CN102465144A - Coxsackie virus A16 type virus-like particle vaccine - Google Patents
Coxsackie virus A16 type virus-like particle vaccine Download PDFInfo
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Abstract
The invention relates to a coxsackie virus A16 type virus-like particle vaccine. The inventor fortuitously finds that proteins which have better spatial configurations and are suitably cut can be obtained by expressing P1 protein of CVA16 and 3CD protein of CVA16 by infecting insect cells with rhabdovirus. The proteins can be automatically assembled into a virus-like particle which has high immunogenicity.
Description
Technical field
The invention belongs to biotechnology and biomedicine field; More specifically, the present invention relates to the coxsackie virus A 16-type virus sample particle vaccines.
Background technology
Hand foot mouth disease is children's a common eqpidemic disease, does not still have effective vaccine and medicine at present.Coxsackie virus A 16 (CVA16) and HEV 71 (EV71) are two main cause of diseases of hand foot mouth disease, belong to the Picornaviridae enterovirus genus, strand justice RNA viruses, the about 7400bp (Xu of genome; J., Y.Qian, S.Wang, J.M.Serrano; W.Li, Z.Huang, and S.Lu.2010.EV71:anemerging infectious disease vaccine target in the Far East Vaccine28:3516-21 and 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 persistentepidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China.J Clin Microbiol 48:619-22).Enterovirus genus genome encoding 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.ApplBioinformatics 1:43-52).Some enteroviruses wherein, like poliovirus, VP0 albumen further is cut into VP2 and VP4 (Kitamura, N.; B.L.Semler, P.G.Rothberg, G.R.Larsen; C.J.Adler, A.J.Dorner, E.A.Emini; R.Hanecak, J.J.Lee, S.van derWerf; C.W.Anderson, and E.Wimmer.1981.Primary structure, gene organizationand polypeptide expression of poliovirus RNA.Nature 291:547-53).
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.EV71 comes to light and is associated with hand foot mouth disease severe and death, and the research and development of therefore existing and ongoing virusology investigation and vaccine for hand-foot-mouth disease mainly concentrate on EV71.But the research of nearest clinical case shows that China is hand foot mouth disease relevant (Zhang, Y., X.J.Tan, the H.Y.Wang with CVA16 and EV71 of outburst recently; D.M.Yan, S.L.Zhu, D.Y.Wang, F.Ji, X.J.Wang; Y.J.Gao, L.Chen, H.Q.An, D.X.Li, S.W.Wang; A.Q.Xu, Z.J.Wang, and W.B.Xu.2009.An outbreak of hand, foot, and mouth diseaseassociated with subgenotype C4 of human enterovirus 71 in Shandong; China.JClin Virol 44:262-7 and 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.Molecularevidence of persistent epidemic and evolution of subgenotype B1 coxsackievirusA16-associated hand, foot, and mouth disease in China.J Clin Microbiol48:619-22); Possible coinfection EV71 of patient and CVA16, and have this two kinds of viruses in vivo simultaneously, thereby cause CVA16 and EV71 to recombinate, cause the appearance of EV71 new subtype D.The independent infection of CVA16 also possibly cause children's death.These researchs show that the multivalence wide spectrum vaccine that anti-CVA16 and EV71 are held concurrently in exploitation could effectively prevent and control hand foot mouth disease.
Can spontaneous assembling assembly virus-like particle after the virus structural proteins of a lot of viruses are recombinant expressed (virus-likeparticle, VLP), its structure and antigenicity and euvirus are as broad as long, but lack viral nucleic acid, be do not have infective.Virus-like particle has become develops an available strategy of virus disease vaccine safely and effectively, for example, has obtained to examine based on hepatitis B and nerpes vinrus hominis's vaccine of virus-like particle.In expressed in insect cells, it can produce the active antibody of neutralization to the virus-like particle of EV71, thus the mouse of protection lethal dose virus attack.
Yet this area does not also have a kind of desirable vaccine that effectively is directed against Coxsackie virus at present.
Summary of the invention
The object of the present invention is to provide a kind of coxsackie virus A 16-type virus sample particle vaccines.
