CN103965350A - Tick cystatin Rhcyst-2, and gene and applications thereof - Google Patents

Abstract

The invention discloses a tick cystatin. The tick cystatin is a Rhipicephalus haemaphysaloide cystatin Rhcyst-2. The invention also discloses a Rhipicephalus haemaphysaloide cystatin Rhcyst-2 gene. The Rhipicephalus haemaphysaloide cystatin Rhcyst-1 gene includes a nucleotide sequence coding an amino acid sequence represented by SEQ ID NO.1. The Rhipicephalus haemaphysaloide cystatin Rhcyst-2 has substantial cysteine protease inhibition activity and toxoplasma infection resistance, and is suitable for preparing anti-parasitical medicines.

Description

Tick L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 and gene and application

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of tick L-Cysteine HCL Anhydrous Inhibitory molecules and gene and application.

Background technology

L-Cysteine HCL Anhydrous has critical function in parasite physiological metabolism, is considered to a promising anti-parasite medicine target.Chemical classes cystatin has and is applied to treat trypanosome and plasmodial report.In organism, also have the Inhibitory molecules similar to chemical classes inhibitor function, they and L-Cysteine HCL Anhydrous produce reversible combining closely and efficient inhibitory enzyme activity, are called as L-Cysteine HCL Anhydrous Inhibitory molecules (cystatin).Research discovery, the L-Cysteine HCL Anhydrous Inhibitory molecules in tick body is very abundant, because it generally has good inhibition active to L-Cysteine HCL Anhydrous, may have the value that is developed as anti-parasite medicine.In addition, research discovery, people source L-Cysteine HCL Anhydrous Inhibitory molecules cystatin C and cystatinM show the inhibition invasion and attack of tumour and the effect of transfer, show that this class biomolecules also has potential cancer therapy drug potentiality to be exploited.

Tick, is commonly called as tick worm, is a kind of common animal body surface parasite, also often bites people, and spreads disease.Molecule Rhipilin-1 (Rhipicephalus haemaphysaloide) is distributed in South East Asia and south China, is China's advantage tick kind, at wildlife body surface parasitisms such as ox, sheep, dog, pig and hares, sucks blood, and also attacks people.Up to the present, the research report that still there is no L-Cysteine HCL Anhydrous Inhibitory molecules in molecule Rhipilin-1 body both at home and abroad.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of tick L-Cysteine HCL Anhydrous Inhibitory molecules and gene thereof, and this L-Cysteine HCL Anhydrous Inhibitory molecules is molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2, can be used for preparing antiparasitic medicine.

In addition, also need to provide the application of a kind of tick L-Cysteine HCL Anhydrous Inhibitory molecules in preparing anti-parasite medicine.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, provide a kind of tick L-Cysteine HCL Anhydrous Inhibitory molecules, described L-Cysteine HCL Anhydrous Inhibitory molecules is molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2, and its aminoacid sequence is selected from:

(1) aminoacid sequence shown in SEQ ID NO.1;

(2) shown in SEQ ID NO.1, in aminoacid sequence, lack, add, insert or replace that one or more amino acid is resulting has L-Cysteine HCL Anhydrous and suppress active, and there is the aminoacid sequence of 95% above homology with SEQ ID NO.1.

Preferably, the aminoacid sequence of described molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 is as shown in SEQ ID NO.1.

In another aspect of this invention, a kind of tick L-Cysteine HCL Anhydrous Inhibitory molecules gene is provided, described gene is molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 gene, comprises: the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1.

Preferably, the nucleotide sequence of described RHcyst-2 gene is as shown in SEQ ID NO.2.

In another aspect of this invention, a kind of recombinant vectors that comprises above-mentioned molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 gene order or its partial sequence is also provided, and described partial sequence is that the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1 is removed the nucleotide sequence after signal coding sequence.

Described recombinant vectors comprises recombinant cloning vector or recombinant expression vector.

In another aspect of this invention, also provide a kind of host cell that comprises above-mentioned recombinant vectors.

