CN101757618B - Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof - Google Patents
Recombined subunit vaccine of haemaphysalis concinna and preparation method thereof Download PDFInfo
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- CN101757618B CN101757618B CN200910263441XA CN200910263441A CN101757618B CN 101757618 B CN101757618 B CN 101757618B CN 200910263441X A CN200910263441X A CN 200910263441XA CN 200910263441 A CN200910263441 A CN 200910263441A CN 101757618 B CN101757618 B CN 101757618B
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Abstract
The invention discloses a recombined subunit vaccine of haemaphysalis concinna and a preparation method thereof. The recombined subunit vaccine is formed by mixing antigenic gene recombined protein of the haemaphysalis concinna and Freund's complete adjuvant (FCA), wherein the content of the antigenic gene recombined protein is 50 mg/ml, that is 50 mL rHc-23 and 950 ml Freund's complete adjuvant are mixed to prepare the recombined subunit vaccine of the haemaphysalis concinna; the amino acid sequence of the antigenic gene recombined protein is shown in the table SEQID NO:1; and the nucleotidesequence of the antigenic gene is shown in the table SEQID NO:2. Through screening and cloning, prokaryotic expression and separation and purification of protective antigen gene of the haemaphysalis concinna and the application effect test of the recombined subunit vaccine, the method shows that an expression product of recombined vector bacteria can be identified by rabbit anti-haemaphysalis concinna positive serum. In an animal immunity test, after rHc-23-FCA is subjected to three immune rabbits, the blood saturation rates of haemaphysalis larva, haemaphysalis middle and haemaphysalis imagoare 40.3 percent, 45.6 percent and 41.3 percent respectively; and compared with the blood saturation rates of the haemaphysalis larva, haemaphysalis middle and haemaphysalis imago in a contrast set: 90.1 percent, 94.3 percent and 97.7 percent, the differences are remarkable. The method promotes the development of anti-haemaphysalis immunity, haemaphysalis control and haemaphysalis disease spread work.
Description
Technical field
The present invention relates to a kind of recombinant subunit vaccine, relate in particular to recombinant subunit vaccine of a kind of haemaphysalis conicinna and preparation method thereof.
Background technology
Haemaphysalis conicinna (Haemaphysalis concinna) is the obligate hematophagus of animal body surface, is distributed widely in countries in the world, on the multiple animal body of more than 10 provinces and regions of China, has found this Ticks at present.Haemaphysalis conicinna is bitten and is sucked a large amount of blood of host, causes the swelling of host's local skin, inflammation, Pruritus and ulcer etc.
[1]In addition, this Ticks also can be propagated the several diseases substance, be one of important Ticks kind of serious harm China animal husbandry [
[2-11]].
Existing known this Ticks is distributed in a plurality of countries and regions, the world
[12-19], report is also all arranged in a lot of provinces and regions of China
[5,20-21]White the nineties is since latter stage, and the epidemic situation of haemaphysalis conicinna has been broken out in China Sichuan successively.Since in March, 1999, the outburst epidemic situation that haemaphysalis conicinna has appearred in 42 small towns such as Bazhong City, Sichuan Province Le Feng, Wan An township causes very big harm to people and domestic animal.Over the past two years; In Sichuan Province the Guangyuan City Cangxi County and on every side the small towns break out this disease again; Cause symptoms such as local pimple, redness, Pruritus after humans and animals is bitten, most of patients causes secondary infection because of Pruritus, and itch behind the wound healing pain and pigmented spots still continue the several months; Cause the frightened outdoor activities of people, had a strong impact on agricultural production and human and livestock health
[22-24,26]The control of ixodism is main with medicine still at present, but the life-time service of tick eradication medicine is prone to cause the appearance of drug resistance worm strain, especially has medicine residual in livestock products and to the problems such as pollution of environment, therefore, presses for the new method of preventing and treating of seeking.The research of past to haemaphysalis conicinna mainly is morphology, biological nature, popular investigation and treatment thereof.At present; Anti-Ticks immunity is considered to control Ticks and Ticks one of the most potential method that spreads disease; And up to the present, still lack research to the haemaphysalis conicinna immunoprophylaxis, still have nothing to do both at home and abroad in the research report of haemaphysalis conicinna protective antigen gene and anti-tick vaccine.Recent years, existing test carried out studying [30-32] to the reorganization parasite antigen as blood tick vaccine candidate gene along with immunology and molecular biological fast development.Research has obtained certain achievement to haemaphysalis longicornis protective antigen gene P27/30 especially.[30]Donnelly?JJ，Ulmer?JF，Shiver?JW，Liu?MA.DNA?vaccination.AnnuRev?Immunol?1997；15：617-48.[32]Geldhof?P，Maere?VD，Vercruysse?J，Claerebout?E.Recombinant?expression?systems：the?obstacle?to?helminth?vaccines?Trends?Parasitol2007；23(11)：527-32.。For this reason, screening and cloning and the prokaryotic expression and the immunoprotection experiment of haemaphysalis conicinna protective antigen gene (Hc-23 gene) carried out in this research, analyzed the immunogenicity of this gene simultaneously, and purpose is for developing a kind of recombinant subunit vaccine of haemaphysalis conicinna.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, promptly overcome the residual and environmental pollution problem of medicine in livestock products, finally develop the good haemaphysalis conicinna recombinant subunit vaccine of a kind of immunogenicity.
