CN108727503A

CN108727503A - VZV recombinates gE- flagellum plain fusion proteins and its preparation method and application

Info

Publication number: CN108727503A
Application number: CN201710449087.4A
Authority: CN
Inventors: 肖杨; 刘昊智; 毛普加; 高媛; 王雨; 李津; 吴克
Original assignee: BRAVOVAX Co Ltd
Current assignee: BRAVOVAX Co Ltd
Priority date: 2017-04-18
Filing date: 2017-06-14
Publication date: 2018-11-02

Abstract

The invention discloses a kind of VZV to recombinate gE- flagellum plain fusion proteins and its preparation method and application, wherein, the fusion protein is including at least the N-terminal conserved region of flagellum fibroin, the C-terminal conserved region of varicella virus (VZV) glycoprotein gE and flagellum fibroin.GE albumen can cause higher antibody titer before VZV recombinations gE- flagellums plain fusion protein provided by the invention more individually recombinates, therefore there is preferable immunocompetence, it can and humoral immunity immune with activated cell, and without the risk for causing latent infection, good amynologic basis has been established for the preparation of novel varicella protein vaccine.

Description

VZV recombinates gE- flagellum plain fusion proteins and its preparation method and application

Technical field

The present invention relates to a kind of VZV to recombinate gE- flagellum plain fusion proteins and its preparation method and application, belongs to bio-pharmaceuticals Field, especially recombinant vaccine field.

Background technology

One, varicella virus is summarized

Varicella virus (varicella-zoster virus, VZV) primary infection can cause varicella (chickenpox) and herpes zoster (herpes zoster, HZ), there is higher infectiousness and incidence.Varicella is children A kind of period common acute, highly infective disease, there is systemic rash in infant, with performances such as fever, malaises.Band Shape bleb often betides the middle-aged and the old, is a kind of hydroa appearing in dermatotome, it is characterized in that nerve pain and with neural Zhi Dan The skin rash of side distribution.The performance of both Disease Clinicals is different, is but caused by same virus, i.e. varicella-zoster disease Poison.VZV is by nervous system infection human body, and experience twice wait by viremia virusemia, and virus reaches skin surface, generates typical bleb Sample skin lesion, subsequent cell entry peripheral nervous system, and hide in spinal ganglion.It waits for deflorescence, after patient's recovery from illness, has few The virus of amount remaining can be hidden all the life in the nerve cell of the spinal cord posterior root ganglion of host, cerebroganglion and enteric nervous section In.

VZV belongs to herpetoviridae Alphaherpesvirinae, is a kind of double-stranded DNA virus, and only there are one serotypes.Virus Nucleocapsid diameter about 80~120nm, is in icosahedron, and surrounding wraps the coating of one layer of inner membrance and a layer segment from cell membrane. Entire VZV particle diameters are 180~200nm.Core DNA includes at least 71 open reading frame, wherein 2/3 is replication in vitro institute Required including important 8 kinds of glycoprotein, DNA replication dna GAP-associated protein GAP, the GAP-associated protein GAPs such as DNA untwists, capsid is packed.VZV is main Air borne is passed through by the virion in cutaneous lesion blister liquid.Enter body after upper respiratory tract mucous membrane or conjunctiva infection, It is replicated in local cells, primary disease toxaemia and premonitory symptom occurs within about 4~6 days, vesicle occur within about 2 weeks.Infect water Acne is generally more with light-duty and medium-sized case, and common complication is the Secondary bacterial infections of skin, accidental a variety of skin appearances It is existing, such as pneumonia, encephalitis, cerebellar ataxia.

For the newborn that immune function is insufficient or without immunity, varicella be it is a kind of it is serious, be related to the lethal of multiple organ Sexuality dye.The state of an illness can aggravate after immunosupress children suffer from varicella.Adult's infection VZV is rare, but symptom is often very serious, mostly concurrently more The lithogenous pneumonia of property is dissipated, case fatality rate is up to 10%~40%.When pregnant woman suffers from varicella, fetus can be infected or even be formed congenital comprehensive Close disease (CVS).

Two, chicken pox vaccine is summarized

For varicella and herpes zoster, mainly anti symptom treatment, no specific treatment method, antiviral drugs, such as Ah former times Lip river Wei, Valaciclovir, famciclovir etc. cannot terminate viral discharge though contributing to varicella and the rapid rehabilitation of HZ patient, can not be pre- Anti- subclinical infection.And give excessive immunoglobulin after exposing VZV, for stop or mitigate Disease Spectrum be also it is limited, Therefore, vaccine plays an important role in terms of controlling varicella infection, and vaccine inoculation is most effective and most reliable prevention and control The means of varicella processed.

Chicken pox vaccine was researched and developed successfully by high bridge (Takahashi) seminar of Japan in 1974 earliest.They are from 13 Year is named as isolating one plant of VZV in the varicella infant body of Oka, by it in 34 DEG C of 11 generations of biography of human embryonic fibroblast, in cavy 37 DEG C of fibroblast passed for 12 generations and 37 DEG C of human diploid fibroblasts pass 5 generations (WI-38 and MRC-5), and successfully attenuation is work Vaccine, referred to as Oka vaccines (vOka).Originally, the safety of vOka is very good, even if in the leukaemia youngster of immunity partial injury Also there is not serious adverse reaction in virgin and HIV infection children, also shows the good immunoprotection effect in place Fruit.But the immunoprotection time that inoculation vOka vaccines are inoculated with successively is not lasting enough, and a small number of individuals fail to reach through being inoculated with successively To effective protection state.Such as hebetic teenager slightly larger to those ages, the immune effect of vOka are less than 1~12 years old youngster Child, therefore for preschool child, need to carry out secondary inoculation to the vaccine.Since vOka is VZV attenuated vaccines, also deposit In certain risk for leading to latent infection.

VZV glycoprotein E subunit vaccines are the mainstream research directions of current chicken pox vaccine, and VZV genomes share 71 Open reading frame (openreadingframe, ORF), coding glycoprotein E (gE), totally 8 kinds of gB, gH, gI, gC, gL, gK and gM Glycoprotein.Wherein gE belongs to I type memebrane proteins by ORF68 gene codes, is the required glycoprotein for generating infectious viral particle, Be also that content is most abundant in VZV coatings and infection cell film, the strongest glycoprotein of antigenicity, be present in virion surface and In the surface and cytoplasm of infected cell, exist in the form of different high sugar content peptides in the viral different stages of ripeness.In extensive In the varicella and Patients with Herpes Zoster serum of multiple phase, VZV antibody is mainly for gE, gB and gH, especially using gE as major target class The cellular immunity and humoral immunity of induction, can protect animal from virus attack.Have studies have shown that VZV gE monoclonal antibodies Can mediate antibody dependence cytotoxicity, in the case where there is exogenous complement in energy and virus infectivity.

