CN102464721A - Recombination broad-spectrum vaccine specific to Human enterovirus 71 - Google Patents
Recombination broad-spectrum vaccine specific to Human enterovirus 71 Download PDFInfo
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Abstract
The invention relates to a recombination broad-spectrum vaccine specific to Human enterovirus 71. The invention constructs a fusion protein which comprises at least one copied peptide SP55 and/or SP70 of Human enterovirus 71 VP1 protein and a hepatitis B virus core antigen. The fusion protein can renature and assemble by self in a solubility manner after being expressed, denatured and purified, thereby forming virus-like particles with strong immunogenicity; and the virus-like particles can induce the generation of a broad-spectrum neutralizing antibody with high valence in vivo, thereby being capable of serving as the vaccine for preventing diseases related to Human enterovirus 71 infection. According to the invention, suitable carriers for forming the virus-like particles are found for antigens deriving from the enterovirus 71 VP1 protein, thereby the formed virus-like particles properly expose the antigens and increase the immunogenicity of the antigens.
Description
Technical field
The invention belongs to biomedicine and biological technical field; More specifically, the present invention relates to be directed against the recombined broad spectrum property vaccine of human enterovirus 71.
Background technology
Hand foot mouth disease (Hand; Foot and mouth disease; HFMD) be a kind of acute infectious disease that is caused by enterovirus, main through contact or digestive tube propagation closely, pilosity is born in the infant below 10 years old; With the Mucocutaneous fash in position, bleb, ulcer such as hand, foot, oral cavities is typical case's performance, and the patient who is in a bad way can cause complication such as myocarditis, wet lung, AME, poliomyelitis.This disease transmission property is strong, and mortality ratio is high, is prone to cause break out or popular.Several years of past, hand foot mouth disease is popular in a lot of countries and regions of the Far East Area, comprises China, Hong Kong, Japan, Singapore and Taiwan.During January 1 to the September 30 in this year, China Report 1,567,254 routine hand foot mouth diseases, wherein 829 examples are dead.
(Enterovirus 71, EV71) are one of main pathogens that causes hand foot mouth disease, and this virus belongs to Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus), are strand justice RNA viruses, and the about 7400bp of genome is (referring to Xu for human enterovirus 71; J., Y.Qian, S.Wang, J.M.Serrano, W.Li; Z.Huang, and S.Lu.2010.EV71:an emerging infectious disease vaccine target in the Far East Vaccine 28:3516-21.11.Zhang, Y., D.Wang; D.Yan, S.Zhu, J.Liu, H.Wang; S.Zhao, D.Yu, L.Nan, J.; An, L.Chen, H.An; A.Xu; And W.Xu.2010.Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China.J Clin Microbiol 48:619-22); Coding sub-thread long-chain polypeptide is made up of structural protein P1 and Nonstructural Protein P2, P3.Virus protease is cut into VP0, VP1 and VP3 with P1; The three assembles and forms viral capsid (Ranganathan; S., S.Singh, C.L.Poh; And V.T.Chow.2002.The hand, foot and mouth disease virus capsid:sequence analysis and prediction of antigenic sites from homology modelling.Appl Bioinformatics 1:43-52).
At present clinically still needleless to the vaccine of EV71.VP1 is the main protection antigen of EV71, can induce neutralizing antibody and virus attack is had provide protection.VP1 is last contains 15 amino acid whose linear peptides section SP55 and SP70 high conservative between the EV71 different subtype, with inducing body to produce the wide spectrum neutralizing antibody behind SP55 or the SP70 polypeptide immune mouse.But the less immunogenic of SP55 and SP70 polypeptide itself need heavy dose of immunity just can induce the ideal immune effect, and the cost of synthetic polypeptide is very high, itself is not suitable for directly using as vaccine.
Therefore, also need further exploitation, and reduce the cost of vaccine production to the immunogenicity height of EV71, the vaccine of good immune effect.
Summary of the invention
The objective of the invention is to express the embedded virus appearance particle of showing SP55 or SP70,, make up new E V71 recombined broad spectrum property vaccine in the hope of improving the ability that SP55 and SP70 induce neutralizing antibody.
Another object of the present invention is to provide a kind of recombined broad spectrum property vaccine to human enterovirus 71.
In first aspect of the present invention, a kind of fusion rotein is provided, comprise following albumen:
(1-10 copy of at least 1 copy; As 8,5,3,2 copies) human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70; With
HBcAg (HBcAg).
In a preference, between described human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70 and the HBcAg, link to each other or coupling each other (preferably linking to each other) through chemical bond through chemical bond; Described chemical bond is covalent linkage or non covalent bond.
