CN104965086A - Dog toxoplasma gondii antibody indirect ELISA detection kit - Google Patents
Dog toxoplasma gondii antibody indirect ELISA detection kit Download PDFInfo
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Abstract
The invention discloses a dog toxoplasma gondii antibody indirect ELISA detection kit, and belongs to the technical field of biological inspection and quarantine. According to the dog toxoplasma gondii antibody indirect ELISA detection kit, toxoplasma gondii ROP5 recombinant protein is taken as a detection antigen. According to the dog toxoplasma gondii antibody indirect ELISA detection kit, toxoplasma gondii recombinant protein ROP5 is obtained via gene cloning and prokaryotic expression; and a detection method comprises following steps: establishment of an indirect ELISA detection method, specificity testing, sensitivity testing, and comparison with other dog toxoplasma gondii detection kits. Large-scale standardized production of an envelope antigen of the dog toxoplasma gondii antibody indirect ELISA detection kit can be realized; detection specificity is high; sensitivity is high; repeatability is high; result determination is objective; operation process is simple; and large-scale detection can be realized. The dog toxoplasma gondii antibody indirect ELISA detection kit is suitable for dog toxoplasmosis epidemiological investigation, and detection and clinical diagnosis of dog toxoplasma gondii infection; and a rapid, simple, and specific detection method is provided for diagnosis and prevention, and epidemiological investigation of dog toxoplasmosis.
Description
Technical field
The invention belongs to biological test Quarantine Techniques field, relate in particular to a kind of dog toxoplasma antibody indirect ELISA testing kit.
Background technology
Toxoplasmosis invades the karyocyte of animal and a kind of global parasitic zoonoses caused by toxoplasma gondii (Toxoplasma gondii).Infection of Toxoplasma Gondii is a kind of opportunistic pathogenesis parasite, manifest symptom can not be produced in normal human, for the colony of immune system defect or larger at immunosuppressant patient (as AIDS patient, malignant tumor patient and the people just having accepted organ transplant) hazard ratio, often causing serious organ injury, is one of its common lethal cause of disease.The growth course of Infection of Toxoplasma Gondii needs two hosts, and cat is unique final host; Intermediate host is widely distributed, and almost comprises all warm-blooded animals of the mankind, part poikilotherm and some arthropods.Two hosts play an important role in the propagation and infection of Infection of Toxoplasma Gondii.Current Infection of Toxoplasma Gondii there is no specific drug and treats it, also prevents without effective vaccine, and therefore the monitoring of animal arch insect infection is significant for the prevention and control of disease.
Infection of Toxoplasma Gondii diagnostic method conventional at present mainly comprises the methods such as aetology, molecular biology and immunology.Wherein aetology method wastes time and energy, and accuracy is lower; Molecular biology comprises the technology such as PCR, DNA probe and genetic chip, although the method Sensitivity and Specificity is high, its operating process is complicated and instrument and equipment is expensive; Immunological method has indirect hemagglutination test (IHA), Sa Bin-Feldman stain test (DT), latex agglutination test (LAT), enzyme linked immunosorbent assay (ELISA) etc.IHA is simple to operate, and the serum longer to infection time has good susceptibility, but poor to the serum sensitive that acute infection is early stage, and sensitized erythrocyte is unstable, poor repeatability.DT is the distinctive serological diagnostic method of toxoplasmosis, the method high specificity and susceptibility is high, is applicable to the early diagnosis of arch insect infection.But diagnose antigen used to be necessary for worm alive, poor stability, therefore in the less application of China.The maximum problem of LAT is poor specificity, and the positive blood through LAT diagnosis is checked up and return and need be done further serological test, extends Diagnostic Time, inhibits the propagation and employment of the method.ELISA is a kind of Labeled immunoassay technology occurred the seventies in last century, and through constantly improving for many years, this technology is very ripe.ELISA is easy and simple to handle, does not need special instruments and equipment, and amount of samples is few, and has hypersensitivity and high specific, and judged result is easy, is suitable for the detection of great amount of samples.
