CN106556693A - A kind of toxoplasma series connection multi-epitope gene ELISA detection kit - Google Patents
A kind of toxoplasma series connection multi-epitope gene ELISA detection kit Download PDFInfo
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- 241000223996 Toxoplasma Species 0.000 title claims abstract description 42
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- 102100024017 Glycerol-3-phosphate acyltransferase 3 Human genes 0.000 claims description 43
- 101000904259 Homo sapiens Glycerol-3-phosphate acyltransferase 3 Proteins 0.000 claims description 43
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/45—Toxoplasma
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The multi-epitope gene ELISA detection kit the present invention relates to a kind of toxoplasma is connected, it is characterised in that：After serum diluted sample to be checked, the holes of 100ul to 96 elisa plate is added（1）In 37 DEG C of incubation 1h, positive control, negative control and blank are set.Cleaned with cleaning mixture 5 times, each 3min；37 DEG C of effect 1h of rabbit-anti sheep IgG HRP conjugates are subsequently adding, cleaning mixture is cleaned 5 times, each 3min；Chromogenic Substrate Solution is subsequently adding, by substrate A and substrate B with 1:1 mixing, during 100ul adds ELISA holes, lucifuge colour developing 15min adds 50ul terminate liquids, surveys its OD490 value.Result of determination is so setting：Suppression ratio PI=(OD negative control OD samples)/OD negative controls * 100%, the corresponding sample of PI >=50% for the positive, less than then for feminine gender.The kit method technology maturation, repeatability are strong, the false positive occurred in the detection that can effectively reduce toxoplasma and nonrepeatability, and general Study personnel can complete.
Description
Technical field
The present invention relates to a kind of new Toxophasma gondii detecting kit, more particularly to a kind of toxoplasma series connection multi-epitope base
Because of ELISA detection kit, which can improve detection efficiency, belong to a kind of new animal epidemic diagnostic reagent, be applied to herding
Veterinary field.
Background technology：
Toxoplasmosiss are a kind of protozoacide of the pandemic infecting both domestic animals and human of global range caused by toxoplasma, and the whole world is at least
There is 1/3 population infection toxoplasma.The cell category of toxoplasma invasion and attack is more, adds up organ extensively, in addition individual variation, clinical manifestation
It is very greatly different.Toxoplasmosiss are broken out in swinery, and case fatality rate may be up to 60%.The prevalence of poultry toxoplasmosiss, on the one hand because of miscarriage
Or death causes heavy economic lossess to animal husbandry, animal husbandry development is had a strong impact on;Another aspect toxoplasma Jing meat, breast, eggs
It is a large amount of to flow into food products market, become the source of infection of the mankind.The rise of pet craze and house pet (cat and dog etc.) to the susceptible of toxoplasma, more
The prevalence of arch insect infection is increased and has propagated.It is reported that arch insect infection can make the abortion ratio of pig, sheep reach 20-30.7%,
And make " innominate high fever " of the large quantities of death of infected pigs, the live pig of China's last century the seventies to cause large quantities of death of pig, Jing
Diagnosis is by caused by arch insect infection.Shandong, Henan, Zhejiang, Gansu etc. save the toxoplasmosiss for having broken out pig in recent years, special
It is not nearly 2 years toxplasmosis in pigs and high pathological form reproductive and respiratory syndrome mixed infection, causes large quantities of death of pig；Four, county of Gansu Province
There is the miscarriage of the toxoplasma of sheep in small towns, only sheep miscarriage is lost up to more than 3,000,000 in recent years every year；Cause seriously to animal husbandry
Loss.
