CN108642058A - Qinghai blood tick aquaporin（AQP）Encoding gene and its application - Google Patents
Qinghai blood tick aquaporin（AQP）Encoding gene and its application Download PDFInfo
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- CN108642058A CN108642058A CN201810325705.9A CN201810325705A CN108642058A CN 108642058 A CN108642058 A CN 108642058A CN 201810325705 A CN201810325705 A CN 201810325705A CN 108642058 A CN108642058 A CN 108642058A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0003—Invertebrate antigens
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- A61P33/14—Ectoparasiticides, e.g. scabicides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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Abstract
Disclosed by the invention is a kind of encoding gene and application thereof of Qinghai blood tick aquaporin (AQPs), and the encoding gene of the AQPs can be used for preparing novel anti-tick drug and anti-tick vaccine.The sequence of the encoding gene of the AQPs such as SEQ ID No：Shown in 1.The present invention also provides the specific primer of the encoding gene of AQPs clone, the specific primer sequence is SEQ ID NO：2 and SEQ ID NO：3, the present invention also provides a kind of methods of the encoding gene of the clone AQPs, can also carry out vitro recombination protein expression and Antibody preparation using the cloned gene product；The antibody can be used for preparing novel anti-tick drug and anti-tick vaccine.There is important theory significance and potential using value for the prevention of tick class.The encoding gene and its cloning process of the Qinghai blood tick aquaporin (AQPs) of the present invention can be used in the hypotype to AQPs, type, expression and distribution, protein structure and biological function etc. and carry out systematic research.
Description
Technical field
The present invention relates to biomedicine technical fields, are specifically related to the coding base of Qinghai blood tick aquaporin (AQP)
Cause and its application.
Background technology
Tick is the obligate epizoa sucked blood, and is the second largest vector for propagating a variety of humans and animals diseases, gives the mankind
Health and animal husbandry bring high risks.The prevention and control of tick are domestic and international focus of attention and research hotspot in recent years.Chemicals control
Tick processed the defects of there are drug resistance and environmental pollutions, novel anti-tick preparation is with a wide range of applications.Tick bites host's propagation
Cause of disease and in life cycle there are a large amount of liquid transportings activity, aquaporin (AQPs) is widely present on biological cell film
A kind of obligate critical function albumen for mediating liquid transmembrane transport, it has therefore proved that key effect, Ke Nengzuo are played in liquid transporting
The key protein of tick in order to control, but AQPs researchs are very few in tick body, and biological function is also unclear.Qinghai blood tick is only in China
A variety of cause of diseases are distributed and are propagated, one of the tick kind of most serious is endangered animal husbandry.
Aquaporin (Aquaporins, AQPs) is the embedded albumen (Major being widely present on biological cell film
Intrinsic protein, MIP) superfamily member, prevailing traffic hydrone can also transmit neutral point of glycerine, urea, ammonia etc.
Son plays key effect in the fluid balance of animal body organ.Expression of the AQPs in different tick kind histoorgans point
Cloth has differences, and the AQPs gene hypotypes expressed by different tick kind are also different, and sucks blood the liquid with the nonbloodsucking stage in tick
AQPs may play very important biological function in body transportational process, such as：Liquid absorption in tick life cycle is participated in, it is extra
The important physiology course such as the discharge of moisture, low-temperature resistance and resist drying.
This research is quasi- using Qinghai blood tick as research object, right with technologies such as molecular biology, immunology and RNA interference
AQPs gene types, protein structure, expression and distribution and biological function of the tick etc. carry out system research.Expected results will
Development for novel anti-tick drug and vaccine provides new theoretical foundation, anti-for disclosing liquid transporting mechanism and tick class in tick body
Jig has important theory significance and potential using value.Moreover, the development of novel anti-tick drug and vaccine has prodigious dive
It can be with better application prospect.Therefore, ideal drug target and guarantor are found with modern molecular biology and immunological technique
Shield property antigen, is of great significance to the prevention and control of tick.
Invention content
For the technical problem present on, the present invention provides the encoding genes of Qinghai blood tick aquaporin (AQP)
And its application.
The technical scheme is that：
A kind of purposes of the encoding gene of Qinghai blood tick aquaporin (AQPs), is used to prepare novel anti-tick drug.
A kind of purposes of the encoding gene of Qinghai blood tick aquaporin (AQPs), is used to prepare novel anti-tick vaccine.
Further, the sequence such as SEQ ID of the encoding gene of a kind of Qinghai blood tick aquaporin (AQPs)
No：Shown in 1.
The present invention also provides a kind of encoding gene of Qinghai blood tick aquaporin (AQPs) clone specific primer,
The specific primer sequence is SEQ ID NO：2 and SEQ ID NO：3, i.e.,：
5’-GSP：5’-TCACCTACGACCGCTCGAACACC-3’
3’-GSP：5’–GCCATCACAGACGCCCGCAACATGGCCG-3’.