Toll-like receptor 5 (TLR5) be in body immune system a kind of important transmembrane receptor Toll-like receptor family at Member, is the specific receptor of flagellum fibroin.Flagellum fibroin is the major structural protein of Gram-negative bacteria flagellum.Recognize at present For flagellin has proinflammatory effect, and locally or systemically inflammatory reaction can be caused by TLR5, is known by its specific receptor TLR5 The congenital immunity reaction and inflammatory reaction of body, induction monocytes/macrophages, epithelial cell activation, before release are not started afterwards Inflammatory factor is that there are potentiality to be applied to clinical, potent whole body and mucosal adjuvant.

Have some literature reports using the gene recombination technology expression subunit vaccine, but most in research and development, and III phase clinical trials are not entered, validity and safety need to be further determined that.GSK companies are main with gE glycoprotein Ingredient, AS01B are adjuvant, develop a kind of novel subunit HZ vaccines, in II phase clinical datas, it was demonstrated that gE glycoprotein is immune Dosage without significant difference, but is proportionate to the promotion of cellular immunity with antibody titer.In terms of chicken pox vaccine, some scholars one Directly it is being dedicated to studying expressing fusion protein gE albumen, but is not making substantial progress also.Therefore, design studies are a can be steady The fusion protein for determining high quality expression varicella gE albumen, is to research and develop the basis of novel chicken pox vaccine, and there is an urgent need to solve at present Certainly the problem of.

Invention content

In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is melted with VZV gE glycoprotein and flagellum fibroin Based on closing gene, a kind of VZV recombination gE- flagellum plain fusion proteins and its preparation method and application are obtained.

One of for achieving the above object, the technical solution for the VZV recombination gE- flagellum plain fusion proteins that the present invention uses It is as follows：The fusion protein includes at least the N-terminal conserved region of flagellum fibroin, varicella virus (VZV) glycoprotein gE With the C-terminal conserved region of flagellum fibroin, wherein：

The N-terminal of the fusion protein is the N-terminal of the N-terminal conserved region of flagellum fibroin, and the C-terminal of the fusion protein is flagellum The C-terminal of the C-terminal conserved region of fibroin；

The amino acid sequence of the N-terminal conserved region of the flagellum fibroin such as sequence table SEQ ID NO:Shown in 4, flagellin egg The amino acid sequence of white C-terminal conserved region such as sequence table SEQ ID NO:Shown in 5, varicella virus (VZV) sugar The amino acid sequence of albumen gE such as sequence table SEQ ID NO:Shown in 2.

The main fusion form of above-mentioned fusion protein is that the N-terminal of the N-terminal conserved region of flagellum fibroin is the fusion egg White N-terminal, the C-terminal of the C-terminal conserved region of flagellum fibroin are the C-terminal of the fusion protein, and the wherein N-terminal of flagellum fibroin is conservative The C-terminal in area is connected with the N-terminal of the varicella virus (VZV) glycoprotein gE, the N of the C-terminal conserved region of flagellum fibroin End is connected with the C-terminal of the varicella virus (VZV) glycoprotein gE.

Preferably, the N-terminal of the N-terminal conserved region of flagellum fibroin is the N-terminal of the fusion protein, the C-terminal of flagellum fibroin The C-terminal of conserved region is the C-terminal of the fusion protein, and varicella virus (VZV) glycoprotein gE connects whip by connecting peptide The hair N-terminal conserved region of fibroin and the C-terminal conserved region of flagellum fibroin.

As a kind of preferable embodiment, fusion protein N-terminal conserved region and varicella virus (VZV) Glycoprotein gE is by connecting peptide I connections, and C-terminal conserved region is with varicella virus (VZV) glycoprotein gE by connecting peptide II Connection, the amino acid sequence such as sequence table SEQ ID NO of the fusion protein:Shown in 11.

Preferably, the amino acid sequence such as sequence table SEQ ID NO of connection peptide I:Shown in 7.The gene sequence of expression connection peptide I Row such as sequence table SEQ ID NO:Shown in 6.

Preferably, the amino acid sequence such as sequence table SEQ ID NO of connection peptide II:Shown in 9.The gene of expression connection peptide II Sequence such as sequence table SEQ ID NO:Shown in 8.

Preferably, expression such as sequence table SEQ ID NO:The nucleotide sequence of fusion protein shown in 11 such as sequence table SEQ ID NO:Shown in 10.

Preferably, the nucleotide sequence such as sequence table SEQ ID NO of complete sequence flagellum fibroin are encoded：Shown in 3.

Preferably, the nucleic acid expression vector for expressing the fusion protein nucleotide sequence is plasmid pET28a.

Preferably, the expression bacterial strain for expressing the plasmid is BL21.

Second object of the present invention is to provide a kind of preparation method of VZV recombinations gE- flagellum plain fusion proteins, system Preparation Method includes the following steps：

S1. design primer expands the primer sequence such as SEQ ID NO of VZV glycoprotein gE gene sequences：5 and SEQ ID NO：Shown in 6, the primer sequence such as SEQ ID NO of amplified band histidine tag VZV glycoprotein gE gene sequences：7 and SEQ ID NO：Shown in 8, the primer sequence such as SEQ ID NO of gE- flagellin fusion gene sequences are expanded：9 and SEQ ID NO：Shown in 10, The primer row such as SEQ ID NO of the gE- flagellin fusion gene sequences of amplified band histidine tag：11 and SEQ ID NO：12 institutes Show, expands VZV glycoprotein gE genes sequence, salmonella flagellin protein gene segment respectively, be used in combination fusion DNA vaccine method in base HIS labels, which are added, after target gene splicing because of end builds pET28a carriers；

S2. the pET28a-gE- flagellins of structure are converted into expression bacterial strain BL21, IPTG inductions obtain fusion egg In vain, thalline is collected；

S3. largely induction pET28a-gE- flagellins/BL21, broken collect are precipitated, are resuspended with inclusion body cleaning solution, from The heart；

S4. precipitation, fusion protein gE- flagellins Ni is resuspended in lysis buffer²⁺Column denaturation, renaturation and purifying, obtain pure GE- flagellum fibroins after change.

Preferably, S3 is specially：

In centrifugation after precipitation inclusion body cleaning solution I resuspensions；Precipitation is resuspended with inclusion body cleaning solution II again, after refrigeration is placed Centrifugation；Precipitation is resuspended with inclusion body cleaning solution II I, is centrifuged after being placed at room temperature for；Precipitation is resuspended with Lysis buffer.