Different types of detection kit that the country has developed is obtained in the mankind, epizootiology investigation and medical diagnosis on disease
Application to a certain extent, but the sensitivity, specificity and the repeatability that detect are still not ideal enough, with external ELISA kit
Recombination rate be only 60% or so, lack clinical fast diagnosis method.As the host range of toxoplasma is extensive and life cycle multiple
It is miscellaneous, effective detection is unable to antibody with the elisa plate of single antigen coat, and there is false positive and false negative.Toxoplasma
SAG1 albumen is the specific proteinses positioned at toxoplasma tachyzoite surface, only occurs in the tachyzoite phase of toxoplasma, therefore is anxious
One of property typical diagnostic antigen gene of infective stage；And toxoplasma MAG1 is a kind of egg positioned at tissue encapsulation substrate position
In vain, can only be detected in the encapsulation extract of bradyzoite stage, therefore MAG1 is the important of Chronic Infection of Toxoplasma stage
Diagnosis candidate antigens；Toxoplasma Microneme protein（MIC）Intersperse among around polypide leading portion clavas, with polypide to host cell
Identification with combine relevant, wherein MIC1 genes are single copy, containing single open reading frame, are that the candidate with potential value is anti-
It is former.As MAG1, SAG1 and MIC1 have excellent immunogenicity, it is capable of the antibody of stimulation of host generation high titre, is not only
Host provides immunity protection, can also provide candidate albumen for the immunology detection of toxoplasma.For this purpose, carrying out to toxoplasmosiss
Research, the especially foundation of the quick detection kit of toxoplasma multi-epitope gene effectively push tool to the sick preventing and controlling
It is of great importance.
The content of the invention：
The multi-epitope gene ELISA detection kit it is an object of the invention to provide a kind of toxoplasma is connected, which utilizes insoluble enzyme
Linked immunosorbent adsorption test（ELISA）Principle, think series connection MIC1, MAG1, SAG1 gene expression purifying protein be diagnostic antigen and
The rabbit-anti sheep IgG of horseradish peroxidase-labeled and other matched reagents are constituted.Its principle is the antibody test using enzyme labelling
Combined with solid phase antigen by inspection antibody；For carrying out the Epidemiological study of toxoplasmosiss, it is beneficial to strengthen the raising of drove
Management and the monitoring to positive flock of sheep and preventing and treating, ensure that people's food safety and aquaculture develop in a healthy way；With it is simple, quick,
The features such as sensitive and specificity is good；Suitable for clinical toxoplasma high efficient detection.
The technical scheme is that what is be achieved in that：A kind of toxoplasma series connection multi-epitope gene ELISA detection kit,
Including 96 hole elisa plates, sample diluting liquid, 20 times of concentrated cleaning solutions, rabbit-anti sheep IgG-HRP conjugates, series connection MIC1, MAG1,
SAG1 gene expression purifying proteins, terminate liquid, substrate A, substrate B, positive, negative sample, cover plate film；It is characterized in that：
1）96 hole elisa plates are that series connection MIC1, MAG1, SAG1 isogeneity albumen is diluted to 10ug/mL, are coated with per hole 100ul
Elisa plate, 37 DEG C of incubation 1h add 5% BSA confining liquids, 37 DEG C of closing 1h, Vacuum Package after washing after washing；
2）Sample diluting liquid is by NaCl 8.0g；KH2PO4 0.2g；Na2HPO4 ·12H2O 2.9g；KCl 0.2g add two and evaporate
Water causes 1000ml;Add 0.5ml Tween-20, last 50ml subpackages；
