CN108503696A - A kind of zika virus subunit vaccine of yeast cell to express - Google Patents

A kind of zika virus subunit vaccine of yeast cell to express Download PDF

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CN108503696A
CN108503696A CN201710108477.5A CN201710108477A CN108503696A CN 108503696 A CN108503696 A CN 108503696A CN 201710108477 A CN201710108477 A CN 201710108477A CN 108503696 A CN108503696 A CN 108503696A
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antigenic peptide
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zika virus
zikv
thr
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CN108503696B (en
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黄忠
张伟
屈攀科
刘庆伟
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Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences
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Institut Pasteur of Shanghai of CAS
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Abstract

The present invention provides a kind of zika virus subunit vaccines of yeast cell to express, specifically the invention discloses the subunit's zika virus vaccines researched and developed using yeast 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 yeast cell to express
Technical field
The invention belongs to biomedicine fields, specifically, the present invention relates to the sub- lists of the zika virus of yeast cell to express 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) amino acid sequence shown in SEQ ID NO.1；
(2) by amino acid sequence shown in SEQ ID NO.1 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 The function of 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 yeast cell to express.
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.1 are encoded；
(b) sequence polynucleotides as shown in SEQ ID NO.3；
(c) multinuclear of nucleotide sequence and homology >=95% (preferably >=98%) of sequence shown in SEQ ID NO.3 Thuja acid；
(d) 5 ' ends and/or 3 ' ends of the polynucleotides as shown in SEQ ID NO.3 truncate or add 1-60 (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 yeast, Drosophila S 2 cells, 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.
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, the yeast single bacterium colony of conversion is inoculated into BMGY cultures respectively in the method step (i) In base, thalline is resuspended with BMMY (containing 1% methanol) culture medium in centrifugation removal supernatant after culture, and 25-35 DEG C (is preferably 30 DEG C), Fiber differentiation 24-72 hours (preferably 48 hours).
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 pPink α-HC-ZIKV-ED III and pPink α-HC-ZIKV-E80 plasmid maps.
Fig. 2 shows a small amount of screening Pichia pastoris height expression strains.(A) the culture medium part of different clones takes 5ul samples SDS-PAGE runs glue, Western-blot testing goal albumen, destination protein (ZIKA-ED III is merged with His-tag) size 12.74KDa.(B) the culture medium part of different clones takes 10ul samples SDS-PAGE to run glue, Western-blot testing goal eggs In vain, destination protein (ZIKA-E80 is merged with His-tag) size 45.39KDa.It is the pPink α-that electricity turns linearisation to compare (ctr) The clone that HC is obtained.
Fig. 3 shows expression and purifying of the ZIKA-ED III in Pichia pastoris.(A) SDS- of the ZIKA-ED III purified PAGE and coomassie brilliant blue staining analysis.(B) it is carried out using the antibody of anti-ZIKV-E80 (left side) and anti-His labels (right side) Western-blot is detected.
Fig. 4 shows that ZIKA-ED III inhibits infection of the ZIKA viruses to Vero cells.The ZIKA-ED III of purifying carries out one ZIKA viruses after being serially diluted respectively with 100PFU are uniformly mixed, and are added to immediately on the Vero cells completed in advance, 37 It spends and cultivates a hour in CO2 incubators, the mixture of ZIKA-ED III and ZIKA viruses are replaced into containing 0.2% agar later The 2%DMEM culture mediums of sugar, are placed in 37 degree of CO2 incubators and cultivate, fixed and tied using 4% paraformaldehyde after plaque formation Crystalviolet dyes.BSA is as negative control.(A) the cell plaque number handled with BSA is compared in the cell handled with ZIKA-ED III Purpose is reduced.(B) standardization plaque reduces the quantitative analysis of number.Average value ± standard error has marked.
Fig. 5 shows that ZIKA-ED III induces neutrality antibody in balb/c mouse.(A) balb/c is immunized in ZIKA-ED III Mouse.Mice serum exempt from respectively two after two weeks and three exempt from after be collected within two weeks, 1:Under 10000 dilutions, ELISA detections The antibody response of III specificity of ZIKA-ED.(B) three plaques for exempting from rear two weeks serum, which are reduced, neutralizes experiment, 24 orifice plate crystal violets dye Color part is shown.(C) data analysis of PRNT50s.The geometrical mean and p value of PRNT50s is as shown in the figure.
