CN103966238A - Vaccine used for infection prevention of toxoplasma gondii, preparation method and application of vaccine - Google Patents

Abstract

The invention firstly provides a nucleotide sequence used for infection prevention of toxoplasma gondii. The nucleotide sequence is selected from nucleotide sequences as follows: 1), a nucleotide sequence represented in a sequence table SEQIDNo.1; 2), a nucleotide sequence which is complementary with the nucleotide sequence represented in 1); and 3), a nucleotide sequence which performs substitution, loss and adding modification on one or more basic groups of the nucleotide sequence represented in 1) or 2) and has a toxoplasma gondii infection prevention function. The invention further provides a vaccine which comprises the nucleotide sequence and a medically acceptable carrier. The plasmid DNA vaccine pVAX-HSP60 can effectively prevent and control infection of a toxoplasma gondii animal model (a Kunming strain mouse), that is, the survival time of the mouse can be effectively prolonged, and worm count of brain cyst is reduced, so that the plasmid pVAX-HSP60 can be used as a DNA vaccine candidate antigen for resisting infection of toxoplasma gondii.

Description

A kind of vaccine and preparation method and application thereof for arch insect infection prevention

Technical field

The invention belongs to immunology and biology field, particularly, relate to a kind of vaccine for arch insect infection prevention.

Background technology

Toxoplasma gondii (Toxoplasma gondii) is that one is universal special sexual cell endoparasitism protozoon, has host range very widely, can infect the nearly all warm-blooded animals including the mankind.This cause of disease can be by congenital or acquired approach infection host, and most infection for inapparent infection, does not show obvious clinical symptom; But be the main lethal cause of disease for the immunosuppression such as malignant tumour, organ transplantation and AIDS patient and immune deficiency crowd.

In human infection colony, pregnant woman's sickness rate is higher, and harm is serious.Toxoplasma gondii can be by placental barrier vertical transmission to fetus, often cause the ill symptomses such as premature labor, miscarriage, monster, stillborn foetus, and the fetus of survival is also often with congenital disorders, as dysnoesia, toxoplasma gondii meningoencephalitis, central nervous system injury and/or toxoplasma gondii illness in eye etc.Infect in colony livestock and poultry, arch insect infection can cause the symptom such as fervescence, diarrhoea, when serious, can cause dam premature labor, miscarriage or stillborn foetus, and can act synergistically with other pathogenic micro-organisms, cause serious public safety problem and can produce and bring huge financial loss to livestock industry, therefore prevention and control toxoplasmosis be very urgent.Larger to the toxic side effect of humans and animals during due to conventional clinically sulfanilamide (SN), miazines medical treatment toxoplasmosis, and easily recurrence, can not kill packing, and often form the problems such as drug residue, therefore vaccine control toxoplasmosis becomes and controls one of popular effective measures of this disease.Because can bringing out the cell of body general and humoral immunoresponse(HI), DNA vaccination becomes the focus of resisting toxoplasmosis vaccine research, and the specific antibody of this effective humoral immunization immunne response generation, it is the important factor in Infection Toxoplasma gondii animal model that the cytotoxic T lymphocyte that the helper T cell reaction that CD4+ relies on and CD8+ rely on is replied.

At present, the research of bow-shaped worm dna vaccine is mainly concentrated on to some to Chinese scholars and toxoplasma gondii is invaded on host and pathogenic virulence factor, as: studies on rhoptry proteins of Toxoplasma gondii (rhoptry protein, ROP) comprise ROP1 and ROP2, ROP18, ROP16, with RON4 etc., dense granule antigen (dense granules antige, GRA) comprises GRA4, GRA6 and GRA7 etc., Microneme protein (microneme protein, MIC) comprises MIC4, MIC8 and MIC6 etc., and film surface antigen (surface antige, SAG) is as SAG1 etc.Exploratory development by immune protective that these vaccine candidate antigens are carried out shows; they all have immunogenicity also can induce generation cell and the humoral immunoresponse(HI) challenge infection with opposing toxoplasma gondii; there is certain immune protective efficiency, but do not reach desired complete protectiveness effect.Trace it to its cause, due to the toxoplasma gondii complexity life history, form is various, host range is extensive, its immunogen of antigen of different nature is also different, and the mechanism of causing a disease of its packing and tachyzoite also there are differences, although can find various single antigenic component is basic vaccine candidate antigen, but because its lymphocyte binding site containing is few, be subject to body major histocompatibility complex (Major histocompatibility complex, MHC) restricted large, and be difficult to form the challenge infection of effective resistibility to resisting toxoplasmosis different shape.Therefore only depend on the candidate antigen genes of these minorities to be also difficult to the effect that reaches desirable, and find more, better derive from the DNA vaccination candidate antigens in different toxoplasma gondii stages, especially the antigen gene relevant to toxoplasma gondii virulence is an important channel of Effect of Anti Toxoplasma gondii vaccine.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of vaccine and preparation method and application thereof for arch insect infection prevention.