In first aspect of the present invention, a kind of expression vector is provided, it comprises:
At least one expression cassette 1 (as 1,2 or 3), described expression cassette 1 contain the proteic encoding sequence of P1 of coxsackie virus A 16-type virus (CVA16); With
At least one expression cassette 2 (as 1,2 or 3), described expression cassette 2 contain the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus.
In another preference, described expression vector is pFastBacTM Dual (pFBD) carrier.
In another aspect of this invention, a kind of recombinant baculovirus is provided, comprises in the genome of said baculovirus:
At least one expression cassette (as 1,2 or 3), described expression cassette 1 contain the proteic encoding sequence of P1 of coxsackie virus A 16-type virus (CVA16); With
At least one expression cassette (as 1,2 or 3), described expression cassette 2 contain the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus.
In another preference, described recombinant baculovirus is used for infected insect cell and obtains recombinant protein.
In another preference, said baculovirus prepares through following steps:
(A) the proteic encoding sequence of P1 of coxsackie virus A 16-type virus (CVA16) and the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus are cloned in the rod granule, obtain the rod granule of reorganization;
The rod granule transfection insect cell of the reorganization that (B) (A) is obtained is cultivated the insect cell after the transfection; With
(C) baculovirus of results reorganization.
In another preference, described rod granule is the Bac-mid rod granule.Preferred, described Bacmid derives from intestinal bacteria DH10Bac.
In another preference, described insect cell is the sf9 insect cell.
In another aspect of this invention, provide described expression vector or the purposes of described recombinant baculovirus, be used to prepare virus-like particle.
In another aspect of this invention, a kind of system that produces virus-like particle is provided, comprises:
(1) described recombinant baculovirus; With
(2) insect cell.
In another preference, described insect cell is the sf9 insect cell.
In another aspect of this invention, a kind of method for preparing virus-like particle is provided, comprises:
(a) with described recombinate shape virus infection insect cell;
(b) from the lysate of insect cell, separate the acquisition virus-like particle.
In another preference, through SDGC isolated viral appearance particle.
In another aspect of this invention, a kind of immunogenic virus-like particle that has is provided, it is produced by described method, and its mean diameter is 20-30nm.
In another aspect of this invention, the purposes of described virus-like particle is provided, is used to prepare the compsn of prevention coxsackie virus A 16-type virus infection relative disease.
In another 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 coxsackie virus A 16-type 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
Fragment structure synoptic diagram in Fig. 1, each rhabdovirus expression vector Tn7.
Fig. 2, with rhabdovirus system expression CVP1 and CV3CD.The Sf9 insect cell is used recombinate shape virus infection, and collecting cell carries out with anti-VP0 antibody after 2 days:
(A) immunofluorescence;
(B)ELISA；
(C) Western blot analyzes.
The SDGC analysis of Fig. 3, VLP.Cell lysate is collected 10 components from top to bottom and is analyzed after being laid down on and carrying out ultracentrifugation on the saccharose gradient of 10-50%.
(A) carry out elisa assay with anti-VP0 antibody;
(B) carrying out Western blot with anti-VP0 antibody analyzes;
(C) carrying out Western blot with anti-VP3 antibody analyzes;
(D) carrying out Western blot with anti-VP1 antibody analyzes.
The electronic microscope photos of Fig. 4, CVA16 virus-like particle.
CVA16 capsid subunit protein specific IgG antibodies reaction in Fig. 5, the immune serum.Serum dilution is 1: 100.The Y axis values is the absorbancy of 450nm.
Fig. 6, immune serum are tired to the neutralization of CVA16 live virus.The Sf9 immune serum did not have neutralizing effect at 1: 32 o'clock detecting minimum extent of dilution, therefore was endowed a virtual neutralization and tired and calculate at 1: 16.
Embodiment
The inventor is through extensive and deep research; Be surprised to find that and adopt the baculovirus infection insect cell to express the P1 albumen of CVA16 and the 3CD albumen of CVA16; Can obtain to have reasonable sterie configuration and albumen through suitably cutting; Said albumen can be assembled into virus-like particle automatically, and said virus-like particle has high immunogenicity.Accomplished the present invention on this basis.
