CN107188935B - A kind of zika virus NS1 antigenic mutants and its application - Google Patents
A kind of zika virus NS1 antigenic mutants and its application Download PDFInfo
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Abstract
A kind of zika virus NS1 antigenic mutants of present invention offer and its application, pass through the comparison analysis to four type NS1 antigen amino acid sequences of zika virus NS1 antigens and dengue virus, find out the high part of homology, carry out the knockout mutations of gene level, gene chemical synthesis, vector construction, prokaryotic expression, antigen purification and immunologic function analysis, screening neither influences the recall rate that the sensitivity that stockaded village's card detects can significantly reduce Dengue positive sample again, that is, reduces zika virus and intersect with what dengue virus antibody detected.
Description
Technical field
The invention belongs to molecular biology and bioinformatics technique fields, and relate to genetic engineering and it is immunologic in
Hold, and in particular, to zika virus antigen and its application.
Background technology
Zika virus category flaviviridae, Flavivirus, single strand plus RNA virus, diameter 20nm, be it is a kind of by mosquito into
The arboviruse that row is propagated, host is indefinite, main raw primate out of office and inhabites mosquito in the tree, such as aedes africanus
Cycle in (Aedes aegypti).The virus is most earlier than nineteen forty-seven accidentally by yellow fever monitoring network in stockaded village of Uganda card clump
It finds in the rhesus macaque of woods, is then found in Uganda and Tanzania crowd in nineteen fifty-two.The viral activity compares always
Concealment, only Africa, America, the Asia-pacific region of surrounding have zika virus to infect Sporadic cases under the line.It is primary earliest
Outbreak of epidemic is to be happened within 2007 the islands Western Pacific Mi Keluoni subgroup Dao Yapu, bigger it is primary be popular in 2013-
It is happened at Oceanian French Polynesia within 2014, has infected about 32000 people.Yellow-fever mosquito also propagates other in flaviviridae
How kind viroid accomplishes the difference detection of two kinds of diseases of stockaded village's card and Dengue, reduces Dengue wherein including just dengue fever virus
The validity of cross-pair zika virus antibody test reagent of the patients serum in the diagnosis of stockaded village's card is of great significance.
The classical symptom of zika virus infection include the low-heat of Acute onset, maculopapule, arthralgia (mainly involve hand,
Sufficient Minor articulus), conjunctivitis, other symptoms include myalgia, headache, eye socket pain and powerless.In addition rare symptom includes abdominal pain, dislikes
The heart, vomiting, mucosa ulcer and pruitus.2013 and 2015 respectively in French Polynesia and Brazilian fork clip epidemic situation phase
Between, it has been reported that zika virus disease is likely to result in nerve and self immune system complication.Brazilian stockaded village's card is broken out within 2015
It is found that the newborn of many microcephaluses (microcephaly) in the groove.Zika virus is transmitted to child, stockaded village via mother
Card fever may have connection with the microcephalus of ewborn infant, can influence nervous system to the infection of adult, and may
There is contact with actue infectious polyradiculoneuritis (Guillain-Barre syndrome).
Zika virus is spherical in shape, and diameter is about 40-70nm, there is coating.ZIKV nucleic acid is that a length is 10,794 bases
Single positive chain RNA, encoding gene sequence be 5 '-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3 '.RNA
It is translated as a polyprotein, is then structural proteins capsid protein (capsid, C), precursor memebrane protein by protease cracking
(precursor membrane, prM), memebrane protein (envelope, E) and 7 non-structural protein (nonstructural
protein,NS).Wherein NS1 is a non-structural protein having multi-functions.Protein structure height in flaviviridae
It is conservative.
Zika virus NS1 albumen is made of three structural areas：N-terminal β-roll, C-terminal β-ladder and pterion (wing
domain).NS1 albumen exists usually in the form of dimer and six aggressiveness.Dimer NS1 is in virus genomic reproduction process
In play an important role.Six aggressiveness NS1 can help the identification of viral escape host immune cell.
Bibliography：
1.Xu X,Song H,Qi J,Liu Y,Wang H,Su C,Shi Y,Gao GF.Contribution of
interwined loop to membrane association revealed by Zika virus full-length
NS1 structure.EMBO J.2016 Oct 17；35(20):2170-2178.
2.Brown WC,Akey DL,Konwerski JR,Tarrasch JT,Skiniotis G,Kuhn RJ,Smith
JL.Extended surface for membrane association in Zika virus NS1 structure.Nat Struct Mol Biol.2016 Sep；23(9):865-7.
