CN110540583B - Sickle rhipicephalus vacuole sortilin molecule and application thereof - Google Patents
Sickle rhipicephalus vacuole sortilin molecule and application thereof Download PDFInfo
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- CN110540583B CN110540583B CN201810527455.7A CN201810527455A CN110540583B CN 110540583 B CN110540583 B CN 110540583B CN 201810527455 A CN201810527455 A CN 201810527455A CN 110540583 B CN110540583 B CN 110540583B
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- rhipicephalus
- falciparum
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- sortilin
- tick
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Abstract
The invention discloses a Rhipicephalus falciparum vacuole sortilin molecule which has an amino acid sequence shown in SEQ ID No. 1. The invention also discloses a Rhipicephalus falciparum vacuole sortilin molecular gene which comprises a nucleotide sequence of an amino acid sequence shown in an encoding SEQ ID NO. 1. After gene silencing or knockout, the Rhipicephalus falciparum vacuole sortilin molecule and the gene thereof can obviously reduce the upper body rate of ticks, obviously influence the blood sucking of ticks, enable the death rate of ticks to reach 100 percent, are expected to become candidate molecules for tick-resistant vaccines and tick-borne disease control, and have wide application prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a Rhipicephalus falciparum vacuole sortilin molecule and application thereof.
Background
Ticks are a group of non-permanent parasites that are exclusively parasitic on vertebrate bodies, and are the second largest vector of transmission worldwide to mosquitoes. Ticks bite and spread pathogens to the host, causing damage to the host, seriously hindering the development of animal husbandry and causing great economic loss. Ticks also transmit various pathogens to humans, such as lyme disease, hemorrhagic fever, and the novel brie virus disease, which cause systemic infections, and in severe cases, death of humans due to multiple organ failure. However, the existing methods for preventing tick bites are limited, mainly chemical drugs are used for dispelling ticks, which cannot fundamentally solve tick and tick-borne diseases, and drug resistance is easy to generate, so that a new method for killing ticks, such as vaccines or biological insecticides like double-stranded RNA, is urgently needed to be found.
During normal physiological cellular metabolism, the endocytotic sorting transporter complex (ESCRT) recognizes and binds ubiquitinated membrane proteins and signaling proteins, encapsulating them into multivesicular bodies and selectively degrading from endosomes into lysosomes. In addition, ESCRT is involved in regulating the mitotic spindle and in the process of entry or budding release of the togavirus in a topologically similar manner. The ESCRT-I complex consists of five proteins, namely a tumor sensitive gene (Tsg101), VPS28, VPS37A, VPS37B and a vesicle 12(Mvb12), wherein vacuolar sortilin 28(VPS28) comprises two domains, namely Vps28_ N and Vps28_ C, and a helical domain (CTD) at the end of Vps28_ C can interact with VPS36 which is a component of ESCRT-II and can also interact with VPS20 which is a subunit of ESCRT-III, so that the VPS28 plays an important role in the processes of vesicle formation and cell physiological metabolism. VPS28 has been studied extensively in other species, but no literature report has been made on the study in ticks.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a Rhipicephalus haemaphysalis vacuolar sortilin molecule and a gene thereof, and the Rhipicephalus haemaphysalis sortilin molecule can be used as a candidate molecule for tick-resistant vaccine and tick-borne disease control.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the invention, a Rhipicephalus falciparum vacuolar sortilin molecule is provided having an amino acid sequence shown in SEQ ID No. 1.
In another aspect of the invention, there is provided a rhipicephalus sorting protein molecular gene comprising: a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the nucleotide sequence of the Rhipicephalus falciparum vacuole sortilin molecule gene is shown as SEQ ID No. 2.
In another aspect of the invention, there is also provided a recombinant vector comprising the nucleotide sequence of the Rhipicephalus falciparum vacuolar sortilin molecule gene described above.
The recombinant vector comprises a recombinant cloning vector or a recombinant expression vector.