Baculovirus
The invention provides a kind of baculovirus of reorganization, it can be used for infected insect cell and obtains the albumen through suitable cutting.Contain the expression cassette 1 that at least one contains the proteic encoding sequence of P1 of coxsackie virus A 16-type virus in the genome of described baculovirus; Contain the expression cassette 2 of the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus with at least one.
The baculovirus of described reorganization can be stablized and gone down to posterity, subzero 70 ℃ can prolonged preservation and have high titre.Can obtain recombinant protein after 1-2 days after utilizing this virus infection insect cell, and express stable.
As optimal way of the present invention, said baculovirus prepares through following steps:
(A) with the proteic encoding sequence of P1 of coxsackie virus A 16-type virus and the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus be cloned in the rod granule, obtain the rod granule of reorganization;
The rod granule transfection insect cell of the reorganization that (B) (A) is obtained is cultivated the insect cell after the transfection; With
(C) baculovirus of results reorganization.
As optimal way of the present invention, described rod granule is the Bac-mid rod granule.Preferred, described Bac-mid derives from intestinal bacteria DH10Bac.
As optimal way of the present invention, described insect cell is selected from the sf9 insect cell, the HighFive insect cell; Most preferred, said insect cell is the sf9 insect cell.
As optimal way of the present invention; With the proteic encoding sequence of P1 of coxsackie virus A 16-type virus and the proteic encoding sequence of 3CD and the two ends recombination sequence thereof of coxsackie virus A 16-type virus, under the effect of transposase, be recombined into the Tn7 site of Bacmid rod granule.Preferred, described transposase is expressed by the helper plasmid that carries among the intestinal bacteria DH10Bac (helper).
As optimal way of the present invention, in step (A), the rod granule of said reorganization obtains through following steps:
(a) with the proteic encoding sequence of P1 of coxsackie virus A 16-type virus and the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus insert in the MCS of shuttle vectors (preferred pFASTBac-Dual carrier), obtain to have inserted the recombinant vectors of outer source coding sequence;
(b) recombinant vectors that (a) is obtained changes host cell DH10Bac over to, obtains transformed host cells, contains the reorganization rod granule in the said transformed host cells, and described reorganization rod granule carries described outer source coding sequence; With
(c) from said transformed host cells, extract the reorganization rod granule.
As optimal way of the present invention, the P1 albumen of described coxsackie virus A 16-type virus has the aminoacid sequence shown in the SEQID NO:1.The CD3 albumen of described coxsackie virus A 16-type virus has the aminoacid sequence shown in the SEQ ID NO:2.
As optimal way of the present invention, the encoding sequence of MCS is also contained at the two ends of the encoding sequence of described foreign protein (the CD3 albumen of the P1 albumen of coxsackie virus A 16-type virus or coxsackie virus A 16-type virus).
As optimal way of the present invention; Described baculovirus is through the proteic encoding sox of P1 of foreign gene coxsackie virus A 16-type virus and the proteic encoding sox clone of CD3 of coxsackie virus A 16-type virus are got in the MCS of pFASTBac; Then pFASTBAC is transformed the DH10Bac genetic engineering bacterium; Under the effect of transposase, recombinate, transfection insect cell obtains behind the acquisition rod granule.With behind this virus infection insect cell several hours, virus can get into insect cell, the beginning replication-competent virus, and transcribe the exogenous nucleic acid sequences in protein translation district in the viral recombination region, in this section sequence, contain the nucleotide sequence of said external source.Infect and express albumen after about 1-2 days with pairing aminoacid sequence in protein translation district in the viral recombination region.
In addition,, can also from culture, reclaim recombinant baculovirus, be used for subinfection down through methods such as for example density gradient centrifugations at described baculovirus infection insect cell and after expressing foreign protein.
Has immunogenic virus-like particle
The inventor provides a kind of method for preparing virus-like particle, and the method comprising the steps of:
(1) with described baculovirus infection insect cell, cultivates metainfective insect cell, make it to express described foreign protein
(2) from the lysate of insect cell, separate the acquisition virus-like particle.
As optimal way of the present invention, described insect cell is selected from: sf9 insect cell, HighFive insect cell; Most preferred, said insect cell is the sf9 insect cell.
