CN105601721A

CN105601721A - Preparation method and application of double-effect vaccine for dengue fever

Info

Publication number: CN105601721A
Application number: CN201510830594.3A
Authority: CN
Inventors: 程功; 刘建英; 刘洋
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2015-11-25
Filing date: 2015-11-25
Publication date: 2016-05-25
Anticipated expiration: 2035-11-25
Also published as: CN105601721B; WO2017088448A1

Abstract

The invention discloses a preparation method and application of a double-effect vaccine for the dengue fever. The invention provides a protein which is protein a) or protein b, wherein protein a) is a protein composed of an amino acid sequence as shown in a sequence 4 in a sequence table, and protein b) is a protein which has same functions as protein a) and is derived from the protein a) by subjecting the sequence 4 in the sequence table to substitution and/or deletion and/or addition of one or more amino acid residues. Experiments in the invention prove that reconstructed dengue virus non-structural protein 1 (DENV delta NS1) can be used as the vaccine; and the double-effect vaccine can protect mankind or mammals from dengue haemorrhagic fever and block propagation of the dengue virus in nature via mosquitoes.

Description

A kind of preparation method of dengue fever dual-purpose vaccine and application thereof

Technical field

The invention belongs to dengue virus prevention and control field, be specially a kind of preparation and application thereof of dengue virus dual-purpose vaccine.

Background technology

Dengue fever (DengueFever, DF) is by her mosquito-borne acute viral infectious disease. Dengue virus (DengueVirus, DENV) spherical in shape, formed by after birth, capsid protein, non-structural protein bletilla sub-thread positive chain RNA. NatureIn, there are four kinds of serotypes (DENV-1～DENV-4) in dengue virus, and the nucleotide sequence of each serotype is widely different,Can reach more than 35%. In each serotype, can be divided into multiple gene hypotypes according to the difference of epidemic regions again. Dengue diseasePoisons are bitten and are infected after human body by yellow-fever mosquito, hide and can show clinical symptoms through 2-3 days. The Clinical symptoms of Dengue isHigh heat, bone and DOMS, hemorrhagic tendency and white blood cell count(WBC) obviously reduce. Dengue fever clinical manifestation is slighter,With the self-healing of course advancement most people, fatal rate is low; But a few peoples' the course of disease can proceed to next stage, and Dengue goes outBlood-head (DengueHemorrhagicFever, DHF) and Dengue shock syndrome (DengueShockSyndrome,DSS), this is the serious type of one of dengue infection, and clinical manifestation is each organ severe haemorrhage, high heat, blood plateletCounting obviously reduces, blood is concentrated, shock, causes the death rate of this course of disease high.

Dengue virus is mainly bitten the propagation of human body postoperative infection by Aedes aegypti (Aedesaegypti). Do not exist Egyptian sheThe area of mosquito, aedes albopictus (Aedesalbopictus) is also considered to the important medium that dengue fever is propagated. At presentThink, the natural reservoir (of bird flu viruses) of dengue virus only has people, primate and the yellow-fever mosquito such as low. At occurring in nature, dengue virus is mainIn two kinds of patterns, circulate: 1) city circulation: in the dengue prevalence at urban type, virus is " people-yellow-fever mosquito-people "Between propagate; 2) jungle circulation: be mainly that this follows by propagating between the yellow-fever mosquito in matto and the primate such as lowRing is the original loop of dengue virus. According to the study, the dengue virus under jungle type dengue virus and city cycling condition is poorDifferent larger, virus is diffused to city and rural possibility by the infection yellow-fever mosquito that still can not get rid of jungle circulation. CityThe dengue fever virus of circulation is strictly followed the route of transmission of " people-yellow-fever mosquito-people ", and the mankind are unique hosts of this virus,Yellow-fever mosquito is unique communication media. In the process of propagating in virus, the yellow-fever mosquito of infection is noted dengue fever virus by bitingBe mapped to (virus is propagated) in human body, virus is bred in horn cell and BMDC under fell; Virus is diffused into bloodAll kinds of immunocytes that further infect in blood after liquid form viremia virusemia; In the viremia virusemia phase, virus can be by inhalingThe mode of blood is inhaled in the mosquito body of biting (virus is obtained). The tissues such as the salivary gland of virus infections mosquito, infectYellow-fever mosquito transmitted virus again.