Fig. 6 shows that EDIII antiserums have the Protective effect for preventing ZIKV infection.Two group of five week old AG6 is set Mouse, every group five.For antiserum 50ul/ only with isometric mixing of viral dilution containing 5PFU, 37 degree are incubated a hours.So Pneumoretroperitoneum injects serum-virus mixture, continuous two weeks record changes of weight and survival rate situation.Mouse weight reduces by more than original Initial body weighs 20% and is euthanized, and is defined as death, does not re-record same day weight.
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 disease of yeast cells research and development Malicious vaccine has the advantages that yield is high, purity is high, stability is good, is easy to purifying, simultaneously as viral nucleic acid ingredient is free of, so There is no the possibility for restoring mutation, safety is higher.Moreover, experiment finds to combine aluminium adjuvant, with the immunogene of very low dosage The neutralizing antibody that (ZIKV EDIII) induction generates just is enough the attack for protecting AG6 mouse from lethal dose zika virus.Body Interior Vitro Experimental Results show that zika virus subunit vaccine ZIKV EDIII provided by the invention are one and prevent stockaded village's card disease The relatively good vaccine of poison infection has notable preferable 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, and the inventors discovered that clipping 20% region of E protein C-terminal is conducive to E eggs White secretion.Main target of the present invention is to develop one kind and can induce body and generate targeting E protein (E80 and EDIII) and neutralize to resist The vaccine of body, 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 Z1106033 strain (the viral amino acid 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 GTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDG AKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVG RLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGS VGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNAKNGSISLMCL ALGGVLIFLSTAVSA, 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 suitble to the nucleic acid encode for the Antigenic Peptide of the present invention expressed in yeast cells Sequence.
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 acatggccgaggtgcgcagctactgctacgaggcctcgatcagcgatatggcctccgactcgcgctgcccaa cccagggcgaggcctacctggataagcagagcgacacccagtacgtgtgcaagcgcaccctggtggatcgcggatgg ggaaatggatgcggactgttcggcaagggatccctggtgacctgcgccaagttcgcctgctccaagaagatgaccgg caagtcgatccagccagagaacctggagtaccgcatcatgctgtcggtgcacggaagccagcactccggcatgatcg tgaacgataccggccacgagaccgacgagaatcgcgccaaggtggagatcaccccgaactccccacgcgccgaggcc accctgggaggattcggatcgctgggcctggattgcgagccacgcaccggcctggatttctccgacctgtactacct gaccatgaacaataagcactggctggtgcacaaggagtggttccacgatatcccactgccctggcacgccggagccg acaccggaaccccacactggaacaataaggaggccctggtggagttcaaggacgcccacgccaagcgccagaccgtg gtggtgctgggaagccaggagggagccgtgcacaccgccctggccggagccctggaggccgagatggatggagccaa gggacgcctgagctccggacacctgaagtgccgcctgaagatggacaagctgcgcctgaagggcgtgagctactccc tgtgcaccgccgccttcaccttcaccaagatcccagccgagaccctgcacggaaccgtgaccgtggaggtgcagtac gccggaaccgatggaccatgcaaggtgccagcccagatggccgtggacatgcagaccctgaccccagtgggacgcct gatcaccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgttcggcg acagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcggcaag gccttcgaggccaccgtgcgcggagccaagcgcatggccgtgctgggcgataccgcctgggacttcggaagcgtggg aggagccctgaacagcctgggcaagggcatccaccagatcttcggagccgccttcaagtccctgttcggaggcatgt cgtggttcagccagatcctgatcggcaccctgctgatgtggctgggcctgaacgccaagaatggctccatctcgctg Atgtgcctggccctgggaggagtgctgatcttcctgagcaccgccgtgtccgccta a, SEQ ID NO.4；The sequence Encode 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 ccgccaatcccgtgatcaccgagtccaccgagaactcgaagatgatgctggagctggatcccccgtt cggcgacagctacatcgtgatcggcgtgggcgagaagaagatcacccaccactggcaccgctcgggaagcaccatcg Gcaag, 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 has the expression cassette for expressing the antigen peptide gene, the table There are following elements successively up to box 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 yeast or 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 pPink α HC or pMT/BiP/V5-HisA.These sides Method includes recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..The DNA sequence dna can be effectively connected to table Up in the appropriate promoter in carrier, to instruct mRNA to synthesize.Expression vector further includes the ribosome binding site of translation initiation Point and transcription terminator.In addition, expression vector preferably comprises one or more selected markers, to provide for selecting to turn The phenotypic character of the host cell of change.