In another preference, described chemical bond is a peptide bond.
In another preference, the sequence of described peptide section SP55 such as SEQ ID NO:2.
In another preference, the sequence of described peptide section SP70 such as SEQ ID NO:3.
In another preference, described fusion rotein has or does not have purification tag, like 6 * His.
In another preference, the human enterovirus 71 VP1 proteic peptide section SP55 of described at least 1 copy and/or SP70 are inserted between any two amino acid between the HBcAg aminoacid sequence 76-84 position; Or
The human enterovirus 71 VP 1 proteic peptide section SP55 and/or the SP70 of described at least 1 copy have replaced any one or several amino acid between the HBcAg aminoacid sequence 76-84 position.
In another preference, described fusion rotein comprises from the aminoterminal to the carboxyl terminal successively:
HBcAg 1-76 amino acids;
The human enterovirus 71 VP1 proteic peptide section SP55 and/or the SP70 of at least 1 copy;
The the 84th～(144-185) of HBcAg (for example: the 84-185 position) amino acid.
In another preference, between HBcAg fragment and human enterovirus 71 VP1 proteic peptide section SP55 or SP70, can comprise or not comprise connection peptides; The length of connection peptides is 2-20aa for example; Preferable like 2-10aa.
In another aspect of this invention, the purposes of described fusion rotein is provided, is used for preparation and has immunogenic virus-like particle.
In another aspect of this invention, a kind of nucleic acid molecule is provided, it is characterized in that, the described fusion rotein of the nucleic acid molecule encoding of stating.
In another aspect of this invention, a kind of carrier is provided, it is characterized in that, described carrier contains described nucleic acid.
In another aspect of this invention, a kind of cell is provided, described cell contains in described carrier or its genome and is integrated with described nucleic acid molecule.
In another preference, described cell is a prokaryotic cell prokaryocyte; Preferred, described cell is a Bacillus coli cells.
In another preference, described cell is eukaryotic cell or vegetable cell.
In another aspect of this invention, a kind of immunogenic virus-like particle that has is provided, described virus-like particle is assembled by described fusion rotein.
In another preference, the mean diameter of described virus-like particle is 20-30nm; Preferably be about 25nm.
In another aspect of this invention, a kind of method for preparing the virus-like particle that claim 8 states is provided, said method comprises:
(1) cultivates described cell, thereby express described fusion rotein;
(2) fusion rotein that separation and purification (1) obtains.
In another aspect of this invention, described purposes with immunogenic virus-like particle is provided, is used to prepare the compsn of prevention human enterovirus 71 virus infection relative disease.
In a preference, described disease is: and hand foot mouth disease (Hand, foot and mouth disease, HFMD).
In another aspect of this invention, a kind of immunogenic compositions that has is provided, described compsn comprises:
(a) described have an immunogenic virus-like particle; With
(b) pharmaceutically acceptable carrier.
In another aspect of this invention; A kind of method for preparing prevention human enterovirus 71 virus infection relative disease is provided; Said method comprises: need the described of object significant quantity of prevention to have immunogenic virus-like particle, or describedly have an immunogenic compositions.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
The signal collection of illustrative plates of Fig. 1, colibacillus expression plasmid pET28b-HBc, pET28b-HBcSP55 and pET28b-HBcSP70.Peptide section SP55 sequence is: PDSRESLAWQTATNP (SEQ ID NO:2); Peptide section SP70 sequence is: YPTFGEHKQEKDLEY (SEQ ID NO:3).
SDS-PAGE electrophoresis behind Fig. 2, the protein purification.
Carrying out Western blot with the anti-His monoclonal antibody behind Fig. 3, the protein purification detects.
Carrying out Western blot with the anti-HBc monoclonal antibody behind Fig. 4, the protein purification detects.
Fig. 5, A are 10 layer components with the HBc-VLP after the sandwich ELISA detection sucrose gradient centrifugation; B, C, D result for carrying out Western blot detection with 10 layer components of anti-His monoclonal antibody after to HBc, HBc-sp55, three kinds of VLP sucrose gradient centrifugations of HBc-sp70.
Fig. 6, A, B, C are respectively the electromicroscopic photograph (80000 times) of HBc, HBc-sp55, three kinds of VLP of HBc-sp70.
Fig. 7, respectively organize the detected result (1: 100 extent of dilution of serum) of the HBc specific antibody in the mice serum.
Fig. 8, respectively organize the detected result (1: 100 extent of dilution of serum) of the SP55 specific antibody in the mice serum.
Fig. 9, respectively organize the detected result (1: 100 extent of dilution of serum) of the SP70 polypeptide antibody in the mice serum.