In another aspect of this invention, also provide a kind of preparation method of tick L-Cysteine HCL Anhydrous Inhibitory molecules recombinant protein, comprised the following steps:

Build the recombinant expression vector containing above-mentioned molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 Gene Partial sequence, described partial sequence is that the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1 is removed the nucleotide sequence after signal coding sequence;

Described recombinant expression vector is transformed into e. coli host cell;

The e. coli host cell that cultivation comprises recombinant expression vector, and under conditions suitable, induce this recombinant expression vector to express molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 recombinant protein.

In another aspect of this invention, also provide the application of a kind of above-mentioned tick L-Cysteine HCL Anhydrous Inhibitory molecules in preparing anti-parasite medicine.

Described parasite comprises toxoplasma gondii, trypanosome or plasmodium.Preferably, described anti-parasite medicine is resisting toxoplasmosis medicine.

In another aspect of this invention, also provide the application of a kind of above-mentioned tick L-Cysteine HCL Anhydrous Inhibitory molecules in preparing cystatin.

Molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules of the present invention, inhibition test by enzyme shows to have significant L-Cysteine HCL Anhydrous inhibition activity, through Infection Toxoplasma gondii evidence, there is certain Infection Toxoplasma gondii ability, be suitable for preparing anti-parasite medicine and cystatin.

Accompanying drawing explanation

Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.

Fig. 1 is 3 ' the RACE amplified production electrophoresis result figure of the RHcyst-2 of the embodiment of the present invention 1;

Fig. 2 takes turns (B) PCR product electrophoresis result figure 5 ' the RACE first round (A) and second of the RHcyst-2 of the embodiment of the present invention 1;

Fig. 3 is the RHcyst-2 full-length gene pcr amplification electrophoresis result figure of the embodiment of the present invention 1;

Fig. 4 is the aminoacid sequence comparison chart of RHcyst-2 and three known Ying Pi 2cystatin of family of the embodiment of the present invention 1;

Fig. 5 is the double digestion evaluation figure of the RHcyst-2/pGEX-4T-1 recombinant plasmid of the embodiment of the present invention 2;

Fig. 6 is the SDS-PAGE electrophorogram of the RHcyst-2 recombinant protein abduction delivering of the embodiment of the present invention 2;

Fig. 7 is the SDS-PAGE electrophorogram after the RHcyst-2 recombinant protein purification of the embodiment of the present invention 2;

Fig. 8 is the BCA canonical plotting of the embodiment of the present invention 2;

Fig. 9 is the RHcyst-2 enzyme of the embodiment of the present invention 3 graphic representation that suppresses alive;

Figure 10 is the RHcyst-2 therapeutic test result figure of the embodiment of the present invention 4 arch insect infection mouse.

Embodiment

In the following example, the experimental technique of unreceipted actual conditions, conventionally condition routinely, as < < molecular cloning experiment guide > > (J. Pehanorm Brooker, D.W. Russell work, Huang Peitang, Wang Jiaxi, Zhu Houchu, waits and translates. and the 3rd edition, Beijing: Science Press, 2002) method described in is carried out.

The present invention obtains a new L-Cysteine HCL Anhydrous Inhibitory molecules first in molecule Rhipilin-1 body, called after RHcyst-2 (molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules), its gene coding region is cloned into expression vector, in intestinal bacteria, carry out recombinant expressed, inhibition test by enzyme shows that RHcyst-2 recombinant protein of the present invention can produce effective restraining effect to six kinds of L-Cysteine HCL Anhydrouss, has certain Infection Toxoplasma gondii ability by Infection Toxoplasma gondii evidence RHcyst-2 recombinant protein of the present invention.

Gene clone and the sequential analysis of embodiment 1 molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2

1. the extraction of the total RNA of the female tick of half-full blood molecule Rhipilin-1

Molecule Rhipilin-1 is inoculated on the rabbit ear in the male and female ratio of 2: 1, after 4 days, from the rabbit ear, takes off the female tick of half-full blood.On the mortar through sterilising treatment, with liquid nitrogen, grind, after be transferred in the EP pipe of a nuclease free after adding the Trizol reagent of 1ml fully to grind.The total RNA that extracts 4 library from feeding female Rhipicephalus haemaphysaloides haemaphysaloides that obtain by classical Trizol method, concentration is 11.3ug/ul, OD260/OD280 numerical value is 1.942, can meet the requirement of subsequent experimental in concentration and purity.