There is the ELISA diagnostic kit of multiple diagnosis Infection of Toxoplasma Gondii in the market, quality uneven being difficult to is selected.Though import Infection of Toxoplasma Gondii ELISA diagnostic kit quality is good, but is subject to the restriction of traffic condition and price, be difficult to large-scale popularization application.Therefore preparing a kind of special, responsive, easy and Infection of Toxoplasma Gondii diagnostic kit that is this area, applicable south China has certain necessary.
Current Infection of Toxoplasma Gondii ELISA detects antigen used and is mostly polypide soluble antigen, and its composition is uncertain, and between antigen preparation batch, difference is comparatively large, not easily standardization.Along with the development of Protocols in Molecular Biology, recombinant antigen is determined with its composition, the easy advantage such as standardization, high specificity, and in selection Infection of Toxoplasma Gondii diagnostic antigen, application is more and more extensive.Infection of Toxoplasma Gondii is under the jurisdiction of the multiple door protozoon in top, its front end all has clava (Rhoptry), microsome (Microsone) and dense granule (Dense granule) 3 kinds of most advanced and sophisticated complexs of difference, can secrete three kinds of different albumen.The studies on rhoptry proteins (rhoptry protein, ROP) of clava secretion invades host cell with Infection of Toxoplasma Gondii close relationship.ROP5 is that Infection of Toxoplasma Gondii invades the important virulence factor of host cell, by ROP5 gene knockout before Infection of Toxoplasma Gondii velogen strain invasion host, found that the virulence of Infection of Toxoplasma Gondii disappears completely, therefore show that ROP5 is the required virulence factor of arch insect infection host.Moreover, ROP5 also has good immunogenicity and immune protective, is the preparation of a kind of potential vaccine, diagnosis candidate antigens molecule.
Summary of the invention
The object of the invention is the deficiency overcoming existing Infection of Toxoplasma Gondii ELISA detection technique, a kind of dog toxoplasma antibody indirect ELISA testing kit is provided, this detection kit utilizes Infection of Toxoplasma Gondii ROP5 recombinant protein as envelope antigen, for exploitation lays the foundation with research Infection of Toxoplasma Gondii ELISA diagnostic kit further.
Object of the present invention is realized by following technical scheme: a kind of dog toxoplasma antibody indirect ELISA testing kit, and detectable antigens used in described detection kit is Infection of Toxoplasma Gondii ROP5 recombinant protein.
Described Infection of Toxoplasma Gondii ROP5 recombinant protein is prepared from by the following method:
(1) DNA of RH strain Infection of Toxoplasma Gondii is extracted;
(2) according to the gene order design primer delivered in GenBank, pcr amplification goes out Infection of Toxoplasma Gondii ROP5 gene, and primer sequence is as follows:
RH strain ROP5F:5 '-TATAGGATCCATGGCGACGAAGCTCG-3 '; (SEQ IDNO 1);
RH strain ROP5R:5 '-TATACTCGAGCTCAAGCGACTGAGGGCG-3 '; (SEQID NO 2).
Wherein, bolded section sequence is restriction enzyme site sequence;
(3) retrieve and purification of the PCR primer increased;
(4) PCR primer is connected with PMD-18T cloning vector, and connect product and proceed to DH5 α competent cell, cut qualification through BamH I and Xho I enzyme, screening positive clone checks order;
(5) insert in expression vector pET32a through the ROP5 genetic fragment of BamH I and Xho I double digestion and build recombinant expression plasmid pET32a-ROP5;
(6) proceeded to by recombinant expression plasmid pET32a-ROP5 in BL21 (DE3) competent cell, picking positive plasmid after cultivating, enzyme cuts qualification;
(7) putting into bacterium to density proliferated culture medium (MDG) cultivation by being accredited as positive clone bacterium, when bacterium liquid little cloudy, bacterium liquid being inoculated in abduction delivering in automatic abduction delivering nutrient culture media (ZYM-5052); Use ni-sepharose purification recombinant protein, obtain recombination fusion protein TgROP5.
Pcr amplification condition described in step (2) is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 1min, 61 DEG C of renaturation 1min, and 72 DEG C extend 2min, and carry out 30 circulations, last 72 DEG C of ends extend 7min.
