CN109001450A - A kind of kit and preparation method detecting chicken Mycoplasma synoviae antibody - Google Patents

Abstract

The present invention provides a kind of ELISA kit for detecting chicken Mycoplasma synoviae antibody.The kit is the ELISA kit for the detection chicken Mycoplasma synoviae antibody established based on chicken Mycoplasma synoviae pyruvate kinase (PYK) recombinant protein for envelope antigen.The present invention also provides be used to prepare the mentioned reagent box envelope antigen i.e. preparation method of PYK albumen, the PYK albumen of the method preparation provided through the invention has good reactionogenicity, and ELISA detection kit established by the present invention provides effective method for the diagnosis of chicken Mycoplasma synoviae and the detection of immune rear antibody level, plays the role of remarkably promoting for the Prevention Research of the disease.

Description

A kind of kit and preparation method detecting chicken Mycoplasma synoviae antibody

Technical field

The present invention relates to a kind of antibacterial agents boxes and preparation method thereof, are specifically a kind of detection chicken Mycoplasma synoviaes The ELISA kit of antibody.

Background technique

Mycoplasma (Mycoplasmal) is a kind of cell-free wall and precursor, and organelle is few, is in height pleomorphism, can be Microorganism without the minimum prokaryotic cell type of growth and breeding in life culture medium.Birds mycoplasma relates generally to poultry, posts extensively It is born in respiratory tract, cloaca, alimentary canal, tunica mucosa tubae uterinae and the joint capsule of poultry.The birds mycoplasma of identification name at present It shares in 29, wherein belonging to the having in 25 of Mycoplasma, 3 kinds of acholeplasma, a kind of urea substance.In these mycoplasmas, simultaneously Energy infected chicken, turkey and pathogenic mycoplasma mainly have chicken virus mycoplasma (Mycoplasma gallisepticum, MG) and slide Liquid capsule mycoplasma (Mycoplasma synoviae, MS).But compared with virulent MG, raiser is to the understanding of MS, detection And prevention and control are much insufficient, and MS infection is caused to turn to serious respiratory system disease from atypical upper respiratory tract inferior clinical symptom Disease, such as air bag inflammation；When merging other virulence factors, such as newcastle disease virus (NDV), infectious bronchitis virus (IBV), low When pathogenic avian influenza virus or coli-infection, it can make that sb.'s illness took a turn for the worse；In addition, infectiousness bursitis, egg caused by MS Hull shape shape deformity and autoimmune disease equally threaten the sound development of global poultry farming.It is big in 18 world's poultry dieases In meeting, just there is expert to propose the viewpoint of " now and the harm of foreseeable future MS can be more than MG ".Newest epidemiology tune Display is looked into, in Eastern Europe, Holland, Mexico, Argentina, MS infection can cause serious respiratory disease and arthrosynovitis； In China, the disease incidence of MS is also in obvious ascendant trend, in addition the unfavorable factors such as environment and chicken group's state are bad, cause MS to infect The phenomenon that breaking out in chicken group is on the rise.

The method of detection MS includes: being separately cultured of mycoplasma, the diagnosis of serology antibody test and molecular gene at present, In to be separately cultured be to diagnose the goldstandard of mycoplasma infection disease, but mycoplasma is high to the nutritional requirement of culture medium, growth is slow Slowly, separating difficulty is big, is only applicable to laboratory testing；Serologic detection such as plate agglutination test (SPA) specificity is low, and blood clotting Inhibit test (HI) sensibility lower, needs multiple repairing weld, be more suitable for the detection of population level, and the difference and antigen bacteria of result The source of strain is closely related；Polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (real-time PCR) are to apply at present Most diagnostic methods, it is more but there is also operating procedures, it is at high cost, the problem of time-consuming (at least 4.5 hours), it is not easy scene It carries out operation and is promoted to base；External commercialization chicken Mycoplasma synoviae indirect ELISA reagent kit at present, mainly from the U.S. And Europe, inadequate to the antibody test susceptibility of different strains infection although specificity is high, especially expensive, a blood The testing cost of sample is up to 11.5 yuan, limits the use of raiser, only limits laboratory research and uses.It is domestic at present still without using The recombinant protein of high efficient expression does envelope antigen and carries out kit ELISA detection, cheap, specificity is good, sensibility is high Development.With going deep into for MS proteomics research, more Mycoplasma synoviaes cause a disease and immune-related protein is reflected Fixed, Bercic et al. (2008) identifies the main immunogenic protein of Mycoplasma synoviae separation strains, identifies Immunogenic protein including MSPA, EF-Tu etc., wherein pyruvate kinase PYK is also the important immunogenic membrane peptide of MS, Sequence in MS analysis shows that have high conservative.Vivoexpression PYK recombinant protein is established ELISA method as envelope antigen and is opened Sending out antibody test reagent and the detection for the disease has realistic meaning.MS infection at present occurs and joins in China's poultry farms Therefore system develops an urgent demand that special, sensitive, cheap, easy kit is the current prevention and control disease in China.