Embodiment
The inventor discloses a kind of fusion rotein first through deep research, and this fusion rotein comprises the human enterovirus 71 VP1 proteic peptide section SP55 and/or the SP70 of at least 1 copy; And HBcAg (HBcAg).This fusion rotein can carry out oneself's assembling through renaturation behind expression and sex change purifying, have strong immunogenic virus-like particle thereby form, and this virus-like particle wide spectrum neutralizing antibody of inducement efficient valency in vivo produces.
As used herein; Term " fusion rotein of the present invention ", " fusion rotein of human enterovirus 71 VP 1 proteic peptide section SP55 and/or SP70 and HBcAg " interchangeable use; All refer to human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70 by at least 1 copy; Merge the albumen form with HBcAg, wherein between human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70, HBcAg, can be connected or coupling through chemical bond; Be connected through chemical bond (like peptide bond) between preferable they, have or do not have the connection peptides sequence between them.
As used herein, it is a kind of by 1 or many virus-like particles that the monomer fusion rotein assembles that term " has immunogenic virus-like particle ".It is described that to have immunogenic virus-like particle be granular.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " belong to " containing ", the subordinate concept of " having " or " comprising " with " by ... formation ".
As used herein, " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other part of linear DNA sequence.For example, if promotor control with the transcribing of encoding sequence, it is exactly operationally to be connected in encoding sequence so.
Fusion rotein
The present invention provides a kind of fusion rotein, and this albumen comprises the human enterovirus 71 VP1 proteic peptide section SP55 and/or the SP70 of at least 1 copy; And HBcAg.This molecular energy is used to form has immunogenic virus-like particle.Preferable, described fusion rotein is a kind of isolating albumen, does not have with other albumen, polypeptide or molecule and gets in touch, and is the purified product cultivated of recombinant host cell or as a kind of extract of purifying.
The described proteic antigen of enterovirns type 71 (EV71) VP1 that derives from comprises peptide section SP55 and/or peptide section SP70.
VP1 is a kind of capsid protein of EV71, can induce neutralizing antibody and virus attack is had provide protection.VP1 is last contains 15 amino acid whose linear peptides section SP55 and SP70 can induce body to produce neutralizing antibody (Foo behind immune mouse; D.G.; S.Alonso, M.C.Phoon, N.P.Ramachandran; V.T.Chow, and C.L.Poh.2007.Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides.Virus Res 125:61-8).But, the less immunogenic of SP55 and SP70 polypeptide itself.Wherein, the sequence of described peptide section SP55 is following: PDSRESLAWQTATNP (SEQ ID NO:2); The sequence of described peptide section SP70 is following: YPTFGEHKQEKDLEY (SEQ ID NO:3).
HBcAg can be self-assembled into virus-like particle, and with derive from the proteic antigen of enterovirns type 71 (EV71) VP1 and form fusion rotein of the present invention after, still can keep self-assembling ability.
The bioactive fragment of HBcAg is meant that as a kind of polypeptide fragment its all or part of oneself that still can keep the total length HBcAg is assembled into the ability of virus-like particle.Generally, described bioactive fragment keeps the self-assemble ability of 50% total length HBcAg at least.Under preferred condition, described bioactive fragment can keep the self-assemble ability of 60%, 70%, 80%, 90%, 95%, 99% or 100% total length HBcAg.
Total length HBcAg or its bioactive fragment comprise a part of conserved amino acid alternative sequence, and said sequence through the amino acid replacement does not influence its self-assemble ability basically.This replaceability often can rule of thumb be confirmed or confirm according to known conserved sequence.
In optimal way of the present invention, the aminoacid sequence of described HBcAg can be substantially the same with the sequence shown in the SEQ ID NO:1.
Described deriving between the proteic antigen of enterovirns type 71 VP1, the HBcAg links to each other or coupling each other through chemical bond; Described chemical bond is covalent linkage or non covalent bond.As optimal way of the present invention, described deriving between the proteic antigen of enterovirns type 71 VP1, the HBcAg links to each other through chemical bond; Better, described chemical bond is a peptide bond.
Described deriving between the proteic antigen of enterovirns type 71 VP1, the HBcAg can directly be connected, and perhaps connects through polypeptide connexon (connection peptides).Described connexon for example comprises 1-50 amino acid; Preferably be 1-30 amino acid.The setting of connection peptides does not influence fusion rotein oneself assembling formation basically and has immunogenic virus-like particle.