The technical solution that realizes the present invention's purpose realizes through following technical measures:
A kind of recombinant subunit vaccine of haemaphysalis conicinna; It is characterized in that; Mix formation by haemaphysalis conicinna antigenic gene recombiant protein with Freund's complete adjuvant (FCA), wherein said antigenic gene recombiant protein content 50mg/ml is that 50mL rHc-23 and 950mL Freund's complete adjuvant mixed preparing form.
Described haemaphysalis conicinna antigenic gene recombiant protein, its aminoacid sequence are sequence shown in the sequence table SEQ ID NO:1.
The nucleotides sequence of described haemaphysalis conicinna antigenic gene is classified sequence shown in the table SEQID NO:2 as.
The method for preparing of the recombinant subunit vaccine antigenic gene recombiant protein of described a kind of haemaphysalis conicinna, its step is following:
(1) according to the nucleotide sequence shown in the described nucleotides sequence tabulation SEQID NO:2, the primer that design contains EcoRI and HindIII restriction enzyme site respectively does
5’-CG
GAATTCATGGGAGACGAGGAGAAGAG-3’；
5’-GGG
TTCGAATTGCCTCGTTGTTTATTCCT-3’
(2) amplify the Hc-23 gene; With the Hc-23 gene is that template is carried out pcr amplification; The PCR product that reclaims is carried out double digestion with restricted enzyme; Be connected with the expression vector of handling through same enzyme action (pMD18-T) then, be transformed in the recipient bacterium, obtain to contain the recombinant expression carrier (pMD18-T-Hc-23 plasmid) of Hc-23 gene;
(3) be that template is carried out pcr amplification with the positive colony plasmid (pMD18-T-Hc-23 plasmid) that checks order correct, reclaim the purpose fragment and carry out sub-clone that be about to the PCR product cloning and go into the pMD18-T carrier, last sub-clone is gone into pET-32a (+) expression vector;
(4) respectively positive recombiant plasmid and expression vector pET32a (+) are carried out double digestion with restriction endonuclease EcoRI and Hind III, obtain to contain the purpose fragment and the wire carrier of cohesive end;
(5) under 16 ℃ of conditions, use T
4Dna ligase connects back transformed competence colibacillus cell DH5a;
(6) will identify that correct recombinant expression plasmid (pET32a-Hc-23) transforms expressive host bacterium BL21 (DE3) and carries out prokaryotic expression, express recombinant protein collection, purification will promptly be got;
(7) carry out the SDS-PAGE electrophoresis detection rHc-23 albumen that obtains recombinating;
(8) expressing protein purification procedures:
The expression bacterium liquid 500mL that the IPTG inducing culture of learning from else's experience is good uses the 50mL centrifuge tube, and the centrifugal 5min of 10000r/min abandons supernatant, stays deposition; Tris-Hcl (PH=8.0) 10mL that in deposition, adds 20mmol/L suspends, and then 1mL lysozyme (10mg/mL) is added in the suspension, and 37 ℃ of joltings are spent the night ,-20 ℃ of overnight incubation; Ultrasonication (200W, 30s/ time, 10 times); The centrifugal 10min of 10000r/min; Supernatant is deposited subsequent use in addition, and deposition washes twice with 2mol/L carbamide+1%TritonX-100+50mmol/LNacl+0.2mmol/LEDTA, and reuse 2mol/L carbamide+1%TritonX-100 washes twice; Abandon supernatant behind the centrifugal 10min of 10000r/min, be precipitated as expressed albumen.
