CN1657617A

CN1657617A - Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes

Info

Publication number: CN1657617A
Application number: CN 200410016414
Authority: CN
Inventors: 周金林; 陈灵芝; 周勇志; 龚海燕; 向飞宇
Original assignee: SHANGHAI INST OF ANIMAL PARASI
Current assignee: SHANGHAI INST OF ANIMAL PARASI
Priority date: 2004-02-18
Filing date: 2004-02-18
Publication date: 2005-08-24
Anticipated expiration: 2024-02-18
Also published as: CN100375785C

Abstract

The sequences of falcate rhipicephalus RhCA and RhcB genes, the cloning of said RhoA and RhcB genes, their expression in colibacillus, and their anti-tick immunoprotection action to rabbit are disclosed. The present invention provides the basis for preparing anti-tick vaccine and medicine from cysteine proteinase moleculae.

Description

The clone of crescent fan head tick RhcA and RhcB gene, expression and anti-tick immanoprotection action

Technical field

The invention belongs to the biological gene technical field.Be specifically related to clone and the expression in intestinal bacteria and the anti-tick immanoprotection action of rabbit body of sequence, crescent fan head tick RhcA and the RhcB gene of crescent fan head tick RhcA and RhcB gene.

Background technology

Tick is the modal animal body surface parasite of a class, and they attack Mammals, birds, reptiles, even batrachians, are distributed widely in all over the world.At present, the tick of having found in the world surpasses 850 kinds, and the tick that has write down in China has 110 kinds.The harm of tick is not only as ecotozoa, the more important thing is the communication media as many humans and animals important diseases.Tick is the second largest class transmission of pathogen person who is only second to mosquito, according to rough Statistics, the virus of propagating and carrying through tick has 126 kinds (as forest encephalitis, hemorrhagic fevers), 20 kinds of Rickettsiaes (as Q heat, tick matchmaker spotted fever), 18 kinds of spirochetes (as Lyme disease), bacterium 14 kinds of (drawing bacterium, Brucella) and 26 kinds of protozoons (as babesia, Taylor worm) as soil.Human health through diseases such as tick-borne people's Lyme disease, Q heat and forest encephalitis serious harm; Tick is at the general parasitism of snake class, birds and wild mammal and often change the host, the disease of wildlife can be passed to the people, and therefore, tick plays very vital role in the propagation of disease of natural focus, brings serious threat for sanitarian safety.At present, the pathophorous control of tick and tick mainly relies on chemical insecticide, but because the drug-fast appearance of tick, environmental pollution and food safety etc. must press for new control strategy.Since Willadsen after entero-antigen Bm86 immune cattle succeeds in for the first time with boophilus microplus, immunoprophylaxis is considered to the most promising tick control method, therefore, screens potential candidate vaccine molecule and be tick and tick one of the focus of research that spreads disease.

Recently many studies show that, L-Cysteine HCL Anhydrous many parasitic play a part aspect pathogenic very important.As, degradable oxyphorase in plasmodium falciparum can cut host's immunoglobulin (Ig) etc. in liver fluke.L-Cysteine HCL Anhydrous also plays important effect in the vital movement of tick, preliminary study shows, may be relevant with degraded host's oxyphorase.Because the importance of parasitic L-Cysteine HCL Anhydrous in its vital movement, and they and mammiferous L-Cysteine HCL Anhydrous difference structurally, therefore, the L-Cysteine HCL Anhydrous molecule is as one of the target spot of pest-resistant medicinal design and the candidate antigens that develops vaccine.For example, can suppress the third phase of heart worm and casting off a skin of fourth phase larva with cystatin, with Schistosoma japonicum L-Cysteine HCL Anhydrous dna vaccination immune mouse, can obtain 24.2% worm reduction rate and 34.9% liver ovum decrement, but the application of L-Cysteine HCL Anhydrous in tick and the sick anti-system of tick biography also lacks research.Two new genes of L-Cysteine HCL Anhydrous of crescent fan head tick are cloned in the present invention, and it is studied as anti-tick vaccine candidate molecules, will carry out anti-tick and tick biography disease vaccine or develop novel drugs the basis is provided for exploring application L-Cysteine HCL Anhydrous molecule.

Summary of the invention

Technical problem to be solved by this invention is to study application L-Cysteine HCL Anhydrous molecule and carries out new control medicines such as anti-tick and tick biography disease vaccine.

The invention provides the sequence of crescent fan head tick RhcA and RhcB gene.

The present invention uses preference design primer according to the avtive spot conservative amino acid sequence and crescent fan head tick amino acid code of L-Cysteine HCL Anhydrous, pcr amplification, order-checking and analysis obtain the cysteine proteinase gene segment of two crescent fan head ticks, and the method by 5 ' RACE obtains full-length gene order again.RhcA total length 1168bp has polyA and a signal sequence AATAAA thereof at 3 ' end, and open reading frame is from 1053 bases of the 58th base to the, 332 aminoacid sequences of encoding, and predicted molecular weight is 36KDa.To amino acid whose signal peptide analysis, finding has 16 amino acid whose signal peptides, and cut point is between the 16th and the 17th amino acid; RhcB total length 1153bp, at 3 ' end polyA and signal sequence AATAAA thereof are arranged, open reading frame is from 1047 bases of the 43rd base to the, 335 amino acid of encoding, predicted molecular weight is about 37KDa, to amino acid whose signal peptide analysis, finding has 18 amino acid whose signal peptides, and cut point is between the 18th and the 19th amino acid.Homology analysis shows that these two sequences all contain cysteine protease activity site C ²⁵, H ¹⁵⁰And N ¹⁷⁵The conservative amino acid sequence at place, and very high homology is arranged with the cathepsin L-sample L-Cysteine HCL Anhydrous of other species, wherein, the homology of L-Cysteine HCL Anhydrous on amino acid levels of RhcA and other tick kinds or species is 58.5%-79.8%; The homology of L-Cysteine HCL Anhydrous on amino acid levels of RhcB and other tick kinds or species is 56.7%～89.8%.Although homology analysis shows that two genes are cathepsin L-sample cysteine proteinase gene, RhcA and RhcB homology of nucleotide sequence have only 63%.Therefore, with RhcA and two new cathepsin L-sample cysteine proteinase genes of RhcB called after crescent fan head tick.

