CN108503697A

CN108503697A - A kind of zika virus subunit vaccine of drosophila cell expression

Info

Publication number: CN108503697A
Application number: CN201710108921.3A
Authority: CN
Inventors: 黄忠; 屈攀科; 张伟; 刘庆伟
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences
Priority date: 2017-02-27
Filing date: 2017-02-27
Publication date: 2018-09-07
Anticipated expiration: 2037-02-27
Also published as: CN108503697B

Abstract

The present invention provides a kind of zika virus subunit vaccines of drosophila cell expression, specifically the invention discloses the subunit's zika virus vaccines researched and developed using Drosophila S 2 cells to have the advantages that yield is high, purity is high, stability is good, is easy to purifying, simultaneously as being free of viral nucleic acid ingredient, so there is no the possibility for restoring mutation, safety is higher.

Description

A kind of zika virus subunit vaccine of drosophila cell expression

Technical field

The invention belongs to biomedicine fields, specifically, the present invention relates to the sub- lists of the zika virus of drosophila cell expression Position vaccine.

Background technology

Zika virus belongs to flaviviridae (Flaviviridae) Flavivirus (Flavivirus) on biological classification, should Belong to virus for single positive chain RNA virus.Zika virus detaches in stockaded village of Uganda Carson woods macaque for the first time since nineteen forty-seven, until It is just noted within 2013, it has been found that the outburst of Green's barye syndrome and the popular phase of french polynesia zika virus It closes.Zika virus mainly bites propagation by yellow-fever mosquito, although infection is that do not have Symptomatic, the more and more microcephalies of discovery mostly Disease case is related to mother's period of gestation infection zika virus, the stockaded village's card disease isolated in microcephaly fetus amniotic fluid and brain tissue Poison has further confirmed that the relationship of the two.The outburst of zika virus, which constitutes global public health, to be seriously threatened.

Therefore, in order to effectively, targetedly prevent and/or treat zika virus infection, there is an urgent need in the art to open Send out zika virus vaccine and its suitable preparation method.

Invention content

The purpose of the present invention is to provide a kind of zika virus subunit vaccine, preparation method and its application.

The first aspect of the present invention provides a kind of Antigenic Peptide, and the Antigenic Peptide is derived from zika virus envelope protein, and And it is selected from the group：

(1) SEQ ID NO.2, amino acid sequence shown in 3；

(2) by amino acid sequence shown in SEQ ID NO.2,3 by it is one or more (≤20, such as 2-10, preferably It is 2-5) amino acid residue replaces, misses or adds the derived peptides to be formed, and the derived peptides have inhibition stockaded village The function of card virus infected cell and/or the function of inducing the immune response for zika virus.

In another preferred example, the Antigenic Peptide is the recombinant protein of Drosophila S 2 cells expression.

The second aspect of the present invention provides a kind of polynucleotides of separation, and the polynucleotide encoding present invention The Antigenic Peptide of one side.

In another preferred example, the polynucleotides are selected from the group：

(a) polynucleotides of the polypeptide as shown in SEQ ID NO.2,3 are encoded；

(b) sequence polynucleotides as shown in SEQ ID NO.5,6；

(c) nucleotide sequence and SEQ ID NO.5, sequence shown in 6 homology >=95% (preferably >=98%) it is more Nucleotide；

(d) 5 ' ends and/or 3 ' ends of the polynucleotides as shown in SEQ ID NO.5,6 truncate or addition 1-60 is a (preferably 1-30, more preferably 1-10) nucleotide polynucleotides；

(e) with the polynucleotides of any polynucleotides complementations of (a)-(d).

The third aspect of the present invention, provides a kind of expression vector, and the expression vector contains second aspect of the present invention institute The polynucleotides stated.

The fourth aspect of the present invention, provides a kind of host cell, and the host cell contains third aspect present invention The expression vector, or it is integrated in genome the polynucleotides described in second aspect of the present invention.

In another preferred example, the host cell includes prokaryotic cell and eukaryocyte.

In another preferred example, the host cell includes Drosophila S 2 cells, yeast, Escherichia coli, Chinese hamster ovary celI, DC Cell etc..

The fifth aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains first aspect present invention Polynucleotides described in the Antigenic Peptide, second aspect of the present invention either the expression vector described in third aspect present invention or Host cell and pharmaceutically acceptable carrier described in fourth aspect present invention and/or auxiliary material.

In another preferred example, the composition is vaccine.

In another preferred example, the vaccine is therapeutic vaccine and/or preventative vaccine.

The sixth aspect of the present invention, provides a kind of vaccine composition, and the composition contains first aspect present invention Polynucleotides described in the Antigenic Peptide, second aspect of the present invention either the expression vector described in three aspect of the present invention or this Acceptable carrier and/or auxiliary material on host cell and immunology described in invention fourth aspect.

In another preferred example, the vaccine composition also contains adjuvant.

In another preferred example, the adjuvant includes aluminium oxide, saponin(e, quil A, muramyl dipeptide, mineral oil or plant Object oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell factor (including IL-1, IL-2, IFN- R, GM-CSF, IL-6, IL-12 and CpG).

The seventh aspect of the present invention, provides the purposes of Antigenic Peptide as described in the first aspect of the invention, and (a) is used to prepare For the antibody of zika virus；And/or it (b) is used to prepare treatment and/or prevents the drug with the relevant disease of zika virus.

In another preferred example, described to include with the relevant disease of zika virus：Zika virus infection, Green's barye are comprehensive Close disease, microcephaly etc..

The eighth aspect of the present invention provides a kind of method preparing the Antigenic Peptide described in first aspect present invention, including Step：

(i) host cell described in fourth aspect present invention is cultivated under optimum conditions, to express first party of the present invention Antigenic Peptide described in face；

(ii) Antigenic Peptide is purified.

In another preferred example, wait for that cell density reaches 2-4x10 in the method step (i)⁶When cell/ml, it is added eventually The chromium chloride induced expression of a concentration of 5uM.

The ninth aspect of the present invention provides a kind of therapy, and first aspect present invention institute is applied to the object needed Polynucleotides described in the Antigenic Peptide stated, the second aspect either expression vector described in third aspect present invention or the present invention the Described in host cell described in four aspects or pharmaceutical composition or sixth aspect present invention described in fifth aspect present invention Vaccine composition.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows the structure of recombinant plasmid pMT/Bip/V5-ZIKV E80 and pMT/Bip/V5-ZIKV EDIII.

Fig. 2 shows the expression and purifying of target protein ZIKV E80 and ZIKV EDIII.(A) the ZIKV E80 purified It detaches by 15% polyacrylamide gel electrophoresis and is dyed with coomassie brilliant blue R_250；(B) Western blot analyses are pure The ZIKV E80 of change are detected with the monoclonal antibody of anti-His-tag；(C) the ZIKV EDIII purified are solidifying by 15% polyacrylamide Gel electrophoresis is detached and is dyed with coomassie brilliant blue R_250；(D) the ZIKV EDIII of Western blot analyses purifying, with anti- The monoclonal antibody of His-tag detects.