This research is carried out bioinformatic analysis to the Hc-23 gene, finds Hc-23 gene 201 aminoacid of encoding altogether, and this aminoacid predicted molecular weight is 23.386kDa, and the isoelectric point, IP theoretical value is 9.77, has stronger hydrophilic.Homology analysis shows; The ORF of the strain in China (EF088685) of this gene and Yang Caiming and You login and the troponin gene P27/30 sequence of Japanese strain haemaphysalis longicornis (AB051079) comparison of comparing; The c-t transversion has only taken place at the 300th bit base place up to 99.83% in haemaphysalis conicinna Hc-23 gene and strain in China haemaphysalis longicornis troponin gene P27/30 homology; With Japanese strain haemaphysalis longicornis troponin gene P27/30 homology up to 99.67%; At the 183rd bit base place the a-g transversion having taken place respectively and the c-t transversion has taken place at the 300th bit base place, is 89% and rise the crescent fan head tick Troponin I dna homolog property of login on GenBank with horse.
Compared with present technology, the present invention has outstanding advantage and significant effect:
1, this research is the existence of finding the Hc-23 antigen gene first in each stage of development of haemaphysalis conicinna, Hc-23 is cloned into expression vector pET-32a (+), and at escherichia expression system it is carried out expression study; Recombinant expression carrier pET-32a (+)-Hc-23 is transformed into abduction delivering in the e. coli bl21 (DE3); Confirm to contain the fusion rotein identical in the cell lysate through SDS-PAGE, have immunogenicity through this fusion rotein of West-blot assay certificate with desired location.
2, in animal immune test, rHc-23-FCA is through three immunize rabbits after, and young Ticks, if Ticks is respectively 40.3%, 45.6% and 41.3% with the full blood rate that becomes Ticks is compared the young Ticks of matched group, as if Ticks and the full blood rate 90.1% that becomes Ticks.94.3 with 97.7%, difference is all remarkable.
3, Ticks is a kind of arthropod of sucking blood liked, and extensively distributes in China, is the important media of human and low etc. a lot of infectious disease of animal.The several diseases substance can carried and propagate to the Ticks class; Spread out of heat in blood, the plague etc. as forest encephalitis, Ticks; Can also propagate protozoacide such as Brucella; Of the present invention open, pathogenetic recombinant subunit vaccine of a kind of effective control animal haemaphysalis conicinna and preparation method thereof will be provided, greatly promote the spread disease development of work of immunity of anti-Ticks and control Ticks and Ticks.
Description of drawings
Fig. 1 is the PCR product electrophoretogram of the described recombiant plasmid of invention (pET32a-Hc-23).
Fig. 2 is the described reorganization rHc-23 albumen SDS-PAGE electrophoresis detection figure that under the IPTG variable concentrations, takes a sample.
Fig. 3 is described reorganization rHc-23 expressing protein western blot figure.
Fig. 4 is the purification SDS-PAGE analysis chart of described recombiant protein rHc-23.
Fig. 5 is that ELISA detects the antibody its growth figure in the rabbit anteserum in the described immunoprotection test.
The specific embodiment
Segmental the obtaining of embodiment 1 haemaphysalis conicinna Hc-23 antigen gene
Utilize the nucleotide sequence of the protective antigen gene P27/30 of known haemaphysalis longicornis strain in China and Japanese strain, the synthetic a pair of Auele Specific Primer of design.Utilize this primer that total RNA of haemaphysalis conicinna is carried out RT-PCR, the cDNA that becomes with reverse transcription is a template, uses the Auele Specific Primer of this gene to increase, and obtains the purpose fragment.
The experiment haemaphysalis conicinna picks up from Cangxi County, Sichuan Province rabbit body; Be accredited as haemaphysalis conicinna through morphology; Laboratory condition is used Ticks as test after breeding for 4 generations through the rabbit body down, and the experiment in non-parasitic stage is all cultivated (Deng 1978) with Ticks in the incubator under the laboratory condition.
