CN105601721B - A kind of preparation method and applications of dengue fever dual-purpose vaccine - Google Patents

A kind of preparation method and applications of dengue fever dual-purpose vaccine Download PDF

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CN105601721B
CN105601721B CN201510830594.3A CN201510830594A CN105601721B CN 105601721 B CN105601721 B CN 105601721B CN 201510830594 A CN201510830594 A CN 201510830594A CN 105601721 B CN105601721 B CN 105601721B
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CN105601721A (en
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程功
刘建英
刘洋
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Tsinghua University
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Abstract

The invention discloses a kind of preparation method and applications of dengue fever dual-purpose vaccine, and the present invention provides a kind of protein, are following protein a) or b)：A) protein that amino acid sequence forms shown in sequence in sequence table 4；B) amino acid sequence of sequence in sequence table 4 by the substitution of one or several amino acid residues and/or is lacked and ored add and the protein with the same function derived from a).The experiment proves that; the present invention obtain transformation dengue virus non-structural protein 1 (DENV Δ NS1) can be used as vaccine, which can protect the mankind or dengue hemorrhagic fever occurs in mammal and block dengue virus by mosquito nature propagation.

Description

A kind of preparation method and applications of dengue fever dual-purpose vaccine
Technical field
The invention belongs to dengue virus prevention and control field, the specially a kind of preparation and its application of dengue virus dual-purpose vaccine.
Background technology
Dengue fever (Dengue Fever, DF) is by her mosquito-borne acute viral infectious disease.Dengue virus (Dengue Virus, DENV) it is spherical in shape, it is made of after birth, capsid protein, non-structural protein bletilla single-stranded positive RNA.In nature, Dengue disease For poison there are four kinds of serotypes (DENV-1~DENV-4), the nucleotide sequence difference of each serotype is very big, up to 35% or more.Often In a serotype, and multiple gene hypotypes can be divided into according to the difference of epidemic regions.Dengue virus is bitten via yellow-fever mosquito infects people After body, clinical symptoms can be shown by hiding through 2-3 days.The Clinical symptoms of Dengue is high fever, bone and DOMS, bleeding is inclined To and white blood cell count(WBC) significantly reduce.Dengue fever clinical manifestation is slighter, and with disease progression most people self-healing, lethality is low； But the course of disease of a few peoples can proceed to next stage, i.e., dengue hemorrhagic fever (Dengue Hemorrhagic Fever, DHF) and step on Shock-syndrome (Dengue Shock Syndrome, DSS) is removed from office, this is a kind of serious types of dengue infection, clinical manifestation It is significantly reduced for each organ severe haemorrhage, high fever, platelet count, pachyemia, shock, causes the death rate pole of the course of disease It is high.
Dengue virus mainly bites the propagation of human body postoperative infection by Aedes aegypti (Aedes aegypti).There is no it is Egyptian she The area of mosquito, aedes albopictus (Aedes albopictus) are also considered as the important medium of Dengue heat propagation.It is now recognized that stepping on Remove from office natural reservoir (of bird flu viruses) only someone, low primate and the yellow-fever mosquito of virus.In nature, dengue virus mainly follows in both of which Ring：1) city recycles：In the dengue prevalence of urban type, virus is propagated between " people-yellow-fever mosquito-people "；2) jungle recycles： Mainly by being propagated between yellow-fever mosquito in matto and low primate, this cycle is the original loop of dengue virus.According to Research, jungle type dengue virus differ greatly with the dengue virus under the cycling condition of city, but can not rule out jungle cycle Virus is diffused to city and rural possibility by infection yellow-fever mosquito.City cycle dengue fever virus follow strictly " people-she The route of transmission of mosquito-people ", the mankind are unique hosts of the virus, and yellow-fever mosquito is unique communication media.In the mistake of viral transmission Cheng Zhong, for the yellow-fever mosquito of infection by biting dengue fever virus injection into the human body (viral transmission), virus cutin under fell is thin It is proliferated in born of the same parents and Dendritic Cells；Virus is diffused into all kinds of immunocytes further infected after blood in blood and forms viral blood Disease；In the viremia virusemia phase, virus can be inhaled into the mosquito body bitten (virus obtains) by way of sucking blood.Virus infection The tissues such as the salivary gland of mosquito, the yellow-fever mosquito of infection can transmitted virus again.
