CN108982849A - The application of EG-TPx and the kit for diagnosing domestic animal echinococcosis granulosa - Google Patents

The application of EG-TPx and the kit for diagnosing domestic animal echinococcosis granulosa Download PDF

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CN108982849A
CN108982849A CN201810587349.8A CN201810587349A CN108982849A CN 108982849 A CN108982849 A CN 108982849A CN 201810587349 A CN201810587349 A CN 201810587349A CN 108982849 A CN108982849 A CN 108982849A
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domestic animal
echinococcosis granulosa
tpx
sheep
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杨光友
梁雨琴
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Sichuan Agricultural University
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Abstract

Kit the invention discloses the application of Eg-TPx and for diagnosing domestic animal echinococcosis granulosa, Eg-TPx can be identified as immunizing antigen by the sheep positive serum of natural infection echinococcosis granulosa, when applied to indirect ELISA detection, with good immunogenicity, higher specificity and sensitivity, sensitivity is 92.6%, specificity 99.0%, lower with the cross reaction of other Taeniidae disease positive serums.There is good diagnosis effect with the ELISA detection method that immunizing antigen made from Echinococcus granulosus thioredoxin peroxidase is established, can be used for the preliminary screening of epidemic-stricken area cattle and sheep echinococcosis granulosa.

Description

The application of EG-TPx and the kit for diagnosing domestic animal echinococcosis granulosa
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Echinococcus granulosus thioredoxin peroxidase (Eg-TPx) application and the kit for diagnosing domestic animal echinococcosis granulosa.
Background technique
Echinococcosis granulosa is a kind of children of middle silk ribbon phase by Echinococcus granulosus (Echinococcus granulosus) Worm colonizes in a kind of Amphixenosis caused by a variety of mammalian livers, lungs etc..The disease is also known as capsule echinococcosis (hydatid disease) is in worldwide distribution, the serious development for having threatened public health service and animal husbandry.Currently, should Disease has infected about 3,000,000 people, and estimation causes damages 7.6 hundred million dollars every year.In addition, the disease causes at least 3,000,000,000 beauty in animal husbandry The problems such as economic loss of member is reduced including the weight of animals, and milk crop reduction and fertility-rate reduce.Therefore, the disease has been Through being classified as " ignored tropical disease " by the World Health Organization.
China is one of the country of echinococcosis Major Epidemic, and agriculture and animal husbandry of the Major Epidemic in the northwestward is regional, at present China Echinococcosis at least 368 popular counties.It is calculated according to scholar, echinococcosis burden in China's accounts for global disease burden 40%.Due to the disease Western, northern masses of farmers and herdsmen's bring heavy losses to China, which has been cited as China, and " long-term animal epidemic is anti-in country Control planning " (2012-the year two thousand twenty) preferentially prevent and treat and the Animal diseases of guard key.
It is examined currently, the monitoring of domestic animal echinococcosis and quarantine rely primarily on to visually observe after the ptomatopsia of slaughterhouse with serology Disconnected method.Due to not doing further histological characterization, only observe by the naked eye butchered animal pathological change rear and usually had it is higher Error rate (15.4%).However the report about the seriously ill group of diagnostic antigen is also less, and there are sensibility and specificities The problems such as not high, therefore, screen it is a kind of effectively, it is sensitive, special recombination diagnostic antigen and establish accurate diagnostic method and be One of focus on research direction for needing to break through from now on.
Summary of the invention
For the above-mentioned problems in the prior art, the present invention provides a kind of Echinococcus granulosus thioredoxin peroxide The application of compound enzyme (Eg-TPx) can be identified by the sheep positive serum of natural infection echinococcosis granulosa, be applied to When indirect ELISA reagent kit detects, there is good immunogenicity, higher specificity and sensitivity, reduce and other band sections The cross reaction of taeniasis positive serum.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
Echinococcus granulosus thioredoxin peroxidase is used to prepare to the recombination of detection domestic animal echinococcosis granulosa Antigen.
