CN104162151A - Vaccine for preventing and/or treating dengue fever - Google Patents

Vaccine for preventing and/or treating dengue fever Download PDF

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CN104162151A
CN104162151A CN201310181920.3A CN201310181920A CN104162151A CN 104162151 A CN104162151 A CN 104162151A CN 201310181920 A CN201310181920 A CN 201310181920A CN 104162151 A CN104162151 A CN 104162151A
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程功
刘洋
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Tsinghua University
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Abstract

The invention discloses a vaccine for preventing and/or treating dengue fever. The active component of the vaccine can inhibit the expression of encoding gene of C-type agglutinin in aedes. The vaccine can be used to inhibit the dengue virus from replicating in aedes, prevent and/or treat dengue fever, and stop the propagation of dengue virus. The invention finds that the replication of dengue virus in aedes can be inhibited through inhibiting the activity of aedes C-type agglutinin protein, thus the ability of obtaining dengue virus from hosts of aedes is reduced, so the virus carrying rate of aedes is prominently reduced, and the goal of preventing and treating dengue fever is achieved. The vaccine has an important value on controlling dengue fever, and has an important meaning for human health.

Description

A kind of vaccine preventing and/or treating for dengue fever
Technical field
The present invention relates to a kind of vaccine preventing and/or treating for dengue fever.
Background technology
Dengue fever (Dengue Fever, DF) is by her mosquito-borne viral acute infectious disease.(Dengue virus, DENV) is spherical in shape for dengue virus, after birth, capsid protein, non-structural protein Pseudobulbus Bletillae (Rhizoma Bletillae) sub-thread positive chain RNA, consists of.Occurring in nature, there are four kinds of serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) in dengue virus.Dengue virus is bitten and is infected after human body via yellow-fever mosquito, through 2-3 days, hides and can show clinical symptoms.The Clinical symptoms of dengue fever is that high heat, skeleton and muscular soreness, bleeding tendency and numeration of leukocyte obviously reduce.Dengue fever clinical manifestation is slighter, with the spontaneous recovery of course advancement most people, fatality rate is low. but a few peoples' the course of disease can proceed to next stage, be dengue hemorrhagic fever (Dengue Hemorrhagic Fever, DHF) and Dengue shock syndrome (Dengue Shock Syndrome, DSS), this is a kind of serious type of dengue infection, clinical manifestation is each organ severe haemorrhage, height is warm, platelet count obviously reduces, blood is concentrated, shock, causes the mortality rate of this course of disease high.
Dengue virus is mainly bitten the propagation of human body postoperative infection by Aedes aegypti (Aedes aegypti).Think at present, the natural reservoir (of bird flu viruses) of dengue virus only has people, primates and the yellow-fever mosquito such as low.At occurring in nature, dengue virus mainly circulates in two kinds of patterns: 1) city circulation (Urban Cycle): in the dengue prevalence at urban type, virus is propagated between " people-yellow-fever mosquito-people "; 2) jungle circulation (Sylvatic Cycle): be mainly that this circulation is the original loop of dengue virus by propagating between the yellow-fever mosquito in matto and the primates such as low.According to the study, the dengue virus under jungle type dengue virus and city cycling condition differs greatly, and the infection yellow-fever mosquito that still can not get rid of jungle circulation diffuses to city and rural probability by virus.
At present, in worldwide, existing more than 100 countries and area occur that the infection of dengue fever is popular.According to the World Health Organization (WHO), estimate, the region that threatened by dengue infection is approximately lived in by 2,500,000,000 people in the whole world, has every year 5000 ten thousand people by dengue virus primary infection or repeated infection, has 500,000 people to be hospitalized for treatment, and cause about 25,000 people's death.Dengue fever become in the world first viral arthropod borne infection ( http:// www.who.int/tdr/publications/publications/dengue).Over nearly 10 years, dengue fever fashion trend aggravation in the world, has become a global public health difficult problem.
C type agglutinin (C-type Lectin, CTL) is that a class has C type agglutinin (C-type lectin) domain, the protein families that can be combined with saccharide, and it is extensively to exist in most eukaryotes.A plurality of genes in mammal in C type lectin family are at dendritic cell (Dendritic cell, DC), a large amount is expressed in the immunocyte such as macrophage (Macrophage) and mononuclear cell (Monocytes), and play an important role in the process that activates host immune response.C type agglutinin has a plurality of subtype expression in yellow-fever mosquito, and mainly with the form of secretory protein, exists.
There is no in the world at present specific medicament and the vaccine for dengue fever.The pathogenesis of dengue fever is special, and low-level dengue antibody can promote the superinfection (antibody relies on enhancement effect, Antibody-dependent Enhancement, ADE) of different serotypes virus significantly.This phenomenon is considered to one of principal element of bringing out dengue hemorrhagic fever.Antibody relies on enhancement effect the safety of traditional dengue vaccine is greatly reduced, and has seriously hindered the research and development of dengue vaccine.So far, finding new method comes the infection of " antagonism " dengue virus extremely urgent with propagation.
Summary of the invention
The object of this invention is to provide a kind of vaccine preventing and/or treating for dengue fever.
The invention provides a kind of product, the material that its active component is expressed for suppressing the encoding gene of the C type agglutinin in yellow-fever mosquito body; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
The present invention also protects a kind of product, thereby its active component is combined and is suppressed the active material of described C type agglutinin for C type agglutinin in yellow-fever mosquito body; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.Described " thereby the C type agglutinin in yellow-fever mosquito body be combined suppress the active material of described C type agglutinin " is the antibody of described C type agglutinin.
The present invention also protects a kind of product, thereby its active ingredient is the material of the active material that impels the C type agglutinin producing in animal body in yellow-fever mosquito body to be combined to suppress described C type agglutinin; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.Described " thereby material of the active material that impels the C type agglutinin producing in animal body with yellow-fever mosquito body in to be combined to suppress described C type agglutinin " is C type agglutinin.
