WO2017088448A1 - 一种登革热双效疫苗的制备方法及其应用 - Google Patents

一种登革热双效疫苗的制备方法及其应用 Download PDF

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WO2017088448A1
WO2017088448A1 PCT/CN2016/085080 CN2016085080W WO2017088448A1 WO 2017088448 A1 WO2017088448 A1 WO 2017088448A1 CN 2016085080 W CN2016085080 W CN 2016085080W WO 2017088448 A1 WO2017088448 A1 WO 2017088448A1
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dengue virus
protein
denv2
dengue
virus
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程功
刘建英
刘洋
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清华大学
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of dengue virus control, and specifically relates to the preparation and application of a dengue virus double-effect vaccine.
  • Dengue Fever is an acute viral infection transmitted by Aedes mosquitoes.
  • Dengue virus (DENV) is spherical and consists of membrane, capsid protein, non-structural protein and single-stranded positive strand RNA.
  • there are four serotypes (DENV-1 to DENV-4) in dengue virus, and the nucleotide sequences of each serotype vary widely, reaching more than 35%. Within each serotype, it can be divided into multiple subtypes of genes according to the different epidemic regions. After the dengue virus infects the human body through the bite of the Aedes mosquito, it can manifest clinical symptoms after 2-3 days of incubation.
  • dengue fever hyperthermia, bone and muscle soreness, bleeding tendency, and a marked reduction in white blood cell count.
  • the clinical manifestations of dengue fever are mild, and most people self-heal as the disease progresses, and the mortality rate is low; however, the disease progression of a few people will progress to the next stage, namely Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome ( Dengue Shock Syndrome (DSS), a serious type of dengue infection, has clinical manifestations of severe bleeding in various organs, high fever, marked reduction in platelet count, blood concentration, and shock, resulting in extremely high mortality rates.
  • DHF Dengue Hemorrhagic Fever
  • DSS Dengue Shock Syndrome
  • the dengue virus is mainly transmitted by the Aedes aegypti in Egypt. In areas where there is no Aedes aegypti, Aedes albopictus is also considered to be an important mediator of dengue fever transmission. It is currently believed that the natural host of dengue virus is only human, lower primate and Aedes. In nature, dengue virus mainly circulates in two modes: 1) urban circulation: in the urban dengue epidemic, the virus spreads between “human-Aedes-human”; 2) the jungle cycle: mainly through It is transmitted between Aedes mosquitoes and lower primates in the tropical jungle. This cycle is the original cycle of dengue virus.
  • the dengue virus in the jungle type and the dengue virus in the urban circulation conditions are quite different, but the possibility that the Aedes mosquitoes in the jungle cycle can spread the virus to cities and villages cannot be ruled out.
  • the urban circulating dengue virus strictly follows the route of “human-Aedes-human”. Human being is the sole host of the virus, and Aedes mosquito is the only vector.
  • infected Aedes mosquitoes inject dengue virus into the human body through the bite (virus transmission), and the virus proliferates in human subcutaneous keratinocytes and dendritic cells; the virus spreads to the blood and further infects the blood.
  • the genome of dengue virus consists of a single-stranded forward RNA that can be translated into a multimeric protein peptide chain in a host cell, after which the peptide chain is hydrolyzed into three structural proteins and seven non-structural proteins.
  • Non-structural protein NS
  • NS1 non-structural protein 1
  • NS1 can be secreted from infected cells and is present in a large amount in the serum of infected hosts.
  • the amount of NS1 in the patient's serum is 70-15,000 ng/ml.
  • the amount of NS1 in the serum can reach 50,000 ng/ml. Therefore, NS1 is one of the main indicators for clinical detection of early dengue virus infection.
  • NS1 plays a very important role in the pathogenesis of dengue fever and dengue hemorrhagic fever.
  • the NS1 antibody binds non-specifically to human vascular endothelial cells, resulting in increased permeability of endothelial cells, leading to bleeding tendency; in addition, antibodies to NS1 can also deposit on the surface of platelets, causing platelet coagulation disorders and leading to increased bleeding.
  • the NS1 protein and the virus can be simultaneously inhaled into the bitten mosquito by blood suction. Therefore, NS1 plays an important role in the process of virus acquisition.
  • Interferon-receptor-deficient mice IFN-alpha/gamma receptor double knock-outmouse, AG mouse
  • the B6 background AG6 mouse IFN-alpha/gamma receptor double knock-out B6 mouse
  • AG6 mice are very sensitive to dengue virus. After inoculation with dengue 2 virus, the virus can be rapidly expanded in AG6 mice. At the same time, Aedes aegypti bites dengue virus-infected AG6 mice, can take the virus and obtain infection. .
  • Dengue fever has been found for more than 200 years, but there are still no specific treatments and safe and effective dengue vaccines. Due to the particularity of the pathogenesis of dengue fever, antibodies produced by primary infection of the virus only neutralize the same type of virus infection, while infection with other serotypes has an infection-enhancing effect. At the same time, T cell activation after viral infection also promotes the incidence of dengue hemorrhagic fever and dengue shock syndrome. This pathogenesis is limiting the dengue virus routine One of the main factors in the development of subunit vaccines, live attenuated vaccines and inactivated vaccines.
  • arthropod-borne infectious diseases infectious diseases
  • the traditional vaccine directly immuno-attenuated or inactivated pathogens or their recombinant proteins
  • it can also block the circulation of vector pathogens in nature, thereby reducing vector transmission.
  • the spread of disease in nature. Therefore, the spread-blocking vaccine is an immune strategy that immunizes an antigenic protein in a host to block pathogen transmission, thereby reducing the rate of insect-borne infection and host morbidity.
  • transmission-blocking vaccines have been widely used in the prevention and treatment of vector-borne diseases such as malaria. Therefore, the use of vaccine methods to reduce the infection rate of vector mosquitoes will be an effective measure for the prevention and treatment of dengue fever.
  • the protein provided by the present invention is a protein of the following a) or b):
  • Substitutions and/or deletions and/or additions of one or more of the above amino acid residues are substitutions and/or deletions and/or additions of no more than 10 amino acid residues.
  • DNA molecules encoding the above proteins are also within the scope of the invention.
  • the above DNA molecule is a DNA molecule of any one of the following 1) to 3):
  • the coding region is the DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;
  • the defined DNA sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least a DNA molecule having 98% or at least 99% homology and encoding the protein of claim 1;
  • the above stringent conditions may be that the mixture is hybridized in a solution of 6 x SSC, 0.5% SDS at 65 ° C, and then washed once with 2 x SSC, 0.1% SDS and 1 x SSC, 0.1% SDS.
  • a recombinant vector, expression cassette, transgenic cell line, recombinant strain or recombinant virus containing the above DNA molecule is also within the scope of protection of the present invention.
  • antibodies prepared from the above proteins are also within the scope of the invention.
  • the mammal is a mouse
  • the mosquito is Aedes aegypti or Aedes albopictus.
  • the product is a kit.
  • Another object of the present invention is to provide a functional product having at least one of the following 1) to 5).
  • the product provided by the invention has an active ingredient of the above protein
  • the mammal is a mouse
  • the mosquito is Aedes aegypti or Aedes albopictus.
  • the above products are drugs.
  • the above dengue virus is DENV2.
  • Figure 1 shows the titer of passive immunization of DENV2 NS1 and DENV2 ⁇ NS1 antibody
  • Figure 2 is a passive immunization of DENV2 NS1 antiserum to block the production of NS1 in AG6 mice;
  • Figure 3 is a passive immunization of DENV2 NS1 antiserum to reduce the incidence of dengue virus infection on Aedes;
  • Figure 4 shows that active DENV2 NS1 and DENV2 ⁇ NS1 proteins block the production of NS1 in vivo in AG6 mice;
  • Figure 5 shows that active DENV2 NS1 and DENV2 ⁇ NS1 proteins reduce the incidence of dengue virus infection on Aedes aegypti;
  • Figure 6 shows that active immunization of DENV2 ⁇ NS1 reduces DENV2 viral load in AG6 mice
  • Figure 7 is a plotting function of the Evans blue content in each tissue
  • Figure 8 shows that active immunization of DENV2 NS1 and DENV2 ⁇ NS1 reduces bleeding symptoms in various organs of AG6 mice during dengue infection;
  • Figure 9 shows that active immunization of DENV2 NS1 and DENV2 ⁇ NS1 reduces the mortality of AG6 mice in dengue infection.