The nucleotide sequence and the corresponding amino acid sequence of crescent fan head tick RhcA gene of the present invention are seen Fig. 1.

The nucleotide sequence and the corresponding amino acid sequence of crescent fan head tick RhcB gene of the present invention are seen Fig. 2.

Through the RT-PCR analysis revealed, RhcA and RhcB differ at the different developmental phases expression of crescent fan head tick.

Another object of the present invention has provided the clone and the expression of crescent fan head tick RhcA and RhcB gene.

In expression plasmid pET32a (+), construction recombination plasmid pET32a/RhcA and pET32a/RhcB are transformed into after BL21 (DE3) expresses bacterium, through the IPTG abduction delivering with RhcA and RhcB gene subclone.SDS-PAGE shows that recombinant plasmid pET32a/RhcA and pET32a/RhcB are all efficiently expressed.The recombination fusion protein size is about 55kD, considers that the Trx fusion rotein is about 20kD, and recombination fusion protein is big or small consistent with predictive analysis.Western Blot shows that two recombinant proteins all can be shown that recombinant protein has good antigenicity, for the anti-tick immunization experiment of rabbit body provides the basis by the anti-salivary antibody identification of tick.

Another purpose of the present invention has provided the anti-tick immanoprotection action of crescent fan head tick RhcA and RhcB gene recombinant protein.

Behind two recombinant protein purifications, immune programme for children immunization experiment rabbit routinely, promptly purifying protein adds isopyknic Freund's complete adjuvant by 300 a μ g/ dosage, and rabbit back intracutaneous multi-point injection carries out head and exempts from.Exempt to be undertaken two by 150 a μ g/ dosage after two weeks, purifying protein adds Freund's incomplete adjuvant intracutaneous multi-point injection.Two exempt from 10 days after, undertaken three by 150 a μ g/ dosage and exempt from, do not add any adjuvant, leg muscle injection.Three exempt to finish a week after, rabbit ear vein blood sampling detects antibody titer, and is carried out to the ixodes infestation experiment.The result shows, compares with control group, and the immune group rabbit attacks and sucks blood the crescent fan head tick and demonstrates significant protective effect.Contrast Trx immune group, the inoculation tick is after 48 hours, and adsorption rate is 68%, and RhcA and RhcB immune group only are 17% and 16%.The full blood rate of control group tick is 54%, and RhcA and RhcB immune group only are 42% and 22%.

To further describe content of the present invention by following embodiment.

Description of drawings

Fig. 1: the nucleotide sequence of crescent fan head tick RhcA gene and corresponding amino acid sequence ↑ locate to be signal peptide cut point, ^*Represent the amino acid of avtive spot, ^*The expression termination codon

Fig. 2: the nucleotide sequence of crescent fan head tick RhcB gene and corresponding amino acid sequence ↑ locate to be signal peptide cut point, ^*Represent the amino acid of avtive spot, ^*Expression termination codon Fig. 3: the homology of the halfcystine (AY208824-1) of the halfcystine homology comparison crescent fan head tick RhcA of crescent fan head tick RhcA and RhcB and other species cathepsin L-samples and the amino acid of RhcB and Rhipicephalus appendicularis cathepsin L-sample is respectively 79.8%and 56.7%, be respectively 89.2%and 66.6% with the homology of boophilus microplus (AF227957-1), be respectively 63.1%and 61.1% with the homology of darkling ground beetle (AY207373-1), be respectively 64.5%and 89.8% with the homology of Rhipicephalus appendicularis (AY208822-1), the homology of sarcophagid (D16533-1) is respectively 58.5%and 61.4%.The aminoacid sequence that box indicating avtive spot place is conservative

Fig. 4: crescent fan head tick RhcA (A) and RhcB (B) gene are at the RT-PCR of different developmental phases expression analysis

Swimming lane 1, ovum; Swimming lane 2, young tick; If swimming lane 3 is tick; Swimming lane 4 is not sucked blood into tick; Swimming lane 5, half-full blood becomes tick; Swimming lane 6, the recombinant plasmid positive control; Swimming lane 7, rabbit white corpuscle negative control

Recombinant expressed in intestinal bacteria of Fig. 5: crescent fan head tick RhcA (A) and RhcB (B) gene

Swimming lane M, expression standard molecular weight albumen, swimming lane 1 is not for inducing tropina, and swimming lane 2 is for inducing the back tropina, and swimming lane 3 is the recombinant protein of purifying, and swimming lane 4 is the empty carrier inducible protein

Fig. 6: Western blot analyzes the recombinant protein identification of anti-tick salivary antibody to RhcA and RhcB

Swimming lane 1, the RhcA recombinant protein, swimming lane 2, the Trx reference protein, swimming lane 3, the RhcB recombinant protein, swimming lane 4 is for dying standard molecular weight albumen in advance

Embodiment

Two cathepsin Ls of the embodiment 1 crescent fan head tick-new gene of sample L-Cysteine HCL Anhydrous

The clone of RhcA and RhcB and sequential analysis

1. materials and methods

1.1. tick

The crescent fan head tick picks up from Wuchang, Hubei, obtains the female tick tick of the list group of tick through the artificial breeding of rabbit body.

1.2. reagent

TRIZOL reagent, 3 '-RACE and 5 '-RACE test kit are available from Invitrogen company; The Taq archaeal dna polymerase is available from TaKaRa company; Granulated glass sphere DNA glue reclaims test kit available from Shanghai bio-engineering corporation; PGEM-T Easy carrier is available from Promega company; The primer is synthetic by match Parkson company.