Above-mentioned dog toxoplasma antibody indirect ELISA testing kit, also comprises:
1) the anti-dog IgG of the rabbit of horseradish peroxidase-labeled;
2) phosphate buffer is 15mM PBS: be specially NaCl 8.0g, KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, KCl 0.2g, adding distil water is settled to 1000mL, regulates the pH value to 7.4 of solution;
3) antigen coated liquid is 50mM Tris hydrochloride buffer (50mM TBS): be specially Tris 0.60g, NaCl 0.88g, add deionized water 90mL, with salt acid for adjusting pH to 7.6 after dissolving completely, adds deionized water and is settled to 100ml;
4) cleansing solution is 15mM PBST: be specially the phosphate buffer containing 0.05%Tween-20;
5) sample diluting liquid is 1%BSA-PBST: be specially BSA 1g and add 100mL PBST;
6) confining liquid is 5% skimmed milk power: be specially 5g skimmed milk power and add 100mL PBS;
7) ELIAS secondary antibody dilution is 10% hyclone-PBST: be specially hyclone 10mL and add 90mLPBST;
8) substrate buffer solution is phosphate-citrate buffer (pH5.0): be specially citric acid 2.553gNa
2hPO
412H
2o 9.2g deionized water is settled to 500mL, 4 DEG C of preservations;
9) TMB stores liquid (10mg/mL): be specially TMB 1g, DMSO 99mL, packing after dissolving, and 4 DEG C keep in Dark Place;
10) tmb substrate nitrite ion: be specially substrate buffer solution 99mL, TMB stores liquid 1mL, 30%H
2o
2100 μ L, now with the current;
11) stop buffer (2M H
2sO
4): be specially concentrated sulphuric acid 21.7mL, deionized water 178.3mL, slowly added in deionized water by the concentrated sulphuric acid, limit edged stirs.
Application mentioned reagent box carries out the method that dog toxoplasma antibody indirect ELISA detects, and comprises the following steps: envelope antigen, close, application of sample, add ELIAS secondary antibody, colour developing, termination.
The method that above-mentioned dog toxoplasma antibody indirect ELISA detects, best condition of work is: antigen diluent 320 times (concentration is 1-25 μ g/mL), Sample Dilution 50 times, ELIAS secondary antibody dilute concentration is 1:2000, antigen coated time 1.5h, antigen coated liquid is 50mM Tris hydrochloride buffer, off-period is 1h, confining liquid is 5% skimmed milk power, sample diluting liquid is 1%BSA-PBST, the sample incubation time is 2.5h, ELIAS secondary antibody dilution is 10% hyclone-PBST, ELIAS secondary antibody incubation time is 30min, substrate developing time 10min, negative and positive critical value is 0.484.
The method that above-mentioned dog toxoplasma antibody indirect ELISA detects, specifically comprises the following steps:
A. wrap quilt: with 50mM Tris hydrochloride buffer, TgROP5 recombinant protein is diluted to 1-25 μ g/mL, wrap by ELISA ELISA Plate, 100 μ L/ holes, hatch 1.5h for 37 DEG C, dry coating buffer, wash 3 times, 2min/ time with cleansing solution PBST;
B. close: every hole is added 300 μ L confining liquid 5% skimmed milk powers, hatch 1h for 37 DEG C, dry confining liquid, wash 3 times with PBST, 2min/ time;
C. add detection serum: by the dilution proportion of serum 1%BSA-PBST by 1:50, if negative and positive control, every hole adds 100 μ L, hatches 2.5h for 37 DEG C, dries, washs 3 times;
D. add two to resist: with 10% hyclone-PBST, anti-for rabbit dog IgG-HRP is done 1:2000 dilution, 100 μ L/ holes, hatch 30min for 37 DEG C, dry, the same washing 3 times;
E. substrate is added: every hole adds the freshly prepared tmb substrate solution of 100 μ L, in 37 DEG C of lucifuge colour developing 10min;
F. stop: every hole 50 μ L adds stop buffer, cessation reaction;
G. result judges: the OD value surveying 450nm place by microplate reader; OD value >=0.484 is positive, and OD value < 0.484 is negative.