3）20 times of concentrated cleaning solutions are by NaCl 160.0g；KH2PO4 4g；Na2HPO4 ·12H2O 58g；KCl 4g add two
Distilled water causes 1000ml;Add 10ml Tween-20, last 50ml subpackages；
4）Rabbit-anti sheep IgG-HRP conjugates are to add 5ul rabbit-anti sheep IgG-HRP bis- to resist 10ml PBST, are subsequently adding 4%PEG,
25ml subpackages；
Series connection MIC1, MAG1, SAG1 gene expression purifying protein is according to toxoplasma MIC1, MAG1, SAG1 sequences in GenBank
Row, design specific primer, and according to toxoplasma MIC1, MAG1, SAG1 sequences in GenBank, design specific primer is as follows：
1. MIC1
-F:GGATCCGCGTCGCATTCTCATTCG BamH I, 24bp, 18, GC% 58.3, Tm 74.2
-R:GAATTCGGCCTTCTCGTAACACCTCC EcoR I,,26bp, GC%53.8, Tm 69.5
2. MAG1
-F:GAATTCAGCCAAAGGGTGCCAGAG EcoR I ,24bp, GC%54.2, Tm 69.0
-R:AAGCTTAGATCCCTGAACCCTTAGAATATACAC Hind III, 33bp, GC% 39.4, Tm 65.5
3. SAG1
-F:AAGCTTGATCCCCCTCTTGTTGCC Hind III, 24bp, GC%54.2, Tm69.4
-R:CTCGAGAAGAGTGCTGTCTGCACCGT Xho I , 26bp, GC%57.7,Tm 71.1
Expanded by RT-PCR technology and expand respectively toxoplasma MIC1, MAG1, SAG1 gene, arch is expanded by RT-PCR technology
Purpose fragment is cloned into carrier T, the identification of Jing enzyme action and sequencing analysis by worm MIC1, MAG1, SAG1 gene, as a result proves to obtain
MIC1, MAG1, SAG1 gene；Multi-epitope gene is connected with prokaryotic expression carrier pET-28a, is converted into escherichia coli,
Induced with IPTG, Jing SDS-PAGE and Western blotting analysis MIC1, MAG1, SAG1 albumen；Using Ni-NTA to table
Purification is carried out up to albumen, the purifying protein of ideal concentration is obtained；Terminate liquid is by 2mol/L H2SO4 Concentrated sulphuric acid 44.5ml, double steamings
Water 355.5ml, 10ml subpackage；
5）Substrate A：TMB 200mg, dehydrated alcohol 100ml, plus distilled water is to 1000ml, 10ml subpackages；
7）Substrate B：（0.1ml/L citric acid -0.2ml/L phosphoric acid hydrogen two is received, pH5.0-5.4）:Na2HPO414.60g, citric acid
9.33g, 0.75% hydrogen peroxide urea 6.4ml, plus tri-distilled water is to 1000ml, is adjusted to pH5.0-5.43,10ml subpackages；
8）Positive：By Standard arch wire worm positive serum 1:5 are diluted in sample diluting liquid, 1ml subpackages；
9）Negative sample：To toxoplasma negative serum 1 after testing:5 are diluted in sample diluting liquid, 1ml subpackages.
Coating series connection MIC1, MAG1, SAG1 gene expression purifying protein on 96 described hole elisa plates, with coating buffering
Liquid is diluted to 10ug/mL, and with the closing 1h of the PBS containing 5% defatted milk powder after 37 DEG C of coating 1h, PBS uses aluminium foil after washing 5 dryings
Vacuum sealing.
The working concentration of described rabbit-anti sheep IgG-HRP conjugates is diluted for 2000 times.
The positive effect of the present invention is：
1st, with biological safety, series connection MIC1, MAG1, SAG1 gene expression purifying protein which is used is antigen, does not contain
Toxoplasma, therefore there is no the danger for dissipating poison.
2nd, high specificity, detection antibody MIC1, MAG1, SAG1 are the preferable antibody of Sensitivity and Specificity, therefore are improved
The Sensitivity and Specificity of test kit.
3rd, detection efficiency can be improved using polygenes antigen, false sun is there may be with the elisa plate of single antigen coat
Property and the problems such as false negative, repeatable difference.This test kit can be applicable to the purification and Epidemiological study of plant.It is prepared
Albumen include three kinds of representational toxoplasma cdnas, can efficiently reduce and extensively and be lived by the host range of toxoplasma
The relatively low problem of detection efficiency caused by history is complicated.