1.3. extraction and the reverse transcription of the total RNA of crescent fan head tick

Be taken at the half-full blood that the rabbit body sucked blood 4 days and become tick, after the cleaning, in liquid nitrogen, fully grind, add TRIZOL reagent, fully homogenate in homogenizer.Step by TRIZOL reagent specification sheets is extracted total RNA that the half-full blood of crescent fan head tick becomes tick.Total RNA of about 5 μ g is used for reverse transcription and gets cDNA first chain.

1.4. the clone of target gene fragment and order-checking

Conservative amino acid sequence according to the cysteine protease activity site ^[6,7]The frequency of utilization of amino acid code of (GQCGSCWA and YWLVKMSW) and tick, designed one couple of PCR primers:

Upstream primer: 5 ' ACC AGG GCC AGT GCG GCT CCT GCT G 3 '

Downstream primer: 5 ' CCC AGC TGT TCT TGA CGA GC 3 '

Becoming the cDNA of tick with the half-full blood of crescent fan head tick is template, carries out pcr amplification, amplification condition with designed upstream and downstream primer: 94 ℃ of pre-sex change of 3min; 94 ℃ of 40s, 50 ℃ of 40s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 7min.

The PCR product is connected on the pGEM-T Easy carrier through 1.5% agarose gel electrophoresis, recovery purpose band, Transformed E .coli DH5 α cell, and after PCR identifies, objective gene sequencing.

1.5. rapid amplifying cDNA 5 ' end (5 ' RACE)

Sequencing result design 5 ' RACE the primer according to target gene fragment, RhcA:GSP1=5 '-AGTGTCCAGCGTCAATGGCTA-3 ', GSP2=5 '-GATGTCTACAAAGCCGGTGTCGG-TTG-3 ', nestedGSP=5 '-ACCAGGGCCAGTGCGGCTCCTGCTG-3 '; RhcB:GSP1=5 '-TAGACAC-CTTCCGAGTAGAAC-3 ', GSP2=5 '-CGTGGCTGGCGTCGATGGCAACTGAGAC-3 ', nestedGSP=5 '-ACCAGGGCCAGTGCGGCTCCTGCTG-3 '.5 ' end according to the method for 5 ' RACE amplification cDNA reclaims the purpose band, is connected on the pGEM-T Easy carrier, Transformed E .coli DH5 α cell, and after PCR identifies, order-checking.

1.6. Full Length cDNA Cloning and order-checking

According to the upstream primer of 5 ' the sequencing result design amplification full-length gene of holding,

(RhcA：GSP＝5’-GAGCTCTCCACCAGGTGCACC-3’，RhcB：GSP＝5’-AGGTCACTTGGGGTCTCCGGC-3’)。According to the method amplification RhcA of 3 ' RACE and the full-length cDNA of RhcB.Amplification condition: 94 ℃ of pre-sex change 4min; 94 ℃ of 40s, 53 ℃ of 40s, 72 ℃ of 2min, 35 circulations; 72 ℃ are extended 7min.The PCR product reclaims, and is connected into pGEM-T Easy carrier, Transformed E .coli DH5 α cell, and after PCR identifies, order-checking.

1.7. sequential analysis

Carry out homology analysis with Blastx; Simultaneously, adopt Gentyx-MAC software that open reading frame, the conservative amino acid sequence of this gene are analyzed.

1.8.RT-PCR analyze RhcA and RhcB expression in the different developmental phases of crescent fan head tick

In order to judge cysA, cysB is at the different developmental phases expression, from the crescent fan head tick of different etap, comprise ovum, young tick, if tick, do not suck blood into tick, half-full blood becomes to extract total RNA in the tick respectively.Synthesize the first chain cDNA with the total RNA reverse transcription of 1 μ g respectively, and get 2 μ l reverse transcription products as the pcr amplification template.

The RhcA gene-specific primer:

The upstream, 5 '-AAGCTTCTTGCCTCCACCATGCTGAG-3 '

The downstream, 5 '-CTCGAGGACGAGTGGGTAGCTGGCC-3 '

The RhcB gene-specific primer:

The upstream, 5 '-GAATTCATGCGGGGGTACATTGTTTTGTGCTGC-3 '

The downstream, 5 '-GCGGCCGCGACGAGAGGATAGCTGGCG-3 '

With the recombinant plasmid of RhcA and RhcB is that the PCR reaction of template is made as positive control, is that the PCR of template reacts and is made as negative control with the reverse transcription product of the total RNA of rabbit white corpuscle.Get 5 μ l PCR products, through 1% agarose gel electrophoresis observations.

2. result and analysis

2.1. the clone of target gene fragment and order-checking

The PCR product detects through agarose gel electrophoresis, and a bright band is arranged in about 500bp position.This product is connected to plasmid vector and transforms, 20 positive colony order-checkings of picked at random, the result obtains 2 visibly different gene fragments of sequence, analyze through NCBI website Blastx, the L-Cysteine HCL Anhydrous sequence of these two gene fragments and listed other species has very high homology, preliminary evaluation is two cysteine proteinase gene fragments, respectively called after RhcA and RhcB.

2.2. rapid amplifying cDNA 5 ' end (5 '-RACE)

5 '-RACE PCR does not for the first time all have special taking out of now, and Nested PCR obtains the gene fragment about 500bp, inserts pGEM-T Easy carrier, after PCR identifies, and the order-checking of picking positive colony, the result obtains 5 ' terminal sequence.