Laboratory animal is selected 12 Chinese large ear rabbits of health for use, and 2.9-3.2kg is available from Sichuan Agricultural University's Experimental Animal Center.
Extract RNA with RNA extraction agent box.The hungry one-tenth of the haemaphysalis longicornis Ticks of peeking and only living, adding 1mLRNAiso Reagent grinds the back in mortar centrifugal, gets supernatant and separate with chloroform; Isopropanol precipitating, after the washing with alcohol with 1mL75%, drying at room temperature; RNA with the extraction of RNase free water dissolution; The PCR product that UNIQ-10DNA glue is reclaimed the test kit recovery is connected on the pMD18-T carrier, transforms the DH5a competent cell then, behind the blue white macula screening positive clone; Extracting positive bacteria plasmid carries out the pcr amplification check, obtains the purpose fragment of 631bp.Find that through the DNAStar software analysis comprising a length in the fragment of 631bp is the complete ORFs (ORF) of 603bp, and with this fragment called after Hc-23 gene (the GenBank accession number is FJ425897) referring to SEQIDNO 2 gene order tables.
The preparation of embodiment 2 haemaphysalis conicinna subunit vaccines
Hc-23 gene order according to obtaining designs a pair of prokaryotic expression primer, and the primer that design contains EcoR I and HindIII restriction enzyme site respectively is:
5’-CG
GAATTCATGGGAGACGAGGAGAAGAG-3’；
5’-GGG
TTCGAATTGCCTCGTTGTTTATTCCT-3’
With the Hc-23 gene is that template is carried out pcr amplification; The PCR product that reclaims is carried out double digestion with restricted enzyme; Be connected with the expression vector of handling through same enzyme action (pMD18-T) then; Be transformed in the recipient bacterium, obtain to contain the recombinant expression carrier (pMD18-T-Hc-23 plasmid) of Hc-23 gene;
Positive colony plasmid (pMD18-T-Hc-23 plasmid) to check order correct is that template is carried out pcr amplification, reclaims the purpose fragment and carries out sub-clone, is about to the PCR product cloning and goes into the pMD18-T carrier, and last sub-clone is gone into pET-32a (+) expression vector; The structure of accomplishing recombinant expression plasmid (pET32a-Hc-23) is referring to Fig. 1.
Respectively positive recombiant plasmid and expression vector pET32a (+) are carried out double digestion with restriction endonuclease EcoRI and Hind III, obtain to contain the purpose fragment and the wire carrier of cohesive end; With identifying that correct recombinant expression plasmid (pMD32-T-Hc-23 plasmid) is transformed into expressive host escherichia coli F-strain BL21 (DE3) and carries out prokaryotic expression; It is extracting positive bacteria plasmid; Be transformed into and express in bacterium BL21 (DE3) recipient bacterium, get the positive plasmid transformed bacteria and the empty plasmid transformed bacteria that are in exponential phase, add in the LB fluid medium with Amp 50 μ g/mL; 37 ℃ of 180rmp/min cultivate 3-6h, reach about 0.6 to OD600.Bacterium liquid add IPTG to final concentration be 1mmol/L, cultivated 4 hours for 37 ℃; Carry out the SDS-PAGE electrophoresis detection rHc-23 albumen that obtains recombinating, the proteic molecular weight of amalgamation and expression is about 43kDa, and exists with the form of inclusion body.Referring to recombinant vector bacterium expression product shown in Figure 2.
Expressing protein separation, purification and evaluation:
The expression bacterium liquid 500mL that the IPTG inducing culture of learning from else's experience is good uses the 50mL centrifuge tube, and the centrifugal 5min of 10000r/min abandons supernatant, stays deposition; Tris-Hcl (PH=8.0) 10mL that in deposition, adds 20mmol/L suspends, and then 1mL lysozyme (10mg/mL) is added in the suspension, and 37 ℃ of joltings are spent the night ,-20 ℃ of overnight incubation; Ultrasonication (200W, 30s/ time, 10 times); The centrifugal 10min of 10000r/min; Supernatant is deposited subsequent use in addition, and deposition washes twice with 2mol/L carbamide+1%TritonX-100+50mmol/LNacl+0.2mmol/LEDTA, and reuse 2mol/L carbamide+1%TritonX-100 washes twice; Abandon supernatant behind the centrifugal 10min of 10000r/min, be precipitated as expressed albumen; Drying precipitated back adds 8mol/L carbamide 5mL, after the vibration dissolving, gets 40 μ L and adds that a kind buffer carries out SDS-PAGE 10 μ L on, identifies as shown in Figure 4.Referring to SEQIDNO 1 aminoacid sequence table.