Description of the drawings
Fig. 1 series connection MIC1, MAG1, SAG1 Primary structures, wherein 1. series connection MIC1, MAG1, SAG1 genes induction tables
Up to 2. empty carrier M. low molecular weight protein (LMWP) Marker of albumen.
Fig. 2 series connection MIC1, MAG1, SAG1 gene western blot detections, wherein M. low molecular weight protein (LMWP) Marker 1.
Series connection MIC1, MAG1, SAG1 gene expression albumen.
Fig. 3 series connection MIC1, MAG1, SAG1 Primary structure purification, wherein 1. purifying proteins, M. low molecular weight protein (LMWP)
Marker。
The optimal anti-and two anti-working concentrations of Fig. 4 ELISA.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and examples：Experimental technique in below example,
If no special instructions, it is conventional method.
The clone of 1 toxoplasma MIC1, MAG1, SAG1 gene of embodiment
According to toxoplasma MIC1, MAG1, SAG1 sequences in GenBank, design specific primer is as follows：
1. MIC1
-F:GGATCCGCGTCGCATTCTCATTCG BamH I, 24bp, 18, GC% 58.3, Tm 74.2
-R:GAATTCGGCCTTCTCGTAACACCTCC EcoR I,,26bp, GC%53.8, Tm 69.5
2. MAG1
-F:GAATTCAGCCAAAGGGTGCCAGAG EcoR I ,24bp, GC%54.2, Tm 69.0
-R:AAGCTTAGATCCCTGAACCCTTAGAATATACAC Hind III, 33bp, GC% 39.4, Tm 65.5
3. SAG1
-F:AAGCTTGATCCCCCTCTTGTTGCC Hind III, 24bp, GC%54.2, Tm69.4
-R:CTCGAGAAGAGTGCTGTCTGCACCGT Xho I , 26bp, GC%57.7,Tm 71.1
Expanded by RT-PCR technology and expand respectively toxoplasma MIC1, MAG1, SAG1 gene, reaction system is as follows：
RT reaction systems：
42 DEG C of 60min, 95 DEG C of 5min, 4 DEG C of 1-5min.
PCR reaction systems：
95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 50s, after 30 circulations, 72 DEG C of 10min, 4 DEG C of preservations.
Purpose fragment is cloned into into carrier T, is attached Jing after enzyme action identification, it is as follows to finally give genes of interest,
Through sequencing analysis, as a result prove to obtain MIC1, MAG1, SAG1 gene of series connection.
Series connection MIC1-MAG1-SAG1 sequences：（477bp+516bp+450bp=1443bp）
gcgtcgcattctcattcgccggcatcgggacgttatatacaacagatgcttgaccaacgctgccaagagattg
ctgcagaactctgccaaagcggacttcgtaaaatgtgtgtgccctctagccggatagtagctcgaaacgccgtgggc
attactcatcaaaatacacttcaatggagatgctttgatacagcctctttgctggagagcaatcaagaaaacaacgg
tgttaattgcgtggacgactgtggccacacgataccgtgtcctggcggcgtacaccggcaaaacagtaatcacgcaa
cgcgccatgagatactgtccaaattggtcgaagaaggagtacaacggttctgcagtccttatcaagcatctgccaac
aagtactgtaacgacaaatttccagggaccattgcgaggaggtcgaagggtttcggaaacaatgtcgaggttgcgtg
gaggtgttacgagaaggccagccaaagggtgccagagctaccagaagtggagtcctttgatgaagtaggcacgggag
ctcgacggtccgggtccattgcgacccttcttccacaagacgctgntttatatgagaactcagaggacgttgccgtt
ccgagtgattcagcatcgaccccgtcatactttcatgtggaatctccaagtgctagtgtggaagccgcgactggcgc
ggtgggagaggtggtgccgnactgtgaagaacgacaggaacagggtgacacgacgttatccgatcacgatttccatt
caggtggaactgaacangagggtttgccggaaacagaggtggcgcatcagcatgagacagaagaacagtacgggact
gaagggatgcccccccctgttctgccacctgcaccggtagtccatccgcgttttattgcagtaccagggccgtcggt
gcctgttccatttttcagtttgccagacatccacccggatcaggttgtgtatattctaagggttcagggatctgatc
cccctcttgttgccaatcaagttgtcacctgcccagataaaaaatcgacagccgcggtcattctcacaccgacggag
aaccacttcactctcaagtgccctaaaacagcgctcacagagcctcccactcttgcgtactcacccaacaggcaaat
ctgcccagcgggtactacaagtagctgtacatcaaaggctgtaacattgagctccttgattcctgaagcagaagata
gctggtggacgggggattctgctagtctcgacacggcaggcatcaaactcacagttccaatcgagaagttccccgtg