Fig. 3 shows the inhibition that ZIKV E80 and ZIKV EDIII albumen infects zika virus.The ZIKV of gradient dilution E80, ZIKV EDIII or BSA are added to the Vero cells completed in advance after being mixed with the zika virus of 100PFU, 37 DEG C of incubations 1 are small When after sop up and be changed to overlay culture mediums, BSA is as negative control.(A) control group is compared, at ZIKV E80, ZIKVEDIII The case where cell plaque managed is reduced；(B) number for counting plaque reduction, is used in combination inhibiting rate to indicate, inhibiting rate (%) indicates The percentage for being plaque number relative to untreated cell, processing shared by the plaque of Leukopenia.

Fig. 4 shows the measurement of serum titer after the immune BALB/c mouse of ZIKV E80, ZIKV EDIII.BALB/c mouse It is immune three times plus aluminium adjuvant with ZIKV E80, ZIKV EDIII or PBS, it takes a blood sample within immune latter two weeks every time, antibody is surveyed with ELISA Potency.Endpoint titers are defined as the dilution of one positive findings of highest (OD450 is higher than preimmune serum potency 0.1).(A) Two weeks serum endpoint potency after PBS, ZIKV E80 groups two are exempted from, three exempt from；(B) two weeks after PBS, ZIKV EDIII groups two are exempted from, three exempt from Serum endpoint potency；Horizontal dashes indicate the geometric mean of every group of serum endpoint potency.

Fig. 5 shows that ZIKV E80, ZIKV EDIII tri- exempt from the measurement of serum neutralizing antibody after BALB/c mouse.After three exempt from ZIKV E80, ZIKV EDIII and the sero-fast neutralising capacity of PBS groups are surveyed, reducing neutralization analysis method with plaque measures.Geometry is flat Mean and P values are shown.

Fig. 6 show ZIKV E80, ZIKV EDIII mouse Immune inducing in vivo cell immune response.After three exempt from four weeks, Each group of mouse boosting cell is separated, and the cell for generating IFN-γ and IL-4 is analyzed with enzyme-linked immunospot assay.(A and B) ZIKV E80 groups splenocyte is added stimulant, while culture medium is as a contrast；(C and D) ZIKV EDIII groups splenocytes are added Stimulant, while culture medium is as negative control.Mean ± SEM is as shown in the figure.

Fig. 7 shows that passive protection is tested.PBS, ZIKV EDIII tri- are exempted to the stockaded village of latter two weeks serum 100ul and 5PFU Block after virus is incubated 1 hour at 37 DEG C through the AG6 mouse big by 5 weeks is injected intraperitoneally.Then it observes 2 weeks, it is small to record each group daily The survival condition (A) and changes of weight (B) of mouse.

Specific implementation mode

The present inventor's in-depth study by extensive by, it has unexpectedly been found that utilize subunit's stockaded village's card of Drosophila S 2 cells research and development Viral vaccine has the advantages that yield is high, purity is high, stability is good, is easy to purifying, simultaneously as being free of viral nucleic acid ingredient, institute With there is no the possibility for restoring mutation, safety is higher.Moreover, using the Antigenic Peptide E80 and EDIII of the present invention, helped in conjunction with aluminium Agent, after mouse is immunized, immune mouse generates stronger immune response, and induction body generation high-titer has neutralization activity Antibody, be just enough to protect AG6 mouse from lethal dose stockaded village card with the antibody of the immunogene ZIKV EDIII induction of low dosage The attack of virus.Experimental result shows zika virus subunit vaccine ZIKV EDIII and E80 provided by the invention to pre- Anti- zika virus infection has significant protecting effect.

Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.

Unless otherwise defined, otherwise whole technologies used herein all have with scientific terminology such as fields of the present invention The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated " about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

Although can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention And material, herein place enumerate preferred method and material.

Zika virus envelope protein

The envelope protein (E protein) of zika virus is the major target of neutralizing antibody, and E protein is divided into three regions, The neutralizing antibody of EDI, EDII, EDIII, most of specific antibodies or partial intersection reaction, the main table identified on EDIII Position.E80 albumen is 80% region of zika virus envelope protein N-terminal in the present invention, is the extracellular section of E protein, be responsible for cell by The combination of body is the Main Antigenic of zika viruses.Main target of the present invention is to develop one kind can induce body generation target To the vaccine of E protein (E80 and EDIII) neutralizing antibody, the infection for preventing zika virus.

The present invention provides a kind of Antigenic Peptides derived from zika virus envelope protein, it is preferable that the stockaded village for the present invention Card virus envelope protein is originated from the Z1106033 strain (viral amino acids of zika virus Asian type South America prevalence in 2015 Genbank：ALX35659, strain nucleotide GenBank:KU312312).

One in the present invention is preferably carried out in mode, the following institute of amino acid sequence of the envelope protein (E protein) Show：

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQ GEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVN DTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADT GTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLC TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDS YIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSW FSQILIGTLLMWLGLNAKNGSISLMCLALGGVLIFLSTAVSA, SEQ ID NO.1.

One in the present invention is preferably carried out in mode, and the Antigenic Peptide includes ZIKV E80 albumen, amino acid sequence Row are as follows：

IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQ GEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVN DTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADT GTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLC TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDS YIVIGVGEKKITHHWHRSGSTIGK, SEQ ID NO.2.

Another in the present invention is preferably carried out in mode, and the Antigenic Peptide includes ZIKV EDIII albumen, amino Acid sequence is as follows：

KLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSK MMLELDPPFGDSYIVIGVGEKKITHHWHRSGST, SEQ ID NO.3.

The optimization of coding for antigens peptide gene sequence

In the present invention, provide optimization, be particularly suitable for the core for the Antigenic Peptide of the present invention expressed in Drosophila S 2 cells Coding sequences.

The present inventor is according to preferred codons, to the DNA sequences of coding E protein under the premise of not changing its amino acid sequence Row are optimized.However, the inventors discovered that, the optimization obtained only in accordance with codon frequency is not fully suitble in place It is expressed in chief cell.Therefore present inventor has performed double optimization, including the G/C content for sequence carried out adjustment and Optimization, eliminates the higher region of G/C content in original series；For the repetitive sequence and cis-acting factors in original series Equal labyrinths (GGGGGG, GGTAAG) optimize；According to Preference for rare codon present in protokaryon thuja acid (AGG, CGG, GGG, ACG) is optimized.

By largely testing and screening, the present inventor obtains an E protein especially optimized in numerous optimizations Coded sequence, polynucleotide sequence are as follows：

atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctgga gcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcga acatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaacccag ggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaa tggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagt cgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaac gataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccct gggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgacca tgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacacc ggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggt gctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggac gcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgc accgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccgg aaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatca ccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagc tacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaaggcctt cgaggccaccgtgcgcggagccaagcgcatggccgtgctgggcgataccgcctgggacttcggaagcgtgggaggag ccctgaacagcctgggcaagggcatccaccagatcttcggagccgccttcaagtccctgttcggaggcatgtcgtgg ttcagccagatcctgatcggcaccctgctgatgtggctgggcctgaacgccaagaatggctccatctcgctgatgtg Cctggccctgggaggagtgctgatcttcctgagcaccgccgtgtccgcctaa, SEQ ID NO.4；The sequential coding E protein shown in SEQ ID NO.1.