Heat shock protein(HSP), conventionally be called again heat stress proteins, one group of special protein that organism stress be synthesized under hostile environment factor stimulates, in immunity system, play the part of molecular chaperones role, play a significant role with intersecting in the activation maturation of submission approach, scavenger cell and lymphocytic activation and dendritic cell at the classical submission of antigen.HSP protein family member participates in multiple biological procedureses of toxoplasma gondii, is its important virulence factor.Toxoplasma gondii HSP60 (heat shockprotein60, TgHSP60) is a member of HSP60 subfamily.This albumen height is conservative, be positioned in the plastosome of toxoplasma gondii, it is the signal of host antigen presenting cell activation, can induce the release of the host cell factor and the generation that immune stimulatory is replied, and there is the effect of molecular chaperones, and can pass through Toll sample acceptor 4(Toll-like receptor4) mediation host's innate immune response and the release of cytokine.Therefore, toxoplasma gondii HSP60 is expected to become desirable vaccine candidate antigen.

The invention provides a kind of nucleotide sequence for arch insect infection prevention, it contains and is selected from following nucleotide sequence:

1) nucleotide sequence shown in SEQ ID No.1 in sequence table;

2) with 1) shown in the nucleotide sequence of nucleotide sequence complementation;

3) to above-mentioned 1) or 2) shown in the nucleotide sequence replacement, disappearance, interpolation of carrying out one or more bases modify, and there is the nucleotide sequence of arch insect infection prophylactic function.

Second object of the present invention is to provide a kind of recombinant protein, and its aminoacid sequence is by claimed in claim 1 nucleotide sequence coded.

Preferably, the aminoacid sequence of described recombinant protein is referring to SEQ ID No.2 in sequence table.

The 3rd object of the present invention is to provide a kind of recombinant vectors, and it comprises nucleotide sequence claimed in claim 1.

The 4th object of the present invention is to provide a kind of recombinant plasmid, and its nucleotides sequence is classified the nucleotide sequence shown in SEQ ID No.3 in sequence table as.

The 5th object of the present invention is to provide a kind of vaccine, and it comprises nucleotide sequence claimed in claim 1, and medically acceptable carrier.

The 6th object of the present invention is to provide above-mentioned nucleotide sequence, recombinant protein, recombinant vectors, recombinant plasmid, the application of vaccine in arch insect infection prevention.

The 7th object of the present invention is to provide a kind of preparation method of above-mentioned vaccine, and step is as follows:

(1) extract the total RNA of RH strain of Toxoplasma gondii;

(2) the total RNA of RT-PCR amplification RH strain;

(3) amplified production in step 2 is connected with carrier;

(4) a small amount of of pVAX I plasmid is extracted;

(5) reclaim pVAX I carrier and object fragment HSP60;

(6) connect pVAX I carrier and object fragment HSP60;

(7) connection product is converted in Host Strains.

Preferably, when described RT-PCR increases the total RNA of RH strain, the primer is:

Upstream primer: 5 '-GGTACCATGCTTGCCCGCGCTTCAGC-3 ';

Downstream primer: 5 '-AAGGAAAAAAGCGGCCGCCTAGTACATGCCTCCCATGCCGC-3 '.

Preferably, when described RT-PCR increases the total RNA of RH strain, PCR reaction conditions is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 1min, 60.4 DEG C of renaturation 45s; 72 DEG C are extended 2min; 35 circulations, last, 72 DEG C are extended 10min.

Plasmid DNA vaccine pVAX-HSP60 of the present invention can prevent and control the infection of toxoplasma gondii animal model (Kunming mouse) effectively, can effectively extend the survival time of mouse, reduces the lotus worm amount of brain packing.Therefore, plasmid pVAX-HSP60 can be served as the infection of DNA vaccination candidate antigens resisting toxoplasmosis.

Brief description of the drawings

Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, for explaining the present invention, is not construed as limiting the invention together with embodiments of the present invention.In the accompanying drawings:

Fig. 1 is that pVAX-HSP60 builds route;

Fig. 2 is RH strain of Toxoplasma gondii death condition;

Fig. 3 is pMD18-T plasmid;

Fig. 4 is pVAX I plasmid.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.

In the present invention experiment material purchased from:

Female KM mouse, about 6w age, body weight 20 ± 2g, purchased from Lanzhou University's Medical experimental center.

Host Strains, toxoplasma gondii RH, PRU strain, the materials such as pVAX I carrier for expression of eukaryon, are parasite functional genomics research department of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences and preserve and provide.

PrimeScript ^tMone Step RT-PCR Kit Ver.2(article No.: RR055B), 10 × PCR Buffer(Mg ²⁺free), MgCl ₂(25mmol/L), dNTP Mixture(2.5mmol/L each), Ex-Taq(5U/ μ L) (article No.: DDR100D), pMD18-T Vector(article No.: D103A), BamH I(article No.: D1010A), XhoI(article No.: D1094A), KpnI(article No.: D1068A), PstI(article No.: D1073A), XbaI(article No.: D1093A), 10 × M Buffer, 10 × K Buffer, 10 × and 6 × Loading Buffer: purchased from TaKaRa company;

sV Genomic DNA Purification System(article No.: A2360), Tris-Cl, EDTA, IPTG, X-gal, T4DNA ligase enzyme: purchased from Promega company;

RNAprep Pure Tissue Kit(article No.: DP431), TIANprep Mini Plasmid Kit(article No.: DP103-02), plain agar sugar gel DNA reclaims test kit (article No.: article No.: DP209-02), common DNA product purification test kit (article No.: DP204-02): purchased from TIANGEN Biotech (Beijing) Co., Ltd.;

Penbritin (Amp), kantlex (Kan), agarose: purchased from Sigma company;

Other chemical reagent as NaCl,, dehydrated alcohol etc. is domestic analytical pure level product.