The genome of dengue virus is made of the RNA of a single-stranded forward direction, can be translated into more than one in host cell The peptide chain of polyprotein, later the peptide chain be hydrolyzed into three structural proteins (Structural protein) and seven non-structural proteins (Non-structural protein, NS) in vain.Wherein non-structural protein 1 (NS1) can be secreted from infection cell, largely be existed In the serum of infection host.In dengue virus infection acute stage, the amount of the NS1 in patients serum in 70-15,000ng/ml, It is certain in patient body, the amount of NS1 can reach 50,000ng/ml in serum.Therefore, NS1 is by as clinically detection early stage One of leading indicator of dengue virus infection.Research has shown that NS1 plays non-in the pathogenic mechanism of dengue fever and dengue hemorrhagic fever Normal important role.The antibody of NS1 can with the nonspecific combination of Human vascular endothelial's cell, so as to cause the logical of endothelial cell Permeability increases, and leads to hemorrhagic tendency；In addition, the antibody of NS1 can also deposit to platelet surface, cause blood platelet blood coagulation disorders And bleeding is caused to increase.There is the epitope of multiple NS1 albumen to be considered pathogenic related to dengue fever at present.Further, since NS1 largely exists jointly with dengue virus in the patients serum of viremia virusemia phase, and NS1 albumen can be by the side sucked blood with virus Formula is inhaled into the mosquito body bitten simultaneously.Therefore NS1 plays an important role during virus obtains.
In common mouse and monkey isotype animal, typical dengue fever clinical condition is not showed after dengue virus infection Shape, or only slight viremia virusemia.Interferon amboceptor deficient mice (IFN-alpha/gamma receptor Double knock-outmouse, AG mouse) be current dengue fever pathogenic mechanism research and medicament research and development primary animal mould Type can be generated more than 106The viremia virusemia of pfu/ml simultaneously leads to animal dead.AG6 mouse (the IFN-alpha/ of B6 backgrounds Gamma receptor double knock-out B6mouse) it is widely used in dengue virus research.AG6 mouse pair Dengue virus is very sensitive, after being inoculated with dengue 2-type virus, virus can in AG6 Mice Bodies rapid amplifying, while Aedes aegypti stings After stinging the AG6 mouse of dengue virus infection, virus can be sucked and infected.
Dengue fever finds have more than 200 years history so far, but the treatment therapy still without special efficacy and safely and effectively at present Dengue vaccine.Due to the particularity of dengue fever pathogenesis, the antibody that virus prime infection generates is only to the virus of homotype Infected with neutralization, and then there is infection humidification to the infection of other serotypes.Meanwhile being generated after virus infection T cell activation also has facilitation to the morbidity of dengue hemorrhagic fever and Dengue shock syndrome.This pathogenesis is that limitation is stepped on The conventional subunit vaccine of leather virus, attenuated live vaccine and one of the principal element of inactivated vaccine development.For arthropod-borne Infectious disease (insect-borne infectious disease) prevention, in addition to traditional vaccine direct immunization be attenuated or inactivate pathogen or its recombinant protein In addition, medium pathogen can also be blocked in the cyclic process of nature, to reduce propagation of the vection disease in nature. Therefore, transmission_blocking vaccine is a kind of immune certain antigen protein blocking pathogen route of transmission in host, to reduce worm The immunization strategy of matchmaker's infection rate and host's incidence.As the important supplement of traditional vaccine, transmission_blocking vaccine is in malaria It is widely applied in the prevention of equal insect-borne infectious diseases.Therefore, it will be Dengue to reduce the infection rate of Mosquito Vectors using vaccine approach One effective measures of heat prevention.
Invention content
It is an object of the present invention to provide a kind of albumen.
Protein provided by the invention is following protein a) or b)：
A) protein that amino acid sequence forms shown in sequence in sequence table 4；
B) substitution by the amino acid sequence of sequence in sequence table 4 by one or several amino acid residues and/or missing And/or addition and the protein with the same function derived from a).
It the substitution of said one or several amino acid residues and/or lacks and ors add residual to be no more than 10 amino acid It the substitution of base and/or lacks and ors add.
The DNA molecular for encoding above-mentioned protein is also the scope of protection of the invention.
Above-mentioned DNA molecular is following 1) -3) in any DNA molecular：
1) code area is DNA molecular shown in sequence 3 in sequence table；
2) with the DNA sequence dna that 1) limits at least with 70%, at least with 75%, at least with 80%, at least have 85%, at least have with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least There is 99% homology and encodes the DNA molecular of protein described in claim 1；
3) hybridize under strict conditions with the DNA sequence dna 1) limited and encode the DNA molecular of above-mentioned protein.
Above-mentioned stringent condition can be to hybridize at 65 DEG C in 6 × SSC, the solution of 0.5%SDS, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
Recombinant vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus containing above-mentioned DNA molecular are also this Invent the range of protection；
Or the antibody prepared by above-mentioned protein is also the scope of protection of the invention.
Above-mentioned protein or above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line, recombinant bacterium, again Group virus or antibody are preparing following 1) -5) at least one of application in product be also the scope of protection of the invention：
1) dengue vaccine；
2) have and block people, other primates or other mammalian infections dengue fever virus work(susceptible to dengue virus Energy；
3) have and mosquito is blocked to obtain dengue fever from people, other primates or other mammals susceptible to dengue virus Viral function；
4) have the function of that mosquito is inhibited to obtain or propagate dengue fever virus；
5) have the function of to suppress the generation of the Hemorrhagic fever caused by dengue fever virus or dengue fever virus NS1 protein antibodies.
In above application, the mammal is mouse；
The mosquito is Aedes aegypti or aedes albopictus.
In above application, the product is kit.