The present invention also protects and suppresses the application in preparing product of material that the encoding gene of the C type agglutinin in yellow-fever mosquito body expresses; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
The present invention also protects the application of C type agglutinin in preparing product; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
Arbitrary described leather fever virus can be dengue fever 2 type viruses above, specifically can be New Guinea C strain.
Arbitrary described C type agglutinin is arbitrary described protein in following (1) to (19) above: the protein that the sequence 1 of (1) sequence table forms from 5 ' end 21-168 amino acids residue; (2) protein shown in the sequence 1 of sequence table; (3) protein that the sequence 3 of sequence table forms from 5 ' end 17-174 amino acids residue; (4) protein shown in the sequence 3 of sequence table; (5) protein that the sequence 5 of sequence table forms from 5 ' end 18-191 amino acids residue; (6) protein shown in the sequence 5 of sequence table; (7) protein that the sequence 7 of sequence table forms from 5 ' end 26-154 amino acids residue; (8) protein shown in the sequence 7 of sequence table; (9) protein that the sequence 9 of sequence table forms from 5 ' end 20-158 amino acids residue; (10) protein shown in the sequence 9 of sequence table; (11) protein that the sequence 11 of sequence table forms from 5 ' end 17-153 amino acids residue; (12) protein shown in the sequence 11 of sequence table; (13) protein that the sequence 13 of sequence table forms from 5 ' end 26-154 amino acids residue; (14) protein shown in the sequence 13 of sequence table; (15) protein that the sequence 15 of sequence table forms from 5 ' end 22-173 amino acids residue; (16) protein shown in the sequence 15 of sequence table; (17) protein that the sequence 17 of sequence table forms from 5 ' end 26-160 amino acids residue; (18) protein shown in the sequence 17 of sequence table; (19) by arbitrary described protein in (1) to (18) through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and have identical function by its derivative protein.
Arbitrary described C type agglutinin also can be the C type agglutinin with HIS label above.
The encoding gene of arbitrary described C type agglutinin is following 1 above) to arbitrary described DNA molecular in (20): the sequence 2 of (1) sequence table is from the DNA molecular shown in 5 ' end 61-504 position nucleotide; (2) DNA molecular shown in the sequence 2 of sequence table; (3) sequence 4 of sequence table is from the DNA molecular shown in 5 ' end 49-522 position nucleotide; (4) DNA molecular shown in the sequence 4 of sequence table; (5) sequence 6 of sequence table is from the DNA molecular shown in 5 ' end 52-573 position nucleotide; (6) DNA molecular shown in the sequence 6 of sequence table; (7) sequence 8 of sequence table is from the DNA molecular shown in 5 ' end 76-462 position nucleotide; (8) DNA molecular shown in the sequence 8 of sequence table; (9) sequence 10 of sequence table is from the DNA molecular shown in 5 ' end 58-474 position nucleotide; (10) DNA molecular shown in the sequence 10 of sequence table; (11) sequence 12 of sequence table is from the DNA molecular shown in 5 ' end 49-459 position nucleotide; (12) DNA molecular shown in the sequence 12 of sequence table; (13) sequence 14 of sequence table is from the DNA molecular shown in 5 ' end 76-462 position nucleotide; (14) DNA molecular shown in the sequence 14 of sequence table; (15) sequence 16 of sequence table is from the DNA molecular shown in 5 ' end 64-519 position nucleotide; (16) DNA molecular shown in the sequence 16 of sequence table; (17) sequence 18 of sequence table is from the DNA molecular shown in 5 ' end 76-480 position nucleotide; (18) DNA molecular shown in the sequence 18 of sequence table; (19) under stringent condition with (1) to (18) in the DNA sequence hybridization of arbitrary restriction and the DNA molecular that coding has the albumen of identical function; (20) with (1) to (18) in the DNA sequence of arbitrary restriction there is the DNA molecular that 90% above homology and coding have the albumen of identical function.Described stringent condition is at 0.1 * SSPE(or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃ of conditions, hybridize and wash film.
Above arbitrary described " suppressing the material that the encoding gene of the C type agglutinin in yellow-fever mosquito body is expressed " specifically can be following 1. to arbitrary described dsRNA in 9.: 1. the sequence 2 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 1st to 333 nucleotide of 5 ' end; 2. the sequence 4 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in 5 ' end 31-381 position nucleotide; 3. the sequence 6 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 79th to 407 nucleotide of 5 ' end; 4. the sequence 8 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 118th to 452 nucleotide of 5 ' end; 5. the sequence 10 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 78th to 471 nucleotide of 5 ' end; 6. the sequence 12 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 1st to 389 nucleotide of 5 ' end; 7. the sequence 14 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 24th to 413 nucleotide of 5 ' end; 8. the sequence 16 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 38th to 415 nucleotide of 5 ' end; 9. the sequence 18 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 149th to 465 nucleotide of 5 ' end.
The present invention finds, suppress yellow-fever mosquito C type agglutinin (mosquito Galactose specific C-type Lectin, mosGCTL) activity of albumen can suppress dengue virus copying in yellow-fever mosquito body, cut off yellow-fever mosquito and in host, obtain the ability of dengue virus in the process of sucking blood, thus significantly reduce mosquito band poison rate, reach the object of control dengue virus.In actual applications, blocking-up vaccine not only can carry out immunity to single hypotype, also can form combination vaccine to multiple hypotype co-immunization, the propagation of blocking-up dengue virus.The present invention has great value for the control of dengue fever, for human health cause, has far reaching significance.
Accompanying drawing explanation
Fig. 1 is the result of the step 2 of embodiment 1 to embodiment 9.
Fig. 2 is the result of embodiment 14.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.Actin gene is as shown in the sequence 20 of sequence table.