One, the preparation method of RH strain of Toxoplasma gondii pVAX-HSP60 vaccine is as follows:

1. the extraction of the total RNA of RH strain of Toxoplasma gondii: by RH strain of Toxoplasma gondii intraperitoneal inoculation KM mouse, after 96h, lethal through cervical vertebra dislocation, aseptic collection mouse ascites 1.0mL, centrifugal 3～5min under 10000 revs/min of rotating speeds, abandons supernatant, for subsequent use.With the total RNA of test kit RNAprep Pure Tissue Kit extraction RH strain of Toxoplasma gondii of TIANGEN Biotech (Beijing) Co., Ltd., the concrete grammar of extraction and step are referring to specification sheets.

2.RT-PCR amplification: taking the total RNA of RH strain of Toxoplasma gondii that extracted as template, with the PrimeScript of Dalian Bao Bio-Engineering Company ^tMone Step RT-PCR Kit Ver.2 test kit carries out the RT-PCR amplification of HSP60 gene, and using method is referring to specification sheets.Wherein,

Upstream primer: FHSP60:5 '-GGTACCATGCTTGCCCGCGCTTCAGC-3 ';

Downstream primer: RHSP60:5 '-AAGGAAAAAAGCGGCCGCCTAGTACATGCCTCCCATGCCGC-3 ';

PCR reaction conditions is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 1min, 60.4 DEG C of renaturation 45s; 72 DEG C are extended 2min; 35 circulations, last, 72 DEG C are extended 10min.

The connection of 3.PCR purified product: the amplified production in step 2 is connected with pMD18, and ligation is used the pMD of TaKaRa company ^tM18-T Vector, using method is referring to specification sheets, and condition of contact is that 16 DEG C of reaction 2～4h or 4 DEG C of connections are spent the night, and connection Product Labeling is pMD18-HSP60.

The sequencing of 4.HSP60: by cut all positive bacterial strains of qualification through bacterium liquid PCR and enzyme, deliver to the order-checking of Shanghai Sheng Gong biotechnology company limited.Sequencing result is referring to SEQ ID No.1 in sequence table, and the protein sequence of its coding is referring to SEQ ID No.2 in sequence table.

The a small amount of of 5.pVAX I plasmid is extracted: carry out pVAX I plasmid extraction with the TIANprep Mini Plasmid Kit of TIANGEN Biotech (Beijing) Co., Ltd., using method is with reference to specification sheets.

The recovery of 6.pVAX I carrier (shown in Fig. 4) and object fragment HSP60: with Kpn I and Not I double digestion pVAXI plasmid and pMD18-HSP60 recombinant plasmid respectively, and reclaim test kit with the plain agar sugar gel DNA of TIANGEN Biotech (Beijing) Co., Ltd. and reclaim carrier and object fragment, using method is with reference to specification sheets.

7.pVAX I carrier is connected with object fragment HSP60's: will reclaim the directed subclone of the HSP60 gene fragment that obtains of purifying to pVAX I carrier through cutting glue.16 DEG C of reaction 4～6h or 4 DEG C of connections are spent the night, and Product Labeling is pVAX-HSP60.Its nucleotide sequence is referring to SEQ ID No.3 in sequence table.

8. connect the conversion and qualification of product: will be connected product 10 μ L and transform in bacillus coli DH 5 alpha, the some single bacterium colonies of picking are done bacterium liquid PCR qualification.Product Labeling is pVAX-HSP60.PVAX-HSP60 builds route and sees Fig. 1.

Two, the preparation of substratum and solution:

The preparation of a, solution:

(1) LB liquid nutrient medium

Take LB powder 5.0g, add appropriate deionized water dissolving, and be settled to 200mL, 121 DEG C of autoclaving 20min, 4 DEG C save backup.

(2) LB solid medium

In LB liquid nutrient medium, add 2.0%(m/V) agar powder, 121 DEG C of autoclaving 20min, pour plate in the time that it is cooled to 55 DEG C～65 DEG C, after solidifying, 4 DEG C save backup.

(3) Kan ⁺lB selects liquid nutrient medium

Be cooled to 50 DEG C～55 DEG C after the LB liquid nutrient medium autoclaving of preparation in (1) time, add Kan(kantlex) (final concentration is 1000IU/mL), rock and mix rear 4 DEG C of preservations.

(4) Kan ⁺lB solid is selected substratum

Be cooled to 50 DEG C～55 DEG C after the LB solid medium sterilizing of preparation in (2) time, adding Kan(final concentration is 1000IU/mL) and shake gently, mixing rear rapid pour plate, room temperature is solidified and is saved backup in 4 DEG C.

The preparation of solution when b, plasmid extract in a large number:

(1) 0.25mol/L Tris-Cl solution (pH8.0)

Take Tris-Cl powder 6.055g, and be dissolved into 100mL ddH ₂in O, regulating pH value is 8.0, and constant volume is to 200mL, and after autoclaving, 4 DEG C save backup.

(2) 0.5mol/L EDTA solution (pH8.0)

Take Na ₂eDTA37.22g, is dissolved in 200mL ddH ₂in O, vibration mixes, and regulating pH value is 8.0, and after autoclaving, 4 DEG C save backup.