It is highly preferred that the inclusion body cleaning solution I used in S3 steps be specially 50mM Tris-HCl, 150mM NaCl, 5mM EDTA and 1mM PMSF；Precipitation is specially again 2M urea with inclusion body cleaning solution II；Precipitation inclusion body cleaning solution II I tools Body is 50mM Tris-HCl, 0.5%Triton X-100 (v/v), 5mM EDTA and 1mM PMSF；Lysis buffer is specially Lysis buffer are specially 100mM NaH2PO4,10mM Tris-HCl and 8M urea, pH8.0.

The concrete operations implementation of above-mentioned experimental procedure can refer to prior art progress, no longer be repeated herein.

Third object of the present invention is to provide a kind of immune composition, and the immune composition includes claim 1 The fusion protein.

Fourth object of the present invention is to provide a kind of VZV vaccine, which includes the above-mentioned VZV recombination gE- whips of power Hair plain fusion protein.

The 5th of the present invention is designed to provide a kind of application of VZV recombinations gE- flagellum plain fusion proteins, the fusion Albumen is used to prepare the drug of immune response of the induction for VZV antigens.

Preferably, the fusion protein is used to prepare the drug that protection individual prevents VZV infection.

Preferably, the fusion protein in the present invention can be used for preparing the medicine for having infected VZV individuals.

As a preferred embodiment, being used to prepare the medicine for having infected VZV human bodies.

Fusion protein in the present invention has the immunogenicity of VZV viruses, can be used as prevention and treatment is each caused by VZV Para-infectious antigenicity substance is not limited to the content described in above application, other aspects can refer to the prior art, not do herein It repeats.

Compared with prior art, the present invention is expressed using pronucleus, successfully induces fusion protein gE- flagellins, SDS-PAGE and Western blot identification fusion protein gE- flagellins are mainly inclusion body expression, the success after denaturation, renaturation It is purified into destination protein.GE albumen can cause before VZV recombinations gE- flagellums plain fusion protein provided by the invention more individually recombinates Higher antibody titer, therefore there is preferable immunocompetence.In flagellin protein activation body adjuvant can be played after TLR5 accesses Effect so that the fusion protein has both the immunogenicity and adjuvanticity of gE albumen, can and body fluid immune with activated cell exempt from Epidemic disease, and without the risk for causing latent infection, good amynologic basis has been established for the preparation of novel varicella protein vaccine.

Description of the drawings

Fig. 1 is the PCR amplification figure of VZV recombinations gE- flagellum plain fusion proteins provided by the invention.

Fig. 2 is the double digestion amplification figure of VZV recombinations gE- flagellum plain fusion proteins provided by the invention.

Fig. 3 is the SDS-PAGE electrophoresis of VZV recombinations gE- flagellum plain fusion proteins provided by the invention.

Fig. 4 is the Western blot result figures of VZV recombinations gE- flagellum plain fusion proteins provided by the invention.

Fig. 5 is VZV recombinations gE- flagellin fusion protein purification figures provided by the invention.

Fig. 6 is the VZV recombinations gE- flagellums plain fusion protein anti-His Tag monoclonal antibodies of mouse provided by the invention Western blot qualification results.

Specific implementation mode

Further detailed to a kind of VZV recombinations gE- flagellum plain fusion proteins work provided by the invention with reference to embodiment, Completely illustrate.The embodiments described below is exemplary, and is only used for explaining the present invention, and should not be understood as to the present invention's Limitation.

Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples It tests material unless otherwise specified, is that market is commercially available.

The reagent and bacterial strain used in the present embodiment is as follows：

Max DNA PolymeragE- flagellins, DL5000 are purchased from takara；

Restriction enzyme NcoI/XhoI is purchased from New England Biolabs；

The used DNA glue recycling of experiment, plasmid be small to be carried, product QIAquick Gel Extraction Kit is purchased from Beijing Tiangeng；

The anti-His Tag of mouse and sheep anti-mouse igg are purchased from the green skies；

The relevant peptone of culture medium, yeast extract and Agar power are purchased from OXIOD；

Relevant antibiotic is purchased from Shanghai life work；

Protein electrophoresis Marker and Ago-Gel are purchased from GENVIEW；

Related strain in embodiment is that this laboratory preserves.

Embodiment 1

1. the design and synthesis of primer

According to Oka plants of VZV glycoprotein gE in NCBI and salmonella flagellin protein gene sequence, it is designed optimization, It selects the sequence of flagellin DNA conservative regions and blocking and splicing for catenation sequence, on structure to pET28a carriers, is named as PET28a-gE- flagellins.

The sequence being related to is as follows：

GE protein DNA sequences such as sequence table SEQ ID NO：It is specific as follows shown in 1：

ATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGT ATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTC CGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGT ACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGG GTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCT TGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAAC GTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAA GAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTT GCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGCTTAAAACATACAACATGCTTTCAAG ACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGT AAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGA GATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCT CCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCA GTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTT TAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCA TCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCAT ATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACA TGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGG GCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCAT GTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCC AACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTATAC GA

GE protein amino acid sequences such as sequence table SEQ ID NO：It is specific as follows shown in 2：

MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGE SSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKI VNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKH TTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYI WNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGA

Salmonella flagellin protein gene sequence such as sequence table SEQ ID NO：It is specific as follows shown in 3：

ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCC GCACTGGGCACTGCTATCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGC GATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTG CGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCG AATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGT ATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACG ACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGAT GCCTACACCCCGAAAGAAACTGCTGTAACCGTTGATAAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCA GAGCAATACTGATATCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATG GTCAATACTATTTAGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAA GTTAATATTGATACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAAC CCACAACCAAATTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGG TTACTGGCGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGT GGCTATGCAGTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAAC CACTACTTATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAG TTGTTACTGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTTGACAAACATAACTTCAGAACAGGCGGTGAGCTT AAAGAGGTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCG TTCTGACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTT CTGCCCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAG GCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTA

The amino acid sequence of the N-terminal conserved region of flagellum fibroin such as sequence table SEQ ID NO：Shown in 4, specially：

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNAN DGISIAQTTEGALNEINNNLQRVRELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTI QVGANDGETIDIDLKEISSKTLGLDKLNVQDAYT

The amino acid sequence of the C-terminal conserved region of flagellum fibroin such as sequence table SEQ ID NO：Shown in 5, specially：

HNFRTGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDS DYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR

