CN106754748A - Aedes albopictus densovirus and its application - Google Patents
Aedes albopictus densovirus and its application Download PDFInfo
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Abstract
The invention discloses a kind of aedes albopictus densovirus, its entitled aedes albopictus densovirus AalDV 6 is preserved in China typical culture collection center, and deposit number is CCTCC NO.V201652.Also disclose application of the above-mentioned aedes albopictus densovirus in biological culicide is prepared.
Description
Technical field
The invention belongs to mosquito densovirus technical field, and in particular to aedes albopictus densovirus and its application.
Background technology
Mosquito matchmaker's infectious disease is the disease with mosquito as media transmission, including malaria, filariasis, dengue fever, popular B-mode
Encephalitis, yellow fever etc..Mosquito matchmaker disease Epidemic Scope is wide, and transmissibility is strong, and the incidence of disease is high, although the effort that have passed through several centuries is prevented
System, the disease that mosquito matchmaker propagates is still flourishing all over the world.The whole world has more than one hundred million people to be infected with malaria every year, and malaria causes 100 every year
The death of many ten thousand children, mainly on the south African the Sahara.The primary vehicle of dengue fever is aedes albopictus and Aedes aegypti,
In the past few decades, with the global diffusion of its communication media, spread scope also gradually expands, Aedes aegypti (Aedes
Aegypti it) is returned in the region that is destroyed of 20th century mid-term and causes pandemic Hemorrhagic fever, and aedes albopictus then originates from
In Southeast Asia.West Nile Virus had turned into epidemic disease between past 10 years in whole America, and CHIK has gone out
Present Indian Ocean basin and Asia continent, have influence on millions of people.Japanese encephalitis virus is also in the big drawing west of the Indian subcontinent and Australia
Asia diffusion, mainly influences child.Mosquito class known to China 18 category, 371 kinds and subspecies, cover malaria, dengue fever, B-mode brain
The scorching, main media of filariasis.The popular serious harm human life and health of mosquito matchmaker's infectious disease, influences the stabilization of society, hinders
Expanding economy.Limitation mosquito disease is faced with control mosquito populations and provides effective public in the effort of the influence of Prevalent district
The double challenge of health program.
The anti-system of Mosquito Vectors is to control the important measures of mosquito matchmaker's disease transmission.At present, for dengue fever, yellow fever, west
The mosquito such as Nile virus disease matchmaker's property viral disease there is no specific treatment method or specific preventive means, and medium competition is preventing it
Importance in terms of occurring and being popular just seems more prominent, and seeks effective controlling party rule and faced as world medical circle
Important topic.The anti-system of medium chemistry once occupies dominant position due to its effect for protruding in the anti-system of mosquito matchmaker, but with
Serious environmental pollution caused by chemical anti-system traditionally, easily causes increasingly manifesting for the drawbacks such as mosquito drug resistance generation,
The biological control of mosquito is more and more favored by people.Biological control is mainly using mosquito pathogen, parasitic animal and plant, predation thing
Prevention effect is reached, harmless to nontarget organism and beneficial organism, free from environmental pollution, mosquito is not likely to produce resistance to it.
Mosquito densovirus (mosquito densovirus, MDV) belongs to Parvoviridae (Parvovirinae) densovirus
Category, is single normal chain DNA virus, and viral capsid is without coating, the regular dodecahedron structure of a diameter of 20nm.The gene of mosquito densovirus
Group leader 4000nt or so, by the structural proteins VP code areas of the non-structural protein NS 1 in left side, NS2 code areas and right side, and two
Repetitive sequence (inverted repeat sequences, the IRS) composition at end.The effect of non-structural protein NS 1 is mainly auxiliary
Virus replication, the transcription of activation structure albumen.Structural proteins VP is the capsid protein IRS of virus then as virus replication start bit
Point and packaging signal.
MDV is the cause of disease of a class specific infection mosquito class, and the fine and close increasing of nucleus of host characteristics is caused after infected mosquito class
Sick change (densonucleosis) causes host to fall ill or dead when serious.People in succession from the Aedes aegypti in nature,
Such disease is separated in various mosquito classes such as aedes albopictus, anopheles minius and kinds of experiments room mosquito cell line/laboratory strains in succession
Poison, isolates obtain more than 20 strains at present, and about 60% is isolated from Wild population.Its host specificity is strong, only infected mosquito class, real
Real its of checking can not infect other species insects, fish, birds and mammal including humans.