The present invention also provides a kind of methods of the encoding gene of clone's Qinghai blood tick aquaporin (AQPs), including with
Lower step：
1) based on the special 5' and 3' end primers of encoding gene design Qinghai blood tick to expand the ends 5' and 3' cDNA
Sequence；The specific primer such as SEQ ID NO：2 and SEQ ID NO：Shown in 3：
2) amplification obtains the ends cDNA (RACE), including 5 ' RACE, 3 ' RACE-1,3 ' RACE-2,3 ' RACE-3,3 '
RACE-4, sequence number is respectively such as SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID
NO：Shown in 8；
3) in the sequence basis of 5 ' RACE of acquisition and 3 ' RACE, design pair for amplification Qinghai blood tick AQPs full length genes
The primer of sequence, sequence number such as SEQ ID NO：9、SEQ ID NO：Shown in 10, i.e.,：
AQP-full-F：5’-ATGTTGGAAAGCGTAAAGATCAAG-3’
AQP-full-R：5’-CATATTCTTGATATCGTTTGTCTTGATG-3’
4) it utilizes the primer amplification of step 3) to obtain 4 AQP and transcribes isomers, cDNA overall lengths are respectively：864bp,
876bp, 879bp and 885bp, sequence number is respectively such as SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID
NO：Shown in 14；4 transcription isomers are separately encoded 288,292,293 and 295 amino acid, and sequence number is respectively such as SEQ ID
NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：Shown in 18.
Further, vitro recombination protein expression and Antibody preparation are carried out using the cloned gene product：External structure
Expression vector, to the recombinant protein induced expression of acquisition, SDS-PAGE and Western-blot analyze protein expression situation, purifying
AQPs recombinant proteins, immune health rabbit or mouse are obtained, the serum containing antibody is obtained.
The present invention also provides a kind of novel anti-tick drugs, including above-mentioned antibody.
The present invention also provides a kind of novel anti-tick vaccines, including above-mentioned antibody.
The beneficial effects of the invention are as follows：The encoding gene of the Qinghai blood tick aquaporin (AQPs) of the present invention and its clone
Method can be used in the hypotype to AQPs, type, expression and distribution, protein structure and biological function etc. and carry out systematic research.
New theoretical foundation can also be provided for the development of novel anti-tick drug or vaccine, it is anti-to disclosing liquid transporting mechanism and tick in tick body
Jig has important theory significance and potential using value.
Specific implementation mode
From the Qinghai blood tick of field acquisition, it is stored in Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences parasite and entomophila epidemic disease
Sick innovation team laboratory, culture, is bitten by rabbit body in growth cabinet (26 ± 1 DEG C of temperature, RH70%, natural lighting)
Full blood simultaneously passes on.
The AQPs gene families announced according to ncbi database are in six kinds of tick (GenBank accession number:FN178519,
CAX48964,XM002399751,XP002399794,DQ904355,ABI53034,KP406519,CV443183,
AIT69684, KJ626366, CAR66115, FM210537, EST651745) in amino acid sequence characterization of molecules, completely
There are two amino acid sequence high conservative regions in 1kb or so, each complete AQPs gene copies for the length of AQPs genes
(NPA), we design the degenerate primer (AQP-F of amplification Qinghai blood tick AQPs Gene Partial sequences according to the two regions:5’-
TCC CAC CTG AAY CCY GCM GTG ACG CTG-3’and AQP-R:5’-GTC TCG DGC CGG GTT SAG
GGG-3 '), Qinghai blood tick total serum IgE is extracted, reverse transcription cDNA is template according to cDNA, and PCR amplification goes out one section of specific fragment
(SEQ ID No：1) and cloning and sequencing is carried out.The sequence of acquisition carry out BLAST analysis shows that, Qinghai blood tick AQPs and blood red fan
Carrapato (GenBank accession number：FM210537), ixodes scapularis (GenBank accession number：XM_002399490 and KT988052) and
Double-edged fine-toothed comb hard tick (GenBank accession number：FN178519 AQPs gene orders) have higher homology.
Partial sequence (SEQ ID No based on acquisition：1) the special 5' and 3' end primers of design Qinghai blood tick are to expand
The ends 5' and 3' cDNA sequence.
According to SMARTER RACE 5'/3' kits user's manuals (Clotech Laboratories of manufacturer
Inc., Mountain View, CA94043, USA) description, use universal primer and AQPs gene-specific primers (5 '-GSP
(5’-TCACCTACGACCGCTCGAACACC-3’)and 3’-GSP(5’–GCCATCACAGACGCCCGCAACATGGCCG-3’)
Rapid amplification of cDNA ends (RACE), the specific primer sequence are SEQ ID NO：2 and SEQ ID NO：3, amplification obtains
The ends cDNA (RACE), including 5 ' RACE, 3 ' RACE-1,3 ' RACE-2,3 ' RACE-3,3 ' RACE-4, sequence number difference
Such as SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：Shown in 8.The sequence of acquisition
It is assembled with DNAstar seqman softwares.
On the basis of the 5 ' of acquisition and 3 ' terminal sequence, design pair for amplification Qinghai blood tick AQPs full length genes sequence is (complete
ORF) primer (AQP-full-F (5 '-ATGTTGGAAAGCGTAAAGATCAAG-3 ', AQP-full-R (5 '-
CATATTCTTGATATCGTTTGTCTTGATG-3 '), sequence number such as SEQ ID NO：9、SEQ ID NO：Shown in 10.Amplification obtains
It obtains 4 AQP and transcribes isomers, cDNA overall lengths are respectively：864bp, 876bp, 879bp and 885bp, sequence number is respectively such as SEQ ID
NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：Shown in 14.Four transcription isomers are separately encoded 288,
292,293 and 295 amino acid, sequence number is respectively such as SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ
ID NO：Shown in 18.Bioinformatic analysis shows that AQPs albumen has typical 6 transmembrane regions and 2 conservative days
Winter amic acid-proline-alanine (NPA) structure, sequential analysis of protein show, Qinghai blood tick AQPs albumen and brown dog tick
AQP albumen homology between 84.5%-88%, with the homology of additional fan head tick AQP 85.7%-86.3% it
Between.The AQP amino acid sequences for all ticks that phylogenetic analysis is announced at present, the results show that AQP sequences are on systematic evolution tree
Display is divided into two big branch, and tetra- transcription isomers of Qinghai blood tick AQP of this research are distributed in the first subbranch propped up greatly, with
Brown dog tick and additional fan head tick are on being distributed in same branch.