According to above-mentioned optimized DNA sequence dna, the DNA sequence dna for encoding E80 is as follows：

atccgctgcatcggcgtgtcgaatcgcgatttcgtggagggaatgagcggaggaacctgggtggacgtggtgctgga gcacggaggatgcgtgaccgtgatggcccaggataagccgaccgtggacatcgagctggtgaccaccaccgtgtcga acatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaacccag ggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatggggaaa tggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccggcaagt cgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcgtgaac gataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggccaccct gggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacctgacca tgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccgacacc ggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtggtggt gctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaagggac gcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccctgtgc accgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtacgccgg aaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcctgatca ccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcgacagc Tacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcggg aagcaccatcggcaag, SEQ ID NO.5；

The DNA sequence dna for encoding EDIII albumen is as follows：

aagctgcgcctgaagggcgtgagctactccctgtgcaccgccgccttcaccttcaccaagatcccagccgagaccct gcacggaaccgtgaccgtggaggtgcagtacgccggaaccgatggaccatgcaaggtgccagcccagatggccgtgg acatgcagaccctgaccccagtgggacgcctgatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaag atgatgctggagctggatcccccgttcggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccacca Ctggcaccgctcgggaagcacc, SEQ ID NO.6.

Carrier and host cell

The present invention also provides a kind of carriers of the antigen peptide-coding sequence of the optimization comprising the present invention, and contain the load The host cell of body.

In the preference of the present invention, the carrier of stating has the expression cassette for expressing the antigen peptide gene, described Expression cassette has following elements successively from 5 ' -3 '：Promoter, antigen peptide gene and terminator.

The conventional method that those skilled in the art can use obtains the above-mentioned optimization gene sequence of the Antigenic Peptide Row, such as complete artificial synthesized or PCR methods synthesis.A kind of preferred synthetic method is asymmetric PCR method.Primer for PCR can root It is properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.It can be such as logical with conventional method Cross the DNA/RNA segments of gel electrophoresis separation and purifying amplification.

Destination protein (antigen can be expressed or be produced to the present invention polynucleotide sequence by the recombinant dna technology of routine Peptide), including step：

(1) with the polynucleotides (or variant) for encoding albumen of the present invention, or with containing the polynucleotide recombinant expression Carrier converts or transduce suitable host cell, preferably Drosophila S 2 cells；

(2) host cell is cultivated in suitable culture medium；

(3) it is separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used to build DNA sequences encoding containing albumen of the present invention and suitable The expression vector of transcription/translation control signal, preferably commercially available carrier such as pMT/BiP/V5-HisA.These methods include external Recombinant DNA technology, DNA synthetic technologys, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in expression vector In appropriate promoter, to instruct mRNA to synthesize.Expression vector further includes the ribosome bind site of translation initiation and transcribes eventually It is only sub.In addition, expression vector preferably comprises one or more selected markers, to provide for selecting the host of conversion thin The phenotypic character of born of the same parents.

Including above-mentioned DNA sequence dna and the carrier of appropriate promoter or control sequence, can be used for converting place appropriate Chief cell, express express target protein.The host cell that Antigenic Peptide of the present invention can be expressed can be prokaryotic cell, such as Escherichia coli； Or low eukaryocyte, such as yeast cells (Pichia pastoris, saccharomyces cerevisiae)；Or higher eucaryotic cells, such as insect cell；It is excellent It is selected as Drosophila S 2 cells.It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.Engineering Cell can quickly utilize methanol type (Mut⁺) or at a slow speed utilize methanol type (Mut^s)。

The culture of engineering cell and destination protein fermenting and producing

After obtaining engineering cell, can culturing engineering cell under the suitable conditions, express the gene order of the present invention Encoded albumen.According to the difference of host cell, culture medium used in culture can be selected from various conventional mediums, suitable for It is cultivated under conditions of host cell growth.After host cell growth is to cell density appropriate, (such as with suitable method Temperature transition or chemical induction) promoter that induces selection, cell is further cultured for a period of time.

In the present invention, conventional fermentation condition can be used.Representative condition includes (but being not limited to)：

(a) for temperature, the fermentation of Antigenic Peptide of the invention and inducing temperature are maintained at 28-30 DEG C；

(b) for the pH value of induction period, induction period pH controls in 3-9；

(c) for dissolved oxygen (DO), DO controls can use oxygen/air mixed gas in 20-90%, the maintenance of dissolved oxygen It is passed through to solve；

(d) for feed supplement, feed supplement type preferably includes the carbon sources such as glycerine, methanol, glucose, can individually feed supplement or mixing be mended Material.

Engineering cell express express target protein may be used chromatographic technique and be purified.Chromatographic technique includes cation exchange layer The technologies such as analysis, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affinity chromatography.Commonly chromatography method includes：

1. anion-exchange chromatography：

Anion-exchange chromatography medium includes but is not limited to：Q-Sepharose、DEAE-Sepharose.If fermentation The salinity of sample is higher, the combination of influence and Ion Exchange Medium, then needs to reduce salinity before carrying out ion-exchange chromatography. Sample can be balanced the replacement of buffer solution with means such as dilution, ultrafiltration, dialysis, gel permeation chromatographies, until with corresponding Ion exchange column equilibrium liquid system is similar, then loading, carries out salinity or the gradient elution of pH.

2. hydrophobic chromatography：

Hydrophobic chromatoghaphy medium includes but is not limited to：Phenyl-Sepharose、Butyl-Sepharose、Octyle- Sepharose.Sample is by adding NaCl, (NH₄)₂SO₄Etc. modes improve salinity, then loading, passing through reduces salinity side Method elutes.Removing hydrophobicity by hydrophobic chromatography has the foreign protein of larger difference.

3. gel permeation chromatography

Hydrophobic chromatoghaphy medium includes but is not limited to：Sephacryl, Superdex, Sephadex class.Pass through gel filtration Chromatography replaces buffer system, or further consummate.

4. affinity chromatography

Affinity chromatography medium includes but is not limited to：HiTrap^TM Heparin HP Columns。

Prepare vaccine composition

The present invention also provides a kind of methods preparing vaccine composition, specifically, including step：

Antigenic Peptide prepared by the present invention is mixed with pharmaceutically acceptable vaccine adjuvant, to form vaccine composition.

In another preferred example, the adjuvant is aluminium adjuvant, GLA adjuvants, preferable GLA adjuvants.

Composition and method of administration

The present invention also provides a kind of composition, the composition contains：(i) recombinant antigen prepared with the method for the present invention Peptide, and (ii) acceptable excipient or adjuvant pharmaceutically or in immunology.In the present invention, term " containing " indicate it is various at Divide in the composition that can be applied to or be present in together the present invention.Therefore, term " mainly by ... form " and " consist of " Included in term " containing ".

The composition of the present invention includes pharmaceutical composition and vaccine composition.The present invention composition can be it is monovalent, It can also be multivalence.

The pharmaceutical composition or vaccine composition of the present invention can be prepared into various regular dosage forms, including (but and it is unlimited In)：Injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..

(i) pharmaceutical composition

The pharmaceutical composition of the present invention includes a effective amount of Antigenic Peptide prepared with the method for the present invention, and the Antigenic Peptide can be with It is monovalent, can also be multivalence.