2.3. the clone of two full length gene cDNA and order-checking

According to 5 '-RACE result, the design gene-specific primer is by the method amplification full-length gene of 3 '-RACE.Single bright band appears in PCR product electrophoresis about 1.2kb, conform to substantially with the size of expection.The purpose band successfully is connected into pGEM-T Easy carrier, after PCR identifies, chooses the positive colony order-checking, and the result obtains full-length gene.RhcA (as Fig. 1) total length 1168bp holds the signal sequence AATAAA that polyA and polyA are arranged 3 ', and an open reading frame is arranged, from 1053 bases of the 58th base to the, and 332 aminoacid sequences of encoding, molecular weight is 36.33KDa; To amino acid whose signal peptide analysis, 16 amino acid whose signal peptides are arranged, cut point is between the 16th and the 17th amino acid; RhcB (as Fig. 2) total length 1153bp, the signal sequence AATAAA that polyA and polyA are arranged at 3 ' end, open reading frame is from 1047 bases of the 43rd base to the, 335 amino acid of encoding, molecular weight is 37.56KDa, to amino acid whose signal peptide analysis, 18 amino acid whose signal peptides are arranged, cut point is between the 18th and the 19th amino acid.Although homology analysis shows that two genes are organizes L-sample cysteine proteinase gene, homology of nucleotide sequence has only 63%.

2.4. homology analysis with other L-Cysteine HCL Anhydrous

The L-Cysteine HCL Anhydrous of the same Rhipicephalus appendicularis of having reported of the aminoacid sequence of RhcA and RhcB, boophilus microplus, darkling ground beetle (Tenebrio molitor), sarcophagid (Sarcophaga peregrina) is carried out homology relatively, and these two sequences all contain cysteine protease activity site C as a result ²⁵, H ¹⁵⁰And N ¹⁷⁵The conservative amino acid sequence (as Fig. 3) at place, and the cathepsin L-sample L-Cysteine HCL Anhydrous of they and other tick kind or species has very high homology, wherein, the homology of L-Cysteine HCL Anhydrous on amino acid levels of RhcA and other tick kinds or species is 58.5%-79.8%; The homology of L-Cysteine HCL Anhydrous on amino acid levels of RhcB and other tick kinds or species is 56.7%-89.8%.

2.5.RhcA and RhcB is at the expression analysis of the different development stage of crescent fan head tick

Extract RNA from the different developmental phases of tick, reverse transcription becomes cDNA, and getting equivalent cDNA is template, carries out RT-PCR respectively with the Auele Specific Primer of two genes.The result as shown in Figure 4, all do not amplify the specific gene fragment among the rabbit white corpuscle cDNA, got rid of RhcA and RhcB and be subjected to rabbit blood contamination of heavy, RhcA is at the young tick of crescent fan head tick, if tick, do not suck blood in tick and the female tick of half-full blood and all have than high expression level, but the expression amount in ovum seldom.RhcB is at the ovum of crescent fan head tick, young tick, if all have than high expression level in the tick, the female tick of half-full blood, but expression amount is extremely not low in sucking blood into tick, this result reflects RhcA and the RhcB differential expression at the different development stage of tick, infers that the biological function different in vivo with two genes is relevant.

Embodiment 2 crescent fan head tick RhcA and RhcB gene in vitro are expressed and the experiment of anti-tick immunoprotection

1. material and method

1.1. reagent

The Taq archaeal dna polymerase is available from BioStar company; T4 dna ligase, restriction enzyme are available from TaKaRa company; PGEM-T easy carrier is available from Promega company; Plasmid DNA is extracted test kit available from match Parkson company; The PCR primer is synthetic by match Parkson company; BugBuster His Bind Purification Kit is a Novagen company product; Low molecular weight protein (LMWP) Marker is supervised by Shanghai Inst. of Biochemistry, Chinese Academy of Sciences, and BCA analysis of protein test kit is a PIERCE company product.

1.2. bacterial strain and plasmid

Coli strain DH5 α, BL21 (DE3) and plasmid pET-32a are this institute laboratory to be provided.

1.3. laboratory animal

New zealand white rabbit is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.

1.4.RhcA and the structure and the vivoexpression of RhcB recombinant expression plasmid

According to RhcA and RhcB dna encoding the protein sequence, design respectively a pair of Auele Specific Primer that contains restriction enzyme site (RhcA:5 '-CACGGATATCTCTCATGAAATCCTACGCACCC-3 ' and 5 '-CACGAAGCTTGACGAGTGGGTAGCTGG-3 ' contains EcoR V and HindIII restriction enzyme site respectively; RhcB, 5 '-GTAGAATTCGAGCTCGTTGGTGCTGAGTGG-3 ' and 5 '-GTAGCGGCCGCGACGAGAGGATAGCTGGCG-3 ' contains EcoR I and Not I restriction enzyme site respectively), recombinant plasmid pGEM-T/RhcA and the pGEM-T/RhcB that makes up with this laboratory is template respectively, the pcr amplification coded protein sequence, subclone is to the pGEM-T carrier, after the recombinant plasmid enzyme is cut digestion, be connected to expression vector pET-32 (a+).After the order-checking conclusive evidence, recombinant plasmid transformed is to e. coli bl21 (DE3).The conversion positive bacteria is inoculated in 3ml contains in the LB liquid nutrient medium of 100mg/L penbritin, 37 ℃, 220rpm, overnight incubation.The bacterium liquid of getting incubated overnight was inoculated in the fresh LB liquid nutrient medium by 1: 100, and 37 ℃ are cultured to bacterium liquid OD ₆₀₀When the 0.6-0.8 left and right sides, in substratum, add IPTG, making final concentration is 1mmol/L, continues to cultivate 6h, centrifugal collection thalline is analyzed through 15%SDS-PAGE.Recombinant expression protein carries out purifying with BugBuster His Bind PurificationKit, and the BCA method is measured protein concentration.

1.5.Western blot analyzes

Two recombinant proteins and reference protein Trx are behind the SDA-PAGE electrophoresis, be transferred on the nitrocellulose filter, the sealing of 5% skim-milk is spent the night, with the anti-tick saliva of rabbit antiserum(antisera) (1: 1000 dilution) is one anti-, the goat anti-rabbit igg of horseradish peroxidase-labeled (1: 1500 dilution) is two anti-ly to hybridize DAB color developing detection signal.