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 yeast cells.It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.Engineering is thin Born of the same parents can quickly utilize methanol type (Mut+) or at a slow speed utilize methanol type (Muts)。
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：HiTrapTM 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) RibiTMAdjuvant 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 yeast cells great expression, therefore manufacturing cost is low, and suitable industrialization is answered With；
(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) after mouse being immunized using the Antigenic Peptide EDIII of the present invention, immune mouse generates stronger immune response, 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；
(4) 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
Vero cells are cultivated with the DMEM culture mediums containing 10% cow's serum.PichiaPinkTMYeast strain is purchased from U.S. Invitrogen companies of state, and cultivated according to shop instruction.
2. virus
The stockaded village's card strain used in this research is ZIKV/SZ-WIV01 plants of (GenBank of Asian type:KU963796), come from Wuhan Virology Institute,Chinan academy of Sciences's microbial bacteria seed culture of viruses collection (preservation number：IVCAS 6.6110).
3. antibody
The monoclonal antibody that the HRP that the antibody of anti-His tag is Proteintech is marked, the antibody sheet of anti-ZIKV-E80 The polyclonal antibody that laboratory is obtained using the E80 protein immunization experimental rabbits of insect cell expression.
4. plasmid construction
According to Z1106033 strains (the viral nucleotide GenBank of zika virus Asian type South America prevalence in 2015: KU312312, amino acid Genbank：ALX35659 E protein coded sequence (SEQ ID NO.1)), carry out codon optimization and Gene chemical synthesis (SEQ ID NO.4), one-step cloning of going forward side by side (on (being purchased from GenScript USA Inc.), are obtained to carrier pUC57 Plasmid pZIKV-E.
Utilize primer (ZIKV-ED3-F:AAGCTGCGCCTGAAGGGC (SEQ ID NO.7) and ZIKV-ED3-KpnI-R: CAACGGTACCTTAATGGTGATGGTGATGATGGGTGCTTCCCGAGCGGTG (SEQ ID NO.8)) from the matter of gene optimization Upper III genes of PCR amplification ED of grain pZIKV-E, generation one end is another for flat end after obtained PCR product I single endonuclease digestions of Kpn End is the segment of cohesive end, is cloned on pPink α-HC (Invitrogen) carrier of I double digestion of Stu I and Kpn, obtains Plasmid pPink α-HC-ZIKV-ED III.
Similarly, primer (ZIKV-E80-F is utilized:ATCCGCTGCATCGGCGTGTCGAAT (SEQ ID NO.9) and ZIKV-E80-KpnI-R:CAACGGTACCTTAATGGTGATGGTGATGATGCTTGCCGATGGTGCTTCC(SEQ ID NO.10)) the PCR amplification E80 segments from plasmid pZIKV-E, and be cloned on pPink α-HC (Invitrogen) carrier, it obtains Plasmid pPink α-HC-ZIKV-E80.
5 Pichia pastoris convert and the detection of bacterial strain expression quantity
In order to convert Pichia pastoris, plasmid pPink α-HC-ZIKV-ED III and pPink α-HC-ZIKV-E80 I lines of EcoN Property, then electricity is transferred to PichiaPinkTMStrain 1(Invitrogen).The conversion of Pichia pastoris and subsequent transformant Screening operated according to shop instruction.A small amount of culture expression screening experiments have been carried out according to specification.In simple terms, turn The yeast single bacterium colony of change is inoculated into respectively in 5ml BMGY culture mediums, and 30 DEG C of 250rpm cultivate r for 24 hours, is then centrifuged for removal supernatant, Thalline, 30 DEG C of 250rpm Fiber differentiations 48hr are resuspended with 1ml BMMY (containing 1% methanol) culture medium.After induction, centrifugation Culture medium supernatant is collected, takes supernatant to carry out Western blotting detections in right amount, specific method is before as described in article.
The preparation of III albumen of 6.ZIKV-ED
In order to prepare III antigens of ZIKV-ED, selected bacterial strain is cultivated and induced.Culture medium supernatant is collected by centrifugation, The membrane filtration for using 0.45uM first, is then concentrated by ultrafiltration using the super filter tube of 3KDa, and the sample after concentration is used Binding buffer wash from super filter tube, are purified using nickel column.It is bright that coomassie finally is carried out to the albumen of purifying Indigo plant dyeing and Western-blot detections.