3.Rastogi M,Sharma N,Singh SK Flavivirus NS1:a multifaceted enigmatic
viral protein.Virol J.2016 Jul 29；13:131.
4.Akey DL,Brown WC,Jose J,Kuhn RJ,Smith JL.Structure-guided insights
on the role of NS1 in flavivirus infection.Bioessays.2015 May；37(5):489-94.
5.Stettler K,Beltramello M,Espinosa DA,et al.Specificity,cross-
reactivity,and function of antibodies elicited by zika virus
infection.Science.2016 Aug 19；353(6301):823-6.
Invention content
An object of the present invention is：It is compared and analyzed by zika virus and dengue virus NS 1 albumen, to homologous region
Deletion mutation is carried out, then zika virus NS1 mutant sequences are expressed and purified, and then obtains the high stockaded village's card of specificity
Virus N S1 antigenic mutants；
An object of the present invention is：A kind of having applied to stockaded village's card antibody test can significantly reduce Dengue intersection is provided
The zika virus NS1 antigenic mutants of effect；
An object of the present invention is：A kind of having applied to stockaded village's card antibody test can significantly reduce Dengue intersection is provided
The application of stockaded village's card NS1 antigenic mutants of effect.
In order to achieve the object of the present invention, the first aspect of the invention provides a kind of zika virus NS1 antigenic mutations
Body：
Specifically, downloading zika virus nucleotide sequence (KX377335.1) first from Genebank databases.It utilizes
Clustal Omega arrange the amino acid sequence of zika virus NS1 antigens and the dengue virus NS 1 antigen of four serotype
Row compare (Fig. 1), find out very high homology region sequence (DGCWYGMEIRP).Since the sequence is in zika virus and dengue virus
In be highly conserved, therefore plan the region carrying out deletion mutation, and test the specific reaction of the mutant protein.It is described
The amino acid sequence of deletion mutant is as shown in SEQ ID NO.2.The homologous region sequence (DGCWYGMEIRP) is located at C-terminal β-
Ladder (Fig. 2).
The second aspect of the invention is to provide the screening technique of the zika virus NS1 antigens, specially：
The mutant sequence is utilized into wild type control before SWISS-MODEL databases progress Blast search, with mutation
The variation for carrying out three-dimensional conformation compares.By homologous region sequence after confirming the front and back protein three-dimensional structure of mutation without significant changes
Deletion mutation is carried out, expression vector pET24_zNS1, transfection Escherichia coli host BL21 of the structure with histidine tag are utilized
Nickel affinity chromatography purifies expression product, and purified product is carried out dialysis operation, pure with SDS-PAGE electrophoresis detection albumen
Degree, obtains corresponding antigen protein.With indirect ELISA verification is carried out after the Zika NS1 coated elisa plates of purifying, stockaded village is detected
Card and Dengue antibody positive sample.Mutant significantly drops while not reducing stockaded village's card positive sample detection known to testing result
The low detection of Dengue positive sample, meets the set goal.
There is provided the expression vectors of the zika virus antigenic mutant for the third aspect of the invention.The expression carries
Nucleotide sequence shown in SEQ ID NO.4 is attached with pET24 commercial carriers and is obtained by body.
The fourth aspect of the invention is to provide the host of the expression vector containing the zika virus antigenic mutant
Cell.The host cell can be Bacillus coli cells strain BL21 (pET24-zNS1).
The fifth aspect of the invention provides the purification process of the zika virus antigen, will pass through DNA analysis and correspondence
The nucleotide sequence that protein structural analysis confirms carries out PCR amplification synthesis (Fig. 3), and introduces Nde I and Xho I restriction enzyme sites,
It is basic carrier so that plasmid pET24 is commercialized, builds zika virus expression vector, the nucleotide sequence such as SEQ ID NO.4
It is shown, it is purified by affinity chromatography, obtains zika virus NS1 mutains.
Belong to the protection of the present invention containing amino acid sequence biological products of zika virus antigen as shown in SEQ ID NO.2
Range.
Preferably, the biological products are detection reagent.
Belong to the present invention containing amino acid sequence detection reagent of zika virus antigenic mutant as shown in SEQ ID NO.2
Protection domain.
The present invention provides amino acid sequences, and the zika virus antigenic mutant as shown in SEQ ID NO.2 is being prepared for examining
Survey the purposes in the detection kit of zika virus.
Optionally, the kit is ELISA kit or the fast check reagent box of colloidal gold method.
The present invention provides amino acid sequences, and the zika virus antigenic mutant as shown in SEQ ID NO.2 is preparing stockaded village's card disease
Purposes in malicious vaccine.