2.3 ' end rapid amplifying

2.1 design of primers

The 2cystatin of family molecule for molecule Rhipilin-1, the QVVAGXNY aminoacid sequence conservative according to the 2cystatin of family of several close relative's edge ticks, take Dermacentor variabilis the 2cystatin of Yi Ge family (accession number: ACF35514) as with reference to design 3 ' end rapid amplifying primer.

3 ' RACE primer: the RHcyst-2-3R-GSP:5 '-CAGGTTGTGGCTGGTACAAACTAC-3 ' (SEQ IDNO.3) of family 2.

Synthesizing of 2.2cDNA the first chain

Press 3 ' RACE system (3 ' RACE System for RapidAmplification of cDNAEnds of cDNA end rapid amplifying, Invitrogen) operation instructions, total RNA that above-mentioned steps 1 is extracted and an anchor primer (AP) that comprises poly thymidylic acid (Oligo dT) reverse and generate cDNA and RNA hybridization chain under the effect of ThermoScript II, then hybridize chain and under the effect of RNA enzyme, form cDNA strand.

2.3 3 ' amplification of end

By operation instructions, 3 ' end of goal gene is increased, 2.2 synthetic cDNA the first chains of take are template, upstream primer is gene-specific primer (GSP) designed in 2.1, and downstream primer is the AUAP primer of the anchor series complementary pairing that can bring into anchor primer (AP).

RHcyst-2PCR reaction conditions: 94 ℃ 3 minutes; Again 94 ℃ 35 seconds, 55 ℃ of 35 seconds and 72 ℃ 45 seconds, carry out 35 circulations; Last 72 ℃ 7 minutes.

Result: use 3 ' end rapid amplifying method through PCR, can obtain the 3 ' end fragment (seeing Fig. 1) of about 373bp left and right of RHcyst-2.

2.4 3 ' cloning and sequencing of end fragment

Product after pcr amplification is after sepharose leakage of electricity swimming, with glue, reclaim test kit (Axygen) DNA fragmentation in blob of viscose is reclaimed to purifying, purify DNA is connected with PMD-18T carrier, be transformed in competent cell DH5 α, on the agarose substratum that contains Amp resistance, select single bacterium colony, by PCR, identify its Insert Fragment size.After definite T carrier Insert Fragment size is errorless, bacterial strain is delivered to order-checking company (Shanghai Invitrogen company) order-checking.TA cloning and sequencing obtains the sequence (SEQ ID NO.4) of the 3 ' end fragment of RHcyst-2.

3.5 ' end rapid amplifying

3.1 design of primers

Utilize the gene-specific primer of 3 ' terminal nucleotide sequence design 5 ' the end rapid amplifying of the RHcyst-2 having obtained.

RHcyst-2-5R-GSP1：5’-CTTGGCAGGCGAAAGACGACAC-3’(SEQ ID NO.5)；

RHcyst-2-5R-GSP2：5’-GACACAGATCGTTCATTCAGC-3’(SEQ ID NO.6)。

Synthesizing of 3.2cDNA the first chain

Press 5 ' RACE system (5 ' RACE System for RapidAmplification of cDNAEnds of cDNA end rapid amplifying, Invitrogen) operation instructions, the gene-specific primer (GSP1) of the total RNA extracting in above-mentioned steps 1 and 3.1 designs is reversed and generates cDNA and RNA hybridization chain under the effect of ThermoScript II, then hybridize chain and under the effect of RNA enzyme, form cDNA strand.

The purifying of 3.3cDNA the first chain

By the operation instructions about cDNA the first chain purifying in 5 ' RACE system (Invitrogen) of cDNA end rapid amplifying, cDNA the first chain is carried out to purifying.

The tailing of 3.4cDNA

Press the operation instructions of 5 ' RACE system (Invitrogen) of cDNA end rapid amplifying, the cDNA of 3.3 purifying and dCTP endways under the effect of deoxynucleotidyl transferase (TdT), are added to the tail of POLY CYTIDYLIC ACID (Oligo dC) at the 5 ' end of cDNA.