Aedes aegypti (Aedes aegypti): list of references: Colpitts TM, Cox J, Vanlandingham DL, Feitosa FM, Cheng G, et al. (2011) Alterations in the Aedes aegypti Transcriptome during Infection with West Nile, Dengue and Yellow Fever Viruses.PLoS Pathog7 (9): e1002189.doi:10.1371/journal.ppat.1002189..
Dengue fever 2 type viruses (dengue virus type-2, DENV-2) used in embodiment are dengue fever 2 type virus N ew Guinea C strains; List of references: Chao Y C, Huang C S, Lee C N, et al.Higher infection of dengue virus serotype 2 in human monocytes of patients with G6PD deficiency[J] .PloS one, 2008,3 (2): e1557..
Plasmid pET-28a (+): Merck Millipore company (former Novagen company, catalog number 69864.
E. coli bl21 (DE3): Merck Millipore company (former Novagen company, catalog number 69450).
Hemotek mosquito feeding device (this system sample of can 37 ℃ of heating of constant temperature feeding, simulation human body environment, capable of attracting mosquitoes picked-up sample blood): Hemotek LIMITED company (Britain company, catalog number 6W1Hemotek membrane feeding system).
MID 50(50%mosquito Infectious dose) is half mosquito infective dose; MID 50numerical value implication for to make half Aedes aegypti infect viral extension rate by thoracic cavity injecting virus diluent 300nL; 1MID 50for virus stock solution used being diluted to its MID 50after numerical value multiple, thoracic cavity injection 300nL can make half Aedes aegypti infect.
The application of the material of embodiment 1, inhibition mosGCTL-3 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 3 of Aedes aegypti claims again mosGCTL-3 albumen, as shown in the sequence 1 of sequence table.The gene of coding mosGCTL-3 albumen claims again mosGCTL-3 gene, and the coding region of its cDNA is as shown in the sequence 2 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-1 that suppresses mosGCTL-3 gene expression), the sequence 2 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 1st to 333 nucleotide of 5 ' end.
Be designed for the dsRNA(dsRNA-2 that suppresses GFP gene expression), i.e. double-stranded RNA shown in the sequence 19 of sequence table.
Two, the application of the material of inhibition mosGCTL-3 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-1 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Dengue fever 2 type virus quantities in Aedes aegypti body are shown in Fig. 1, and the unit of vertical coordinate is " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing ".Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.109ng(20 meansigma methods), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods).
Result shows, import for suppressing after the dsRNA of mosGCTL-3 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-3 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-3 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 2, inhibition mosGCTL-26 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 26 of Aedes aegypti claims again mosGCTL-26 albumen, as shown in the sequence 3 of sequence table.The gene of coding mosGCTL-26 albumen claims again mosGCTL-26 gene, and the coding region of its cDNA is as shown in the sequence 4 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-3 that suppresses mosGCTL-26 gene expression), the sequence 4 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in 5 ' end 31-381 position nucleotide.
Two, the application of the material of inhibition mosGCTL-26 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-3 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.252g(20 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-26 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-26 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-26 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 3, inhibition mosGCTL-15 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 15 of Aedes aegypti claims again mosGCTL-15 albumen, as shown in the sequence 5 of sequence table.The gene of coding mosGCTL-15 albumen claims again mosGCTL-15 gene, and the coding region of its cDNA is as shown in the sequence 6 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-4 that suppresses mosGCTL-15 gene expression), the sequence 6 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 79th to 407 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-15 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-4 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.182ng(23 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-15 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-15 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-15 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 4, inhibition mosGCTL-19 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 19 of Aedes aegypti claims again mosGCTL-19 albumen, as shown in the sequence 7 of sequence table.The gene of coding mosGCTL-19 albumen claims again mosGCTL-19 gene, and the coding region of its cDNA is as shown in the sequence 8 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-5 that suppresses mosGCTL-19 gene expression), the sequence 8 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 118th to 452 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-19 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-5 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.194ng(15 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-19 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-19 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-19 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 5, inhibition mosGCTL-20 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 20 of Aedes aegypti claims again mosGCTL-20 albumen, as shown in the sequence 9 of sequence table.The gene of coding mosGCTL-20 albumen claims again mosGCTL-20 gene, and the coding region of its cDNA is as shown in the sequence 10 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-6 that suppresses mosGCTL-20 gene expression), the sequence 10 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 78th to 471 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-20 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-6 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.268ng(15 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-20 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-20 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-20 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 6, inhibition mosGCTL-22 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 22 of Aedes aegypti claims again mosGCTL-22 albumen, as shown in the sequence 11 of sequence table.The gene of coding mosGCTL-22 albumen claims again mosGCTL-22 gene, and the coding region of its cDNA is as shown in the sequence 12 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-7 that suppresses mosGCTL-22 gene expression), the sequence 12 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 1st to 389 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-22 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-7 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.227ng(17 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-22 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-22 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-22 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 7, inhibition mosGCTL-23 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 23 of Aedes aegypti claims again mosGCTL-23 albumen, as shown in the sequence 13 of sequence table.The gene of coding mosGCTL-23 albumen claims again mosGCTL-23 gene, and the coding region of its cDNA is as shown in the sequence 14 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-8 that suppresses mosGCTL-23 gene expression), the sequence 14 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 24th to 413 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-23 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-8 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.199ng(15 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-23 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-23 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-23 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 8, inhibition mosGCTL-24 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 24 of Aedes aegypti claims again mosGCTL-24 albumen, as shown in the sequence 15 of sequence table.The gene of coding mosGCTL-24 albumen claims again mosGCTL-24 gene, and the coding region of its cDNA is as shown in the sequence 16 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-9 that suppresses mosGCTL-24 gene expression), the sequence 16 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 38th to 415 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-24 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-9 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.191ng(15 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-24 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-24 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-24 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The application of the material of embodiment 9, inhibition mosGCTL-32 gene expression in inhibition dengue virus copies
The C type agglutinin hypotype 32 of Aedes aegypti claims again mosGCTL-32 albumen.As shown in the sequence 17 of sequence table.The gene of coding mosGCTL-32 albumen claims again mosGCTL-32 gene, and the coding region of its cDNA is as shown in the sequence 18 of sequence table.