Further, domestic animal is cattle and sheep.
A kind of ELISA kit detecting domestic animal echinococcosis granulosa, including it is above-mentioned recombinant antigen, antigen coat liquid, dilute Release secondary antibody, TMB developing solution and the terminate liquid of liquid, cleaning solution, enzyme standard liquid, confining liquid, HRP label.
The preparation method of the recombinant antigen of above-mentioned detection domestic animal echinococcosis granulosa, comprising the following steps:
(1) design primer extracts Echinococcus granulosus total serum IgE, and reverse transcription carries out PCR at cDNA, and as template Amplification；
Wherein, upstream primer (TPx-F) sequence are as follows:
5'-CGCGGATCCATGGCTGCTGTTGTTGG-3'；
Downstream primer (TPx-R) sequence are as follows: 5'-CCGGAATTCTCACGAGCTCATGAACGA-3'；
(2) building and identification of recombinant plasmid；
(3) expression and purity of recombinant protein.
Further, PCR amplification system in step (1) are as follows: 0.5 μ L of PCR MasterMix, RNase Free dH2O 0.35 μ L, 0.5 μ L of cDNA template, 0.5 μ L of upstream primer, 0.5 μ L of downstream primer；Amplification program are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C denaturation 40s, 58 DEG C of annealing 45s, 72 DEG C of extension 45s, 35 recycle, last 72 DEG C of extensions 10min.
Further, step (2) detailed process are as follows: PCR product is connect with pMD19-T carrier, converts DH5 α competence Then cell, screening bacterium colony, culture, PCR identification are sequenced, errorless bacterium solution will be sequenced and expand culture, extract plasmid, to plasmid into Row digestion recycling, the purpose band of recycling are connect with expression vector pET32a (+), are converted, and are screened positive bacterium colony and are extracted plasmid, double Digestion is identified and is sequenced, and the recombinant expression plasmid for being sequenced errorless is transformed into e. coli bl21.
Further, step (3) detailed process are as follows: will expression bacterium be inoculated in the LB culture medium containing AMP, in 37 DEG C, IPTG inducing expression 5-6h is added after cultivating 5-6h under the conditions of 160r/min, the bacterium solution after collecting induction carries out SDS-PAGE electrophoresis Analysis is then centrifuged for collecting thallus, is resuspended with lysate, then ultrasound cracking, centrifugation, takes supernatant precipitating to carry out respectively soluble Analysis, and recombinant protein purification is carried out to the bacterium solution after induction.
Further, confining liquid is 5% skimmed milk power.
Further, the secondary antibody of HRP label is goat or the sheep anti-rabbit secondary antibody of HRP label.
The invention has the benefit that Echinococcus granulosus thioredoxin peroxidase (Eg- provided by the invention TPx) the application in the recombinant antigen of preparation detection domestic animal (especially cattle and sheep) echinococcosis granulosa can be used as immune anti- The enough sheep positive serums by natural infection echinococcosis granulosa of proper energy identify, when being applied to indirect ELISA detection, have good Immunogenicity, it is higher specificity and sensitivity, sensitivity 92.6%, specificity 99.0%, with other Taeniidaes The cross reaction of sick positive serum is lower.It follows that exempt from made from Echinococcus granulosus thioredoxin peroxidase The ELISA detection method that epidemic disease antigen is established has good diagnosis effect, can be used for the first of epidemic-stricken area cattle and sheep echinococcosis granulosa Step screening.
Detailed description of the invention
Fig. 1 is the PCR amplification gel figure of Eg-TPx gene.
The western blot that Fig. 2 is r Eg-TPx schemes；Wherein, M is Protein standards, and 1 is rEg- after purification TPx；2: the sheep positive serum of natural infection echinococcosis granulosa identifies rEg-TPx；3: healthy sheep serum identifies rEg- TPx。
Fig. 3 is indirect ELISA to the specificity analysis of rEg-TPx and clinical test results figure.