Expand the primer such as sequence table SEQ ID NO of VZV glycoprotein gE gene sequences：5 and SEQ ID NO：Shown in 6, gE- F sequences are SEQ ID NO：5, gE-R sequences are SEQ ID NO：6：

gE-F：GGAATTCATGGGGACAGTTAATAAACCTGT

gE-R：TTATCGTATAAGTGGTGACGTT

The primer such as sequence table SEQ ID NO of amplified band histidine tag VZV glycoprotein gE gene sequences：7 and SEQ ID NO：Shown in 8, gE-F sequences are SEQ ID NO：7, gE-F-R (HIS) sequences are SEQ ID NO：8：

gE-F：GGAATTCATGGGGACAGTTAATAAACCTGT

gE-F-R(HIS)：

CCGCTCGAGTTAGTGGTGGTGGTGGTGGTGTTATCGTATAAGTGGTGACGTT

Expand the primer such as sequence table SEQ ID NO of gE- flagellin fusion gene sequences：9 and SEQ ID NO：Shown in 10, GE (RH)-F sequences are SEQ ID NO：9, gE (RH)-R sequences are SEQ ID NO：10：

gE(RH)-F：GGAATTCATGGCACAAGTCATTAATACA

gE(RH)-R：CCGCTCGAG TTA ACGCAGTAAAGAGAGGACGTTTTGC

The primer such as sequence table SEQ ID NO of the gE- flagellin fusion gene sequences of amplified band histidine tag：11 Hes SEQ ID NO：Shown in 12, gE-F- (RH)-F sequences are SEQ ID NO：11, gE-F- (RH)-R (HIS) SEQ ID NO：12：

gE-F-(RH)-F：GGAATTCATGGCACAAGTCATTAATACA

gE-F-(RH)-R(HIS)：CCGCTCGAG TTAGTGGTGGTGGTGGTGGTGACG

By the above primer pair, VZV glycoprotein gE genes sequence and gE- flagellin fusion gene sequences are expanded, fusion is used in combination PCR method adds HIS labels in gene end, then builds pET28a carriers.

The DNA sequence dna of Fusion albumen after fusion such as sequence table SEQ ID NO：It is specific as follows shown in 10：

ATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCC GCACTGGGCACTGCTATCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGC GATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTG CGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCG AATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGT ATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACG ACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGAT GCCTACACCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTAT GGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATC CGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAG CCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAA CTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCACGGGGTGTATAATC AGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGAT ACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGG TGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACC CGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTA ACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGCTTAAAACATACAACATGCTTTCAAGACGTGGTGGT GGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAG CGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCG GGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTAC GTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTC AACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCA ATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTAC ATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCG GTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAAC TACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTT AAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCG TAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGT CAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTATACGAGGTGGTGG TGGTTCTGGTGGTGGTGGTTCTGGATCCCATAACTTCAGAACAGGCGGTGAGCTTAAAGAGGTTAATACAGATAAGA CTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTGACCTGGGTGCGGTTCAG AACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGCCCGTAGCCGTATCGAAGA TTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGC AGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT

The protein sequence that fusion protein DNA sequence dna gives expression to such as sequence table SEQ ID NO：Shown in 11, protein structure is specific For the flagellin conservative region-protein domains connection peptide-gE-connection peptide-flagellin conservative region-His labels.Specifically such as Under：

MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNAN DGISIAQTTEGALNEINNNLQRVRELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTI QVGANDGETIDIDLKEISSKTLGLDKLNVQDAYTGGGGSGGGGSASGGGGSGGGGSMGTVNKPVVGVLMGFGIITGT LRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEH HGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVS VEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRF QGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPT PAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCL SHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFN GHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLIRGGGGSGGGGSGSHNFRTGGELKE VNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDSDYATEVSNMSRAQILQQAG TSVLAQANQVPQNVLSLLRHHHHHH

Wherein underscore part is the gene order for connecting peptide and the amino acid sequence of expression.

It is sequenced after the fusion of synthesis is identified with PCR and digestion.According to synthesis fusion gene sequence design primer, expand The fusion of gaining.After fusion pET28a-gE- flagellins are cut with NcoI/XhoI is bis-, seen through 1% agarose electrophoresis It examines.Agarose electrophoresis result is as shown in Figures 1 and 2, and Fig. 1 is PCR amplification electrophoresis result, and Fig. 2 is double digestion electrophoresis result, from electricity It is found that PCR amplification and double digestion obtain expected size, about 2600bp, by the result and original series of sequencing in swimming result It compares, no gene mutation.

2. the expression and purifying of destination protein

Correct pET28a-gE- flagellins will be sequenced to convert into expression bacterial strain BL21.By pET28a-gE- flagellins/ BL21 is seeded to 5ml LB (AMP⁺) in culture medium, 37 DEG C, 220rps shaking table cultures stay overnight；1ml is taken to be forwarded to 100ml LB (AMP⁺) in culture medium, 37 DEG C, 220rps shaking table cultures about 3h, OD be approximately equal to 0.4；The IPTG of the 1mM of final concentration is added, continues 37 DEG C, 220rps shaking table cultures 4h；The bacterium solution of induction is centrifuged into 10min with 10000rpm, collects thalline.

Precipitation is resuspended with the PBS of 30ml, with the method 300W of conventional ultrasonic disruption, work 5s gap 5s, it is total when Long 1h；The bacterium solution of ultrasonic disruption is centrifuged into 10min with 12000rpm, collects precipitation and supernatant respectively；Pass through protein electrophoresis point S fibroin expressions are analysed, using zero load as control group.

3.Western blot detect protein expression

Broken gE- flagellums fibroin will be induced with unloaded, full bacterium, the sequence loading of supernatant precipitation, through 10% After SDS-PAGE protein isolates, electricity is gone on pvdf membrane, with 5% skimmed milk power (5g skimmed milk powers are added in 100mlPBST) in 4 It is closed overnight in DEG C；It is added through 5% skimmed milk power with 1：The anti-HIS of mouse after 2000 dilutions, places in 37 DEG C and is incubated 1h, use PBST Washing 3 times, each 5min；It is added through 5% skimmed milk power with 1：Rabbit anti-mouse igg after 2000 dilutions, places in 37 DEG C and is incubated 1h, It is washed 3 times with PBST, each 5min；It is developed the color with DAB, tap water terminates.