One, design dsRNA
Be designed for the dsRNA(dsRNA-10 that suppresses mosGCTL-32 gene expression), the sequence 18 of sequence table is from double-stranded RNA corresponding to double-stranded DNA shown in the 149th to 465 nucleotide of 5 ' end.
Two, the application of the material of inhibition mosGCTL-32 gene expression in suppressing dengue fever 2 type virus replications
1, Aedes aegypti is divided into two groups, is handled as follows respectively:
Experimental group: every Aedes aegypti is injected 2 microgram dsRNA-10 by thoracic cavity;
Matched group: every Aedes aegypti is injected 2 microgram dsRNA-2 by thoracic cavity;
2, the injection of completing steps 1 is after 3 days, and to Aedes aegypti inoculation dengue fever 2 type viruses, (every Aedes aegypti is 10MID by thoracic cavity injected dose 50dENV-2 virus, volume injected is 300nL).
3, the inoculation of completing steps 2, after 6 days, utilizes Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body.
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
Adopt " for the mRNA that often contains 1ng Actin gene, the ng number of the E gene containing " to characterize dengue fever 2 type virus quantities.Dengue fever 2 type virus quantities in experimental group Aedes aegypti body are 0.180ng(16 meansigma methods only), the dengue fever 2 type virus quantities in matched group Aedes aegypti body are 0.494ng(22 meansigma methods only).
Result shows, import for suppressing after the dsRNA of mosGCTL-32 gene expression, obviously declining appears in the virus quantity of the dengue fever 2 type viruses in Aedes aegypti body, suppress mosGCTL-32 gene expression and can suppress dengue fever 2 type viruses copying in Aedes aegypti body, mosGCTL-32 gene can promote dengue fever 2 type viruses copying in Aedes aegypti body.
The preparation of embodiment 10, mosGCTL-3 protein maturation peptide
1, total RNA the reverse transcription of extraction Aedes aegypti obtain cDNA.
2, take the cDNA that step 1 obtains is template, with the primer pair that F1 and R1 form, carries out pcr amplification, obtains pcr amplification product.
F1：5’-tctcta GCTAGCCAGCCCATATGCAGTGAT-3’；
R1：5’-tatccg CTCGAGAAACTCCTGGATTGAAT-3’。
3, with the pcr amplification product of restricted enzyme Nhe I and Xho I double digestion step 2, reclaim enzyme action product.
4,, with restricted enzyme Nhe I and Xho I double digestion plasmid pET-28a (+), reclaim the carrier framework of about 5290bp.
5, the enzyme action product of step 3 and the carrier framework of step 4 are connected, obtain recombiant plasmid (in recombiant plasmid, the coded sequence of the His label on external source fragment and carrier merges, expression has the mosGCTL-3 protein maturation peptide of His label, and (mosGCTL-3 protein maturation peptide is comprised of from 5 ' end 21-168 amino acids residue the sequence 1 of sequence table, and N end and C that His label is positioned at mature peptide hold; N has held His label and mosGCLT-3 mature peptide midfeather following 13 amino acid residues " Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Ala Ser "; Two aminoacid of C end His label and mature peptide midfeather Xho I restriction endonuclease recognition sequence coding).
6, recombiant plasmid step 5 being obtained imports e. coli bl21 (DE3), obtains recombinant bacterium.
The monoclonal colony inoculation of the recombinant bacterium 7, step 6 being obtained (containing 25 μ g/ml kanamycin) to 5ml LB fluid medium, 37 ℃, 220rpm concussion are cultivated 16 hours; Then by the volume ratio of 1:100, be seeded to fresh LB fluid medium (containing 25 μ g/ml kanamycin), cumulative volume is 500ml, and 37 ℃, 220rpm concussion are cultivated 4 hours; Then adding IPTG and making its concentration is 1mM, and 37 ℃, 220rpm concussion are cultivated 6 hours; Room temperature, the centrifugal 10min of 4000rpm, collect thalline.
8, get the thalline that step 7 obtains, with inclusion body cleaning mixture, (solvent is the Tris-HCl buffer of pH8.0,50mM, contains 100mM NaCl, 5mM EDTA, 0.1%NaN 3, 0.5%Triton-X100, with front adding final concentration, be the PMSF of 0.1mM and the DTT of 1mM) suspension thalline, (60% power of ultrasonication on ice, ultrasonic 3s, stops 9s, total supersound process time 5min), the centrifugal 15min of 6000rpm, collecting precipitation (inclusion body).
9, the precipitation obtaining by inclusion body cleaning mixture suspension step 8, adds excessive MgSO 4with in and inclusion body cleaning mixture in EDTA, then adding final concentration is the DNA enzyme (the catalog number (Cat.No.) DN25 of Sigma company) of 0.01mg/ml and the lysozyme (the catalog number (Cat.No.) L6876 of Sigma company) that final concentration is 0.1mg/ml, room temperature treatment 20 minutes, the centrifugal 15min of 6000rpm, collects inclusion body; Then, the precipitation obtaining by inclusion body cleaning mixture suspension step 8, adds excessive MgSO 4with in and EDTA in inclusion body cleaning mixture, room temperature treatment 20 minutes, the centrifugal 15min of 6000rpm, collects inclusion body; Then, the precipitation obtaining by inclusion body cleaning mixture suspension step 8, adds excessive MgSO 4with in and EDTA in inclusion body cleaning mixture, room temperature treatment 20 minutes, the centrifugal 15min of 6000rpm, collects inclusion body.