Above-mentioned dog toxoplasma antibody indirect ELISA Cleaning Principle figure as shown in Figure 7.
The present invention has following advantage and effect relative to prior art:
1. the detectable antigens described in the present invention is recombinant protein ROP5-PET32a.The studies on rhoptry proteins of Infection of Toxoplasma Gondii clava secretion and Infection of Toxoplasma Gondii invade host cell close relationship.Research finds, ROP5 is that Infection of Toxoplasma Gondii invades the important virulence factor of host cell, and ROP5 also has good immunogenicity and immune protective.Therefore detect toxoplasma antibody with recombinant protein ROP5-PET32a as envelope antigen, there is very strong specificity and susceptibility.
2. the present invention is by optimizing reaction system and reaction conditions; establish a kind of new dog toxoplasma antibody indirect ELISA detection method; the method envelope antigen can scale standardization be produced; and method specificity, susceptibility and repeatability are strong; it is objective that result judges; operating process is simple, can carry out mass detection.The method can realize epidemiology survey to dog toxoplasmosis, the detection of dog arch insect infection and clinical diagnosis, for dog Infection of Toxoplasma Gondii Diagnosis and treatment technology etc. further research lay a good foundation.
Accompanying drawing explanation
The pcr amplification result figure of Fig. 1: RH strain Infection of Toxoplasma Gondii ROP5 gene.Wherein, M is DL2000DNAMarker; 1 is ROP5PCR amplified production; 2 is negative control.
The enzyme of Fig. 2: PET32a-ROP5 recombinant plasmid cuts qualification result figure.Wherein, M is DL5000DNAMarker; 1 is ROP5PCR amplified production; 2-4 is recombinant plasmid pMD18-ROP5 double digestion product; 5-6 is recombinant plasmid pMD18-ROP5.
Fig. 3: the toxoplasma recombinant protein ROP5 SDS-PAGE electrophoresis result figure expressed.Wherein, M is 170kDa albumen marker; 1 is induction 12h PET32a empty carrier bacterium; 2 is PET32a-ROP5 induction 12h; 3 is PET32a-ROP5 induction 13h; 4 is PET32a-ROP5 induction 15h; 5 is PET32a-ROP5 induction 17h; 6 is PET32a-ROP5 induction 19h.
Fig. 4: the result figure of expression of recombinant proteins form.Wherein, M is 170kDa albumen marker; 1 induces full bacterium for PET32a-ROP5; 2-3 is that PET32a-ROP5 induces supernatant; 4-5 is PET32a-ROP5 induced precipitation.
Fig. 5: toxoplasma recombinant protein ROP5 purification result.Wherein, M is 170kDa albumen marker; 1 is sample before loading; 2 wear liquid for sample stream; 3 is foreign protein cleansing solution; 4 is 80mM imidazole elution; 5 is 160mM imidazole elution; 6 is 200mM imidazole elution; 7 is 320mM imidazole elution; 8 is 500mM imidazole elution.
The western-blot of Fig. 6: ROP5 purifying protein analyzes.Wherein, M is 170kDa albumen marker; 1 is 160mM imidazole elution; 2 is 200mM imidazole elution; 3 is PET32a empty carrier induction bacterium
Fig. 7: indirect ELISA Cleaning Principle figure.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The structure of embodiment 1 expression vector ROP5-PET32a
1, the Design and synthesis of primer
According to the gene order delivered in GenBank, the primer of design amplification Infection of Toxoplasma Gondii ROP5 gene order.Needed for clone, introduce BamH I restriction enzyme site at the 5 ' end of primer ROP5F, introduce Xho I restriction enzyme site at the 5 ' end of ROP5R.Primer is synthesized by Invitrogen Corp., and its nucleotide sequence is composed as follows:
RH strain ROP5F:5 '-TATA
gGATCCaTGGCGACGAAGCTCG-3 '
RH strain ROP5R:5 '-TATA
cTCGAGcTCAAGCGACTGAGGGCG-3 '
2, the amplification of ROP5 genes of interest
The RH strain toxoplasma tachyzoite preserved in this laboratory liquid nitrogen container is recovered in BALB/c mouse body, collects mouse ascites extraction RH strain Infection of Toxoplasma Gondii and entirely organize DNA.With the Toxoplasma DNA extracted for masterplate, use F and R two primers, carry out the pcr amplification of ROP5 gene, PCR reacts the reaction system of employing 25 μ L.Reaction system is as follows:
Amplification condition is:
By the 1.0% agarose gel electrophoresis analysis of above-mentioned pcr amplification product, voltage 120V, time 30min, be placed in ultraviolet transilluminator and observe, and the results are shown in accompanying drawing 1.