(3) TE solution (pH8.0)

First by 0.5mol/L EDTA solution (pH8.0) dilution of preparation in the 0.25mol/L Tris-Cl solution of preparation in (1) and (2), and draw respectively 10mmol/L Tris-Cl l.0mL with 1mmol/L EDTA0.2mL, add aseptic ddH ₂after O, constant volume is to 100mL, and 4 DEG C save backup.

(4) 10%SDS solution (pH7.2)

Take SDS(sodium laurylsulfonate) powder 10g, add 60 DEG C of water-baths after deionized water 90mL to dissolve, its pH value is adjusted to 7.2 with dense HCl, and constant volume is to 100mL, now with the current, should not preserve for a long time.

(5) 2mol/L NaOH solution

Prior to adding deionized water 160mL in beaker, take NaOH powder 16.0g slowly joining in deionized water, limit edged mixes, and constant volume is to 4 DEG C of preservations in Plastic Bottle after 200mL.

（6）Solution I

Get the 0.25mol/L Tris-C110mL of preparation in (1), in (2), the 0.5mol/LEDTA2mL of preparation, adds deionized water constant volume to 100mL, 4 DEG C of preservations after high pressure steam sterilization.

（7）Solution II

Get 2mol/L NaOH10mL, 10%SDS10mL, adds deionized water constant volume to 100mL, now with the current, and normal temperature is preserved, unsuitable long storage time.

（8）Solution III

Take KAc powder 58.884g, 23mL mixes with glacial acetic acid, adds appropriate amount of deionized water to dissolve rear constant volume to 200mL, 4 DEG C of preservations.

(9) 3mol/L NaAc solution (pH5.2)

Take anhydrous Na Ac49.22g, add deionized water constant volume to 200mL, 4 DEG C of preservations after high pressure steam sterilization.

(10) 5mol/L LiCl solution

Take anhydrous LiCl42.4g, add deionized water constant volume to 200mL, 4 DEG C of preservations after high pressure steam sterilization.

(11) 10mg/mL N,O-Diacetylmuramidase

Before use, take the N,O-Diacetylmuramidase of 10mg, add the Tris-Cl(PH8.0 of 10mmol/L) at once dissolve and be settled to 1mL ,-20 DEG C of preservations.

Three, prepare in a large number plasmid

In picking step () and (two) preparation be stored in the positive bacteria liquid that contains object plasmid (pVAX I, pVAX-HSP60) of-20 DEG C in Kan ⁺lB solid is selected to rule on substratum, 37 DEG C of overnight incubation.A single bacterium colony on picking plate is placed in 12mL Kan ⁺in LB liquid selective medium, 37 DEG C of shaking tables, cultivate 12～16h with the rotating speeds of 180～220 revs/min.Above-mentioned cultivation bacterium liquid is inoculated into 400mL Kan is housed with the ratio of volume ratio 1:100 ⁺in the 1000mL Erlenmeyer flask of LB liquid selective medium, 37 DEG C of shaking tables, overnight incubation under 180～220 revs/min of rotating speeds.

2. bacterial cultures step 1 being obtained is placed in ice or 4 DEG C of refrigerator numbers minute, and 4 DEG C, with 9000 revs/min of centrifugal 5min of rotating speed, collect bacterial cultures precipitation, abandon supernatant (utilizing the careful sucking-off supernatant of pipettor), be inverted centrifuge tube supernatant is flowed out as far as possible.With the resuspended thalline of Solution I of 10mL4 DEG C of precooling, vortex vibration disperses bacterium completely in Solution I.(owing to being enough bacterium liquid cracking dispersion adding of vortex vibration and Solution I, in order to simplify the operation, save the step that in prior art, further cracking disperses herein: add the 10mg/mL lysozyme soln (pH8.0) of the new preparation of 1mL, concussion mixes).

3. in the bacterium liquid obtaining in step 2, add the Solution II of the new preparation of 20mL, jog also turns upside down and mixes 2～4 times.The Solution III that adds again 15mL4 DEG C of precooling, jog mixes immediately, in order to avoid produce localized precipitation.On ice or-20 DEG C of refrigerators place 10min.Add the time interval controls of Solution II and Solution III in 5min, in order to avoid undue cracking.4 DEG C or room temperature, centrifugal 10min under 11000 revs/min, utilizes 4 layers of gauze that supernatant is filtered, and transfers in two new 50mL centrifuge tubes.

4. supernatant step 3 being obtained and 14-16mL Virahol (need the method for the Virahol that adds 0.6 times of volume herein in prior art, the applicant found through experiments, the add-on of Virahol is at 0.6 times of volume up and down in the scope of each 1ml, the extraction Quality and yield that does not affect product) mix, room temperature is placed 10min.Under room temperature, under 11000 revs/min, centrifugal 10min, abandons supernatant, and blank pipe is inverted on filter paper to several minutes, removes remnants.Add 3mL ddH ₂o fully dissolves, then adds the 5mol/L LiCl solution of 3mL precooling (now to need first to add ddH ₂o, after add LiCl, just can make to precipitate abundant dissolving, put upside down step and easily cause the resolution of precipitate insufficient or do not dissolve), fully mix, the mode of unavailable piping and druming herein mixes.Piping and druming mixes and easily makes the plasmid extracting rupture, and two centrifuge tubes are merged into a pipe, in ice or-20 DEG C of refrigerators placement 10min.4 DEG C or room temperature, centrifugal 10min under 11000 revs/min, transfers to supernatant respectively in one new 50mL centrifuge tube, adds the Virahol of equal-volume (12mL) precooling, fully mixes, and room temperature is placed 10min.4 DEG C or room temperature, centrifugal 10min under 11000 revs/min, carefully abandons supernatant, is inverted the dry liquid in pipe of control, is generally inverted 10～15min.Now to there will be white depositions be plasmid to tube wall.