The inclusion body of collection is carried out to polyacrylamide gel electrophoresis, the object band of clear display (about 18kD).
10, use protein purification liquid (solvent is the Tris buffer of pH8.0,100mM, contains 50mM glycine and the 8M carbamide) inclusion body that dissolving step 9 obtains, obtain crude protein solution.
11, use TALON purification Kit (Clontech company catalog number (Cat.No.) 635515), purification mosGCTL-3 protein maturation peptide from crude protein solution.4 ℃ of 1ml Resin and 50ml crude protein solution are hatched 2 hours altogether, then be transferred in filter post, by 20ml cleaning mixture first (the 1X balance liquid in TALON test kit adds 8M carbamide), wash, use again 20ml cleaning mixture second (the 1X balance liquid in TALON test kit adds 8M carbamide and 20mM imidazoles) to wash, finally use 5ml eluent third (the 1X balance liquid in TALON test kit adds 8M carbamide and 150mM imidazoles) to wash and collected solution after post, (purity of protein is greater than 95% to be the solution that contains mosGCTL-3 protein maturation peptide, concentration is greater than 1mg/ml), be called for short mosGCTL-3 mature peptide solution.
Embodiment 11, the application of the anti-mosGCTL-3 albumen of rabbit serum in suppressing dengue fever 2 type virus replications
One, the preparation of the anti-mosGCTL-3 serum of rabbit
Laboratory animal is new zealand rabbit: Jin Sirui (Genescript) company.
Idiographic flow is as follows:
The 0th day, get rabbit serum, be control serum.
The 1st day (initial immunity), every rabbit skin hemostasis embodiment 10 mosGCTL-3 mature peptide solution of preparation and the equal-volume mixture of Freund's complete adjuvant (containing 1mg mosGCTL-3 mature peptide).
The 14th day (booster immunization for the first time), every rabbit skin hemostasis embodiment 10 mosGCTL-3 mature peptide solution of preparation and the equal-volume mixture of Freund's complete adjuvant (containing 0.5mg mosGCTL-3 mature peptide).
The 24th day, every rabbit was got 100 serum, and ELISA detects and confirms that immunne response is normal.
The 35th day (booster immunization for the second time), every rabbit skin hemostasis embodiment 10 mosGCTL-3 mature peptide solution of preparation and the equal-volume mixture of Freund's complete adjuvant (containing 0.5mg mosGCTL-3 mature peptide).
The 56th day (booster immunization for the third time), every rabbit skin hemostasis embodiment 10 mosGCTL-3 mature peptide solution of preparation and the equal-volume mixture of Freund's complete adjuvant (containing 0.5mg mosGCTL-3 mature peptide).
The 70th day, every rabbit gathered 40ml serum, is the anti-mosGCTL-3 serum of rabbit.
Two, the application of the anti-mosGCTL-3 albumen of rabbit serum in suppressing dengue fever 2 type virus replications
1, use the blood of heparin blood taking tube extraction normal healthy people (volunteer of informed consent), 4 ℃, the centrifugal 10min of 1000g, separated plasma and hemocyte.
2, get the hemocyte that step 1 obtains, use pre-cooling PBS buffer washing 3 times, to remove residual adsorption at the heparin (heparin can affect the normal physiological function of mosquito) on hemocyte surface.
3, get the blood plasma that step 1 obtains, 55 ℃ of water bath processing 1 hour, with the activity of complement system in deactivation blood plasma (complement can kill virus, suppress viral infectivity).
4, the blood plasma that hemocyte step 2 being obtained and step 3 obtain mixes, and places standby on ice.
5, the preparation of mixed feeding sample
Mixed feeding sample one: the mixing blood that 490 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) the anti-mosGCTL-3 serum of and 10 μ l rabbit (diluting 100 times) mix homogeneously is standby.
Mixed feeding sample two: the mixing blood that 499 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) the anti-mosGCTL-3 serum of and 1 μ l rabbit (diluting 1000 times) mix homogeneously is standby.
Mixed feeding sample three: the mixing blood that 499.8 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) the anti-mosGCTL-3 serum of and 0.2 μ l rabbit (diluting 5000 times) mix homogeneously is standby.
Mixed feeding sample four: the mixing blood that 490 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 10 μ l control serums (diluting 100 times) mix homogeneously is standby.
Mixed feeding sample five: the mixing blood that 499 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 1 μ l control serum (diluting 1000 times) mix homogeneously is standby.
Mixed feeding sample six: the mixing blood that 499.8 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 0.2 μ l control serum (diluting 5000 times) mix homogeneously is standby.
Use the Hemotek mosquito feeding device Aedes aegypti 30 minutes (every kind of sample feed approximately 50 Aedes aegyptis) of respectively above six kinds of samples being fed.The mosquito that confirm to suck mixes blood is picked out and puts into special container and feed with the rayon balls that contains 1% sucrose solution in standard conditions (70% humidity, 28 ℃).Raise and put to death afterwards for 8 days, total RNA the reverse transcription of extracting mosquito are cDNA, utilize Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body, and calculate infection rate (positive that is designated as that is greater than 0.0002 with the ratio ratio of dengue virus ng number and internal contrast Actin gene ng number).
The primer pair that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
Forward primer: 5 '-CATTCCAAGTGAGAATCTCTTTGTCA-3 ';
Downstream primer: 5 '-CAGATCTCTGATGAATAACCAACG-3 '.
The probe that detects dengue fever 2 type viruses for Taqman RT-QPCR is as follows:
5’-FAM-ATGCTGAAACGCGAGAGAAACCGC-TRAMA-3’。
The results are shown in Table 1.