The genome of dengue virus is made up of the RNA of a strand forward, in host cell, can translate into more than oneThe peptide chain of polyprotein, afterwards this peptide chain be hydrolyzed into three structural proteins (Structuralprotein) and seven non-Structural proteins (Non-structuralprotein, NS). Wherein non-structural protein 1 (NS1) can be from infection cellMiddle secretion, is present in the serum of infection host in a large number. In dengue virus infection acute stage, the NS1 in patients serumAmount at 70-15,000ng/ml, in some patient body, in serum, the amount of NS1 can reach 50,000ng/ml.Therefore, NS1 is used as one of leading indicator detecting clinically early stage dengue virus infection. Studies have shown that, NS1 existsIn the mechanism of causing a disease of dengue fever and dengue hemorrhagic fever, play very important effect. The antibody of NS1 can with mankind's blood vessel inThe nonspecific combination of chrotoplast, thus cause the permeability of endothelial cell to increase, cause hemorrhagic tendency; In addition NS1,Antibody can also deposit to platelet surface, cause blood platelet blood coagulation disorders and cause hemorrhage increase. Have at present multipleThe epitope of NS1 albumen is considered to pathogenic relevant to dengue fever. In addition, due to the Blood of Patients in the viremia virusemia phaseCommon a large amount of existence of NS1 and dengue virus in clear, NS1 albumen can be inhaled into by the mode of sucking blood with virus simultaneouslyIn the mosquito body of biting. Therefore in the process that NS1 obtains in virus, play an important role.

In common mouse and monkey isotype animal, after dengue virus infection, do not show typical dengue fever clinical symptoms,Or only there is a slight viremia virusemia. Interferon amboceptor deficient mice (IFN-alpha/gammareceptorDoubleknock-outmouse, AGmouse) be the main moving of the research of current dengue fever mechanism of causing a disease and medicament research and developmentObject model, can produce and exceed 10⁶The viremia virusemia of pfu/ml also causes animal dead. The AG6 mouse of B6 background(IFN-alpha/gammareceptordoubleknock-outB6mouse) in dengue virus research, quilt is extensiveApplication. AG6 mouse is very responsive to dengue virus, and after inoculation dengue 2-type virus, virus can be in AG6 Mice BodyRapid amplifying, Aedes aegypti bites after the AG6 mouse of dengue virus infection simultaneously, can suck virus and obtain and infect.

Dengue fever is found the history of existing more than 200 year so far, but at present still without the treatment therapy of special efficacy and safe and effectiveDengue vaccine. Due to the particularity of dengue fever pathogenesis, the antibody that viral primary infection produces is only to homotypeVirus infections has neutralization, and the infection of other serotype viruses is had to infection humidification. Meanwhile, virus senseThe T cell activation that dyes rear generation also has facilitation to the morbidity of dengue hemorrhagic fever and Dengue shock syndrome. This causingAnttdisease Mechanism is one of principal element of the conventional subunit vaccine of restriction dengue virus, attenuated live vaccine and inactivated vaccine development.For the prevention of arthropod-borne infectious disease (insect-borne infectious disease), except traditional vaccine direct immunization attenuation or go outBeyond the pathogen or its recombinant protein of living, can also block medium pathogen in natural cyclic process, thereby fallLow vection disease is in natural propagation. Therefore, propagating blocking-up vaccine is one certain antigen egg of immunity in hostWhite blocking-up pathogen propagation approach, thereby the immunization strategy of reduction insect-borne infection rate and host's incidence of disease. As traditional epidemic diseaseThe important supplement of seedling, propagates the application widely in the control of the insect-borne infectious diseases such as malaria of blocking-up vaccine. Therefore,Utilize the infection rate of vaccine method reduction Mosquito Vectors by effective measures that are dengue control.

Summary of the invention

An object of the present invention is to provide a kind of albumen.

Protein provided by the invention is following protein a) or b):

A) protein being formed by the amino acid sequence shown in sequence in sequence table 4;

B) replacement and/or the disappearance through one or several amino acid residue by the amino acid sequence of sequence in sequence table 4And/or add and have identical function by a) derivative protein.

The replacement of above-mentioned one or several amino acid residue and/or disappearance and/or be added to that to be no more than 10 amino acid residualThe replacement of base and/or disappearance and/or interpolation.

The above-mentioned protein DNA molecule of encoding 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 the DNA molecular shown in sequence 3 in sequence table;

2) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, tool at leastHave 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98%Or at least have 99% homology and coding claim 1 described in protein DNA molecule;

3) under stringent condition with 1) the DNA sequence dna hybridization limiting and the above-mentioned protein DNA molecule of encoding.

Above-mentioned stringent condition can be at 6 × SSC, and in the solution of 0.5%SDS, at 65 DEG C, hybridization, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.

The recombinant vector, expression cassette, transgenic cell line, recombinant bacterium or the recombinant virus that contain above-mentioned DNA molecular are alsoThe scope of protection of the invention;

Or be also the scope of protection of the invention by the antibody of above-mentioned protein preparation.