  • DENV2 NS1 and DENV2 ⁇ NS1 proteins were expressed in prokaryotic cells as follows:
  • the total RNA of DENV2 virus was extracted and reverse-transcribed to obtain cDNA as a template. PCR amplification was performed using F1 and R1 primer pairs to obtain a 1056 bp PCR product, which is the DENV2 NS1 gene. After sequencing, the nucleotide sequence is sequence 1.
  • the encoded protein is DENV2 NS1, which has an amino acid length of 352AA and an amino acid sequence of sequence 2.
  • the total RNA of the DENV2 virus was extracted, and the cDNA was reverse transcribed to obtain a cDNA as a template for amplification.
  • the first step PCR amplification of the F2 and R2 primer pairs using the cDNA of DENV2 virus as a template to obtain a PCR product of 1-115AA; the second step: using the cDNA of DENV2 virus as a template and the pair of F3 and R3 primers Amplification, 120-220AA PCR product was obtained; Step 3: PCR amplification of F4 and R4 primer pairs using DENV2 virus cDNA as template, 267-310AA PCR product was obtained; Step 4: DENV2 virus cDNA as a template to F5 Amplification with the R5 primer pair to obtain a PCR product of 331-352AA; the fifth step: using the PCR products obtained in the first step and the second step as a template, and amplifying the F6 and R6 primer pairs to obtain 1-220AA ( PCR product of ⁇ 116-119AA); the sixth step: using the PCR products obtained in the third step and the fourth step as a template, and amplifying the F7 and R7 primer pairs to obtain a
  • F1 5'-GGCATTCCATATGGATAGTGGTTGCGTTG-3';
  • R1 5'-TAATTCCTCGAGGGCTGTGACCAAGGA-3';
  • F2 5'-GGCATTCCATATGGATAGTGGTTGCGTTG-3';
  • R2 5'-GAGAGCATTTTCGCTTTCCATGAATACTTCAGC-3';
  • F3 5'-GCTGAAGTATTCATGGAAAGCGAAAATGCTCTC-3';
  • R3 5'-TACCTAGATGCCATGGAACTTCGATGAAAGAG-3';
  • F4 5'-CTCTTTCATCGAAGTTCCATGGCATCTAGGTA-3';
  • R4 5'-CTGATTTCCATCCCGTATTCTGTTATGAGTTTTCC-3';
  • F5 5'-GGAAAACTCATAACAGAATACGGGATGGAAATCAG-3';
  • R5 5'-TAATTCCTCGAGGGCTGTGACCAAGGA-3';
  • F6 5'-GGCATTCCATATGGATAGTGGTTGCGTTG-3';
  • R6 5'-TACCTAGATGCCATGGAACTTCGATGAAAGAG-3'.
  • F7 5'-CTCTTTCATCGAAGTTCCATGGCATCTAGGTA-3';
  • R7 5'-TAATTCCTCGAGGGCTGTGACCAAGGA-3';
  • F8 5'-GGCATTCCATATGGATAGTGGTTGCGTTG-3';
  • R8 5'-TAATTCCTCGAGGGCTGTGACCAAGGA-3'.
  • the DENV2 NS1 gene shown in the above Sequence 1 was inserted into the restriction endonuclease site of NdeI and XhoI of the pET-28a (+) vector, and designated as pET-28a(+)-DENV2 NS1.
  • pET-28a(+)-DENV2 NS1 and pET-28a(+)-DENV2 ⁇ NS1 were introduced into Escherichia coli BL21 (DE3), respectively, to obtain recombinant bacteria BL21(DE3)/pET-28a(+)-DENV2 NS1 and BL21. (DE3)/pET-28a(+)-DENV2 ⁇ NS1.
  • the monoclonal colonies of the above recombinant bacteria were inoculated separately into 5 ml of LB liquid medium (containing 25 ⁇ g/ml kanamycin), cultured at 37 ° C, shaking at 220 rpm for 16 hours; then inoculated to fresh LB at a volume ratio of 1:100 Liquid medium (containing 25 ⁇ g/ml kanamycin), total volume 200 ml, shaking culture at 37 ° C, 220 rpm for 4 hours; then adding IPTG and making it 1 mM, shaking at 37 ° C, 220 rpm for 6 hours; room temperature, 4000 rpm The cells were collected by centrifugation for 10 min.
  • LB liquid medium containing 25 ⁇ g/ml kanamycin
  • the obtained cells were treated with inclusion body washing solution (solvent pH 8.0, 50 mM Tris-HCl buffer containing 100 mM NaCl, 5 mM EDTA, 0.1% NaN3, 0.5% Triton-X 100, and the final concentration was added before use).
  • the cells were suspended by 0.1 mM PMSF and 1 mM DTT), sonicated on ice (60% power, sonicated for 3 s, stopped for 9 s, total sonication time 5 min), centrifuged at 6000 rpm for 15 min, and the precipitate (inclusion body) was collected.
  • the precipitate obtained above was suspended with an inclusion body washing solution, excess MgSO 4 was added to neutralize the EDTA in the inclusion body washing solution, and then a final concentration of 0.01 mg/ml of DNase (Sigma Catalog No. DN25) was added and the final concentration was 0.1 mg/ml lysozyme (Sigma catalog number L6876), treated at room temperature for 20 minutes, centrifuged at 6000 rpm for 15 min, and collected inclusion bodies; then, the precipitate obtained in step 8 was suspended with the inclusion body washing solution, and excess MgSO 4 was added to neutralize The EDTA in the inclusion body washing solution was treated at room temperature for 20 minutes, centrifuged at 6000 rpm for 15 min, and the inclusion bodies were collected to obtain DENV2 NS1 inclusion bodies and DENV2 ⁇ NS1 inclusion bodies.
  • DENV2 NS1 was about 42 kD
  • DENV2 ⁇ NS1 was about 31 kD.
  • the inclusion bodies obtained above were dissolved using a protein purification solution (Tris buffer of pH 8.0, 100 mM, containing 50 mM glycine and 8 M urea) to obtain a DENV2 NS1 crude protein solution and a DENV2 ⁇ NS1 crude protein solution.
  • a protein purification solution Tris buffer of pH 8.0, 100 mM, containing 50 mM glycine and 8 M urea
  • the DENV2 NS1 mature peptide was purified from the crude protein solution using the TALON purification kit (Clontech Cat. No. 635515) as follows: 1 ml Resin and 50 ml thick The protein solution was incubated at 4 ° C for 2 hours, then transferred to a filter column, washed with 20 ml of washing solution A (1X equilibration solution in TALON kit was added to 8 M urea), and then washed with 20 ml of washing solution B (1X in TALON kit) The equilibration solution was washed with 8 M urea and 20 mM imidazole), and finally washed with 5 ml of eluent C (1X equilibration solution in TALON kit, 8 M urea and 150 mM imidazole), and 5 ml of eluate was collected as DENV2 NS1 protein;
  • DENV2 ⁇ NS1 was purified by the same method, and 5 ml of the eluate was collected as DENV2 ⁇ NS1 protein.
  • the above protein has a purity greater than 95% and a concentration greater than 1 mg/ml.
  • Example 2 Application of DENV2 NS1 and DENV2 ⁇ NS1 in passive immune obstruction of dengue virus acquisition
  • the experimental animals were 8-week-old Balb/c female mice.
  • the experimental procedure was as follows:
  • each mouse was intraperitoneally injected with an equal volume mixture of DENV2NS1 and DENV2 ⁇ NS1 protein prepared in the above one with Freund's complete adjuvant (containing 40 ug of DENV2 NS1 or DENV2 ⁇ NS1 protein) .
  • each mouse was injected subcutaneously with an equal volume mixture of DENV2 NS1 and DENV2 ⁇ NS1 protein and Freund's incomplete adjuvant (containing 40 ug DENV2 NS1 or DENV2 ⁇ ). NS1 protein).
  • each mouse was injected subcutaneously with an equal volume mixture of DENV2 NS1 and DENV2 ⁇ NS1 protein and Freund's incomplete adjuvant (containing 40 ug DENV2 NS1 or DENV2 ⁇ ). NS1 protein).
  • mice On day 42, normal-fed, non-immune mice collected 500 ul of serum, which was an anti-Pre-immune control antibody.