5. in the white depositions of preparing in step 4, add 3mL ddH ₂o or TE solution (pH8.0) dissolution precipitation, and add 30 μ L10mg/mL RNase A(Takara company products), 37 DEG C of water-bath 1h remove RNA.Add 2 times of volume (8mL) dehydrated alcohols and 1/10 volume (400 μ L).With 3mol/L NaAc solution, fully mix, in ice or-20 DEG C of refrigerators place 20min.4 DEG C, centrifugal 10min under 11000 revs/min, carefully supernatant sucking-off (is not removed to supernatant by the mode of toppling over, easily cause the precipitation that is adsorbed in tube wall is disperseed, but not supernatant is poured out), and centrifuge tube is inverted on filter paper to control dry (main control dry remaining reagent, as ethanol and NaAc, reduces impurity, improves the purity of plasmid).Add 100 μ L ddH ₂o or TE solution (pH8.0), fully wash-out plasmid precipitation.Utilize concentration the record of spectrophotometer measurement gained plasmid, the concentration of measuring is 10000mg/mL.Therefore, we by taking preserve concentration as ten times of dilutions of 1000mg/mL() amount be sub-packed in 1.5mL centrifuge tube, every pipe is preserved 1000mg plasmid, volume is 1mL, after coating-dividing sealing, saves backup in-20 DEG C.

Four, the purity detecting of plasmid DNA

With ddH ₂o or TE(pH8.0) be blank, by nucleic acid solution optical density(OD) (OD) value under spectrophotometric determination wavelength 260nm and 280nm.The OD of double-stranded DNA sterling ₂₆₀/ OD ₂₈₀value is 1.8, if sample OD ₂₆₀/ OD ₂₈₀value is lower than 1.8, and interpret sample may, by protein contamination, need be further purified.

Five, the Efficacy evaluation of recombinant plasmid

The immunity of 1.KM mouse and grouping: in order to reduce stress reaction, KM mouse needs to raise after buying back 1 week, then it is divided at random to 4 groups, 35 every group, specifically divides into groups in table 1.

Table 1 immunization experiment grouping situation

Group	The immunoreagent adding
		Group I	Control (being left intact)
Group II	PBS
		Group III	pVAXI
Group IV	pVAX-HSP60

Carry out immunity with left back leg tibialis posterior injecting pathway, first by empty carrier pVAX I and extract the recombinant plasmid pVAX-HSP60 PBS(pH7.4 of purifying) solution dilution to 100 μ g/100 μ L, give the 3rd, 4 groups respectively in every mouse respectively inject 100 μ L.After initial immunity, according to same dosage, in the 2nd week and the 4th week to III group to the IV group booster immunization once.

2. abdominal injection toxoplasma gondii infection RH strain: the recovery of RH strain of Toxoplasma gondii need be carried out attacking for before worm 2 weeks.From liquid nitrogen container, take out the cryopreservation tube of preserving RH strain of Toxoplasma gondii polypide, exist side by side to put it in 37 DEG C of warm water and thaw.By the liquid blending in cryopreservation tube, every mouse peritoneal is injected 200 μ L, goes down to posterity for the first time.Treat infecting mouse morbidity (4～6d), rear aseptic collection mouse ascites 1～2mL is put to death in cervical vertebra dislocation.After mixing, draw 10 μ L ascites and carry out microscopy.Carry out in the manner described above second and go down to posterity for the third time.Will be after the purifying that goes down to posterity for the third time gained polypide by 1 × 10 ³individual tachyzoite/dosage only, intraperitoneal inoculation is respectively organized each 10 of mouse, observes and record its survival time.In table 2.

3. gavage toxoplasma gondii infection PRU strain: the preparation of toxoplasma gondii PRU strain need be carried out attacking worm for first 30 days.The mouse being infected by toxoplasma gondii PRU strain, 75% alcohol-pickled 2～3min are put to death in cervical vertebra dislocation.Take out and in Bechtop, aseptic collection cerebral tissue is also placed in mortar, adds 1mL physiological saline to grind.To be ground completely after, draw 10 μ L and carry out microscopy, and observe counting.By 10 packings/dosage only, infect each 10 of each group of mouse in the mode of gavage, within 30 days, carry out brain packing counting after attacking worm.In table 3.

Beneficial effect of the present invention will be by following data declaration:

Table 2 RH strain of Toxoplasma gondii death condition:

Group (n=15; Every group 15 for attacking worm)	Survival number of days (mean value ± S.D.)
		Control	9.0±0 A
PBS	9.0±0 A
		pVAXI	9.0±0 A
pVAX-HSP60	19.4±4.88 C

Table 3PRU brain packing counting and worm reduction rate:

Grouping (n=4; Assist counting for bag for every group 4)	Bag is assisted counting (mean value ± S.D.)	Alkali worm rate (%)
			Control	3489±144 A	-
PBS	3276±167 A	6.11
			pVAXI	3022±142 A	13.38
pVAX-HSP60	1711±124 B	50.96

As shown in table 2, compare with control group (Control, PBS and pVAX I), experimental group (pVAX-HSP60) the mouse survival time, equal significance increased, the survival time of pVAX-HSP60 is respectively 19.4 ± 4.88, can significance increase the survival number of days after experimental mouse infection T.gondii RH strain; 9d internal reference group mouse is without survival.