Above-mentioned protein or above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line, recombinant bacterium,Recombinant virus or antibody are being prepared as follows 1)-5) in application at least one product be also the scope of protection of the invention:

1) dengue vaccine;

2) there is blocking-up people, other primates or other mammalian infections dengue fever virus to dengue virus susceptibleFunction;

3) there is blocking-up mosquito and obtain Dengue from people, other primates or other mammals to dengue virus susceptibleFever virus function;

4) there is the mosquito of inhibition and obtain or propagate the function of dengue fever virus;

5) there is the generating function of preventing the Hemorrhagic fever being caused by dengue fever virus or dengue fever virus NS1 protein antibodies.

In above-mentioned application, described mammal is mouse;

Described mosquito is Aedes aegypti or aedes albopictus.

In above-mentioned application, described product is kit.

Another object of the present invention is to provide one and has following 1)-5) at least one functional product.

Product provided by the invention, its active component is above-mentioned protein;

1) dengue vaccine;

In the said goods, described mammal is mouse;

Described mosquito is Aedes aegypti or aedes albopictus.

The said goods is medicine.

Above-mentioned dengue virus is DENV2.

The present invention is based on dengue virus non-structural protein 1 (DENVNS1), reject harmful epitope and obtain transforming DengueVirus non-structural protein 1 (DENV Δ NS1), sets it as antigen and carries out the immune mankind or mammalian hosts. ImmunityImproved dengue virus non-structural protein 1 plays simultaneously and suppresses mosquito band poison and animal infected double effects, can fallThe animal reservoir that low dengue virus infection causes is hemorrhage and dead, reduces the band poison rate of mosquito medium simultaneously, reaches controlThe object of dengue fever. This dual-purpose vaccine can to protect mankind or mammal there is dengue hemorrhagic fever and block dengue virus logicalCross mosquito in natural propagation.

Brief description of the drawings

Fig. 1 is the qualification of tiring of passive immunity DENV2NS1 and DENV2 △ NS1 protein antibodies;

Fig. 2 is the generation of NS1 in passive immunity DENV2NS1 antiserum blocking-up AG6 Mice Body;

Fig. 3 is that passive immunity DENV2NS1 antiserum reduces the upper Infection Rate with Dengue Virus of yellow-fever mosquito;

Fig. 4 is the generation of NS1 in body in active immunity DENV2NS1 and DENV2 △ NS1 albumen blocking-up AG6 mouse;

Fig. 5 is that active immunity DENV2NS1 and DENV2 △ NS1 albumen reduce the upper Infection Rate with Dengue Virus of Aedes aegypti;

Fig. 6 is that active immunity DENV2 △ NS1 reduces the DENV2 virus load in AG6 Mice Body;

Fig. 7 is that the mark of the Yi Wensilan content in each tissue is got function;

Fig. 8 is that active immunity DENV2NS1 and DENV2 △ NS1 reduce AG6 mouse each organ in the time of dengue infectionBleeding;

Fig. 9 is that active immunity DENV2NS1 and DENV2 △ NS1 reduce the death of AG6 mouse in the time of dengue infectionRate.

Detailed description of the invention

The experimental technique using in following embodiment if no special instructions, is conventional method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

The acquisition of embodiment 1, DENV2 △ NS1 albumen

By prokaryotic expression DENV2NS1 and DENV2 △ NS1 albumen, specific as follows:

1, the acquisition of DENV2NS1 gene and DENV2 △ NS1 gene

1) DENV2NS1 gene

The total RNA that extracts DENV2 virus, reverse transcription obtains cDNA as template, with F1 and R1 primer pair PCR expansionIncrease, obtain the PCR product of 1056bp, be DENV2NS1 gene, through order-checking, its nucleotides sequence is classified sequence as1, the albumen of its coding is DENV2NS1, and the amino acid length of this albumen is 352AA, and amino acid sequence is sequence 2.

2) DENV2 △ NS1 gene

The total RNA that extracts DENV2 virus, reverse transcription obtains cDNA and increases as template.

The first step: using the cDNA of DENV2 virus as template, carry out pcr amplification with F2 and R2 primer pair,To the PCR product of 1-115AA; Second step: using the cDNA of DENV2 virus as template, with F3 and R3 primer pairIncrease, obtain the PCR product of 120-220AA; The 3rd step: using the cDNA of DENV2 virus as template, withF4 and R4 primer pair carry out pcr amplification, obtain the PCR product of 267-310AA; The 4th step: with DENV2 virusCDNA as template, increase with F5 and R5 primer pair, obtain the PCR product of 331-352AA; The 5th step:The PCR product obtaining using the first step and second step, as template, increases with F6 and R6 primer pair, obtains 1-220AAThe PCR product of (△ 116-119AA); The 6th step: the PCR product obtaining using the 3rd step and the 4th step is as template,Increase with F7 and R7 primer pair, obtain the PCR product of 267-352AA (△ 311-330AA); The 7th step:The PCR product obtaining using the 5th step and the 6th step, as template, increases with F8 and R8 primer pair, obtains 1-352AAThe PCR product of (△ 116-119AA, △ 221-266AA, △ 311-330AA), finally obtains the PCR of 846bpProduct, is DENV2 △ NS1 gene, and through order-checking, its nucleotides sequence is classified sequence 3 as, and the albumen of its coding is DENV2△ NS1, the amino acid sequence of this albumen 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