4. fusion protein gE- flagellins renaturation and purifying

A large amount of induced expression bacterium, and broken collect is precipitated.Precipitation inclusion body cleaning solution I (50mM Tris-HCl, 150mM NaCl, 5mM EDTA, 1mM PMSF) be resuspended after in 4 DEG C 6000g centrifuge 15min；Precipitation is again with inclusion body cleaning solution II (2M Urea) it is resuspended, 15min is placed in 4 DEG C, 6000g centrifuges 30min in 4 DEG C；Precipitation inclusion body cleaning solution II I (50mM Tris- HCl, 0.5%Triton X-100 (v/v), 5mM EDTA, 1mM PMSF) be resuspended, be placed at room temperature for 20min, in 4 DEG C 6000g from Heart 15min；Precipitation ddH₂Washing is resuspended in O, and 6000g centrifuges 15min in 4 DEG C；Finally precipitation Lysis buffer (100mM NaH₂PO₄, 10mM Tris-HCl, 8M urea, pH8.0) be resuspended.

By the liquid after Lysis buffer are resuspended, 6000g centrifuges 15min in 4 DEG C, supernatant is removed, with 0.45 μm of filter membrane After filtering renaturation and purifying are lived in purifying：1. 2ml Ni will be taken⁺Filler is in purification column, with 5 times of column volume ddH₂O is washed, then With 5 times of column volume Lysis buffer activation；2. filtered supernatant is added in purification column, 4 DEG C of slow shaking tables adsorb 2h；③ The Lysis buffer for configuring the urea containing 0-7M carry out albumen on-column refolding according to high concentration to low concentration；4. with 5 times of cylinders Product Wash buffer (50mM NaH₂PO₄, 30mM NaCl, 20mM imidazoles, pH8.0) washing；5. finally using Elution buffer(50mM NaH₂PO₄, 30mM NaCl, 250mM imidazoles, pH8.0) elution destination protein；6. SDS-PAGE and Western Blot analyzes albumen.

SDS-PAGE electrophoresis showeds, there is clearly band in 95KD, 117KD in recombinant strains, as shown in figure 3, weight The albumen of group expression matches with prediction albumen size.As shown in figure 4, recombinant protein and Western blot detected magnitudes one It causes, shows fusion protein and exist with inclusion bodies.

5.Western blot detect fusion protein sample after purification

According to the detection method of Western blot in 3.Western blot detection protein expressions, after Purification Sample is detected.

A large amount of induced expression bacterium, through denaturation, renaturation and purifying, after protein sample is eluted with 250mM imidazoles, SDS-PAGE inspections It surveys result and is shown at 95KD and apparent band occur, its purity reaches 85% (Fig. 5) after software analysis.Using the anti-His Tag of mouse Monoclonal antibody Western blot identifications, as a result visible about 95KD have specific purpose band (Fig. 6).

Zoopery detects the antigenicity of VZV recombination gE- flagellum plain fusion proteins

6-8 weeks BALB/C female mice is chosen, is raised at standard animal care center.Using protein quantification kit to pure GE albumen and recombination gE- flagellum plain fusion proteins after change carry out protein quantification, and adjust a concentration of 0.5mg/ml and 0.8mg/ ml.Experiment mice is divided into 6 groups, every group 8, primary, specific immunization ways and experimental result such as 1 institute of table are immunized every two weeks Show, mouse vein blood is taken before being immunized every time, ELISA is utilized to detect Serum Antibody.With the gE albumen and recombination gE- whips of purifying Hair plain fusion protein coating adds the immune serum of different dilutions, after secondary antibody HRP-IgG, ABTS colour developing, warp OD410nm measures neutralizing antibody content, and physiological saline is negative control.

From 1 experimental result of table：To recombinate individual gE albumen as the immune serum neutralizing antibody titers of antigen 3,5 weeks after immune are respectively 1:91,1:102；And gE- flagellums plain fusion protein is recombinated as the immune mouse blood of antigen using VZV Clear neutralizing antibody titers 3,5 weeks respectively 1 after immune:216,1:267；It can be seen that being anti-to recombinate gE- flagellum plain fusion proteins Former immune mouse can obviously generate the neutralizing antibody of more high titre.

Table 1：Mouse experiment antibody titer (GMT)

	Individual gE albumen	Recombinate gE- flagellum plain fusion proteins	Negative control
				It is 3 weeks immune	1：91	1：216	0
It is 5 weeks immune	1：102	1：267	0

To sum up, recombination gE- flagellum plain fusion proteins provided by the invention, which can be used for preparing, prevents or treats live body due to sense Contaminate the drug of body reaction caused by varicella virus, especially human body.

It is it is necessary to described herein finally：Above example is served only for making technical scheme of the present invention further detailed Ground illustrates, should not be understood as limiting the scope of the invention, those skilled in the art's the above according to the present invention Some the nonessential modifications and adaptations made all belong to the scope of protection of the present invention.

SEQUENCE LISTING

<110>The Wuhan bio tech ltd Bo Wo

<120>VZV recombinates gE- flagellum plain fusion proteins and its preparation method and application