In another aspect of the present invention, there is also provided a host cell comprising the above recombinant vector.
In another aspect of the invention, a substance for inhibiting the activity of the Rhipicephalus falcatus vacuolar sortilin molecule is also provided, and the substance comprises various protein activity inhibitors and the like.
In another aspect of the invention, there is also provided a substance which inhibits the expression of the above mentioned rhipicephalus sorting protein molecule gene. The substance comprises: dsRNA for inhibiting the gene expression of the Rhipicephalus falciparum vacuole sortilin molecule.
Preferably, the dsRNA is double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO.18 and a reverse complementary sequence thereof.
In another aspect of the invention, there is also provided an anti-tick vaccine comprising the rhipicephalus haemaglobus sortilin molecule described above or a partially active fragment thereof.
In another aspect of the present invention, there is also provided a product comprising a substance that inhibits the activity of a Rhipicephalus falciparum vacuolar sortilin molecule or inhibits the expression of a Rhipicephalus falciparum vacuolar sortilin molecule gene, which has any one of the following functions (1) to (3): (1) preventing and treating tick-borne diseases; (2) preventing the bite of ticks; (3) inhibiting the egg laying of female ticks.
In another aspect of the invention, the invention also provides an application of the Rhipicephalus falciparum vacuole sortilin molecule or the gene of the Rhipicephalus falciparum vacuole sortilin molecule, which is used for preparing the biological insecticide for preventing and treating tick-borne diseases.
In another aspect of the invention, the invention also provides an application of the substance for inhibiting the activity of the Rhipicephalus falciparum vacuolar sortilin molecule or inhibiting the gene expression of the Rhipicephalus falciparum vacuolar sortilin molecule, which is used for preparing the biological insecticide for preventing and treating tick-borne diseases.
The gene of the Rhipicephalus falciparum vacuole sortilin molecule vps28 is mainly expressed in ovaries, has specificity of ovarian organs, and RNA interference experiments of the vps28 gene prove that the vps28 gene is silent, can obviously reduce the upper body rate of ticks, obviously influence blood sucking of ticks, enable the death rate of ticks to reach 100%, and the vps28 gene is expected to become a candidate molecule for tick-resistant vaccine and tick-borne disease control, so that the Rhipicephalus falciparum shiponensis and has wide application prospects.
Drawings
The invention is described in further detail below with reference to the drawings and the detailed description.
FIG. 1 is a sequence analysis diagram of Rhipicephalus falcatus VPS28 gene in example 1 of the present invention;
FIG. 2 is a graph showing the expression profile of Rhipicephalus falciparum VPS28 gene in different tissues and organs under a state of partial satiety according to example 2 of the present invention;
FIG. 3 is a graph showing the effect of Rhipicephalus falciparum VPS28 gene silencing on tick somatotroph rate in example 3 of the present invention.
Detailed Description
In order to find candidate molecules for resisting tick vaccines or tick-borne disease control, vacuole sortilin molecule VPS28 gene is obtained from Rhipicephalus falciparum for the first time, and the results of experiments on cloning, expression distribution and influence on biological characteristics such as tick blood sucking of the VPS28 gene show that after the VPS28 gene is silenced or knocked out, the size of the upper body of the tick and the size of the tick can be obviously reduced, the saturated blood rate of the tick is reduced to 0 percent, and the death rate is up to 100 percent, so that the VPS28 gene is suitable for serving as candidate molecules for resisting tick vaccines and tick-borne disease control.
Example 1 Gene cloning and sequence analysis of the Rhipicephalus falciparum vacuolar sortilin molecule (VPS28)
1. Materials and methods
1.1. Tick and experimental animal
Rhipicephalus falciparum (collected from Wuhan Hubei, and propagated and passaged in the laboratory) is mainly prepared by inoculating tick onto ear of New Zealand white rabbit, allowing it to absorb blood, allowing it to fall off automatically after full blood, culturing in biochemical incubator at 25 deg.C and relative humidity of 95% to molt into the next development stage, and storing.