The impact of the anti-mosGCTL-3 serum of rabbit of table 1 blood meal variable concentrations on Aedes aegypti dengue virus infection rate
? Infection rate
Mixed feeding sample four 39%
Mixed feeding sample one 22%
Mixed feeding sample five 41%
Mixed feeding sample two 31%
Mixed feeding sample six 41%
Mixed feeding sample three 32%
The preparation of embodiment 12, multiple protein mature peptide
Primer pair for the mosGCTL-26 gene that increases is as follows
F#：5’-TCTCTAGCTAGC?ACACATTTTTGCTCCC-3’；
R#:5’-TATTCGAAGCTT?CGACCAGTTTGGAGTGAC-3’。
Primer pair for the mosGCTL-15 gene that increases is as follows:
F3：5’-ctgccgGCTAGC?CAACGTATCACCACAATCC-3’；
R3：5’-tataccCTCGAG?CCCCTGCGCCGACGA-3’；
Primer pair for the mosGCTL-19 gene that increases is as follows:
F4：5’-cgcctaGCTAGC?CAATTCAACTTCCAT-3’；
R4：5’-tcaccgCTCGAG?GATGCAATACCTCAAT-3’。
Primer pair for the mosGCTL-20 gene that increases is as follows:
F5：5’-tcatcgGCTAGC?TCATCGACTAAACCAAAAT-3’；
R5：5’-ttatccCTCGAG?AAACTCCTCTATGCACGTC-3’。
Primer pair for the mosGCTL-22 gene that increases is as follows:
F6：5’-cgcctaGCTAGC?ATAAGCGAATACTTTATTCCT-3’；
R6：5’-tcaccgCTCGAG?CCGAAATTCACTAATGCA-3’。
Primer pair for the mosGCTL-23 gene that increases is as follows:
F7：5’-cgcctaGCTAGC?GATAGACGATTCTTCATACCCA-3’；
R7：5’-ttatccCTCGAG?CAGACATTGTCGTTCGA-3’。
Primer pair for the mosGCTL-24 gene that increases is as follows:
F8：5’-tcatcgGCTAGC?GATCCCCTAAGAAATCAGAC-3’；
R8：5’-ttatccCTCGAG?GTTCAACCTTTCACAAA-3’。
Primer pair for the mosGCTL-32 gene that increases is as follows:
F9：5’-cgcctaGCTAGC?CAATCCAAGTATCAAAT-3’；
R9：5’-tatccgCTCGAG?AAACTCTTGGATGC-3’。
MosGCTL-26 protein maturation peptide is comprised of from 5 ' end 17-174 amino acids residue the sequence 3 of sequence table.MosGCTL-15 protein maturation peptide is comprised of from 5 ' end 18-191 amino acids residue the sequence 5 of sequence table.MosGCTL-19 protein maturation peptide is comprised of from 5 ' end 26-154 amino acids residue the sequence 7 of sequence table.MosGCTL-20 protein maturation peptide is comprised of from 5 ' end 20-158 amino acids residue the sequence 9 of sequence table.MosGCTL-22 protein maturation peptide is comprised of from 5 ' end 17-153 amino acids residue the sequence 11 of sequence table.MosGCTL-23 protein maturation peptide is comprised of from 5 ' end 26-154 amino acids residue the sequence 13 of sequence table.MosGCTL-24 protein maturation peptide is comprised of from 5 ' end 22-173 amino acids residue the sequence 15 of sequence table.MosGCTL-32 protein maturation peptide is comprised of from 5 ' end 26-160 amino acids residue the sequence 17 of sequence table.
Other operating procedure is completely with embodiment 10(wherein mosGCTL-26C end His label and 7 aminoacid of mature peptide midfeather, for Lys Leu Ala Ala Ala Leu Glu, other is identical), obtain respectively mosGCTL-26 mature peptide solution, mosGCTL-15 mature peptide solution, mosGCTL-19 mature peptide solution, mosGCTL-20 mature peptide solution, mosGCTL-22 mature peptide solution, mosGCTL-23 mature peptide solution, mosGCTL-24 mature peptide solution and mosGCTL-32 mature peptide solution, purity is all greater than 90%.
Embodiment 13, the application of pooled serum in inhibition dengue virus copies
One, the preparation of each serum
According to the method in embodiment 11, prepare respectively the anti-mosGCTL-26 serum of rabbit, the anti-mosGCTL-15 serum of rabbit, the anti-mosGCTL-19 serum of rabbit, the anti-mosGCTL-20 serum of rabbit, the anti-mosGCTL-22 serum of rabbit, the anti-mosGCTL-23 serum of rabbit, the anti-mosGCTL-24 serum of rabbit and the anti-mosGCTL-32 serum of rabbit.
8 kinds of serum equal-volumes of the anti-mosGCTL-3 serum of rabbit of embodiment 11 preparations and above preparation are mixed, obtain pooled serum, called after mosGCTL MIX9 antiserum.
Control serum in embodiment 11 and above 8 kinds of serum control serum equal-volume are separately mixed, obtain pooled serum, called after Pre MIX9 control serum.
Two, the application of pooled serum in inhibition dengue virus copies
Step 1 is to 4 steps 1 with embodiment 11 to 4.
5, the preparation of mixed feeding sample
Mixed feeding sample one: the mixing blood that 490 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 10 μ l mosGCTL MIX9 antiserum (diluting 100 times) mix homogeneously are standby.
Mixed feeding sample two: the mixing blood that 499 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 1 μ l mosGCTL MIX9 antiserum (diluting 1000 times) mix homogeneously is standby.
Mixed feeding sample three: the mixing blood that 499.8 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 0.2 μ l mosGCTL MIX9 antiserum (diluting 5000 times) mix homogeneously is standby.
Mixed feeding sample four: the mixing blood that 490 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 10 μ l Pre MIX9 control serum (diluting 100 times) mix homogeneously are standby.