Specific embodiment
1 preparation and reorganization antigen of embodiment
1, the extraction of particulate spine tapeworm total serum IgE
The Echinococcus Granulosus Cysts for taking out Liquid nitrogen storage, are ground with mortar, are extracted referring next to the animal tissue RNA of Tiangeng Kit specification extracts total serum IgE.
2, the synthesis of the first chain cDNA
It is public referring to Thermo with Oligo dT (18) for reverse transcription primer using the Echinococcus Granulosus Cysts total serum IgE of extracting as template Department's reverse transcription reagent box specification is operated.
3, the amplification of Eg-TPx gene
According to the gene order of the Eg-TPx (EgrG_000791700) of announcement, 5.0 software of Primer Premier is utilized Design primer:
TPx-F:5'-CGCGGATCCATGGCTGCTGTTGTTGG-3'(SEQ ID NO:1), underscore BamHI；
TPx-R:5'-CCGGAATTCTCACGAGCTCATGAACGA-3'(SEQ ID NO:2), underscore EcoRI.
Amplification system (10 μ L): 0.5 μ L of PCR MasterMix, RNase Free dH20.35 μ L of O, cDNA template 0.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L of downstream primer；Amplification program are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 40s, 58 DEG C are annealed 45s, 72 DEG C of extension 45s, 35 circulations, last 72 DEG C of extensions 10min.
Amplification program are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 40s, 58 DEG C of annealing 45s, 72 DEG C of extension 45s, 35 are followed Ring, last 72 DEG C of extensions 10min.
To go out the band of 582bp or so using the cDNA of Echinociccus granulosus protoscolex as template amplification, such as Fig. 1 institute Show.
4, gene cloning, identification and conversion
(1) gene cloning is sequenced
PCR product is run into gel electrophoresis, then recycles target fragment, target fragment is connect with pMD19-T carrier, 4 DEG C Overnight.
Linked system (8 μ L) are as follows: 14 μ L of Solution, 3.5 μ L of DNA profiling, 0.5 μ L of PMD19-T carrier.
Take 8 μ L of connection product that 30 μ L DH5 α competent cells are added, soft to mix, ice-water bath 30min, 42 DEG C of heat shocks After then adding 600 μ L fluid nutrient mediums, 200r/min to cultivate 2h, the solid that bacterium solution is applied to Amp resistance is trained by 90s, ice bath 5min It supports on base, 37 DEG C of culture 12h.
Picking single bacterium colony is inoculated in LB meat soup, 37 DEG C of shaken cultivation 6h, is that template carries out bacterium solution PCR mirror with bacterium solution It is fixed, the bacterium solution of PCR test positive is sent to biotech firm's sequencing.
Sequence similarity through Eg-TPx (EgrG_000791700) in obtained sequence and GeneDB is sequenced reaches 100%.
(2) plasmid extracts
Sequencing result compare it is correct after, bacterial strain is expanded and is cultivated, small mentions reagent referring to the plasmid of Tiangeng biochemistry Co., Ltd Box operating instruction carries out:
1. taking 5mL bacterium solution, precipitating is collected by centrifugation；
2. suspension P1 suspension thalline is added, and lysate P2 is added, bacterium solution is cracked；
3. addition P1 generates precipitating, overturning is mixed, and is centrifuged 15min in 12,000r/min after standing 10min.
4. drawing supernatant into collecting pipe, add 600 μ L rinsing solution PW, stand 2min, is centrifuged in 12,000r/min 1min abandons waste liquid；
5. repeating 4.；
6. recycling plasmid with 75 μ L eluents after drying rinsing liquid, it is stored in -20 DEG C.
(3) plasmid enzyme restriction recycles
By the pMD19-T-Eg-TPx of extraction Takara BamH I and the fast enzyme cutting double digestion of Xho I, 37 DEG C of digestion 10min, After digestion products are carried out agarose gel electrophoresis, the single band of gel extraction.