MDV has lethal effect in the case of high titre to aedes albopictus individuality, and in the case of low concentration, individual to mosquito have
Have a sublethal effect, including extension Aedes Albopictus Larva larval phase, extension pupates the time, extension emergence time etc..Mosquito multiplies
Virus in unboiled water body can invade the Various Tissues organs such as mosquito fat-body, imaginal discs, tracheae peripheral cell, Malpighian tube.Mosquito children
Energy is substantially reduced after insect infection MDV, distortion, spasmodic tic after being upset, Mortality.Survival larva pupa and plumage
Change time delay, it is easy to dead when casting off a skin.The life-span of adult mosquito can shorten after virus infection, so as to reduce the machine that mosquito propagates cause of disease
Meeting, and metainfective female mosquito spawning rate and the vigor laid eggs also all greatly decline.Virus not only can be via larva, adult water
Flat pass and broadcast, can also be by female mosquito vertical transmission, so that virus can be sent out and various multiply water body and constantly prolong in nature
It is continuous.
The Characteristic of pathogenic bacteria such as the host specificity of mosquito densovirus MDV, biological safety make it have the latent of mosquito control
In the advantage that value and other species cause of diseases can not compare, there is the application prospect as efficient mosquito class biological insecticides, have
Potential value in terms of mosquito matchmaker's infectious disease prevention and control.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of aedes albopictus densovirus and its preparation method and its
Prepare the application in biological culicide.
Above-mentioned technical problem of the invention is achieved by the following technical solution：A kind of aedes albopictus densovirus,
Its entitled aedes albopictus densovirus AalDV-6, is preserved in China typical culture collection center, and deposit number is CCTCC
NO.V201652。
The viral aedes albopictus densovirus AalDV-6 is waited first by Guangzhou by Nanfang Medical Univ associate professor Gu Jinbao
The aedes albopictus of field capture is isolated, and to its Cell culture invitro, morphosis, and the aspect such as security is goed deep into
Research, Organization of viral genome is shown in Fig. 4.
Present invention also offers the complete sequence of above-mentioned aedes albopictus densovirus AalDV-6, its nucleotide sequence such as sequence
Shown in table SEQ ID NO.1, the sequence includes the inverted repeats ITR of aedes albopictus densovirus, two kinds of non-structural proteins
White coded sequence NS1 and NS2, and a kind of structural proteins coded sequence VP.
The preparation method of above-mentioned aedes albopictus densovirus AalDV-6 includes the culture cell line proliferation mosquito densovirus
AalDV-6 plants and raising mosquito larvae propagation mosquito densovirus AalDV-6.Mosquito is bred in the wherein method culture of mass rearing mosquito larvae
Densovirus can significantly improve viral yield, reduce the cost of virus production, operate also relative ease.
Specifically, the method for described mosquito cell line culture propagation is：
(1) aedes albopictus densovirus AalDV-6 (present invention separates what is obtained) is inoculated in susceptible mosquito cell line, and is trained
Support postvaccinal cell, harvesting culture；
(2) by the cell culture multigelation in step (1) repeatedly, after centrifugation, it is mosquito desonucleosis to collect supernatant
Malicious AalDV-6 suspensions.
Preferably by the cell culture multigelation 3 times in step (1) in step (2), preferably 3750 revs/min of centrifugation from
The heart 15 minutes.
Wherein permissive cell can be continuous cell line, or primary cell.It is suitable for the susceptible of mosquito densovirus
Cell includes but is not limited to aedes albopictus C6/36 cell lines, and (ATCC is numbered：CRL-1660), the passage such as Aedes aegypti aag2 cells
Cell line；Primary cell can be separated and prepared with mosquito larvae or its tissue by means commonly known in the art.
The permissive cell, refers to the permissive cell for being suitable for the infection of mosquito densovirus and propagation.It is including but not limited to white
The mosquito class continuous cell lines such as line yellow-fever mosquito C6/36 cells, Aedes aegypti aag2 cells.
The method that the breeding grub prepares mosquito densovirus is：
(1) about 200 susceptible mosquitoes are exposed to 10 by every group8(derived from the mosquito densovirus AalDV-6 of individual/milliliter
Mosquito densovirus AalDV-6 suspensions), and the mosquito larvae of infection is raised, dead larvae is collected, whole larvas are collected after 7 days；
(2) larva obtained in step (1) is ground with 1 milliliter of PBS, 3750 revs/min are centrifuged 15 minutes, and supernatant is used
0.22 μm of membrane filtration is degerming, and filtrate is mosquito densovirus suspension.
The susceptible mosquito, refers to the susceptible mosquito for being suitable for infected mosquito densovirus.Including but not limited to aedes albopictus,
The mosquito larvaes such as Aedes aegypti, Culex quinquefasciatus.
The preparation method of further above-mentioned aedes albopictus densovirus AalDV-6 is comprised the following steps：
(1) virus infection larva
The Aedes Albopictus Larva just casted off a skin is placed in mosquito densovirus AalDV-6 (present invention separates what is obtained), 24 is small
Shi Hou, then use the raising of tortoise grain instead, gradually breed in vivo after virus infection larva；
(2) virus purification
In feeding process, the corpse of dead larvae is collected daily, Cord blood grinds whole larvas with PBS, centrifugation
Afterwards, supernatant is degerming using 0.22 μm of membrane filtration, draws 50 μ L and extracts DNA and virus is entered using real-time fluorescence quantitative PCR
Row is quantitative, and remaining low-temperature preservation obtains the suspension containing mosquito densovirus AalDV-6.