7.ED III inhibits virus infection tests in vitro
In order to which whether III conformations of ZIKV-ED for verifying expression are correct, the present inventor carries out ED III and inhibits virus infection in vitro Experiment.In simple terms, it is exactly to dilute expression ZIKV-ED III totally for five with a concentration of starting point of 200ug/ml successively 5 times of dilutions Degree, viral (100PFU) mixings of ZIKV of III solution of ZIKV-ED and 100ul after 100ul dilutions, the mixture of 200ul is added Into a hole of 24 orifice plates for being covered with Vero cells, 37 degree of CO2Incubator sops up mixed liquor after being incubated 1h, is added per hole The DMEM of 2%FBSs of the 700ul containing 0.2% agarose, four degree are placed 15 minutes, and 24 orifice plates are finally transferred to 37 degree of CO2Culture Case culture fixes cell, 0.1% violet staining after 3-4 days using 4% paraformaldehyde.
8. mouse immune and vaccine specific antibody test
By III albumen of ED of purifying with(Invivogen, the U.S.) adjuvant mixes, and makes tentative Vaccine, each dosage contain the aluminum hydroxide adjuvant of 10ug antigens (ED III) and 500ug.PBS is mixed with adjuvant as negative Control.(every group six, the 6-8 week old) purchases of Balb/c mouse were from Shanghai Experimental Animal Center (SLAC) company, at the 0th week, 2 weeks It with 4 weeks intraperitoneal injection test vaccines or PBS negative controls, then took a blood sample at the 4th week and the 6th week, is used for antibody test.
The assay method of III specific antibodies of ZIKV-ED is as follows：It is coated with elisa plate with the ZIKV-ED III of Yeast expression (holes 50ng/) is required for PBST board-washings three times to remove non-specific binding after often step operation later, with 5% defatted milk After powder closing, add the 1 of 50ul per hole:10000 dilute serums, then plus the sheep anti-mouse igg (sigma) of HRP couplings is used as secondary antibody, TMB developing solutions develop the color, and 1N phosphoric acid solutions terminate, and measure 0D450.
9. the micro- neutralization test of virus
The micro- neutralization experimental implementation of virus is as follows：It tests 24 well culture plate of the previous day and spreads Vero cells, 10 are spread per hole5Cell； Neutralization experiment is done within second day, ZIKA virus liquids and the 100 μ l gradients that 100 μ l are contained to 100PFU first in 1.5mlEP pipes are dilute The serum mixing released, 37 DEG C of incubation 1h；Then culture medium in 24 orifice plates is sopped up, is cleaned one time with serum-free DMEM, in 24 holes 200 μ l serum virus mixed liquors, 37 DEG C of CO is added in plate per hole2Mixed liquor is sopped up after incubator culture 1h, is added per hole The DMEM of 2%FBSs of the 700ul containing 0.2% agarose, four degree are placed 15 minutes, and 24 orifice plates are finally transferred to 37 degree of CO2Culture Case culture fixes cell, 0.1% violet staining after 3-4 days using 4% paraformaldehyde.The dilution factor of serum sample is determined Justice is, compared to only plus viral hole, can inhibit the highest serum dilution (PRNT50) corresponding to 50% plaque number.
10. mouse protest test
Protest test carries out in I types and the double AG6 mouse struck of II types interferon receptors.Two weeks after three are exempted from respectively III group of the ED of acquisition or PBS groups antiserum contain respectively with the isometric mixing of ZIKV/SZ-WIV01 viruses per 100ul mixtures Then 50ul antiserums and 5PFU zika virus are incubated 1h at 37 degree.Two group of 5 week old AG6 mouse (every group 5) respectively note by abdominal cavity Penetrate 100ul/ ED III or PBS antiserums-virus mixed liquors.Continuous 15 days progress mouse are weighed after attacking before poison and attacking poison, and Observe Survival.
11. statistics
All statistical analyses are carried out using GraghPad Prism versions 5.Kaplan-Meier survivorship curves make It is compared with log-rank inspections, other experimental datas are come using two-tailed student ' s t-test methods Analysis.The significant difference of statistics is defined as follows：Ns, P >=0.05；*0.01≤P<0.05；**P<0.01；***P<0.001.