The inventor is surprised to find, and when HBcAg formed virus-like particle, the amino acid of its 77-83 position was in a position preferably, and it is exposed on the skin of three-dimensional structure.Therefore; As optimal way of the present invention; In the described fusion rotein, the human enterovirus 71 VP1 proteic peptide section SP55 of described at least 1 copy and/or SP70 are inserted between any two amino acid between the HBcAg aminoacid sequence 76-84 position; Or the human enterovirus 71 VP1 proteic peptide section SP55 of described at least 1 copy and/or SP70 have replaced any one or several amino acid between the HBcAg aminoacid sequence 76-84 position.
As optimal way of the present invention, described fusion rotein comprises from the aminoterminal to the carboxyl terminal successively: HBcAg 1-76 amino acids; The human enterovirus 71 VP1 proteic peptide section SP55 and/or the SP70 of at least 1 copy; HBcAg the 84th～(144-185) amino acids.
On the other hand, the present invention also provides the isolating nucleic acid of the described fusion rotein of encoding, and also can be its complementary strand.The nucleic acid of the described fusion rotein of any coding all is applicable to the present invention.
The dna sequence dna of code book invention fusion rotein; Can the complete sequence synthetic; Also the method for available pcr amplification obtains respectively to encode and derives from the dna sequence dna of the proteic antigen of enterovirns type 71 VP1, HBcAg; Then it is stitched together, forms the dna sequence dna of code book invention fusion rotein.
The present invention also provides the carrier of the nucleic acid molecule that comprises encoding said fusion protein.Described carrier also can comprise the expression regulation sequence that links to each other with the series of operations property of said nucleic acid molecule, so that said Expression of Fusion Protein.
In the present invention, any suitable carriers can be used, and is used for the clone of bacterium, fungi, yeast and mammalian cell and the carrier of expression such as some, like Pouwels etc., and cloning vector: described in the laboratory manual.
In addition, the reconstitution cell that contains the nucleotide sequence of encoding said fusion protein is also included among the present invention.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.; For example can be Bacillus coli cells (E.coli), like intestinal bacteria HMS174 (DE3) or BL21 (DE3).Eukaryotic host cell commonly used comprises yeast cell, insect cell, vegetable cell and mammalian cell.In preferred implementation of the present invention, adopt prokaryotic cell prokaryocyte as host cell.
The method of producing fusion rotein also comprises in the present invention.Said method comprises cultivates the reconstitution cell that contains the fusion rotein coding nucleic acid.Said method can comprise the fusion rotein that lets cell expressing encode, and the renaturation that makes the fusion rotein of expression.Said method also can comprise the separation and/or the purifying of the fusion rotein of renaturation.
Can be the character of basic homogeneous with the above-mentioned fusion rotein purifying for preparing, for example on the SDS-PAGE electrophoresis, be single band.
Fusion rotein of the present invention can be used for preparation and has immunogenic virus-like particle, and described macromole can cause immunoreation in vivo, and described immunoreation comprises cell-mediated immunoreation.
Has immunogenic virus-like particle
The present invention also provides a kind of immunogenic virus-like particle that has, and it is polymerized by described fusion rotein basically.
The MHC I type of body immune system and MHC II type to the exogenous antigen that exists with Granular forms offer antigenic the offering of soluble and monomeric is eager to excel 1000 or 10000 times; The antigen that promptly exists with Granular forms has stronger immunogenicity than soluble and monomeric antigen; The MHC I type of body immune system and MHC II approach to the exogenous antigen that exists with Granular forms offer antigenic the offering of soluble and monomeric is eager to excel 1000 or 10000 times, the antigen that promptly exists with Granular forms has stronger immunogenicity than soluble and monomeric antigen.
Of the present invention have immunogenic virus-like particle and have very strong immunogenicity, and this is also different relevant with antigen or antigenic determinant that virus-like particle is exposed.The present invention derives from enterovirns type 71 VP 1 proteic antigen to have found the suitable carrier that is used to form virus-like particle, thereby the virus-like particle that forms has suitably exposed said antigen, has improved immunogenicity of antigens.
The present invention also provides described purposes with immunogenic virus-like particle, is used to prepare the compsn of prevention human enterovirus 71 virus infection relative disease.Described disease for example is: hand foot mouth disease.
Compsn
The present invention also provides a kind of immunogenic compositions (preventative or therapeutic vaccine) that has, and described compsn comprises: significant quantity of the present invention has immunogenic virus-like particle and pharmaceutically acceptable carrier.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and does not have excessive bad side reaction (like toxicity), promptly has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the carrier of therapeutical agent administration, comprises various vehicle and thinner.This term refers to some medicament carriers like this: they itself are not necessary activeconstituents, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find proving absolutely about pharmaceutically acceptable carrier in N.J.1991) at Remington ' s Pharmaceutical Sciences.Acceptable carrier can contain liquid on combination of traditional Chinese medicine is learned, like water, salt solution, glycerine and sorbyl alcohol.In addition, also possibly there is complementary material in these carriers, like lubricant, glidant, wetting agent or emulsifying agent, pH buffer substance and stablizer, like BSA etc.