  • Anti-DENV2 NS1 antibody Anti-DENV2 NS1
  • Anti-DENV2 ⁇ NS1 antibody Anti-DENV2 ⁇ NS1
  • control Pre-immune antibody were diluted 1/50 to 1/10,000,000 with a 1:15 BSA gradient, per well. Add 100ul and combine at room temperature for 2 hours;
  • PBST was washed 6 times, and color development was carried out by adding 100 ul of TMB (52-00-01 and 50-85-04, Kirkegaard & Perry Laboratories) color developing solution, and color development was stopped with sulfuric acid, and OD450 was read.
  • TMB 52-00-01 and 50-85-04, Kirkegaard & Perry Laboratories
  • pfu DENV2 (AF204178, Guo et al., 2013) virus was injected into the AG6 (IFN-alpha/gamma receptor double knock-out B6 mouse) by intraperitoneal injection, and the anti-DENV2 prepared above was prepared 12 hours later.
  • NS1 antibody anti-DENV2 NS1
  • Pre-immune control serum was injected into AG6 mice infected with dengue virus by two-point intraperitoneal injection; 12 hr, 2 days, 3 days, 4 days, 5 days after serum injection. Blood was taken from the tail vein of the mouse and serum samples were collected.
  • the NS1 content in mouse serum was measured using an NS1 detection kit (Diagnoser automata; 8404-25) to identify the blocking effect of NS1 in mice.
  • Aedes aegypti acquires dengue virus by infecting mice
  • mice were anesthetized by intraperitoneal injection of sodium pentobarbital (130 ul/10 g body weight), and the anesthetized mice were placed in the rear to be bitten. On the container of the mosquito, let Aedes suck blood for 30 minutes;
  • the blood-sucking Aedes mosquitoes were sacrificed after 8 days of feeding, and frozen at -80 degrees for 5 minutes.
  • a single Aedes mosquito was placed in an EP tube equipped with an RNA extract, ground with a grinding rod and thoroughly homogenized.
  • RNA extraction kit The total RNA of Aedes aegypti was extracted by RNA extraction kit and reverse transcribed into cDNA. Taqman RT-QPCR was used to detect the DENV2 virus infection rate in Aedes aegypti.
  • Aedes mosquito infection rate is calculated as: number of infected Aedes / total number of Aedes in the experiment; statistical DENV2 NS1 antiserum group and Pre-immune group Aedes mosquito DENV2 virus infection rate from the first to fourth days .
  • the infection rate of Aedes mosquitoes in the anti-DENV2 NS1 antibody group was significantly lower than that in the Pre-immune group, indicating that the anti-DENV2 NS1 antibody can block Aedes mosquitoes from acquiring viruses from AG6 mice.
  • Example 3 DENV2 NS1 and DENV2 ⁇ NS1 in the application as a dengue vaccine
  • the DENV2 NS1 protein and DENV2 ⁇ NS1 protein prepared in Example 1 were actively immunized in AG6 mice, and the mice were immunized into 6-week-old AG6 mice.
  • the experiment was divided into three groups, PBS control group, DENV2 NS1 and DENV2 ⁇ NS1.
  • PBS control group In the experimental group, 12 AG6 mice in each group were immunized as follows:
  • DENV2 NS1 experimental group each AG6 mouse was intraperitoneally injected with an equal volume mixture of DENV2 NS1 prepared in Example 1 and Freund's complete adjuvant (40ug DENV2 NS1 per mouse);
  • DENV2 ⁇ NS1 experimental group Each AG6 mouse was intraperitoneally injected with an equal volume mixture of DENV2 ⁇ NS1 prepared in Example 1 and Freund's complete adjuvant (40 ug DENV2 ⁇ NS1 per mouse);
  • Control group Each AG6 mouse was injected intraperitoneally with an equal volume mixture of PBS and Freund's complete adjuvant.
  • 1x10 6 pfu DENV2 (AF204178) virus was injected into the above two groups by two intraperitoneal injections; the virus was injected for 1 day, 2 days, 3 days, 4 days, 5 days, respectively. Blood was taken from the tail vein of the mouse and serum samples were collected.
  • the NS1 test kit was used to detect the NS1 content in mouse serum to identify the blocking effect of NS1 in mice.
  • the results are shown in Figure 4, DENV2 NS1 group and DENV2 ⁇ NS1 group, and NS1 in AG6 mice.
  • the content was almost undetectable; in the PBS control group, the NS1 content in the AG6 mice was normal, and gradually increased with dengue virus infection.
  • DENV2 NS1 protein and DENV2 ⁇ NS1 protein block Aedes aegypti to obtain dengue virus from animals
  • mice injected with DENV2 NS1 group, DENV2 ⁇ NS1 group and PBS control group for 1 day, 2 days, 3 days and 4 days in the above 4 steps were intraperitoneally injected with sodium pentobarbital (130 ul / 10g body weight) anesthetized mice, place the anesthetized mice on a container for mosquitoes to be bitten, let Aedes suck blood for 30 minutes; place the blood-sucking Aedes on 4 degrees ice for 10 minutes for anesthesia, from anesthetized mosquitoes Picking up a full-blooded Aedes mosquito and transferring it to a new container for feeding;
  • sodium pentobarbital 130 ul / 10g body weight
  • AIDS DENV2 NS1 group was given 1 day, 2 days, 3 days and 4 days of virus injection, 1 day, 2 days, 3 days and 4 days of DENV2 ⁇ NS1 group Aedes and virus injection 1 day, 2 days, 3 Day and 4 days PBS control group Aedes.
  • RNA extraction kit to extract the total RNA of the above 2 Aedes aegypti and reverse transcription into cDNA, and using Taqman RT-QPCR to detect the infection rate of DENV2 virus in Aedes aegypti;
  • Primer pairs for detection of DENV2 virus by Taqman RT-QPCR are as follows:
  • Upstream primer 5'-CATTCCAAGTGAGAATCTCTTTGTCA-3';
  • Downstream primer 5'-CAGATCTCTGATGAATAACCAACG-3'.
  • the probes used to detect dengue type 2 virus by Taqman RT-QPCR are as follows:
  • the Aedes aegypti Actin gene (AAEL011197) was used as an internal reference, and the primer pairs of Taqman RT-QPCR were as follows:
  • Upstream primer 5'-GAACACCCAGTCCTGCTGACA-3';
  • Downstream primer 5'-TGCGTCATCTTCTCACGGTTAG-3'.
  • the probes used to detect the Aedes Actin gene by Taqman RT-QPCR are as follows:
  • the ⁇ Ct value (Ct value of Actin minus the Ct value of DENV2) detected by RT-QPCR using Actin and DENV2 was calculated. When the value of 2 - ⁇ Ct was greater than 0.0002, the Aedes mosquito was identified as positive for DENV2 infection.
  • the infection rate of Aedes mosquito is calculated as: the number of infected Aedes / the total number of Aedes in the experiment;
  • the results are shown in Fig. 5.
  • the infection rate of Aedes aegypti in the DENV2 NS1 and DENV2 ⁇ NS1 groups was significantly lower than that in the PBS control group, demonstrating that both active DENV2 NS1 and DENV2 ⁇ NS1 can block Aedes mosquitoes from AG6.
  • the virus was obtained from the mouse; in the immunized DENV2 ⁇ NS1 group, the Aedes mosquito infection rate was additionally decreased by 2-3 times compared with the DENV2NS1 group, showing a better blocking effect.
  • DENV2 NS1 protein and DENV2 ⁇ NS1 protein suppress the occurrence of hemorrhagic fever
  • the DENV2 NS1 protein and DENV2 ⁇ NS1 protein prepared in Example 1 were actively immunized in AG6 mice, and the mice were immunized into 6-week-old AG6 mice.
  • the experiment was divided into three groups, PBS control group, DENV2 NS1 and DENV2 ⁇ NS1.
  • PBS control group In the experimental group, 12 AG6 mice in each group were immunized as follows:
  • DENV2 NS1 experimental group each AG6 mouse was intraperitoneally injected with an equal volume mixture of DENV2 NS1 prepared in Example 1 and Freund's complete adjuvant (40 ug DENV2 NS1 per mouse);
  • DENV2 ⁇ NS1 experimental group Each AG6 mouse was intraperitoneally injected with an equal volume mixture of DENV2 ⁇ NS1 prepared in Example 1 and Freund's complete adjuvant (40 ug DENV2 ⁇ NS1 per mouse);
  • Control group Each AG6 mouse was injected intraperitoneally with an equal volume mixture of PBS and Freund's complete adjuvant.