Another object of the present invention, which is to provide, a kind of having following 1) -5) at least one of functional product.
Product provided by the invention, active constituent are above-mentioned protein；
1) dengue vaccine；
2) have and block people, other primates or other mammalian infections dengue fever virus work(susceptible to dengue virus Energy；
3) have and mosquito is blocked to obtain dengue fever from people, other primates or other mammals susceptible to dengue virus Viral function；
4) have the function of that mosquito is inhibited to obtain or propagate dengue fever virus；
5) have the function of to suppress the generation of the Hemorrhagic fever caused by dengue fever virus or dengue fever virus NS1 protein antibodies.
In the said goods, the mammal is mouse；
The mosquito is Aedes aegypti or aedes albopictus.
The said goods are drug.
Above-mentioned dengue virus is DENV2.
The present invention is based on dengue virus non-structural protein 1 (DENV NS1), reject harmful antigens epitope and obtain transformation Dengue Virus nonstructural protein 1 (DENV Δ NS1) carries out the immune mankind or mammalian hosts as antigen.After immunologic reconstitution Dengue virus non-structural protein 1 simultaneously play inhibit mosquito band poison and animal infected double effects, dengue virus can be reduced Animal reservoir's bleeding and death caused by infection, while the band poison rate of mosquito medium is reduced, achieve the purpose that prevent dengue fever.It should Dual-purpose vaccine can protect the mankind or dengue hemorrhagic fever occurs in mammal and block dengue virus by mosquito nature biography It broadcasts.
Description of the drawings
Fig. 1 identifies for passive immunity DENV2 NS1 and DENV2 △ NS1 protein antibodies potency；
Fig. 2 is the generation that passive immunity DENV2 NS1 antiserums block NS1 in AG6 Mice Bodies；
Fig. 3, which is passive immunity DENV2 NS1 antiserums, reduces the upper Infection Rate with Dengue Virus of yellow-fever mosquito；
Fig. 4 is that active immunity DENV2 NS1 and DENV2 △ NS1 albumen blocks the generation of NS1 in vivo in AG6 mouse；
Fig. 5, which is active immunity DENV2 NS1 and DENV2 △ NS1 albumen, reduces the upper Infection Rate with Dengue Virus of Aedes aegypti；
Fig. 6 is the DENV2 virus loads in active immunity DENV2 △ NS1 reduction AG6 Mice Bodies；
Fig. 7 is that the mark of the Yi Wensilan contents in each tissue takes function；
Fig. 8, which is active immunity DENV2 NS1 and DENV2 △ NS1, reduces AG6 mouse each organ in dengue infection Bleeding；
Fig. 9, which is active immunity DENV2 NS1 and DENV2 △ NS1, reduces the death rate of the AG6 mouse in dengue infection.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The acquisition of embodiment 1, DENV2 △ NS1 albumen
It is specific as follows by prokaryotic expression DENV2 NS1 and DENV2 △ NS1 albumen：
1, the acquisition of DENV2 NS1 genes and DENV2 △ NS1 genes
1) DENV2 NS1 genes
The total serum IgE of DENV2 viruses is extracted, reverse transcription obtains cDNA and obtained with F1 and R1 primer pair PCR amplifications as template To the PCR product of 1056bp, as DENV2 NS1 genes, by sequencing, nucleotides sequence is classified as sequence 1, the albumen of coding Amino acid length for DENV2 NS1, the albumen is 352AA, and amino acid sequence is sequence 2.
2) DENV2 △ NS1 genes
The total serum IgE of DENV2 viruses is extracted, reverse transcription obtains cDNA and expanded as template.
The first step：Using the cDNA of DENV2 viruses as template, PCR amplification is carried out with F2 and R2 primer pairs, obtains 1- The PCR product of 115AA；Second step：Using the cDNA of DENV2 viruses as template, is expanded, obtained with F3 and R3 primer pairs The PCR product of 120-220AA；Third walks：Using the cDNA of DENV2 viruses as template, PCR expansions are carried out with F4 and R4 primer pairs Increase, obtains the PCR product of 267-310AA；4th step：Using the cDNA of DENV2 viruses as template, carried out with F5 and R5 primer pairs Amplification, obtains the PCR product of 331-352AA；5th step：Using the PCR product that the first step and second step obtain as template, with F6 It is expanded with R6 primer pairs, obtains the PCR product of 1-220AA (△ 116-119AA)；6th step：With third step and the 4th step Obtained PCR product is expanded as template with F7 and R7 primer pairs, and the PCR of 267-352AA (△ 311-330AA) is obtained Product；7th step：Using the PCR product that the 5th step and the 6th step obtain as template, is expanded, obtained with F8 and R8 primer pairs The PCR product of 1-352AA (△ 116-119AA, △ 221-266AA, △ 311-330AA), finally obtains the PCR product of 846bp, As DENV2 △ NS1 genes, by sequencing, nucleotides sequence is classified as sequence 3, and the albumen of coding is DENV2 △ NS1, the egg White amino acid sequence is sequence 4.