Step (1)~(2) rearing conditions are preferably 28 ± 1 DEG C of constant temperature, and humidity is 70%~80%, illumination and black in one day
Dark each 12h.
Just casted off a skin about 200 Aedes Albopictus Larvas are preferably added 200 milliliter 10 in step (1)8Individual/milliliter concentration
In mosquito densovirus AalDV-6.
In step (2) after preferably 7 days, whole larvas are ground with 1 milliliter of PBS, and 3750 revs/min are centrifuged 15 minutes, supernatant
Liquid is degerming using 0.22 μm of membrane filtration.
The quantitative method of above-mentioned virus concentration, using the method for real-time quantitative fluorescence PCR absolute quantitation well known in the art,
Taqman sonde methods can be also used according to the SYBR Green methods described in embodiment 2.Used in the method for embodiment 2
Primer is as follows：
Forward primer：5 '-CAGGAGGAAACAGCACAAGA-3 ',
Reverse primer：5 '-GTTTCGATACCGTAACGGATGC-3 ',
Annealing temperature is 55 DEG C, and quantitative fluorescent PCR reaction system is formulated as follows：
| 2 SuperReal Green PreMix Plus | 10μL |
| Template | 1μL |
| Forward primer (10 μm) | 0.6μL |
| Reverse primer (10 μm) | 0.6μL |
| 50 ROX Reference Dye | 0.4μL |
| RNase-free | 7.4μL |
Quantitative real time PCR Instrument uses the ABI 7500 of life companies, and response procedures are as follows：
Application of the above-mentioned aedes albopictus densovirus in biological culicide is prepared.
Present invention also offers a kind of biological culicide, it includes aedes albopictus densovirus AalDV-6, by aedes albopictus
Larva is exposed to 1010The aedes albopictus densovirus AalDV-6 of individual/milliliter, the LD50 time LT50 of aedes albopictus are
11.54 days.
The invention has the advantages that：
(1) the aedes albopictus densovirus that the present invention is provided is a kind of virus of infection mosquito, and can not infect other
Species insect, fish, birds and mammal including humans, and parasitics of the virus with height, are completely dependent on place
The energy and metabolic system of chief cell, the matter and energy needed for obtaining vital movement.
(2) the aedes albopictus densovirus in the present invention leaves host cell, and it is a big chemical molecular, stops living
It is dynamic, can be made into crystallization of protein, be a non-life body, only run into mosquito class and its cell just can by absorption, into, it is multiple
Make, assemble, discharge progeny virus and show typical life body characteristicses, its security is indicated according to the feature, and compare
In the biological culicide of other species such as bacterial fungus exploitation, to the requirement of the conditions such as the temperature in storage and transportation process, time
Lower, transport and storage cost are lower.
(3) with the prevalence of mosquito matchmaker's virus spreads such as dengue fever, stockaded village's card heat and Chikungunya fever, mosquito kills for chemistry
The enhanced problem of the worm agent resistance to the action of a drug is gradually highlighted, and the invention of new mosquito matchmaker control method is particularly important, and in the present invention
Mosquito densovirus can be developed into the biological culicide of a class highly effective and safe.
Brief description of the drawings
Fig. 1 is C6/36 intracellular in infected mosquito densovirus AalDV-6 7 days proliferative conditions in embodiment 2；
Fig. 2 is propagation feelings of the mosquito densovirus AalDV-6 in Aedes Albopictus Larva body and in multiplying water body in embodiment 3
Condition；
Fig. 3 is the electromicroscopic photograph of the mosquito densovirus AalDV-6 purified after C6/36 cell culture in embodiment 4；
Fig. 4 is mosquito densovirus AalDV-6 genomes in embodiment 5, and wherein AalDV-6 full-length genomes 3961nt is compiled
Two kinds of non-structural protein NS 1s of code and NS2, and a kind of structural proteins VP, viral genome both sides are terminal repeat
(Inverted terminal repeat sequence, ITR)；
Fig. 5 be in embodiment 6 mosquito densovirus AalDV-6 1010Under the dosage of individual virus/milliliter, aedes albopictus it is tired
The product death rate.
Specific embodiment
The present invention is given specific description in each of the embodiments described below.These embodiments be all it is illustrative, not
The present invention is limited in any way.It will be understood by those skilled in the art that without departing from the spirit and scope of the invention
The details of technical solution of the present invention and form can be modified or replaced, but these modification and replacement each fall within it is of the invention
In protection domain.
Definition：
" coded sequence " refers in this application a kind of DNA sequence dna, and it can be transcribed and obtain corresponding RNA sequence.