Expression and identification of the 1 ZIKV ED III and E80 of embodiment in Pichia pastoris
It is recombinantly expressed in Pichia pastoris to probe into zika virus ED III and E80, the present inventor constructs plasmid pPink α-HC-ZIKV-ED III and plasmid pPink α-HC-ZIKV-E80, plasmid construct are as shown in Figure 1.Plasmid pPink α-HC-ZIKV- ED III is used for III albumen of secreting, expressing ED, and alpha-amylase secreting signal peptide is merged at the ends N- and His tag are merged at the ends C-.It is similar Ground, plasmid pPink α-HC-ZIKV-E80 are used for secreting, expressing E80 albumen, and alpha-amylase secreting signal peptide and C- are merged in the ends N- End fusion His tag.
Plasmid pPink α-HC-ZIKV-ED III and plasmid pPink α-HC-ZIKV-E80 are distinguished transformed yeast by the present inventor Cell, obtained each recombination yeast clone detect the expression quantity of ED III and E80, empty carrier with western-blot methods The yeast cells of pPink α-HC conversions is as negative control.The results show that III transformed yeasts of pPink α-HC-ZIKV-ED are cloned There is very strong anti-His tag monoclonal antibodies detection signal (Fig. 2A), prompts the expression quantity in yeast of ED III very high；On the contrary, pPink α-HC-ZIKV-E80 transformed yeasts, which are cloned at expected molecular weight, only has small-signal (Fig. 2 B), prompts tables of the E80 in yeast It is relatively low up to measuring.
No. 2 bacterial strain conducts of No. 4 bacterial strains and pPink α-HC-ZIKV-E80 of pPink α-HC-ZIKV-ED III are selected respectively Height expression strain carries out great expression purifying, obtained purified product SDS-PAGE coomassie brilliant blue stainings and Western Blotting is analyzed and identified.Convert the albumen purified in No. 4 bacterium from pPink α-HC-ZIKV-ED III is in SDS-PAGE It is now the band (Fig. 3 A) of about 13KDa, it is consistent with gained molecular weight of albumen is calculated according to III sequences of ED；It is anti-with the anti-His tag of mouse Body and rabbit-anti ZIKV-E80 antibody also detect positive signal (Fig. 3 B) at coomassie brilliant blue staining protein band respectively, show Purified albumen is strictly ED III, calculates yield after purification and reaches 4.5mg/l.No. 2 bacterium are converted from pPink α-HC-ZIKV-E80 Purified albumen is seldom in strain, without clear band (data not shown) in SDS-PAGE, it may be possible to since E80 is expressed It measures low and there is degradation situation.
The above result shows that E80 expressions in yeast are poor, recombinant vaccine research and development are not suitable for.And ED III is in ferment Correct, high efficient expression in mother, has great vaccine development potentiality.Therefore, follow-up study concentrates on ED III.
Using Drosophila S 2 cells express above-mentioned ZIKV E80 and ZIKV EDIII's the experimental results showed that, destination protein ZIKV Although E80 can not in yeast cells high efficient expression, really can in Drosophila S 2 cells successful expression.And ZIKV EDIII exist Expression quantity in yeast is significantly higher than the expression quantity in Drosophila S 2 cells, and the expression quantity in yeast has reached Drosophila S 2 cells 1.7 times of (2.6mg/l).
Embodiment 2.ED III can inhibit ZIKV to infect in vitro
Vero cells are inoculated with after the Yeast expression ED III of purifying and ZIKV viruses are mixed, plaque number is observed after three days；It is right It is set as BSA and ZIKV viruses mixing and Vero cell incubations according to group.The results show that control group is reduced with BSA concentration, plaque There was no significant difference for number, shows that BSA cannot inhibit viral infection；On the contrary, the plaque number of III processing groups of ED increases with III concentration of ED And (Fig. 4 A) is gradually decreased, show that ED III being capable of infection (Fig. 4 B) of the dose-dependent inhibition virus to cell.It is bent according to inhibition 50% inhibition concentration (IC50) that line computation has obtained ED III is 7.597ug/ml.The result prompts, the tool of Yeast expression ED III There is correct functional conformation, can be combined with the virus receptor of cell surface, to which Reverse transcriptase virus knot is combined into Cell.