The concrete preparation of vaccine according to the invention is by there being described haemaphysalis conicinna antigenic gene recombiant protein to mix formation with Freund's complete adjuvant (FCA); Antigenic gene recombiant protein content 50mg/ml is that 50mL rHc-23 and 950mL Freund's complete adjuvant mixed preparing form the vaccine physicochemical property that is obtained: vaccine is the milky oil preparation.
Method for using: the neck subcutaneous injection, injected once respectively again in 7 days, 14 days after injection for the first time, inject altogether 3 times, 1mL/ is only.
(1) the anti-haemaphysalis conicinna immune serum preparation of rabbit
With haemaphysalis conicinna artificial challenge China large ear rabbit, every ear inoculates into 30 of Tickss, if 100 of 60 of Tickss and young Tickss repeat once after full blood falls, and totally twice, the blood sampling separation of serum places serum-20 ℃ of preservations subsequent use.
(2) immunoblotting
Purifying protein is behind SDS-PAGE, and the transfer printing cellulose nitrate film is hatched 30min with 1%BSA, and the PBS-T with 0.05%Tween-20 washes film then; The anti-haemaphysalis conicinna positive serum of adding rabbit (by dilution in 1: 100) is anti-as one, more than 4 ℃ of reaction 12h, abandons one and resists; Wash film respectively with 0.1M PBS-T, add two anti-goat anti-rabbit igg-HRP (Chengdu Bo Ruike biotech firm) (diluted concentration is 1: 1000), incubated at room 2h; Abandon two and resist, wash film, add the colour developing of colour developing liquid with 0.01M PBS-T.Referring to Fig. 3.
(3) immunoprotection test
12 Chinese large ear rabbits are divided into four groups, 3 every group.First group of subcutaneous vaccination recombiant protein (rHc-23) and Freund's complete adjuvant mixed liquor
Three inoculations contain the Freund's complete adjuvant of 50 μ g rHc-23, and 1mL/ only; Second group of subcutaneous vaccination recombiant protein (rHc-23) and Freund's complete adjuvant mixed liquor, twice inoculation contain the Freund's complete adjuvant of 75 μ g rHc-23, and 1mL/ only; The 3rd group of subcutaneous vaccination recombiant protein (rHc-23) and Freund's complete adjuvant mixed liquor, once inoculation contains the Freund's complete adjuvant of 150 μ grHc-23, and 1mL/ is only; The 4th group of subcutaneous injection 1mLPBS (pH 7.5), 0.1mL/ are only.Each inoculation time is 14d at interval.Last inoculation back 14d; Every rabbit is with becoming 30 of Tickss, infecting as if 50 of Tickss and 100 of young Tickss; Infect the back ixodes infestation situation on the rabbit body is checked that recording parameters is: full blood time, full blood rate, full blood body weight, ecdysis and the quantity or lay eggs weight of laying eggs.After the full blood of Ticks falls, Ticks is positioned over separately in the hatching bottle of 29 ± 3 ℃ of temperature and humidity 80 ± 4%, it can normally be hatched or lay eggs.(the full blood time: the time from infecting to falling; Full blood rate: the quantity of the quantity/infection of recovery; Full blood body weight: the body weight after full blood falls; Ecdysis: full blood drops to the time of casting off a skin for the next stage of development).