The amount that the term as used herein " effective quantity " refers to therapeutic agent treatment, alleviates or prevent target disease or situation, or Show the detectable amount for treating or preventing effect.The effect can be detected for example, by antigen levels.Therapeutic effect is also wrapped Include the reduction of physical symptoms.The build and health status, illness of the object are depended on for the accurate effective quantity of certain an object Property and degree and the combination of therapeutic agent and/or therapeutic agent given of selection.Therefore, accurate effective quantity is preassigned It is useless.However, for the situation that Mr. Yu gives, the effective quantity can be determined with routine experiment.

For the purposes of the present invention, effective dosage is to give individual about 0.2 micro- g kg to 2 micro- g kgs.

Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to for controlling Treat the carrier that agent (such as Antigenic Peptide or other therapeutic agents) is administered.The term refers to some such medicament carriers：Themselves is not lured Artificial delivery life does not have excessive toxicity to receiving the harmful antibody of individual of the composition after being administered.Suitable carrier can be Big, the slow macromolecular of metabolism, such as protein, polysaccharide, polylactic acid (polylactic acid), polyglycolic acid.These are carried Body is well known to those of ordinary skill in the art.In Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J.1991) in can find discussing fully about pharmaceutically acceptable carrier or excipient.

The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, brine, glycerine and ethyl alcohol.In addition, these are carried There is likely to be complementary substances, such as wetting agent or emulsifier, pH buffer substance in body.In general, composition can be made Injectable agent, such as liquid solution or suspension；It may also be fabricated which and be suitble to supplying solution or suspension, liquid excipient before the injection Solid form.Liposome is also included in the definition of pharmaceutically acceptable carrier.

(ii) vaccine composition

The vaccine composition of the present invention can be preventative (preventing infection), can also be therapeutic.Described Vaccine composition include immunising antigen (including albumen of the present invention or virus-like particle of self assembly), and usually with " pharmacy Upper acceptable carrier " combination, these carriers include itself not inducing the harmful antibody of individual generated to receiving the composition Any carrier.Suitable carrier is typically big, the slow macromolecular of metabolism, such as protein, polysaccharide, polylactic acid, polyethanol Acid, amino acid polymer, amino acid copolymer, lipid aggregates (such as oil droplet or liposome).These carriers are that this field is general Known to logical technical staff.In addition, these carriers can play immunostimulant (" adjuvant ").In addition, antigen can also be with Bacterial toxoid (such as toxoid of diphtheria, lockjaw, cholera, helicobacter pylori pathogen) is coupled.

Enhancing immune composition effect preferred adjuvant include but not limited to：(1) aluminium salt (alum), such as aluminium hydroxide, phosphorus Sour aluminium, aluminum sulfate etc.；(2) oil-in-water emulsion formula, for example, (a) MF59 (referring to WO 90/14837), (b) SAF, and (c) Ribi^TMAdjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant；(4) Freund Freund's complete adjuvants (CFA) and Freund Freund's incomplete adjuvants (IFA)；(5) cell factor, as interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferon (such as interferon), macrophage colony stimulating factor (M-CFS), tumor necrosis factor (TNF) etc.；(6) bacterial ADPribosylating toxin (such as cholera toxin CT, pertussis toxin PT or Escherichia coli thermally labile poison Plain LT) detoxification variant, see, for example, WO93/13302 and WO92/19265；And (7) enhance as immunostimulant The other materials of composition effect.

Including immunogenic composition vaccine composition (such as, it may include antigen, pharmaceutically acceptable carrier And adjuvant), usually contain diluent, such as water, brine, glycerine, ethyl alcohol etc..In addition, auxiliary substances, such as wetting agent or emulsification Agent, pH buffer substance etc. may be present in this kind of carrier.

More specifically, the vaccine including immunogenic composition, includes the immunogenic polypeptide of immunological effective amount, And above-mentioned other required components." immunological effective amount " refers to gives the amount of individual to treatment with single dose or a continuous agent part Or prevent to be effective.The dosage can according to the health status and physiological status for treating individual, treat individual classification (such as People), the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician is to medical conditions Depending on assessment and other correlative factors.It is expected that the dosage is by relatively wide range, it can be by routine experiment come really It is fixed.

In general, can injectable agent, such as liquid solution or suspension be made for vaccine composition or immunogenic composition；Also It can be made into the solid form for being suitble to supplying solution or suspension, liquid excipient before the injection.Said preparation is also emulsifiable or is encapsulated in In liposome, to enhance adjuvant effect.

(iii) administration route and dosage

The composition can directly give object.Object can be people or non-human mammal, be preferably people.When with When making vaccine, can the virus-like particle of the present invention be directly applied to individual by known method.Generally use and conventional vaccine These vaccines are applied in identical administration method and/or simulation pathogenic infection path.

The approach for giving pharmaceutical composition or vaccine composition of the present invention includes (but being not limited to)：Intramuscular, subcutaneous, skin Interior, intrapulmonary, intravenous, intranasal, intravaginal, by oral administration or other parenteral route of administration.If desired, can be with combination medicine-feeding way Diameter, or be adjusted according to disease event.Vaccine composition can be given with single dose or multi-dose, and may include giving to reinforce Dosage is to cause and/or maintain immunity.

Virus sample particle vaccines should be given with " effective quantity ", i.e., the amount of virus-like particle is in selected administration routes mesopodium To cause immune response, can effectively promote that host is protected to resist zika virus infection.

The amount of selected virus-like particle in each vaccine dose part, be by can cause protective immune response and without apparent Side effect amount depending on.In general, after infecting host cell, each dose of vaccine is enough containing about 1 μ g-1000 μ g, preferably For 1 μ g-100 μ g, more preferably 10 μ g-50 μ g proteins or VLP.Can use include observe object in IgG titers and it is other instead The standard research techniques answered determine the optimum amount of specific vaccine.It can be determined by monitoring the immunity level that vaccine provides Whether need to enhance dosage.After the IgG titers in having evaluated serum, it may be necessary to select enhancing dose immunizations.It applies The immune response of the protein to the present invention just can be improved with adjuvant and/or immunostimulant.Preferred method is from parenteral (skin It is lower or intramuscular) approach gives immunogenic composition by injection.

Main advantages of the present invention are：

(1) Antigenic Peptide of the invention can in Drosophila S 2 cells great expression, therefore manufacturing cost is low, is suitble to industrialization Using；

(2) present invention carries out redesign and sequence optimisation, optimized gene order to the gene of zika virus E protein In host cell inner expression amount height, stability is good, is suitble to high density fermentation；

(3) using the Antigenic Peptide E80 and EDIII of the present invention, in conjunction with aluminium adjuvant, after mouse is immunized, immune mouse produces Raw stronger immune response, induction body generates the antibody with neutralization activity of high-titer, with the immunogene ZIKV of low dosage The antibody of EDIII inductions is just enough the attack for protecting AG6 mouse from lethal dose zika virus, zika virus subunit vaccine ZIKV EDIII and E80 are the candidate vaccines for preventing zika virus infection, have significant protecting effect.

(3) present invention is compared with traditional attenuated live vaccine, DNA vaccination and inactivated vaccine, and the candidate vaccine is not because having There is viral nucleic acid, so very safe.