The NdeI of DENV2NS1 gene insertion pET-28a (+) carrier shown in above-mentioned sequence 1 and the enzyme of XhoI are cutThe recombinant vector that site obtains, called after pET-28a (+)-DENV2NS1.

DENV2 △ NS1 gene shown in above-mentioned sequence 3 is inserted to the NdeI of pET-28a (+) carrier and the enzyme of XhoICut the recombinant vector that site obtains, called after pET-28a (+)-DENV2 △ NS1.

3, the expression and purification of albumen

Above-mentioned pET-28a (+)-DENV2NS1 and pET-28a (+)-DENV2 △ NS1 are imported respectively to Escherichia coliBL21 (DE3), obtain recombinant bacterium BL21 (DE3)/pET-28a (+)-DENV2NS1 andBL21(DE3)/pET-28a(+)-DENV2△NS1。

The monoclonal bacterium colony of above-mentioned recombinant bacterium is seeded to respectively in 5mlLB fluid nutrient medium (containing 25 μ g/ml cards thatMycin), 37 DEG C, 220rpm concussion are cultivated 16 hours; Then be seeded to fresh LB liquid by the volume ratio of 1:100Body culture medium (containing 25 μ g/ml kanamycins), cumulative volume is 200ml, 37 DEG C, 220rpm concussion are cultivated 4 hours;Then adding IPTG and making its concentration is 1mM, and 37 DEG C, 220rpm concussion are cultivated 6 hours; Room temperature, 4000rpmCentrifugal 10min, collects respectively thalline.

The thalline of obtaining, with inclusion body cleaning solution, (solvent is the Tris-HCl buffer solution of pH8.0,50mM, containsHaving 100mMNaCl, 5mMEDTA, 0.1%NaN3,0.5%Triton-X100, is 0.1mM with front adding final concentrationPMSF and the DTT of 1mM) suspension thalline, (60% power, ultrasonic 3s, stops 9s, always super in ultrasonication on iceSonication time 5min), the centrifugal 15min of 6000rpm, collecting precipitation (inclusion body).

By the inclusion body cleaning solution precipitation obtained above that suspends, add excessive MgSO₄With in and inclusion body cleaning solution inEDTA, then adding final concentration is that DNA enzyme (the catalog number (Cat.No.) DN25 of Sigma company) and the final concentration of 0.01mg/ml isThe lysozyme (the catalog number (Cat.No.) L6876 of Sigma company) of 0.1mg/ml, room temperature treatment 20 minutes, the centrifugal 15min of 6000rpm,Collect inclusion body; Then, the precipitation obtaining by inclusion body cleaning solution suspension step 8, adds excessive MgSO₄With inWith the EDTA in inclusion body cleaning solution, room temperature treatment 20 minutes, the centrifugal 15min of 6000rpm, collects inclusion body,To DENV2NS1 inclusion body and DENV2 △ NS1 inclusion body.

The inclusion body of collection is carried out to polyacrylamide gel electrophoresis, the object band of clear display, DENV2NS1 is approximatelyFor 42kD, DENV2 △ NS1 is about 31kD.

(solvent is the Tris buffer solution of pH8.0,100mM, contains 50mM glycine to use respectively protein purification liquidWith 8M urea) dissolve inclusion body obtained above, obtain DENV2NS1 crude protein solution and the thick egg of DENV2 △ NS1White solution.

Use TALONpurificationKit (Clontech company catalog number (Cat.No.) 635515), from crude protein solutionPurifying DENV2NS1 mature peptide, specific as follows: 4 DEG C of 1mlResin and 50ml crude protein solution to be hatched altogether 2 littleTime, be then transferred in filter post, by 20ml cleaning solution first (the 1X equilibrium liquid in TALON kit adds 8M urea)Wash, then (the 1X equilibrium liquid in TALON kit adds 8M urea and 20mM miaow to use 20ml cleaning solution secondAzoles) wash, (the 1X equilibrium liquid in TALON kit adds 8M urea and 150mM finally to use 5ml eluent thirdImidazoles) washing, collecting 5ml eluent is DENV2NS1 albumen;

Adopt the purifying DENV2 △ NS1 that uses the same method, collecting 5ml eluent is DENV2 △ NS1 albumen.