(4) target fragment connects expression vector
The Eg-TPx target fragment and pET32a (+) carrier that double digestion is obtained are in 22 DEG C of connection 2h, conversion, gained bacterium solution PCR identification is carried out, while double digestion identification is carried out to recombinant plasmid.
5, the expression and purification of recombinant protein
(1) expression of recombinant protein
1. correct recombinant plasmid pET32a (+)-TPx, which will be sequenced, is transferred to E.coli BL21 (DE3) expression bacterium；
2. by expression bacterium be inoculated in one bottle of fresh LB (the 100 μ g/mL containing AMP) culture solution containing 20mL, in 37 DEG C, Then inducer IPTG (1mmol/L) is added in 160r/min shaking table culture 6h, induce 5h in 37 DEG C, 160r/min；
3. taking the bacterium solution 1.5mL after induction in two new EP pipes respectively, it is centrifuged under the conditions of 4 DEG C, 12,000r/min 1min collects thallus, is separately added into 10 μ L 5 × SDS loading Buffer and 40 μ L PBS solutions, mixes well；
4. boiling water boiling 10min takes supernatant to carry out SDS-PAGE after centrifugation；
5. observing expression after decoloration with coomassie brilliant blue staining 1h.
(2) soluble analysis of recombinant protein
It will be inoculated in fluid nutrient medium of the 500mL containing AMP containing the expression bacterium bacterium of recombinant plasmid pET32a (+)-TPx, 37 DEG C, 1700r/min cultivate to bacterium solution muddiness, be added best IPTG concentration, induce 6h.By bacterium solution under the conditions of 8000r/min It is centrifuged 10min, precipitating is suspended with lysate (20mM Tris-HCl, pH8.0), ultrasonication thallus.Broken thallus is split Solution liquid is centrifuged 10min, precipitation and separation and supernatant under the conditions of 4 DEG C, 12,000r/min；Precipitating is added molten in suitable 8M urea Solution.Supernatant precipitating respectively takes 40 μ L, respectively plus 10 μ L ddH2O, boils 10min, and 12,000r/min centrifugation 10min are carried out SDS-PAGE electrophoresis, whether analysis rEg-TPx is solubility expression.
(3) purifying of recombinant protein
According to above-mentioned steps to recombinant bacterium inducing expression, a large amount of recombinant protein bacterium solutions are obtained, are then proceeded as follows:
1. taking 1L bacterium solution, 8,000r/min centrifugation 10min abandon supernatant, collect thallus, lysate is added；
2. ultrasonic disruption, until all bacterial cell disruptions are complete；
3. 12,000r/min centrifugation 15min, stay supernatant；
4. by sample after 0.22 μm of membrane filtration loading；
5. it is steady to be washed till baseline with Binding Buffer；
6. using 50mM, 100mM, 200mM respectively, 300mM, 400mM imidazole elution is eluted, and collects eluting peak；
7. being 0 with 20% ethanol washing to ion concentration after being cleaned with 400mM imidazole elution, pillar is protected in 4 DEG C It deposits；
8. albumen is carried out ultrafiltration and concentration with super filter tube, the displacement that PBS carries out solution is repeatedly added, it is original to remove Imidazoles, and albumen after purification is stored in -80 DEG C；
9. after protein concentration, carrying out SDS-PAGE inspection, and with BCA quantification of protein kit measurement protein concentration.
As a result: Eg-TPx is successfully connected on pET-32a carrier, and conversion enters inducing expression in BL21 Escherichia coli, table The recombinant protein size reached is 39kDa or so, meets expected size；Soluble analysis is the results show that rEg-TPx expression is can Dissolubility albumen；Recombinant protein after purification is single band.