The present invention also provides a kind of immunogenic virus-like particle that has, and it is obtained by method for preparing 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.The mean diameter (particle diameter) of the virus-like particle that the present invention obtains is 20-30nm.
The present invention also provides described purposes with immunogenic virus-like particle, is used to prepare the compsn that prevention human coxsackievirus A16 C-type virus C infects 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 PharmaceuticalSciences.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.
Major advantage of the present invention is:
The present invention has prepared the recombinant virus appearance particle (CVA16-LP) of CVA16 first, has obtained senior middle school and active antibody behind the immune mouse, shows that CVA16-LP is the desirable vaccine of prevention Coxsackie virus.
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.
Material
Cell and virus
RD (human rhabdomyosarcoma's cell is available from ATCC) cell: contain 37 ℃ of the DMEM substratum of 10%FBS, 100unit/mL penicillium mould and 100ug/mL Streptomycin sulphate, 5%CO
2Cultivate.
Virus CVA16 shzh05-1 (genome sequence is referring to the GenBank accession number: and EU262658) (referring to Wu, Z., Y.Gao; L.Sun; P.Tien, and Q.Jin.2008.Quick identification of effectivesmall interfering RNAs that inhibit the replication of coxsackievirus A16.AntiviralRes 80:295-301.Yang, F.; Q.Jin; Y.He, L.Li, and Y.Hou.2001.The completegenome of Enterovirus 71 China strain.Sci China C Life Sci 44:178-83) in the RD cell, breed; Microtitrimetry is measured the CVA16 virus titer, measures half tissue infection lethal quantity (TCID according to the Reed-Muench method
50) (Reed, L.J.M., H.1938.A simple method ofestimating 50 percent endpoints.Am J Hyg 27:493-499).
(CA USA) cultivates insect cell Sf9 in 27 ℃ for Invitrogen, Carlsbad to adopt SF90 0II SFM substratum.
Antibody
The mouse monoclonal antibody MAB979 of anti-EV71 (cross reaction being arranged) with CVA16 purchase in Millipore (Billerica, MA, USA); The antiserum(antisera) of anti-VP0, VP1 or the VP3 of cavy prepares for three times through reorganization VP0, VP1 or VP3 immunity (routine immunization method) cavy with escherichia coli expression (ordinary method expression).
The preparation recombinant baculovirus
CVA16 shzh05-1 infects RD cell, Trizol (Invitrogen, Carlsbad; CA, USA) method is extracted viral RNA, and oligo (dT) is a primer; M-MLV (Invitrogen, Carlsbad, CA; USA) ThermoScript II is carried out reverse transcription, is P1 albumen and the proteic gene fragment of 3CD of template amplification coding CVA16, called after CV P1 and CV 3CD respectively with the first chain cDNA that obtains.
Adopt: 5 '-CACTCTAGAATGGGGTCACAAGTCTCC-3 ' (SEQ ID NO:3) is a upstream primer, and 5 '-CCAAAGCTTTTACAATCTTGTTATCTTGTC-3 ' (SEQ ID NO:4) is a downstream primer, amplification CV P1; Behind Xba I and the Hind III double digestion, subclone is cut the pFBD of processing to same enzyme, and (abbreviation of pFastBacTM Dual (Invitrogen, Carlsbad, CA, USA)) makes up pFBD-CVP1.
With 5 '-TCACCATGGGACCGAGCTTAGACTT-3 ' (SEQ ID NO:5) is upstream primer, and 5 '-CACATGCATCTAAAATAATTCGAGCCA-3 ' (SEQ ID NO:6) is a downstream primer, amplification CV 3CD; Behind Nco I and the Nsi I double digestion, subclone is cut the pFBD of processing to same enzyme, makes up pFBD-CV3CD.
Xba I and Hind III double digestion pFBD-CVP1 obtain CVP1, and the pFBD-CV3CD that subclone is cut to same enzyme makes up pFBD-CVP1/CV3CD.