Above-mentioned structure primer is as follows：
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, the structure of expression vector
DENV2 NS1 genes shown in above-mentioned sequence 1 are inserted into the digestion position of the NdeI and XhoI of pET-28a (+) carrier The recombinant vector that point obtains, is named as pET-28a (+)-DENV2 NS1.
DENV2 △ NS1 genes shown in above-mentioned sequence 3 are inserted into the digestion position of the NdeI and XhoI of pET-28a (+) carrier The recombinant vector that point obtains, is named as pET-28a (+)-DENV2 △ NS1.
3, the expression and purification of albumen
Above-mentioned pET-28a (+)-DENV2 NS1 and pET-28a (+)-DENV2 △ NS1 are directed respectively into e. coli bl21 (DE3), recombinant bacterium BL21 (DE3)/pET-28a (+)-DENV2 NS1 and BL21 (DE3)/pET-28a (+)-DENV2 △ are obtained NS1。
The monoclonal colonies of above-mentioned recombinant bacterium are seeded in 5ml LB liquid mediums to (containing 25 μ g/ml cards, that is mould respectively Element), 37 DEG C, 220rpm shake cultures 16 hours；Then 1 is pressed:100 volume ratio is seeded to fresh LB liquid medium and (contains 25 μ g/ml kanamycins), total volume 200ml, 37 DEG C, 220rpm shake cultures 4 hours；Then IPTG is added and keeps its dense Degree is 1mM, 37 DEG C, 220rpm shake cultures 6 hours；Room temperature, 4000rpm centrifuge 10min, collect thalline respectively.
The thalline obtained, with inclusion body cleaning solution, (the Tris-HCl buffer solutions of solvent pH8.0,50mM, contain 100mM NaCl, 5mM EDTA, 0.1%NaN3,0.5%Triton-X 100, with the preceding PMSF that final concentration of 0.1mM is added and The DTT of 1mM) suspension thalline, ultrasonication on ice (60% power, ultrasonic 3s stop 9s, total sonication treatment time 5min), 6000rpm centrifuges 15min, collects precipitation (inclusion body).
With inclusion body cleaning solution suspension precipitation obtained above, excessive MgSO is added4To neutralize in inclusion body cleaning solution EDTA, the DNA enzymatic (Sigma corporate directory DN25) of final concentration of 0.01mg/ml and final concentration of 0.1mg/ is then added The lysozyme (Sigma corporate directory L6876) of ml, room temperature are handled 20 minutes, and 6000rpm centrifuges 15min, collect inclusion body； Then, the precipitation obtained with inclusion body cleaning solution resuspending step 8, is added excessive MgSO4To neutralize in inclusion body cleaning solution EDTA, room temperature are handled 20 minutes, and 6000rpm centrifuges 15min, are collected inclusion body, are obtained DENV2 NS1 inclusion bodys and DENV2 △ NS1 inclusion bodys.
The inclusion body of collection is subjected to polyacrylamide gel electrophoresis, clearly purpose band, DENV2 NS1 are about for display 42kD, DENV2 △ NS1 are about 31kD.
Using protein purification liquid respectively, (the Tris buffer solutions of solvent pH8.0,100mM are urinated containing 50mM glycine and 8M Element) dissolving inclusion body obtained above, obtain DENV2 NS1 crude protein solution and DENV2 △ NS1 crude protein solution.
It is pure from crude protein solution using TALON purification Kit (Clontech corporate directories number 635515) Change DENV2 NS1 mature peptides, it is specific as follows：1ml Resin and 50ml crude protein solution is incubated 2 hours for 4 DEG C altogether, is then shifted Into filter column, washed with 20ml cleaning solutions first (8M urea is added in the 1X equilibrium liquids in TALON kits), then washed with 20ml It washs liquid second (8M urea and 20mM imidazoles is added in the 1X equilibrium liquids in TALON kits) to be washed, finally uses 5ml eluents third (8M urea and 150mM imidazoles is added in the 1X equilibrium liquids in TALON kits) washing, collection 5ml eluents are DENV2 NS1 eggs In vain；
DENV2 △ NS1 are purified using same method, collection 5ml eluents are DENV2 △ NS1 albumen.
Above-mentioned purity of protein is more than 95%, and concentration is more than 1mg/ml.
Embodiment 2, DENV2 NS1 and DENV2 △ NS1 hinder the application that dengue virus obtains in passive immunity
One, DENV2 NS1 mouse source antiserum and DENV2 △ NS1 mouse source antiserum are prepared
1. the sero-fast preparation in mouse source.
Experimental animal is the Balb/c female mices of 8 weeks sizes, and experiment flow is as follows：
(1) the 1st day (initial immunity), every mouse peritoneal inject DENV2 NS1 and the DENV2 △ prepared in above-mentioned one NS1 albumen respectively with isometric mixture of Freund's complete adjuvant (NS1 containing 40ugDENV2 or DENV2 △ NS1 albumen).