3.5 5 ' amplification of end

By operation instructions, 5 ' end of goal gene is increased, in order to improve specificity and the output of goal gene, adopt the mode of nested PCR to increase.First round PCR is that to take the cDNA of 3.4 tailings be template, upstream primer is an anchor primer (AAP) that comprises poly guanylic acid (Oligo dG), and downstream primer is gene-specific primer (GSP2) designed according to 3 ' terminal sequence in 3.1.It is that to take first round PCR product be template that nested type second is taken turns PCR, and upstream primer is the AUAP primer of the anchor series complementary pairing that can bring into anchor primer (AAP), downstream primer or gene-specific primer (GSP2).

RHcyst-2 first round PCR reaction conditions: 94 ℃ 3 minutes; Again 94 ℃ 35 seconds, 52 ℃ of 35 seconds and 72 ℃ 45 seconds, carry out 35 circulations; Last 72 ℃ 7 minutes.

RHcyst-2 second takes turns PCR reaction conditions: 94 ℃ 3 minutes; Again 94 ℃ 35 seconds, 50 ℃ of 35 seconds and 72 ℃ 45 seconds, carry out 35 circulations; Last 72 ℃ 7 minutes.

Result: use 5 ' end rapid amplifying method through two-wheeled PCR, can obtain the 5 ' end fragment (seeing Fig. 2) of about 566bp left and right of RHcyst-2.

The cloning and sequencing of 3.65 ' end fragment

Method is with 2.4.TA cloning and sequencing obtains the sequence (SEQ ID NO.7) of the 5 ' end fragment of RHcyst-2.

4. the amplification of full-length gene

4.1 design of primers

By the sequence that can obtain full-length gene to 3 ' end of goal gene and the splicing of 5 ' terminal sequence, at 5 ' tip designs upstream primer of sequence so that amplify full-length gene in synthetic cDNA from 2.2.

RHcyst-2：5’-ACTCGAAGACGACGCCGTTC-3’(SEQ ID NO.8)。

4.2 full-length gene amplifications

2.2 3 ' synthetic RACE cDNA the first chains of take are template, and the primer of 4.1 designs is upstream primer, and AUAP is downstream primer, and the total length of RHcyst-2 is increased.

RHcyst-2PCR reaction conditions: 94 ℃ 3 minutes; Again 94 ℃ 40 seconds, 56 ℃ of 40 seconds and 72 ℃ 50 seconds, carry out 35 circulations; Last 72 ℃ 7 minutes.

Pcr amplification result as shown in Figure 3, obtains the RHcyst-2 full-length gene order of about 750bp left and right.

The cloning and sequencing of 4.3 full-length genes

Method is with 2.4.

The sequential analysis of 4.4 full-length genes

Application Genetyx (version7) software carries out sequence alignment and predicted protein molecular size and iso-electric point.

Utilize the CBS server of Denmark University of Science and Technology (DTU) to carry out signal peptide (signal peptide) prediction, enter the PredictionServes page.Network address: http://www.cbs.dtu.dk/services/SignalP/

The ORF of the ORF Finder software analysis gene of application NCBI (American National biotechnology information center) website, the homology of BLAST online services analyzing gene.Network address: http://www.ncbi.nlm.nih.gov/

The functional domain of application smart Main page software analysis gene.Network address: http://smart.embl-heidelberg.de/

By the full-length gene order to RHcyst-2, analyze, can learn: RHcyst-2 full-length gene is 773bp (SEQID NO.2), have coding 139 amino acid whose open reading frame (SEQ ID NO.1), wherein having 23 amino acid is signal peptide sequence.RHcyst-2 has the feature of the very typical 2cystatin of family, the conservative glycine of N end, the conservative site of QXVX6, the conservative site of PW and two disulfide linkage bridges.The albumen size of inferring RHcyst-2 is about 15kDa, and iso-electric point is 5.20.The homology of the 2cystatin of the family aminoacid sequence of RHcyst-2 and other hard tick is not high, with haemaphysalis longicornis (Hlcyst-2, accession number: ABC94582), brown dog tick (RScyst-2, accession number: ACX53862) and amblyomma americanum (AAcyst, accession number: the homology of the 2cystatin of family AEO35688) is respectively 41%, 62% and 66% (see Fig. 4, remove the aminoacid sequence comparison chart of signal peptide).