Can described compsn be processed the various formulations that are suitable for the Mammals administration, said formulation includes but not limited to: injection, capsule, tablet, emulsion, suppository; It preferably is injection.
Experimentation on animals shows, utilize of the present invention have the vaccine immunity animal that immunogenic virus-like particle processes after, can produce the antibody that height is tired in animal body.
In use; Be safe and effective amount of the present invention to be had immunogenic virus-like particle be applied to Mammals (like the people); Wherein this safe and effective amount is usually at least about 1 microgram/kg body weight; And in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 1 microgram/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
The present invention adopts embedded virus appearance particle to show the strategy of SP55 and/or SP70, improves the immunogenicity of SP55 and/or SP70, has developed the wide spectrum vaccine to EV71.Particularly, the inventor has made up the fusion rotein of HBcAg (HBcAg) with SP55 or SP70, in intestinal bacteria, efficiently expresses, and has obtained to show the embedded virus appearance particle of SP55 or SP70.The SP55 or the SP70 specific antibody that have height to tire with this virus-like particle immune serum, this antiserum(antisera) multiple EV71 strain isolated that can effectively neutralize.These results show that the embedded virus appearance particle of showing SP55 or SP70 can be used as the wide spectrum vaccine to EV71.
Major advantage of the present invention is:
(1) the polypeptide SP55 of utilization of the present invention, the SP70 proteic neutralizing epitope of VP1 that is EV71; Wherein SP70 is more conservative in each hypotype of Enterovirus 71; SP55 has two amino acid whose differences between A, B, three hypotypes of C, induce neutralizing antibody and have broad spectrum.And more existing deactivation vaccines, subunit vaccine or the VLP that depends on VP1 be because VP1 difference to some extent between each hypotype, its cross protection ability a little less than.
(2) virus-like particle of HBcAg formation can be enriched in polypeptide SP55, SP70 on the surperficial furcella of virus-like particle; Being convenient to antigen is the identification of carrying cell and catches; Compare with other carrier link coupled polypeptide immune laboratory animal and to have certain advantage, be expected to produce higher antibody horizontal.
(3) security after treatment of the albumen of escherichia coli expression is higher.With respect to insect baculovirus expression system, the cycle of escherichia expression system is shorter, and commercial promise is preferably arranged.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
PET28b (+) plasmid, the plasmid pIBT-HBc that contains the HBcAg gene, restriction enzyme, Taq enzyme, T4 ligase enzyme are all available from NEB.The Ni-NTA resin can lottery industry reagent company available from the Shen, Shanghai.Western blot analyzes used anti-His monoclonal antibody available from Ai Bimate company, and anti-HBcAg monoclonal antibody mAb8638 is available from BiosPacific company.(in 6 ages in week, 18～22g) available from west, Shanghai pul-Bi Kai laboratory animal ltd for female BALb/c mouse.
The structure of embodiment 1, expression plasmid
The preparation primer is following:
SP55-Xba?I-F(SEQ?ID?NO：4)：
5’-CTAGAGCCAGATTCTAGGGAATCTCTTGCATGGCAAACTGCTACTAACCCTGGA-3’，
SP55-BglII-R(SEQ?ID?NO：5)：
5’-GATCTCCAGGGTTAGTAGCAGTTTGCCATGCAAGAGATTCCCTAGAATCTGGCT-3’，
SP70-Xba?I-F(SEQ?ID?NO：6)：
5’-CTAGAGTACCCAACATTCGGAGAACATAAACAGGAGAAAGACCTTGAATATGGA-3’，
SP70-BglII-R(SEQ?ID?NO：7)：
5’-GATCTCCATATTCAAGGTCTTTCTCCTGTTTATGTTCTCCGAATGTTGGGTACT-3’，
HBc-F-NcoI(SEQ?ID?NO：8)：
5’-CTGCCATGGACATTGACCCTTACAAAG-3’，
HBc-R-XhoI(SEQ?ID?NO：9)：
5’-GGCCTCGAGACATTGAGATTCCCTAGA-3’。
Get primer SP55-Xba I-F and the SP55-BglII-R of isopyknic 10uM respectively, behind SP70-Xba I-F and the SP70-BglII-R mixing, behind 94 ℃ of sex change 4min, ice bath 10min.Product with carry out ligation through restriction enzyme Xba I, the postdigestive pIBT-HBc of BglII; Connect with the T4DNA ligase enzyme; The ligation condition is 16 ℃, and 6h is with ligation product transformed into escherichia coli DH5 α; Extracting plasmid pIBT-HBc-SP55, pIBT-HBc-SP70 are after employing bacterium colony PCR, restriction enzyme Nco I, Sac I double digestion and order-checking etc. are identified correctly.With pIBT-HBc, pIBT-HBc-SP55 and three plasmids of pIBT-HBc-SP70 is template; With primer HBc-F-Nco I and HBc-R-Xho I are carried out pcr amplification; Amplified production and pET28b (+) are simultaneously after restriction enzyme Nco I, Xho I digestion; Connect with the T4DNA ligase enzyme, the ligation condition is 16 ℃, 6h.With ligation product transformed into escherichia coli DH5 α; Adopt methods such as bacterium colony PCR, restriction enzyme Nco I, Sac I double digestion and order-checking to identify; Can get plasmid pET28b-HBc, pET28b-HBc-SP55, pET28b-HBc-SP70, each plasmid signal collection of illustrative plates is seen Fig. 1.