" ITR " refers in this application the inverted terminal repeat sequence of viral genome both sides, is tiny with ITR structures
One of feature of Viraceae, the structure has various functions such as the duplication of regulation and control viral gene, transcription, virus rescue.
" NS1 and NS2 " refers to virus nonstructural protein 1 and 2 in this application, and the structure primarily serves helper virus and turns
The effects such as record, packaging.
" VP " refers to virus structural protein in this application, and the structure mainly expresses the capsid of virus.
" carrier " refers in this application artificial constructed, for the recombinant plasmid cloned.
" AalDV-6 " refers in this application one plant of isolated mosquito densovirus from aedes albopictus body, due to existing
Reported in worldwide, isolated 5 plants of mosquitos in the internal or aedes albopictus C6/36 cell lines of aedes albopictus
Densovirus, so the strain virus for separating acquisition in the present invention at present are by the 6th isolated strain virus of aedes albopictus, root
According to ICTV (International Committee on Taxonomy of Viruses, ICTV) 2015
The strain virus are named as AalDV-6 by the virus taxis naming rule that year promulgates, the virus is in Chinese Typical Representative microbial preservation
The preserving number of the heart is CCTCC V201652.
" LT50 " refers in this application the time of half quantity mosquito death in every group.
The separation of the mosquito densovirus AalDV-6 of embodiment 1 and identification
Materials and methods：
Aedes albopictus densovirus AalDV-6 (Aedes albopictus densovirus AalDV-6), in Chinese allusion quotation
Type culture collection (CCTCC) preservation, preserving number：CCTCC V 201652.Preservation date：On November 29th, 2016, protect
Hide place：Wuhan, China, Wuhan University.
The pH7.2 phosphate buffers PBS of this experiment 0.01mM used is purchased from life bio tech ltd of the U.S.,
Viral genome extracts kit TaKaRa miniBEST Viral RNA/DNA Extraction Kit are public purchased from Takara
Department, Thermo Scientific Maxima Hot Start Green PCR Master Mix enzymes are purchased from U.S. Thermo
Fisher companies, carrier T is purchased from Promega companies, and Trans1-T1 Competents cell is purchased from the full formula gold biotechnology in Beijing
Co., Ltd, primer synthesis and DNA sequencing are completed in Shanghai Invitrogen trade Co., Ltd.
The aedes albopictus that Guangzhou field catches, for one group, is ground, 3750 with 2mL PBSs respectively with 20
Rev/min centrifugation 15 minutes, collects supernatant, and 0.22 μm of membrane filtration is degerming, takes 100 μ L of supernatant and extracts viral genome,
The NS1 code areas design primer guarded in mosquito densovirus, detects mosquito densovirus, and primer uses NIH
The design of primers instrument Primer-blast of NCBI is designed, and primer sequence is：
Forward primer：5 '-CAGGAGGAAACAGCACAAGA-3 ',
Reverse primer：5’-GTTTCGATACCGTAACGGATGC-3’
Response procedures：95 DEG C of predegeneration, anneals 55 DEG C, extends 72 DEG C, and extension of time is 15 seconds.
Agarose gel electrophoresis detection and analysis containing ethidium bromide, PCR primer length is 150bp or so, and PCR is detected
The PCR primer of positive sample, purifying is reclaimed, and connects carrier T, converts Trans1-T1 Competent cells, bacterium colony PCR identifications
Afterwards, the sequencing of Shanghai Invitrogen Bioisystech Co., Ltd is submitted to.
After each sample PCR detections, a positive sample is obtained, be sequenced through company after PCR primer connection carrier T, with NCBI
On the densovirus that has found carry out blast, it is found that it is similar to the mosquito densovirus sequence having found.
The aedes albopictus densovirus -6 of embodiment 2 is in aedes albopictus C6/36 cellular proliferative situations
Materials and methods：
This experiment viral genome extracts kit TaKaRa miniBEST Viral RNA/ used
DNAExtraction Kit are purchased from Takara companies, and EcoRV restriction enzymes are purchased from Thermo Fisher companies,
SuperReal PreMix Plus are purchased from Beijing Tiangeng bio tech ltd.
C6/36 passages are to 25cm2Tissue Culture Flask, cell coverage about 70~90%, culture medium is abandoned after 24h
Fall, the PBS rinsings cell of 28 DEG C of preheatings of 1mL once adds the AalDV-6 detected in embodiment 1, supplies and contains 5% tire ox
1640 culture mediums of serum are placed in 28 DEG C of biochemical cultivation case cultures to 5mL.200 μ L cell suspensions are collected daily, and -80 DEG C store,
Last till the 7th day.Remaining cell suspension multigelation 3 times, 4 DEG C 3750 leaves the heart 10 minutes, supernatant be transferred to new 15mL from
Heart pipe, for electron microscopic observation morphology of virus in embodiment 4.