(CA USA) prepares corresponding baculovirus to above-mentioned three plasmids for Invitrogen, Carlsbad, respectively called after Bac-CVP1, Bac-CV3CD and Bac-CVP1/3CD through the Bac-to-Bac system.With recombinant plasmid transformed DH10Bac E.coli, identify recombinant plasmid Bac-mid according to the Invitrogen specification sheets, Bac-mid transfection Sf9 cell, screening amplification recombinant baculovirus.Press the specified MOI of Invitrogen specification sheets with recombinate shape virus infection Sf9 cell, express target protein.Infect back 48h and collect cell and the supernatant that infects, go to the test of biochemical analysis and Electronic Speculum.
Immunofluorescent test
Behind the recombinant virus infection Sf9 cell 48h, PBS washes cell twice, 4% (w/v) Paraformaldehyde 96 room temperature fixed cell 20min, and PBS gives a baby a bath on the third day after its birth inferior; 1% (v/v) NP40 effect room temperature 10min, PBS give a baby a bath on the third day after its birth inferior, 37 ℃ of effects of 10% (v/v) normal sheep serum (NGS) 30min; PBS gives a baby a bath on the third day after its birth inferior, 37 ℃ of effects of the anti-CVVP0 of cavy (1: 100) 30min, and PBS gives a baby a bath on the third day after its birth inferior; 37 ℃ of effects of the goat-anti cavy (1: 500) of FITC mark 30min, PBS gives a baby a bath on the third day after its birth inferior, and the DAPI room temperature is dyed nuclear 5min; PBS washes five times, 50% (v/v) glycerine mounting, fluorescence microscope.
ELISA and Western blot
Collect the cell of baculovirus infection, the PBS damping fluid re-suspended cell of precooling, FastprepFP120 (Bio101, Vista, CA, USA) smudge cells, 4 ℃ of centrifugal 15min of 12000rpm collect supernatant.With the bovine serum albumin is standard protein, Bradford analysis of protein test kit (Bio-Rad, Hercules, CA, USA) concentration of solubility total protein in the mensuration supernatant.
Directly ELSA detects, and PBS is a coating buffer, supernatant 50uL/ hole, the centrifugal back of cell lysate; 37 ℃ encapsulate 96 hole elisa plate 2h, and PBST (contains 1 * PBS) rinsing 3 times of 0.05% (v/v) tween 20,37 ℃ of sealings of 5% (w/v) skim-milk 1h; PBST rinsing 3 times, one anti-be 37 ℃ of effect 2h of 1: 2000 anti-CVVP0 of cavy (the recombinant C VA16 VP0 immune guinea pig of escherichia coli expression three times after antiserum(antisera)), PBST rinsing 3 times; Two anti-be 37 ℃ of effects of goat-anti cavy IgG (sigma) 1h of 1: 5000 HRP mark; PBST rinsing 5 times, TMB (TMB)-hydrogen peroxide system is the substrate colour developing, 1M H
3PO
4End, ELIASA 450nm detects, and establishes negative control and blank simultaneously.
Western bloting detects, and the preparation sample carries out 12% gel SDS-PAGE electrophoresis, transfer printing pvdf membrane then earlier; 5% (w/v) skim-milk room temperature sealing 2h; PBST (polysorbas20 of 0.05% (v/v)) rinsing 3 times, anti-(antiserum(antisera) of anti-VP0, VP1 or the VP3 of cavy prepares for three times through reorganization VP0, VP1 or VP3 immune guinea pig with escherichia coli expression) incubated at room 2h of dilution in 1: 1000; PBST rinsing 3 times; Corresponding two anti-(goat-anti cavy IgG) incubated at room 1h of HRP mark, PBST rinsing 5 times, the BeyoECL luminescence reagent LAS-4000 cold light analyser (Fujifilm Life Science) that adds appropriate amount develops.
Sucrose density gradient and partial purification VLP
4 ℃ of centrifugal 15min of cells infected lysate 12000rpm; The collection supernatant is added to the saccharose gradient upper strata of 10%-50%; 4 ℃ of centrifugal 3h of Beckman whizzer 39000rpm collect 10 saccharose gradients (0.4mL/ gradient), do ELISA and Western blot check and analysis.According to ELISA and Western blot result, collect the gradient of CV VP0 enrichment, Centrifugal Filter Units (Millipore; UFC800396; USA) protein concentrate sample according to the virus-like particle of the Western blot method quantitative collection of having set up, is prepared immune animal.