Mixed feeding sample five: the mixing blood that 499 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 1 μ l Pre MIX9 control serum (diluting 1000 times) mix homogeneously is standby.
Mixed feeding sample six: the mixing blood that 499.8 μ l steps 4 are obtained, 500 μ l DENV-2 virus liquids mix that (virus titer is 2 * 10 7pfu/ml) and 0.2 μ l Pre MIX9 control serum (diluting 5000 times) mix homogeneously is standby.
Use the Hemoteck blood system of the feeding Aedes aegypti 30 minutes (every kind of sample feed approximately 100 Aedes aegyptis) of respectively above two kinds of samples being fed.The mosquito that confirm to suck mixes blood is picked out and puts into special container and with the rayon balls that contains 1% sucrose solution, feed standard conditions (70% humidity, 28 ℃) are lower.Raise and put to death afterwards for 8 days, total RNA the reverse transcription of extracting mosquito are cDNA, utilize Taqman RT-QPCR to detect the dengue fever 2 type virus quantities in Aedes aegypti body, and calculate infection rate (correlation method in the step 2 of method and embodiment 11 is identical).
The results are shown in Table 2.
The impact of table 2 blood meal pooled serum on Aedes aegypti dengue virus infection rate
? Infection rate
Mixed feeding sample four 38%
Mixed feeding sample one 6.1%
Mixed feeding sample five 36%
Mixed feeding sample two 16%
Mixed feeding sample six 35%
Mixed feeding sample three 20%
Embodiment 14, Analysis on Mechanism
One, eucaryon S2 drosophila cell expression system is expressed mosGCTL-3 albumen
1, total RNA the reverse transcription of extraction Aedes aegypti obtain cDNA.
2, take the cDNA that step 1 obtains is template, with the primer pair that F10 and R10 form, carries out pcr amplification, obtains pcr amplification product.
F10：5’-tatagg GGTACCtCAGCCCATATGCAGTGAT-3’；
R10：5’-cgctgc TCTAGAAAACTCCTGGATTGAATC-3’。
3, with the pcr amplification product of restricted enzyme Kpn I and Xba I double digestion step 2, reclaim enzyme action product.
4, with restricted enzyme Kpn I and Xba I double digestion vector plasmid pMT/Bip/V5/His A(Invitrogen company, catalog number V4130-20), reclaim the carrier framework of about 4200bp.
5, the enzyme action product of step 3 and the carrier framework of step 4 are connected, obtain recombiant plasmid (in recombiant plasmid, the coded sequence of the coded sequence of the head end Bip secreting signal peptide on external source fragment and carrier and tail end V5 His label merges, and expresses the mosGCTL-3 protein maturation peptide with signal peptide and His label).
6, recombiant plasmid step 5 being obtained and pCohygro plasmid (contain resistance screening gene; Invitrogen company, catalog number K413001) use Effectene transfection reagent (QIAGEN company catalog number (Cat.No.) 301425), be transfected into Drosophila S 2 cells (purchased from Invitrogen company, catalog number R69007), by HYG, screen that (final concentration of HYG is 300ng/ul, and cell culture medium is the Sneijder Drosophila S 2 cells culture medium that the Sigma company catalog number that contains 10%FBS is S9865; Within every four days, change a cell culture medium), approximately after 1 month, obtain can stably express mosGCTL-3 albumen S2 surely turn cell line.
7, S2 is surely turned to cell line and adds serum-free medium Express Five SFM(Invitrogen company catalog number (Cat.No.) 10485), by the large culture bottle suspension amplification culture (about 500ml cultivating system) of 2L, after 3 days, adding final concentration is the CuSO of 500 μ M 4induced protein is expressed (pMT/Bip/V5His A carrier can be expressed foreign protein and be secreted into supernatant in the situation that Cu ion exists).
8, add Cu ion induction to express after 3 days, 4 ℃, the centrifugal 5min of 4000rpm, collect supernatant, adopts 0.22um filter membrane to remove residual cell debris, usings 5KD size microporous membrane as baffle plate, by the supernatant concentration of 500ml to about 50ml final volume.
9, use TALON Purification Kit (Clontech company article No. 635515) to carry out purification to the protein concentrated solution obtaining.4 ℃ of refrigerators rotations of concentrated solution that 1ml Resin and 50ml step 8 are obtained 2 hours, then be transferred in filter post, with 20ml 1X balance liquid, wash, with 20ml, containing the 1X balance liquid of 20mM imidazoles, wash again, finally with 5ml, containing the 1X balance liquid of 150mM imidazoles, wash and collected solution after post.
10, shifted after post solution to the super filter tube of 10kD size, 4 ℃, the centrifugal 15min of 4000rpm, 5ml filtrate is concentrated into about 1ml, add the aseptic PBS buffer of 4ml to super filter tube, 4 ℃, the centrifugal 15min(of 4000rpm are removed residual imidazoles), the solvent of replacing albumen is PBS buffer subpackage (being mosGCTL-3 mature peptide solution), and-80 degree refrigerators are frozen, and spectrophotometric determination protein concentration is also identified protein concentration and purity with SDS PAGE electrophoresis and Western Blot.
The protein concentration of mosGCTL-3 protein liquid is about 300ng/ul, and purity is greater than 85%.
Two, use ELISA experimental check mosGCTL-3 mature peptide and the surperficial E albumen of DENV-2 virus and the interaction of DENV-2 virion.
1, get mosGCTL-3 mature peptide solution, with the protein content of 1 μ g/well, be taped against in ELISA Plate hole, matched group spreads the BSA of 1 μ g/well, and background hole spreads BSA albumen equally.The 10X coating Buffer(catalog number (Cat.No.) 50-84-00 of the bed board Ye Wei KPL company using), every pore system amount is 100ul.ELISA Plate is positioned over to 4 and spends night.