  • 1x10 6 pfu DENV2 (AF204178) virus was injected into the above two groups by two intraperitoneal injections; the virus was injected for 1 day, 2 days, 3 days, 4 days, 5 days, respectively. 15 ul of blood was taken from the tail vein of the mouse, placed in an EP tube preset with an RNA extract, and thoroughly homogenized.
  • RNA in whole blood of AG6 mice was extracted and reverse transcribed into cDNA using RNA extraction kit.
  • the DENV2 viral load in the blood of AG6 mice was detected by Taqman RT-QPCR (the expression level of mouse Actin gene was used as an internal reference).
  • Primer pairs for detection of DENV2 virus by Taqman RT-QPCR are as follows:
  • Upstream primer 5'-CATTCCAAGTGAGAATCTCTTTGTCA-3';
  • Downstream primer 5'-CAGATCTCTGATGAATAACCAACG-3'.
  • the probes used to detect dengue type 2 virus by Taqman RT-QPCR are as follows:
  • the mouse Actin gene (NM_007393) was used as an internal reference, and the primer pairs of Taqman RT-QPCR were as follows:
  • Upstream primer 5'-AGCCATGTACGTAGCCATCCA-3';
  • Downstream primer 5'-TCTCCGGAGTCCATCACAATG-3'.
  • the probes used to detect the mouse Actin gene by Taqman RT-QPCR are as follows:
  • Actin and DENV2 calculated using (DENV2 subtracting Ct value Ct value of Actin) by RT-QPCR ⁇ Ct value obtained in the detection, defined 2 - viral load DENV2 AG6 ⁇ Ct value of the mice.
  • the dengue virus load of the DENV2 NS1 group and the PBS control group was substantially the same, and the dengue virus load of the DENV2 ⁇ NS1 group was decreased by 3-10 times, demonstrating the deletion of the recombination site which produces the side effect site.
  • DENV2 ⁇ NS1 can reduce the infection of dengue virus in AG6 mice.
  • mice Two hours after the injection of Evans blue, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (130 ul/10 g body weight), the mice were dissected, and the mice were perfused with PBS until the blood was completely removed.
  • sodium pentobarbital 130 ul/10 g body weight
  • the kidney, liver, spleen, small intestine, large intestine and stomach were collected from the mouse, and the tissue was soaked in the tissue (2 ml/100 mg tissue) to extract the Evans orchid infiltrated into the tissue.
  • the tissue fragments were removed by centrifugation, and the absorbance of Evans blue in the supernatant at 610 nm was measured using a spectrophotometer, and the amount of Evans blue in each tissue was calculated from the standard curve (the standard curve is shown in Fig. 7).
  • the amount of Evans blue in the tissues of DENV2 NS1 mice and DENV2 ⁇ NS1 mice was significantly lower than that of the control PBS group, indicating that both proteins can be reduced to reduce AG6 mice.
  • the tendency to hemorrhage when infected with dengue virus; the amount of Evansland in the DENV2 NS1 group was lower than that in the DENV2 NS1 group, indicating that it has a better protective effect.
  • mice The condition of the mice was monitored daily from the first day to the 40th day of the virus infection, and the death time of each mouse was recorded.
  • the survival curves of the mice in each group were analyzed by Kaplan-Meier method.
  • DENV2 NS1 and DENV2 ⁇ NS1 proteins can be used as dengue vaccines for the prevention and treatment of dengue fever, and can block Aedes mosquitoes from dengue virus infection in animals.
  • the present invention is based on dengue virus non-structural protein 1 (DENV NS1), which eliminates harmful antigenic epitopes and obtains engineered dengue virus non-structural protein 1 (DENV ⁇ NS1), which is used as an antigen to immunize a human or mammalian host.
  • the immune-engineered dengue virus non-structural protein 1 also plays a dual role in inhibiting mosquito poisoning and animal infection, reducing the bleeding and death of animal hosts caused by dengue virus infection, and reducing the rate of mosquito vectors.