Electronic microscope photos
The above-mentioned partially purified virus-like particle of the water-soluble uranyl acetate negative staining of 0.5% (w/v), Philip CM-12S scanning electron microscope analysis.
Mouse immune program and antibody titer detect
(Pierce, Rockford IL) carry process specifications according to aluminium adjuvant and mix with antigen protein and aluminium adjuvant; 6 female Balb/c mouse of peritoneal immunity (6-8 age in week); 1ug/ virus-like particle (quantitative according to VP0) handled blank cell Sf9 equally, sets up negative control group.The 3rd and 6 weeks were carried out second and three immunity respectively, blood sampling before each immunity, and serum is collected in the 8th all eyeball of mouse blood samplings, and-80 ℃ of packing are preserved.
Each 100ng/ hole wrapper sheet of the VP0 of the CVA16 of prokaryotic expression, VP1 and VP3; Measure special IgG in the serum according to the ELISA method of having set up (6); With the detection hole greater than 0.1 OD value of corresponding negative hole (preimmune serum) is that yin and yang attribute judges that the inverse of its serum diluting multiple is serum ELISA and tires.
Neutralization test
Through on the RD cell, carrying out TCID
50In the subtrahend analysis of experiments serum and titre, be summarized as follows: RD cell inoculation 96 orifice plates, 1 * 10
4When/porocyte, incubated overnight reach 70% degree of converging to cell, carry out neutralization test.The DMEM gradient dilution serum sample of 2% (v/v) FBS, the CVA16 viral dilution is to 2TCID
50/ uL, the CVA16 virus (100TCID of the serum sample of 50uL dilution and 50uL dilution
50) mix 37 ℃ and hatch 1h, the serum-virus mixture is added in the long hole that the RD cell arranged 37 ℃ and cultivates 3d, the serum dilution that can reduce 50% cytopathy (CPE) be this serum in and titre.
Embodiment
The structure of embodiment 1, baculovirus
The coding P1 of CVA16/shzh05 and the gene fragment of 3CD are synthetic by pcr amplification after through the reverse transcription first chain cDNA, are cloned into the pFBD carrier then, respectively called after pFBD-CVP1 and pFBD-CV3CD.The CVP1 gene is through the polyhedrin promoter regulation among the pFBD-CVP1, and the CV3CD gene is then by the p10 promotor direct regulation and control in the pFBD-CV3CD.The pFBD-CVP1/3CD (Fig. 1) that has loaded two genes simultaneously also successfully makes up, its can be in allogenic cell common two kinds of albumen of effective expression.
Above-mentioned three kinds of structures are building up to corresponding baculovirus more subsequently, difference called after Bac-CVP1, Bac-3CD, Bac-CVP1/3CD.
Embodiment 2, at insect cell inner expression CVA16VLPs
Use Bac-CVP1, Bac-3CD, Bac-CVP1/3CD infect the Sf9 insect cell respectively.At first detect target protein expression in the infected insect cell through immunofluorescence method with the how anti-of anti--CVA16VP0.The result is shown in Fig. 2 A, and Bac-CVP1 and Bac-CVP1/3CD have bright green fluorescence; Blank control group and Bac-3CD infected group do not detect fluorescent signal.Bac-CVP1 also is confirmed through the ELISA experiment in the expression of insect cell simultaneously: the antibody test of anti-CVA16 VP0 is to the expression of the lysis of Bac-CVP1 and Bac-CVP1/3CD, and blank control group and Bac-3CD infected group do not detect signal (Fig. 2 B).
Secondly, anti-VP0 antibody Western blot has analyzed P1 by the shear history of 3CD.Shown in Fig. 2 C; Blank control group and Bac-3CD infected group do not detect band; And the lysate of Bac-CVP1 has a part amount to be about the band of 60KDa; Bac-CVP1/3CD has two bands that are respectively 39KDa and 28KDa, and these bands are consistent by the purpose stripe size that correct cutting produced with the VP0 and the VP2 of expection.Same strips with on wild-type CVA16, found consistent.