1.2. Bacteria and plasmids
Plasmid construction Using E.coli Top 10 cells (TIANGEN). pMD-19T Easy (Takara) was used as a cloning sequencing vector.
1.3. Collecting different blood sucking stages and different tissue and organ materials of Rhipicephalus falciparum Linn
In order to further study the expression characteristics of the Rhipicephalus falciparum vacuole sortilin gene, eggs, young ticks and adult ticks in a non-blood-sucking state and young ticks, young ticks and adult ticks in a semi-full blood state are collected, adult ticks in a non-blood-sucking state and a semi-full blood state are dissected, and ovary tissues, salivary glands, fat bodies and midgut tissues are obtained. The dissection method is as follows:
female ticks were glued onto plates with the back facing up and after fixation ticks were soaked in 1 x PBS buffer. Under an optical microscope or a stereomicroscope, the belly and the back of the tick are separated by a scalpel, fat bodies exist on a dorsal shell, the fat bodies are scraped by a bent forceps, then salivary glands, intestinal tracts and ovaries are separated by the forceps and a fine needle, and the tick is placed in an EP (EP) tube containing Trizol liquid after being cleaned for total RNA extraction.
1.4. Extraction of Total RNA and Synthesis of first Strand cDNA
Total RNA from Rhipicephalus falciparum at different blood-sucking stages and different tissues and organs was extracted by TRIzol reagent (Invitrogen), and was first subjected to DNase I (TAKARA) digestion to remove genomic DNA therefrom. Total RNA was synthesized into first strand cDNA by Reverse transcriptase, followed by gene amplification and fluorescent quantitative PCR, and the whole procedure was performed according to the instructions of Reverse Transcription Kit Reverse Transcription System Kit with gDNA applicator (perfect real time, Takara, Dalian, China).
Molecular cloning of VPS28
Amplification of VPS28 open reading frame was performed by taking ovary cDNA of Rhipicephalus falciparum Segor or Haemophilus saticus as a template, using primers VPS28F1: 5'-ATGGCTTCTTTCGCAGCTCTCA-3' (SEQ ID NO.3) and VPS28R1: 5'-CGTAGAGTTGTGAAGGATCCG-3' (SEQ ID NO.4), and connecting the purified PCR amplification product to cloning vector pMD-19T Easy (Takara) for sequencing.
2. As a result, the
From mRNA of the female tick ovary of Rhipicephalus falciparum, an open reading frame 672bp (SEQ ID NO.2) of a gene vps28 is obtained, and the gene codes for 223 amino acids (SEQ ID NO. 1). The predicted protein has a molecular weight of 25.6kDa and an isoelectric point of 5.08. The signal peptide sequence was predicted and the vps28 gene was found to be free of signal peptide, as shown in FIG. 1. BLAST non-redundant database analysis of the predicted amino acid sequence of the vps28 gene revealed that the predicted protein has some similarity to vacuolar sortilin 28(vps28) from other species. The amino acid sequence of vps28 resembled Anemophilus omarginialis (Ixodes scapularis) vps28(XM _002402646.1) at 90.63%. Activity prediction revealed that the Vps28 gene has two domains, i.e., Vps28_ N (13-120 amino acids) and Vps28_ C (124-220 amino acids).
Example 2RT-qPCR detection of expression distribution of Rhipicephalus falciparum VPS28 Gene
Primers for fluorescent quantitative PCR were designed according to the ORF sequence of VPS28 gene, and the primer sequences were as follows:
VPS28-qRTPCR-F:5'-CCAGTACAAGGCAGCATTCAAG-3'(SEQ ID NO.5)；
VPS28-qRTPCR-R:5'-GATTCGTTCCATGGCAGCAG-3'(SEQ ID NO.6)。
primers were designed based on the full-length sequence of the reference gene as follows:
ELFⅠA-QRT-PCR-F:5’-CGTCTACAAGATTGGTGGCATT-3’(SEQ ID NO.7)；
ELFⅠA-QRT-PCR-R:5’-CTCAGTGGTCAGGTTGGCAG-3’(SEQ ID NO.8)。
the reaction system of the fluorescent quantitative PCR is operated according to the instruction of the kit, cDNA obtained by reverse transcription of different materials of the Rhipicephalus falciparum is taken as a template, and the two-step real-time quantitative PCR method analysis is carried out. According to Takara operating instructions, ABI7500Real-time PCR system, the two-step PCR amplification standard program is: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s, for 40 cycles, followed by addition of the dissolution profile.