Above-mentioned purity of protein is greater than 95%, and concentration is greater than 1mg/ml.

Embodiment 2, DENV2NS1 and DENV2 △ NS1 hinder at passive immunity the application that dengue virus obtains

One, preparation DENV2NS1 mouse source antiserum and DENV2 △ NS1 mouse source antiserum

1. the sero-fast preparation in mouse source.

Animal used as test is the female mouse of Balb/c of 8 weeks sizes, and experiment flow is as follows:

(1). the 1st day (initial immunity), every mouse peritoneal is injected DENV2NS1 and the DENV2 of preparation in above-mentioned△ NS1 albumen (contains 40ugDENV2NS1 or DENV2 △ NS1 with the equal-volume mixture of Freund's complete adjuvant respectivelyAlbumen).

(2). the 14th day (booster immunization for the first time), the DENV2NS1 of every above-mentioned preparation of mouse subcutaneous injection(contain 40ugDENV2NS1 or DENV2 △ with the equal-volume mixture of DENV2 △ NS1 albumen and incomplete Freund's adjuvantNS1 albumen).

(3). the 28th day (booster immunization for the second time), the DENV2NS1 of every above-mentioned preparation of mouse subcutaneous injection(contain 40ugDENV2NS1 or DENV2 △ with the equal-volume mixture of DENV2 △ NS1 albumen and incomplete Freund's adjuvantNS1 albumen).

(4). the 42nd day, every mouse gathered 500ul serum, is anti-DENV2NS1 antibody (DENV2NS1The serum that immunity obtains) and anti-DENV2 △ NS1 antibody (serum that DENV2 △ NS1 immunity obtains).

(5). the 42nd day, the normal mouse without immunity of raising gathered 500ul serum, is anti-Pre-immuneControl antibodies.

2. detect antibody titer

(1). 1ugDENV2NS1 albumen is taped against on 96 orifice plates, and 4 spend night;

(2) .300ul1%BSA room temperature sealing 1 hour;

(3). will resist DENV2NS1 antibody (Anti-DENV2NS1) and anti-DENV2 △ NS1 antibody (Anti-DENV2△ NS1) and contrast Pre-immune antibody 1:15BSA gradient dilution 1/250 to 1/10,000,000, everyHole adds 100ul, and room temperature was in conjunction with 2 hours;

(4) .PBST washes plate 5 times, adds the mouse two anti-(JM-6402-05, MBL, Japan) of 100ul1/5000 dilution,Incubated at room 1 hour;

(5) .PBST washes plate 6 times, adds 100ulTMB (52-00-01and50-85-04, Kirkegaard&PerryLaboratories) nitrite ion develops the color, and uses sulfuric acid color development stopping, reads OD450.

As shown in Figure 1, after immune mouse, anti-DENV2NS1 antibody and anti-DENV2 △ NS1 are anti-for the first time for resultBody produces in a large number, and to gathering serum (the 42nd day) when, the antiserum in mouse blood has reached peak value. Anti-DENV2NS1 antibody and anti-DENV2 △ NS1 antibody can detect the DENV2NS1 albumen of total length completely, prove quiltMoving immune mouse has been prepared successful anti-DENV2NS1 antibody and anti-DENV2 △ NS1 antibody.

Two, anti-DENV2NS1 antibody and anti-DENV2 △ NS1 antibody obtain the application in dengue virus blocking-up yellow-fever mosquito

1, passive immunity DENV2NS1 antiserum verify the barrier effect of NS1.

By lumbar injection by 1x10⁶PfuDENV2 (AF204178, Guoetal., 2013) virus injection arrivesIn AG6 (IFN-alpha/gammareceptordoubleknock-outB6mouse) Mice Body, 12 hours itAfter by logical to the anti-DENV2NS1 antibody (anti-DENV2NS1) of an above-mentioned preparation or Pre-immune control serumCrossing 2 lumbar injections squeezes in the AG6 Mice Body that infects dengue virus; 12hr after serum injection, 2 days, 3My god, 4 days, 5 days, get blood by mouse tail vein, and collect blood serum sample.

Use NS1 detection kit (Diagnoserautomata; 8404-25) detect NS1's in mice serumContent, identifies the barrier effect of the NS1 in Mice Body.

Result as shown in Figure 2, is injected anti-DENV2NS1 antibody group, and in AG6 mouse, the content of NS1 almost detects notArrive; The control group of injection Pre-immune serum, the NS1 content in AG6 mouse is normal, along with dengue virus infectionRise gradually.