In a word, these results prove that P1 albumen can effectively produce and cut by correct in the cell that Bac-CVP1/3CD infects.Bac-CVP1/3CD is used for next step research.
The formation of virus-like particle behind ELISA and the Western blot analysis Bac-CVP1/3CD cells infected, SDGC isolated cell lysate, ELISA detects the differential protein of different saccharose gradient CVA16.Anti-CVVP0 polyclonal antibody detects target protein and mainly has gradient 6# (Fig. 3 A), proves the existence of virus-like particle.Utilize three anti-CVVP0 of polyclonal antibody of CVA16, anti-CVVP1 and anti-CVVP3 Western bloting further to analyze different saccharose gradients respectively and find that the strong signal to these three antibody all appears in component #6, with ELISA result consistent (Fig. 3 B, 3C and 3D).Molecular weight that the more important thing is the purpose band is consistent with the wild strain substructure molecular weight of albumen of having reported, and promptly the VP0 size is 39KDa, and VP2 is 28KDa, and VP1 is 33KDa, and VP3 is 26KDa.The result shows that virus-like particle is to be formed by the capsid substructure albumen that correct cutting is modified.Be the directly formation of definite virus-like particle, after the gradient 6# sample negative staining, the electron microscopic observation analysis, the result observes the virus-like particle (Fig. 4) of the about 25nm of diameter.
Embodiment 3, the immunogenicity of CVA16 virus-like particle in mouse
The immunogenicity of CVA16 virus-like particle is estimated in experimentation on animals, 1ug/ the mouse of virus-like particle abdominal injection in the 0th, 3 and 6 weeks with thick purification, and aluminium adjuvant is an adjuvant, the same Sf9 cell of handling that does not infect is as contrast.Two weeks were collected mice serums after the last immunity, encapsulated ELISA with recombinant antigen and detected special antibody, and the result shows that VLP immune group serum can react with subunit's coat protein of reorganization, and contrast Sf9 serum does not react (Fig. 5).
The neutralization of embodiment 4, VLP immune serum is active
The neutralization of neutralization test assessment VLP immune serum is active, and the serum of twice gradient dilution mixes with the CVA16 of 100TCID50 virus, adds the RD cell, according in the serum dilution judgement serum that can cause minimizing 50% cytopathy (CPE) and titre.
The result shows, the serum that Sf9 cell lysate immune mouse obtains is in the minimum extent of dilution (1: 32) that the detects activity that neutralize, and the neutralization of virus-like particle immune serum tire up to 1: 4096 (Fig. 6).This result shows that the CVA16 virus-like particle of reorganization can cause that body produces the active antibody of higher neutralization, thereby can be used as the vaccine of control CVA16.
Discuss
In the world wide, Coxsackie virus (CVA16) and Enterovirus 71 (EV71) virus are the main cause of diseases of hand foot mouth disease.Nearest discovers, coinfection and reorganization take place for CVA16 and EV71 virus, and this possibly be the major cause of past few years China's Mainland hand foot mouth disease large-scale outbreak.Therefore, CVA16 and EV71 virus all should be the target spots of vaccine for hand-foot-mouth disease research, thereby extensively prevent and treat hand foot mouth disease effectively.
Reported first of the present invention preparation and the ability of inducement efficient valency neutralizing antibody thereof of CVA16 virus-like particle.Utilize the baculovirus of recombinating to prepare the virus-like particle of CVA16 at insect cell coexpression P1 and 3CD; Biochemical test and electronic microscope photos are found the globosity of CVA16 virus-like particle for about 25nm, mainly are made up of capsid protein VP0, VP1, VP3 and a spot of VP2 substructure albumen of virus.The antiserum(antisera) and the capsid subunit protein that prepare behind the CVA16 virus-like particle immune mouse have special reaction, more meaningfully, this serum can external effectively in the CVA16 live virus, the neutralization tire up to 1: 4096.The present invention explains that the antibody that the CVA16 virus-like particle is induced generation has strong effectively provide protection to CVA16, so the CVA16 virus-like particle can be used as the vaccine of the hand foot mouth disease that prevention CVA16 causes.