Prokaryotic expression and the purifying of embodiment 2 molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2

1. expression plasmid builds

According to the analytical results of RHcyst-2 full length sequence, in the ORF two ends design of RHcyst-2 gene removal signal peptide, be added with the gene-specific primer of suitable restriction enzyme site.Primer sequence is as follows:

RHcyst-2express F：5’-TA GAATTCCAACCGCTGGTTGGCGG-3’(SEQ ID NO.9)；

RHcyst-2express R：5’-TA CTCGAGCTAGGTGGATGCGCTGCT-3’(SEQ ID NO.10)。

Pcr amplification RHcyst-2 gene is removed the coding region of signal peptide, is cloned into pGEX-4T-1 carrier, and construction expression recombinant plasmid RHcyst-2/pGEX-4T-1 accurately inserts through double digestion (Xho I and EcoR I) and order-checking evaluation object fragment.Fig. 5 is the double digestion evaluation figure of RHcyst-2/pGEX-4T-1 recombinant plasmid, and RHcyst-2 object fragment is inserted accurately as shown in Figure 5.

2. expression of recombinant proteins form analysis

The abduction delivering of 2.1IPTG

By the RHcyst-2/pGEX-4T-1 recombinant plasmid transformed building, to BL21 (DE3) competent cell, the method by PCR filters out positive strain.BL21 (DE3) positive strain that transforms recombinant plasmid is seeded in two 50ml centrifuge tubes that 20ml LB substratum (Amp resistance) be housed, 37 ℃ 200 turns the about 2h of shaking culture again.Measure bacterium liquid OD600 numerical value, in the time of between numerical value reaches 0.4 to 0.6, the bacterium liquid that inoculation transforms RHcyst-2 recombinant plasmid adds 10ul1M IPTG, and an other pipe does not add as not induction contrast.Then by expressing at 37 ℃ of the nutrient solutions of RHcyst-2 recombinant protein 200, turn shaking culture 10h.At last 4 ℃, 10000rpm5min is centrifugal, goes supernatant to collect thalline.

2.2SDS-PAGE identifies recombinant protein

Cleer and peaceful precipitation on ultrasonic degradation microorganism collection, with the expression of conventional 12%SDS-PAGE electrophoretic analysis albumen, as shown in Figure 6, RHcyst-2 recombinant protein is mainly expressed as master with supernatant to result, in Fig. 6,1: do not induce cellular lysate supernatant; 2: induction cellular lysate supernatant; 3: do not induce cellular lysate precipitation; 4: induction cellular lysate precipitation.

3. protein purification and quantitatively

3.1 protein purification

The RHcyst-2 recombinant protein of expressing is with GST label, and available GST resin carries out purifying to it.The protein liquid of purifying packs into through deionized water and boils in the dialysis tubing of processing, and two clamps with dialysis sackholder, is immersed in 1L PBS damping fluid, and 4 ℃ of dialysis 12h, then change the PBS damping fluid 12h that dialyses again.Through dialysis treatment protein liquid, carry out SDS-PAGE electrophoresis and identify albumen size and purity.The results are shown in Figure 7, in Fig. 7,1: do not induce cellular lysate supernatant; 2: induction cellular lysate supernatant; 3: the RHcyst-2 recombinant protein of purifying.

3.2 protein quantification

According to BCA determination of protein concentration test kit (BCA Protein Assay Kit, Pierce) operation instructions, RHcyst-2 recombinant protein is carried out quantitatively, concrete steps are as follows:

1. A liquid and B liquid are mixed with to reaction solution by the ratio row of 50: 1.

2. the standard protein of getting 25ul testing sample and various gradients drips in elisa plate hole, and each sample is done a multiple hole.

3. with 8 road pipettors, add the reaction solution of 200ul to every hole, vibration mixes.

4. cover elisa plate upper cover, 37 ℃ of reaction 30min.

5. by microplate reader, measure the OD value of 562nm wavelength.

6. according to the standard protein of various gradients, survey OD value, make typical curve, output function relational expression, the OD value of input testing sample, tries to achieve the concentration of testing sample.

As shown in Figure 8, according to the functional relation of this typical curve and derivation thereof, the RHcyst-2 recombinant protein concentration that calculates purifying is 381ug/ml to typical curve.