The result
Expression vector pET28-HBc, pET28-HBcSP55 and pET28-HBcSP70 have successfully been made up.PET28-HBc is used to express nonfused HBcAg; PET28-HBcSP55 is the fusion protein expression vector that SP55 inserts the 77-83 amino acids place of HBcAg; PET28-HBcSP70 is the fusion protein expression vector that SP70 inserts the 77-83 amino acids place of HBcAg.Three carriers all contain T7 promotor and His label, are used in the expression in escherichia coli fusion rotein.
With recombinant plasmid pET28b-HBc, pET28b-HBc-SP55, pET28b-HBc-SP70 transformed into escherichia coli BL21, be inoculated in LB agar plate (containing kantlex 50mg/L), 37 ℃ of cultivations; After waiting to grow single bacterium colony, picking list colony inoculation is in LB (containing kantlex 50mg/L) nutrient solution, and 37 ℃ of shaking culture are spent the night; At 1: 100 next day, ratio was inoculated in the cultivation of the identical nutrient solution relaying of 500mL persistent oscillation; Treat that culture OD value reaches at 0.6 o'clock and adds IPTG (final concentration 0.8mmoL/L) and induce that the centrifugal 10min results of 12000rpm thalline behind 37 ℃ of 5h is with 50mL damping fluid I (0.5M NaCl; 20mM Tris; 10mM imidazole, pH7.9) resuspended thalline, sonicated 5min.The centrifugal 10min of 12000rpm abandons supernatant, with 30mL damping fluid II (0.5M NaCl, 20mM Tris, 10mM imidazole, 6M Urea, pH7.9) dissolving inclusion body deposition is spent the night.
Adopt Ni-NTA affinity chromatography column purification inclusion body solution next day.Albumen behind the purifying is removed urea through progressively dialysing.Products therefrom can carry out Western blot with anti-His monoclonal antibody and anti-HBcAg monoclonal antibody and detect.
Western blot detects
Protein sample carries out 12% gel SDS-PAGE electrophoresis earlier, and the transfer printing pvdf membrane places confining liquid (the PBST solution that contains 5% milk) sealing to spend the night film then; Add anti-(an anti-His monoclonal antibody or an anti-HBcAg monoclonal antibody; 1: 1000) hatch 2h, adding two, anti-(HRP-goat anti mouse IgG or AP-a-mouse Ab 1:5000) are hatched 2h again; Use chromogenic reagent 1min at last, observations.
SDGC
Protein sample is added to the saccharose gradient upper strata of 10%-50%, and 4 ℃ of centrifugal 2.5h of Beckman whizzer 39000rpm collect 10 saccharose gradients (0.4mL/ gradient), do ELISA and Western blot check and analysis.
Electronic microscope photos
Get protein 20 μ L after the renaturation on the copper mesh that carbon film encapsulates, room temperature is placed 5min, and careful the suction abandoned excess liquid, again through 10g/L phospho-wolframic acid (pH 7.0) negative staining 90s, H-600 transmission electron microscope observation.
The result
The HBc of escherichia coli expression, HBcSP55, HBcSP70 all mainly exist with the form of inclusion body; Through obtaining purifying protein (Fig. 2) behind combination of Ni-NTA post and the wash-out; Wherein the size of three protein moleculars has difference slightly, maybe with insert behind the polypeptide proteic structure to some extent due to the difference.
Detect albumen through Western Blot and all can combine (Fig. 3, Fig. 4) with the monoclonal antibody of anti-HBc and anti-His.
