CN101921781A - Xinjiang hemorrhagic fever virus nucleoprotein antigen gene and recombinant protein thereof and application - Google Patents
Xinjiang hemorrhagic fever virus nucleoprotein antigen gene and recombinant protein thereof and application Download PDFInfo
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- CN101921781A CN101921781A CN2010101094934A CN201010109493A CN101921781A CN 101921781 A CN101921781 A CN 101921781A CN 2010101094934 A CN2010101094934 A CN 2010101094934A CN 201010109493 A CN201010109493 A CN 201010109493A CN 101921781 A CN101921781 A CN 101921781A
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Abstract
The present invention relates to the antigen gene technical field, is a kind of Xinjiang hemorrhagic fever virus nucleoprotein antigen gene (XHFNP235) and recombinant protein and application, and this gene has the nucleotide sequence of sequence 1.The present invention obtains Xinjiang hemorrhagic fever virus nucleoprotein antigen gene XHFNP235 and recombinant protein thereof from xinjiang hemorrhagic fever virus.Pass through pcr amplification, the clone, transform, expression plasmid makes up, abduction delivering, immunology detection, the Xinjiang hemorrhagic fever epitope is positioned at the proteic 235-305 amino acids of NP zone, this zone also is the amino acid region of the proteic high conservative of NP, shows that further the XHFNP235 recombinant protein can become the candidate diagnosis antigen of Xinjiang hemorrhagic fever disease, for the diagnosis and the application of Xinjiang hemorrhagic fever disease provides a new approach.
Description
One, technical field
The present invention relates to the antigen gene technical field, is a kind of Xinjiang hemorrhagic fever virus nucleoprotein antigen gene (XHFNP235) and recombinant protein and application.
Two, background technology
Crimean-Congo hemorrhagic fever (Crimean-Congo Hemorrhagic Fever, CCHF) be that a kind of area such as be widely current in Africa, Asia, Eastern Europe and the Middle East is through tick-borne viral disease of natural focus, its pathogenic agent is crimean-Congo hemorrhagic fever virus (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), average case fatality rate is at 10%-50%.
Crimean-Congo hemorrhagic fever claims Xinjiang hemorrhagic fever (Xinjiang Hemorrhagic Fever again in China, XHF), this pathogenic agent xinjiang hemorrhagic fever virus (Xinjiang Hemorrhagic Fever Virus, XHFV) be unique Biosafety level Four (Laboratory Biosafety Level 4, BSL-4) pathogenic agent that is proved the nature outbreak of epidemic that present China exists.
Because this virus can be propagated in people-human world, it is less to fall ill at ordinary times, and the clinician does not diagnose this sick experience, and mistaken diagnosis very easily causes outbreak of epidemic in the hospital, and similar SARS is very harmful; Simultaneously, economy of large scale exploitation and the bio-terrorism in war, the unknown plague area attacks (this virus is listed in one of the terrified war of important biomolecule agent) all can cause the serious popular of this disease and harm.Local outbreak of epidemic has for several times taken place in Xinjiang hemorrhagic fever in Xinjiang.At present, also there be not effectively prevention and treatment means at CCHF.
CCHFV belongs to bunyaviridae (Bunyaviridae) Nairovirus (Nairovirus), viral genome by big (L gene), in (M gene), little (S gene) three bursts of strand RNA fragments forms, distinguish coding RNA polysaccharase (RNA-dependent-polymerase), glycoprotein (GP) and nucleoprotein (NP).The Xinjiang hemorrhagic fever virus nucleoprotein antigen site is linear distribution and stable in properties, is the major antigen that the virus induction body produces early stage humoral immunization and cellular immunization.The nucleoprotein of S genes encoding has been expressed in different systems, and proof can be as the diagnostic antigen of Crimean-Congo hemorrhagic fever disease.Method by gene recombination obtains to have same or similar antigen-specific, and no longer has the toxic antigen fragment of intact proteins, has become prevention and the sick ideal candidate diagnosis of diagnosis Xinjiang hemorrhagic fever antigen reagent.