As a result: to examine the expression profile of the vps28 gene, the RT-qPCR method was used to detect the transcript levels of the vps28 gene in different tissue organs in the state of Rhipicephalus falciparum satiety. The results found that the vps28 gene was highly expressed predominantly in the ovary (fig. 2) and not transcribed in the intestine, indicating that the vps28 gene has ovarian organ specificity.
Example 3 Synthesis and RNA interference of Rhipicephalus falciparum VPS28dsRNA
vps28 is mainly transcribed in ovary, if vps28 gene is silenced or knocked out, whether the ovarian development of ticks is affected, and the embodiment is verified by a vps28 gene RNA interference experiment.
VPS28-pMD-19T easy plasmid (the length of RNAi sequence is about 630 bp) is taken as a template, VPS28RNAi-1F, VPS28RNAi-1R, VPS28RNAi-2F, VPS28RNAi-2R is taken as a primer (the bold part is T7 promoter sequence), the VPS28 sequence containing the T7 promoter sequence is amplified by PCR, and then PCR products are recovered and purified by glue. RNAi uses luciferase gene RNAi (RNAi sequence is about 600 bp) as a control group. Subsequently, using the VPS28 sequence containing the T7 promoter sequence obtained by the above PCR amplification as a template (DNA sequence shown in SEQ ID NO. 17), T7Ribomax was usedTMAn Express RNAi System (Promega) kit for in vitro transcription to generate VPS28 double-stranded RNA (dsRNA), wherein the double-stranded RNA consists of a sense strand and an antisense strand, the nucleotide sequence of the sense strand is shown as SEQ ID NO.18, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of SEQ ID NO. 18.
VPS28RNAi-1F:5’-TAATACGACTCACTATAGGCAAGAGCACCGTCCTGAGCT-3’(SEQ ID NO.9)；
VPS28RNAi-1R:5’-CGTAGAGTTGTGAAGGATCCG-3’(SEQ ID NO.10)；
VPS28RNAi-2F:5’-CAAGAGCACCGTCCTGAGCT-3’(SEQ ID NO.11)；
VPS28RNAi-2R:5’-TAATACGACTCACTATAGGCGTAGAGTTGTGAAGGATCCG-3’(SEQ ID NO.12)；
Luciferase
RNAi-1F:5’-GGATCCTAATACGACTCACTATAGGGCTTCCATCTTCCAGGGATAC-3’(SEQ ID NO.13)；
Luciferase RNAi-1R:5’-CGTCCACAAACACAACTCCTCC-3’(SEQ ID NO.14)；
Luciferase RNAi-2F:5’-GCTTCCATCTTCCAGGGATACG-3’(SEQ ID NO.15)；
Luciferase
RNAi-2R:5’-GGATCCTAATACGACTCACTATAGGCGTCCACAAACACAACTCCTC-3’(SEQ ID NO.16)。
The same batch of adult falciparum (female tick) and male tick) in a non-blood-sucking state are selected and divided into an experimental group and a control group, wherein the female tick of the experimental group is injected with dsRNA of VPS28 gene, and the female tick of the control group is injected with dsRNA of Luciferase gene. Finding out the 4 th limb basal segment part of the tick under an optical microscope, and respectively injecting the dsRNA of the VPS28 gene and the dsRNA of the luciferase gene into the tick body in a micro-injection way without any treatment on the male tick; the injected ticks were placed in a biochemical incubator at 25 ℃ and 95% relative humidity for 24h to observe the living status and mortality of the ticks. The next day, male and female ticks of the experimental group were inoculated on one ear of the rabbit, the control group was inoculated on the other ear of the rabbit, and 24h of the ticks, the upper body rate, mortality, weight of saturated blood, rate of saturated blood, and oviposition rate were observed to determine the effect of VPS28 gene silencing on the biological characteristics of the ticks.