Vitro recombination protein expression and Antibody preparation are carried out using the cloned gene product：External structure expression vector,
To the recombinant protein induced expression of acquisition, SDS-PAGE and Western-blot analyze protein expression situation, and purifying obtains AQPs
Recombinant protein, immune health rabbit or mouse obtain the serum containing antibody.The antibody can be used for preparing novel anti-tick drug
And anti-tick vaccine.There is important theory significance and potential using value for the prevention of tick class.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of Qinghai blood tick aquaporin（AQPs）Encoding gene and application thereof
<130>Nothing
<141> 2018-04-12
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 416
<212> DNA
<213> Specific fragment
<220>
<221> misc_feature
<222> (1)..(416)
<400> 1
tcccacctga atcctgcagt gacgctgtca ccacggtcgg caagttttcc tggcgcaaga 60
tcgtgcccta catggtggcg cagtacctgg gagccttcat tgcgtccgtc atcctcttcc 120
tgacatacag gggagccctg gacaacttcg acggtggtgt tcgagcggtc gtaggtgaaa 180
atgccactgc ggggatcttc gcttcatacc ccaaggaatt cgtcagcacc ggaaatggcc 240
ttgttgatca gatcgtgggc acagcgctgc tggtgctcgc catcttggcc atcacagacg 300
cccgcaacat ggccgttcca cagggtgtgc agcccctgtt cattggcttc gctctcggcg 360
cacttatcct gtgcttcggg tacaactgcg gtgctcccct gaacccggca cgagac 416
<210> 2
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<213> Specific primers
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<222> (1)..(23)
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tcacctacga ccgctcgaac acc 23
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actattccta ctcacgtgcg agttcacgtc gcgactcggc gacactgatc ttcgttatct 60
cctttttttc ccctcctctt aatcgttgag cagtcctgaa ttattccccc cgtgttagga 120
cgcagaggcc ggcatttaag ccaccggcgg gtcggtcgaa acgatctgta tagaagccag 180
acgtgttcgc gtcgtcgacg tctagacgag cccgcgtgtg gcttggttat tcctgctgcc 240
cttttgccat ctgtcgcgtc gccggcgagt gaagtgcgtg ttttcgacgt ctgcgaatac 300
cggacacaat gttggaaagc gtaaagatca agaacgccgt cgtccgggaa gtcctggcgg 360
agttcttggg gactttcgtg cttgtgcttt tcggcaacac agtactggcc gtgctgacgt 420
tcgacaaggt cggcatttcc ggctacgggg ccgccttctg gggctggggc ctggcgctca 480
cgctgggggt cctcgtcgct ggaggggtct cgggggccca cctgaacccg gcgatcactg 540
tcgctgtcac cacggtcggc aagttttcct ggcgcaaaat cgtgccctac atggtggcgc 600
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atcgtgggca cagcgctgct ggtgctcgcc atcttggcca tcacagatgc ccgcaacatg 60
gccgttccac agggtgtgca gcccctgttc attggcttcg ctctcggcgc acttatcctg 120
tgcttcgggt acaactgcgg tgctccgctc aacccggccc gagacctggg gccgcgtgtc 180
ttcactgcca tggctggatg gggaggcgag gtcttcagct tccgggacta caactggttc 240
tgggtgccca tcgtgggtcc ccacattggc gccatcatcg gcgcctggct gtacatgctg 300
gccgtggagc tgcactggcc cggcgcgagc tacgacatcg acagcggtaa ctccgtgtcc 360
gccaaggacg ttgaaaacgt catccaaatg aggggcatca agacaaacga tatcaagaat 420
taggtgccga gaaacgccaa tattaatatc aagaggaatc acacacatgc gcgcgtacaa 480
gggatgcaca cagccggtcc gtgcttacgg tgccctctac acaaggttca gctctcttcg 540
agaaacccgc acctcatcgc aacgattcta gcctcggtgt ctggaggccc aagtcacaga 600
caacgaatcg cggagtgcct catacagact cttcttacaa ctgttcttgc cccttttctc 660
tttttgtcgt tgttgttgtt tgattgtgtt tcacgcttgt gcgggtttgc gatgaaaagt 720
cctcccgtcc atgatgcacg tgcgagcacc aggtgtttat tgctacccaa gttgttgtag 780
ctccgtcttg tgcctgctta gctatcataa taatcaagag ccttactcta aaaaaaaaaa 840
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<210> 6
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<212> DNA