acaacgcagacgtttgtggtcggttgcatcaagggagacgacgcacagagttgtatggtcacggtgacagtacaagc
cagagcctcatcggtcgtcaataatgtcgcaaggtgctcctacggtgcagacagcactctt
The expression and purification of series connection MIC1, MAG1, SAG1 gene protein of embodiment 2
Multi-epitope gene is connected with prokaryotic expression carrier pET-28a, is converted to escherichia coli using DE3 competent cells
In, by be accredited as the positive recombinant bacterium by 1:100 ratios are inoculated in the LB culture fluid of 50ml, and 37 DEG C of 200r/min are used after 3h
The IPTG abduction deliverings of final concentration of 1mM, analyze expression conditions with SDS-PAGE and Western blotting.Such as Fig. 1,
Shown in Fig. 2, it is consistent with expection.Then recombiant protein is produced in a large number, the 12000g centrifugations Jing after ultrasonication by the thalline collected
10min, cracking supernatant are used for ni-sepharose purification recombiant protein.According to Sangon Biotech (Shanghai) Co., Ltd. Ni-
NTA-Sefinose Column description carries out purification to expressing protein, and the purifying protein for obtaining ideal concentration is as shown in Figure 3.
The foundation of 3 indirect ELISA method of embodiment
1. the foundation of response procedures：
Coating：Series connection MIC1, MAG1, SAG1 gene expression purifying protein is diluted to into 10 μ g/ml with pH9.6 carbonic acid buffers,
Added in ELISA Plate with the amount of 100 μ L of every hole, 4 DEG C overnight.Drying coating buffer, PBST (PBS containing 0.05%Tween-20,
PH7.2) board-washing 3 times, 200 μ L/ holes, 5min/ time.Closing：Add 5% skim milk confining liquid, 200 μ L/ holes, 37 DEG C of 60min,
Dry, cleaning mixture is washed 3 times.Add serum to be checked：After diluting serum to be checked in proportion, per 100 μ L of hole, 37 DEG C of effects 60
min；Cleaning mixture is washed 3 times.Add ELIAS secondary antibody：The rabbit-anti sheep two of HRP labellings is added to resist, per 100 μ L of hole, 37 DEG C of effects 45
min；Get rid of liquid, board-washing.Colour developing：Tmb substrate liquid is added, per 100 μ L of hole, room temperature acts on 15 min；Add 1 mol/L
H2SO450 μ L terminating reactions, determine OD490nm values.Decision method：Suppression ratio PI=(OD negative control-OD samples)/OD is negative
The corresponding sample of control * 100%, PI >=50% is the positive, less than being then feminine gender.
2. the determination of antigen and serum best effort concentration:
Doubling dilution is done as antigen with series connection MIC1, MAG1, SAG1 gene recombinant protein for determining concentration, concentration is respectively
40ug/mL, 20ug/mL, 10 ug/mL, 5g/mL, 2.5 ug/mL, 100ul/ holes, 4 DEG C of coated elisa plates are overnight.Positive and negative blood
It is clear also to do serial doubling dilution 1 simultaneously：20, l：40, l：80,1：160, square formation is constituted, is carried out indirectly by above-mentioned response procedures
ELISA.Sample OD values are determined at 490 nm wavelength on enzyme-linked detector.Positive serum OD values are selected 1 or so and positive OD
Antigen coat concentration and antibody dilution when value/feminine gender OD values ratio is maximum is best effort concentration.It is determined that optimal antigen bag
It is 1ug/mL by concentration.Qualification result Fig. 3.