1.6. the anti-tick immunoprotection test of recombinant protein

6 new zealand white rabbits are used for immunization experiment, and wherein 2 with reference protein Trx immunity, and 2 with the immunity of RhcA recombinant protein, and 2 are used for the immunity of RhcB recombinant protein.Behind Trx albumen and two recombinant protein purifications, immune programme for children immunization experiment rabbit routinely, promptly antigen adds isopyknic Freund's complete adjuvant by 300 a μ g/ dosage, and rabbit back intracutaneous multi-point injection carries out head and exempts from.Exempt to be undertaken two by 150 a μ g/ dosage after two weeks, purifying protein adds Freund's incomplete adjuvant intracutaneous multi-point injection.Two exempt from 10 days after, undertaken three by 150 a μ g/ dosage and exempt from, do not add any adjuvant, leg muscle injection.Three exempt to finish a week after, rabbit ear vein blood sampling detects antibody titer, and is carried out to the ixodes infestation experiment.In the ixodes infestation test, every left and right ear of rabbit is inoculated 50 respectively and is become tick (male and female half and half), and after 48 hours, the inspection tick is stung the suction situation, calculates adsorption rate.Day by day the full blood tick number of statistics calculates full blood rate.

2. result

2.1.RhcA and the structure of RhcB dna recombinant expression plasmid

With recombinant plasmid pGEM-T/RhcA and pGEM-T/RhcB is template, and the pcr amplification coded protein sequence is for being about 1000bp, and is consistent with the expection size.Recovery, enzyme by dna fragmentation cut, purifying, with being connected of carrier pET32a, construct recombinant expression plasmid pET-32a/RhcA and pET32-a/RhcB, PCR and enzyme are cut and are identified and find the big or small consistent of the gene segment that inserts and expection, sequencing has been proved conclusively the external source fragment and has correctly been inserted, and shows the construction of recombinant expression plasmid success.

2.2.cysA and cysB expression of gene and purifying

Recombinant plasmid pET-32a/RhcA and pET32-a/RhcB and empty plasmid pET32a induce all through IPTG to efficiently express in E.coli BL21.SDS-PAGE shows that recombinant plasmid pET32a/RhcA and pET32a/RhcB are all efficiently expressed (Fig. 5).The recombination fusion protein size is about 55kD, considers that the Trx fusion rotein is about 20kD, and recombination fusion protein is big or small consistent with predictive analysis.These two kinds of fusion roteins all exist with insoluble inclusion body form, and after expression product was used the urea-denatured dissolving of 6M, through the Ni column purification of BugBuster HisBind Purification Kit, purifying protein was after the dialysis renaturation again, and the BCA method records protein concentration.

2.3 Western blot result

Western Blot shows that reference protein Trx does not have detection signal, and two recombinant proteins all can be shown that recombinant protein has good antigenicity by the anti-salivary antibody identification of tick (Fig. 6), for the anti-tick immunization experiment of rabbit body provides the basis.

2.4. immunity test result

Three rabbit anteserums after exempting from, agar diffusion test detects and tires is 1: 16.Anti-tick immunoprotection result shows, compares with control group, and the immune group rabbit attacks and sucks blood the crescent fan head tick and demonstrates significant protective effect.Contrast Trx immune group, the inoculation tick is after 48 hours, and adsorption rate is 68%, and RhcA and RhcB immune group only are 17% and 16%.The full blood rate of control group tick is 54%, and RhcA and RhcB immune group only are 42% and 22%.

Sequence table

＜110〉Shanghai Inst. of Livestock Parastitic Disease, Chinese Academy of Agricultural

Ministry of Agriculture animal parasitology emphasis open laboratory

＜120〉clone of crescent fan head tick RhcA and RhcB gene, expression and anti-tick immanoprotection action