Because MDV belongs to DNA virus, Dnase I except in virus removal mixed liquor it is unpackaged enter virion DNA after, virus
Liquid is extracted with TaKaRa miniBEST Viral RNA/DNA viral genome extracts kits, 50 μ L without enzyme water elution,
Concrete operations are referring to reagent specification.The concentration of fluorescence quantitative PCR detection virus, qPCR primers are as follows：
Forward primer：5 '-CAGGAGGAAACAGCACAAGA-3 ',
Reverse primer：5’-GTTTCGATACCGTAACGGATGC-3’
The preparation of quantitative fluorescent PCR reaction system：
| 2 SuperReal Green PreMix Plus | 10μL |
| Template | 1μL |
| Forward primer (10 μm) | 0.6μL |
| Reverse primer (10 μm) | 0.6μL |
| 50 ROX Reference Dye | 0.4μL |
| RNase-free | 7.4μL |
It is template by the carrier T that PCR primer is connected in embodiment 1, EcoRV carries out single endonuclease digestion, and the linear plasmid of recovery is surveyed
Determine concentration, calculate every microlitre of copy number of linear plasmid in recovery product, computing formula is as follows, copy number=(DNA mass ×
6.022×1023Length × 1 × 10 of)/(template DNA9× 660), DNA mass is the quality of qPCR Plays product, mould in formula
Plate DNA length is the length of standard items.Doubling dilution is carried out successively with multiplying power as 10, chooses wherein 5 points as viral absolute
Quantitative standard items, total copy number of virus in detection virus liquid.
Response procedures are as follows：
Virus concentration is tried to achieve according to following equation：Virus concentration (individual/milliliter)=copy number × 50 μ L/ (200 μ L × 10- 3ML), 50 μ L are elution volume when extracting viral genome in formula, and 200 μ L refer to extracting the viral suspension body of genome
Product.Result is shown in Fig. 1, after adding AalDV-6 in C6/36, from the 1.34 × 10 of the 1st day9Individual/milliliter starts, and virus concentration is presented
The trend for gradually rising, reaches highest 9.32 × 10 on the 5th day9Individual/milliliter, virus concentration increases 5.96 compared with the 1st day
Times, after illustrate AalDV-6 infection C6/36 cells, can in C6/36 cellular proliferatives, after 5 days, cell start it is aging, it is viral dense
Degree begins to decline.
Proliferative conditions of the aedes albopictus densovirus -6 of embodiment 3 in the aedes albopictus body and in multiplying water body
Materials and methods：This experiment TaKaRa miniBEST Viral RNA/DNA Extraction Kit used,
SuperReal PreMix Plus are purchased from Beijing Tiangeng bio tech ltd.
Take the about 1000 aedes albopictus first-instar youngs for just casting off a skin and be divided into one group with every 200, totally five groups, be added separately to
2ml 1010In the AalDV-6 viruses of individual/mL concentration, for the 1st, 3,6,9,12 day Aedes Albopictus Larva body to be interior after infection and multiplies
The detection of virus concentration in unboiled water body.After 24 hours, the water that addition dechlorinates to 200mL is normally raised.Simultaneously in taking-up wherein
Group, collecting 200 microlitres of water bodys is used to multiply the detection of virus concentration in water body.All larvas are then ground with 1mL PBSs,
3750 revs/min are centrifuged 15 minutes, and supernatant is degerming by 0.22 μm of membrane filtration, take out 50 μ L and extract viral genome,
Concentration for detecting virus in larva body, remaining virus is stored in -80 DEG C.Sample collection ginseng after infecting 1,3,6,9,12 days
According to the above method.
Result as shown in Fig. 2 from figure 2 it can be seen that after the instar larvae of aedes albopictus 1 infection AalDV-6, virus infection the
Begin within one day invade in larva body, and breed in larva body, from the 1.86 × 10 of the 1st day10Individual/milliliter starts, to the 9th
It reaches highest 1.65 × 1012Individual/milliliter, virus concentration increases 88.71 times, and dead larva, corpse collapses in water body
Solution is nibbled by other larvas, causes the virus in larva body to be released into water, and virus concentration increased in water body, from the 1st day
2.51×108Individual/milliliter is increased to the 3.65 × 10 of the 9th day10Individual/milliliter, virus concentration increases 145.42 times, compared to implementation
Aedes albopictus C6/36 cell lines production virus in example 2, aedes albopictus first-instar young production has obvious advantage.No matter in children
Still multiplied in water body in polypide, virus suffers from yield higher, and wherein can be enriched to virus higher in larva body can
To reach 1012The rank of individual/milliliter.
The aedes albopictus densovirus Morphological Identification of embodiment 4
Materials and methods：The virus liquid reclaimed in Example 3,4 DEG C of 35000 revs/min of centrifugations, 75 minutes precipitate virus
Particle.The virus of precipitation is further purified for 120 minutes with 1M Sucrose buffers in 4 DEG C of 39000 revs/min of centrifugations.Add chlorination
Caesium (0.3 grams per milliliter), with density gradient separation virus, is examined in 8 DEG C of 60000 revs/min of centrifuged overnights for extracting DNA and Electronic Speculum
Survey.The virion 2%PTA negative stainings that will be purified, shoot under Philips CM12 electron microscopes and amplify 37000.