Three, summary of the invention
The invention provides a kind of Xinjiang hemorrhagic fever virus nucleoprotein antigen gene that from xinjiang hemorrhagic fever virus, obtains (XHFNP235) and recombinant protein and application, particularly the application in the diagnostic antigen reagent of preparation prevention or diagnosis Xinjiang hemorrhagic fever disease.
One of technical scheme of the present invention realizes by following measure: a kind of Xinjiang hemorrhagic fever virus nucleoprotein antigen gene that obtains from xinjiang hemorrhagic fever virus is the XHFNP235 antigen gene, and it has the nucleotide sequence of sequence 1.
Be further optimization or improvement below to one of foregoing invention technical scheme: by designing a pair of primer, the Xinjiang hemorrhagic fever virus nucleoprotein antigen gene XHFNP235 that from xinjiang hemorrhagic fever virus, obtains, this primer is respectively:
Upstream primer (Primer) 5 '-ccggatcccttgccaagcttgcagagactgaagggaagggagtg-3 ',
Downstream primer (Primer) 5 '-ccgaattcctatcaatctgtgcaccctgtgcacgaagtgcagacgagtttttgt-3 '.
Two of technical scheme of the present invention realizes by following measure: above-mentioned Xinjiang hemorrhagic fever virus nucleoprotein antigen gene is the recombinant protein of XHFNP235 antigen gene.
Three of technical scheme of the present invention realizes by following measure: above-mentioned Xinjiang hemorrhagic fever virus nucleoprotein antigen gene is the application of XHFNP235 antigen gene in the diagnostic antigen reagent of preparation prevention or diagnosis Xinjiang hemorrhagic fever disease.
Four of technical scheme of the present invention realizes by following measure: the application of above-mentioned recombinant protein in the diagnostic antigen reagent of preparation prevention or diagnosis Xinjiang hemorrhagic fever disease.
The present invention obtains Xinjiang hemorrhagic fever virus nucleoprotein antigen gene XHFNP235 and recombinant protein thereof from xinjiang hemorrhagic fever virus.Pass through pcr amplification, the clone, transform, expression plasmid makes up, abduction delivering, immunology detection, the Xinjiang hemorrhagic fever epitope is positioned at the proteic 235-305 amino acids of NP zone, this zone also is the amino acid region of the proteic high conservative of NP, shows that further the XHFNP235 recombinant protein can become the candidate diagnosis antigen of Xinjiang hemorrhagic fever disease, for the diagnosis and the application of Xinjiang hemorrhagic fever disease provides a new approach.
Four, description of drawings
Accompanying drawing 1 is Xinjiang hemorrhagic fever virus nucleoprotein antigen XHFNP235 recombinant plasmid agarose gel electrophoresis result of the present invention, and wherein, M is DL2000; 1 for the recombinant plasmid double digestion EcoR I of XHFNP235 of the present invention and BamH I product.
Accompanying drawing 3 for the multi-clone rabbit serum of anti-xinjiang hemorrhagic fever virus to XHFNP235 recombinant protein Western-blot immunology detection analytical results of the present invention, wherein, 1 is the pGEX-KG expressing protein, pGEX-KGNP expressing protein, pGEX-KG XHFNP235 recombinant protein.
Five, embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to technical scheme of the present invention and practical situation:
Embodiment 1: utilize polymerase chain reaction (PCR) technology and gene recombination technology to obtain Xinjiang hemorrhagic fever virus nucleoprotein N235 antigen gene of the present invention in a large number.
Designing primer respectively according to xinjiang hemorrhagic fever virus S gene order, is template with cDNA, pcr amplification purpose fragment.The PCR reaction system is: cDNA2 μ L, 10 * Ex Taq Buffer2 μ L, Sterilized dH
2O12.9 μ L, Ex Taq (5U/ μ L) 0.5 μ L, P1primer (20 μ M) 0.3 μ L, P2primer (20 μ M) 0.3 μ L, dNTP Mixture (each 10mM) 2 μ L.