With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip The works such as part such as U.S. Sambrook.J《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experiment material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

Material and method

1. cell

Drosophila Schneider 2 (S2) cell is purchased from Invitrogen companies, is incubated at 10% fetal calf serum of addition (Gibco), in Schneider ' the s Drosophila Media (Gibco) of 1% dual anti-(Gibco) or addition 1%L- paddy ammonia Amide (Gibco), the Express of 1% dual anti-(Gibco)In SFM culture mediums (Gibco), 28 DEG C of incubator cultures.

2. virus

The stockaded village's card strain used in this research is ZIKV/SZ-WIV01 plants of (GenBank:KU963796), China is derived from Wuhan institute of viruses of academy of sciences microbial bacteria seed culture of viruses collection (preservation number：IVCAS6.6110).

3. antibody

The monoclonal antibody (10E2) of the anti-His-tag of mouse is bought in Abmart (article No.s：264160), horseradish peroxidase (HRP) is marked In Sigma, the mountain sheep anti-mouse igg of alkaline phosphatase (AP) label is bought in Promega (article No.s for the mountain sheep anti-mouse igg purchase of note： S3728)。

4. plasmid construction

Drosophila cell expression vector pMT/BiP/V5-HisA, screening plasmid pCoBlast and calcium phosphate transfection kit are all Purchased from Invitrogen companies.According to the Z1106033 strain (antiviral nucleosides of zika virus Asian type South America prevalence in 2015 Sour GenBank:KU312312, amino acid Genbank：ALX35659 E protein coded sequence) carries out codon optimization and base Because of synthesis (GenScript companies), goes forward side by side on one-step cloning to carrier pUC57 (being purchased from GenScript USA Inc.), obtain Plasmid pZIKV-E.Using pZIKV-E as masterplate, after specific primer PCR amplification, both ends carry Bgl II and Xba I digestions position Point, be connected to containing Bgl II and Xba I restriction enzyme sites insect expression vector pMT/Bip/V5-His A (containing His labels, Be conducive to the detection and purifying of target protein), obtain carrying 80% region (ZIKV E80) of zika virus envelope protein N-terminal and Recombinant plasmid the pMT/Bip/V5-ZIKV E80 and pMT/ of envelope protein region III (ZIKV EDIII) target gene fragment Bip/V5-ZIKV EDIII。

Gene magnification special primer

Primer	Primer sequence	SEQ ID NO.
			ZIKV E80-F	CCAAGATCTATCCGCTGCATCGGCGTGTCGAA	7
ZIKV E80-R	AAATCTAGACTTGCCGATGGTGCTTCCCGAGCG	8
			ZIKV EDIII-F	AAAAGATCTAAGCTGCGCCTGAAGGGCGTGAGC	9
ZIKV EDIII-R	AAATCTAGAGGTGCTTCCCGAGCGGTGCCAGTG	10

5. polyacrylamide gel electrophoresis and western blots

The protein solution sample of expression is acted on by polyacrylamide gel electrophoresis (SDS-PAGE) sample-loading buffer, and 100 DEG C are boiled 5 minutes, are then separated by electrophoresis on 15% polyacrylamide gel, and protein passes through Coomassie brilliant blue It dyes visible or is transferred on PVDF for protein imprinted (Western Blotting) analysis, Western Blotting are used The monoclonal antibody of anti-His-tag detects, and is then incubated with the secondary antibody of the sheep anti mouse of HRP couplings, finally develops the color to detect specific band Position.

6. expression of destination protein ZIKV E80 and the ZIKV EDIII in Drosophila S 2 cells

1) expression of testing goal gene is transiently transfected

It will be in Transfected Recombinant Plasmid to S2 cells using calcium robin.Paving wild type S2 cells first are in six orifice plates, 3x10⁶ Cells/well, 28 DEG C of 6~16 hours of culture, when cell density reaches 2-4x10⁶It is transfected when cell/ml.By 36ul 2M CaCl₂, 19ug recombinant plasmids and sterile water add to 300ul and be configured to A liquid, 300ul 2xHEPES (50mM HEPES, 1.5mM Na₂HPO4,280mM NaCl, pH 7.1) it is configured to B liquid.B liquid is positioned in concussion instrument, A liquid is slowly added dropwise in B liquid, and will This mixed liquor stands 30 minutes at room temperature, drops evenly mixed liquor on the cell in 6 orifice plates, and it is small that 16~24 are cultivated at 28 DEG C Cell 800rpm is centrifuged 5 minutes, three times cells is washed with the complete medium containing 10% serum, to remove calcium by Shi Hou Grain mitigates the damage to cell.Cell is positioned over 28 DEG C to cultivate 72 hours, the table of 5uM chromium chlorides induction target gene is added It reaches, and with the expression of immunoblotting testing goal albumen.

2) great expression of the screening of stability series cell and destination protein

If transiently transfecting the expression that can detect target protein, rice is killed with regard to use and accumulates rhzomorph screening express express target protein Steady turn cell line.1ug pCoBlast screening plasmids are pressed into transient transfection method (36ul 2M CaCl with recombinant plasmid₂、19ug Recombinant plasmid and sterile water add to 300ul and are configured to A liquid) corotation is in S2 cells, when cell density to 2-4x10⁶Cell/ml, Rice is killed with 25ug/ml accumulate rhzomorph screening pressure and filter out positive cell, and detect its destination gene expression.

If detecting destination protein, just constantly expands culture and surely transfer from one department to another cell, use serum free medium instead at this time ExpressSFM (Gibco) (rice that kills of addition 10ug/ml accumulates rhzomorph).It is expanded to one from a T25flask T75flask is finally transferred in rolling bottle and cultivates, and cell densities is waited to reach 2-4x10⁶When cell/ml, final concentration of 5uM is added Chromium chloride induced expression.

Induction 7 days or so, centrifugation are collected supernatant, are filtered with the filter membrane of 0.45um.Then to the ultrafiltration of 3kDa from Heart pipe (Millipore) is concentrated, and is concentrated to 1/20 of original volume or so.Nickel column (Novagen) is used to purify afterwards, before upper prop First binding buffer (0.5M NaCl, 20mM Tris, 10mM imidazoles, pH7.9) the not strong site of binding specificity, then Loading then uses binding buffer and washing buffer (0.5M NaCl, 20mM Tris, 40mM imidazoles, pH7.9) After washing off foreign protein, then with eluting buffer (0.5M NaCl, 20mM Tris, 250mM imidazoles, pH7.9) elute purpose Albumen.Finally quantifying for target protein is carried out with SDS-PAGE and Brandford.

7. virus plaque is tested

The titre of virus is analyzed with Plaque Technique Detected.Briefly, in 24 orifice plate upper berth Vero-E6 cell monolayers, 37 DEG C of cultures Overnight, 10 times of diluted zika virus on cell 37 DEG C be incubated 1 hour, then viral sample be removed and add overlay training It supports base and (contains 1:The 0.2%agarose and 2%FBS-DMEM of 1 mixing).Cell is transferred to 4 DEG C, places 15 minutes.Then 37 DEG C are cultivated about 80 hours.0.1% violet staining is finally fixed and used with 4% paraformaldehyde.It counts plaque and calculates disease The titre of poison.