No matter can find out from above table, be to reduce PRU brain packing number, still delays the death time of RH, all illustrates that recombinant plasmid pVAX-HSP60 can play good immune effect effectively, and the vaccine that can be used as arch insect infection prevention uses.

Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Sequence table

<110> Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120> vaccine and preparation method and application thereof for an arch insect infection prevention

<170> PatentIn version 3.5

<210> 1

<211> 1728

<212> DNA

<213> toxoplasma gondii HSP60

<400> 1

atgcttgccc gcgcttcagc gagagttgcc aagtgcaacc ctggaaacgt ctttcaggtt 60

cgccatgcca gcagcaaaga aatccggttc ggctgtgacg ccagaaacca gatgcttgca 120

ggatgcaacc gcctggcaga cgcagtcgga gtcactctcg gcccgaaggg acgcaacgtg 180

gtaattgagc aaccgtacgg ctcaccaaag atcacgaaag atggagtgac agttgccaag 240

tcgattgagt tgggaaaccg gatgatgaat ctgggagccc agttggtgaa gcaagttgcc 300

tcgacgacca acgacatcgc cggtgacggg acaaccacag cgacgcttct cgcccgcgcc 360

accttcagag aaggctgcaa agccgtggat gcgggaatga accctatgga tctcctgaga 420

ggcatcaacc ttgcggtgga gaaggtcctg gcgcatttga actccgtgac gaagaacgtg 480

acgacatctg aggagatttt caacgtcgca acgatctcgg cgaacggaga caaagtgatt 540

ggaaagctga ttgcagatgc gatggagaag gtgggccgcg acggcaccat cactgtgtcc 600

gaagggaaga ccctgacaca tgagctggag ctcgtggaag gcctcaaatt cgacagaggc 660

tacatttccc cgtactttat cacgaattcg aaggagcaga aggtggagct ggagaaaccc 720

ttcgttctcc tctacgacaa acgcatctcg tctgtcaaaa gcattctccc tgtcctcgaa 780

ttcatcgtcc aaaaccaagg ctctctcctc attatcgcgg aagacgtcga cagcgaagct 840

ctcgcaacga tggtcgtcaa caagctgcgt ctgggcctga agatctgcgc ggtaaaagct 900

cccggcttcg gcgaccacag aaaggccatg cttcacgaca tcgccgtcat gacaggaggc 960

caagtcgtga ctgaggagac cggtggcagt ctcgaggacg cccatcagat gcctcagatg 1020

cttggacgcg ccaagtcggt gacagtgacg aaggacacaa ctctggtgat tgagggtggc 1080

ggcgagaagg cgacgattga cgagcgctgc gaccagattc gagtgtcgat ggagcagact 1140

cactcggatt acgagaagga gaagctgcag gagcgcctgg cccgcatgac aggaggagtc 1200

gcggtgacca aggtgggcgg agcctcagag gtggaggtgg gcgaggcaaa ggacaggatc 1260

caagacgctc tgtgtgcgac gaaggccgcc gtcgaagagg gtattgtgcc cggaggcggt 1320

acggcgctgc tgtacgcgag cgagactctc aagacgatcg agacgaccaa ctacgaccag 1380

aaagtcggtg tgggcattgt acggaacgca tgcaaacagc cttgcaaaac gatcgcggac 1440

aacgccggcc acgagggtgc cgtcgtcgtc ggaaacctct tgagagaagc agacccaacc 1500

aagggcttca acgctcaaac cggcgaatat gttgacatga tggctgccgg catcatcgac 1560

cccaccaaag tcgtcaagac cgccctctcc gacgccgctt ccgtcgcgtc actcatgacc 1620

acaacagagg ccgcggtggt ggaagccaag gaggagaaac ccgacgagcc tatgggaggt 1680

ggcgtgccca tggggggcat gggaggcggc atgggaggca tgtactag 1728

<210> 2

<211> 575

<212> PRT

<213> artificial protein

<400> 2

Met Leu Ala Arg Ala Ser Ala Arg Val Ala Lys Cys Asn Pro Gly Asn

1 5 10 15

Val Phe Gln Val Arg His Ala Ser Ser Lys Glu Ile Arg Phe Gly Cys