The above results shows, the antiserum of passive immunity DENV2NS1 can be successfully in and DENV2 in AG6 Mice BodyThe NS1 that virus produces, reaches the effect of blocking the effect of NS1 albumen.

2, Aedes aegypti obtains dengue virus by biting infecting mouse

1) by lumbar injection by 1x10⁶PfuDENV2 (AF204178, Guoetal., 2013) virus injectionIn AG6 Mice Body;

2), after 12 hours, the anti-DENV2NS1 antibody of lumbar injection 100ul or 100ulPre-immune serum arriveInfect in the AG6 Mice Body of dengue virus;

3) behind 12 hours of antiserum injection (be dengue virus injection one day after), compares by lumbar injection penta barAppropriate sodium (130ul/10g body weight) anesthetized mice, is placed on postanesthetic mouse on the container of raising mosquito to be bitten,Allow yellow-fever mosquito suck blood 30 minutes;

4) yellow-fever mosquito of sucking blood is placed on to 4 degree and within 10 minutes, anaesthetizes on ice, from the mosquito of anesthesia, picking is fully inhaledThe yellow-fever mosquito of blood is transferred in new container and raises;

5) second day after virus injection, the 3rd day and the 4th day, repeating step 3-4, allows yellow-fever mosquito from AG6 mouseSuck blood with it, and the picking yellow-fever mosquito of successfully sucking blood;

6) infection rate of detection yellow-fever mosquito, method is as follows:

The yellow-fever mosquito of A. sucking blood was raised after 8 days, and-80 freezing 5 minutes of degree are put to death, and single yellow-fever mosquito has been put into RNA presetIn the EP pipe of extract, grind and fully homogenate with grinding rod.

B. the total RNA the reverse transcription that use RNA extraction agent box to extract yellow-fever mosquito are cDNA, utilize TaqmanRT-QPCRDetect the DENV2 viral infection rate in Aedes aegypti body.

C. the infection rate computing formula of yellow-fever mosquito is: the yellow-fever mosquito total number in yellow-fever mosquito number/experiment of having infected; StatisticsDENV2NS1 antiserum group and Pre-immune group yellow-fever mosquito first was to the DENV2 viral infection rate of the 4th day.

Result as shown in Figure 3, compare with Pre-immune group by the infection rate of the yellow-fever mosquito of anti-DENV2NS1 antibody group,The reduction of conspicuousness, shows that anti-DENV2NS1 antibody can block yellow-fever mosquito and obtain virus in AG6 Mice Body.

Embodiment 3, DENV2NS1 and DENV2 △ NS1 are in the application as in dengue vaccine

One, DENV2NS1 albumen and DENV2 △ NS1 albumen active immunity AG6 mouse

DENV2NS1 albumen and the DENV2 △ NS1 albumen in AG6 mouse, in active immunity embodiment 1, prepared,Immune mouse is the AG6 mouse of 6 weeks sizes, and experiment is divided into three groups, PBS control group, DENV2NS1 and DENV2△ NS1 experimental group, every group of 12 AG6 mouse, immunization method is as follows:

1. the 1st day (initial immunity)

DENV2NS1 experimental group: DENV2NS1 and Fu Shi prepared by every AG6 mouse peritoneal injection embodiment 1 are completeThe equal-volume mixture (every mouse 40ugDENV2NS1) of full adjuvant;

DENV2 △ NS1 experimental group: DENV2 △ NS1 prepared by every AG6 mouse peritoneal injection embodiment 1 with notThe equal-volume mixture (every mouse 40ugDENV2 △ NS1) of family name's Freund's complete adjuvant;

Control group: every AG6 mouse peritoneal is injected the equal-volume mixture of PBS and Freund's complete adjuvant.

Obtain each group of initial immunity mouse.

2. the 14th day (booster immunization for the first time): the method according to above-mentioned 1 is carried out immunity to each group of initial immunity mouse,Obtain each group of booster immunization mouse for the first time;

3. the 28th day (booster immunization for the second time): the method according to above-mentioned 1 to each group for the first time booster immunization mouse enterRow immunity, obtains each group of booster immunization mouse for the second time;

4. within the 42nd day, inject 1x10 by 2, abdominal cavity⁶PfuDENV2 (AF204178) virus to above-mentioned each group twiceIn booster immunization Mice Body; Virus injection 1 day, 2 days, 3 days, 4 days, 5 days, pass through mouse tail vein respectivelyGet blood, and collect blood serum sample.

Use NS1 detection kit to detect the content of the NS1 in mice serum, identify NS1's in Mice BodyBarrier effect, result as shown in Figure 4, DENV2NS1 group and DENV2 △ NS1 group, NS1 in AG6 mouseContent almost can't detect; PBS control group, the NS1 content in AG6 mouse is normal, along with dengue virus infection graduallyRise.