The inhibition that 8.ZIKV E80 and ZIKV EDIII albumen infects zika virus

An evening paving Vero-E6 cells are in 24 orifice plates in advance, and 10⁵Cells/well, second day, by ZIKV E80, ZIKV EDIII Gradient dilution, is incubated one hour with 100PFU zika virus at 37 DEG C respectively, and then viral sample is removed and overlay is added to train It supports base and (contains 1:The 0.2%agarose and 2%FBS-DMEM of 1 mixing).Cell is transferred to 4 DEG C, places 15 minutes.Then 37 DEG C are cultivated about 80 hours.0.1% violet staining is finally fixed and used with 4% paraformaldehyde.It counts plaque and calculates The inhibiting rate that ZIKV E80 and ZIKV EDIII albumen infects zika virus, not plus the hole of destination protein is with reference to (0%).

9. animal immune

Using ZIKV E80 and ZIKV EDIII as immunogene, 500ug aluminium hydroxides (Alhydrogel) (are purchased from Invivogen it is) adjuvant, 10ug immunogenes are injected intraperitoneally in the BALB/c female mices big to 6 weeks (every group 6) respectively, in addition Using PBS groups as control group.It is immunized respectively when the 0th week, 2 weeks, 4 weeks primary and first before second week, 4th week are immune Retroorbital venous is taken a blood sample.The blood adopted first is placed at room temperature for 1-3 hours, then 4 spends night, 4 DEG C of centrifugations in second day, 3000rpm Centrifugation 30 minutes, 56 DEG C of the serum separated are handled 30 minutes.Antibody titers from serum and neutralizing antibody effect are detected later Valence.

10. the measurement of serum antibody titer

1) ZIKV E80 group serum antibody titers are measured

96 orifice plates are coated with the ZIKV E80 being purified, the holes 50ng/, 4 DEG C overnight, and the closing 1 of 5% 37 DEG C of skim milk is small When, by treated, 2 times of gradient dilutions of serum are washed 5 times with PBST then with the incubation 2 hours of 37 DEG C of coated antigen, use horseradish 37 DEG C of the secondary antibody of the sheep anti mouse of peroxidase coupling is incubated 1 hour, is developed the color with TMB, and room temperature is protected from light effect, is terminated with 1M phosphoric acid anti- Light absorption value of the multi-functional readout instrument determination samples of Ying Hou, Thermo Scientific Varioskan Flash in A450.

2) ZIKV EDIII group serum antibody titers are measured

With measuring ZIKV E80 groups, but with ZIKV EDIII wrapper sheets.

11. the measurement of serum neutralize antibody titers

Vero-E6 cells are pressed 10⁵Cells/well is taped against in 24 orifice plates, overnight incubation, and cells is waited to cover 90% or so, is used The serum gradient of inactivation is diluted to 100ul by plasma-free DMEM medium, and 1 hour is incubated for 37 DEG C with 100PFU zika virus, 200ul mixtures are added on Vero-E6 cells, 37 DEG C are cultivated 1 hour, and cell conditioned medium is discarded, and add 700ul overlay per hole Culture medium, 37 DEG C are cultivated 72 hours or so.Culture medium then is discarded, is washed twice with PBS, 30 are fixed with 4% paraformaldehyde room temperature Minute, ultraviolet irradiation 30 minutes.Paraformaldehyde is discarded, is dyed 30 minutes with 0.1% crystal violet room temperature, twice with distillation washing, Dry counting.After GraphPad Prism software matched curves, statistics makes the titre (PRNT of plaque reduction half₅₀)。

12. elisa detects

1) in advance with 35% ethyl alcohol by 96 orifice plates (Mi llipore MultiScreen 96-well ELISPOT Plates it) soaks, is then coated with the IFN-γ capture antibody (10ug/ml) and IL-4 capture antibody (4ug/ in diluted people source Ml), the holes 50ul/, overnight 4 DEG C of incubations；

2) coating buffer is discarded, is washed 4 times with PBS, is closed 3 hours with the culture medium of RPMI-1640；

3) by diluted ZIKV E80, ZIKV EDIII and positive control (Concanavalin A), negative control (RPMI-1640) it is added in corresponding hole；

4) it separating Morr. cell and counts, is then added into corresponding hole, 37 DEG C are cultivated 24-48 hours；

5) cell is discarded, with cold ddH₂O is washed, then is washed 5 times with PBST；

6) plus the diluted anti-IFN-γ of mouse (2ug/ml), IL-4 (4ug/ml) detection antibody, be incubated at room temperature 3 hours；With PBST is washed three times；The Streptavidin (1 for adding AP to be coupled:1000) after, being washed three times with PBST, BCIP/NBT is added to develop the color, And add ddH₂O is terminated；

7) it is counted with ELISPOT plate reader after drying.

13. passive protection is tested

100ul tri- is first exempted from into mice serum (serum of each group of each mouse is blended in one piece) and stockaded village's card disease of 5PFU Poison is incubated 1 hour at 37 degree, and AG6 mouse (I types, II type interferon receptors knock out) body to 5 week old is then injected intraperitoneally It is interior；The situation of observation mouse is carried out within continuous 2 weeks, and is weighed；Epidemic disease is finally judged according to survival condition and changes of weight situation The protecting effect of seedling.

14. statistical analysis

All data analyses all use GraphPad Prism software v 5.0 to analyze.Kaplan-Meier survive Curve compares with log-rank test.The Student's 2-tailed t check analyses of other results.Data significant difference It is expressed as：Ns (is not significantly different, P >=0.05)；*0.01≤P<0.05；**P<0.01；***P<0.001.

Experiment material used in the embodiment of the present invention can obtain unless otherwise specified from commercially available channel.

Expression of 1 destination protein ZIKV E80 and the ZIKV EDIII of embodiment in Drosophila S 2 cells

Recombinant plasmid pMT/Bip/V5-ZIKV E80 and the pMT/Bip/V5-ZIKV EDIII (such as Fig. 1) that will be built, Drosophila cell is transiently transfected, western blot are carried out after chromium chloride induces and detect culture medium supernatant, detect purpose egg In vain.Then recombinant plasmid and pCoBlast are screened plasmid corotation, after screening stability series cell, carries out induced expression, obtained thin It is purified after born of the same parents' supernatant, SDS-PAGE shows that ZIKV E80 albumen sizes are 54KD (such as Fig. 2A), with the anti-His-tag antibody of mouse As primary antibody, western blot detect that ZIKV E80 (such as Fig. 2 B) size is consistent with SDS-PAGE results.Similarly, ZIKV EDIII sizes are 15KD (such as Fig. 2 C), and the band detected with western blot (Fig. 2 D) is in the same size.Result above prompts Target protein E80 and EDIII are expressed, and calculate the expression yield difference of ZIKV E80 and ZIKV EDIII Antigenic Peptides after purification Reach 10mg/l and 2.6mg/l.

Yeast expression the experimental results showed that, destination protein ZIKV E80 can not be expressed in yeast cells, but can be in fruit High efficient expression in fly S2 cells, yield reach 10mg/l.

The inhibition that embodiment 2.ZIKV E80 and ZIKV EDIII albumen infects zika virus

For the biological function of Preliminary Identification ZIKV E80 and ZIKV EDIII, We conducted ZIKV E80 and ZIKV The inhibition analysis that EDIII albumen infects zika virus.The results are shown in Figure 3, zika virus to the infection of Vero cells by ZIKV E80(IC₅₀=54.63ug/ml), ZIKV EDIII (IC₅₀=71.85ug/ml) inhibition, and in dose-dependent Mode, and reference protein BSA can not inhibit infection of the zika virus to cell.And ZIKV E80 Antigenic Peptides are to zika virus sense The inhibitory activity of dye is significantly better than ZIKV EDIII albumen, IC₅₀Value reduces 30% or more compared with ZIKV EDIII.