  • the purpose of preventing dengue fever The double-acting vaccine protects dengue hemorrhagic fever in humans or mammals and blocks the spread of dengue virus in nature through mosquitoes.

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Abstract

本发明提供了一种蛋白质及其用途。蛋白质是如下a)或b)：a)由序列表中序列4所示的氨基酸序列组成的蛋白质；b)将序列表中序列4的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由a)衍生的蛋白质；以及改造登革病毒非结构蛋白1(DENVΔNS1)作为疫苗的用途。

Description

一种登革热双效疫苗的制备方法及其应用 技术领域
本发明属于登革病毒防治领域，具体为一种登革病毒双效疫苗的制备及其应用。
背景技术
登革热(Dengue Fever，DF)是由伊蚊传播的急性病毒性传染病。登革病毒(Dengue virus，DENV)呈球形，由胞膜、衣壳蛋白、非结构蛋白及单股正链RNA组成。自然界中，登革病毒存在四种血清型(DENV-1～DENV-4)，各血清型的核苷酸序列差异很大，可达35％以上。每个血清型内，又可根据流行区域的不同分为多个基因亚型。登革病毒经由伊蚊叮咬传染人体后，经2-3天潜伏即可表现出临床症状。登革的临床特征为高热、骨骼及肌肉酸痛、出血倾向及白细胞计数明显减少。登革热临床表现较轻微，随病程进展大多数人自愈，致死率低；但少数人的病程会进展到下一阶段，即登革出血热(Dengue Hemorrhagic Fever,DHF)及登革休克综合症(Dengue Shock Syndrome,DSS)，这是登革热感染的一种严重类型，临床表现为各器官严重出血、高热、血小板计数明显减少、血液浓缩、休克，造成该病程的死亡率极高。
登革病毒主要由埃及伊蚊(Aedes aegypti)叮咬人体后感染传播。不存在埃及伊蚊的地区，白纹伊蚊(Aedes albopictus)也被认为是登革热传播的重要媒介。目前认为，登革病毒的自然宿主仅有人、低等灵长类及伊蚊。在自然界中，登革病毒主要在两种模式中循环：1)城市循环：在城市型的登革热流行中，病毒在“人－伊蚊－人”之间传播；2)丛林循环：主要是通过热带丛林中的伊蚊及低等灵长类之间传播，此循环为登革病毒的原始循环。据研究，丛林型登革病毒与城市循环条件下的登革病毒差异较大，但是不能排除丛林循环的感染伊蚊将病毒扩散至城市及乡村的可能性。城市循环的登革热病毒严格遵循“人－伊蚊－人”的传播途径，人类是该病毒的唯一宿主，伊蚊是唯一的传播媒介。在病毒传播的过程中，感染的伊蚊通过叮咬将登革热病毒注射到人体内(病毒传播)，病毒在人皮下角质细胞及树突状细胞中增殖；病毒扩散到血液后进一步感染血液中的各类免疫细胞形成病毒血症；在病毒血症期，病毒可通过吸血的方式被 吸入到叮咬的蚊虫体内(病毒获取)。病毒感染蚊虫的唾液腺等组织，感染的伊蚊可再次传播病毒。
登革病毒的基因组由一条单链正向的RNA组成，在宿主细胞中可以翻译成一条多聚蛋白的肽链，之后该肽链被水解成三个结构蛋白(Structural protein)及七个非结构蛋白(Non-structural protein，NS)。其中非结构蛋白1(NS1)可从感染细胞中分泌，大量存在于感染宿主的血清中。在登革病毒感染急性期，病人血清中的NS1的量在70-15,000ng/ml，在某些病人体内，血清中NS1的量可以达到50,000ng/ml。因此，NS1被作为临床上检测早期登革病毒感染的主要指标之一。研究证明，NS1在登革热及登革出血热的致病机制中起到非常重要的作用。NS1的抗体可与人类血管内皮细胞非特异性的结合，从而导致内皮细胞的通透性增加，导致出血倾向；此外，NS1的抗体还可以沉积到血小板表面，引起血小板凝血障碍并导致出血增加。目前有多个NS1蛋白的抗原表位被认为与登革热的致病相关。此外，由于在病毒血症期的病人血清中NS1与登革病毒共同大量存在，NS1蛋白与病毒可通过吸血的方式同时被吸入到叮咬的蚊虫体内。因此NS1在病毒获取的过程中起到重要作用。
在普通小鼠及猴子等模式动物中，登革病毒感染后不表现典型的登革热临床症状，或仅有轻微的病毒血症。干扰素双受体缺陷型小鼠(IFN-alpha/gamma receptor double knock-outmouse，AG mouse)是目前登革热致病机制研究及药物研发的主要动物模型，可以产生超过106pfu/ml的病毒血症并导致动物死亡。B6背景的AG6小鼠(IFN-alpha/gamma receptor double knock-out B6 mouse)在登革病毒研究中被广泛应用。AG6小鼠对登革病毒非常敏感，接种登革2型病毒后，病毒可在AG6小鼠体内快速扩增，同时埃及伊蚊叮咬登革病毒感染的AG6小鼠后，可以吸食病毒并获得感染。
登革热发现至今已有200多年的历史，但是目前仍无特效的治疗疗法及安全有效的登革热疫苗。由于登革热致病机理的特殊性，病毒初次感染产生的抗体只对同型的病毒感染有中和作用，而对其他血清型病毒的感染则有感染增强作用。同时，病毒感染后产生的T细胞活化对登革出血热及登革休克综合症的发病也有促进作用。这种致病机理是限制登革病毒常规 亚单位疫苗、减毒活疫苗及灭活疫苗研制的主要因素之一。对于节肢动物传播的传染病(虫媒传染病)的预防，除了传统疫苗直接免疫减毒或灭活的病原体或其重组蛋白以外，还可以阻断媒介病原体在自然界的循环过程，从而降低媒介传染病在自然界的传播。因此，传播阻断疫苗是一种在宿主中免疫某种抗原蛋白阻断病原体传播途径，从而降低虫媒感染率及宿主发病率的免疫策略。作为传统疫苗的重要补充，传播阻断疫苗已经在疟疾等虫媒传染病的防治中广泛的应用。因此，利用疫苗方法降低媒介蚊虫的感染率将是登革热防治的一个有效措施。
发明公开
本发明的一个目的是提供一种蛋白。
本发明提供的蛋白质，是如下a)或b)的蛋白质：
a)由序列表中序列4所示的氨基酸序列组成的蛋白质；
b)将序列表中序列4的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由a)衍生的蛋白质。
上述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
编码上述蛋白质的DNA分子也是本发明保护的范围。
上述DNA分子为如下1)-3)中任一种的DNA分子：
1)编码区为序列表中序列3所示的DNA分子；
2)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码权利要求1所述蛋白质的DNA分子；
3)在严格条件下与1)限定的DNA序列杂交且编码上述蛋白质的DNA分子。
上述严格条件可为在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
含有上述DNA分子的重组载体、表达盒、转基因细胞系、重组菌或重组病毒也是本发明保护的范围；
或由上述蛋白质制备的抗体也是本发明保护的范围。
上述蛋白质或上述DNA分子或上述的重组载体、表达盒、转基因细胞 系、重组菌、重组病毒或抗体在制备如下1)-5)中至少一种产品中的应用也是本发明保护的范围：
1)登革热疫苗；
2)具有阻断人、其他灵长类或其他对登革病毒易感的哺乳动物感染登革热病毒功能；
3)具有阻断蚊虫从人、其他灵长类或其他对登革病毒易感的哺乳动物获取登革热病毒功能；
4)具有抑制蚊虫获取或传播登革热病毒的功能；
5)具有阻抑由登革热病毒或登革热病毒NS1蛋白抗体引起的出血热的发生功能。
上述应用中，所述哺乳类动物为小鼠；
所述蚊虫为埃及伊蚊或白纹伊蚊。
上述应用中，所述产品为试剂盒。
本发明另一个目的是提供一种具有如下1)-5)中至少一种功能产品。
本发明提供的产品，其活性成分为上述蛋白质；
1)登革热疫苗；
2)具有阻断人、其他灵长类或其他对登革病毒易感的哺乳动物感染登革热病毒功能；
3)具有阻断蚊虫从人、其他灵长类或其他对登革病毒易感的哺乳动物获取登革热病毒功能；
4)具有抑制蚊虫获取或传播登革热病毒的功能；
5)具有阻抑由登革热病毒或登革热病毒NS1蛋白抗体引起的出血热的发生功能。
上述产品中，所述哺乳类动物为小鼠；
所述蚊虫为埃及伊蚊或白纹伊蚊。
上述产品为药物。
上述登革病毒为DENV2。
附图说明
图1为被动免疫DENV2 NS1和DENV2 △NS1蛋白抗体效价鉴定；
图2为被动免疫DENV2 NS1抗血清阻断AG6小鼠体内NS1的产生；
图3为被动免疫DENV2 NS1抗血清降低伊蚊上登革病毒感染率；