(2) the 14th day (first time booster immunization), every mouse subcutaneous injection it is above-mentioned one prepare DENV2 NS1 and Isometric mixture (NS1 containing 40ugDENV2 or the DENV2 △ NS1 eggs of DENV2 △ NS1 albumen and incomplete Freund's adjuvant In vain).
(3) the 28th day (second of booster immunization), every mouse subcutaneous injection it is above-mentioned one prepare DENV2 NS1 and Isometric mixture (NS1 containing 40ugDENV2 or the DENV2 △ NS1 eggs of DENV2 △ NS1 albumen and incomplete Freund's adjuvant In vain).
(4) the 42nd day, every mouse acquire 500ul serum, and (DENV2 NS1 are immunized as anti-DENV2 NS1 antibody To serum) and anti-DENV2 △ NS1 antibody (DENV2 △ NS1 immune obtained serum).
(5) the 42nd day, the mouse that the nothing normally raised was immunized acquire 500ul serum, as anti-Pre-immune controls Antibody.
2. detecting antibody titer
(1) 1ug DENV2 NS1 albumen is taped against on 96 orifice plates by, and 4 spend night；
(2) .300ul 1%BSA room temperatures are closed 1 hour；
(3) is by anti-DENV2 NS1 antibody (Anti-DENV2 NS1) and anti-DENV2 △ NS1 antibody (Anti-DENV2 △ NS1) and Pre-immune antibody is compareed with 1:15BSA gradient dilutions 1/250 to 1/10,000,000, it is added per hole 100ul, room temperature combine 2 hours；
(4) 1/5000 diluted mouse secondary antibodies (JM-6402-05, MBL, Japan) of 100ul, room is added in .PBST board-washings 5 times Temperature is incubated 1 hour；
(5) 100ul TMB (52-00-01and 50-85-04, Kirkegaard＆Perry is added in .PBST board-washings 6 times Laboratories) developing solution develops the color, and sulfuric acid color development stopping is used in combination, and reads OD450.
The results are shown in Figure 1, is immunized after mouse for the first time, anti-DENV2 NS1 antibody and anti-DENV2 △ NS1 antibody are big Amount generates, until when acquisition serum (the 42nd day), the antiserum in mouse blood has reached peak value.Anti- DENV2 NS1 are anti- Body and anti-DENV2 △ NS1 antibody can detect the DENV2 NS1 albumen of overall length completely, it was demonstrated that passive immunity mouse is prepared for into The anti-DENV2 NS1 antibody of work(and anti-DENV2 △ NS1 antibody.
Two, the application of anti-DENV2 NS1 antibody and anti-DENV2 △ NS1 antibody in blocking yellow-fever mosquito to obtain dengue virus
1, passive immunity DENV2NS1 antiserums and the barrier effect of NS1 is verified.
By being injected intraperitoneally 1x106Pfu DENV2 (AF204178, Guo et al., 2013) virus injection is to AG6 It, will be above-mentioned after 12 hours in (IFN-alpha/gamma receptor double knock-out B6mouse) Mice Body The one anti-DENV2 NS1 antibody (anti-DENV2 NS1) prepared or Pre-immune control serums are beaten by 2 points of intraperitoneal injections Enter to infect in the AG6 Mice Bodies of dengue virus；12hr after serum injection 2 days, 3 days, 4 days, 5 days, passes through mouse tail vein Blood is taken, and collects blood serum sample.
Use NS1 detection kits (Diagnoser automata；8404-25) detect containing for the NS1 in mice serum Amount, to identify the barrier effect of the NS1 in Mice Body.
The results are shown in Figure 2, injects anti-DENV2 NS1 antibody groups, and the content of NS1 is nearly no detectable in AG6 mouse；Note The control group of Pre-immune serum is penetrated, the NS1 contents in AG6 mouse are normal, as dengue virus infection is gradually increasing.
The above results show that the antiserum of passive immunity DENV2 NS1 can successfully neutralize DENV2 diseases in AG6 Mice Bodies The NS1 that poison generates achievees the effect that block the effect of NS1 albumen.
2, Aedes aegypti obtains dengue virus by biting infecting mouse
1) by being injected intraperitoneally 1x106Pfu DENV2 (AF204178, Guo et al., 2013) virus injection is to AG6 In Mice Body；
2) after 12 hours, the anti-DENV2 NS1 antibody of 100ul or 100ul Pre-immune serum is injected intraperitoneally to infection In the AG6 Mice Bodies of dengue virus；
3) behind 12 hours of antiserum injection (i.e. behind one day of dengue virus injection), by the way that amobarbital is injected intraperitoneally Sodium (130ul/10g weight) anesthetized mice, postanesthetic mouse is placed on the container for raising mosquito to be bitten, yellow-fever mosquito is allowed to inhale Blood 30 minutes；
4) yellow-fever mosquito sucked blood is placed on 4 degree to anaesthetize within 10 minutes on ice, picking is fully sucked blood from the mosquito of anesthesia Yellow-fever mosquito be transferred in new container and raise；
5) second day after virus injection, third day and the 4th day repeat step 3-4, allow yellow-fever mosquito from AG6 mouse It sucks blood, and the yellow-fever mosquito that picking is successfully sucked blood；
6) infection rate of yellow-fever mosquito is detected, method is as follows：
A. the yellow-fever mosquito sucked blood after raising 8 days, put to death for 5 minutes, and single yellow-fever mosquito is put into and is prefixed RNA pumpings by -80 degree freezings In the EP pipes of extract, is ground with grinding rod and be fully homogenized.