(4) ELISA detects the proteic specific IgG antibodies level of the anti-Hc-23 of rabbit anteserum
Be diluted to 5 μ g/ml r Hc-23 albumen with 0.1M carbonate (pH9.6) and encapsulate 96 hole elisa plates, 50 μ L/ holes place 4 ℃ of refrigerators to hatch 16h; With 0.05%Tween 20 (PBS-T) washing 3 times, add 1% bovine serum albumin again, 100 μ L/ holes; At 37 ℃ of sealing 1h, with PBS-T washing 5 times.Use 0.1M PBS (pH 7.5) to do to add ELISA Plate behind the serial dilution rabbit anteserum by 1: 100 then, 1h is hatched for 37 ℃ in 100 μ L/ holes; With PBS-T washing 3 times, add ELISA Plate, 100 μ L/ holes by 1: 1000 dilution goat anti Mus IgG-HRP with 0.1M PBS (pH 7.5); Hatch 1h for 37 ℃,, add substrate solution with PBS-T washing 3 times; Hatch 5-10min for 37 ℃, add the stop buffer cessation reaction, the OD of test sample article
490
The result of the test statistical analysis
The random experiment design is adopted in this test.Use SPSS software that test data is carried out arithmetical average, standard error, Duncan (Duncan) multiple comparisons, it is remarkable to be designated as significant difference with P≤0.05.
(1) immunoblotting assay
Immunoblotting assay finds that empty carrier bacterium expression product can not be discerned by the anti-haemaphysalis conicinna positive serum of rabbit, and recombinant vector bacterium expression product then can be discerned by the anti-haemaphysalis conicinna positive serum of rabbit.
(2) immune response that causes of vaccine
ELISA detects the antibody result demonstration in the mice serum: the IgG level that immunity back 7d～56d, rHc-23 organize rabbit is higher than the PBS group, except that immunity back 14d, all the other detection times of interior all differences remarkable (P＜0.05).
(3) immunoprotection experiment
RHc-23-FCA is behind three immunize rabbits, and young Ticks, if Ticks is respectively 40.3%, 45.6% and 41.3% with the full blood rate that becomes Ticks is compared the young Ticks of matched group, as if Ticks and the full blood rate 90.1% that becomes Ticks.94.3 with 97.7%, difference is all remarkable.The young Ticks that reclaims on the immune group rabbit body, if Ticks, has just directly influenced young Ticks, casting off a skin and further grow and become laying eggs of Ticks as if Ticks with to become the full blood rate of Ticks low.
Generally speaking; The pET32a (+) that makes up-Hc-23 expression vector is transformed expression strain BL21 (DE3) carry out abduction delivering; And with recombiant protein and Freund's complete adjuvant (FCA) combination immunization in rabbit; Immunity back is attacked rabbit with the haemaphysalis conicinna of different developmental phases, the full blood early stage of each period of development Ticks of immune group, full blood phase and full blood body weight and matched group (PBS) significant difference (P≤0.05), one-tenth Ticks egg laying amount and matched group (PBS) significant difference (P≤0.05).
The antibody result that ELISA as shown in Figure 5 detects in the rabbit anteserum shows: immune back 7 days～56 days, the IgG level of rHc-23 group rabbit is higher than PBS to be organized, and removes back 14 days of immunity, all the other detection times of interior all differences remarkable (P≤0.05).