3. the determination of the two anti-best effort concentration of rabbit-anti sheep of HRP labellings：
Indirect ELISA is carried out with fixed envelope antigen and the most suitable working concentration of serum, is done the rabbit-anti sheep two of HRP labellings anti-
Dilute in various degree, indirect ELISA is carried out by above-mentioned response procedures.Sample OD values are determined at 490 nm wavelength.OD values are selected 1
The two anti-dilution factor of rabbit-anti sheep of HRP labellings when left and right and maximum positive OD values/feminine gender OD value ratio is best effort concentration.Really
The two anti-best effort concentration of rabbit-anti sheep for determining HRP labellings is diluted for 2000 times.
4. the determination of confining liquid and action time：
Indirect ELISA is carried out with the most suitable working concentration of fixed antigen, antibody and ELIAS secondary antibody.Respectively with containing 2% defatted milk
The PBST of powder, the PBST of 5% defatted milk powder, 10% serum, the PBST of 0.05%Tween-20 are used as the confining liquid for reacting.37 DEG C points
Feng Bi not 30min, 1h, 2h.Each sample OD values are determined at 490 nm wavelength.Compare the OD490 values and P/N values of each group, it is determined that
The most suitable confining liquid of ELISA reactions and off-period.The PBST of 5% defatted milk powder is selected to act on 1h for optimal confining liquid and effect
Time.
5. sensitivity testss
The determination of criterion：Take 20 parts of Jing Toxoplasma Gondi IgG antibody detection kit and be detected as the positive serum of toxoplasma antibody,
Determine OD490nm values by indirect ELISA being carried out after 1: 100 dilution, it is stipulated that add 3 times with the average OD490nm of 20 parts of serum
Marginal value of the standard deviation as yin and yang attribute.
6. specific test
This method detects that positive serum carries out ELISA detections simultaneously with neospora, coccidiosiss, giardia lamblia, healthy animal serum.Knot
Fruit explanation and these viral no cross reactions.
7. repeatability test
Take the coated ELISA Plate of 4 pieces of different batches, each dilution factor sets 4 repetitions, carry out in same ELISA Plate batch in weight
It is multiple, repeat between carrying out criticizing between different ELISA Plate, determine OD values, calculate its coefficient of variation, if the coefficient of variation<10% is said
Bright its repeatability and stability are fine.The ELISA Plate of 4 different batches, as a result unanimously.Illustrate the repeatability of detection method preferably.
The assembling of 4 ELISA kit of embodiment
By test kit material used in the blocking ELISA method for establishing：96 hole elisa plates（1）, sample diluting liquid（2）、20
Times concentrated cleaning solution（3）, rabbit-anti sheep IgG-HRP conjugates（4）, series connection MIC1, MAG1, SAG1 gene expression purifying protein（5）, eventually
Only liquid（6）, substrate A(7), substrate B(8), positive（9）, negative sample（10）, cover plate film（11）Respectively with corresponding wide mouthed bottle in addition
Encapsulation, it is labelled, test kit is assembled into, concrete operations as shown in Figure 4 are as follows：
（1）96 hole elisa plates：Will series connection MIC1, MAG1, SAG1 isogeneity albumen（5）10ug/mL is diluted to, per hole 100ul
Coating elisa plate, 37 DEG C of incubation 1h add 5% BSA confining liquids, 37 DEG C of closing 1h after washing.Vacuum Package after washing.
（2）Sample diluting liquid（PBST PH7.4）:NaCl 8.0g；KH2PO4 0.2g；Na2HPO4 ·12H2O 2.9g；
KCl 0.2g add two distilled waters and cause 1000ml;Add 0.5ml Tween-20, last 50ml subpackages.