Summary of the invention

The present invention overcomes the deficiencies of the prior art and provide a kind of kit of accurate detection chicken Mycoplasma synoviae antibody, And the preparation method of this detection kit.

Preparation and purification method including a breeder Mycoplasma synoviae PYK albumen, amino acid sequence such as SEQ ID Shown in No.2；The study found that the albumen has good immunogenic response, it is chicken Mycoplasma synoviae antibody assay kit The envelope antigen of offer.

The ELSIA kit of above-mentioned detection chicken Mycoplasma synoviae comprising the coated elisa plate of PYK albumen, enzyme mark two Anti-, positive control, negative control, cleaning solution, sample diluting liquid, the TMB of commercialization and the component of terminate liquid and proportion be as follows:

Coating buffer: anhydrous Na₂CO₃1.59g NaHCO₃2.93g adds distilled water to 1L；

Cleaning solution: NaCl 8.0g, KH₂PO₄0.2g, Na₂HPO₄·12H₂O 2.9g, KCl 0.2g, Tween-20 0.5mL adds distilled water to 1L；

Confining liquid: the cleaning solution containing 5% serum；

Sample dilution: the cleaning solution containing 5%BSA；

Terminate liquid: for the sulfuric acid solution of 2M.

The preparation method that PYK albumen is recombinated used in above-mentioned dose of box, it is characterized in that artificial synthesized chicken Mycoplasma synoviae The gene order of PYK albumen is that template amplification obtains sequence shown in SEQ ID No.1, and said gene is imported into prokaryotic expression In carrier PET-30a (+), recombinant expression carrier is obtained；Competent escherichia coli cell DH5 α is converted with recombinant expression carrier, is mentioned Recombinant plasmid is taken, is identified through PCR and digestion, filters out positive colony PET-30a-pyk；By above-mentioned recombinant plasmid transformed to BL21 (DE3) after competent cell inducing expression, albumen is expressed in SDS-PAGE electrophoretic analysis, then takes Ni column affinity chromatography method, right Destination protein is purified, then respectively with SDS-PAGE and Western blot detection protein purification as a result, and analyzing PYK egg White antigenicity.

Specifically, the preparation of chicken Mycoplasma synoviae PYK albumen, may comprise steps of:

1) amplifying target genes: using artificial synthesized chicken Mycoplasma synoviae PYK protein gene sequence as template, pass through PCR method expands its pyk gene, clones to obtain target gene, and upstream primer adds BamH I restriction enzyme site, and downstream primer adds Xhol I restriction enzyme site, primer are as follows:

Pyk upstream region of gene primer: 5 '-GGCTTGGATCCATGTATAAAGAAACCG-3’

Pyk downstream of gene primer: 5 '-GCCCTCGAGTTGAACTGTATATTC-3’

2) double digestion is carried out with BamH I, Xhol I after the recycling of Pyk gene and PET-30a (+) plasmid extract, recycles mesh Segment, link 12-16 at 16 DEG C in the ratio that T4DNA connection enzyme effect declines target gene and carrier 5:1 in molar ratio Hour, conversion is extracted plasmid by state cell DH5 α, is obtained positive restructuring after BamH I, Xhol digestion identification are correct and is expressed matter Grain PET-30a-pyk

3) then will extract plasmid PET-30a-pyk and convert BL21 (DE3) competent cell, the positive plasmid bacterium of acquisition in 37 DEG C of cultures are added IPTG to final concentration of 1mmol/L and carry out inducing expression, collect induction when A600 value reaches 0.6-0.8 4 hours thallus after expression, ultrasonic disruption take precipitating to carry out SDS-PAGE electrophoretic analysis after centrifugation.