20 25 30

Asp Ala Arg Asn Gln Met Leu Ala Gly Cys Asn Arg Leu Ala Asp Ala

35 40 45

Val Gly Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Ile Glu Gln

50 55 60

Pro Tyr Gly Ser Pro Lys Ile Thr Lys Asp Gly Val Thr Val Ala Lys

65 70 75 80

Ser Ile Glu Leu Gly Asn Arg Met Met Asn Leu Gly Ala Gln Leu Val

85 90 95

Lys Gln Val Ala Ser Thr Thr Asn Asp Ile Ala Gly Asp Gly Thr Thr

100 105 110

Thr Ala Thr Leu Leu Ala Arg Ala Thr Phe Arg Glu Gly Cys Lys Ala

115 120 125

Val Asp Ala Gly Met Asn Pro Met Asp Leu Leu Arg Gly Ile Asn Leu

130 135 140

Ala Val Glu Lys Val Leu Ala His Leu Asn Ser Val Thr Lys Asn Val

145 150 155 160

Thr Thr Ser Glu Glu Ile Phe Asn Val Ala Thr Ile Ser Ala Asn Gly

165 170 175

Asp Lys Val Ile Gly Lys Leu Ile Ala Asp Ala Met Glu Lys Val Gly

180 185 190

Arg Asp Gly Thr Ile Thr Val Ser Glu Gly Lys Thr Leu Thr His Glu

195 200 205

Leu Glu Leu Val Glu Gly Leu Lys Phe Asp Arg Gly Tyr Ile Ser Pro

210 215 220

Tyr Phe Ile Thr Asn Ser Lys Glu Gln Lys Val Glu Leu Glu Lys Pro

225 230 235 240

Phe Val Leu Leu Tyr Asp Lys Arg Ile Ser Ser Val Lys Ser Ile Leu

245 250 255

Pro Val Leu Glu Phe Ile Val Gln Asn Gln Gly Ser Leu Leu Ile Ile

260 265 270

Ala Glu Asp Val Asp Ser Glu Ala Leu Ala Thr Met Val Val Asn Lys

275 280 285

Leu Arg Leu Gly Leu Lys Ile Cys Ala Val Lys Ala Pro Gly Phe Gly

290 295 300

Asp His Arg Lys Ala Met Leu His Asp Ile Ala Val Met Thr Gly Gly

305 310 315 320

Gln Val Val Thr Glu Glu Thr Gly Gly Ser Leu Glu Asp Ala His Gln

325 330 335

Met Pro Gln Met Leu Gly Arg Ala Lys Ser Val Thr Val Thr Lys Asp

340 345 350

Thr Thr Leu Val Ile Glu Gly Gly Gly Glu Lys Ala Thr Ile Asp Glu

355 360 365

Arg Cys Asp Gln Ile Arg Val Ser Met Glu Gln Thr His Ser Asp Tyr

370 375 380

Glu Lys Glu Lys Leu Gln Glu Arg Leu Ala Arg Met Thr Gly Gly Val

385 390 395 400

Ala Val Thr Lys Val Gly Gly Ala Ser Glu Val Glu Val Gly Glu Ala

405 410 415

Lys Asp Arg Ile Gln Asp Ala Leu Cys Ala Thr Lys Ala Ala Val Glu

420 425 430

Glu Gly Ile Val Pro Gly Gly Gly Thr Ala Leu Leu Tyr Ala Ser Glu

435 440 445

Thr Leu Lys Thr Ile Glu Thr Thr Asn Tyr Asp Gln Lys Val Gly Val

450 455 460

Gly Ile Val Arg Asn Ala Cys Lys Gln Pro Cys Lys Thr Ile Ala Asp

465 470 475 480

Asn Ala Gly His Glu Gly Ala Val Val Val Gly Asn Leu Leu Arg Glu

485 490 495

Ala Asp Pro Thr Lys Gly Phe Asn Ala Gln Thr Gly Glu Tyr Val Asp

500 505 510

Met Met Ala Ala Gly Ile Ile Asp Pro Thr Lys Val Val Lys Thr Ala

515 520 525

Leu Ser Asp Ala Ala Ser Val Ala Ser Leu Met Thr Thr Thr Glu Ala

530 535 540

Ala Val Val Glu Ala Lys Glu Glu Lys Pro Asp Glu Pro Met Gly Gly

545 550 555 560

Gly Val Pro Met Gly Gly Met Gly Gly Gly Met Gly Gly Met Tyr

565 570 575

<210> 3

<211> 4671

<212> DNA

<213> artificial sequence

<400> 3

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccatgct tgcccgcgct tcagcgagag ttgccaagtg caaccctgga 780