（3）20 times of concentrated cleaning solutions：NaCl 160.0g；KH2PO4 4g；Na2HPO4 ·12H2O 58g；KCl 4g are added
Two distilled waters cause 1000ml;Add 10ml Tween-20, last 50ml subpackages.
（4）Rabbit-anti sheep IgG-HRP conjugates:10ml PBST add 5ul rabbit-anti sheep IgG-HRP bis- to resist, and are subsequently adding 4%
PEG, 25ml subpackage.
（6）Terminate liquid：2mol/L H2SO4Concentrated sulphuric acid 44.5ml, distilled water 355.5ml, 10ml subpackages.
（7）Substrate A：TMB 200mg, dehydrated alcohol 100ml, plus distilled water is to 1000ml, 10ml subpackages.
（8）Substrate B：（0.1ml/L citric acid -0.2ml/L phosphoric acid hydrogen two is received, pH5.0-5.4）:Na2HPO414.60g, lemon
Lemon acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml, plus tri-distilled water is to 1000ml, is adjusted to pH5.0-5.43,10ml subpackages.
（9）Positive：By Standard arch wire worm positive serum 1:5 are diluted in sample diluting liquid, 1ml subpackages.
（10）Negative sample：To toxoplasma negative serum 1 after testing:5 are diluted in sample diluting liquid, 1ml subpackages.
The determination of the shelf-life of 5 ELISA kit of embodiment
4 DEG C of the blocking ELISA kit that assembles is placed determine within one month, two months, four months, six months its specificity and
Sensitivity.Determine the test kit shelf-life be 6 months.
The testing result of part sample of 6 ELISA kit of embodiment to gathering
The test kit assembled in Example 4, carries out following operation：
1. will be using sample diluting liquid（2）By 1:After 100 dilutions, add（1）In 96 hole elisa plates, per 50 μ l of hole, 37 DEG C, 1h；
2. by 20 times of concentrated cleaning solutions（3）20 times of dilutions, wash 5 times, each 3min；
3. the rabbit-anti sheep IgG-HRP conjugates of 2000 times of dilutions are added（4）, per 50 μ l of hole, 37 DEG C, 1h；
4. 20 times of concentrated cleaning solutions after dilution are added（5）, wash 5 times, each 3min；
5. substrate A is taken（7）And substrate B（8）1：1 mixing, adds 50 μ l, 37 DEG C of lucifuges colour developing 20min per hole；
6. terminate liquid is added（6）, per 50 μ l of hole；
7. using microplate reader measure OD490nm values, suppression ratio PI=(OD negative control-OD samples)/OD negative controls * 100%,
The corresponding sample of PI >=50% for the positive, less than then for feminine gender.Carry out result judgement as follows：Positive rate occupies 16.4%（25/
152）.