According to the Xinjiang hemorrhagic fever virus nucleoprotein gene order, the design primer.
Upstream primer P1 (Primer):
5′-ccggatcccttgccaagcttgcagagactgaagggaagggagtg-3′，
Downstream primer P2 (Primer):
5′-ccgaattcctatcaatctgtgcaccctgtgcacgaagtgcagacgagtttttgt-3′。
The reaction conditions of pcr amplification XHFNP235 gene of the present invention is: 94 ℃, and 10 minutes; 32 the circulation comprise 94 ℃ 30 seconds, 61 ℃ 30 seconds, 72 ℃ 1 minute; Last 72 ℃ continue 10 minutes.Utilize the amplification of pcr amplification instrument, amplified production detects through 1.0% agarose gel electrophoresis, and after the order-checking correctly, pcr amplification is obtained the multiple clone site (EcoR I and BamH I) of fragment XHFNP235 gene fragment clone to pGEX-KG.Recombinant plasmid pGEX-KG-XHFNP235 cuts evaluation through enzyme, and size is 5110bp, and enzyme slitting band conforms to the expection size.As shown in Figure 1.
Embodiment 2: obtain XHFNP235 recombinant protein of the present invention.
With XHFNP235 gene fragment clone of the present invention to the pGEX-KG prokaryotic expression carrier, be converted in the e. coli bl21 (having expressive function), be inoculated in the 3mL LB substratum that contains acillin (50 μ g/mL), 37 ℃, after the 220rpm/min shaking culture is spent the night, be inoculated in the LB substratum that contains the same concentrations acillin by 1: 100,37 ℃, 220rpm/min is behind the cultivation 3h, to OD
600During ≈ 0.4, adding IPTG is 0.4mmol/L to final concentration, continuation shaking culture 3h, and in 4 ℃, 4000rpm/min, centrifugal 12min collects bacterium.With an amount of PBS suspension thalline, add sample-loading buffer [0.08M Tris/HCl (pH 6.8), 2%SDS, 10% glycerine, 5% beta-mercaptoethanol, 0.005% tetrabromophenol sulfonphthalein], boil 10min, centrifugal 10 minutes of 12000 * g gets 5 μ L supernatant liquors and carries out the 12%SDS-PAGE electrophoresis, Xylene Brilliant Cyanine G R-250 dyeing.The visible XHFNP235 expression of recombinant proteins of the present invention at the 32KDa place of electrophorogram shown in its accompanying drawing 2, shows XHFNP235 recombinant protein successful expression of the present invention.
Embodiment 3: adopting XHFNP235 recombinant protein of the present invention is antigen, adopts the ELISA method to detect the application of the animal serum sample that picks up from the popular district of Xinjiang hemorrhagic fever.
3.1 with the XHFNP235 recombinant protein is the ELISA detection method that antigen is set up XHFV antibody
(1) preparation antigen plate: with coating buffer (carbonate buffer solution of 0.05M pH9.6) dilution XHFNP235 recombinant protein to 1 μ g/ml, the every hole 0.2ml of enzyme plate, go to 4 ℃ of refrigerator overnight after putting 37 ℃ of incubator 2h, pat dry standby after the PBST washing lotion flushing on next day 5 times.
(2) the every hole of antigen plate adds the sheep blood serum 100 μ l of 1/100 dilution, and enzyme plate is put 37 ℃ of incubator reaction 1h in the wet box, need do the positive, feminine gender, blank simultaneously; The PBST washing lotion pats dry after washing 4-5 time, adds the anti-sheep IgG-HRP of the rabbit enzyme labelling thing 100 μ l of 1/500 dilution, puts 37 ℃ of incubators reaction 1h in the wet box; The PBST washing lotion pats dry after washing 4-5 time, adds TMB (tetramethyl benzidine) colour developing liquid, and each 100 μ l of A liquid B liquid are hatched 10min for 37 ℃, add 2M H
2SO
4Stop buffer, every hole 200 μ l put microplate reader OD
450, OD
630Dual wavelength reads the result.