The purifying protein urea of dialysing, the albumen after the renaturation detects the 9th layer (Fig. 5 A) that HBcAg mainly concentrates on 10 saccharose gradients through sandwich ELISA, and is close with the description of HBcAg standard protein.Western blot result shows that HBc concentrates on the 9th layer, and HBcSP55, HBcSP70 then concentrate on the tenth layer and the 9th layer (Fig. 5 B, C, D) respectively.
Through electron microscopic observation, each fusion rotein has the due external appearance characteristic of virus-like particle (Fig. 6 A, B, C), the about 25nm of diameter.
Virus-like particle behind the purifying is diluted to 0.1 μ g/ μ L with PBS, respectively gets 100 μ L and equal-volume aluminium adjuvant vibration mixing 1h.3 experimental group, every group of 6 mouse are all given 200 μ L/ abdominal injection BALb/c mouse, and 0,3,5 each immunity of week are once established 1 PBS control group simultaneously, and PBS and aluminium adjuvant mixture 200 μ L/ * 6 are given in the abdominal cavity.Two weeks got blood through the eye socket vein respectively with the immunity back before the immunity, all serum all are stored in-20 ℃.
Get the serum of three exempting from (0,3,5 each immunity of week are once) back two weeks (i.e. the 7th week) and carry out the ELISA detection, respectively with HBc, SP55, SP70 encapsulate 96 orifice plates, the 100ng/ hole, and 37 ℃ encapsulate 2h; Milk sealing back 2h with 5%.With seized serum dilution, PBS mice in control group serum is done negative control, does same processing.Every part of serum all with above-mentioned three albumen effects, 50 μ L/ holes, 37 ℃ of effect 2h; Two is anti-with conventional HRP-goat-anti-mouse Ab (be diluted at 1: 5000 PBST that contains 1% milk), 37 ℃ of effect 2h, colour developing, reading.
Behind 56 ℃ of deactivation 30min of each serum with embodiment 3 acquisitions.Carry out virus neutralization tests: RD cell (available from the ATCC) suspension that will dispel with nutritive medium after will washing adds 96 porocyte culture plates, 100 μ L/ holes (2 * 10
5Individual cell/ml), in 37 ℃ of 5%CO
2Cultivate 24h in the incubator, to cell density be 70%, discard enchylema; Every part of seized serum is done dilution in 1: 8,1: 16,1: 32,1: 64, and each dilution serum is got the viral suspension (100TCID of 200 μ L and equivalent
50/ 0.1mL; Measure half tissue infection lethal quantity (TCID according to the Reed-Muench method
50) (Reed, L.J.M., H.1938.A simple method of estimating 50 percent endpoints.Am J Hyg 27:493-499)) mixing, 37 ℃ of effect 1h, each extent of dilution is done 4 holes, and every hole adds 100 μ L mixed solutions.Contrast is set up: every part of seized serum of serum toxicity contrast must be established 4 hole toxicity contrasts by high dilution, and every hole adds each extent of dilution serum 50 μ L and 50 μ L cell culture fluids.8 hole normal cell contrasts are established in the cell contrast, and every hole adds cell nutrient solution 100 μ L.Virus returns contrast, with viral liquid be diluted to 1000,100,10,1,0.1TCID
50/ 50 μ l, each extent of dilution is done 4 holes, and every hole adds viral suspension 50 μ L and 50 μ L cell culture fluids.Put 5% CO2gas incubator in 37 ℃ of cultivations, after 3 days, observe go forward side by side line item, result of CPE and judge.
The result
The Balb/c mouse is used HBc, HBcSP55 or the immunity of HBcSP70 virus-like particle respectively.The mice serum that obtains after to immunity through ELISA carries out antibody test, and the result shows in three experimental mice serum all can detect the HBc specific antibody, in the HBcSP55 group mice serum SP55 specific antibody is arranged and have only; Equally, have only HBcSP70 to organize in the mice serum SP70 specific antibody (Fig. 7,8,9) is arranged.
The neutralizing antibody level of serum is measured through neutralization test, and the result sees table 1.The mice serum that HBc group and PBS organize at minimum extent of dilution (1: 8) for the EV71 live virus activity that do not neutralize; It is 1: 64 that the neutralization of HBcSP55 group mice serum has been tired 4, and remaining 2 is 1: 32; It is 1: 64 that the mice serum of HBcSP70 group has been tired 3 for the neutralization of this virus, and 3 is 1: 128 in addition.