The enzyme activity assay experiment of embodiment 3 recombinant protein rRHcyst-2

1. method: verify the inhibition activity of rRHcyst-2 to papoid sample L-Cysteine HCL Anhydrous by measuring with the remaining activity that after rRHcyst-2 effect, cathepsin L, B, C, H, S, papoid and its corresponding fluorogenic substrate react.Concrete grammar is as follows:

(1) press the formulated L-Cysteine HCL Anhydrous reaction solution of 100mM NaAC, 100mM NaCl, 1mM EDTA, 1mg/ml halfcystine and 0.005%TritonX-100, regulate pH to 5.5.

(2) with L-Cysteine HCL Anhydrous reaction solution, six kinds of L-Cysteine HCL Anhydrouss are mixed with to 1.5uM, their corresponding fluorogenic substrate is mixed with to 0.5mM.

(3) with PBS, the good RHcyst-2 recombinant protein of purifying is mixed with to the concentration gradient of 12uM, 6uM, 3uM, 1.5uM, 0.75uM, 0.375uM and 0uM.

(4) in 96 hole black microwell plates, add successively the RHcyst-2 recombinant protein 20ul (each concentration is done 3 repeating holes) of seven concentration gradients, then the 1.5uM L-Cysteine HCL Anhydrous to be measured that adds respectively 20ul, delivers to 37 ℃ of reaction 30min of thermostat container.

(5) under lucifuge condition, to every hole, add the corresponding fluorogenic substrate of 100ul, add 60ul reaction solution, add a hole that only adds 100ul reaction solution and 100ul substrate solution as a setting, 37 ℃ of reaction 15min, deliver to microplate reader and detect every hole OD value under the conditions of excitation wavelength (λ ex) 360nm and emission wavelength (λ em) 460nm.

(6) respectively organize the OD value in the equal subtracting background of OD numerical value hole, by the OD value of each concentration group, calculate the remaining activity of L-Cysteine HCL Anhydrous except the OD value of the 0uM in rRHcyst-2, with this, draw the RHcyst-2 enzyme inhibition curve of living.

2. the enzyme of result: RHcyst-2 is lived and is suppressed curve as shown in Figure 9, as shown in Figure 9, rRHcyst-2 can produce and effectively suppress and present significant concentration dependence six kinds of L-Cysteine HCL Anhydrouss, but inhibition is not quite similar, wherein the highest to the inhibition activity of cathepsin S, the in the situation that of 0.25 times of volumetric molar concentration, just can reach more than 90% inhibition to its enzymic activity.

The Infection Toxoplasma gondii experiment of embodiment 4 recombinant protein rRHcyst-2

(1) experimental animal is kunming mouse, and 25 grams of left and right body weight are male.Toxoplasma gondii is international standard strain RH strain, collects ascites and gather polypide after mouse infection.

(2) RHcyst-2 recombinant protein is dissolved in PBS damping fluid, test is divided into high dose group (200ug/ only), middle dosage group (100ug/ only), low dose group (50ug/ only), albumen control group (GST albumen) and blank group (PBS), every group of 20 tests mouse, 10000 toxoplasma gondii of every mouse intraperitoneal inoculation.Recombinant protein is the 2nd day after toxoplasma gondii infection and treating through abdominal injection for the 4th day respectively, then observes mouse death rate and death time, and between group, mean time to death is assessed curative effect through the t check analysis significance of difference and had or not.

The mouse model of arch insect infection is the common model of toxoplasma gondii research and evaluating drug effect, considers Proteometabolism feature, and successive administration is carried out in this test for the 2nd day and the 4th day after infection, with each group mouse death rate and death time evaluate efficacy.RHcyst-2 recombinant protein treatment result is shown in Figure 10, and each dosage group and control group mice are all dead in 16 days, mortality ratio 100%; But the death time between 5-16 days not etc., GST albumen control group and not treating between blank group without without significant difference (mean time to death is respectively 6.5 days and 6.4 days), but between control group and recombinant protein treatment group there is significant difference (50ug/ group, a 100ug/ group, a 200ug/ group in mouse survival time span, mean time to death is respectively 8.5 days, 10 days and 10.2 days), RHcyst-2 significant prolongation mouse survival time that shows to recombinate, there is the ability of certain Infection Toxoplasma gondii.