Above-mentioned zika virus antigenic mutant immunogenicity provided by the invention is good, and high specificity can be used as zika virus
The active princlple of Serology test, additionally it is possible to be used to prepare zika virus vaccine.
Description of the drawings
Fig. 1 is the Amino acid sequences alignment of zika virus NS1 and dengue virus NS 1
Fig. 2 is the tomograph of zika virus NS1 albumen；The structure is dimer.Lack the amino acid sequence deleted
Row DGCWYGMEIRP is indicated by space-filling model.
Fig. 3 is the nucleotide fragments electrophoresis photographs of zika virus NS1 of the present invention.Well M is nucleic acid Marker, molecular weight
It is from top to bottom：3,000bp,2,000bp,1,500bp,1,000bp,800bp,600bp,400bp,300bp,200bp.Sample-adding
Hole 1 is Zika NS1 wild-type sequence amplified productions.Well 2 is Zika NS1 mutant sequences amplified productions.
Fig. 4 is the induced expression of zika virus NS1 wild types and mutant in host cell, 4a. wild-type proteins
Induced expression；The induced expression of 4b. muteins；
Fig. 5 is the protein purification electrophoresis photographs of zika virus NS1 wild types and mutant, 5a wild-type proteins it is pure
Change；The purifying of 5b mutant proteins.S,supernatant；F,flowthrough；Bf,wash down with binding
buffer；Wf,wash down with washing buffer；E,wash down with elution buffer；M,
marker.
Fig. 6 is the baseline results that zika virus antigen protein of the present invention is used for ELISA detections.
Specific implementation mode：
The acquisition and screening of 1 zika virus NS1 antigenic mutants of embodiment
The zika virus nucleotide sequence KX377335.1 downloaded from Genebank databases is analyzed, it will wherein
Amino acid sequence (the UnitProtKB of the amino acid sequence (876-1202) of NS1 antigens and four serotype Dengue NS1 antigens:
P17763, P12823, Q5UB51, Q2YHF0) amino acid alignment is carried out, sequence alignment is carried out by Clustal Omega
Find out homologous region (Fig. 1), carry out sequence (shown in DGCWYGMEIRP, SEQ ID NO.1, the nucleotide sequence such as SEQ of coding
Shown in ID NO.3) mutation removal.The amino acid sequence of the NS1 antigenic mutants of acquisition is as shown in SEQ ID NO.2.To it
Three dimensional analysis is carried out, as shown in Figure 2.
The preparation and purification of 2 zika virus NS1 antigenic mutants of embodiment
Disappeared respectively to amplification oligonucleotide product shown in SEQ ID NO.4 with restriction enzyme Nde I and Xho I
Change, collect target fragment, and be equally attached with the pET24a carriers of restriction enzyme enzymatic treatment, builds recombinant expression plasmid
pET24_zNS1；Transfection Escherichia coli BL21 carries out protein expression, SDS-PAGE electrophoresis is used in combination to determine expression and yield.
1, the plasmid pET24_zNS1 completed will be rebuild and is transferred to BL21 (de3) e. coli strains, spread plate, picking Dan Ke
It is grand in the middle test tube of 5ml LB culture mediums (Kana+, 50 μ g/ml), 37 DEG C of 240rpm are incubated overnight.
2, the bacterial strain that will be incubated overnight is seeded in containing in 300ml LB culture mediums (Kana+, 50 μ g/ml), 37 DEG C
240rpm continues to cultivate, and when waiting for that bacterium solution OD600 reaches 0.6-0.8, IPTG derivants is added and induce 3 hours.
3,4000rpm is centrifuged, and thalline is collected, with 10mm Tris-Hcl pH 8.0, the buffering of 0.5%Triton X-10
Liquid is resuspended according to the ratio of every liter of culture solution of 30ml buffer solutions, and 200W ultrasonic grind instrument smudge cells are used in combination.
The SDS-PAGE protein electrophoresis glue of cell pyrolysis liquid shows (Fig. 4), the big portion of expression product of zika virus NS1 albumen
Part is that supernatant is purified using affinity chromatography after soluble protein cell pyrolysis liquids are centrifuged with 15,000g.Base fluid is adopted when purifying
With 10mM Tris pH8.0 buffer solutions, eluent adds final concentration of 30mm/L, and the imidazole gradient of 60mm/L, 300mm/L are washed
It is de-.Collect the peak value that the imidazole elution of 300mm/L flows through, the purity (Fig. 5) of SDS-page electrophoresis detection destination proteins.