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 (10)
1. expression vector, it comprises:
Expression cassette 1, described expression cassette 1 contain the proteic encoding sequence of P1 of coxsackie virus A 16-type virus; With
Expression cassette 2, described expression cassette 2 contain the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus.
2. recombinant baculovirus comprises in the genome of said baculovirus:
Expression cassette 1, described expression cassette 1 contain the proteic encoding sequence of P1 of coxsackie virus A 16-type virus; With
Expression cassette 2, described expression cassette 2 contain the proteic encoding sequence of 3CD of coxsackie virus A 16-type virus.
The described expression vector of claim 1 or the purposes of the described recombinant baculovirus of claim 3, be used to prepare virus-like particle.
4. system that produces virus-like particle comprises:
(1) the described recombinant baculovirus of claim 2; With
(2) insect cell.
5. system as claimed in claim 4 is characterized in that, described insect cell is the sf9 insect cell.
6. method for preparing virus-like particle comprises:
(a) with the described recombinate shape virus infection insect cell of claim 2;
(b) from the lysate of insect cell, separate the acquisition virus-like particle.
7. one kind has immunogenic virus-like particle, and it is produced by the described method of claim 6, and its mean diameter is 20-30nm.
8. the purposes of the described virus-like particle of claim 7 is used to prepare the compsn that prevents coxsackie virus A 16-type virus infection relative disease.
9. purposes as claimed in claim 8 is characterized in that described disease is: hand foot mouth disease.
10. 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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| CN108823245A (en) * | 2015-06-01 | 2018-11-16 | 长春百克生物科技股份公司 | A kind of purification process of virus-like particle |
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| CN102827846A (en) * | 2012-09-19 | 2012-12-19 | 北京工业大学 | Preparation method and application of recombination coxsackie virus B3-type virus-like particles |
| CN103436553A (en) * | 2013-08-22 | 2013-12-11 | 上海博唯生物科技有限公司 | Method for preparing recombinant coxsackievirus A16 type virus-like particles |
| CN103436553B (en) * | 2013-08-22 | 2015-11-18 | 上海博唯生物科技有限公司 | A kind of method preparing restructuring coxsackie virus A 16-type virus-like particle |
| CN104513310B (en) * | 2013-09-27 | 2017-12-08 | 中国科学院上海巴斯德研究所 | A kind of preparation and its application of the monoclonal antibody of anti-coxsackie virus A 16-type |
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| CN103642760B (en) * | 2013-11-14 | 2016-08-17 | 北京万泰生物药业股份有限公司 | A kind of method preparing the complete full particle of COxsackie A16 type virus |
| CN104745606A (en) * | 2013-12-26 | 2015-07-01 | 上海泽润生物科技有限公司 | Coxsackie A16 type virus-like particles |
| CN106661102A (en) * | 2014-05-28 | 2017-05-10 | 财团法人卫生研究院 | Viral particles as immunogens against enterovirus infection and production thereof |
| CN106661102B (en) * | 2014-05-28 | 2021-01-26 | 财团法人卫生研究院 | Viral particles as immunogens against enterovirus infection and their manufacture |
| CN108823245A (en) * | 2015-06-01 | 2018-11-16 | 长春百克生物科技股份公司 | A kind of purification process of virus-like particle |
| CN109777819A (en) * | 2019-01-14 | 2019-05-21 | 深圳鑫泰康生物科技有限公司 | Standard plasmid and preparation method use the method and application of the plasmids detection recombinant C VA16 vaccine copy number of foreign gene |
| CN109777819B (en) * | 2019-01-14 | 2022-12-02 | 深圳鑫泰康生物科技有限公司 | Standard plasmid for detecting copy number of foreign gene of recombinant CVA16 vaccine, detection method and application thereof |
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| CN113564133A (en) * | 2021-09-23 | 2021-10-29 | 北京民海生物科技有限公司 | Coxsackie virus A16 type strain and immunogenic composition and application thereof |
| CN113564133B (en) * | 2021-09-23 | 2022-01-07 | 北京民海生物科技有限公司 | Coxsackie virus A16 type strain and immunogenic composition and application thereof |
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