Result of this example indicate that ZIKV E80, ZIKV EDIII can be competed with zika virus enters cell, therefore The potentiality of neutralizing antibody are generated with induction animal.

The specific antibody of embodiment 3.ZIKV E80 and ZIKV EDIII Immune inducing in vivo in mouse reacts

In order to evaluate the immunogenicity of ZIKV E80, ZIKV EDIII, three groups of BALB/c respectively by peritoneal immunity three times, ZIKV E80+Alum、ZIKV EDIII+Alum、PBS+Alum.The anti-blood of mouse that ZIKV E80, ZIKV EDIII induction generate It is detected with ELISA clearly.As shown in Figure 4 A, the specific antibody titres two that ZIKV E80 inductions generate are exempted to exempt from geometric mean with three Respectively 252.0,1007.9；As shown in Figure 4 B, the specific antibody titres two that ZIKV EDIII inductions generate are exempted to exempt from more than three What average is respectively 178.2,5701.8, and control group serum does not show binding ability.

Embodiment 4.ZIKV E80, ZIKV EDIII tri- exempt from the measurement of serum neutralizing antibody after BALB/c mouse

In order to evaluate neutralising capacity of the mouse resisting anteserum to zika virus, we reduce neutralization analysis with plaque (PRNT).As shown in figure 5, ZIKV E80 groups serum can neutralize zika virus, PRNT₅₀It is 365.5, and ZIKV EDIII group serum Neutralising capacity is stronger, PRNT₅₀It is 1633.8, and control group serum is almost without neutralising capacity, PRNT₅₀It is 49.1.

The cell immune response of embodiment 5.ZIKVE80 and ZIKV EDIII Immune inducing in vivo in mouse

In order to probe into the t cell responses that can immunogene induce generation special, we are thin by mice spleen after exempting from four weeks three Born of the same parents separate, and the cell of IFN-γ and IL-4 is generated with enzyme-linked immunospot assay.As shown in fig. 6, ZIKV E80 groups, ZIKV EDIII groups can induce the cell for generating number more secretion of gamma-IFN and IL-4, and PBS groups not can induce generation and compare The cell of more secretion of gamma-IFN and IL-4.Therefore, ZIKV E80, ZIKV EDIII can be induced in mouse generates antigen-specific IFN-γ and IL-4 memory t cells.

6. passive protection of embodiment is tested

According to serum neutralization test as a result, the serum after being exempted from using ZIKV EDIII tri- does passive protection experiment.Serum and After zika virus is incubated, intraperitoneal injection observes the situation of two weeks mouse and weighs daily.As a result (Fig. 7) is shown, control group A G6 Mouse starts weight loss in the 5th day after attacking poison, and the 9th day completely dead, and ZIKV EDIII group AG6 mouse are in addition to one the Death at 12 days, other 4 were still all survived at the 14th day.These results indicate that the antiserum of anti-ZIKV EDIII with Zika virus can protect the attack of lethal dose zika virus after being incubated.

Conclusion

The present inventor is obtained using drosophila S2 systems surely turns cell line, and expresses the envelope protein that zika virus blocks E80 and EDIII.Destination protein ZIKV E80 and the ZIKV EDIII that the present invention obtains is inhibiting the experiment of zika virus infection cell In all illustrate relatively good inhibiting effect.After BALB/c mouse is immunized in third time, either also from serum antibody titer It is from the point of view of special t cell responses, the immune mouse of two kinds of Antigenic Peptides generates stronger immune response.Most of all, knot Aluminium adjuvant is closed, is just enough to protect AG6 mouse from lethal dose stockaded village with the antibody of the immunogene ZIKV EDIII inductions of low dosage Block the attack of virus.In terms of the preparation of destination protein, the applicant has found under study for action, is carried out using different host cells Expression, under conditions of gene order is just the same, the expression difference of Antigenic Peptide is huge and also has in terms of protein active Prodigious difference, for example ZIKV E80 Antigenic Peptides active in the present invention can not successfully be prepared using yeast cells, and fruit Not only yield is high for the ZIKA E80 Antigenic Peptides of fly expression, but also in the Zhong Heshui of inhibitory activity and induction of antibodies to ZIKA viruses Preferable antiviral effect is shown on flat, the ZIKA E80 Antigenic Peptides of drosophila expression are the ideal candidate epidemic diseases of ZIKA viruses Seedling.

Compared with traditional attenuated live vaccine, DNA vaccination and inactivated vaccine, the candidate vaccine is because without viral core Acid, so very safe.In addition purifying is convenient, does not need complicated technology, easy to operate, has bigger potentiality to be exploited.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Institut Pasteur of Shanghai, Chinese Academy of Sciences

<120>A kind of zika virus subunit vaccine of drosophila cell expression

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Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val