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<220>
<221> misc_feature
<222> (1)..(1001)
<400> 6
gccactgcgg ggatcttcgc ttcatacccc aaggaattcg tcagcaccgg aaatggcctt 60
gttgatcaga tcgtgggcac agcgctgctg gtgctcgcca tcttggccat cacagatgcc 120
cgcaacatgg ccgttccaca gggtgtgcag cccctgttca ttggcttcgc tctcggcgca 180
cttatcctgt gcttcgggta caactgcggt gctccgctca acccggcccg agacctgggg 240
ccgcgtgtct tcactgccat ggctggatgg ggaggcgagg tcttcagctt ccgggactac 300
aactggttct gggtgcccat cgtgggtccc cacattggcg ccatcatcgg cgcctggctg 360
tacatgctgg ccgtggagct gcactggccc ggcgcgagct acgacatcga cagcggtaac 420
cccgtgtcca ccaaggacga tgcttatgga caggatggta ttcgaatgtg aaattgaaaa 480
cgtcatccaa atgaggggca tcaagacaaa cgatatcaag aattaggtgc cgagaaacgc 540
caatattaat atcaagagga atcacacaca tgcgcgtgta caagggatgc acacagccgg 600
tccgtgttta cggtgccctc tcacaaggtt cagctctctt caagaaaccc gcacctcatc 660
gcaacgattc tagcctgggt gtctggaggc ccaagtcaca gacaacgaat cgcggagtgc 720
ctcatacaga ctcttcttac aactgttctt gccccttttt ttctttttgt cgttgttgtt 780
gtttgattgt gtttcacgct tgtgcgggtt tgcggtgaaa agtcctcccg tccatgatgc 840
acgtgcgagc accaggtgtt tattgctacc caagttgttg tagctccgtc ttctgcctcc 900
ttagctatca tataatcaga gccttactct acagaagaaa taaaaataaa aaaaagtact 960
ctgcgttgat accactgctt gccctatagt gagtcgtatt a 1001
<210> 7
<211> 755
<212> DNA
<213> End fragment
<220>
<221> misc_feature
<222> (1)..(755)
<400> 7
gccactgcgg ggatcttcgc ttcatacccc aaggaattcg tcagcaccgg aaatggcctt 60
gttgatcaga tcgtgggcac agcgctgctg gtgctcgcca tcttggccat cacagacgcc 120
cgcaacatgg ccgttccaca gggtgtgcag cccctgttca ttggcttcgc tctcggcgcg 180
cttatcctgt gcttcgggta caactgcggt gctccactca acccggcccg agacctgggg 240
ccgcgtgtct tcactgccat ggctggatgg ggaggcgagg tctccagctt ccgggactac 300
aactggttct gggtgcccat cgtgggtccc cacattggcg ccatcatcgg cgcctggctg 360
tacatgctgg ccgtggagct gcactggccc ggcgcgagct acgacatcga cagcggtaac 420
cccgtgtccg ccaaggacga tgcttatgga caggatggta ttcgaatgtg aaattgaaaa 480
cgtcatccaa atgaggggca tcaagacaaa cgatatcaag aattaggtgc cgagaaacgc 540
caatattaat atcaagagga atcacacaca tgcgcgtgta caagggatgc acacagccgg 600
tccgtgttta cggtgccctc tcacaaggtt cagctctctt caagaaaccc gcacctcatc 660
gcaacgattc tagcctgggt gtctggaggc ccaagtcaca gacaacgaat cgcggagtgc 720
ctcatacaga ctcttcttac aactgttctt gcccc 755
<210> 8
<211> 764
<212> DNA
<213> End fragment
<220>
<221> misc_feature
<222> (1)..(764)
<400> 8
gccactgcgg ggatcttcgc ttcatacccc aaggaattca tcagcaccgg gaatggcctt 60
gttgatcaga tcgtgggcac agcgctgctg gtgctcgcca tcttggccat cacagatgcc 120
cgcaacatgg ccgttccaca gggtgtgcag cccctgttca ttggcttcgc tctcggcgca 180
catatcctgt gcttcgggta caactgcggt gccccgctca acccggcccg agacctgggg 240
ccgcgtgtct tcactgccat ggctggatgg ggaggcgagg tcttcagctt ccgggactac 300
aactggttct gggtgcccat cgtgggcccc cacattggcg ccatcatcgg cgcctggctg 360
tacatgctgg ccgtggagct gcactggccg gcgcgagcta cgacatcgac agcggtaacc 420
ccgtgtccac caaggacggt gcgtacaacg gtcatcaacc gtccgctatc gagacgggca 480
gttgaaaacg tcatccaaat gaggggcatc aagacaaacg atatcaagaa ttaggtgccg 540
agaaacgcca atattaatat caagaggaat cacacacatg cgcgcgtaca agggatgcac 600
acagccggtc cgtgtttacg gtgccctcta cacaaggttc agctctcttc gagaaacccg 660
cacctcatcg caacgattct agcctcggtg tctggaggcc caagtcacag acaacgaatc 720
gcggagtgcc tcatacagac tcttcttaca actgttcttg cccc 764
<210> 9
<211> 24
<212> DNA
<213> Specific primers
<220>
<221> misc_feature
<222> (1)..(24)
<400> 9
atgttggaaa gcgtaaagat caag 24
<210> 10
<211> 28
<212> DNA
<213> Specific primers
<220>
<221> misc_feature
<222> (1)..(28)
<400> 10
catattcttg atatcgtttg tcttgatg 28
<210> 11
<211> 864