As a result: after the gene vps28 is silenced or knocked out, the upper body and the size of the tick are obviously reduced (figure 3); in the vps28 gene interference group, 0 ticks reached a full blood state with a full blood rate as low as 0% and a mortality rate as high as 100% (table 1).
TABLE 1
The results show that after the gene vps28 is silenced or knocked out, the blood sucking of ticks is obviously influenced, and the gene vps28 can be used as a candidate molecule for anti-tick vaccine and tick-borne disease control.
The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is specific and detailed, but not to be understood as the limitation of the patent scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Sequence listing
<110> Shanghai institute of veterinary medicine of Chinese academy of agricultural sciences
<120> Rhipicephalus falciparum vacuole sortilin molecule and application thereof
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 223
<212> PRT
<213> Fusarium fancifolium (Rhipicephalus haemaphysioides)
<400> 1
Met Ala Ser Phe Ala Ala Leu Met Ser Gln Thr Asn Asn Gln Glu His
1 5 10 15
Arg Pro Glu Leu Tyr Glu Glu Val Arg Leu Gln Arg Thr Ser Arg Glu
20 25 30
Arg Glu Lys Tyr Asp Asn Met Ala Asp Leu Tyr Ala Val Ile Asn Thr
35 40 45
Leu Gln Cys Leu Glu Lys Ala Tyr Ile Lys Asp Cys Val Thr Pro Lys
50 55 60
Glu Tyr Thr Ala Ala Cys Ser Lys Leu Leu Val Gln Tyr Lys Ala Ala
65 70 75 80
Phe Lys Gln Val Gln Gly Pro Glu Phe Ser Thr Val Glu Ala Phe Met
85 90 95
Ala Ala Phe Arg Leu Asp Cys Pro Ala Ala Met Glu Arg Ile Arg Glu
100 105 110
Asp Arg Pro Ile Thr Ile Lys Asp Asp Lys Gly Asn Thr Ser Lys Cys
115 120 125
Ile Ala Asp Ile Val Ser Leu Phe Ile Thr Ile Ile Asp Lys Leu Arg
130 135 140
Leu Glu Ile Lys Ser Met Asp Glu Leu Gln Pro Asp Leu Arg Glu Leu
145 150 155 160
Ser Glu Thr Met Ser Arg Leu Ser Leu Ile Pro Ala Asn Phe Glu Gly
165 170 175
Lys Ala Lys Val Asp Glu Trp Leu Gln Thr Met Ser Ser Met Ala Ala
180 185 190
Ser Asp Glu Leu Asn Glu Ser Gln Val Arg Gln Leu Ile Phe Asp Leu
195 200 205
Glu Ser Ser Tyr Asn Ala Phe Asn Arg Ile Leu His Asn Ser Thr
210 215 220
<210> 2
<211> 672
<212> DNA
<213> Fusarium fancifolium (Rhipicephalus haemaphysioides)
<400> 2
atggcttctt tcgcagctct catgtcgcaa accaacaacc aagagcaccg tcctgagctg 60
tacgaagaag tgcggcttca gaggacgtca cgggagcgtg agaagtatga caacatggct 120
gatttgtatg ctgtaatcaa cacacttcaa tgcctagaga aggcctacat taaagattgc 180
gtgacgccaa aagagtacac tgctgcttgt tcaaagcttc tggtccagta caaggcagca 240
ttcaagcagg tgcaaggacc agaattctct acggttgaag ccttcatggc tgcctttcgg 300
ctggactgtc ctgctgccat ggaacgaatc cgtgaggaca ggcctattac catcaaggat 360
gacaaaggga acacaagcaa atgcattgct gacattgtgt cgctgttcat cacaattatt 420
gataagctac gcctggagat caaatccatg gatgagctgc aaccagatct gagagagttg 480
agtgagacca tgagccggct cagtctcata ccagccaact ttgaaggcaa agccaaggta 540
gatgaatggc tgcagaccat gtcgtcaatg gccgcttcgg atgagctgaa tgagagccaa 600
gtgcgccaac tcatctttga cttggaatct tcgtacaatg ccttcaaccg gatccttcac 660