Claims (3)
1. a kind of toxoplasma is connected multi-epitope gene ELISA detection kit, including 96 hole elisa plates, sample diluting liquid, 20 times
Concentrated cleaning solution, rabbit-anti sheep IgG-HRP conjugates, series connection MIC1, MAG1, SAG1 gene expression purifying protein, terminate liquid, substrate
A, substrate B, positive, negative sample, cover plate film；It is characterized in that：1）96 hole elisa plates be will series connection MIC1, MAG1,
SAG1 isogeneity albumen is diluted to 10ug/mL, and elisa plate is coated with per hole 100ul, and 37 DEG C of incubation 1h add 5% after washing
BSA confining liquids, 37 DEG C of closing 1h, Vacuum Package after washing；
2）Sample diluting liquid is by NaCl 8.0g；KH2PO4 0.2g；Na2HPO4 ·12H2O 2.9g；KCl 0.2g add two
Distilled water causes 1000ml;Add 0.5ml Tween-20, last 50ml subpackages；
3）20 times of concentrated cleaning solutions are by NaCl 160.0g；KH2PO4 4g；Na2HPO4 ·12H2O 58g；KCl 4g are added
Two distilled waters cause 1000ml;Add 10ml Tween-20, last 50ml subpackages；
4）Rabbit-anti sheep IgG-HRP conjugates are to add 5ul rabbit-anti sheep IgG-HRP bis- to resist 10ml PBST, are subsequently adding 4%PEG,
25ml subpackages；
Series connection MIC1, MAG1, SAG1 gene expression purifying protein is according to toxoplasma MIC1, MAG1, SAG1 sequences in GenBank
Row, design specific primer, and according to toxoplasma MIC1, MAG1, SAG1 sequences in GenBank, design specific primer is as follows：
MIC1
-F:GGATCCGCGTCGCATTCTCATTCG BamH I, 24bp, 18, GC% 58.3, Tm 74.2
-R:GAATTCGGCCTTCTCGTAACACCTCC EcoR I,,26bp, GC%53.8, Tm 69.5
MAG1
-F:GAATTCAGCCAAAGGGTGCCAGAG EcoR I ,24bp, GC%54.2, Tm 69.0
-R:AAGCTTAGATCCCTGAACCCTTAGAATATACAC Hind III, 33bp, GC% 39.4, Tm 65.5
SAG1
-F:AAGCTTGATCCCCCTCTTGTTGCC Hind III, 24bp, GC%54.2, Tm69.4
-R:CTCGAGAAGAGTGCTGTCTGCACCGT Xho I , 26bp, GC%57.7,Tm 71.1
Expanded by RT-PCR technology and expand respectively toxoplasma MIC1, MAG1, SAG1 gene, arch is expanded by RT-PCR technology
Purpose fragment is cloned into carrier T, the identification of Jing enzyme action and sequencing analysis by worm MIC1, MAG1, SAG1 gene, as a result proves to obtain
MIC1, MAG1, SAG1 gene；Multi-epitope gene is connected with prokaryotic expression carrier pET-28a, is converted into escherichia coli,
Induced with IPTG, Jing SDS-PAGE and Western blotting analysis MIC1, MAG1, SAG1 albumen；Using Ni-NTA to table
Purification is carried out up to albumen, the purifying protein of ideal concentration is obtained；Terminate liquid be by 2mol/L H2SO4 concentrated sulphuric acid 44.5ml, it is double
Steam water 355.5ml, 10ml subpackages；
5）Substrate A：TMB 200mg, dehydrated alcohol 100ml, plus distilled water is to 1000ml, 10ml subpackages；
7）Substrate B：（0.1ml/L citric acid -0.2ml/L phosphoric acid hydrogen two is received, pH5.0-5.4）:Na2HPO4 14.60g, citric acid
9.33g, 0.75% hydrogen peroxide urea 6.4ml, plus tri-distilled water is to 1000ml, is adjusted to pH5.0-5.43,10ml subpackages；
8）Positive：By Standard arch wire worm positive serum 1:5 are diluted in sample diluting liquid, 1ml subpackages；
9）Negative sample：To toxoplasma negative serum 1 after testing:5 are diluted in sample diluting liquid, 1ml subpackages.
2. a kind of toxoplasma according to claim 1 is connected multi-epitope gene ELISA detection kit, it is characterised in that institute
On the 96 hole elisa plates stated, coating series connection MIC1, MAG1, SAG1 gene expression purifying protein, is diluted to coating buffer
10ug/mL, with the closing 1h of the PBS containing 5% defatted milk powder after 37 DEG C of coating 1h, PBS is sealed with aluminum foil under vacuum after washing 5 dryings.
3. a kind of toxoplasma according to claim 1 is connected multi-epitope gene ELISA detection kit, it is characterised in that institute
The working concentration of the rabbit-anti sheep IgG-HRP conjugates stated is diluted for 2000 times.
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