2, use 0.05%PBST solution (adding 0.05%Tween20 in PH7.4PBS solution) washing sample hole 3 times, add the catalog number (Cat.No.) 50-61-00 of 100μ l2%BSA(KPL company) room temperature sealing 1 hour.
3, use 0.05%PBST solution washing sample well 3 times, in mosGCTL-3 mature peptide hole and BSA control wells, add respectively the surperficial E albumen of DENV-2 virus (to buy from L2 Diagnostic, New Haven, the United States) and the DENV-2 virion of deactivation (buy from Microbix, Canada, article No. EL-22-02-001), use 1%BSA as diluent.Here be divided into two parallel group, one group adds final concentration in reactant liquor is the Ca ion of 5mM, it is that the interaction of 5mM EDTA(mosGCTL-3 albumen and virion needs the auxiliary of Ca ion that another group adds final concentration, and EDTA can chelating divalent ion, as the matched group of Ca group).Incubated at room 2 hours (what add in background hole is BSA albumen, is equally also divided into Ca group and EDTA group).
4, use 0.05%PBST solution washing sample well 5 times, in every hole, add the monoclonal antibody 4G2(of the surperficial E albumen of the anti-DENV-2 virus of diluting with 1%BSA to buy from L2 Diagnostic, New Haven, the United States), 1:3000 dilution, incubated at room 2 hours.
5, use 0.05%PBST solution washing sample well 5 times, to the anti-mouse IgG goat polyclonal antibody (the catalog number (Cat.No.) M330 of MBL company) that adds the HRP coupling of 1%BSA dilution in every hole, 1:5000 dilution, incubated at room 1 hour.
6, use 0.05%PBST solution washing sample well 7 times, in every hole, add SureBlue tmb substrate nitrite ion (the catalog number (Cat.No.) 52-00-01 of KPL company), after the about 15min of color development at room temperature, use TMB stop buffer cessation reaction (the catalog number (Cat.No.) 50-95-05 of KPL company).
7, finally use ELISA Plate plate reading machine to obtain each hole in the OD of 450nm value, and the numerical value mapping of hole and control wells per sample, the results are shown in Figure 2.Surperficial E albumen and the DENV-2 virion of mosGCTL-3 mature peptide and DENV-2 virus have direct combination, simultaneously the combination of mosGCTL-3 mature peptide and DENV-2 virion is Ca ionic dependent, but the combination of the surperficial E albumen of mosGCTL-3 mature peptide and DENV-2 virus can be not auxiliary by Ca ion.
According to mosGCTL-3 albumen, can mutually combine with dengue virus granule.Proof yellow-fever mosquito C type lectin family is the crucial cofactor of dengue virus infection Aedes aegypti.

Claims (9)

1. a product, the material that its active component is expressed for suppressing the encoding gene of the C type agglutinin in yellow-fever mosquito body; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
2. a product, thus its active component is combined and is suppressed the active material of described C type agglutinin for the C type agglutinin in yellow-fever mosquito body; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
3. product as claimed in claim 2, is characterized in that: described " thereby the C type agglutinin in yellow-fever mosquito body be combined suppress the active material of described C type agglutinin " is the antibody of described C type agglutinin.
4. a product, thus its active ingredient is the material of the active material that impels the C type agglutinin producing in animal body in yellow-fever mosquito body to be combined to suppress described C type agglutinin; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
5. product as claimed in claim 4, is characterized in that: described " thereby material of the active material that impels the C type agglutinin producing in animal body with yellow-fever mosquito body in to be combined to suppress described C type agglutinin " is described C type agglutinin.
6. as the product as described in arbitrary in claim 1 to 5, it is characterized in that: described dengue virus is dengue fever 2 types viruses.
7. as the product as described in arbitrary in claim 1 to 6, it is characterized in that: described C type agglutinin is arbitrary described protein in following (1) to (19):
(1) protein that the sequence 1 of sequence table forms from 5 ' end 21-168 amino acids residue;
(2) protein shown in the sequence 1 of sequence table;
(3) protein that the sequence 3 of sequence table forms from 5 ' end 17-174 amino acids residue;
(4) protein shown in the sequence 3 of sequence table;
(5) protein that the sequence 5 of sequence table forms from 5 ' end 18-191 amino acids residue;
(6) protein shown in the sequence 5 of sequence table;
(7) protein that the sequence 7 of sequence table forms from 5 ' end 26-154 amino acids residue;
(8) protein shown in the sequence 7 of sequence table;
(9) protein that the sequence 9 of sequence table forms from 5 ' end 20-158 amino acids residue;
(10) protein shown in the sequence 9 of sequence table;
(11) protein that the sequence 11 of sequence table forms from 5 ' end 17-153 amino acids residue;
(12) protein shown in the sequence 11 of sequence table;
(13) protein that the sequence 13 of sequence table forms from 5 ' end 26-154 amino acids residue;
(14) protein shown in the sequence 13 of sequence table;
(15) protein that the sequence 15 of sequence table forms from 5 ' end 22-173 amino acids residue;
(16) protein shown in the sequence 15 of sequence table;
(17) protein that the sequence 17 of sequence table forms from 5 ' end 26-160 amino acids residue;
(18) protein shown in the sequence 17 of sequence table;
(19) by arbitrary described protein in (1) to (18) through replacement and/or disappearance and/or the interpolation of one or several amino acid residue and have identical function by its derivative protein.
8. the application of the material that the encoding gene of the C type agglutinin in inhibition yellow-fever mosquito body is expressed in preparing product; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
9.C the application of type agglutinin in preparing product; The purposes of described product is for following (a) or (b) or (c): (a) suppress dengue virus and copy in yellow-fever mosquito body; (b) prevent and/or treat dengue fever; (c) propagation of blocking-up dengue virus.
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