245 250 255

Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala

260 265 270

Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly His

275 280 285

Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser

290 295 300

Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu

305 310 315 320

Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp

325 330 335

Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu

340 345 350

Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser

355 360 365

Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp

370 375 380

Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp

385 390 395 400

His Arg Ser Gly Ser Thr Ile Gly Lys

405

<210> 3

<211> 110

<212> PRT

<213>Zika virus

<400> 3

Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe

1 5 10 15

Thr Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr Val Thr Val

20 25 30

Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln

35 40 45

Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly Arg Leu Ile Thr

50 55 60

Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys Met Met Leu

65 70 75 80

Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly

85 90 95

Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser Thr

100 105 110

<210> 4

<211> 1515

<212> DNA

<213>Artificial sequence

<400> 4

atccgctgca tcggcgtgtc gaatcgcgat ttcgtggagg gaatgagcgg aggaacctgg 60

gtggacgtgg tgctggagca cggaggatgc gtgaccgtga tggcccagga taagccgacc 120

gtggacatcg agctggtgac caccaccgtg tcgaacatgg ccgaggtgcg cagctactgc 180

tacgaggcct cgatcagcga tatggcctcc gactcgcgct gcccaaccca gggcgaggcc 240

tacctggata agcagagcga cacccagtac gtgtgcaagc gcaccctggt ggatcgcgga 300

tggggaaatg gatgcggact gttcggcaag ggatccctgg tgacctgcgc caagttcgcc 360

tgctccaaga agatgaccgg caagtcgatc cagccagaga acctggagta ccgcatcatg 420

ctgtcggtgc acggaagcca gcactccggc atgatcgtga acgataccgg ccacgagacc 480

gacgagaatc gcgccaaggt ggagatcacc ccgaactccc cacgcgccga ggccaccctg 540

ggaggattcg gatcgctggg cctggattgc gagccacgca ccggcctgga tttctccgac 600

ctgtactacc tgaccatgaa caataagcac tggctggtgc acaaggagtg gttccacgat 660

atcccactgc cctggcacgc cggagccgac accggaaccc cacactggaa caataaggag 720

gccctggtgg agttcaagga cgcccacgcc aagcgccaga ccgtggtggt gctgggaagc 780

caggagggag ccgtgcacac cgccctggcc ggagccctgg aggccgagat ggatggagcc 840

aagggacgcc tgagctccgg acacctgaag tgccgcctga agatggacaa gctgcgcctg 900

aagggcgtga gctactccct gtgcaccgcc gccttcacct tcaccaagat cccagccgag 960

accctgcacg gaaccgtgac cgtggaggtg cagtacgccg gaaccgatgg accatgcaag 1020

gtgccagccc agatggccgt ggacatgcag accctgaccc cagtgggacg cctgatcacc 1080

gccaatcccg tgatcaccga gtccaccgag aactcgaaga tgatgctgga gctggatccc 1140

ccgttcggcg acagctacat cgtgatcggc gtgggcgaga agaagatcac ccaccactgg 1200

caccgctcgg gaagcaccat cggcaaggcc ttcgaggcca ccgtgcgcgg agccaagcgc 1260

atggccgtgc tgggcgatac cgcctgggac ttcggaagcg tgggaggagc cctgaacagc 1320

ctgggcaagg gcatccacca gatcttcgga gccgccttca agtccctgtt cggaggcatg 1380

tcgtggttca gccagatcct gatcggcacc ctgctgatgt ggctgggcct gaacgccaag 1440

aatggctcca tctcgctgat gtgcctggcc ctgggaggag tgctgatctt cctgagcacc 1500

gccgtgtccg cctaa 1515

<210> 5

<211> 1227

<212> DNA

<213>Artificial sequence

<400> 5

atccgctgca tcggcgtgtc gaatcgcgat ttcgtggagg gaatgagcgg aggaacctgg 60

gtggacgtgg tgctggagca cggaggatgc gtgaccgtga tggcccagga taagccgacc 120

gtggacatcg agctggtgac caccaccgtg tcgaacatgg ccgaggtgcg cagctactgc 180

tacgaggcct cgatcagcga tatggcctcc gactcgcgct gcccaaccca gggcgaggcc 240

tacctggata agcagagcga cacccagtac gtgtgcaagc gcaccctggt ggatcgcgga 300

tggggaaatg gatgcggact gttcggcaag ggatccctgg tgacctgcgc caagttcgcc 360

tgctccaaga agatgaccgg caagtcgatc cagccagaga acctggagta ccgcatcatg 420

ctgtcggtgc acggaagcca gcactccggc atgatcgtga acgataccgg ccacgagacc 480

gacgagaatc gcgccaaggt ggagatcacc ccgaactccc cacgcgccga ggccaccctg 540

ggaggattcg gatcgctggg cctggattgc gagccacgca ccggcctgga tttctccgac 600

ctgtactacc tgaccatgaa caataagcac tggctggtgc acaaggagtg gttccacgat 660

atcccactgc cctggcacgc cggagccgac accggaaccc cacactggaa caataaggag 720

gccctggtgg agttcaagga cgcccacgcc aagcgccaga ccgtggtggt gctgggaagc 780

caggagggag ccgtgcacac cgccctggcc ggagccctgg aggccgagat ggatggagcc 840

aagggacgcc tgagctccgg acacctgaag tgccgcctga agatggacaa gctgcgcctg 900

aagggcgtga gctactccct gtgcaccgcc gccttcacct tcaccaagat cccagccgag 960

accctgcacg gaaccgtgac cgtggaggtg cagtacgccg gaaccgatgg accatgcaag 1020

gtgccagccc agatggccgt ggacatgcag accctgaccc cagtgggacg cctgatcacc 1080

gccaatcccg tgatcaccga gtccaccgag aactcgaaga tgatgctgga gctggatccc 1140

ccgttcggcg acagctacat cgtgatcggc gtgggcgaga agaagatcac ccaccactgg 1200

caccgctcgg gaagcaccat cggcaag 1227

<210> 6

<211> 330

<212> DNA

<213>Artificial sequence

<400> 6

aagctgcgcc tgaagggcgt gagctactcc ctgtgcaccg ccgccttcac cttcaccaag 60

atcccagccg agaccctgca cggaaccgtg accgtggagg tgcagtacgc cggaaccgat 120

ggaccatgca aggtgccagc ccagatggcc gtggacatgc agaccctgac cccagtggga 180

cgcctgatca ccgccaatcc cgtgatcacc gagtccaccg agaactcgaa gatgatgctg 240

gagctggatc ccccgttcgg cgacagctac atcgtgatcg gcgtgggcga gaagaagatc 300

acccaccact ggcaccgctc gggaagcacc 330

<210> 7

<211> 32

<212> DNA

<213>Artificial sequence

<400> 7

ccaagatcta tccgctgcat cggcgtgtcg aa 32

<210> 8

<211> 33

<212> DNA

<213>Artificial sequence

<400> 8

aaatctagac ttgccgatgg tgcttcccga gcg 33

<210> 9

<211> 33

<212> DNA

<213>Artificial sequence

<400> 9

aaaagatcta agctgcgcct gaagggcgtg agc 33

<210> 10

<211> 33

<212> DNA

<213>Artificial sequence

<400> 10

aaatctagag gtgcttcccg agcggtgcca gtg 33

Claims

1. a kind of Antigenic Peptide, which is characterized in that the Antigenic Peptide is derived from zika virus envelope protein, and is selected from the group：

(1) SEQ ID NO.2, amino acid sequence shown in 3；

(2) by amino acid sequence shown in SEQ ID NO.2,3 by it is one or more (≤20, such as 2-10, preferably 2- 5) replacing, missing or adding for amino acid residue and the derived peptides that are formed, and the derived peptides have and inhibit stockaded village's card disease The function of malicious infection cell and/or the function of inducing the immune response for zika virus.

2. Antigenic Peptide as described in claim 1, which is characterized in that the Antigenic Peptide is the recombination egg of Drosophila S 2 cells expression In vain.

3. a kind of polynucleotides of separation, which is characterized in that the Antigenic Peptide described in the polynucleotide encoding claim 1.

4. a kind of expression vector, which is characterized in that the expression vector contains the polynucleotides described in claim 3.

5. a kind of host cell, which is characterized in that the host cell contains the expression vector described in claim 4, or The polynucleotides described in claim 3 are integrated in genome.

6. host cell as claimed in claim 5, which is characterized in that the host cell includes Drosophila S 2 cells, yeast, big Enterobacteria, Chinese hamster ovary celI, DC cells etc..

7. a kind of pharmaceutical composition, which is characterized in that the composition contains Antigenic Peptide described in claim 1, right is wanted Polynucleotides described in the 3 either expression vector described in claim 4 or the host cell described in claim 5 are asked, and Pharmaceutically acceptable carrier and/or auxiliary material.

8. a kind of vaccine composition, which is characterized in that the composition contains Antigenic Peptide described in claim 1, right is wanted Polynucleotides described in the 3 either expression vector described in claim 4 or the host cell described in claim 5 are asked, and Acceptable carrier and/or auxiliary material in immunology.

9. the purposes of Antigenic Peptide as described in claim 1, (a) is used to prepare the antibody for zika virus；And/or it (b) uses In the drug for preparing treatment and/or prevention and the relevant disease of zika virus.

10. a kind of method preparing the Antigenic Peptide described in first aspect present invention, including step：

(i) host cell described in fourth aspect present invention is cultivated under optimum conditions, to express first aspect present invention institute The Antigenic Peptide stated；

(ii) Antigenic Peptide is purified.