The eluent containing destination protein that affinity chromatography is collected is transferred in semi-permeable membrane, is stirred in PBS buffer solution
It dialysed liquid, and changed liquid twice within second day, and concentration mensuration is carried out to the protein solution after dialysis.With bovine serum albumin(BSA)
(BSA) it is standard, using the content of BIORAD companies quantification of protein reagent (protein assay) colorimetric estimation protein.
Above-mentioned experimental result is as follows：1) E.coli BL21 (DE3) engineering bacteria containing pET24_zNS1 can effective expression purpose
Albumen；2) pass through Ni-NTA affinitive layer purifications, zika virus NS1 purity of protein can reach 95%-98%.
The IgM ELISA methods and kit of 3 antigenic mutants of NS1 containing zika virus of embodiment
Zika virus antigen protein is diluted to 1 μ g/ml, coated elisa plate with the carbonate buffer solution (pH9.6) of 0.05M
(per 200 μ 1 of hole) sets 2 DEG C -8 DEG C overnight.Second day, coating buffer is abandoned, it is primary with 300ml washing lotion board-washings.Confining liquid is added (per hole
300 μ, 1 washing lotions), 2 DEG C -8 DEG C are overnight.Later, confining liquid is abandoned, is dried overnight in stove room of the relative humidity less than 25%.Detect sample
This when, is separately added into the negative controls N1-N8 after dilution, stockaded village card positive sample zika 1-zika 7, Dengue positive sample
Denv 1-denv 8, per 100 μ l of hole, 37 DEG C are reacted 120 minutes.Reaction solution is abandoned, 5 times are washed (per 300 μ l of hole with PBST buffer solutions
Washing lotion).Washing lotion is abandoned, the anti-human IgMHRP100 μ l of mouse are added per hole, 37 DEG C are reacted 20 minutes.Reaction solution is abandoned, with PBST buffer solutions
It washes 5 times (300 μ l washing lotions are added per hole).Washing lotion is abandoned, substrate A liquid, each 50 μ l of B liquid are added per hole, 37 DEG C are protected from light 15 minutes.
100 μ l of terminate liquid are added per hole and terminate reaction.450nm measures each hole A values (absorption value).The present embodiment uses horseradish peroxide
Compound enzyme Color Appearance System, but other Color Appearance Systems can also be used, as alkaline phosphoric acid enzyme system or biotin and Avidin develop the color
System.
This experiment sample is arranged and testing result is shown in Fig. 6.Experimental data shows, stockaded village's card NS1 mutant antigens of this patent
The cross reaction with Dengue sample can be eliminated, while the mutant has no significantly the sensitivity of stockaded village's card IgM detection kits
It influences.
The preferred embodiment of the present invention is described in detail above in association with attached drawing, still, the present invention is not limited to above-mentioned realities
The detail in mode is applied, within the scope of the technical concept of the present invention, a variety of letters can be carried out to technical scheme of the present invention
Monotropic type, these simple variants all belong to the scope of protection of the present invention.
Sequence table
<120>A kind of zika virus NS1 antigenic mutants and its application
<160>4
<210>1
<211>11
<212>Amino acid
<213>Lack the zika virus NS1 amino acid sequences deleted
<400>
DGCWYGMEIRP
<210>2
<211>342
<212>Amino acid
<213>Zika virus NS1 deletion mutant amino acid sequences
<400>
VGCSVDFSKKETRCGTGVFIYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEEGICGISSVSRMENIMWKS
VEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLEH
RAWNSFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGREAAHSDLGYWIESEKNDTWRLKRAHLIEMKTCE