图4为主动免疫DENV2 NS1和DENV2 △NS1蛋白阻断AG6小鼠中体内NS1的产生；
图5为主动免疫DENV2 NS1和DENV2 △NS1蛋白降低埃及伊蚊上登革病毒感染率；
图6为主动免疫DENV2 △NS1降低AG6小鼠体内的DENV2病毒载量；
图7为各组织中的伊文斯兰含量的标取函数；
图8为主动免疫DENV2 NS1和DENV2 △NS1降低AG6小鼠在登革热感染时各器官的出血症状；
图9为主动免疫DENV2 NS1和DENV2 △NS1降低AG6小鼠在登革热感染时的死亡率。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、DENV2 △NS1蛋白的获得
通过原核表达DENV2 NS1和DENV2 △NS1蛋白，具体如下：
1、DENV2 NS1基因和DENV2 △NS1基因的获得
1)DENV2 NS1基因
提取DENV2病毒的总RNA，反转录得到cDNA作为模板，用F1和R1引物对PCR扩增，得到1056bp的PCR产物，即为DENV2 NS1基因，经过测序，其核苷酸序列为序列1，其编码的蛋白为DENV2 NS1，该蛋白的氨基酸长度为352AA，氨基酸序列为序列2。
2)DENV2 △NS1基因
提取DENV2病毒的总RNA，反转录得到cDNA作为模板进行扩增。
第一步：以DENV2病毒的cDNA作为模板，以F2和R2引物对进行PCR扩增，得到1-115AA的PCR产物；第二步：以DENV2病毒的cDNA作为模板，以F3和R3引物对进行扩增，得到120-220AA的PCR产物；第三步：以DENV2病毒的cDNA作为模板，以F4和R4引物对进行PCR扩增，得到267-310AA的PCR产物；第四步：以DENV2病毒的cDNA作为模板，以F5 和R5引物对进行扩增，得到331-352AA的PCR产物；第五步：以第一步和第二步得到的PCR产物作为模板，以F6和R6引物对进行扩增，得到1-220AA(△116-119AA)的PCR产物；第六步：以第三步和第四步得到的PCR产物作为模板，以F7和R7引物对进行扩增，得到267-352AA(△311-330AA)的PCR产物；第七步：以第五步和第六步得到的PCR产物作为模板，以F8和R8引物对进行扩增，得到1-352AA(△116-119AA，△221-266AA，△311-330AA)的PCR产物，最终得到846bp的PCR产物，即为DENV2△NS1基因，经过测序，其核苷酸序列为序列3，其编码的蛋白为DENV2△NS1，该蛋白的氨基酸序列为序列4。
上述构建引物如下：
F1：5’-GGCATTCCATATGGATAGTGGTTGCGTTG-3’；
R1：5’-TAATTCCTCGAGGGCTGTGACCAAGGA-3’；
F2：5’-GGCATTCCATATGGATAGTGGTTGCGTTG-3’；
R2：5’-GAGAGCATTTTCGCTTTCCATGAATACTTCAGC-3’；
F3：5’-GCTGAAGTATTCATGGAAAGCGAAAATGCTCTC-3’；
R3：5’-TACCTAGATGCCATGGAACTTCGATGAAAGAG-3’；
F4：5’-CTCTTTCATCGAAGTTCCATGGCATCTAGGTA-3’；
R4：5’-CTGATTTCCATCCCGTATTCTGTTATGAGTTTTCC-3’；
F5：5’-GGAAAACTCATAACAGAATACGGGATGGAAATCAG-3’；
R5：5’-TAATTCCTCGAGGGCTGTGACCAAGGA-3’；
F6：5’-GGCATTCCATATGGATAGTGGTTGCGTTG-3’；
R6：5’-TACCTAGATGCCATGGAACTTCGATGAAAGAG-3’。
F7：5’-CTCTTTCATCGAAGTTCCATGGCATCTAGGTA-3’；
R7：5’-TAATTCCTCGAGGGCTGTGACCAAGGA-3’；
F8：5’-GGCATTCCATATGGATAGTGGTTGCGTTG-3’；
R8：5’-TAATTCCTCGAGGGCTGTGACCAAGGA-3’。
2、表达载体的构建
将上述序列1所示的DENV2 NS1基因插入pET-28a(+)载体的NdeI和XhoI的酶切位点得到的重组载体，命名为pET-28a(+)-DENV2 NS1。
将上述序列3所示的DENV2△NS1基因插入pET-28a(+)载体的NdeI 和XhoI的酶切位点得到的重组载体，命名为pET-28a(+)-DENV2△NS1。
3、蛋白的表达纯化
将上述pET-28a(+)-DENV2 NS1和pET-28a(+)-DENV2△NS1分别导入大肠杆菌BL21(DE3)，得到重组菌BL21(DE3)/pET-28a(+)-DENV2 NS1和BL21(DE3)/pET-28a(+)-DENV2△NS1。
将上述重组菌的单克隆菌落分别接种至5ml LB液体培养基中(含25μg/ml卡那霉素)，37℃、220rpm震荡培养16小时；然后按1:100的体积比接种至新鲜的LB液体培养基(含25μg/ml卡那霉素)，总体积为200ml，37℃、220rpm震荡培养4小时；然后加入IPTG并使其浓度为1mM，37℃、220rpm震荡培养6小时；室温、4000rpm离心10min，分别收集菌体。
取得到的菌体，用包涵体洗涤液(溶剂为pH8.0、50mM的Tris-HCl缓冲液，含有100mM NaCl、5mM EDTA、0.1％NaN3、0.5％Triton-X 100,用前加入终浓度为0.1mM的PMSF和1mM的DTT)悬浮菌体，冰上超声破碎(60％功率，超声3s，停止9s，总超声处理时间5min)，6000rpm离心15min，收集沉淀(包涵体)。
用包涵体洗涤液悬浮上述得到的沉淀，加入过量的MgSO4以中和包涵体洗涤液中的EDTA，然后加入终浓度为0.01mg/ml的DNA酶(Sigma公司目录号DN25)和终浓度为0.1mg/ml的溶菌酶(Sigma公司目录号L6876)，室温处理20分钟，6000rpm离心15min，收集包涵体；然后，用包涵体洗涤液悬浮步骤8得到的沉淀，加入过量的MgSO4以中和包涵体洗涤液中的EDTA，室温处理20分钟，6000rpm离心15min，收集包涵体，得到DENV2 NS1包涵体和DENV2 △NS1包涵体。
将收集的包涵体进行聚丙烯酰胺凝胶电泳，显示清晰的目的条带，DENV2 NS1约为42kD，DENV2 △NS1约为31kD。
分别使用蛋白纯化液(溶剂为pH8.0、100mM的Tris缓冲液，含有50mM甘氨酸和8M尿素)溶解上述得到的包涵体，得到DENV2 NS1粗蛋白溶液和DENV2 △NS1粗蛋白溶液。
使用TALON purification Kit(Clontech公司目录号635515)，从粗蛋白溶液中纯化DENV2 NS1成熟肽，具体如下：将1ml Resin和50ml粗 蛋白溶液4℃共孵育2小时，然后转移至滤柱中，用20ml洗涤液甲(TALON试剂盒中的1X平衡液加入8M尿素)进行洗涤，再用20ml洗涤液乙(TALON试剂盒中的1X平衡液加入8M尿素和20mM咪唑)进行洗涤，最后用5ml洗脱液丙(TALON试剂盒中的1X平衡液加入8M尿素和150mM咪唑)洗涤，收集5ml洗脱液为DENV2 NS1蛋白；
采用同样的方法纯化DENV2 △NS1，收集5ml洗脱液为DENV2 △NS1蛋白。
上述蛋白纯度大于95％，浓度大于1mg/ml。
实施例2、DENV2 NS1和DENV2△NS1在被动免疫阻碍登革病毒获取的应用
一、制备DENV2 NS1鼠源抗血清和DENV2 △NS1鼠源抗血清
1.鼠源抗血清的制备。
实验动物为8周大小的Balb/c雌鼠，实验流程如下：
(1).第1天(初次免疫)，每只小鼠腹腔注射上述一中制备的DENV2NS1和DENV2 △NS1蛋白分别与弗氏完全佐剂的等体积混合物(含40ugDENV2 NS1或DENV2 △NS1蛋白)。
(2).第14天(第一次加强免疫)，每只小鼠皮下注射上述一制备的DENV2 NS1和DENV2 △NS1蛋白与弗氏不完全佐剂的等体积混合物(含40ugDENV2 NS1或DENV2 △NS1蛋白)。
(3).第28天(第二次加强免疫)，每只小鼠皮下注射上述一制备的DENV2 NS1和DENV2 △NS1蛋白与弗氏不完全佐剂的等体积混合物(含40ugDENV2 NS1或DENV2 △NS1蛋白)。
(4).第42天，每只小鼠采集500ul血清，即为抗DENV2 NS1抗体(DENV2 NS1免疫得到的血清)和抗DENV2 △NS1抗体(DENV2 △NS1免疫得到的血清)。
(5).第42天，正常饲养的无免疫过的小鼠采集500ul血清，即为抗Pre-immune对照抗体。
2.检测抗体效价
(1).将1ug DENV2 NS1蛋白铺到96孔板上，4度过夜；