6, Analysis of Immunogenicity
It is analyzed using Western blot, concrete operations are as follows:
1. preparing protein electrophorese gel, SDS-PAGE electrophoresis is carried out to albumen after purification；
2. after protein electrophorese, take the corresponding gel position where protein, be put into transferring film buffer carry out it is flat Weighing apparatus, totally 3 times, each 4min；
3. nitrocellulose filter (NC film) and 24 layers of filter paper to be placed in transfering buffering liquid and impregnate 5min；
4. cathode electrode plate, 24 layers of filter paper, gel, NC film, 24 layers of filter paper are placed in the transfer of Bio-Rad half dry type in order In slot, anode electrode plate, 35mA transferring film 30min are covered；
After 5., NC film is taken out, is immersed in the TBST of 3%BSA, 4 DEG C of closings are overnight；
6. after closing, cutting off NC film, the negative and positive is separated, and the diluted sheep positive serum of 1:200 is added, After being incubated at room temperature 2h, primary antibody is outwelled, is quickly washed with TBST film 3 times, 5min/ times；
7. NC film is added after the IgG of HRP label is diluted by 1:1000, after being incubated at room temperature 2h, secondary antibody is outwelled, is washed；
8. NC film is placed in plate, rinsed with fresh substrate developing solution until developing the color；
9. after colour developing, rinsing NC film color development stopping with distilled water, and photograph to record to result.
Immunoblotting shows that rEg-TPx can be identified by echinococcosis granulosa positive sheep serum, and is single band (figure 2), illustrate that rEg-TPx has preferable immunogenicity.
The foundation of 2 indirect ELISA method of embodiment
1, indirect ELISA operating procedure
(1) it is pressed with antigen coat liquid and dilutes recombinant antigen rEg-TPx than column, every 100 μ L of hole is added in 96 ELISA Plates and carries out Coating；
(2) coating buffer is outwelled, liquid in hole is patted dry, is washed with PBST, each 5min is repeated four times；
(3) confining liquid closing is added；
(4) with after PBS in proportion dilute serum after washing, every 100 μ L of hole is added enzyme mark hole and is incubated for, and outwells liquid；
(5) goat or the sheep anti-rabbit secondary antibody for the HRP label that 100 μ L have diluted is added in every hole after washing, and is incubated for；
(6) solubility one pack system substrate TMB is added into hole under the conditions of being protected from light and carries out chromogenic reaction；
(7) 100 μ L 2M H are added in hole2SO4Reaction is terminated, measures its OD value when ultraviolet absorptivity is 450nm；
(8) liquid is outwelled, after patting dry, is washed with PBST, each 5min is repeated four times；
(9) liquid is outwelled, after patting dry, color reaction is carried out under the conditions of being protected from light, soluble one pack system bottom is added into hole Object TMB, every 100 μ L of hole are incubated at room temperature 10-20min；
(10) 100 μ L 2M H are added into hole again2SO4Reaction is terminated, 96 orifice plates are placed in microplate reader immediately, ultraviolet Absorbance measures its OD value when being 450nm.
2, best operating condition is determined
(1) best antigen and serum diluted concentration are determined with Checkerboard titration method, 6 antigen concentration gradients are set, serum from 1:20 to 1:640 makees doubling dilution, using the maximum condition of P/N as most preferably.
(2) 1%BSA, 5%BSA, 1% skim milk are used in the determination of best confining liquid respectively, and 5% skim milk is closed, Using the maximum condition of P/N as most preferably.
(3) most preferably incubating for positive and negative serum is screened in the determination of positive and negative seroreaction time according to determining condition The time is educated, 3 groups of 37 DEG C of 0.5h, 1h, 1.5h are set as, using the maximum concentration of P/N as optimum reacting time.
(4) determination of the optium concentration of secondary antibody effect sets 1:2000,1:3000,1:4000,1:5000 tetra- dilutions respectively Concentration is groped, using the maximum condition of P/N as optium concentration.
The results show that it is the 0.9 every hole μ g/ that P/N value, which reaches highest antigen optium concentration, serum most preferably dilutes dense ELISA Degree is 1:40, and best sealing condition is 5% skimmed milk power, and it is 37 DEG C of 60min that serum, which is incubated for Best Times, and secondary antibody most preferably dilutes dense Degree is 1:3000.