<212> DNA
<213> Transcript isomers
<220>
<221> misc_feature
<222> (1)..(864)
<400> 11
atgttggaaa gcgtaaagat caagaacgcc gtcgtccggg aagtcctggc ggagttcttg 60
gggactttcg tgcttgtgct tttcggcaac acagtactgg ccgtgctgac gttcgacaag 120
gtcggcattt ccggctacgg ggccgccttc tggggctggg gcctggcgct cacgctgggg 180
gtcctcgtcg ctggaggggt ctcgggggcc cacctgaacc cggcgatcac tgtcgctgtc 240
accacggtcg gcaagttttc ctggcgcaag atcgtgccct acatggtggc gcagtacctg 300
ggagccttca ttgcgtccgt catcctcttc ctgacataca ggggagccct ggacaacttc 360
gacggcggtg ttcgagcggt cgtaggtgaa aatgccactg cggggatctt cgcttcatac 420
cccaaggaat tcgtcagcac cggaaatggc cttgttgatc agatcgtggg cacagcgctg 480
ctggtgctcg ccatcttggc catcacagat gcccgcaaca tggccgttcc acagggtgtg 540
cagcccctgt tcattggctt cgctctcggc gcacttatcc tgtgcttcgg gtacaactgc 600
ggtgctccgc tcaacccggc ccgagacctg gggccgcgtg tcttcactgc catggctgga 660
tggggaggcg aggtcttcag cttccgggac tacaactggt tctgggtgcc catcgtgggt 720
ccccacattg gcgccatcat cggcgcctgg ctgtacatgc tggccgtgga gctgcactgg 780
cccggcgcga gctacgacat cgacagcggt aaccccgtgt ccaccaagga cgatgcttat 840
ggacaggatg gtattcgaat gtga 864
<210> 12
<211> 876
<212> DNA
<213> Transcript isomers
<220>
<221> misc_feature
<222> (1)..(876)
<400> 12
atgttggaaa gcgtaaagat caagaacgcc gtcgtccggg aagtcctggc ggagttcttg 60
gggactttcg tgcttgtgct tttcggcaac acagtactgg ccgtgctgac gttcgacaag 120
gtcggcattt ccggctacgg ggccgccttc tggggctggg gcctggcgct cacgctgggg 180
gtcctcgtcg ctggaggggt ctcgggggcc cacctgaacc cggcgatcac tgtcgctgtc 240
accacggtcg gcaagttttc ctggcgcaag atcgtgccct acatggtggc gcagtacctg 300
ggagccttca ttgcgtccgt catcctcttc ctgacataca ggggagccct ggacaacttc 360
gacggcggtg ttcgagcggt cgtaggtgaa aatgccactg cggggatctt cgcttcatac 420
cccaaggaat tcgtcagcac cggaaatggc cttgttgatc agatcgtggg cacagcgctg 480
ctggtgctcg ccatcttggc catcacagat gcccgcaaca tggccgttcc acagggtgtg 540
cagcccctgt tcattggctt cgctctcggc gcacttatcc tgtgcttcgg gtacaactgc 600
ggtgctccgc tcaacccggc ccgagacctg gggccgcgtg tcttcactgc catggctgga 660
tggggaggcg aggtcttcag cttccgggac tacaactggt tctgggtgcc catcgtgggt 720
ccccacattg gcgccatcat cggcgcctgg ctgtacatgc tggccgtgga gctgcactgg 780
cccggcgcga gctacgacat cgacagcggt aaccccgtgt ccaccaagga cgcacctcga 840
ttccaattgg cgaatcaaag tgatacgttg ttttag 876
<210> 13
<211> 879
<212> DNA
<213> Transcript isomers
<220>
<221> misc_feature
<222> (1)..(879)
<400> 13
atgttggaaa gcgtaaagat caagaacgcc gtcgtccggg aagtcctggc ggagttcttg 60
gggactttcg tgcttgtgct tttcggcaac acagtactgg ccgtgctgac gttcgacaag 120
gtcggcattt ccggctacgg ggccgccttc tggggctggg gcctggcgct cacgctgggg 180
gtcctcgtcg ctggaggggt ctcgggggcc cacctgaacc cggcgatcac tgtcgctgtc 240
accacggtcg gcaagttctc ctggcgcaag atcgtgccct acatggtggc gcagtacctg 300
ggagccttca ttgcgtccgt catcctcttc ctgacataca ggggagccct ggacaacttc 360
gacggcggtg ttcgagcggt cgtaggtgaa aatgccactg cggggatctt cgcttcatac 420
cccaaggaat tcgtcagcac cggaaatggc cttgttgatc agatcgtggg cacagcgctg 480
ctggtgctcg ccatcttggc catcacagat gcccgcaaca tggccgttcc acagggtgtg 540
cagcccctgt tcattggctt cgctctcggc gcacttatcc tgtgcttcgg gtacaactgc 600
ggtgctccgc tcaacccggc ccgagacctg gggccgcgtg tcttcactgc catggctgga 660
tggggaggcg aggtcttcag cttccgggac tacaactggt tctgggtgcc catcgtgggt 720
ccccacattg gcgccatcat cggcgcctgg ctgtacatgc tggccgtgga gctgcactgg 780
cccggcgcga gctacgacat cgacagcggt aaccccgtgt ccaccaagga cggtgcgtac 840
aacggtcatc aaccgtccgc tatcgagacg ggcagttga 879
<210> 14
<211> 885
<212> DNA
<213> Transcript isomers
<220>
<221> misc_feature
<222> (1)..(885)
<400> 14
atgttggaaa gcgtaaagat caagaacgcc gtcgtccggg aagtcctggc ggagttcttg 60