Table 1
Discuss
The present invention is with intestinal bacteria system expression HBc and SP55, SP70 fusion rotein, and forms virus-like particle.From result of experiment, the serum of HBc-SP55 or HBc-SP70 group mouse has higher antibody horizontal, and in all having preferably for EV71 and provide protection (respectively up to 1: 64; 1: 128), and latter's effect is better.And tiring with the neutralization of the serum that obtains behind KLH link coupled SP55, the SP70 immune mouse of Damian Guang Wei Foo was respectively 1: 8; 1: 32 (Foo; D.G., S.Alonso, M.C.Phoon; N.P.Ramachandran; V.T.Chow, and C.L.Poh.2007.Identification of neutralizinglinear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides.Virus Res 125:61-8), the survival rate of nascent mouse is 80% (Foo in the animal protection test of the serum of its relevant anti-SP70; D.G.; S.Alonso, V.T.Chow, and C.L.Poh.2007.Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide.Microbes Infect9:1299-306).
Therefore the serum that the present invention obtained has better effect in the protection of animal experiment.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (12)
1. fusion rotein comprises following albumen:
Human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70; With
HBcAg.
2. fusion rotein as claimed in claim 1 is characterized in that, described human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70 are inserted between any two amino acid between the HBcAg aminoacid sequence 76-84 position; Or
Described human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70 have replaced any one or several amino acid between the HBcAg aminoacid sequence 76-84 position.
3. fusion rotein as claimed in claim 2 is characterized in that, described fusion rotein comprises from the aminoterminal to the carboxyl terminal successively:
HBcAg 1-76 amino acids;
Human enterovirus 71 VP1 proteic peptide section SP55 and/or SP70;
HBcAg the 84th～(144-185) amino acids.
4. the purposes of the described fusion rotein of claim 1 is used for preparation and has immunogenic virus-like particle.
5. a nucleic acid molecule is characterized in that, the arbitrary described fusion rotein of described nucleic acid molecule encoding claim 1-3.
6. a carrier is characterized in that, described carrier contains the described nucleic acid of claim 4.
7. a cell is characterized in that, described cell contains and is integrated with the described nucleic acid molecule of claim 5 in the described carrier of claim 5 or its genome.
8. one kind has immunogenic virus-like particle, it is characterized in that, described virus-like particle is assembled by the arbitrary described fusion rotein of claim 1-3.
9. a method for preparing the described virus-like particle of claim 8 is characterized in that, said method comprises:
(1) cultivates the described cell of claim 6, thereby express the described fusion rotein of claim 1;
(2) fusion rotein that separation and purification (1) obtains.
10. the described purposes with immunogenic virus-like particle of claim 8 is used to prepare the compsn that prevents human enterovirus 71 virus infection relative disease.
11. purposes as claimed in claim 10 is characterized in that, described disease is: and hand foot mouth disease (Hand, foot and mouth disease, HFMD).
12. one kind has immunogenic compositions, it is characterized in that, described compsn comprises:
(a) claim 8 is described has an immunogenic virus-like particle; With
(b) pharmaceutically acceptable carrier.
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| CN105085626A (en) * | 2014-05-22 | 2015-11-25 | 厦门大学 | Broad-spectrum affinity epitope polypeptide and antibody for human enteroviruses and use thereof |
| CN106220738A (en) * | 2016-07-31 | 2016-12-14 | 中国医学科学院医学生物学研究所 | The structure of EV71 Neutralization and crystallization and norovirus P-structure territory chimeric vector and expression |
| CN107365762A (en) * | 2017-07-03 | 2017-11-21 | 东莞市第八人民医院 | It can secrete and the hybridoma cell strain and its construction method of the antibody of CA16, EV71 Neutralization and crystallization simultaneous reactions |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102925476A (en) * | 2012-11-15 | 2013-02-13 | 中国科学院上海巴斯德研究所 | Preparation method and application of self-combined immunogenic enterovirus-like particles in yeast |
| CN105085626A (en) * | 2014-05-22 | 2015-11-25 | 厦门大学 | Broad-spectrum affinity epitope polypeptide and antibody for human enteroviruses and use thereof |
| CN105085626B (en) * | 2014-05-22 | 2019-08-13 | 厦门大学 | The wide spectrum of human enterovirus is affine epitope polypeptide, antibody and application thereof |
| CN106220738A (en) * | 2016-07-31 | 2016-12-14 | 中国医学科学院医学生物学研究所 | The structure of EV71 Neutralization and crystallization and norovirus P-structure territory chimeric vector and expression |
| CN107365762A (en) * | 2017-07-03 | 2017-11-21 | 东莞市第八人民医院 | It can secrete and the hybridoma cell strain and its construction method of the antibody of CA16, EV71 Neutralization and crystallization simultaneous reactions |
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