(2).300ul 1％BSA室温封闭1小时；
(3).将抗DENV2 NS1抗体(Anti-DENV2 NS1)和抗DENV2 △NS1抗体(Anti-DENV2 △NS1)以及对照Pre-immune抗体用1:15BSA梯度稀释1/250至1/10,000,000，每孔加入100ul，室温结合2小时；
(4).PBST洗板5次，加入100ul 1/5000稀释的鼠二抗(JM-6402-05,MBL,Japan)，室温孵育1小时；
(5).PBST洗板6次，加入100ul TMB(52-00-01and 50-85-04,Kirkegaard&Perry Laboratories)显色液进行显色，并用硫酸终止显色，读取OD450。
结果如图1所示，第一次免疫小鼠之后，抗DENV2 NS1抗体和抗DENV2△NS1抗体大量产生，至采集血清(第42天)的时候，小鼠血液中的抗血清已经达到峰值。抗DENV2 NS1抗体和抗DENV2 △NS1抗体都能完全检测全长的DENV2 NS1蛋白，证明被动免疫小鼠制备了成功的抗DENV2 NS1抗体和抗DENV2 △NS1抗体。
二、抗DENV2 NS1抗体和抗DENV2 △NS1抗体在阻断伊蚊获取登革病毒中的应用
1、被动免疫DENV2NS1抗血清并验证NS1的阻断效果。
通过腹腔注射将1x106pfu DENV2(AF204178，Guo et al.,2013)病毒注射到AG6(IFN-alpha/gamma receptor double knock-out B6 mouse)小鼠体内,12小时之后将上述一制备的抗DENV2 NS1抗体(anti-DENV2 NS1)或Pre-immune对照血清通过两点腹腔注射打入感染登革病毒的AG6小鼠体内；在血清注射后的12hr，2天，3天，4天，5天，通过小鼠尾静脉取血，并收集血清样品。
使用NS1检测试剂盒(Diagnoser automata；8404-25)检测小鼠血清中的NS1的含量，来鉴定在小鼠体内的NS1的阻断效果。
结果如图2所示，注射抗DENV2 NS1抗体组，AG6小鼠中NS1的含量几乎检测不到；注射Pre-immune血清的对照组，AG6小鼠中的NS1含量正常,随着登革病毒感染逐渐上升。
上述结果表明，被动免疫DENV2 NS1的抗血清可以成功中和AG6小鼠体内DENV2病毒产生的NS1，达到阻断NS1蛋白作用的效果。
2、埃及伊蚊通过叮咬感染小鼠获取登革病毒
1)通过腹腔注射将1x106pfu DENV2(AF204178，Guo et al.,2013)病毒注射到AG6小鼠体内；
2)12个小时后，腹腔注射100ul抗DENV2 NS1抗体或100ul Pre-immune血清到感染登革病毒的AG6小鼠体内；
3)在抗血清注射的12小时后(即登革病毒注射的一天后)，通过腹腔注射戊巴比妥钠(130ul/10g体重)麻醉小鼠，将麻醉后的小鼠放置在饲养待叮咬蚊子的容器上，让伊蚊吸血30分钟；
4)将吸血的伊蚊放置在4度冰上10分钟进行麻醉，从麻醉的蚊子中挑取充分吸血的伊蚊转移至新的容器中饲养；
5)在病毒注射后的第二天、第三天和第四天，重复步骤3-4，让伊蚊从AG6小鼠身上吸血，并挑取成功吸血的伊蚊；
6)检测伊蚊的感染率，方法如下：
A.吸血的伊蚊在饲养8天后，-80度冷冻5分钟处死，将单个伊蚊放入预置了RNA抽提液的EP管中，用研磨棒磨碎并充分匀浆。
B.使用RNA抽提试剂盒抽取伊蚊的总RNA并反转录为cDNA，利用Taqman RT-QPCR检测埃及伊蚊体内的DENV2病毒感染率。
C.伊蚊的感染率计算公式为：感染了的伊蚊数目/实验中的伊蚊总数目；统计DENV2 NS1抗血清组和Pre-immune组伊蚊第一到第四天的DENV2病毒感染率。
结果如图3所示，抗DENV2 NS1抗体组的伊蚊的感染率和Pre-immune组相比，显著性的降低，表明抗DENV2 NS1抗体可以阻断伊蚊从AG6小鼠体内获取病毒。
实施例3、DENV2 NS1和DENV2 △NS1在作为登革热疫苗中的应用
一、DENV2 NS1蛋白和DENV2 △NS1蛋白主动免疫AG6小鼠
在AG6小鼠中主动免疫实施例1中制备的DENV2 NS1蛋白和DENV2 △NS1蛋白，免疫小鼠为6周大小的AG6小鼠，实验分为三组，PBS对照组，DENV2 NS1以及DENV2 △NS1实验组，每组12只AG6小鼠，免疫方法如下：
1、第1天(初次免疫)
DENV2 NS1实验组：每只AG6小鼠腹腔注射实施例1制备的DENV2 NS1与弗氏完全佐剂的等体积混合物(每只小鼠40ugDENV2 NS1)；
DENV2 △NS1实验组：每只AG6小鼠腹腔注射实施例1制备的DENV2△NS1与弗氏完全佐剂的等体积混合物(每只小鼠40ugDENV2△NS1)；
对照组：每只AG6小鼠腹腔注射PBS与弗氏完全佐剂的等体积混合物。
得到各组初次免疫小鼠。
2、第14天(第一次加强免疫)：按照上述1的方法对各组初次免疫小鼠进行免疫，得到各组第一次加强免疫小鼠；
3、第28天(第二次加强免疫)：按照上述1的方法对各组第一次加强免疫小鼠进行免疫，得到各组第二次加强免疫小鼠；
4、第42天通过腹腔两点注射1x106pfu DENV2(AF204178)病毒到上述各组两次加强免疫小鼠体内；分别在病毒注射1天，2天，3天，4天，5天，通过小鼠尾静脉取血，并收集血清样品。
使用NS1检测试剂盒检测小鼠血清中的NS1的含量，来鉴定在小鼠体内的NS1的阻断效果，结果如图4所示，DENV2 NS1组以及DENV2 △NS1组，AG6小鼠中NS1的含量几乎检测不到；PBS对照组，AG6小鼠中的NS1含量正常,随着登革病毒感染逐渐上升。
二、DENV2 NS1蛋白和DENV2 △NS1蛋白阻断埃及伊蚊从动物体内获取登革病毒
1、分别将上述一中4步骤中DENV2 NS1组、DENV2 △NS1组和PBS对照组病毒注射1天、2天、3天和4天的小鼠，通过腹腔注射戊巴比妥钠(130ul/10g体重)麻醉小鼠，将麻醉后的小鼠放置在饲养待叮咬蚊子的容器上，让伊蚊吸血30分钟；将吸血的伊蚊放置在4度冰上10分钟进行麻醉，从麻醉的蚊子中挑取充分吸血的伊蚊转移至新的容器中饲养；
得到病毒注射1天、2天、3天和4天DENV2 NS1组伊蚊、病毒注射1天、2天、3天和4天DENV2 △NS1组组伊蚊和病毒注射1天、2天、3天和4天PBS对照组伊蚊。
2、将上述各组吸血的伊蚊子在饲养8天后，-80度冷冻5分钟处死，将单个伊蚊放入预置了RNA抽提液的EP管中，用研磨棒磨碎并充分匀浆；
3.使用RNA抽提试剂盒抽取上述2伊蚊的总RNA并反转录为cDNA，利用Taqman RT-QPCR检测埃及伊蚊体内的DENV2型病毒感染率；
用于Taqman RT-QPCR检测DENV2型病毒的引物对如下：
上游引物：5’-CATTCCAAGTGAGAATCTCTTTGTCA-3’；
下游引物：5’-CAGATCTCTGATGAATAACCAACG-3’。
用于Taqman RT-QPCR检测登革热2型病毒的探针如下：
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
采用埃及伊蚊Actin基因(AAEL011197)作为内参，其Taqman RT-QPCR的引物对如下：
上游引物：5’-GAACACCCAGTCCTGCTGACA-3’；
下游引物：5’-TGCGTCATCTTCTCACGGTTAG-3’。
用于Taqman RT-QPCR检测伊蚊Actin基因的探针如下：
5’-FAM-AGGCCCCGCTCAACCCGAAG-TRAMA-3’。
使用Actin和DENV2通过RT-QPCR检测得到的△Ct值(Actin的Ct值减去DENV2的Ct值)进行计算，当2-△Ct的值大于0.0002时，该伊蚊被认定为DENV2感染阳性。
伊蚊的感染率计算公式为：感染了的伊蚊数目/实验中的伊蚊总数目；
统计DENV2 NS1组，DENV2 △NS1和PBS对照组在伊蚊第一到第四天的DENV2病毒感染率；
结果如图5所示，DENV2 NS1和DENV2 △NS1组的伊蚊的感染率和PBS对照组相比，显著性的降低，证明主动免疫DENV2 NS1和DENV2 △NS1均可以阻断伊蚊从AG6小鼠体内获取病毒；其中免疫DENV2 △NS1组与DENV2NS1组相比，伊蚊的感染率额外下降了2-3倍，显示出更好的阻断效果。
三、DENV2 NS1蛋白和DENV2 △NS1蛋白阻抑出血热的发生
A、DENV2病毒载量的检测
在AG6小鼠中主动免疫实施例1中制备的DENV2 NS1蛋白和DENV2 △NS1蛋白，免疫小鼠为6周大小的AG6小鼠，实验分为三组，PBS对照组，DENV2 NS1以及DENV2 △NS1实验组，每组12只AG6小鼠，免疫方法如下：
1、第1天(初次免疫)
DENV2 NS1实验组：每只AG6小鼠腹腔注射实施例1制备的DENV2 NS1与弗氏完全佐剂的等体积混合物(每只小鼠40ug DENV2 NS1)；
