fusion protein of any one of 4-17.

24. the carrier of the isolated nucleic acid comprising claim 23.

25. the host cell of the carrier of the isolated nucleic acid and/or claim 24 comprising claim 23.

26. a kind of composition, it includes 8 albumen of truncated rotavirus vp or claim 4- of any one of claim 1-3 The isolated nucleic acid of any one of 17 fusion protein or the conjugate of any one of claim 18-22 or claim 23, or The carrier of claim 24 or the host cell of claim 25.

27. a kind of pharmaceutical composition or vaccine, it includes 8 albumen of truncated rotavirus vp of any one of claim 1-3, or The fusion protein of any one of claim 4-17 or the conjugate of any one of claim 18-22.

28. the pharmaceutical composition or vaccine of claim 27, wherein described pharmaceutical composition or vaccine also include pharmaceutically acceptable Carrier and/or excipient.

29. the pharmaceutical composition or vaccine of claim 27, wherein described pharmaceutical composition or vaccine also include adjuvant.

30. the pharmaceutical composition or vaccine of claim 29, wherein the adjuvant is aluminium adjuvant.

31. the pharmaceutical composition or vaccine of claim 27, wherein truncated 8 albumen of rotavirus vp or the fusion Albumen or the conjugate are to prevent or treat rotavirus infection or by the effective quantity of the disease caused by rotavirus infection In the presence of.

32. the pharmaceutical composition or vaccine of any one of claim 27-31, wherein described pharmaceutical composition or vaccine also include Other active constituent.

33. the pharmaceutical composition or vaccine of claim 32, wherein the other active constituent can prevent or treat colyliform Virus infection or by the disease caused by rotavirus infection.

34. obtaining the fusion of 8 albumen of truncated rotavirus vp or any one of claim 4-17 of any one of claim 1-3 The method of albumen comprising, under conditions of allowing truncated 8 albumen of rotavirus vp or the expressing fusion protein, Cultivate the host cell of claim 25；With recycle expressed 8 albumen of truncated rotavirus vp or fusion protein.

35. the method for claim 34, wherein the method includes the steps: it is expressed using escherichia expression system described Truncated 8 albumen of rotavirus vp or the fusion protein, then crack Escherichia coli, and purifying obtains institute from lysate State truncated 8 albumen of rotavirus vp or the fusion protein.

36. the method for claim 35, wherein the purifying includes chromatography.

37. a kind of method for preparing vaccine comprising by 8 albumen of truncated rotavirus vp of any one of claim 1-3, or The fusion protein of any one of claim 4-17 or the conjugate of any one of claim 18-22, or pass through claim 8 albumen of truncated rotavirus vp or fusion protein and pharmaceutically acceptable carrier that the method for any one of 34-36 obtains and/or Excipient mixing.

38. the method for claim 37, wherein the method also includes by truncated 8 albumen of rotavirus vp or described Fusion protein or the conjugate are mixed with adjuvant and/or other active constituent.

39. the method for claim 38, wherein the adjuvant is aluminium adjuvant；Alternatively, the other active constituent is can be pre- Anti- or treatment rotavirus infection or other active constituent by the disease caused by rotavirus infection.

40. the fusion protein of any one of 8 albumen of truncated rotavirus vp of any one of claim 1-3 or claim 4-17 Or any one of claim 18-22 conjugate or the truncated wheel that is obtained by the method for any one of claim 34-36 The purposes of shape virus VP8 albumen or fusion protein in preparation pharmaceutical composition or vaccine, described pharmaceutical composition or vaccine are used Disease caused by the prevention in subject or treatment rotavirus infection or by rotavirus infection.

41. the purposes of claim 40, wherein the disease by caused by rotavirus infection is selected from rotaviral stomach and intestine Scorching and diarrhea.

42. the purposes of claim 40 or 41, wherein the subject is mammal.

43. the purposes of claim 42, wherein the mammal is selected from mouse and people.