<130> 2017

<160> 19

<170> PatentIn version 3.5

<210> 1

<211> 1611

<212> DNA

<213> Unknown

<220>

<223>VZV glycoprotein gE gene sequences

<400> 1

atggggacag ttaataaacc tgtggtgggg gtattgatgg ggttcggaat tatcacggga 60

acgttgcgta taacgaatcc ggtcagagca tccgtcttgc gatacgatga ttttcacatc 120

gatgaagaca aactggatac aaactccgta tatgagcctt actaccattc agatcatgcg 180

gagtcttcat gggtaaatcg gggagagtct tcgcgaaaag cgtacgatca taactcacct 240

tatatatggc cacgtaatga ttatgatgga tttttagaga acgcacacga acaccatggg 300

gtgtataatc agggccgtgg tatcgatagc ggggaacggt taatgcaacc cacacaaatg 360

tctgcacagg aggatcttgg ggacgatacg ggcatccacg ttatccctac gttaaacggc 420

gatgacagac ataaaattgt aaatgtggac caacgtcaat acggtgacgt gtttaaagga 480

gatcttaatc caaaacccca aggccaaaga ctcattgagg tgtcagtgga agaaaatcac 540

ccgtttactt tacgcgcacc gattcagcgg atttatggag tccggtacac cgagacttgg 600

agctttttgc cgtcattaac ctgtacggga gacgcagcgc ccgccatcca gcatatatgc 660

ttaaaacata caacatgctt tcaagacgtg gtggtggatg tggattgcgc ggaaaatact 720

aaagaggatc agttggccga aatcagttac cgttttcaag gtaagaagga agcggaccaa 780

ccgtggattg ttgtaaacac gagcacactg tttgatgaac tcgaattaga cccccccgag 840

attgaaccgg gtgtcttgaa agtacttcgg acagaaaaac aatacttggg tgtgtacatt 900

tggaacatgc gcggctccga tggtacgtct acctacgcca cgtttttggt cacctggaaa 960

ggggatgaaa aaacaagaaa ccctacgccc gcagtaactc ctcaaccaag aggggctgag 1020

tttcatatgt ggaattacca ctcgcatgta ttttcagttg gtgatacgtt tagcttggca 1080

atgcatcttc agtataagat acatgaagcg ccatttgatt tgctgttaga gtggttgtat 1140

gtccccatcg atcctacatg tcaaccaatg cggttatatt ctacgtgttt gtatcatccc 1200

aacgcacccc aatgcctctc tcatatgaat tccggttgta catttacctc gccacattta 1260

gcccagcgtg ttgcaagcac agtgtatcaa aattgtgaac atgcagataa ctacaccgca 1320

tattgtctgg gaatatctca tatggagcct agctttggtc taatcttaca cgacgggggc 1380

accacgttaa agtttgtaga tacacccgag agtttgtcgg gattatacgt ttttgtggtg 1440

tattttaacg ggcatgttga agccgtagca tacactgttg tatccacagt agatcatttt 1500

gtaaacgcaa ttgaagagcg tggatttccg ccaacggccg gtcagccacc ggcgactact 1560

aaacccaagg aaattacccc cgtaaacccc ggaacgtcac cacttatacg a 1611

<210> 2

<211> 537

<212> Protein

<213> Unknown

<220>

<223>VZV glycoprotein gE amino acid sequences

<400> 2

mgtvnkpvvg vlmgfgiitg tlritnpvra svlryddfhi dedkldtnsv yepyyhsdha 60

esswvnrges srkaydhnsp yiwprndydg flenahehhg vynqgrgids gerlmqptqm 120

saqedlgddt gihviptlng ddrhkivnvd qrqygdvfkg dlnpkpqgqr lievsveenh 180

pftlrapiqr iygvrytetw sflpsltctg daapaiqhic lkhttcfqdv vvdvdcaent 240

kedqlaeisy rfqgkkeadq pwivvntstl fdeleldppe iepgvlkvlr tekqylgvyi 300

wnmrgsdgts tyatflvtwk gdektrnptp avtpqprgae fhmwnyhshv fsvgdtfsla 360

mhlqykihea pfdlllewly vpidptcqpm rlystclyhp napqclshmn sgctftsphl 420

aqrvastvyq ncehadnyta yclgishmep sfglilhdgg ttlkfvdtpe slsglyvfvv 480

yfnghveava ytvvstvdhf vnaieergfp ptagqppatt kpkeitpvnp gtsplir 537

<210> 3

<211> 1520

<212> DNA

<213> Salmonella

<220>

<223>Salmonella flagellin protein gene sequence

<400> 3

atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60

tcccagtccg cactgggcac tgctatcgag cgtttgtctt ccggtctgcg tatcaacagc 120

gcgaaagacg atgcggcagg acaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180

ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240

gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgcg 300

aatggtacta actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360

aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420

gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tattgattta 480

aaagaaatca gctctaaaac actgggactt gataagctta atgtccaaga tgcctacacc 540

ccgaaagaaa ctgctgtaac cgttgataaa actacctata aaaatggtac agatcctatt 600

acagcccaga gcaatactga tatccaaact gcaattggcg gtggtgcaac gggggttact 660

ggggctgata tcaaatttaa agatggtcaa tactatttag atgttaaagg cggtgcttct 720

gctggtgttt ataaagccac ttatgatgaa actacaaaga aagttaatat tgatacgact 780

gataaaactc cgttggcaac tgcggaagct acagctattc ggggaacggc cactataacc 840

cacaaccaaa ttgctgaagt aacaaaagag ggtgttgata cgaccacagt tgcggctcaa 900

cttgctgcag caggggttac tggcgccgat aaggacaata ctagccttgt aaaactatcg 960

tttgaggata aaaacggtaa ggttattgat ggtggctatg cagtgaaaat gggcgacgat 1020

ttctatgccg ctacatatga tgagaaaaca ggtgcaatta ctgctaaaac cactacttat 1080

acagatggta ctggcgttgc tcaaactgga gctgtgaaat ttggtggcgc aaatggtaaa 1140

tctgaagttg ttactgctac cgatggtaag acttacttag caagcgacct tgacaaacat 1200

aacttcagaa caggcggtga gcttaaagag gttaatacag ataagactga aaacccactg 1260

cagaaaattg atgctgcctt ggcacaggtt gatacacttc gttctgacct gggtgcggtt 1320

cagaaccgtt tcaactccgc tatcaccaac ctgggcaata ccgtaaataa cctgtcttct 1380

gcccgtagcc gtatcgaaga ttccgactac gcaaccgaag tctccaacat gtctcgcgcg 1440

cagattctgc agcaggccgg tacctccgtt ctggcgcagg cgaaccaggt tccgcaaaac 1500

gtcctctctt tactgcgtta 1520

<210> 4

<211> 180

<212> Protein

<213> Unknown

<220>

<223>Salmonella flagellin albumen n end guards region amino acid sequence

<400> 4

maqvintnsl slltqnnlnk sqsalgtaie rlssglrins akddaagqai anrftanikg 60

ltqasrnand gisiaqtteg alneinnnlq rvrelavqsa ngtnsqsdld siqaeitqrl 120

neidrvsgqt qfngvkvlaq dntltiqvga ndgetididl keissktlgl dklnvqdayt 180

<210> 5

<211> 107

<212> Protein

<213> Unknown

<220>

<223>Guard region amino acid sequence in salmonella flagellin PROTEIN C end

<400> 5

hnfrtggelk evntdktenp lqkidaalaq vdtlrsdlga vqnrfnsait nlgntvnnls 60

sarsriedsd yatevsnmsr aqilqqagts vlaqanqvpq nvlsllr 107

<210> 6

<211> 66

<212> DNA

<213> Unknown

<220>

<223>Connect peptide I gene sequence

<400> 6

ggtggtggtg gttctggtgg tggtggttct gctagcggtg gtggtggttc tggtggtggt 60

ggttct 66

<210> 7

<211> 22

<212> Protein

<213> Unknown

<220>

<223>Connect peptide I amino acid sequences

<400> 7

ggggsggggs asggggsggg gs 22

<210> 8

<211> 36

<212> DNA

<213> Unknown

<220>

<223>Connect peptide II gene orders

<400> 8

ggtggtggtg gttctggtgg tggtggttct ggatcc 36

<210> 9

<211> 12

<212> Protein

<213> Unknown

<220>

<223>Connect peptide II amino acid sequences

<400> 9

ggggsggggs gs 12

<210> 10

<211> 2574

<212> DNA

<213> Unknown

<220>

<223>Antigen-4 fusion protein gene sequence

<400> 10

atggcacaag tcattaatac aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60

tcccagtccg cactgggcac tgctatcgag cgtttgtctt ccggtctgcg tatcaacagc 120

gcgaaagacg atgcggcagg acaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180