The present embodiment the experimental results showed that, the destination protein ZIKV EDIII of Yeast expression according to the present invention are to stockaded village's card The inhibitory activity IC of virus infection50For 7.597ug/ml；And the destination protein ZIKV EDIII of Drosophila S 2 cells expression are to stockaded village's card The inhibitory activity IC of virus infection50For 71.85ug/ml, the two has differed about 10 times.
Certainly, the result of the present embodiment also indicates that ZIKV EDIII can be competed with zika virus into cell, therefore has There is induction animal to generate the potentiality of neutralizing antibody.
Mice produced high titers neutralizing antibody is immunized in embodiment 3.ED III
In order to study the immunogenicity of ED III, 2 groups of (every group 6) Balb/c mouse difference at the 0th week, the 2nd week and the 4th week Immune ED III or PBS, PBS is negative control group.Blood serum sample was collected at the 4th week and the 6th week, with the ED III of Pichia anomala expression As envelope antigen, ELISA experiments are carried out to detect specific antibody reaction.As shown in Figure 5A, the antigen of PBS groups mice serum Antibody response is background level, and the immune serum of III group of mouse of ED has apparent antibody response, and three exempt from rear two weeks serum The antibody titer for exempting from rear two weeks serum than two is high.ELISA the result shows that, ED III be immunized mouse generate specific antibody.
The present inventor assesses the ability that immune serum inhibits ZIKV to infect in vitro by the micro- neutralization test of virus.Such as figure Shown in 5B and 5C, the sero-fast neutralization titer of PBS groups and III group of significant difference of antiserum neutralization titer of ED；III group of anti-blood of ED Clear geometric mean neutralization titer is 3608.The above result shows that the immunogenicity of III vaccines of ED is good, tool can be induced strongly There is the neutralizing antibody of protection potentiality.
The present embodiment the experimental results showed that, the destination protein ZIKV EDIII of Yeast expression according to the present invention are to stockaded village's card The neutralization activity of virus infection is 3608；And what the destination protein ZIKV EDIII of Drosophila S 2 cells expression infected zika virus Neutralization activity is 1633.8, and the two has differed about 3 times.
III immune serums of embodiment 4.ED have the function of interior resisting virus.
In order to evaluate immune serum in mouse Protective effect, it is dry to choose the I type and II type sensitive to wild type ZIKV It disturbs plain receptor defects AG6 mouse and carries out protest test.Every AG6 mouse peritoneals injection ED III or PBS antiserums and ZIKV Mixture is observed continuously 14 days, the variation of record mouse weight and survival rate.As shown in fig. 6, PBS group mouse are the 5th after attacking poison Celestial body is begun to decline again, all dead successively at the 9-10 days；On the contrary, III antiserum processing group mouse weight one after attacking poison of ED Straight gentle rising, survival rate reach 100%.Should statistics indicate that, III vaccine group serum of ED in Mice Body have protecting effect, energy Enough prevent the zika virus attack of lethal dose.
Expression of 1 destination protein ZIKV E80 and the ZIKV EDIII of comparative example in Drosophila S 2 cells
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.
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 (viral nucleotides of zika virus Asian type South America prevalence in 2015 GenBank:KU312312, amino acid Genbank：ALX35659 E protein coded sequence (SEQ ID NO.1)) carries out password Son optimization and gene chemical synthesis go 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 restriction enzyme sites, Being connected to the insect expression vector pMT/Bip/V5-His A containing Bgl II and Xba I restriction enzyme sites (containing His labels, has Conducive to the detection and purifying of target protein), it obtains carrying 80% region (ZIKV E80) of zika virus envelope protein N-terminal and packet Recombinant plasmid the pMT/Bip/V5-ZIKV E80 and pMT/Bip/ of memebrane protein region III (ZIKV EDIII) target gene fragment V5-ZIKV EDIII。
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, consistent with the stripe size that western blot are detected.Result above prompts target protein E80 and EDIII are expressed, and the expression yield for calculating ZIKV E80 and ZIKV EDIII Antigenic Peptides after purification respectively reaches 10mg/l and 2.6mg/l.
Conclusion
The present inventor is obtained using yeast cell system surely turns cell line, and expresses the truncated coating egg of zika virus White EDIII.The destination protein ZIKV EDIII that the present invention obtains are illustrated in inhibiting the experiment of zika virus infection cell Relatively good inhibiting effect.After BALB/c mouse is immunized in third time, either from serum antibody titer or special T cell From the point of view of reaction, immune mouse generates stronger immune response.Most of all, in conjunction with aluminium adjuvant, with the immunogene of low dosage The antibody of ZIKV EDIII inductions is just enough the attack for protecting AG6 mouse from lethal dose zika virus.