aactctacgt ga 672
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
atggcttctt tcgcagctct ca 22
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
cgtagagttg tgaaggatcc g 21
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 5
ccagtacaag gcagcattca ag 22
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 6
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 7
cgtctacaag attggtggca tt 22
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 8
<210> 9
<211> 39
<212> DNA
<213> Artificial sequence (Artificial)
<400> 9
taatacgact cactataggc aagagcaccg tcctgagct 39
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 10
cgtagagttg tgaaggatcc g 21
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 11
<210> 12
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 12
taatacgact cactataggc gtagagttgt gaaggatccg 40
<210> 13
<211> 46
<212> DNA
<213> Artificial sequence (Artificial)
<400> 13
ggatcctaat acgactcact atagggcttc catcttccag ggatac 46
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 14
cgtccacaaa cacaactcct cc 22
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 15
gcttccatct tccagggata cg 22
<210> 16
<211> 46
<212> DNA
<213> Artificial sequence (Artificial)
<400> 16
ggatcctaat acgactcact ataggcgtcc acaaacacaa ctcctc 46
<210> 17
<211> 630
<212> DNA
<213> Artificial sequence (Artificial)
<400> 17
caagagcacc gtcctgagct gtacgaagaa gtgcggcttc agaggacgtc acgggagcgt 60
gagaagtatg acaacatggc tgatttgtat gctgtaatca acacacttca atgcctagag 120
aaggcctaca ttaaagattg cgtgacgcca aaagagtaca ctgctgcttg ttcaaagctt 180
ctggtccagt acaaggcagc attcaagcag gtgcaaggac cagaattctc tacggttgaa 240
gccttcatgg ctgcctttcg gctggactgt cctgctgcca tggaacgaat ccgtgaggac 300
aggcctatta ccatcaagga tgacaaaggg aacacaagca aatgcattgc tgacattgtg 360
tcgctgttca tcacaattat tgataagcta cgcctggaga tcaaatccat ggatgagctg 420
caaccagatc tgagagagtt gagtgagacc atgagccggc tcagtctcat accagccaac 480
tttgaaggca aagccaaggt agatgaatgg ctgcagacca tgtcgtcaat ggccgcttcg 540
gatgagctga atgagagcca agtgcgccaa ctcatctttg acttggaatc ttcgtacaat 600
gccttcaacc ggatccttca caactctacg 630
<210> 18
<211> 630
<212> RNA
<213> Artificial sequence (Artificial)
<400> 18
caagagcacc guccugagcu guacgaagaa gugcggcuuc agaggacguc acgggagcgu 60
gagaaguaug acaacauggc ugauuuguau gcuguaauca acacacuuca augccuagag 120
aaggccuaca uuaaagauug cgugacgcca aaagaguaca cugcugcuug uucaaagcuu 180
cugguccagu acaaggcagc auucaagcag gugcaaggac cagaauucuc uacgguugaa 240
gccuucaugg cugccuuucg gcuggacugu ccugcugcca uggaacgaau ccgugaggac 300
aggccuauua ccaucaagga ugacaaaggg aacacaagca aaugcauugc ugacauugug 360
ucgcuguuca ucacaauuau ugauaagcua cgccuggaga ucaaauccau ggaugagcug 420
caaccagauc ugagagaguu gagugagacc augagccggc ucagucucau accagccaac 480
uuugaaggca aagccaaggu agaugaaugg cugcagacca ugucgucaau ggccgcuucg 540
gaugagcuga augagagcca agugcgccaa cucaucuuug acuuggaauc uucguacaau 600
gccuucaacc ggauccuuca caacucuacg 630
Claims (8)