3, ROP5-PMD-18T cloning vector plasmids is built
Adopt and cut the PCR primer of glue purification method to amplification and cut glue and reclaim, be connected on cloning vector PMD-18T by the ROP5 genetic fragment of recovery, with the qualification of BamHI and Xho I double digestion, it is as follows that enzyme cuts system:
Enzyme is cut and be the results are shown in accompanying drawing 2, and enzyme is cut the PMD-18T plasmid being accredited as the positive and deliver to the order-checking of Shanghai Sheng Gong company, its ROP5 enzyme cuts the nucleotide sequence of order-checking as shown in SEQ ID NO:3.
4, ROP5-PET32a expression vector plasmid is built
By the order-checking correct ROP5-PMD-18T plasmid of qualification and PET32a empty carrier BamHI and XhoI double digestion, enzyme cut after ROP5 genetic fragment is connected with linearizing PET32a, structure ROP5-PET32a recombinant plasmid, linked system is:
Of short duration centrifugal after mixing, spend the night in 4 DEG C of connections.Product conversion will be connected cultivate to DH5 α competent cell, transfer vector plasmid is cloned in a large number, extract ROP5-PET32a vector plasmid.
Embodiment 2 ROP5 destination protein abduction delivering and purifying
1, the abduction delivering of genes of interest ROP5 in Escherichia coli
Recombinant expression plasmid ROP5-PET32a is transformed into and expresses in bacterium BL21 (DE3), coated plate, cultivate 12 ~ 14h.The single white colony of picking from the flat board cultivated, be inoculated in bacterium proliferation of high-density nutrient culture media (MDG), 37 DEG C, 200rpm cultivates 6 ~ 8h, when little cloudy appears in bacterium, is inoculated in containing 100 μ g/mL Amp by bacterium liquid with the ratio of 1:1000
+in automatic abduction delivering nutrient culture media (ZYM-5052), 28 DEG C, 200rpm jolts cultivation, and respectively at the time sampling that induction is different, in contrast, carry out SDS-PAGE electrophoresis detection expression product, electrophoresis result is shown in accompanying drawing 3 to the empty carrier bacterium arranging induction.
2, the detection of expression product existence form
Bacterium liquid in bacterium proliferation of high-density nutrient culture media is inoculated in abduction delivering in automatic abduction delivering nutrient culture media with the ratio of 1:1000, and the time the highest to expressing quantity stops induction.Bacterium liquid after induction is placed in 5min on ice, 4 DEG C, the centrifugal 10min of 8000rpm, collects thalline.Heavily thalline is revolved with sample buffer, with ultrasonic cell disruptor ultrasonic degradation thalline, power 200w, ultrasonic 3s, interval 5s, ultrasonic number of times 120 times.4 DEG C, 8000rpm, centrifugal 10min, get cleer and peaceful precipitation respectively, carries out SDS-PAG electrophoresis, detects recombinant protein and be present in supernatant or in inclusion body, the results are shown in accompanying drawing 4.
3, the purifying of destination protein
Select supernatant through SDS-PAG electroresis appraisal, carry out purifying destination protein with the Ni-Agarose His label protein purification kit (soluble type albumen) of Beijing Quan Shi King Company.
(1) fill post: heavily revolve protein purification nickel column packing, 1mL filler is added chromatographic column, leave standstill and treat that filler is separated with alcohol conserving liquid, the lid opened below chromatographic column makes liquid flow out.
(2) balance: with 5-10 times of column volume equilibration buffer pillar, in order to improve specific binding, low concentration imidazoles (10-20mM) in level pad, can be added.