4) affinitive layer purification is carried out using Ni-NTA, destination protein after purification carries out SDS-PAGE analysis, as a result table The IPTG of bright 1mmol/L induces 4h, and genetic engineering bacterium obtains efficient expression, generates the specific protein band of about 57KD, with The molecular mass of prediction is consistent, and sees attached drawing 1.

5) antigenicity of elisa assay gel extraction albumen.

The present invention also provides the preparation method of mentioned reagent box, the system including chicken Mycoplasma synoviae PYK protein ELISA It is standby, method are as follows: PYK albumen is diluted to 2.6 μ g/mL with above-mentioned coating buffer, is added in ELISA reaction plate by 100 holes μ L/, 37 DEG C are closed 1 hour, and 4 DEG C of coatings overnight, pat dry, and are closed 1 hour with 37 DEG C of 5% horse serum, to contain 0.05% tween PBST washing pats dry and is saved with aluminium film vacuum sealing, is spare.

Based on kit of the present invention, following ELISA method can be used, detect chicken Mycoplasma synoviae serum antibody:

(1) it closes: confining liquid, every hole 100 μ L, 37 DEG C of closing 1h being added into enzyme mark version micropore；

(2) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(3) it is loaded: to be detected blood serum sample of the addition after 1:200 times of sample diluting liquid dilution into enzyme mark version micropore 100 μ L, 37 DEG C of closing 1h.

(4) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(5) add ELIAS secondary antibody: every hole, which is added, has diluted enzyme labelled antibody working solution 100 μ L, 37 DEG C of incubation 45min；

(6) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(7) TMB developing solution is added in every hole, and every 100 μ L of hole, 37 DEG C are protected from light incubation 10min；

(8) the every 100 μ L of hole of terminate liquid is added in every hole, reads OD in microplate reader immediately₄₅₀。

The present invention provides soluble PET-30a (+) plasmid is used, chicken Mycoplasma synoviae PYK albumen, and benefit are expressed With 6 × His label that it is carried, the fusion protein of expression is purified, Western-blot detection proves the albumen of purifying With sound response originality, attached drawing 2 is seen；In addition the protein sequence through NCBI Blast software analysis shows, protein sequence is in chicken It is highly conserved in Mycoplasma synoviae, therefore the detection kit invented has very high specificity and sensibility.

The kit for the antibody test of chicken Mycoplasma synoviae that the present invention constructs, easy to operation, detection is quick Sensitivity, especially suitable for the quick detection clinically to chicken Mycoplasma synoviae antibody, for the disease Prevention Research play it is aobvious Write facilitation.

(1 is protein molecule to expression electrophoretogram of Fig. 1 recombinant plasmid PET-30a/PYK in E.coliBL21 (DE3) Amount standard；2 be not induce engineering bacteria；3-8 be genetic engineering bacterium 0,0.2,0.4,0.6,0.8,1.0mmol/L difference IPTG it is dense Expression product under degree induction；9 be supernatant after ultrasound；10 for ultrasound after precipitate)

(1 is molecular weight of albumen Marker to the purifying electrophoretogram of Fig. 2 recombinant protein PYK；2 be loading sample；3 be 80mM B Eluent；4 be 250mM B eluent)

(1 is chicken Mycoplasma synoviae negative serum to the Western-blot detection figure of Fig. 3 .PYK purifying protein；2 is sliding for chicken Liquid capsule mycoplasma positive serum)

Specific embodiment

One, experimental material

1, key instrument, serum and reagent: ultrasound cracking instrument (U.S.), Bio-Rad680 type enzyme mark detector are purchased from upper Extra large genome company, 96 hole high-affinity ELISA Plates are Costar Products, and recombinant protein PYK is the preparation of this laboratory；Chicken is sliding Liquid capsule mycoplasma standard positive, negative serum are purchased from Chinese animal doctor and supervise institute；Chicken virus mycoplasma positive serum, Mycoplasma bovis are positive Serum, mycoplasma ovine pneumoniae positive serum, Salmonella gallinarum positive serum, white diarrhea positive serum are laboratory preservation；45 Part chicken Mycoplasma synoviae feminine gender chicken picks up from Gansu；Detection 93 parts, 91 parts of chicken serums pick up from Jiangsu and Guangxi respectively, be placed in- 20 DEG C save backup.The SPA of horseradish peroxidase-labeled, tetramethyl benzidine (TMB) and BSA are the product of Sigma company； Horseradish peroxidase-labeled rabbit anti-chicken IgG is purchased from Beijing Suo Laibao Biotechnology Co., Ltd；The production of IDEXX company of the U.S. Chicken Mycoplasma synoviae antibody assay kit is purchased from Beijing Ai Deshi Yuan Heng Biotechnology Co., Ltd；Other conventional reagents are equal It is pure for import or domestic analysis.