Sequence is following shown in the aminoacid sequence SEQID NO:1 of recombinant antigen protein according to the invention:
< 110>Sichuan Agricultural University
< 120>recombinant subunit vaccine of a kind of haemaphysalis conicinna and preparation method thereof
< 130>invention
< 140>do not have temporarily
<141>2009-12-16
<160>1
<170>PatentIn?version?3.3
<210>1
<211>200
<212>PRT
< 213>haemaphysalis conicinna
<400>1
Met?Gly?Asp?Glu?Glu?Lys?Arg?Lys?Met?Glu?Glu?Lys?Glu?Arg?Lys?Lys
1 5 10 15
Ala?Glu?Val?Arg?Lys?Arg?Leu?Glu?Glu?Ala?Ala?Lys?Ala?Lys?Lys?Ala
20 25 30
Gly?Gly?Lys?Arg?Gly?Phe?Met?Thr?Pro?Glu?Arg?Lys?Lys?Lys?Leu?Arg
35 40 45
Asn?Leu?Leu?Arg?Lys?Lys?Ala?Ala?Glu?Glu?Leu?Lys?Lys?Glu?Gln?Glu
50 55 60
Arg?Lys?Ala?Glu?Gln?Arg?Arg?Lys?Ile?Ile?Ala?Glu?Arg?Ile?Gly?Gln
65 70 75 80
Pro?Lys?Pro?Leu?Asp?Asn?Cys?Asn?Glu?Ala?Thr?Leu?Val?Gly?Ile?Leu
85 90 95
Lys?Gln?Tyr?His?Ala?Arg?Ile?Ala?Gln?Leu?Glu?Asp?Ala?Lys?Tyr?Asp
100 105 110
Leu?Glu?Tyr?Glu?Val?Arg?Gln?Lys?Asp?Phe?Val?Ile?Asn?Glu?Leu?Thr
115 120 125
Ile?Gln?Val?Asn?Asp?Leu?Arg?Gly?Lys?Phe?Val?Lys?Pro?Ala?Leu?Lys
130 135 140
Lys?Val?Ser?Lys?Phe?Asp?Lys?Leu?Lys?Met?Val?Val?Lys?Ser?Thr?Ser
145 150 155 160
Glu?Val?Asp?Phe?Arg?Ser?Ser?Leu?Lys?Ser?Val?Lys?Lys?Asp?Ala?Phe
165 170 175
Lys?Leu?Asp?Glu?Glu?Asn?Lys?Ala?Asp?Lys?Lys?Pro?Glu?Trp?Ala?Leu
180 185 190
Gly?Ser?Lys?Lys?Glu?Asn?Glu?Glu
195 200
The nucleotide sequence SEQ ID NO:2 sequence that contains antigenic gene in the said haemaphysalis conicinna is following:
< 110>Sichuan Agricultural University
< 120>recombinant subunit vaccine of a kind of haemaphysalis conicinna and preparation method thereof
< 130>invention
< 140>do not have temporarily
<141>2009-12-16
<160>1
<170>PatentIn?version?3.3
<210>1
<211>603
<212>DNA
< 213>haemaphysalis conicinna
<400>1
atgggagacg?aggagaagag?gaagatggag?gagaaggagc?ggaaaaaggc?cgaggtgcgc 60
aaacgtctcg?aggaggccgc?taaggcgaag?aaggctggcg?gaaagcgcgg?cttcatgacc 120
cccgagcgca?agaagaagct?caggaacttg?ctgaggaaga?aggccgctga?ggagttgaaa 180
aaggagcagg?agcgcaaggc?tgagcaacgt?aggaagatca?tcgcggaacg?catcggccag 240
cccaagcctc?tggataactg?caacgaagcc?actctggtgg?gaatcctcaa?gcagtaccat 300
gcccggatag?cgcaactaga?ggacgccaag?tacgacctgg?agtacgaagt?caggcagaag 360
gacttcgtga?tcaacgagct?gacaatccag?gtgaacgatc?ttcgtggcaa?gttcgtgaag 420
ccggctctca?agaaagtttc?caagttcgac?aagctcaaaa?tggtcgtcaa?gagcacctcg 480
gaggttgact?tcaggtccag?ccttaagtcc?gtcaagaagg?atgctttcaa?gctcgatgaa 540
gagaacaagg?cggacaagaa?gcctgaatgg?gctcttggct?ccaagaagga?gaacgaggaa 600
taa
603
Claims (3)
1. the recombinant subunit vaccine of a haemaphysalis conicinna; It is characterized in that; Mix with Freund's complete adjuvant by haemaphysalis conicinna antigenic gene recombiant protein and to constitute; Wherein said antigenic gene recombiant protein content 50mg/mL is formed by 50mL rHc-23 and 950mL Freund's complete adjuvant mixed preparing, and its aminoacid sequence is a sequence shown in the sequence table SEQ ID NO:1.
2. the nucleotides sequence of haemaphysalis conicinna antigenic gene according to claim 1 is classified sequence shown in the table SEQID NO:2 as.