3.2 to from XHFV detection of antibodies in the popular district of the Xinjiang hemorrhagic fever animal serum
Altogether to from the popular district of Xinjiang hemorrhagic fever Weili County experimental group lamb (adopt medicine drive the tick group) blood part 85,37 parts of control groups (not adopting medicine to drive the tick group), and 10 parts of XHFV antibody positive control group sheep blood serums amount to 132 parts and detect.The positive controls positive rate is 100%, several 19 parts of experimental group CCHFV antibody positive, positive rate 22.3%, positive 17 parts of control group, positive rate 45.9%.Have utmost point significant difference through chi square test, show that medicine drives tick and suppresses to have XHFV invasion and attack lamb and can effectively control lamb and infect Xinjiang hemorrhagic fever.
Show that by above experiment XHFNP235 recombinant protein of the present invention can detect humans and animals serum and whether infect hemorrhagic fever virus is arranged, and can become the candidate diagnosis antigen that detects the Xinjiang hemorrhagic fever disease.
In sum, by the clone of XHFNP235 gene, transform abduction delivering and serum test experience, show that further the XHFNP235 recombinant protein can become the candidate diagnosis antigen that detects the Xinjiang hemorrhagic fever disease, for the diagnosis and the application of Xinjiang hemorrhagic fever disease provides a new approach.
Xinjiang hemorrhagic fever virus nucleoprotein antigen gene of the present invention is that the sequence 1 of the Nucleotide of XHFNP235 antigen gene is seen sequence table.
SEQUENCE LISTING-sequence 1
<110>The?Center?for?Disease?Control?and?Prevention?of?Xinjiang
Uygur Autonomous Region Xinjiang Uygur Autonomous Region Disease Control ﹠ Prevention Center
Zhang，Yu?Jiang
<120>The?application?of?Nucleoprotein?antigen?gene?from?Xinjiang
Hemorrhagic?Fever?Virus?and?its?recombinant
Protein Xinjiang hemorrhagic fever virus nucleoprotein antigen gene and recombinant protein thereof and application
<130>1
<140>1
<141>2010-02-11
<160>2
<170>PatentIn?version?3.3
<210>1
<211>210
<212>DNA
<213>Crimean-Congo?hemorrhagic?fever?virus
<220>
<221>exon
<222>(1)..(210)
<220>
<221>CDS
<222>(124)..(210)
<400>1
ctt?gcc?aag?ctt?gca?gag?act?gaa?ggg?aag?gga?gtg?ttt?gat?gaa?gca 48
Leu?Ala?Lys?Leu?Ala?Glu?Thr?Glu?Gly?Lys?Gly?Val?Phe?Asp?Glu?Ala
1 5 10 15
aaa?aag?act?gtg?gag?gct?ctc?aac?ggg?tac?ttg?aac?aaa?cat?aag?gat 96
Lys?Lys?Thr?Val?Glu?Ala?Leu?Asn?Gly?Tyr?Leu?Asn?Lys?His?Lys?Asp
20 25 30
gag?gtt?gat?aaa?gca?agt?gcc?gac?agc?atg?ata?aca?aac?ctt?ctt?aaa 144
Glu?Val?Asp?Lys?Ala?Ser?Ala?Asp?Ser?Met?Ile?Thr?Asn?Leu?Leu?Lys
35 40 45
cac?att?gcc?aag?gca?cag?gag?ctt?tac?aaa?aac?tcg?tct?gca?ctt?cgt 192
His?Ile?Ala?Lys?Ala?Gln?Glu?Leu?Tyr?Lys?Asn?Ser?Ser?Ala?Leu?Arg
50 55 60
gca?cag?ggt?gca?cag?att 210
Ala?Gln?Gly?Ala?Gln?Ile
65 70
<210>2
<211>29
<212>PRT
<213>Crimean-Congo?hemorrhagic?fever?virus
<400>2
Met?Ile?Thr?Asn?Leu?Leu?Lys?His?Ile?Ala?Lys?Ala?Gln?Glu?Leu?Tyr
1 5 10 15
Lys?Asn?Ser?Ser?Ala?Leu?Arg?Ala?Gln?Gly?Ala?Gln?Ile
20 25
Claims (5)
1. an Xinjiang hemorrhagic fever virus nucleoprotein antigen gene that obtains from xinjiang hemorrhagic fever virus is the XHFNP235 antigen gene, it is characterized in that having the nucleotide sequence of sequence 1.