gggactttcg tgcttgtgct ttttggcaac acagtactgg ccgtgctgac gttcgacaag 120
gtcggcattt ccggctacgg ggccgccttc tggggctggg gcctggcgct cacgctgggg 180
gtcctcgtcg ctggaggggt ctcgggggcc cacctgaacc cggcgatcac tgtcgctgtc 240
accacggtcg gcaagttttc ctggcgcaaa atcgtgccct acatggtggc gcagtacctg 300
ggagccttca ttgcgtccgt catcctcttc ttgacataca ggggagccct ggacaacttc 360
gacggcggtg ttcgagcggt cgtaggtgaa aatgccactg cggggatctt cgcttcatac 420
cccaaggaat tcgtcagcac cgggaatggc cttgttgatc agatcgtggg cacagcgctg 480
ctggtgctcg ccatcttggc catcacagat gcccgcaaca tggccgttcc acagggtgtg 540
cagcccctgt tcattggctt tgctctcggc gcgcttatcc tgtgcttcgg gtacaactgc 600
ggtgctccgc tcaacccggc ccgagacctg gggccgcgtg tcttcactgc catggctgga 660
tggggaggcg aggtcttcag cttccgggac tacaactggt tctgggtgcc catcgtgggt 720
ccccacattg gcgccatcat cggcgcctgg ctgtacatgc tggccgtgga gctgcactgg 780
cccggcgcga gctacgacat cgacagcggt aaccccgtgt ccaccaagga cgttgaaaac 840
gtcatccaaa tgaggggcat caagacaaac gatatcaaga attag 885
<210> 15
<211> 287
<212> PRT
<213> Amino acid
<220>
<221> LIPID
<222> (1)..(287)
<400> 15
Met Leu Glu Ser Val Lys Ile Lys Asn Ala Val Val Arg Glu Val Leu
1 5 10 15
Ala Glu Phe Leu Gly Thr Phe Val Leu Val Leu Phe Gly Asn Thr Val
20 25 30
Leu Ala Val Leu Thr Phe Asp Lys Val Gly Ile Ser Gly Tyr Gly Ala
35 40 45
Ala Phe Trp Gly Trp Gly Leu Ala Leu Thr Leu Gly Val Leu Val Ala
50 55 60
Gly Gly Val Ser Gly Ala His Leu Asn Pro Ala Ile Thr Val Ala Val
65 70 75 80
Thr Thr Val Gly Lys Phe Ser Trp Arg Lys Ile Val Pro Tyr Met Val
85 90 95
Ala Gln Tyr Leu Gly Ala Phe Ile Ala Ser Val Ile Leu Phe Leu Thr
100 105 110
Tyr Arg Gly Ala Leu Asp Asn Phe Asp Gly Gly Val Arg Ala Val Val
115 120 125
Gly Glu Asn Ala Thr Ala Gly Ile Phe Ala Ser Tyr Pro Lys Glu Phe
130 135 140
Val Ser Thr Gly Asn Gly Leu Val Asp Gln Ile Val Gly Thr Ala Leu
145 150 155 160
Leu Val Leu Ala Ile Leu Ala Ile Thr Asp Ala Arg Asn Met Ala Val
165 170 175
Pro Gln Gly Val Gln Pro Leu Phe Ile Gly Phe Ala Leu Gly Ala Leu
180 185 190
Ile Leu Cys Phe Gly Tyr Asn Cys Gly Ala Pro Leu Asn Pro Ala Arg
195 200 205
Asp Leu Gly Pro Arg Val Phe Thr Ala Met Ala Gly Trp Gly Gly Glu
210 215 220
Val Phe Ser Phe Arg Asp Tyr Asn Trp Phe Trp Val Pro Ile Val Gly
225 230 235 240
Pro His Ile Gly Ala Ile Ile Gly Ala Trp Leu Tyr Met Leu Ala Val
245 250 255
Glu Leu His Trp Pro Gly Ala Ser Tyr Asp Ile Asp Ser Gly Asn Pro
260 265 270
Val Ser Thr Lys Asp Asp Ala Tyr Gly Gln Asp Gly Ile Arg Met
275 280 285
<210> 16
<211> 291
<212> PRT
<213> Amino acid
<220>
<221> LIPID
<222> (1)..(291)
<400> 16
Met Leu Glu Ser Val Lys Ile Lys Asn Ala Val Val Arg Glu Val Leu
1 5 10 15
Ala Glu Phe Leu Gly Thr Phe Val Leu Val Leu Phe Gly Asn Thr Val
20 25 30
Leu Ala Val Leu Thr Phe Asp Lys Val Gly Ile Ser Gly Tyr Gly Ala
35 40 45
Ala Phe Trp Gly Trp Gly Leu Ala Leu Thr Leu Gly Val Leu Val Ala
50 55 60
Gly Gly Val Ser Gly Ala His Leu Asn Pro Ala Ile Thr Val Ala Val
65 70 75 80
Thr Thr Val Gly Lys Phe Ser Trp Arg Lys Ile Val Pro Tyr Met Val
85 90 95
Ala Gln Tyr Leu Gly Ala Phe Ile Ala Ser Val Ile Leu Phe Leu Thr
100 105 110
Tyr Arg Gly Ala Leu Asp Asn Phe Asp Gly Gly Val Arg Ala Val Val
115 120 125
Gly Glu Asn Ala Thr Ala Gly Ile Phe Ala Ser Tyr Pro Lys Glu Phe
130 135 140
Val Ser Thr Gly Asn Gly Leu Val Asp Gln Ile Val Gly Thr Ala Leu
145 150 155 160
Leu Val Leu Ala Ile Leu Ala Ile Thr Asp Ala Arg Asn Met Ala Val
165 170 175
Pro Gln Gly Val Gln Pro Leu Phe Ile Gly Phe Ala Leu Gly Ala Leu
180 185 190
Ile Leu Cys Phe Gly Tyr Asn Cys Gly Ala Pro Leu Asn Pro Ala Arg
195 200 205