＜130〉specification sheets, claims

<160>4

<170>PatentIn?version?3.2

<210>1

<211>1168

<212>DNA

＜2 13〉RhcA nucleotide sequence

<400>1

gcagggtgca?ttgaaagagt?cgagctctcc?accaggtgca?ccgtccttgc?ctccaccatg 60

ctgagattaa?gcctactttg?cgccattgtg?gcggtaaccg?ttgccgcaaa?ctctcatgaa 120

atcctacgca?cccaatggga?ggcattcaaa?actacacaca?agaaatccta?cgaatcgcac 180

atggaggagc?tcctgaggtt?caaaattttc?actgaaaaca?gcctgatcat?tgccaagcac 240

aacgcgaagt?atgccaaggg?tctcgtttct?tacaagctcg?gaatgaacca?gttcggagat 300

ctgctggcgc?acgaattcgc?caagatcttc?aacggttacc?gtggtcagcg?cacatcccgc 360

ggatccacat?tcatgccacc?agcaaacgtc?aacgacagca?gcctgccaag?cactgtcgac 420

tggcgcaaga?aaggagctgt?cacacctgtc?aaggaccagg?gccagtgcgg?ctcctgctgg 480

gcgttcagtg?ccactggatc?tctggaggga?cagcattttc?tgaaggacgg?cgagctggtt 540

tcactcagtg?agcaaaactt?ggtcgactgc?tctcagtcct?ttggcaacaa?tggttgcgag 600

ggcggcctca?tggacaacgc?tttcaagtac?attaaggcta?acgatggtat?cgacgccgag 660

gagagctatc?catatgaggc?tatggatgac?aagtgtcgct?tcaagaagga?agacgttggt 720

gcaaccgaca?ccggctttgt?agacatcgag?gggggatctg?aggatgactt?gaaaaaagcc 780

gtcgctaccg?ttggcccaat?ttccgtagcc?attgacgctg?gacactcgtc?attccagctg 840

tattccgaag?gagtgtacga?tgagcccgag?tgtagcagcg?aagaactgga?ccacggtgtg 900

cttgctgtcg?gctatggtgt?taaggacgga?aagaagtatt?ggctcgtcaa?gaacagctgg 960

ggtgggtcct?ggggagacaa?cggctacatc?ctgatgtccc?gtgacaagaa?caaccagtgc 1020

ggcattgcct?cagcggccag?ctacccactc?gtctaaacaa?gtgatttcac?gtagtgacgt 1080

tcgcctgaat?gtttcctaaa?atgtacataa?cttattaacc?taaataaatt?gcccatattt 1140

cgccactgtg?aaaaaaaaaa?aaaaaaaa 1168

<210>2

<211>332

<212>PRT

＜213〉RhcA aminoacid sequence

<400>2

Met?Leu?Arg?Leu?Ser?Leu?Leu?Cys?Ala?Ile?Val?Ala?Val?Thr?Val?Ala

1 5 10 15

Ala?Asn?Ser?His?Glu?Ile?Leu?Arg?Thr?Gln?Trp?Glu?Ala?Phe?Lys?Thr

20 25 30

Thr?His?Lys?Lys?Ser?Tyr?Glu?Ser?His?Met?Glu?Glu?Leu?Leu?Arg?Phe

35 40 45

Lys?Ile?Phe?Thr?Glu?Asn?Ser?Leu?Ile?Ile?Ala?Lys?His?Asn?Ala?Lys

50 55 60

Tyr?Ala?Lys?Gly?Leu?Val?Ser?Tyr?Lys?Leu?Gly?Met?Asn?Gln?Phe?Gly

65 70 75 80

Asp?Leu?Leu?Ala?His?Glu?Phe?Ala?Lys?Ile?Phe?Asn?Gly?Tyr?Arg?Gly

85 90 95

Gln?Arg?Thr?Ser?Arg?Gly?Ser?Thr?Phe?Met?Pro?Pro?Ala?Asn?Val?Asn

100 105 110

Asp?Ser?Ser?Leu?Pro?Ser?Thr?Val?Asp?Trp?Arg?Lys?Lys?Gly?Ala?Val

115 120 125

Thr?Pro?Val?Lys?Asp?Gln?Gly?Gln?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser

130 135 140

Ala?Thr?Gly?Ser?Leu?Glu?Gly?Gln?His?Phe?Leu?Lys?Asp?Gly?Glu?Leu

145 150 155 160

Val?Ser?Leu?Ser?Glu?Gln?Asn?Leu?Val?Asp?Cys?Ser?Gln?Ser?Phe?Gly

165 170 175

Asn?Asn?Gly?Cys?Glu?Gly?Gly?Leu?Met?Asp?Asn?Ala?Phe?Lys?Tyr?Ile

180 185 190

Lys?Ala?Asn?Asp?Gly?Ile?Asp?Ala?Glu?Glu?Ser?Tyr?Pro?Tyr?Glu?Ala

195 200 205

Met?Asp?Asp?Lys?Cys?Arg?Phe?Lys?Lys?Glu?Asp?Val?Gly?Ala?Thr?Asp

210 215 220

Thr?Gly?Phe?Val?Asp?Ile?Glu?Gly?Gly?Ser?Glu?Asp?Asp?Leu?Lys?Lys

225 230 235 240

Ala?Val?Ala?Thr?Val?Gly?Pro?Ile?Ser?Val?Ala?Ile?Asp?Ala?Gly?His

245 250 255

Ser?Ser?Phe?Gln?Leu?Tyr?Ser?Glu?Gly?Val?Tyr?Asp?Glu?Pro?Glu?Cys

260 265 270

Ser?Ser?Glu?Glu?Leu?Asp?His?Gly?Val?Leu?Ala?Val?Gly?Tyr?Gly?Val

275 280 285

Lys?Asp?Gly?Lys?Lys?Tyr?Trp?Leu?Val?Lys?Asn?Ser?Trp?Gly?Gly?Ser

290 295 300

Trp?Gly?Asp?Asn?Gly?Tyr?Ile?Leu?Met?Ser?Arg?Asp?Lys?Asn?Asn?Gln

305 310 315 320

Cys?Gly?Ile?Ala?Ser?Ala?Ala?Ser?Tyr?Pro?Leu?Val

325 330

<210>3

<211>1153

<212>DNA

＜213〉RhcB nucleotide sequence

<400>3

gaggtcactt?ggggtgtccg?gctccacacg?cttcctaaaa?aaatgcgggg?gtacattgtt 60

ttgtgctgct?tgttcgtgac?tgctgctgca?attacgcatc?aagagctcgt?tggtgctgag 120

tggtcagcgt?tcaaggcatt?acacggaaag?gactatgcgt?ctgacacaga?agaatactac 180

aggctgaaga?tctacatgga?aaacaggttg?aagattgcgc?ggcacaatga?gaagtacgcc 240

aaaagccagg?tctcgtacaa?gcttgcgatg?aatgaatttg?gcgacttgct?acaccacgag 300

ttcgtcagca?cacgcaacgg?cttcaagcgc?aactacaggg?actctccacg?agagggcagc 360

ttcttcgtcg?agcctgaggg?ctttgaggat?ttgcagctgc?ccaagactgt?cgactggagg 420

aagaaaggcg?ctgtgacccc?tgtcaagaac?cagggacagt?gcggctcctg?ctgggcattc 480

agcactacgg?gatccctgga?gggcccgcac?ttccgcaaga?cacgcaagct?tgtgtccctc 540

agtgagcaga?acctggtcga?ctgctccaga?agcttcggca?acaacggctg?cgagggtggt 600

ctcatggaca?atgctttcaa?gtacatcaag?tccaacaagg?gcattgacac?agagtggagc 660

tacccttaca?atgccacgga?cggtgtctgc?cacttcaaca?ggagtgatgt?gggtgccacc 720

gacactggct?tcgtcgacat?accagaaggg?gacgagaaca?agctgaagaa?ggctgtggcc 780

gctgttggac?ccgtctcagt?tgccatcgac?gccagccacg?agtcattcca?gttctactcg 840

gaaggtgtct?atgatgaacc?agagtgcagc?agtgagcagc?ttgaccacgg?tgtccttgtt 900

gttggctatg?gaaccaagga?tggccaggac?tattggctcg?tcaagaacag?ctggggcaca 960

acctggggag?acgagggcta?catctacatg?acaaggaaca?aggacaacca?gtgcggaatc 1020

gccagctccg?ccagctatcc?tctcgtctag?ttacgcgcgg?ttttttacat?gctcgtcttt 1080

gtttcatatt?gcatccccgc?tcactcggca?caaataaact?ttttttttgt?acagtgaaaa 1140

aaaaaaaaaa?aaa 1153

<210>4

<211>335

<212>PRT

＜213〉RhcB aminoacid sequence

<400>4

Met?Arg?Gly?Tyr?Ile?Val?Leu?Cys?Cys?Leu?Phe?Val?Thr?Ala?Ala?Ala

1 5 10 15

Ile?Thr?His?Gln?Glu?Leu?Val?Gly?Ala?Glu?Trp?Ser?Ala?Phe?Lys?Ala

20 25 30

Leu?His?Gly?Lys?Asp?Tyr?Ala?Ser?Asp?Thr?Glu?Glu?Tyr?Tyr?Arg?Leu

35 40 45

Lys?Ile?Tyr?Met?Glu?Asn?Arg?Leu?Lys?Ile?Ala?Arg?His?Asn?Glu?Lys

50 55 60

Tyr?Ala?Lys?Ser?Gln?Val?Ser?Tyr?Lys?Leu?Ala?Met?Asn?Glu?Phe?Gly

65 70 75 80

Asp?Leu?Leu?His?His?Glu?Phe?Val?Ser?Thr?Arg?Asn?Gly?Phe?Lys?Arg

85 90 95

Asn?Tyr?Arg?Asp?Ser?Pro?Arg?Glu?Gly?Ser?Phe?Phe?Val?Glu?Pro?Glu

100 105 110

Gly?Phe?Glu?Asp?Leu?Gln?Leu?Pro?Lys?Thr?Val?Asp?Trp?Arg?Lys?Lys

115 120 125

Gly?Ala?Val?Thr?Pro?Val?Lys?Asn?Gln?Gly?Gln?Cys?Gly?Ser?Cys?Trp

130 135 140

Ala?Phe?Ser?Thr?Thr?Gly?Ser?Leu?Glu?Gly?Pro?His?Phe?Arg?Lys?Thr

145 150 155 160

Arg?Lys?Leu?Val?Ser?Leu?Ser?Glu?Gln?Asn?Leu?Val?Asp?Cys?Ser?Arg

165 170 175

Ser?Phe?Gly?Asn?Asn?Gly?Cys?Glu?Gly?Gly?Leu?Met?Asp?Asn?Ala?Phe

180 185 190

Lys?Tyr?Ile?Lys?Ser?Asn?Lys?Gly?Ile?Asp?Thr?Glu?Trp?Ser?Tyr?Pro

195 200 205

Tyr?Asn?Ala?Thr?Asp?Gly?Val?Cys?His?Phe?Asn?Arg?Ser?Asp?Val?Gly

210 215 220

Ala?Thr?Asp?Thr?Gly?Phe?Val?Asp?Ile?Pro?Glu?Gly?Asp?Glu?Asn?Lys

225 230 235 240

Leu?Lys?Lys?Ala?Val?Ala?Ala?Val?Gly?Pro?Val?Ser?Val?Ala?Ile?Asp

245 250 255

Ala?Ser?His?Glu?Ser?Phe?Gln?Phe?Tyr?Ser?Glu?Gly?Val?Tyr?Asp?Glu

260 265 270

Pro?Glu?Cys?Ser?Ser?Glu?Gln?Leu?Asp?His?Gly?Val?Leu?Val?Val?Gly

275 280 285

Tyr?Gly?Thr?Lys?Asp?Gly?Gln?Asp?Tyr?Trp?Leu?Val?Lys?Asn?Ser?Trp

290 295 300

Gly?Thr?Thr?Trp?Gly?Asp?Glu?Gly?Tyr?Ile?Tyr?Met?Thr?Arg?Asn?Lys

305 310 315 320

Asp?Asn?Gln?Cys?Gly?Ile?Ala?Ser?Ser?Ala?Ser?Tyr?Pro?Leu?Val

325 330 335

Claims

1. a crescent fan head tick RhcA gene order is characterized in that total length 1168bp, and the signal sequence AATAAA of polyA and polyA is arranged at 3 ' end, an open reading frame is arranged, from 1053 bases of the 58th base to the, 332 aminoacid sequences of encoding, molecular weight is 36.33Kda; To amino acid whose signal peptide analysis, 16 amino acid whose signal peptides are arranged, cut point is between the 16th and the 17th amino acid;

1?gcagggtgcattgaaagagtcgagctctccaccaggtgcaccgtccttgcctccaccatgctgagattaagcctactttgcgccattgtggcggtaaccg 101

M L R L S L L C A I V A V T V

101?ttgccgcaaactctcatgaaatcctacgcacccaatgggaggcattcaaaactacacacaagaaatcctacgaatcgcacatggaggagctcctgaggtt 201

A A N S H E I L R T Q W E A F K T T H K K S Y E S H M E E L L R F

↑

201?caaaattttcactgaaaacagcctgatcattgccaagcacaacgcgaagtatgccaagggtctcgtttcttacaagctcggaatgaaccagttcggagat 301

K I F T E N S L I I A K H N A K Y A K G L V S Y K L G M N Q F G D

301?ctgctggcgcacgaattcgccaagatcttcaacggttaccgtggtcagcgcacatcccgcggatccacattcatgccaccagcaaacgtcaacgacagca 401

L L A H E F A K I F N G Y R G Q R T S R G S T F M P P A N V N D S S

401?gcctgccaagcactgtcgactggcgcaagaaaggagctgtcacacctgtcaaggaccagggccagtgcggctcctgctgggcgttcagtgccactggatc 501