ctgactcagg cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc 240

gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt tcagtctgcg 300

aatggtacta actcccagtc tgacctcgac tccatccagg ctgaaatcac ccagcgcctg 360

aacgaaatcg accgtgtatc cggccagact cagttcaacg gcgtgaaagt cctggcgcag 420

gacaacaccc tgaccatcca ggttggtgcc aacgacggtg aaactatcga tattgattta 480

aaagaaatca gctctaaaac actgggactt gataagctta atgtccaaga tgcctacacc 540

ggtggtggtg gttctggtgg tggtggttct gctagcggtg gtggtggttc tggtggtggt 600

ggttctatgg ggacagttaa taaacctgtg gtgggggtat tgatggggtt cggaattatc 660

acgggaacgt tgcgtataac gaatccggtc agagcatccg tcttgcgata cgatgatttt 720

cacatcgatg aagacaaact ggatacaaac tccgtatatg agccttacta ccattcagat 780

catgcggagt cttcatgggt aaatcgggga gagtcttcgc gaaaagcgta cgatcataac 840

tcaccttata tatggccacg taatgattat gatggatttt tagagaacgc acacgaacac 900

cacggggtgt ataatcaggg ccgtggtatc gatagcgggg aacggttaat gcaacccaca 960

caaatgtctg cacaggagga tcttggggac gatacgggca tccacgttat ccctacgtta 1020

aacggcgatg acagacataa aattgtaaat gtggaccaac gtcaatacgg tgacgtgttt 1080

aaaggagatc ttaatccaaa accccaaggc caaagactca ttgaggtgtc agtggaagaa 1140

aatcacccgt ttactttacg cgcaccgatt cagcggattt atggagtccg gtacaccgag 1200

acttggagct ttttgccgtc attaacctgt acgggagacg cagcgcccgc catccagcat 1260

atatgcttaa aacatacaac atgctttcaa gacgtggtgg tggatgtgga ttgcgcggaa 1320

aatactaaag aggatcagtt ggccgaaatc agttaccgtt ttcaaggtaa gaaggaagcg 1380

gaccaaccgt ggattgttgt aaacacgagc acactgtttg atgaactcga attagacccc 1440

cccgagattg aaccgggtgt cttgaaagta cttcggacag aaaaacaata cttgggtgtg 1500

tacatttgga acatgcgcgg ctccgatggt acgtctacct acgccacgtt tttggtcacc 1560

tggaaagggg atgaaaaaac aagaaaccct acgcccgcag taactcctca accaagaggg 1620

gctgagtttc atatgtggaa ttaccactcg catgtatttt cagttggtga tacgtttagc 1680

ttggcaatgc atcttcagta taagatacat gaagcgccat ttgatttgct gttagagtgg 1740

ttgtatgtcc ccatcgatcc tacatgtcaa ccaatgcggt tatattctac gtgtttgtat 1800

catcccaacg caccccaatg cctctctcat atgaattccg gttgtacatt tacctcgcca 1860

catttagccc agcgtgttgc aagcacagtg tatcaaaatt gtgaacatgc agataactac 1920

accgcatatt gtctgggaat atctcatatg gagcctagct ttggtctaat cttacacgac 1980

gggggcacca cgttaaagtt tgtagataca cccgagagtt tgtcgggatt atacgttttt 2040

gtggtgtatt ttaacgggca tgttgaagcc gtagcataca ctgttgtatc cacagtagat 2100

cattttgtaa acgcaattga agagcgtgga tttccgccaa cggccggtca gccaccggcg 2160

actactaaac ccaaggaaat tacccccgta aaccccggaa cgtcaccact tatacgaggt 2220

ggtggtggtt ctggtggtgg tggttctgga tcccataact tcagaacagg cggtgagctt 2280

aaagaggtta atacagataa gactgaaaac ccactgcaga aaattgatgc tgccttggca 2340

caggttgata cacttcgttc tgacctgggt gcggttcaga accgtttcaa ctccgctatc 2400

accaacctgg gcaataccgt aaataacctg tcttctgccc gtagccgtat cgaagattcc 2460

gactacgcaa ccgaagtctc caacatgtct cgcgcgcaga ttctgcagca ggccggtacc 2520

tccgttctgg cgcaggcgaa ccaggttccg caaaacgtcc tctctttact gcgt 2574

<210> 11

<211> 864

<212> Protein

<213> Unknown

<220>

<223>Fusion protein amino acid sequence

<400> 11

maqvintnsl slltqnnlnk sqsalgtaie rlssglrins akddaagqai anrftanikg 60

ltqasrnand gisiaqtteg alneinnnlq rvrelavqsa ngtnsqsdld siqaeitqrl 120

neidrvsgqt qfngvkvlaq dntltiqvga ndgetididl keissktlgl dklnvqdayt 180

ggggsggggs asggggsggg gsmgtvnkpv vgvlmgfgii tgtlritnpv rasvlryddf 240

hidedkldtn svyepyyhsd haesswvnrg essrkaydhn spyiwprndy dgflenaheh 300

hgvynqgrgi dsgerlmqpt qmsaqedlgd dtgihviptl ngddrhkivn vdqrqygdvf 360

kgdlnpkpqg qrlievsvee nhpftlrapi qriygvryte twsflpsltc tgdaapaiqh 420

iclkhttcfq dvvvdvdcae ntkedqlaei syrfqgkkea dqpwivvnts tlfdeleldp 480

peiepgvlkv lrtekqylgv yiwnmrgsdg tstyatflvt wkgdektrnp tpavtpqprg 540

aefhmwnyhs hvfsvgdtfs lamhlqykih eapfdlllew lyvpidptcq pmrlystcly 600

hpnapqclsh mnsgctftsp hlaqrvastv yqncehadny tayclgishm epsfglilhd 660

ggttlkfvdt peslsglyvf vvyfnghvea vaytvvstvd hfvnaieerg fpptagqppa 720

ttkpkeitpv npgtsplirg gggsggggsg shnfrtggel kevntdkten plqkidaala 780

qvdtlrsdlg avqnrfnsai tnlgntvnnl ssarsrieds dyatevsnms raqilqqagt 840

svlaqanqvp qnvlsllrhh hhhh 864

<210> 12

<211> 30

<212> DNA

<213> Unknown

<220>

<223> gE-F

<400> 12

ggaattcatg gggacagtta ataaacctgt 30

<210> 13

<211> 22

<212> DNA

<213> Unknown

<220>

<223> gE-R

<400> 13

ttatcgtata agtggtgacg tt 22

<210> 14

<211> 30

<212> DNA

<213> Unknown

<220>

<223> gE-F

<400> 14

ggaattcatg gggacagtta ataaacctgt 30

<210> 15

<211>

<212> DNA

<213> Unknown

<220>

<223> gE-F-R（HIS）

<400> 15

ccgctcgagt tagtggtggt ggtggtggtg ttatcgtata agtggtgacg tt 52

<210> 16

<211> 28

<212> DNA

<213> Unknown

<220>

<223> gE-F-（RH）-F

<400> 16

ggaattcatg gcacaagtca ttaataca 28

<210> 17

<211> 37

<212> DNA

<213> Unknown

<220>

<223> gE-（RH）-R

<400> 17

ccgctcgagt taacgcagta aagagaggac gttttgc 37

<210> 18

<211> 28

<212> DNA

<213> Unknown

<220>

<223> gE-F-（RH）-F

<400> 18

ggaattcatg gcacaagtca ttaataca 28

<210> 19

<211> 33

<212> DNA

<213> Unknown

<220>

<223> gE-F-（RH）-R（HIS）

<400> 19

ccgctcgagt tagtggtggt ggtggtggtg acg 33

Claims