Two, DENV2NS1 albumen and DENV2 △ NS1 albumen blocking-up Aedes aegypti obtain dengue virus in animal body

1, respectively by DENV2NS1 group, DENV2 △ NS1 group and PBS control group virus note in 4 steps in above-mentionedPenetrate the mouse of 1 day, 2 days, 3 days and 4 days, anaesthetize by lumbar injection yellow Jackets (130ul/10g body weight)Mouse, is placed on postanesthetic mouse on the container of raising mosquito to be bitten, and allows yellow-fever mosquito suck blood 30 minutes; To suck bloodYellow-fever mosquito be placed on 4 degree and within 10 minutes, anaesthetize on ice, the yellow-fever mosquito that picking is fully sucked blood from the mosquito of anesthesia is transferred toIn new container, raise;

Obtain virus injection 1 day, 2 days, 3 days and 4 days DENV2NS1 group yellow-fever mosquito, virus injection 1 day, 2 days,3 days and 4 days DENV2 △ NS1 group group yellow-fever mosquitos and virus injection 1 day, 2 days, 3 days and 4 days PBS control group yellow-fever mosquitos.

2, above-mentioned each group yellow-fever mosquito of sucking blood raised after 8 days ,-80 freezing 5 minutes of degree are put to death, by single yellow-fever mosquitoPut into the EP pipe of RNA extract preset, ground and fully homogenate with grinding rod;

3. the total RNA the reverse transcription that use RNA extraction agent box to extract above-mentioned 2 yellow-fever mosquitos are cDNA, utilize TaqmanRT-QPCR detects the DENV2 type viral infection rate in Aedes aegypti body;

The primer pair that detects DENV2 type virus for TaqmanRT-QPCR is as follows:

Upstream primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';

Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.

The probe that detects dengue fever 2 type viruses for TaqmanRT-QPCR is as follows:

5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。

Adopt Aedes aegypti Actin gene (AAEL011197) as internal reference, the primer pair of its TaqmanRT-QPCRAs follows:

Upstream primer: 5 '-GAACACCCAGTCCTGCTGACA-3 ';

Downstream primer: 5 '-TGCGTCATCTTCTCACGGTTAG-3 '.

The probe that detects yellow-fever mosquito Actin gene for TaqmanRT-QPCR is as follows:

5’-FAM-AGGCCCCGCTCAACCCGAAG-TRAMA-3’。

(the Ct value of Actin deducts DENV2 to use Actin and DENV2 to detect by RT-QPCR the △ Ct value obtainingCt value) calculate, when 2^-△CtValue be greater than at 0.0002 o'clock, this yellow-fever mosquito is identified as DENV2 and infects positive.

The infection rate computing formula of yellow-fever mosquito is: the yellow-fever mosquito total number in yellow-fever mosquito number/experiment of having infected;

Statistics DENV2NS1 group, DENV2 △ NS1 and PBS control group be the DENV2 disease by the 4th day yellow-fever mosquito firstPoison infection rate;

Result as shown in Figure 5, infection rate and the PBS control group phase of the yellow-fever mosquito of DENV2NS1 and DENV2 △ NS1 groupRatio, the reduction of conspicuousness, proves that active immunity DENV2NS1 and DENV2 △ NS1 all can block yellow-fever mosquito from AG6In Mice Body, obtain virus; Wherein immune DENV2 △ NS1 group is compared with DENV2NS1 group, the infection rate volume of yellow-fever mosquitoDecline 2-3 doubly outward, demonstrated better barrier effect.

Three, DENV2NS1 albumen and DENV2 △ NS1 albumen are prevented the generation of Hemorrhagic fever

The detection of A, DENV2 virus load

1. the 1st day (initial immunity)

Obtain each group of initial immunity mouse.

4. within the 42nd day, inject 1x10 by 2, abdominal cavity⁶PfuDENV2 (AF204178) virus to above-mentioned each group twiceIn booster immunization Mice Body; Virus injection 1 day, 2 days, 3 days, 4 days, 5 days, pass through mouse tail vein respectivelyGet blood 15ul, put into the EP pipe of RNA extract preset, and fully homogenate.

In use RNA extraction agent box extraction AG6 mouse whole blood, total RNA reverse transcription are cDNA, utilize TaqmanRT-QPCR detects DENV2 virus load (expression of mouse Actin gene is as internal reference) in AG6 mouse blood.

The primer pair that detects DENV2 type virus for TaqmanRT-QPCR is as follows:

Upstream primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';

Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.