Asp Leu Gly Pro Arg Val Phe Thr Ala Met Ala Gly Trp Gly Gly Glu
210 215 220
Val Phe Ser Phe Arg Asp Tyr Asn Trp Phe Trp Val Pro Ile Val Gly
225 230 235 240
Pro His Ile Gly Ala Ile Ile Gly Ala Trp Leu Tyr Met Leu Ala Val
245 250 255
Glu Leu His Trp Pro Gly Ala Ser Tyr Asp Ile Asp Ser Gly Asn Pro
260 265 270
Val Ser Thr Lys Asp Ala Pro Arg Phe Gln Leu Ala Asn Gln Ser Asp
275 280 285
Thr Leu Phe
290
<210> 17
<211> 292
<212> PRT
<213> Amino acid
<220>
<221> LIPID
<222> (1)..(292)
<400> 17
Met Leu Glu Ser Val Lys Ile Lys Asn Ala Val Val Arg Glu Val Leu
1 5 10 15
Ala Glu Phe Leu Gly Thr Phe Val Leu Val Leu Phe Gly Asn Thr Val
20 25 30
Leu Ala Val Leu Thr Phe Asp Lys Val Gly Ile Ser Gly Tyr Gly Ala
35 40 45
Ala Phe Trp Gly Trp Gly Leu Ala Leu Thr Leu Gly Val Leu Val Ala
50 55 60
Gly Gly Val Ser Gly Ala His Leu Asn Pro Ala Ile Thr Val Ala Val
65 70 75 80
Thr Thr Val Gly Lys Phe Ser Trp Arg Lys Ile Val Pro Tyr Met Val
85 90 95
Ala Gln Tyr Leu Gly Ala Phe Ile Ala Ser Val Ile Leu Phe Leu Thr
100 105 110
Tyr Arg Gly Ala Leu Asp Asn Phe Asp Gly Gly Val Arg Ala Val Val
115 120 125
Gly Glu Asn Ala Thr Ala Gly Ile Phe Ala Ser Tyr Pro Lys Glu Phe
130 135 140
Val Ser Thr Gly Asn Gly Leu Val Asp Gln Ile Val Gly Thr Ala Leu
145 150 155 160
Leu Val Leu Ala Ile Leu Ala Ile Thr Asp Ala Arg Asn Met Ala Val
165 170 175
Pro Gln Gly Val Gln Pro Leu Phe Ile Gly Phe Ala Leu Gly Ala Leu
180 185 190
Ile Leu Cys Phe Gly Tyr Asn Cys Gly Ala Pro Leu Asn Pro Ala Arg
195 200 205
Asp Leu Gly Pro Arg Val Phe Thr Ala Met Ala Gly Trp Gly Gly Glu
210 215 220
Val Phe Ser Phe Arg Asp Tyr Asn Trp Phe Trp Val Pro Ile Val Gly
225 230 235 240
Pro His Ile Gly Ala Ile Ile Gly Ala Trp Leu Tyr Met Leu Ala Val
245 250 255
Glu Leu His Trp Pro Gly Ala Ser Tyr Asp Ile Asp Ser Gly Asn Pro
260 265 270
Val Ser Thr Lys Asp Gly Ala Tyr Asn Gly His Gln Pro Ser Ala Ile
275 280 285
Glu Thr Gly Ser
290
<210> 18
<211> 294
<212> PRT
<213> Amino acid
<220>
<221> LIPID
<222> (1)..(294)
<400> 18
Met Leu Glu Ser Val Lys Ile Lys Asn Ala Val Val Arg Glu Val Leu
1 5 10 15
Ala Glu Phe Leu Gly Thr Phe Val Leu Val Leu Phe Gly Asn Thr Val
20 25 30
Leu Ala Val Leu Thr Phe Asp Lys Val Gly Ile Ser Gly Tyr Gly Ala
35 40 45
Ala Phe Trp Gly Trp Gly Leu Ala Leu Thr Leu Gly Val Leu Val Ala
50 55 60
Gly Gly Val Ser Gly Ala His Leu Asn Pro Ala Ile Thr Val Ala Val
65 70 75 80
Thr Thr Val Gly Lys Phe Ser Trp Arg Lys Ile Val Pro Tyr Met Val
85 90 95
Ala Gln Tyr Leu Gly Ala Phe Ile Ala Ser Val Ile Leu Phe Leu Thr
100 105 110
Tyr Arg Gly Ala Leu Asp Asn Phe Asp Gly Gly Val Arg Ala Val Val
115 120 125
Gly Glu Asn Ala Thr Ala Gly Ile Phe Ala Ser Tyr Pro Lys Glu Phe
130 135 140
Val Ser Thr Gly Asn Gly Leu Val Asp Gln Ile Val Gly Thr Ala Leu
145 150 155 160
Leu Val Leu Ala Ile Leu Ala Ile Thr Asp Ala Arg Asn Met Ala Val
165 170 175
Pro Gln Gly Val Gln Pro Leu Phe Ile Gly Phe Ala Leu Gly Ala Leu
180 185 190
Ile Leu Cys Phe Gly Tyr Asn Cys Gly Ala Pro Leu Asn Pro Ala Arg
195 200 205
Asp Leu Gly Pro Arg Val Phe Thr Ala Met Ala Gly Trp Gly Gly Glu
210 215 220
Val Phe Ser Phe Arg Asp Tyr Asn Trp Phe Trp Val Pro Ile Val Gly
225 230 235 240
Pro His Ile Gly Ala Ile Ile Gly Ala Trp Leu Tyr Met Leu Ala Val
245 250 255
Glu Leu His Trp Pro Gly Ala Ser Tyr Asp Ile Asp Ser Gly Asn Pro
260 265 270
Val Ser Thr Lys Asp Val Glu Asn Val Ile Gln Met Arg Gly Ile Lys
275 280 285
Thr Asn Asp Ile Lys Asn
290
Claims (9)
1. a kind of purposes of the encoding gene of Qinghai blood tick aquaporin (AQPs), it is characterised in that：It is used to prepare novel anti-
Tick drug.