(3) loading: supernatant and the sample buffer 1:1 of ultrasonication dilute, with the metre filter sample of 0.45 μm.Sample is slowly added chromatographic column, blocks column outlet, sample and filler hatch 30min, slowly flow out, and collect sample stream and wear liquid.
(4) wash: after loading, with 5-10 times of column volume equilibration buffer solution pillar, collect cleansing solution.
(5) wash-out: the 80mM imidazoles wash-out pillar of 5 times of column volumes 1 time, collects eluent; The 120mM imidazoles wash-out pillar of 5 times of column volumes one time, collects eluent; The 160mM imidazoles wash-out pillar of 5 times of column volumes one time, collects eluent; The 200mM imidazoles wash-out pillar of 5 times of column volumes one time, collects eluent; The 320mM imidazoles wash-out pillar of 5 times of column volumes one time, collects eluent; The 500mM imidazoles wash-out pillar of 5 times of column volumes one time, collects eluent.
(6) SDS-PAGE: the imidazole elution that sample before getting appropriate loading respectively, sample stream wear liquid, cleansing solution and variable concentrations adds 5 × SDS-PAGE sample-loading buffer, carries out SDS-PAGE electrophoresis detection protein purification result, the results are shown in accompanying drawing 5.
4, purifying protein Western blot detects
Destination protein after purifying and PET32a empty carrier induction bacterium are carried out SDS-PAGE electrophoresis, operate with reference to conventional Western blot method, after an anti-mouse anti-His antibody and two anti-sheep anti-Mouse HRP-IgG are hatched, develop the color in chemiluminescence image analytic system (Tanon Fine-do X6) luminescence with after the process of ECL luminescent solution, to take pictures observation, result shows that purifying protein occurs band at about 81kD, there is band at about 20kD in PET32a empty carrier induction bacterium, result is as accompanying drawing 6.
Embodiment 3 indirect ELISA detects the foundation of dog toxoplasma antibody method
Set up indirect ELISA method with the expressing protein ROP5 after purifying as envelope antigen, adopt chessboard method to grope best antigen coated concentration, best serum diluting multiple, and the optimum reaction condition of ELISA method.Finally determine that condition is as follows:
(1) bag quilt: by purifying protein Tris-HCl damping fluid (50mM pH7.6, TBS) dilute in the ratio (namely albumen bag is 2.06 μ g/mL by concentration) of 1:320, add 96 hole ELISA Plate, 100 μ L/ holes, hatch 1.5h for 37 DEG C.Dry, wash 3 times with cleansing solution PBST, 2min/ time.
(2) close: confining liquid adopts 5% skimmed milk power-PBST, and 300 μ L/ holes, hatch 1h for 37 DEG C, dry confining liquid, wash 3 times with PBST.
(3) increase serum: by the multiple dilutions of serum 1%BSA-PBST according to 1:50, every hole adds 100 μ L, hatches 2.5h for 37 DEG C, dries, washs 3 times with PBST.
(4) add two to resist: with 10% hyclone-PBST, anti-for rabbit dog IgG-HRP is done 1:2000 dilution, 100 μ L/ holes, hatch 30min for 37 DEG C, dry, wash 3 times.
(5) substrate is added: every hole adds freshly prepared tmb substrate solution, 100 μ L/ holes, lucifuge colour developing 10min.
(6) stop: add stop buffer, 50 μ L/ holes, cessation reaction.
(7) OD is surveyed
450value: at 450nm place, measure each hole OD value by automatic microplate reader.
Above-mentioned dog toxoplasma antibody indirect ELISA Cleaning Principle figure as shown in Figure 7.
The determination of embodiment 4 critical value:
Detect with the dog serum of the indirect ELISA method set up to 15 parts of Infection of Toxoplasma Gondii feminine genders, calculate the OD of serum
450nmmean value (X) be 0.326, standard deviation (SD) is 0.079, according to formula: yin and yang attribute critical value=X+2SD show that the critical value of this test is 0.484.As serum OD
450nmwhen>=0.484, result is judged to be the positive; As serum OD
450nmduring < 0.484, result is judged to be feminine gender.