In terms of the preparation of destination protein, the applicant has found under study for action, is expressed using different host cells, 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 4.5mg/l can be reached using the expression quantity of the ZIKV EDIII Antigenic Peptides of the yeast cell to express present invention, and fruit The expression quantity of fly S2 cells is only 2.6mg/l.Compared with Drosophila S 2 cells, the expression quantity of yeast improves 70% or more.In work Property aspect, the inhibitory activity (IC that the ZIKV EDIII Antigenic Peptides of Yeast expression of the present invention infect zika virus50=7.597ug/ Ml) inhibitory activity (the IC that zika virus is infected with the ZIKV EDIII Antigenic Peptides of Drosophila S 2 cells table50=71.85ug/ml) Compare, the ZIKV EDIII Antigenic Peptides of Yeast expression its nearly 10 times are improved to the inhibitory activity of zika virus, it is often more important that, The ZIKV EDIII Antigenic Peptides of yeast cell to express achieve unexpected excellent technique effect, yeast table in neutralization levels The ZIKV EDIII group serum neutralising capacities reached are stronger, PRNT50Reach 3608, and the ZIKV EDIII of Drosophila S 2 cells expression Group serum neutralising capacity PRNT50It is 1633.8.Compared with the ZIKV EDIII Antigenic Peptides of Drosophila S 2 cells expression, Yeast expression The ability of its induction neutralizing antibody of ZIKV EDIII Antigenic Peptides improves 120%.It is anti-in expression yield, antigen peptide activity and induction In general, the ZIKV EDIII Antigenic Peptides of Yeast expression have significantly the neutralization levels of body as the candidate vaccine of zika virus Advantage.
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 yeast cell to express
<130> P2017-0061
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 504
<212> PRT
<213>Zika virus
<400> 1
Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser
1 5 10 15
Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr
20 25 30
Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr
35 40 45
Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser
50 55 60
Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala
65 70 75 80
Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu
85 90 95
Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser
100 105 110
Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys
115 120 125
Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His
130 135 140
Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu Thr
145 150 155 160
Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala
165 170 175
Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro
180 185 190
Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn
195 200 205
Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro
210 215 220
Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu
225 230 235 240
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 Ala Phe Glu Ala Thr Val Arg
405 410 415
Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly
420 425 430
Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln Ile
435 440 445
Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser
450 455 460
Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Ala Lys
465 470 475 480
Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu Ile
485 490 495
Phe Leu Ser Thr Ala Val Ser Ala
500
<210> 2
<211> 409
<212> PRT
<213>Zika virus
<400> 2
Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser
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Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr
20 25 30
Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr
35 40 45
Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser
50 55 60
Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala
65 70 75 80
Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu
85 90 95
Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser
100 105 110
Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys
115 120 125
Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His
130 135 140
Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu Thr
145 150 155 160
Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala
165 170 175
Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro
180 185 190
Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn
195 200 205
Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro
210 215 220
Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu
225 230 235 240
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> 18
<212> DNA
<213>Artificial sequence
<400> 7
aagctgcgcc tgaagggc 18
<210> 8
<211> 49
<212> DNA
<213>Artificial sequence
<400> 8
caacggtacc ttaatggtga tggtgatgat gggtgcttcc cgagcggtg 49
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence
<400> 9
atccgctgca tcggcgtgtc gaat 24
<210> 10
<211> 49
<212> DNA
<213>Artificial sequence
<400> 10
caacggtacc ttaatggtga tggtgatgat gcttgccgat ggtgcttcc 49

Claims (10)

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) amino acid sequence shown in SEQ ID NO.1；
(2) by amino acid sequence shown in SEQ ID NO.1 by it is one or more (≤20, such as 2-10, preferably 2-5 It is a) replacing, missing or adding for amino acid residue and the derived peptides that are formed, and the derived peptides have and inhibit zika virus The function of 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 recombinant protein of yeast cell to express.
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. 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.
7. 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.
8. vaccine composition as claimed in claim 7, which is characterized in that the vaccine composition also contains adjuvant.
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 Antigenic Peptide described in claim 1, including step：
(i) host cell described in claim 5 is cultivated under optimum conditions, to express Antigenic Peptide described in claim 1；
(ii) Antigenic Peptide is purified.
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