2. a kind of purposes of the encoding gene of Qinghai blood tick aquaporin (AQPs), it is characterised in that：It is used to prepare novel anti-
Tick vaccine.
3. a kind of purposes of the encoding gene of Qinghai blood tick aquaporin (AQPs) according to claim 1 or 2, special
Sign is：Its gene order such as SEQ ID No：Shown in 1.
4. the specific primer of the encoding gene clone of Qinghai blood tick aquaporin (AQPs) a kind of, which is characterized in that described
Specific primer sequence is SEQ ID NO：2 and SEQ ID NO：3, i.e.,：
5’-GSP：5’-TCACCTACGACCGCTCGAACACC-3’
3’-GSP：5’–GCCATCACAGACGCCCGCAACATGGCCG-3’.
5. a kind of method of the encoding gene of clone's Qinghai blood tick aquaporin (AQPs) as claimed in claim 3, special
Sign is, includes the following steps：
1) based on the special 5' and 3' end primers of encoding gene design Qinghai blood tick to expand the ends 5' and 3' cDNA sequence；
The specific primer such as SEQ ID NO：2 and SEQ ID NO：Shown in 3：
2) amplification obtains the ends cDNA (RACE), including 5 ' RACE, 3 ' RACE-1,3 ' RACE-2,3 ' RACE-3,3 ' RACE-4,
Its sequence number is respectively such as SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8 institutes
Show；
3) in the sequence basis of 5 ' RACE of acquisition and 3 ' RACE, design pair for amplification Qinghai blood tick AQPs full length gene sequences
Primer, sequence number such as SEQ ID NO：9、SEQ ID NO：Shown in 10, i.e.,：
AQP-full-F：5’-ATGTTGGAAAGCGTAAAGATCAAG-3’
AQP-full-R：5’-CATATTCTTGATATCGTTTGTCTTGATG-3’
4) it utilizes the primer amplification of step 3) to obtain 4 AQP and transcribes isomers, cDNA overall lengths are respectively：864bp,876bp,
879bp and 885bp, sequence number is respectively such as SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14
It is shown；4 transcription isomers are separately encoded 288,292,293 and 295 amino acid, and sequence number is respectively such as SEQ ID NO：15、
SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：Shown in 18.
6. according to the method described in claim 5, it is characterized in that, carrying out vitro recombination albumen using the cloned gene product
Expression and Antibody preparation：External structure expression vector, to the recombinant protein induced expression of acquisition, SDS-PAGE and Western-
Blot analyzes protein expression situation, and purifying obtains AQPs recombinant proteins, immune health rabbit or mouse, obtains the blood containing antibody
Clearly.
7. a kind of novel anti-tick drug, which is characterized in that include the antibody described in claim 6.
8. a kind of novel anti-tick vaccine, which is characterized in that include the antibody described in claim 6.
9. a kind of novel anti-tick drug, including the antibody described in claim 6.
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|---|---|---|---|
| CN201810325705.9A CN108642058A (en) | 2018-04-12 | 2018-04-12 | Qinghai blood tick aquaporin（AQP）Encoding gene and its application |
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| CN201810325705.9A CN108642058A (en) | 2018-04-12 | 2018-04-12 | Qinghai blood tick aquaporin（AQP）Encoding gene and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576233A (en) * | 2018-12-03 | 2019-04-05 | 中国农业科学院兰州兽医研究所 | A kind of tick source property recombinant vaccine |
| CN114853883A (en) * | 2022-05-06 | 2022-08-05 | 青海大学 | Haematococcus qinghaiensis serine protease inhibitor and polyclonal antibody thereof |
| WO2022155084A3 (en) * | 2021-01-15 | 2022-08-25 | Us Biologic, Inc. | Method to reduce tick population with a universal tick antigen |
-
2018
- 2018-04-12 CN CN201810325705.9A patent/CN108642058A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576233A (en) * | 2018-12-03 | 2019-04-05 | 中国农业科学院兰州兽医研究所 | A kind of tick source property recombinant vaccine |
| WO2022155084A3 (en) * | 2021-01-15 | 2022-08-25 | Us Biologic, Inc. | Method to reduce tick population with a universal tick antigen |
| US11771746B2 (en) | 2021-01-15 | 2023-10-03 | Us Biologic, Inc | Method to reduce tick population with a universal tick antigen |
| CN114853883A (en) * | 2022-05-06 | 2022-08-05 | 青海大学 | Haematococcus qinghaiensis serine protease inhibitor and polyclonal antibody thereof |
| CN114853883B (en) * | 2022-05-06 | 2023-05-16 | 青海大学 | Qinghai blood tick serine protease inhibitor and polyclonal antibody thereof |
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