L P S T V D W R K K G A V T P V K D Q G Q C G S C W A F S A T G S

**

501?tctggagggacagcattttctgaaggacggcgagctggtttcactcagtgagcaaaacttggtcgactgctctcagtcctttggcaacaatggttgcgag 601

L E G Q H F L K D G E L V S L S E Q N L V D C S Q S F G N N G C E

601?ggcggcctcatggacaacgctttcaagtacattaaggctaacgatggtatcgacgccgaggagagctatccatatgaggctatggatgacaagtgtctct 701

G G L M D N A F K Y I K A N D G I D A E E S Y P Y E A M D D K C R F

701?tcaagaaggaagacgttggtgcaaccgacaccggctttgtagacatcgaggggggatctgaggatgacttgaaaaaagccgtcgctaccgttggcccaat 801

K K E D V G A T D T G F V D I E G G S E D D L K K A V A T V G P I

801?ttccgtagccattgacgctggacactcgtcattccagctgtattccgaaggagtgtacgatgagcccgagtgtagcagcgaagaactggaccacggtgtg 901

S V A I D A G H S S F Q L Y S E G V Y D E P E C S S E E L D H G V

**

901?cttgctgtcggctatggtgttaaggacggaaagaagtattggctcgtcaagaacagctggggtgggtcctggggagacaacggctacatcctgatgtccc 1001

L A V G Y G V K D G K K Y W L V K N S W G G S W G D N G Y I L M S R

**

1001?gtgacaagaacaaccagtgcggcattgcctcagcggccagctacccactcgtctaaacaagtgatttcacgtagtgacgttcgcctgaatgtttcctaaa 1101

D K N N Q C G I A S A A S Y P L V *

1101?atgtacataacttattaaccta aataaattgcccatatttcgccactgtgaaaaaaaaaaaaaaaaaa 1168

2. crescent fan head tick RhcB gene order, it is characterized in that total length 1153bp, the signal sequence AATAAA that polyA and polyA are arranged at 3 ' end, open reading frame is from 1047 bases of the 43rd base to the, 335 amino acid of encoding, molecular weight is about 37.56KDa, to amino acid whose signal peptide analysis, 18 amino acid whose signal peptides are arranged, and cut point is between the 18th and the 19th amino acid;

1?gaggtcacttggggtgtccggctccacacgcttcctaaaaaaatgcgggggtacattgttttgtgctgcttgttcgtgactgctgctgcaattacgcatc 101

M R G Y I V L C C L F V T A A A I T H Q

↑

101?aagagctcgttggtgctgagtggtcagcgttcaaggcattacacggaaaggactatgcgtctgacacagaagaatactacaggctgaagatctacatgga 201

E L V G A E W S A F K A L H G K D Y A S D T E E Y Y R L K I Y M E

201?aaacaggttgaagattgcgcggcacaatgagaagtacgccaaaagccaggtctcgtacaagcttgcgatgaatgaatttggcgacttgctacaccacgag 301

N R L K I A R H N E K Y A K S Q V S Y K L A M N E F G D L L H H E

301?ttcgtcagcacacgcaacggcttcaagcgcaactacagggactctccacgagagggcagcttcttcgtcgagcctgagggctttgaggatttgcagctgc 401

F V S T R N G F K R N Y R D S P R E G S F F V E P E G F E D L Q L P

401?ccaagactgtcgactggaggaagaaaggcgctgtgacccctgtcaagaaccagggacagtgcggctcctgctgggcattcagcactacgggatccctgga 501

K T V D W R K K G A V T P V K N Q G Q C G S C W A F S T T G S L E

**

501?gggcccgcacttccgcaagacacgcaagcttgtgtccctcagtgagcagaacctggtcgactgctccagaagcttcggcaacaacggctgcgagggtggt 601

G P H F R K T R K L V S L S E Q N L V D C S R S F G N N G C E G G

601?ctcatggacaatgctttcaagtacatcaagtccaacaagggcattgacacagagtggagctacccttacaatgccacggacggtgtctgccacttcaaca 701

L M D N A F K Y I K S N K G I D T E W S Y P Y N A T D G V C M F N R

701?ggagtgatgtgggtgccaccgacactggcttcgtcgacataccagaaggggacgagaacaagctgaagaaggctgtggccgctgttggacccgtctcagt 801

S D V G A T D T G F V D I P E G D E N K L K K A V A A V G P V S V

801?tgccatcgacgccagccacgagtcattccagttctactcggaaggtgtctatgatgaaccagagtgcagcagtgagcagcttgaccacggtgtccttgtt 901

A I B A S N E S F Q F Y S E G V Y D E P E C S S E Q L D H G V L V

**

901?gttggctatggaaccaaggatggccaggactattggctcgtcaagaacagctggggcacaacctggggagacgagggctacatctacatgacaaggaaca 1001

V G Y G T K D G Q D Y W L V K N S W G T T W G D E G Y I Y M T R N K

**

1001?aggacaaccagtgcggaatcgccagctccgccagctatcctctcgtctagttacgcgcggttttttacatgctcgtctttgtttcatattgcatccccgc 1101

N N Q C G I A S S A S Y P L V *

1101?tcactcggcaca aataaactttttttttgtacagtgaaaaaaaaaaaaaaaaa 1153

3. the cloning process of crescent fan head tick RhcA and RhcB gene, it is characterized in that using preference design primer according to the avtive spot conservative amino acid sequence and crescent fan head tick amino acid code of L-Cysteine HCL Anhydrous, pcr amplification, order-checking and analysis obtain the cysteine proteinase gene segment of two crescent fan head ticks, and the method by 5 ' RACE obtains full-length gene order again.

4. crescent fan head tick RhcA and the expression method of RhcB gene in intestinal bacteria is characterized in that employed host bacterium is BL21 (DE3), plasmid pET-32a (+).

5. crescent fan head tick RhcA and RhcB gene have application in the immunological reagent of anti-tick immanoprotection action in preparation.