Embodiment 5 sensitivity tests
The positive serum of 37 parts of dog Infection of Toxoplasma Gondiis and the positive serum of 15 parts of dog Infection of Toxoplasma Gondiis is detected with the indirect ELISA detection method set up, detect positive serum 36 parts, negative serum 15 parts, according to formula: susceptibility=(detecting number/total number) × 100%, can show that the susceptibility of the method is 98.08%.
Embodiment 6 specific test
Detect by the indirect ELISA top condition set up the positive serum that dog is tiny, canine distemper, hepatitis infectiosa canis virus sick and canine parainfluenza virus is sick respectively, set up positive control, negative control and blank simultaneously, often kind is repeated 4 holes, measures OD by microplate reader
450nmvalue, shown in result table 1:
Table 1 microplate reader OD value testing result
Result shows, except dog Infection of Toxoplasma Gondii positive serum OD
450value is for outside the positive, and all the other serum are negative entirely.This recombinant protein antigen is described and dog is tiny, canine distemper, hepatitis infectiosa canis virus are sick and canine parainfluenza virus is sick positive serum does not all have cross reaction, this indirect ELISA method of foundation has good specificity.
Embodiment 7 compares with other ELISA kit testing results
37 parts of dog Infection of Toxoplasma Gondii positive serum samples are detected with the indirect ELISA reagent kit of this research assembling, the ELISA kit of Haitai bio tech ltd, Zhuhai purchase and the direct hemagglutination test kit of Lanzhou veterinary institute respectively, compare the correlativity between them and meet situation, result is as shown in table 2:
The testing result of the different ELISA kit of table 2
From result, 37 parts of positive serums, have 35 parts to be positive by the indirect ELISA detection method testing result that this research is set up, positive coincidence rate is 94.59%; The ELISA kit that Haitai bio tech ltd, Zhuhai buys detects has 36 parts to be positive, and positive coincidence rate is 97.3%; Lanzhou veterinary institute HIA kit testing result has 35 parts to be positive, and positive coincidence rate is 94.59%.Can find out that the testing result of ELISA detection kit of the present invention and the kit testing result of other companies are more or less the same, even much better than the testing result of some companies.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. a dog toxoplasma antibody indirect ELISA testing kit, is characterized in that: detectable antigens used in described detection kit is Infection of Toxoplasma Gondii ROP5 recombinant protein.
2. dog toxoplasma antibody indirect ELISA testing kit according to claim 1, is characterized in that: described Infection of Toxoplasma Gondii ROP5 recombinant protein is prepared from by the following method:
(1) DNA of RH strain Infection of Toxoplasma Gondii is extracted;
(2) according to the gene order design primer delivered in GenBank, pcr amplification goes out Infection of Toxoplasma Gondii ROP5 gene, and primer sequence is as follows:
RH strain ROP5F is as shown in SEQ ID NO1;
RH strain ROP5R is as shown in SEQ ID NO2;
(3) retrieve and purification of the PCR primer increased;
(4) PCR primer is connected with PMD-18T cloning vector, and connect product and proceed to DH5 α competent cell, cut qualification through BamH I and Xho I enzyme, screening positive clone checks order;
(5) insert in expression vector pET32a through the ROP5 genetic fragment of BamH I and Xho I double digestion and build recombinant expression plasmid pET32a-ROP5;
(6) proceeded to by recombinant expression plasmid pET32a-ROP5 in BL21 (DE3) competent cell, picking positive plasmid after cultivating, enzyme cuts qualification;
(7) the clone bacterium being accredited as the positive is put into bacterium to cultivate to density proliferated culture medium, when bacterium liquid little cloudy, bacterium liquid is inoculated in abduction delivering in automatic abduction delivering nutrient culture media; Use ni-sepharose purification recombinant protein, obtain recombination fusion protein TgROP5.
3. dog toxoplasma antibody indirect ELISA testing kit according to claim 2, it is characterized in that: the pcr amplification condition described in step (2) is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 1min, 61 DEG C of renaturation 1min, 72 DEG C extend 2min, carry out 30 circulations, last 72 DEG C of ends extend 7min.