B. it is cDNA to use RNA extraction agent boxes to extract the total serum IgE of yellow-fever mosquito and reverse transcription, is examined using Taqman RT-QPCR Survey the DENV2 viral infection rates in Aedes aegypti body.
C. the infection rate calculation formula of yellow-fever mosquito is：Yellow-fever mosquito total number in the yellow-fever mosquito number/experiment infected；Statistics DENV2 NS1 antiserums groups and Pre-immune groups yellow-fever mosquito first were to the 4th day DENV2 viral infection rate.
The results are shown in Figure 3, and the infection rate of the yellow-fever mosquito of anti-DENV2 NS1 antibody groups is compared with Pre-immune groups, significantly The reduction of property, shows that anti-DENV2 NS1 antibody can block yellow-fever mosquito to obtain virus out of AG6 Mice Bodies.
Embodiment 3, DENV2 NS1 and DENV2 △ NS1 are as the application in dengue vaccine
One, DENV2 NS1 albumen and DENV2 △ NS1 albumen active immunity AG6 mouse
The DENV2 NS1 albumen and DENV2 △ NS1 albumen prepared in active immunity embodiment 1 in AG6 mouse is immunized Mouse is the AG6 mouse of 6 weeks sizes, tests and is divided into three groups, PBS control group, DENV2 NS1 and DENV2 △ NS1 experimental groups, Every group of 12 AG6 mouse, immunization method are as follows：
1. the 1st day (initial immunity)
DENV2 NS1 experimental groups：DENV2 NS1 prepared by every AG6 mouse peritoneals injection embodiment 1 are helped completely with Freund Isometric mixture (every mouse 40ugDENV2 NS1) of agent；
DENV2 △ NS1 experimental groups：DENV2 △ NS1 and Freund prepared by every AG6 mouse peritoneals injection embodiment 1 is complete Isometric mixture (every mouse 40ugDENV2 △ NS1) of full adjuvant；
Control group：Isometric mixture of every the injection of AG6 mouse peritoneals PBS and Freund's complete adjuvant.
Obtain each group initial immunity mouse.
2. the 14th day (first time booster immunization)：Each group initial immunity mouse is immunized according to above-mentioned 1 method, is obtained To each group first time booster immunization mouse；
3. the 28th day (second of booster immunization)：Each group first time booster immunization mouse is carried out according to above-mentioned 1 method It is immune, obtain second of booster immunization mouse of each group；
4. the 42nd day passes through 2 points of abdominal cavity injection 1x106Pfu DENV2 (AF204178) viruses add twice to above-mentioned each group By force in immune Mice Body；Respectively in virus injection 1 day, 2 days, 3 days, 4 days, 5 days, blood is taken by mouse tail vein, and collect blood Final proof product.
The content that the NS1 in mice serum is detected using NS1 detection kits, to identify the resistance of the NS1 in Mice Body Disconnected effect, the results are shown in Figure 4, DENV2 NS1 groups and DENV2 △ NS1 groups, and the content of NS1 almost detects in AG6 mouse Less than；PBS control group, the NS1 contents in AG6 mouse are normal, as dengue virus infection is gradually increasing.
Two, DENV2 NS1 albumen and DENV2 △ NS1 albumen block Aedes aegypti to obtain dengue virus out of animal body
1, respectively by DENV2 NS1 groups, DENV2 △ NS1 groups and PBS control group virus injection 1 in 4 steps in above-mentioned one It, 2 days, the mouse of 3 days and 4 days, by the way that yellow Jackets (130ul/10g weight) anesthetized mice is injected intraperitoneally, after anesthesia Mouse be placed on the container for raising mosquito to be bitten, allow yellow-fever mosquito to suck blood 30 minutes；The yellow-fever mosquito sucked blood is placed on 4 degree on ice It is anaesthetized within 10 minutes, is transferred in new container and raises from the yellow-fever mosquito that picking is fully sucked blood in the mosquito of anesthesia；
Obtain 1 day, 2 days, 3 days and 4 days DENV2 NS1 groups yellow-fever mosquito of virus injection, virus injection 1 day, 2 days, 3 days and 4 days 1 day, 2 days, 3 days and 4 days PBS control group yellow-fever mosquito of DENV2 △ NS1 group group yellow-fever mosquitos and virus injection.