The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Sequence table

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<400>5

cttggcaggc gaaagacgac ac 22

<210>6

<211>21

<212>DNA

<213> artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

<223> primer

<400>6

gacacagatc gttcattcag c 21

<210>7

<211>566

<212>DNA

<213>Rhipicephalus haemaphysaloide

<400>7

actcgaagac gacgccgttc ccggttcaga cgtctggcac ttgagagcgg ctcacgccac 60

ggccattcct gtctcttgaa ctggaggtcc tggctacgca tcggggttcc cggcggtcgc 120

tgcgtaagca gaactagaac acggttgctg tcggccaaca ccgctgaaat tttgaaagcg 180

accatggcat ctttgagaat cgccacgtgc gggttcgcat tcctgattgc catctgccag 240

ttcggcgcca ctcaaccgct ggttggcgga tggcataggc acagcgtcgg cgacaacgcc 300

ctgttcgagg agctcgcgca cttcgccata tcacgacagg ttggtgaccg ggagtacttc 360

gacaccgtgc tcgaactggt cgacgtggaa agtcaggttg tggccggtac aaactaccga 420

atcaagttca aggtgggcga atctacatgc agggttacag aaacatacac caaagaggcc 480

tgtgttccac agtccaggga gacggtcaag gatacctgta cagcagtcat atacgacgtg 540

ccctggctga atgaacgatc tgtgtc 566

<210>8

<211>20

<212>DNA

<213> artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

<223> primer

<400>8

actcgaagac gacgccgttc 20

<210>9

<211>25

<212>DNA

<213> artificial sequence

<220>

<221>misc_feature

<222>(1)..(25)

<223> primer

<400>9

tagaattcca accgctggtt ggcgg 25

<210>10

<211>26

<212>DNA

<213> artificial sequence

<220>

<221>misc_feature

<222>(1)..(26)

<223> primer

<400>10

tactcgagct aggtggatgc gctgct 26

Claims (10)

1. a tick L-Cysteine HCL Anhydrous Inhibitory molecules, is characterized in that, described L-Cysteine HCL Anhydrous Inhibitory molecules is molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2, and its aminoacid sequence is selected from:

(1) aminoacid sequence shown in SEQ ID NO.1;

(2) shown in SEQ ID NO.1, in aminoacid sequence, lack, add, insert or replace that one or more amino acid is resulting has L-Cysteine HCL Anhydrous and suppress active, and there is the aminoacid sequence of 95% above homology with SEQ ID NO.1.

2. tick L-Cysteine HCL Anhydrous Inhibitory molecules according to claim 1, is characterized in that, the aminoacid sequence of described molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 is as shown in SEQ ID NO.1.

3. a tick L-Cysteine HCL Anhydrous Inhibitory molecules gene, is characterized in that, described gene is molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 gene, comprises: the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1.

4. tick L-Cysteine HCL Anhydrous Inhibitory molecules gene according to claim 3, is characterized in that, the nucleotide sequence of described RHcyst-2 gene is as shown in SEQ ID NO.2.

5. a recombinant vectors, it is characterized in that, comprise: molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 gene order claimed in claim 3 or its partial sequence, described partial sequence is that the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1 is removed the nucleotide sequence after signal coding sequence.

6. a host cell, is characterized in that, comprises recombinant vectors claimed in claim 5.

7. a preparation method for tick L-Cysteine HCL Anhydrous Inhibitory molecules recombinant protein, is characterized in that, comprises the following steps:

Build the recombinant expression vector containing molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 Gene Partial sequence described in claim 3, described partial sequence is that the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.1 is removed the nucleotide sequence after signal coding sequence;

Described recombinant expression vector is transformed into e. coli host cell;

The e. coli host cell that cultivation comprises recombinant expression vector, and under conditions suitable, induce this recombinant expression vector to express molecule Rhipilin-1 L-Cysteine HCL Anhydrous Inhibitory molecules RHcyst-2 recombinant protein.

8. the application of tick L-Cysteine HCL Anhydrous Inhibitory molecules claimed in claim 1 in preparing anti-parasite medicine.

9. application according to claim 8, is characterized in that, described parasite comprises toxoplasma gondii, trypanosome or plasmodium.

10. the application of tick L-Cysteine HCL Anhydrous Inhibitory molecules claimed in claim 1 in preparing cystatin.