1. A Rhipicephalus falciparum vacuole sortilin molecule has an amino acid sequence shown in SEQ ID NO. 1.
2. A Rhipicephalus falciparum vacuole sortilin molecular gene has a nucleotide sequence of an amino acid sequence shown as a coding SEQ ID NO. 1.
3. The Rhipicephalus falciparum vacuolar sortilin molecule gene of claim 2, wherein the nucleotide sequence of the Rhipicephalus falciparum vacuolar sortilin molecule gene is set forth in SEQ ID No. 2.
4. A substance that inhibits the expression of the rhipicephalus falciparum vacuolar sortilin molecule gene of claim 2, the substance comprising: dsRNA inhibiting the expression of the Rhipicephalus falciparum vacuolar sortilin molecule gene of claim 2.
5. The substance according to claim 4, wherein the dsRNA is a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID No.18 and the reverse complement thereof.
6. An anti-tick vaccine comprising the rhipicephalus falciparum vacuolar sortilin molecule of claim 1.
7. A product comprising the substance of claim 4 or 5, which has any one of the following functions (1) to (3):
(1) preventing and treating tick borne diseases;
(2) preventing the bite of ticks;
(3) inhibit the oviposition of the female ticks.
8. Use of a Rhipicephalus falciparum vacuolar sortilin molecule as defined in claim 1, or a Rhipicephalus falciparum vacuolar sortilin molecule gene as defined in claim 2, or a substance as defined in claim 4 or 5, for the preparation of a biopesticide for the control of tick-borne diseases.
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| WO2004071462A2 (en) * | 2003-02-12 | 2004-08-26 | Johns Hopkins University | Methods and compositions for treatment of viral infections based on tsg101-vps28 interaction |
| JP2009060797A (en) * | 2007-09-04 | 2009-03-26 | National Research Inst Of Brewing | Method for selecting strain for secreting and highly producing foreign protein |
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| CN103965350A (en) * | 2013-02-04 | 2014-08-06 | 中国农业科学院上海兽医研究所 | Tick cystatin Rhcyst-2, and gene and applications thereof |
| CN103965351A (en) * | 2013-02-04 | 2014-08-06 | 中国农业科学院上海兽医研究所 | Tick cystatin Rhcyst-1, and gene and applications thereof |
| CN109312344A (en) * | 2016-01-26 | 2019-02-05 | 孟山都技术公司 | For controlling the composition and method of insect pest |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101406696A (en) * | 2000-04-25 | 2009-04-15 | 梅瑞尔有限公司 | Vaccine comprising a tick cement protein |
| WO2004071462A2 (en) * | 2003-02-12 | 2004-08-26 | Johns Hopkins University | Methods and compositions for treatment of viral infections based on tsg101-vps28 interaction |
| JP2009060797A (en) * | 2007-09-04 | 2009-03-26 | National Research Inst Of Brewing | Method for selecting strain for secreting and highly producing foreign protein |
| CN103965350A (en) * | 2013-02-04 | 2014-08-06 | 中国农业科学院上海兽医研究所 | Tick cystatin Rhcyst-2, and gene and applications thereof |
| CN103965351A (en) * | 2013-02-04 | 2014-08-06 | 中国农业科学院上海兽医研究所 | Tick cystatin Rhcyst-1, and gene and applications thereof |
| CN109312344A (en) * | 2016-01-26 | 2019-02-05 | 孟山都技术公司 | For controlling the composition and method of insect pest |
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| Gene silencing in parasites: current status and future prospects;Manzano-Román R等;《Advances in parasitology》;20120418;第78卷;1-55 * |
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