3. the method for preparing of the recombinant subunit vaccine antigenic gene recombiant protein of a kind of haemaphysalis conicinna according to claim 1, its step is following:
(1) according to the nucleotide sequence shown in the described nucleotides sequence tabulation SEQID NO:2, the primer that design contains EcoRI and Hind III restriction enzyme site respectively does
5’-CG
GAATTCATGGGAGACGAGGAGAAGAG-3，；
5’-GGG
TTCGAATTGCCTCGTTGTTTATTCCT-3’
(2) amplify the Hc-23 gene; With the Hc-23 gene is that template is carried out pcr amplification; The PCR product that reclaims is carried out double digestion with restricted enzyme; With capable connection of pMD18-T expression vector of handling, be transformed in the recipient bacterium then, obtain to contain the pMD18-T-Hc-23 recombinant expression plasmid of Hc-23 gene through same enzyme action;
(3) be that template is carried out pcr amplification with the positive colony pMD18-T-Hc-23 plasmid that checks order correct, reclaim the purpose fragment and carry out sub-clone that be about to the PCR product cloning and go into the pMD18-T carrier, last sub-clone is gone into pET-32a (+) expression vector;
(4) respectively positive recombiant plasmid and expression vector pET32a (+) are carried out double digestion with restriction endonuclease EcoR I and Hind III, obtain to contain the purpose fragment and the wire carrier of cohesive end;
(5) under 16 ℃ of conditions, use T
4Dna ligase connects back transformed competence colibacillus cell DH5a;
(6) will identify that correct pET32a-Hc-23 recombinant expression plasmid transforms expressive host bacterium BL21 (DE3) and carries out prokaryotic expression, express recombinant protein collection, purification will promptly be got;
(7) carry out the SDS-PAGE electrophoresis detection rHc-23 albumen that obtains recombinating;
(8) expressing protein purification procedures:
The expression bacterium liquid 500mL that the IPTG inducing culture of learning from else's experience is good uses the 50mL centrifuge tube, and the centrifugal 5min of 10000r/min abandons supernatant, stays deposition; In deposition, adding 20mmol/L pH and be 8.0 Tris-HCl 10mL and suspend, is that the 1mL lysozyme of 10mg/mL adds in the suspension with concentration then, and 37 ℃ of joltings are spent the night ,-20 ℃ of overnight incubation; At power is under the Ultrasonic Cell Disruptor of 200W; Press 30s/ time; Break process 10 times, the centrifugal 10min of 10000r/min, supernatant is deposited subsequent use in addition; Deposition washes twice with 2mol/L carbamide+1%TritonX-100+50mmol/L NaCl+0.2mmol/L EDTA, and reuse 2mol/L carbamide+1%TritonX-100 washes twice; Abandon supernatant behind the centrifugal 10min of 10000r/min, be precipitated as expressed albumen.
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| CN105463069B (en) * | 2015-04-02 | 2020-02-07 | 中国检验检疫科学研究院 | Target sequence, primer, identification method and kit for identifying haemaphysalis concinna |
| 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 |
| CN117045780B (en) * | 2023-10-13 | 2023-12-15 | 成都大熊猫繁育研究基地 | Application of brown yellow tick salivary gland protein in anti-tick vaccine |
Citations (2)
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| CN1438896A (en) * | 2000-04-25 | 2003-08-27 | 发展技术有限公司 | Vaccine comprising a tick cement protein |
| CN1657617A (en) * | 2004-02-18 | 2005-08-24 | 中国农业科学院上海家畜寄生虫病研究所 | Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1438896A (en) * | 2000-04-25 | 2003-08-27 | 发展技术有限公司 | Vaccine comprising a tick cement protein |
| CN1657617A (en) * | 2004-02-18 | 2005-08-24 | 中国农业科学院上海家畜寄生虫病研究所 | Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes |
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| Myung-Jo You.Immunization of mice with recombinant P27/30 protein confers protection against hard tick Haemaphysalis longicornis (Acari: Ixodidae) infestation.《Journal of Veterinary Science》.AGRIS,2005,第6卷(第1期),第47-51页. * |
| You M.等.AB051079 (Haemaphysalis longicornis mRNA for troponin I-like protein, complete cds).《GenBank》.NCBI,2001, * |
| You M.等.BAB55451.1 (troponin I-like protein [Haemaphysalis longicornis]).《GenBank》.2001, * |
| YouM.等.AB051079(HaemaphysalislongicornismRNAfortroponinI-likeprotein complete cds).《GenBank》.NCBI |
| 吴孔田等.趋化因子受体CCR5胞外段重组蛋白的克隆、表达、纯化及鉴定.《第四军医大学学报》.2006,第27卷(第5期),第412-415页. * |
| 杨彩明等.长角血蜱保护性抗原基因P27/30的克隆和原核表达.《畜牧兽医学报》.2008,第39卷(第10期),第1406-1410页. * |
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