WPKSHTLWTDGVEESDLIIPKSLAGPLSHHNTREGYRTQVKGPWHSEELEIRFEECPGTKVYVEETCGTRGPSLRST
TASGRVIEEWCCRECTMPPLSFRAKRKEPESNLVRSMVTAGS
<210>3
<211>33
<212>DNA
<213>Lack the nucleotide sequence of the zika virus NS1 deleted
<400>
GACGGCTGCTGGTATGGAATGGAGATAAGGCCC
<210>4
<211>1038
<212>DNA
<213>Zika virus NS1 deletion mutant nucleotide sequences
<400>
CATATGGTTGGCTGCTCTGTTGACTTCTCTAAGAAAGAAACCCGTTGTGGTACTGGCGTGTTCATCTAC
AACGATGTAGAAGCCTGGCGTGACCGTTACAAGTACCACCCGGACTCTCCACGTCGTCTGGCTGCTGCTGTTAAACA
GGCGTGGGAAGAAGGTATTTGTGGTATCTCTTCTGTTTCTCGCATGGAGAATATCATGTGGAAGAGCGTTGAAGGTG
AGCTGAACGCTATCCTGGAAGAGAACGGCGTACAGCTGACTGTTGTAGTTGGCTCCGTGAAGAATCCGATGTGGCGT
GGTCCGCAGCGTCTGCCGGTTCCAGTTAACGAGCTGCCGCACGGCTGGAAGGCATGGGGTAAGTCCTACTTTGTACG
TGCGGCTAAGACCAATAACTCTTTCGTTGTTGACGGTGACACCCTGAAAGAATGTCCGCTGGAGCACCGTGCATGGA
ACTCTTTCCTGGTGGAAGATCACGGTTTCGGTGTATTCCACACTTCTGTGTGGCTGAAGGTTCGTGAGGATTACTCC
CTGGAATGCGATCCGGCTGTGATCGGCACCGCAGTGAAGGGTCGTGAGGCTGCGCATAGCGATCTGGGTTATTGGAT
CGAGTCTGAGAAGAACGATACTTGGCGTCTGAAGCGTGCTCATCTGATCGAGATGAAGACCTGTGAGTGGCCGAAAT
CTCATACTCTGTGGACTGACGGTGTGGAGGAATCTGACCTGATCATCCCAAAGTCTCTGGCAGGTCCGCTGTCTCAT
CACAACACCCGTGAGGGTTATCGTACCCAGGTTAAAGGTCCGTGGCATTCCGAGGAGCTGGAGATTCGTTTCGAGGA
GTGTCCGGGTACTAAAGTATACGTGGAGGAAACTTGCGGCACTCGTGGTCCGTCTCTGCGTTCTACCACTGCGAGCG
GTCGTGTTATCGAAGAGTGGTGCTGTCGTGAGTGCACCATGCCACCACTGTCCTTCCGTGCTAAACGCAAAGAGCCG
GAAAGCAACCTGGTTCGCAGCATGGTAACTGCTGGTTCTCTCGAG
Claims (6)
1. one section of zika virus NS1 antigenic mutant nucleotide sequence synthesized by outer-gene engineering method, feature exist
In the nucleotide sequence such as SEQ ID NO:Shown in 4.
2. the expression vector pET24-zNS1 containing zika virus NS1 antigenic mutant nucleotide sequences described in claim 1.
3. containing the expression described in zika virus NS1 antigenic mutants nucleotide sequence described in claim 1 or claim 2
The e. coli host cell BL21 of carrier.
4. the expression and purification method of zika virus NS1 antigenic mutants described in claim 1, it is characterised in that：It will be by DNA points
Analysis DNA sequence dna corresponding with the zika virus NS1 protein sequences that protein structural analysis confirms carries out PCR synthesis, and introduces Nde I
It is basic carrier so that plasmid pET24a is commercialized with Xho I restriction enzyme sites, builds zika virus expression vector, stockaded village's card disease
The amino acid sequence of malicious NS1 antigenic mutants is as shown in SEQ ID NO.2, corresponding nucleotide sequence such as SEQ ID NO.4
It is shown, transfection Escherichia coli BL21 host cells, by affinity chromatography, the zika virus NS1 mutains that are purified.
5. the detection reagent containing zika virus antigenic mutant described in claim 1, the zika virus antigenic mutant
Amino acid sequence as shown in SEQ ID NO.2.
6. use of the zika virus antigenic mutant described in claim 1 in preparing the kit for detecting zika virus
On the way, the amino acid sequence of the zika virus NS1 antigenic mutants is as shown in SEQ ID NO.2.
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| CN110441521A (en) * | 2018-05-03 | 2019-11-12 | 深圳市疾病预防控制中心 | The test strips and kit and method of detection Zika virus N S1 antigen |
| CN110568178B (en) * | 2019-09-18 | 2022-12-13 | 中国检验检疫科学研究院 | Zika virus NS1 antigen and application thereof in preparation of fluorescent immunochromatography reagent |
| WO2021173080A1 (en) * | 2020-02-25 | 2021-09-02 | Agency For Science, Technology And Research | Serological test for zika virus |
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| CN112921124A (en) * | 2021-04-08 | 2021-06-08 | 北京瀚梅生物科技有限公司 | Kit for rapidly detecting viruses |
| CN112921124B (en) * | 2021-04-08 | 2021-09-28 | 广东创晟控股集团有限公司 | Kit for rapidly detecting viruses |
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