Result is shown in Fig. 3, from figure 3, it can be seen that after separating-purifying, a large amount of a diameter of 22~25nm can be observed under Electronic Speculum
Icosahedral virion, the virion surface is smooth；A small number of particle centers can be dyeed by negative staining toner, illustrate its
Nucleic acid has flowed out, and is empty viral capsid.
The genome sequence determination of 5 aedes albopictus densovirus of embodiment -5
Materials and methods：Viral genome extracts kit TaKaRa miniBEST Viral RNA/DNA
Extraction Kit are purchased from Takara companies, and T4 DNA ligase, PCR purification kit, klenow enzymes are purchased
From Thermo fisher companies, Trans Stbl3 Competents cell is purchased from Beijing Quan Shijin bio tech ltd；
DNA sequencing transfers to Shanghai Invitrogen bio tech ltd.
TaKaRa miniBEST Viral RNA/DNA Extraction Kit extract the genomic DNA of AalDV-6.Carry
The klenow enzymes of 10 units are added in the AalDV-6 DNA for taking, normal temperature is incubated makes the flat ends of DNA for 15 minutes.By flat end
DNA purified with PCR purification kit.Cloning vector pKMV plasmids containing card that resistance are then restricted with EcoRV
Endonuclease digestion, by linear plasmid gel extraction.AalDV-6 DNA and linear plasmid pKMV the T4 DNA of flat end
Ligase enzymes are connected, and convert Competent Trans-Stbl3, and picking monoclonal bacterium colony carries out bacterium colony PCR identifications, and primer is used
M13F/M13R, primer sequence is as follows：
Forward primer M13-F20 5'-TGTAAAACGACGGCCAGT-3'
Reverse primer M13-Rev 5'-CAGGAAACAGCTATGACC-3'
Positive colony is selected, submits to Shanghai Invitrogen bio tech ltd to carry out DNA sequencing, using two-way survey
It is logical.
As a result：After bacterium colony PCR identifications, positive colony is obtained, carrying out two-way survey using M13-F20 and M13-Rev leads to.Sequencing
Result carries out splicing the complete sequence for obtaining AalDV-6 as follows (see sequence table 1) with the Seqman instruments of DNAstar：
Bioinformatic analysis are carried out to the sequence, it is known that its genome structure is thin with other Aedes aegyptis and aedes albopictus
Born of the same parents be or Mosquito population in it is isolated similar, with more than 90% homology, show that this virus is one plant of new presence
Densovirus in mosquito body.Coding two kinds of non-structural protein NS 1s and NS2, and a kind of structural proteins VP, viral genome two
Side is terminal repeat (Inverted terminal repeat sequence, ITR), wherein AalDV-6 full-length genomes
3981nt (see Fig. 4).
The Strain Designation is aedes albopictus densovirus AalDV-6 (Aedes albopictus densovirus
AalDV-6), China typical culture collection center is preserved in, preservation date is on November 29th, 2016, and deposit number is
CCTCC NO.V201652, preservation address is Wuhan, China, Wuhan University.
The insecticide virulence of 6 aedes albopictus densovirus of embodiment -6 is tested
Materials and methods：The AalDV-6 particles of densovirus containing aedes albopictus according to the method in embodiment 3, by AalDV-6
With 108It is individual/milliliter concentration infection aedes albopictus first-instar young, after 7 days in separation and Extraction Aedes Albopictus Larva body virus and make
Standby, the method according to qPCR absolute quantitations can accurately determine the concentration of AalDV-6.
400 phase larvas of new hatching, are divided into 2 groups, respectively AalDV-6 infected groups and blank control group.At virus
Reason group is added containing viral suspension in the running water of dechlorination so that AalDV-6 viruses are 1010Individual/milliliter, while setting right
According to group, 24h is kept in the case of no food.Add tortoise grain to raise, and monitor larva quantity until pupating, pupa is put into mosquito
In cage.Larva, pupa and adult quantity are recorded daily, and experiment terminates for 28 days after infection.Probit method calculates aedes albopictus half
Number lethal time LT50.
Result is shown in Fig. 5, from figure 5 it can be seen that aedes albopictus is exposed to 1010The densovirus AalDV-6 of individual/milliliter,
Initially enter peak mortality within 7th day, it was observed that 28 days, the death rate of aedes albopictus reaches 96.5%.Carried out generally by SPSS 22.0
It is 11.54 days that rate per unit system is calculated the LD50 time of densovirus AalDV-6,95% confidential interval is 10.848~
12.217。
By the present invention in detailed description above, for those of ordinary skill in the art person, the present invention
Modifications and variations will be apparent, without departing from the spirit and scope of the present invention, the method for the present invention and
Instrument can carry out numerous modifications.These modifications and variations are all included within the scope of the disclosure by claims.