5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。

Adopt mouse Actin gene (NM_007393) as internal reference, the primer pair of its TaqmanRT-QPCR is as follows:

Upstream primer: 5 '-AGCCATGTACGTAGCCATCCA-3 ';

Downstream primer: 5 '-TCTCCGGAGTCCATCACAATG-3 '.

The probe that detects mouse Actin gene for TaqmanRT-QPCR is as follows:

5’-FAM-TGTCCCTGTATGCCTCTGGTCGTACCAC-TRAMA-3’。

(the Ct value of Actin deducts DENV2 to use Actin and DENV2 to detect by RT-QPCR the △ Ct value obtainingCt value) calculate, definition 2^-△CtValue be the DENV2 virus load of this AG6 mouse.

As shown in Figure 6, the dengue virus carrying capacity of DENV2NS1 group and PBS control group is basic identical for result, and DENV2The dengue virus carrying capacity of △ NS1 group has declined 3-10 doubly, has proved to lack the restructuring DENV2 △ in site of having side effectsNS1, can reduce the infection of dengue virus on AG6 mouse.

The bleeding of AG6 mouse is infected in B, detection

1. injected 1x10 at above-mentioned the 42nd day by 2, abdominal cavity⁶PfuDENV2 (AF204178) virus is to above-mentionedIn twice booster immunization Mice Body of each group;

2. in the time of the 18th day, by mouse tail vein injection 150ul Yi Wensilan, (Evansblue, 0.5%PBS is moltenLiquid) in each group of Mice Body.

3., after 2 hours, anaesthetize by lumbar injection yellow Jackets (130ul/10g body weight) in Yi Wensilan injectionMouse, dissects mouse, uses PBS to pour into mouse, 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)The Yi Wensilan in tissue extracts by infiltration to soak tissue (2ml/100mg tissue).

5. centrifugal removal fragment of tissue, uses spectrophotometer to detect Yi Wensilan in supernatant at 610nm placeAbsorbance, calculates the amount (calibration curve is shown in Fig. 7) of the Yi Wensilan in each tissue according to standard items opisometer.

Result as shown in Figure 8, is compared with contrasting PBS group, DENV2NS1 mouse and DENV2 △ NS1 mouse tissueIn the reduction of amount conspicuousness of Yi Wensilan, show these two kinds of albumen of immunity, all can reduce AG6 mouse and infectThe hemorrhagic tendency producing when dengue virus; Wherein the ratio DENV2NS1 of immune DENV2 △ NS1 group organizes Yi Wensilan'sMeasure lowlyer, show that it has better protection effect.

C, the death rate of observing infecting mouse

2. within the 1st day to the 40th day, monitor the situation of mouse from virus infections every day, record the death time of every mouse,Use Kaplan-Meier method to analyze the survivorship curve of each group of mouse.

Result as shown in Figure 9, is compared with contrast PBS group, DENV2NS1 group mouse and DENV2 △ NS1 group mouseSurvival rate all obviously improves; 40 days survival rates of PBS group are that 0, DENV2NS1 group is that 33%, DENV2 △ NS1 is 75%,Prove that active immunity DENV2NS1 and DENV2 △ NS1 albumen all can protect AG6 mouse to exempt dead; WhereinDENV2 △ NS1 immune group has better protection effect.

The above results all shows, DENV2NS1 and DENV2 △ NS1 albumen can be used as dengue vaccine, for preventionWith treatment dengue fever, and can block yellow-fever mosquito and infect dengue fever virus from animal body.

Claims

1. a protein is following protein a) or b):

2. protein DNA molecule described in coding claim 1.

3. DNA molecular according to claim 2, is characterized in that: described DNA molecular is following 1)-3)In any DNA molecular:

1) code area is the DNA molecular shown in sequence 3 in sequence table;

4. contain recombinant vector, expression cassette, transgenic cell line, the weight of DNA molecular described in claim 2 or 3Group bacterium or recombinant virus;

Or by the antibody of protein preparation described in claim 1.

5. DNA molecular or claimed in claim 4 described in protein or claim 2 or 3 described in claim 1Recombinant vector, expression cassette, transgenic cell line, recombinant bacterium, recombinant virus or antibody are being prepared as follows 1)-5) inApplication at least one product:

1) dengue vaccine;

6. application according to claim 5, is characterized in that: described mammal is mouse;

Described mosquito is Aedes aegypti or aedes albopictus.

7. according to the application described in claim 5 or 6, it is characterized in that: described product is kit.

8. have following 1)-5) at least one functional product, its active component is described in claim 1Protein;

1) dengue vaccine;

9. product according to claim 8, is characterized in that: described mammal is mouse;

Described mosquito is Aedes aegypti or aedes albopictus.

10. product according to claim 8 or claim 9, is characterized in that: described product is medicine.