2, yellow-fever mosquito that above-mentioned each group is sucked blood after raising 8 days, put to death for 5 minutes, and single yellow-fever mosquito is put by -80 degree freezings It is prefixed in the EP pipes of RNA extracts, is ground with grinding rod and is fully homogenized；
3. it is cDNA to extract the total serum IgE of above-mentioned 2 yellow-fever mosquito and reverse transcription using RNA extraction agent boxes, Taqman RT- are utilized QPCR detects the DENV2 type viral infection rates in Aedes aegypti body；
The primer pair that DENV2 type viruses are detected for Taqman RT-QPCR is as follows：
Sense primer：5’-CATTCCAAGTGAGAATCTCTTTGTCA-3’；
Downstream primer：5’-CAGATCTCTGATGAATAACCAACG-3’.
The probe that dengue fever 2 type virus is detected for Taqman RT-QPCR is as follows：
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Internal reference is used as using Aedes aegypti Actin genes (AAEL011197), the primer pair of Taqman RT-QPCR is such as Under：
Sense primer：5’-GAACACCCAGTCCTGCTGACA-3’；
Downstream primer：5’-TGCGTCATCTTCTCACGGTTAG-3’.
The probe that yellow-fever mosquito Actin genes are detected for Taqman RT-QPCR is as follows：
5’-FAM-AGGCCCCGCTCAACCCGAAG-TRAMA-3’。
(the Ct values of Actin subtract the Ct of DENV2 to the △ Ct values detected by RT-QPCR using Actin and DENV2 Value) it is calculated, when 2-△CtValue when being more than 0.0002, it is positive which is identified as DENV2 infection.
The infection rate calculation formula of yellow-fever mosquito is：Yellow-fever mosquito total number in the yellow-fever mosquito number/experiment infected；
Count DENV2 NS1 groups, DENV2 △ NS1 and PBS control group are in yellow-fever mosquito first to DENV2 virus senses in the 4th day Dye rate；
The results are shown in Figure 5, and DENV2 NS1 are compared with the infection rate of the yellow-fever mosquito of DENV2 △ NS1 groups with PBS control group, The reduction of conspicuousness, it was demonstrated that active immunity DENV2 NS1 and DENV2 △ NS1 can block yellow-fever mosquito to be obtained out of AG6 Mice Bodies Take virus；For wherein immune DENV2 △ NS1 groups compared with DENV2 NS1 groups, the infection rate of yellow-fever mosquito additionally has dropped 2-3 times, shows Better barrier effect is shown.
Three, DENV2 NS1 albumen and DENV2 △ NS1 albumen suppress the generation of Hemorrhagic fever
A, the detection of DENV2 virus loads
The DENV2 NS1 albumen and DENV2 △ NS1 albumen prepared in active immunity embodiment 1 in AG6 mouse is immunized Mouse is the AG6 mouse of 6 weeks sizes, tests and is divided into three groups, PBS control group, DENV2 NS1 and DENV2 △ NS1 experimental groups, Every group of 12 AG6 mouse, immunization method are as follows：
1. the 1st day (initial immunity)
DENV2 NS1 experimental groups：DENV2 NS1 prepared by every AG6 mouse peritoneals injection embodiment 1 are helped completely with Freund Isometric mixture (every mouse 40ug DENV2 NS1) of agent；
DENV2 △ NS1 experimental groups：DENV2 △ NS1 and Freund prepared by every AG6 mouse peritoneals injection embodiment 1 is complete Isometric mixture (every mouse 40ugDENV2 △ NS1) of full adjuvant；
Control group：Isometric mixture of every the injection of AG6 mouse peritoneals PBS and Freund's complete adjuvant.
Obtain each group initial immunity mouse.
2. the 14th day (first time booster immunization)：Each group initial immunity mouse is immunized according to above-mentioned 1 method, is obtained To each group first time booster immunization mouse；
3. the 28th day (second of booster immunization)：Each group first time booster immunization mouse is carried out according to above-mentioned 1 method It is immune, obtain second of booster immunization mouse of each group；
4. the 42nd day passes through 2 points of abdominal cavity injection 1x106Pfu DENV2 (AF204178) viruses add twice to above-mentioned each group By force in immune Mice Body；Respectively in virus injection 1 day, 2 days, 3 days, 4 days, 5 days, blood 15ul is taken by mouse tail vein, is put into It is prefixed in the EP pipes of RNA extracts, and is fully homogenized.
Total serum IgE in AG6 Mouse whole bloods is extracted using RNA extraction agent boxes and reverse transcription is cDNA, utilizes Taqman RT- QPCR detects DENV2 virus loads in AG6 mouse bloods (expression quantity of mouse Actin genes is as internal reference).
The primer pair that DENV2 type viruses are detected for Taqman RT-QPCR is as follows：
Sense primer：5’-CATTCCAAGTGAGAATCTCTTTGTCA-3’；
Downstream primer：5’-CAGATCTCTGATGAATAACCAACG-3’.
The probe that dengue fever 2 type virus is detected for Taqman RT-QPCR is as follows：
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
It is used as internal reference, the primer pair of Taqman RT-QPCR as follows using mouse Actin genes (NM_007393)：
Sense primer：5’-AGCCATGTACGTAGCCATCCA-3’；
Downstream primer：5’-TCTCCGGAGTCCATCACAATG-3’.