2. Xinjiang hemorrhagic fever virus nucleoprotein antigen gene according to claim 1 is the XHFNP235 antigen gene, it is characterized in that by designing a pair of primer, the Xinjiang hemorrhagic fever virus nucleoprotein antigen gene XHFNP235 that from xinjiang hemorrhagic fever virus, obtains, this primer is respectively:
Upstream primer (Primer) 5 '-ccggatcccttgccaagcttgcagagactgaagggaagggagtg-3 ',
Downstream primer (Primer) 5 '-ccgaattcctatcaatctgtgcaccctgtgcacgaagtgcagacgagtttttgt-3 '.
3. one kind contains claim 1 or 2 described Xinjiang hemorrhagic fever virus nucleoprotein antigen genes are the recombinant protein of XHFNP235 antigen gene.
4. an Xinjiang hemorrhagic fever virus nucleoprotein antigen gene according to claim 1 and 2 is the application of XHFNP235 antigen gene in the diagnostic antigen reagent of preparation prevention or diagnosis Xinjiang hemorrhagic fever disease.
5. the application of recombinant protein according to claim 3 in the diagnostic antigen reagent of preparation prevention or diagnosis Xinjiang hemorrhagic fever disease.
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| CN2010101094934A CN101921781B (en) | 2010-02-12 | 2010-02-12 | Xinjiang hemorrhagic fever virus nucleoprotein antigen gene as well as recombinant protein and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188285A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院武汉病毒研究所 | A kind of single domain antibody neutralizing xinjiang hemorrhagic fever virus |
| CN109266661A (en) * | 2018-09-18 | 2019-01-25 | 新疆大学 | A kind of building and application of xinjiang hemorrhagic fever virus multi-epitope carrier for expression of eukaryon pVAX-MEPX2 |
| CN111398579A (en) * | 2020-01-18 | 2020-07-10 | 新疆大学 | Indirect E L ISA detection method for constructing palygorskite virus antibody and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101498727A (en) * | 2009-02-13 | 2009-08-05 | 广东出入境检验检疫局检验检疫技术中心 | Xinjiang hemorrhagic fever virus immunochromatography fast detection test paper |
-
2010
- 2010-02-12 CN CN2010101094934A patent/CN101921781B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188285A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院武汉病毒研究所 | A kind of single domain antibody neutralizing xinjiang hemorrhagic fever virus |
| CN106188285B (en) * | 2016-07-21 | 2019-07-23 | 中国科学院武汉病毒研究所 | A kind of single domain antibody neutralizing xinjiang hemorrhagic fever virus |
| CN109266661A (en) * | 2018-09-18 | 2019-01-25 | 新疆大学 | A kind of building and application of xinjiang hemorrhagic fever virus multi-epitope carrier for expression of eukaryon pVAX-MEPX2 |
| CN111398579A (en) * | 2020-01-18 | 2020-07-10 | 新疆大学 | Indirect E L ISA detection method for constructing palygorskite virus antibody and application |
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| CN101921781B (en) | 2012-07-04 |
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