4. dog toxoplasma antibody indirect ELISA testing kit according to claim 1, is characterized in that: also comprise:
1) the anti-dog IgG of the rabbit of horseradish peroxidase-labeled;
2) phosphate buffer is 15mM PBS: be specially NaCl 8.0g, KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, KCl 0.2g, adding distil water is settled to 1000mL, regulates the pH value to 7.4 of solution;
3) antigen coated liquid is 50mM Tris hydrochloride buffer: be specially Tris 0.60g, and NaCl 0.88g adds deionized water 90mL, with salt acid for adjusting pH to 7.6 after dissolving completely, adds deionized water and is settled to 100ml;
4) cleansing solution is 15mM PBST: be specially the phosphate buffer containing 0.05%Tween-20;
5) sample diluting liquid is 1%BSA-PBST: be specially BSA 1g and add 100mL PBST;
6) confining liquid is 5% skimmed milk power: be specially 5g skimmed milk power and add 100mL PBS;
7) ELIAS secondary antibody dilution is 10% hyclone-PBST: be specially hyclone 10mL and add 90mLPBST;
8) substrate buffer solution is phosphate-citrate buffer: be specially citric acid 2.553gNa
2hPO
412H
2o 9.2g deionized water is settled to 500mL, pH5.0,4 DEG C of preservations;
9) TMB stores liquid: be specially TMB 1g, DMSO 99mL, packing after dissolving, and 4 DEG C keep in Dark Place;
10) tmb substrate nitrite ion: be specially substrate buffer solution 99mL, TMB stores liquid 1mL, 30%H
2o
2100 μ L, now with the current;
11) stop buffer: be specially concentrated sulphuric acid 21.7mL, deionized water 178.3mL, slowly adds the concentrated sulphuric acid in deionized water, and limit edged stirs.
5. the application rights method that requires kit described in 1 ~ 4 any one to carry out dog toxoplasma antibody indirect ELISA to detect, is characterized in that: comprise the following steps: envelope antigen, close, application of sample, add ELIAS secondary antibody, colour developing, termination;
The condition of work of the best of the method that dog toxoplasma antibody indirect ELISA detects is: antigen concentration is 1-25 μ g/mL, Sample Dilution 50 times, ELIAS secondary antibody dilute concentration is 1:2000, antigen coated time 1.5h, antigen coated liquid is 50mM Tris hydrochloride buffer, off-period is 1h, confining liquid is 5% skimmed milk power, sample diluting liquid is 1%BSA-PBST, the sample incubation time is 2.5h, and ELIAS secondary antibody dilution is 10% hyclone-PBST, and ELIAS secondary antibody incubation time is 30min, substrate developing time 10min, negative and positive critical value is 0.484.
6. the method for dog toxoplasma antibody indirect ELISA detection according to claim 5, is characterized in that: specifically comprise the following steps:
A. wrap quilt: with 50mM Tris hydrochloride buffer, TgROP5 recombinant protein is diluted to 1-25 μ g/mL, wrap by ELISA ELISA Plate, 100 μ L/ holes, hatch 1.5h for 37 DEG C, dry coating buffer, wash 3 times, 2min/ time with cleansing solution PBST;
B. close: every hole is added 300 μ L confining liquid 5% skimmed milk powers, hatch 1h for 37 DEG C, dry confining liquid, wash 3 times with PBST, 2min/ time;
C. add detection serum: by the dilution proportion of serum 1%BSA-PBST by 1:50, if negative and positive control, every hole adds 100 μ L, hatches 2.5h for 37 DEG C, dries, washs 3 times;
D. add two to resist: with 10% hyclone-PBST, anti-for rabbit dog IgG-HRP is done 1:2000 dilution, 100 μ L/ holes, hatch 30min for 37 DEG C, dry, the same washing 3 times;
E. substrate is added: every hole adds the freshly prepared tmb substrate solution of 100 μ L, in 37 DEG C of lucifuge colour developing 10min;
F. stop: every hole 50 μ L adds stop buffer, cessation reaction;
G. result judges: the OD value surveying 450nm place by microplate reader; OD value >=0.484 is positive, and OD value < 0.484 is negative.
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