3, the determination of critical value
At optimum conditions, the OD of 24 parts of sheep echinococcosis granulosa negative serums is measured450.Three repetitions are set, according to Critical value=+ 3 times of average value standard deviation calculates.
With the OD of 24 parts of sheep negative serum samples450It is worth and determines that critical value calculates 24 parts of silk flosses by statistical analysis Sheep negative serum sample OD450The average value of value is 0.619, standard deviation 0.058.According to calculation formula: critical value=feminine gender sample This OD450+ 3 times of standard deviations of average value show that critical value is 0.792, i.e. OD450When > 0.792, theoretically it can determine that as the positive, OD450< 0.792 can determine that as feminine gender.
4, repetitive test between being criticized in criticizing
The coated plank of same batch is taken, 5 parts of detection is known as the sheep serum of the echinococcosis granulosa positive, every part of setting 3 repeating holes carry out batch interior repetition and test according to the ELISA method having built up.
Take 3 coated planks of batch, at optimum conditions, detect the sheep serum of 3 parts of echinococcosis granulosa positives, often Part 3 repeating holes of setting carry out repeating to test between criticizing according to the ELISA method having built up.The coefficient of variation is calculated, detection should The repeatability between criticizing interior criticize of method.
The results show that the coefficient of variation in plate is between 0.354%-0.874%, average value is repetitive test in batch 0.564%；Repetitive test is the results show that between 0.453%-1.23%, average value is the coefficient of variation between plate between batch 0.815%.
5, clinical test
27 parts of echinococcosis granulosa positive sheep serums are used to detect each egg respectively with the indirect ELISA method that the present invention establishes White sensibility；Afterwards with 21 parts of cysticercus tenuicollis goat positive serums, 20 parts of cenurus cerebralis goat positive serums, 15 parts of not Buddhist nuns time Tapeworm sheep positive serum, 16 parts of haemonchus contortus goat positive serums, 7 parts of Fasciola hepatica goat positive serums and 24 parts are thin Grain hydatidosis feminine gender sheep serum detection specificity；60 parts of lowlenthal serums that Eg95 vaccine has been immunized finally are detected again.
Control group: with two commercial reagents boxes to above-mentioned cross reaction serum, 27 parts of sheep positive serums and 30 parts it is immune The lowlenthal serum of Eg95 vaccine is detected.
Calculation formula: sensibility=ELISA detects number positive/analyses detection number positive * 100%；
Specificity=ELISA detects negative number/analyses the negative number * 100% of detection.
Detected using the indirect ELISA method established of the present invention, testing result as shown in figure 3, as the result is shown with mountain Sheep brain Echinococcus hydatid cyst serum has 1 cross reaction, 24 parts of negative serum OD450Respectively less than critical value, specificity are 99.0%；27 parts of silk flosses The OD of sheep echinococcosis granulosa positive serum450It is all larger than critical value, sensibility is up to 92.6%.
Clinical inspection is carried out to above-mentioned serum (27 parts of positive serums, 79 parts of cross reaction serum) with two kinds of commercial reagents boxes It surveys, kit A and sheep moniezia positive serum have 1 cross reaction as the result is shown, positive with goat cysticercus tenuicollis Serum has 3 cross reactions, has 4 cross reactions with goat haemonchus contortus positive serum, with goat brain Echinococcus hydatid cyst positive blood There are 3 cross reactions clearly, therefore, specificity is 81.0%, and to having in the 30 parts of sheep serum for being immunized Eg95 detections 11 cross reactions, coincidence rate only have 63.3%；In to 27 parts of sheep echinococcosis granulosa positive serum detections, 19 parts aobvious It is shown as positive, therefore its sensibility is 70.4%；Kit B and sheep moniezia positive serum have 7 cross reactions, with Goat cysticercus tenuicollis positive serum has 8 cross reactions, has 6 cross reactions with goat haemonchus contortus positive serum, There are 4 cross reactions with goat brain Echinococcus hydatid cyst positive serum, there are 3 cross reactions with Fasciola hepatica positive serum, it is therefore, special Property be 64.6%, and to having 22 cross reactions in the 30 parts of sheep serum for being immunized Eg95 detections, coincidence rate only has 26.7%；In to 27 parts of sheep echinococcosis granulosa positive serum detections, 21 parts are shown as positive, therefore its sensibility is 77.8%.