1. a kind of VZV recombinates gE- flagellum plain fusion proteins, it is characterised in that：The fusion protein includes at least flagellum fibroin N-terminal conserved region, varicella virus (VZV) glycoprotein gE and flagellum fibroin C-terminal conserved region, wherein：

The N-terminal of the fusion protein is the N-terminal of the N-terminal conserved region of flagellum fibroin, and the C-terminal of the fusion protein is flagellin egg The C-terminal of white C-terminal conserved region；

The amino acid sequence of the N-terminal conserved region of the flagellum fibroin such as sequence table SEQ ID NO:Shown in 4, flagellum fibroin The amino acid sequence of C-terminal conserved region such as sequence table SEQ ID NO:Shown in 5, varicella virus (VZV) glycoprotein The amino acid sequence of gE such as sequence table SEQ ID NO:Shown in 2.

2. VZV as described in claim 1 recombinates gE- flagellum plain fusion proteins, it is characterised in that：Encode the varicella The nucleotide sequence such as sequence table SEQ ID NO of herpesviral (VZV) glycoprotein gE：Shown in 1.

3. VZV as described in claim 1 recombinates gE- flagellum plain fusion proteins, it is characterised in that：The N-terminal of flagellum fibroin is protected The N-terminal of defending zone is the N-terminal of the fusion protein, and the C-terminal of the C-terminal conserved region of flagellum fibroin is the C-terminal of the fusion protein, water Acne-herpes zoster virus (VZV) glycoprotein gE connects N-terminal conserved region and the flagellum fibroin of flagellum fibroin by connecting peptide C-terminal conserved region.

4. VZV as claimed in claim 3 recombinates gE- flagellum plain fusion proteins, it is characterised in that：The fusion protein N-terminal is protected Defending zone is with varicella virus (VZV) glycoprotein gE by connecting peptide I connections, C-terminal conserved region and varicella-zoster Virus (VZV) glycoprotein gE is by connecting peptide II connections, the amino acid sequence such as sequence table SEQ ID NO of the fusion protein: Shown in 11.

5. VZV as claimed in claim 4 recombinates gE- flagellum plain fusion proteins, it is characterised in that：Expression such as sequence table SEQ ID NO:The nucleotide sequence of fusion protein shown in 11 such as sequence table SEQ ID NO:Shown in 10.

6. preparing the preparation method for recombinating gE- flagellum plain fusion proteins according to any VZV of Claims 1 to 5, feature It is, includes the following steps：

S1, design primer expand the primer sequence such as SEQ ID NO of VZV glycoprotein gE gene sequences：12 and SEQ ID NO：13 It is shown, the primer sequence such as SEQ ID NO of amplified band histidine tag VZV glycoprotein gE gene sequences：14 and SEQ ID NO： Shown in 15, the primer sequence such as SEQ ID NO of gE- flagellin fusion gene sequences are expanded：16 and SEQ ID NO：Shown in 17, expand Increase the primer row such as SEQ ID NO of the gE- flagellin fusion gene sequences with histidine tag：18 and SEQ ID NO：19 institutes Show, expands VZV glycoprotein gE genes sequence, salmonella flagellin protein gene segment respectively, be used in combination fusion DNA vaccine method in base HIS labels, which are added, after target gene splicing because of end builds pET28a carriers；

S2, the pET28a-gE- flagellins of structure are converted into expression bacterial strain BL21, IPTG inductions obtain fusion protein, receive Collect thalline；

S3, a large amount of induction pET28a-gE- flagellins/BL21, are crushed and collect precipitation, be resuspended with inclusion body cleaning solution, centrifugation；

Precipitation is resuspended in S4, lysis buffer, and fusion protein gE- flagellins obtain after purification after denaturation, renaturation and purification step GE- flagellum fibroins.

7. the preparation method of VZV recombinations gE- flagellum plain fusion proteins according to claim 6, which is characterized in that S3 is specific For：In centrifugation after precipitation inclusion body cleaning solution I resuspensions；Precipitation is resuspended with inclusion body cleaning solution II again, and refrigeration centrifuges after placing； Precipitation is resuspended with inclusion body cleaning solution II I, is centrifuged after being placed at room temperature for；Precipitation is resuspended with Lysis buffer.

8. the preparation method of VZV recombinations gE- flagellum plain fusion proteins according to claim 6, it is characterised in that：S3 steps In inclusion body cleaning solution I be specially 50mM Tris-HCl, 150mM NaCl, 5mM EDTA and 1mM PMSF；Precipitation is again with packet It is specially 2M urea to contain body cleaning solution II；Precipitation is specially 50mM Tris-HCl, 0.5%Triton with inclusion body cleaning solution II I X-100 (v/v), 5mM EDTA and 1mM PMSF；Lysis buffer is specially that Lysis buffer are specially 100mM NaH2PO4,10mM Tris-HCl and 8M urea, pH8.0.

9. a kind of immune composition, it is characterised in that：The immune composition includes any fusion of claim 1~8 Albumen.

10. a kind of VZV vaccine, it is characterised in that：Including any VZV recombination gE- flagellins fusions of claim 1~9 Albumen.