2, test solution used and its configuration

1) coating buffer (pH=9.6): 2.93g NaHCO₃, 1.59g Na₂CO₃, pH value is adjusted to 9.6, distilled water added to be settled to 1L mixes 4 DEG C of postposition preservations.

2) cleaning solution (PBST that 0.01mol/L pH value is 7.4): 8.0g NaCl, 0.22g KH₂PO₄, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O, 0.5mL Tween-20, add distilled water to be settled to 1L.

3) confining liquid (5% horse serum cleaning solution): 5mL horse serum, 95mL cleaning solution set 4 DEG C of preservations after evenly mixing.

4) tmb substrate buffer: 4 DEG C be kept in dark place or brown bottle save.

5) terminate liquid (2M H₂SO₄): the 22.2mL98% concentrated sulfuric acid is slowly dropped in 150mL distilled water and is stirred continuously, Finally plus distilled water is to 200mL.

Two, preparation method

1, main material used in antigen preparation includes:

Express bacterium: E.coliBL21 (DE3) is purchased from the vast Tyke biological gene technology Co., Ltd in Beijing.

PET-30a (+) expression vector: it is purchased from the vast Tyke biological gene technology Co., Ltd in Beijing.

Chicken Mycoplasma synoviae standard positive and negative serum: it is purchased from China Veterinary Drugs Supervisory Inst..

Primer: amplimer is by this lab design, and by Beijing, handsome biotech firm is synthesized.

Enzyme and reagent: various restriction endonuclease, Taq archaeal dna polymerase, Pyrobest archaeal dna polymerase, T4DNA Ligase is that Dalian treasured biotech firm product SDS, EB are Sigma Products；Tris is Gibco Products；Agarose, TEMED is Promega Products；Acrylamide, methylene diacrylamide, ammonium persulfate are Biomol Products；Pancreas egg White peptone and yeast powder are OXOID Products；IPTG is Dalian treasured biotech firm product；Glycine, DTT are the production of magnificent company Product；Low molecular weight standard protein is Shanghai Biochemical Research institute of Chinese Academy of Sciences product；Other reagents are domestic analytical reagents.

The following are the specific experiment steps of antigen preparation:

This experiment is using artificial synthesized chicken Mycoplasma synoviae PYK protein gene sequence as template, by PCR method to it Pyk gene is expanded, clones to obtain target gene, and upstream primer adds BamH I restriction enzyme site, and downstream primer adds Xhol I enzyme Enzyme site, primer are as follows:

Pyk upstream region of gene primer: 5 '-GGCTTGGATCCATGTATAAAGAAACCG-3’

Pyk downstream of gene primer: 5 '-GCCCTCGAGTTGAACTGTATATTC-3’

2, target gene is cloned:

PCR product is connect with PET-30a (+) expression vector first, then connection product is transferred to DH5 α competent cell, To obtain the recombinant plasmid PET-30a-pyk for having target gene, positive colony is selected by PCR and digestion identification, and will The recombinant plasmid for being inserted into correct target fragment send Beijing handsome Bioisystech Co., Ltd's sequencing.Sequencing shows sequencing result table Bright, the homology for other chicken Mycoplasma synoviae strain sequences announced in the pyk gene and Gene-Bank expanded is 100%, illustrate the gene in inter-species than more conservative.

3, plasmid is extracted:

From wet, smooth, neat in edge the white of picking on the LB solid medium containing kanamycins of culture 12-16h Bacterium colony is transferred in LB liquid medium of the 5mL containing kanamycins, and 37 DEG C of shaking table cultures are stayed overnight.It is public according to the vast Tyke in Beijing The small-sized rapidly extracting kit of A type plasmid for taking charge of production extracts plasmid, operating procedure:

1) 1.5-3mL bacterium solution is collected, 8000r/m is centrifuged 1min, discards supernatant, and 100 μ L solution 1 are added, and vibrates to thorough It suspends.