Sequence table
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<120>application of EG-TPx and the kit for diagnosing domestic animal echinococcosis granulosa
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ccggaattct cacgagctca tgaacga 27

Claims (9)

1. Echinococcus granulosus thioredoxin peroxidase is in the recombinant antigen of preparation detection domestic animal echinococcosis granulosa Application.
2. application according to claim 1, which is characterized in that domestic animal is cattle and sheep.
3. detecting the ELISA kit of domestic animal echinococcosis granulosa, which is characterized in that anti-including recombination as claimed in claim 1 or 2 Secondary antibody, TMB developing solution and the terminate liquid of original, antigen coat liquid, dilution, cleaning solution, enzyme standard liquid, confining liquid, HRP label.
4. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 3, which is characterized in that described heavy Group antigen is prepared by the following method to obtain:
(1) design primer extracts Echinococcus granulosus total serum IgE, and reverse transcription carries out PCR expansion at cDNA, and as template Increase；
Wherein, upstream primer sequence are as follows: 5'-CGCGGATCCATGGCTGCTGTTGTTGG-3'；
Downstream primer sequence are as follows: 5'-CCGGAATTCTCACGAGCTCATGAACGA-3'；
(2) building and identification of recombinant plasmid；
(3) expression and purity of recombinant protein.
5. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 4, which is characterized in that step (1) Middle PCR amplification system are as follows: 0.5 μ L of PCR MasterMix, RNase Free dH20.35 μ L of O, 0.5 μ L of cDNA template, upstream 0.5 μ L of primer, 0.5 μ L of downstream primer；Amplification program are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 40s, 58 DEG C of annealing 45s, 72 DEG C extend 45s, 35 circulation, last 72 DEG C of extensions 10min.
6. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 4, which is characterized in that step (2) Detailed process are as follows: PCR product is connect with carrier, transformed competence colibacillus cell, then screening bacterium colony, culture, PCR identification are sequenced, Errorless bacterium solution will be sequenced and expand culture, extract plasmid, digestion recycling, the purpose band and expression vector of recycling are carried out to plasmid Connection, conversion screen positive bacterium colony and extract plasmid, and double digestion is identified and is sequenced, and errorless recombinant expression plasmid will be sequenced and convert Into Escherichia coli.
7. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 4, which is characterized in that step (3) Detailed process are as follows: expression bacterium is inoculated in the LB culture medium containing AMP, adds after 5-6h is cultivated under the conditions of 37 DEG C, 160r/min Enter IPTG inducing expression 5-6h, the bacterium solution after collecting induction carries out SDS-PAGE electrophoretic analysis, is then centrifuged for collecting thallus, with splitting It solves liquid to be resuspended, then ultrasound cracking, centrifugation, takes supernatant precipitating to carry out soluble analysis respectively, and carry out to the bacterium solution after induction Recombinant protein purification.
8. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 3, which is characterized in that the envelope Closing liquid is 5% skimmed milk power.
9. the ELISA kit of detection domestic animal echinococcosis granulosa according to claim 3, which is characterized in that the HRP The secondary antibody of label is goat or the sheep anti-rabbit secondary antibody of HRP label.
CN201810587349.8A 2018-06-06 2018-06-06 The application of EG-TPx and the kit for diagnosing domestic animal echinococcosis granulosa Pending CN108982849A (en)

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CN110655564A (en) * 2019-10-29 2020-01-07 四川农业大学 Combined protein and application thereof
CN111796090A (en) * 2020-04-21 2020-10-20 沈阳农业大学 Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof
CN111796090B (en) * 2020-04-21 2023-02-07 沈阳农业大学 Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof

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Application publication date: 20181211