DENV2 △NS1实验组：每只AG6小鼠腹腔注射实施例1制备的DENV2△NS1与弗氏完全佐剂的等体积混合物(每只小鼠40ugDENV2△NS1)；
对照组：每只AG6小鼠腹腔注射PBS与弗氏完全佐剂的等体积混合物。
得到各组初次免疫小鼠。
2、第14天(第一次加强免疫)：按照上述1的方法对各组初次免疫小鼠进行免疫，得到各组第一次加强免疫小鼠；
3、第28天(第二次加强免疫)：按照上述1的方法对各组第一次加强免疫小鼠进行免疫，得到各组第二次加强免疫小鼠；
4、第42天通过腹腔两点注射1x106pfu DENV2(AF204178)病毒到上述各组两次加强免疫小鼠体内；分别在病毒注射1天，2天，3天，4天，5天，通过小鼠尾静脉取血15ul，放入预置了RNA抽提液的EP管中，并充分匀浆。
使用RNA抽提试剂盒抽取AG6小鼠全血中总RNA并反转录为cDNA，利用Taqman RT-QPCR检测AG6小鼠血液中DENV2病毒载量(小鼠Actin基因的表达量作为内参)。
用于Taqman RT-QPCR检测DENV2型病毒的引物对如下：
上游引物：5’-CATTCCAAGTGAGAATCTCTTTGTCA-3’；
下游引物：5’-CAGATCTCTGATGAATAACCAACG-3’。
用于Taqman RT-QPCR检测登革热2型病毒的探针如下：
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
采用小鼠Actin基因(NM_007393)作为内参，其Taqman RT-QPCR的引物对如下：
上游引物：5’-AGCCATGTACGTAGCCATCCA-3’；
下游引物：5’-TCTCCGGAGTCCATCACAATG-3’。
用于Taqman RT-QPCR检测小鼠Actin基因的探针如下：
5’-FAM-TGTCCCTGTATGCCTCTGGTCGTACCAC-TRAMA-3’。
使用Actin和DENV2通过RT-QPCR检测得到的△Ct值(Actin的Ct值减去DENV2的Ct值)进行计算，定义2-△Ct的值为该AG6小鼠的DENV2病毒载量。
结果如图6所示，DENV2 NS1组和PBS对照组的登革病毒载量基本相同，而DENV2 △NS1组的登革病毒载量下降了3-10倍，证明缺失了产生副作用位点的重组DENV2 △NS1，可以降低登革病毒在AG6小鼠上的感染。
B、检测感染AG6小鼠的出血症状
1、在上述第42天通过腹腔两点注射1x106pfu DENV2(AF204178)病毒到上述各组两次加强免疫小鼠体内；
2、在第18天时，通过小鼠尾静脉注射150ul伊文斯兰(Evans blue，0.5％PBS溶液)到各组小鼠体内。
3、在伊文斯兰注射2小时后，通过腹腔注射戊巴比妥钠(130ul/10g体重)麻醉小鼠，解剖小鼠，使用PBS对小鼠进行灌注，直至血液被完全清除。
4、收取小鼠的肾，肝，脾脏，小肠，大肠和胃等器官，使用甲酰胺(Formamide)浸泡组织(2ml/100mg组织)将渗透在组织中的伊文斯兰萃取出来。
5.离心去除组织碎片，使用分光光度计检测上清液中的伊文斯兰在610nm处的吸光度，根据标准品曲线计算出各组织中的伊文斯兰的量(标准曲线见图7)。
结果如图8所示，与对照PBS组相比，DENV2 NS1小鼠和DENV2 △NS1小鼠组织中的伊文斯兰的量显著性的降低，表明免疫这两种蛋白，均可以降低AG6小鼠在感染登革病毒时产生的出血倾向；其中免疫DENV2 △NS1组的比DENV2 NS1组伊文斯兰的量更低，表明它具有更好的保护效果。
C、观察感染小鼠的死亡率
1、在上述第42天通过腹腔两点注射1x106pfu DENV2(AF204178)病毒到上述各组两次加强免疫小鼠体内；
2、从病毒感染第1天到第40天每天监控小鼠的状况，记录每只小鼠的死亡时间，使用Kaplan-Meier法对各组小鼠的生存曲线进行分析。
结果如图9所示，和对照PBS组相比，DENV2 NS1组小鼠和DENV2 △NS1组小鼠生存率均明显提高；PBS组40天生存率为0，DENV2 NS1组为33％，DENV2 △NS1为75％，证明主动免疫DENV2 NS1和DENV2 △NS1蛋白均可以保护AG6小鼠免除死亡；其中DENV2 △NS1免疫组有着更好的保护效果。
上述结果均表明，DENV2 NS1和DENV2 △NS1蛋白可作为登革热疫苗，用于预防和治疗登革热，且可以阻断伊蚊从动物体感染登革热病毒。
工业应用
本发明基于登革病毒非结构蛋白1(DENV NS1)，剔除有害抗原表位得到改造登革病毒非结构蛋白1(DENV ΔNS1)，将其作为抗原进行免疫人类或哺乳动物宿主。免疫改造后的登革病毒非结构蛋白1同时起到抑制蚊虫带毒和动物感染的双效作用，可降低登革病毒感染引起的动物宿主出血及死亡，同时降低蚊虫媒介的带毒率，达到防治登革热的目的。该双效疫苗可保护人类或哺乳动物出现登革出血热并阻断登革病毒通过蚊子在自然界的传播。

Claims (15)

  1. 一种蛋白质，是如下a)或b)的蛋白质：
    a)由序列表中序列4所示的氨基酸序列组成的蛋白质；
    b)将序列表中序列4的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由a)衍生的蛋白质。
  2. 编码权利要求1所述蛋白质的DNA分子。
  3. 根据权利要求2所述的DNA分子，其特征在于：所述DNA分子为如下1)-3)中任一种的DNA分子：
    1)编码区为序列表中序列3所示的DNA分子；
    2)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码权利要求1所述蛋白质的DNA分子；
    3)在严格条件下与1)限定的DNA序列杂交且编码上述蛋白质的DNA分子。
  4. 含有权利要求2或3所述DNA分子的重组载体、表达盒、转基因细胞系、重组菌或重组病毒。
  5. 由权利要求1所述蛋白质制备的抗体。
  6. 根据权利要求5所述的抗体，其特征在于：所述抗体为多克隆抗体。
  7. 权利要求1所述蛋白质或权利要求2或3所述DNA分子或权利要求4所述的重组载体、表达盒、转基因细胞系、重组菌或重组病毒或权利要求5或6所述的抗体在制备如下1)-6)中至少一种产品中的应用：
    1)登革热疫苗；
    2)具有阻断人、其他灵长类或其他对登革病毒易感的哺乳动物感染登革热病毒功能的产品；
    3)具有阻断蚊虫从人、其他灵长类或其他对登革病毒易感的哺乳动物获取登革热病毒功能的产品；
    4)具有抑制蚊虫获取或传播登革热病毒的功能的产品；
    5)具有阻抑由登革热病毒或登革热病毒NS1蛋白抗体引起的出血热的发生功能的产品；
    6)具有抑制蚊虫带毒和抑制动物感染登革热病毒的双效功能的产品。
  8. 根据权利要求7所述的应用，其特征在于：
    所述哺乳类动物为小鼠；
    所述蚊虫为埃及伊蚊或白纹伊蚊。
  9. 根据权利要求7或8所述的应用，其特征在于：所述产品为试剂盒或药品。
  10. 权利要求1所述蛋白质或权利要求2或3所述DNA分子或权利要求4所述的重组载体、表达盒、转基因细胞系、重组菌或重组病毒或权利要求5或6所述的抗体在如下1)-5)中至少一种中的应用：
    1)阻断人、其他灵长类或其他对登革病毒易感的哺乳动物感染登革热病毒；
    2)阻断蚊虫从人、其他灵长类或其他对登革病毒易感的哺乳动物获取登革热病毒；
    3)抑制蚊虫获取或传播登革热病毒；
    4)阻抑由登革热病毒或登革热病毒NS1蛋白抗体引起的出血热；
    5)抑制蚊虫带毒和抑制动物感染登革热病毒。
  11. 根据权利要求10所述的应用，其特征在于：
    所述哺乳类动物为小鼠；
    所述蚊虫为埃及伊蚊或白纹伊蚊。
  12. 一种具有如下1)-6)中至少一种功能产品，其活性成分为权利要求1所述蛋白质或权利要求5或6所述的抗体；
    1)登革热疫苗；
    2)具有阻断人、其他灵长类或其他对登革病毒易感的哺乳动物感染登革热病毒功能的产品；
    3)具有阻断蚊虫从人、其他灵长类或其他对登革病毒易感的哺乳动物获取登革热病毒功能的产品；
    4)具有抑制蚊虫获取或传播登革热病毒的功能的产品；
    5)具有阻抑由登革热病毒或登革热病毒NS1蛋白抗体引起的出血热 的发生功能的产品；
    6)具有抑制蚊虫带毒和抑制动物感染登革热病毒的双效功能的产品。
  13. 根据权利要求12所述的产品，其特征在于：
    所述哺乳类动物为小鼠；
    所述蚊虫为埃及伊蚊或白纹伊蚊。
  14. 根据权利要求12或13所述的产品，其特征在于：所述产品还包括佐剂。
  15. 根据权利要求12-14任一所述的产品，其特征在于：所述产品为药物或试剂盒。
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