The probe that mouse Actin genes are detected for Taqman RT-QPCR is as follows：
5’-FAM-TGTCCCTGTATGCCTCTGGTCGTACCAC-TRAMA-3’。
(the Ct values of Actin subtract the Ct of DENV2 to the △ Ct values detected by RT-QPCR using Actin and DENV2 Value) it is calculated, define 2-△CtValue be the AG6 mouse DENV2 virus loads.
The results are shown in Figure 6, and the dengue virus carrying capacity of DENV2 NS1 groups and PBS control group is essentially identical, and DENV2 △ The dengue virus carrying capacity of NS1 groups has dropped 3-10 times, it was demonstrated that the recombination DENV2 △ NS1 for generating side effect site have been lacked, it can To reduce infection of the dengue virus on AG6 mouse.
B, the bleeding of detection infection AG6 mouse
1. passing through 2 points of abdominal cavity injection 1x10 at above-mentioned 42nd day6Pfu DENV2 (AF204178) viruses arrive above-mentioned each group Twice in booster immunization Mice Body；
2. at the 18th day, pass through mouse tail vein injection 150ul Yi Wensilan (Evans blue, 0.5%PBS solution) Into each group Mice Body.
3. small by the way that yellow Jackets (130ul/10g weight) anesthesia is injected intraperitoneally after Yi Wensilan is injected 2 hours Mouse is dissected mouse, is irrigated to mouse using PBS, until blood is completely removed.
4. collect the kidney of mouse, liver, spleen, small intestine, the organs such as large intestine and stomach use formamide (Formamide) to impregnate Tissue (2ml/100mg tissues) extracts the Yi Wensilan of infiltration in the tissue.
5. centrifugation removal fragment of tissue detects suctions of the Yi Wensilan at 610nm in supernatant using spectrophotometer Luminosity calculates the amount of the Yi Wensilan in each tissue according to standard items curve (standard curve is shown in Fig. 7).
The results are shown in Figure 8, compared with compareing PBS groups, in DENV2 NS1 mouse and DENV2 △ NS1 mouse tissues The reduction of the amount conspicuousness of Yi Wensilan, shows that both albumen are immunized, and can reduce AG6 mouse when infecting dengue virus The hemorrhagic tendency of generation；The amount that Yi Wensilan is organized than DENV2 NS1 of wherein immune DENV2 △ NS1 groups is lower, shows that it has There is better protecting effect.
C, the death rate of infecting mouse is observed
1. passing through 2 points of abdominal cavity injection 1x10 at above-mentioned 42nd day6Pfu DENV2 (AF204178) viruses arrive above-mentioned each group Twice in booster immunization Mice Body；
2. from the 1st day to the 40th day situation for monitoring mouse daily of virus infection, the death time of every mouse is recorded, is made The survivorship curve of each group mouse is analyzed with Kaplan-Meier methods.
The results are shown in Figure 9, is compared with control PBS groups, DENV2 NS1 groups mouse and DENV2 △ NS1 group mouse survivals Rate significantly improves；It is 33%, DENV2 △ NS1 is 75% that 40 days survival rates of PBS groups, which are 0, DENV2 NS1 groups, it was demonstrated that actively It is dead that immune DENV2 NS1 and DENV2 △ NS1 albumen can protect AG6 mouse to exempt；Wherein DENV2 △ NS1 immune groups There is better protecting effect.
The above results show that DENV2 NS1 and DENV2 △ NS1 albumen can be used as dengue vaccine, for prevent and Dengue fever is treated, and yellow-fever mosquito can be blocked to infect dengue fever virus from animal body.

Claims (18)

1. a kind of protein, the protein that amino acid sequence forms shown in sequence in sequence table 4.
2. encoding the DNA molecular of protein described in claim 1.
3. DNA molecular according to claim 2, it is characterised in that：The DNA molecular is that code area is sequence in sequence table DNA molecular shown in 3.
4. the recombinant vector containing DNA molecular described in Claims 2 or 3.
5. the expression cassette containing DNA molecular described in Claims 2 or 3.
6. the transgenic cell line containing DNA molecular described in Claims 2 or 3.
7. the recombinant bacterium containing DNA molecular described in Claims 2 or 3.
8. the recombinant virus containing DNA molecular described in Claims 2 or 3.
9. application of the protein described in claim 1 in preparing dengue vaccine product.
10. application of the DNA molecular described in Claims 2 or 3 in preparing dengue vaccine product.
11. application of the recombinant vector in preparing dengue vaccine product described in claim 4.
12. application of the expression cassette in preparing dengue vaccine product described in claim 5.
13. the transgenic cell described in claim 6 ties up to the application prepared in dengue vaccine product.
14. application of the recombinant bacterium in preparing dengue vaccine product described in claim 7.
15. application of the recombinant virus according to any one of claims 8 in preparing dengue vaccine product.
16. according to any application in claim 9-15, it is characterised in that：The product is kit.
17. it is protein described in claim 1 that one kind, which having dengue vaccine functional product, active constituent,.
18. product according to claim 17, it is characterised in that：The product is drug.
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