<110 >Chen Xiao patronizes golden guarantor
<120 >Aedes albopictus densovirus and its application
<210 > 1
<211> 3981
<212> DNA
<221>The complete sequence of aedes albopictus densovirus AalDV-6
<400 >1
tataagtcca tattccatat aagaaatatt atttcgtgat acggatactg taagatacag 1
tttctattag aaacgatgta ttacatctgt atcttacagt atccgtatca cgaaataata 61
tttcttatat ggaatatgga cttatatcaa agttctatat ggatcactgg aggtggaaaa 121
taagagagaa acataaggtg gaaaataact tattatccac atacaaatac atccttaatt 181
tccactacca catggtccac ccctatataa ggagtacaaa aggagggcca aatcgagtga 241
tgaattcagt ctgcgttgaa cattcaccgt gtgaacacgg aaatctattt tgtgagtgca 301
tatattgttg ggagcatgac ggtcagtgca gggggagaaa attggatttg ggagcatcaa 361
ctggaatcga aagaagattg gccaacgata accaacaacc agggctctca gatttatatt 421
gcaccgagac aatacatctt gcaactgcaa taccagaaag gagaaccatc gatcgagaaa 481
attacgtcaa agatttcgct ggtcaaaccg ttggtgacct ctacccacaa ttacaaggca 541
gcaccggagc ctctgaacca attgatttcg catttccaac tgttggctca ggaagctggg 601
aaatacttgt acgtgaatct cacaaacatt tcgagccaaa ttatacggaa gaagcttatc 661
aatcacatat tagaagtgta cgaagaagat tattccccga agaaactatg gataataacg 721
ggtcacaggc aagcacgacc gaaatgctac gagacgctgt ccaaagatgc ggttttgaag 781
gccctcctaa cagcccaagc gaaaataaca gagatggaat tgatggaacg tgtatatcaa 841
ccgtggacat acaaagcaat tgtattgtta acgcacattg cccaaaacaa ggaccaagca 901
gtcaaaccaa taagagaaag aaatcaaccg atacaacaga atcaagcgga tccaaaaaaa 961
ataaaagcag caataatcaa caaaatatac aagaacaaag cagttccagc atcaccgacg 1021
cactcgatct cgtcgacgga gagttggatg gatcaactgg atcgaatcga gaaacagcat 1081
actacacatt cgtcctccac aaaaacaacg ttaaagagga ctggagatac atcgccacaa 1141
ccagggccaa gcaagcgccg agtttcatca cattcgatca cggagaccac atccatatcc 1201
tcttctcctc gtccaataca ggaggaaaca gcacaagagt cagaaccaga atcaccaagt 1261
ttcttagtgc aacaagcgca ggaagtgcag aagcgactat cactttttcc aaagttaaat 1321
ttctcaggaa ctacattctc tattgcatcc gttacggtat cgaaacagtc aatatctatg 1381
gaaataaaat ccaacaacaa ttaaccgaag caatggatac atttaaaata ttatttgaaa 1441
atagagaccc aaatgacgta atattagaag ccggatgcaa attatatcat gaagaaaaaa 1501
aggataataa acaaaaaaga tgcggacaac ggaagcaaca aaatctaacg gacattatat 1561
tggaaaaaat taaagaaaag aaaattacaa cggcacaaca atgggaaaat caaattgaac 1621
cggaattcaa aatacaatta atgaaagagt ttggattaaa tgtggacagt tatgtaacaa 1681
gaatagtacg catcgaaaga acacgtatac aacaattgat aaaagcaaaa acgcttacgg 1741
aaataatgct tgaaatatta aatgatgact atattaaaca cttcacacca ggagaagaca 1801
acagcaaaac aacaaaatgt attgaatgga tagaatatct attcaaagaa aataacatca 1861
atataatcca cttcttggca tggaatgaaa ttataaaaac aaaaagatat aaaaaaataa 1921
acggaatggt actagaaggt atcacaaacg caggaaaatc actaatatta gacaacttat 1981
tggccatggt taaaccagaa gaaataccac gagaacgaga caacagtgga ttccaccttg 2041
accaagtacc aggagcagga tcgatcctat ttgaagaacc aatgatcaca ccagtaaacg 2101
tcggaacatg gaaattatta ttagaaggaa aaaccataaa aacggatgta aaaaacaaag 2161
acaaggaacc gatagaacgc acaccaacgt ggatcacaac agcaactcca ataacaaaca 2221
acattgatat gaatgaaaca tcacaaatac tacaaagaat aaaattatat atattcaaaa 2281
aaagtatcca acacagagac gacaaatata ctataaatgc gcaaattcaa aataaattaa 2341
tcagtcgtcc tccaactctc attgagccaa tacatatggc catagtgttt ataaaaaatt 2401
tcacaaaaat atataatcta atcgcagaag aagacaaggc acacacagta aacgaaaagg 2461
caatacaaat aagcaacgaa gtgaaagaag aagcagaatc atggcagaca gcactacaat 2521
ggaccatgat ggagaacaac gaggaacaaa acgaaaacga gacgcaggag ctggaggatc 2581
aggtgctgga attggcaaag gagcaagcaa ctacgtaaaa gaaggatatg gaccaaatat 2641
gaccgaaatg gtaccaagaa acatattgaa taaaggaaat cacacagtct atcatgtggt 2701