aacgtctttc aggttcgcca tgccagcagc aaagaaatcc ggttcggctg tgacgccaga 840

aaccagatgc ttgcaggatg caaccgcctg gcagacgcag tcggagtcac tctcggcccg 900

aagggacgca acgtggtaat tgagcaaccg tacggctcac caaagatcac gaaagatgga 960

gtgacagttg ccaagtcgat tgagttggga aaccggatga tgaatctggg agcccagttg 1020

gtgaagcaag ttgcctcgac gaccaacgac atcgccggtg acgggacaac cacagcgacg 1080

cttctcgccc gcgccacctt cagagaaggc tgcaaagccg tggatgcggg aatgaaccct 1140

atggatctcc tgagaggcat caaccttgcg gtggagaagg tcctggcgca tttgaactcc 1200

gtgacgaaga acgtgacgac atctgaggag attttcaacg tcgcaacgat ctcggcgaac 1260

ggagacaaag tgattggaaa gctgattgca gatgcgatgg agaaggtggg ccgcgacggc 1320

accatcactg tgtccgaagg gaagaccctg acacatgagc tggagctcgt ggaaggcctc 1380

aaattcgaca gaggctacat ttccccgtac tttatcacga attcgaagga gcagaaggtg 1440

gagctggaga aacccttcgt tctcctctac gacaaacgca tctcgtctgt caaaagcatt 1500

ctccctgtcc tcgaattcat cgtccaaaac caaggctctc tcctcattat cgcggaagac 1560

gtcgacagcg aagctctcgc aacgatggtc gtcaacaagc tgcgtctggg cctgaagatc 1620

tgcgcggtaa aagctcccgg cttcggcgac cacagaaagg ccatgcttca cgacatcgcc 1680

gtcatgacag gaggccaagt cgtgactgag gagaccggtg gcagtctcga ggacgcccat 1740

cagatgcctc agatgcttgg acgcgccaag tcggtgacag tgacgaagga cacaactctg 1800

gtgattgagg gtggcggcga gaaggcgacg attgacgagc gctgcgacca gattcgagtg 1860

tcgatggagc agactcactc ggattacgag aaggagaagc tgcaggagcg cctggcccgc 1920

atgacaggag gagtcgcggt gaccaaggtg ggcggagcct cagaggtgga ggtgggcgag 1980

gcaaaggaca ggatccaaga cgctctgtgt gcgacgaagg ccgccgtcga agagggtatt 2040

gtgcccggag gcggtacggc gctgctgtac gcgagcgaga ctctcaagac gatcgagacg 2100

accaactacg accagaaagt cggtgtgggc attgtacgga acgcatgcaa acagccttgc 2160

aaaacgatcg cggacaacgc cggccacgag ggtgccgtcg tcgtcggaaa cctcttgaga 2220

gaagcagacc caaccaaggg cttcaacgct caaaccggcg aatatgttga catgatggct 2280

gccggcatca tcgaccccac caaagtcgtc aagaccgccc tctccgacgc cgcttccgtc 2340

gcgtcactca tgaccacaac agaggccgcg gtggtggaag ccaaggagga gaaacccgac 2400

gagcctatgg gaggtggcgt gcccatgggg ggcatgggag gcggcatggg aggcatgtac 2460

tagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 2520

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2580

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2640

ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 2700

gctggggatg cggtgggctc tatggcttct actgggcggt tttatggaca gcaagcgaac 2760

cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga 2820

tggctttctc gccgccaagg atctgatggc gcaggggatc aagctctgat caagagacag 2880

gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 2940

gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 3000

ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 3060

gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg 3120

ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 3180

gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 3240

tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 3300

accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 3360

aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 3420

aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 3480

atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 3540

cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 3600

aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 3660

ccttctatcg ccttcttgac gagttcttct gaattattaa cgcttacaat ttcctgatgc 3720

ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacaggtg gcacttttcg 3780

gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 3840

gctcatgaga caataaccct gataaatgct tcaataatag cacgtgctaa aacttcattt 3900

ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 3960

acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 4020

agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 4080

ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4140

cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 4200

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 4260

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 4320

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 4380

caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 4440

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 4500

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 4560

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 4620

ggccttttta cggttcctgg gcttttgctg gccttttgct cacatgttct t 4671

Claims (10)

1. for the nucleotide sequence of arch insect infection prevention, it contains and is selected from following nucleotide sequence:

1) nucleotide sequence shown in SEQ ID No.1 in sequence table;

2) with 1) shown in the nucleotide sequence of nucleotide sequence complementation;

3) to above-mentioned 1) or 2) shown in the nucleotide sequence replacement, disappearance, interpolation of carrying out one or more bases modify, and there is the nucleotide sequence of arch insect infection prophylactic function.

2. a recombinant protein, its aminoacid sequence is by claimed in claim 1 nucleotide sequence coded.

3. recombinant protein according to claim 2, is characterized in that: the aminoacid sequence of described recombinant protein is referring to SEQ ID No.2 in sequence table.

4. a recombinant vectors, it comprises nucleotide sequence claimed in claim 1.

5. a recombinant plasmid, its nucleotides sequence is classified the nucleotide sequence shown in SEQ ID No.3 in sequence table as.

6. a vaccine, it comprises nucleotide sequence claimed in claim 1, and medically acceptable carrier.

7. the recombinant protein described in nucleotide sequence claimed in claim 1, claim 2 or 3, recombinant vectors claimed in claim 4, recombinant plasmid claimed in claim 5, the application of vaccine claimed in claim 6 in arch insect infection prevention.

8. a preparation method for vaccine claimed in claim 6, step is as follows:

(1) extract the total RNA of RH strain of Toxoplasma gondii;

(2) the total RNA of RT-PCR amplification RH strain;

(3) amplified production in step 2 is connected with carrier;

(4) a small amount of of pVAX I plasmid is extracted;

(5) reclaim pVAX I carrier and object fragment HSP60;

(6) connect pVAX I carrier and object fragment HSP60;

(7) connection product is converted in Host Strains.

9. preparation method according to claim 8, is characterized in that: when the total RNA of described RT-PCR amplification RH strain, the primer is:

Upstream primer: 5 '-GGTACCATGCTTGCCCGCGCTTCAGC-3 ';

Downstream primer: 5 '-AAGGAAAAAAGCGGCCGCCTAGTACATGCCTCCCATGCCGC-3 '.

10. preparation method according to claim 8, is characterized in that: when the total RNA of described RT-PCR amplification RH strain, PCR reaction conditions is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 1min, 60.4 DEG C of renaturation 45s; 72 DEG C are extended 2min; 35 circulations, last, 72 DEG C are extended 10min.