aaaacaacaa aaatatctag acttcaatta cgtaacaaat caaaacccat acataattcc 2761
atatcaaaca gcaggattct gggcatcaat gtgggaccaa acagacatcg gatccaataa 2821
cagcattaat ataatgaaag cattaaacaa cgtatcacta ggagtaacat ggatcaaagg 2881
agaaatcaca ttcgaagtat attcagtaac aagacaacgc ttgctaacag gaacaacaaa 2941
ccaaactaca tgggactttg aaacaagtca aaacatgttc atcgcagatg cagacagaga 3001
accagaaaac ttcaacttag caacagcagc agcaactgga ccacttgcac aacaaacaac 3061
acaaacatta ctattcaatg caaacaatga cagatataca aaatatgaat taccacaaag 3121
aaaccaatat acaagggaaa ttaacttcca acaattaaca aacaactata tgtggaaacc 3181
attggacatc agcgctgcag caaactttag aagattgatc ccaatggcag aaggagtata 3241
tacaacatca aatgcaacaa gtaaaatgac agaattaaca aatcaaacta cagcatacgc 3301
cacatcaggc aaaacaacac aagcaacact attcagaaat agaacatcat accctagaat 3361
gcatatggca caaccacaag ttccagatga aaccggatac atgaaattca gataccaagt 3421
acgaatgagc acaaaactac atctcgaatt ccatctctac ccagattacg gcacatcaac 3481
aaacatagaa tatatgcaga gacaagtact ggaattacca gaagtaacag caacaggagg 3541
agtggtaaca tgtatgccgt atgaaatcaa aacttaaata ttaaattcaa cttgtatcaa 3601
ctataacaca tatataatca ataaagcatt caaaaaacat ataagtcaaa ttaatatata 3661
tcacaataaa aatccacctt aaaacataag cttaatttcc acctccgtat tccacctcag 3721
aatattggct taaaatccac ctccaatgat acagttagga agctaatatt agtccgggat 3781
ccccgtgtgg ccgataggcg aggatcgaaa gcccaaattt tgatgacgtc acctcacaca 3841
cataccaaaa gctttagttt ctaatagaaa cagcgtatta cgcttaaagc ttttggtatg 3901
tgtgtgaggt gacgtcatca a 3961。
Claims (2)
1. a kind of aedes albopictus densovirus, it is characterized in that：Its entitled aedes albopictus densovirus AalDV-6, in being preserved in
State's Type Tissue Collection, deposit number is CCTCC NO.V201652.
2. application of the aedes albopictus densovirus described in claim 1 in biological culicide is prepared.
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| CN109536464A (en) * | 2018-12-10 | 2019-03-29 | 中国科学院武汉病毒研究所 | It is a kind of lack capsid protein gene chikungunya virus infection clones and construction method and preparation attenuated vaccine in application |
| CN110110945A (en) * | 2019-05-23 | 2019-08-09 | 信阳农林学院 | A kind of insect pest forecast method and system based on population model |
| CN110946150A (en) * | 2019-12-27 | 2020-04-03 | 武汉市拜乐卫生科技有限公司 | Compound mosquito killing oil suspension and preparation method thereof |
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| CN109536464A (en) * | 2018-12-10 | 2019-03-29 | 中国科学院武汉病毒研究所 | It is a kind of lack capsid protein gene chikungunya virus infection clones and construction method and preparation attenuated vaccine in application |
| CN109536464B (en) * | 2018-12-10 | 2022-06-10 | 中国科学院武汉病毒研究所 | Chikungunya virus infectious clone with deletion of capsid protein gene, construction method and application in preparation of attenuated vaccine |
| CN110110945A (en) * | 2019-05-23 | 2019-08-09 | 信阳农林学院 | A kind of insect pest forecast method and system based on population model |
| CN110110945B (en) * | 2019-05-23 | 2021-04-09 | 信阳农林学院 | Pest prediction method and system based on population model |
| CN110946150A (en) * | 2019-12-27 | 2020-04-03 | 武汉市拜乐卫生科技有限公司 | Compound mosquito killing oil suspension and preparation method thereof |
| CN110946150B (en) * | 2019-12-27 | 2021-08-27 | 武汉市拜乐卫生科技有限公司 | Compound mosquito killing oil suspension and preparation method thereof |
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