2) 150 μ L solution 2 are added, gently overturns centrifuge tube for several times immediately, cracks thallus sufficiently, the thallus after cracking becomes Must be limpid, centrifuge tube is then put into 1-2min on ice.

3) 150 μ L solution 3 are added, mildly reverse centrifuge tube for several times, is placed at room temperature for 5min, 12000r/m centrifugation immediately 12min。

4) 420 μ L combination buffers are added in centrifugal adsorbing column, the supernatant in 3) is then poured into the centrifugation absorption of A type It in column, mixes, 12000r/m is centrifuged 30s, outwells the waste liquid in waste collection pipe.

5) 750 μ L concentrated bleaching liquid are added in centrifugal adsorbing column, after standing 1min, 12000r/m is centrifuged 15s, outwells useless Waste liquid in liquid collecting pipe, is repeated once.After outwelling waste liquid, it is centrifuged 2min in 12000r/m again, removes rinsing buffering as far as possible Liquid.

6) centrifugal adsorbing column is carefully taken out, is inserted in a clean 1.5mL Eppendorf centrifuge tube, is added 30 μ L elution buffer, after being placed at room temperature for 2-5min, 12000r/m is centrifuged 1min.

7) 20 μ L elution buffers are taken again, are placed at room temperature for 1-3min, and 12000r/m is centrifuged 1min, is finally combined into a pipe.

4, the identification of recombinant plasmid PET-30a-pyk

1) electroresis appraisal

5 μ L of recombinant plasmid is taken to carry out agarose gel electrophoresis analysis result.

2) digestion is identified

Double digestion is carried out with BamH I and Xhol I, reaction system is 40 μ L:

After mixing well, 37 DEG C of water-bath 4h.Using DNA 2000Marker as standard, agarose gel electrophoresis enzyme analysis is carried out Cut result.

3) sequencing identification

The positive recombinant plasmid identified through digestion, takes 20 μ L, send Beijing handsome Bioisystech Co., Ltd's sequencing.5, chicken is sliding Expression of the liquid capsule mycoplasma pyk gene in Escherichia coli

Bacterium solution is inoculated in the LB liquid medium containing corresponding antibiotic (kanamycins), 37 DEG C of oscillation trainings by 1:100 It supports to OD₆₀₀When being 0.6~0.8, it is respectively 1mmol/L, 37 DEG C of inducing expression 4h that IPTG, which is added, to final concentration, harvests bacterium, 12000r/m is centrifuged 2min, discards supernatant, and the precipitating that centrifugation obtains is dissolved in 100 μ L 0.01mol/LPBS the mixing that suspends, is added Enter 30 μ L 4 × Protein SDS PAGELoading Buffer, boil 10min, then carries out SDS-PAGE analysis, detection Expression.

6, the detection of expression product

(1)SDS-PAGE

1) glue

By the cleaning of electrophoresis glass plate, do it is baked after, be fixed on according to operating instruction on glue frame, determine between two glass plates Start in the case where liquid-tight with glue.12% separation gel for first matching lower layer, is rapidly added between two glass plates after preparing mixing, Then at once on it layer be carefully added into a small amount of deionized water bind (at this time can with finger gently attack glass plate to exclude to generate Bubble, and to guarantee that the boundary between water and glue is parallel to horizontal table top as far as possible).30min is stood, after gelling to be separated is solid It discards upper layer to bind water, and is inverted and drains residual liquid, configure 5% concentration glue immediately, be added in immediately after preparing mixing Separation gel upper layer, and quick insertion comb (careful insertion, to prevent generating bubble), to its solidification.

2) it installs

Comb carefully is removed, after rinsing loading slot several times with deionized water, electrophoresis tank is put into, is then added into electrophoresis tank 1 × Tris- glycine running buffer.

3) preparation and loading of sample

Take appropriate above-mentioned full bacterium mycoprotein liquid 12000r/m centrifugation 2min, discard supernatant, later with PBS suspend washing from Heart sediment obtains mycoprotein also according to above-mentioned centrifugal condition.Then, it with 100 μ L PBS suspended sediments, and is added 30 μ L 4 × SDS sample-loading buffers mix the two, and 10min is boiled in water-bath, after to be cooled, with 10 μ of micro sample adding appliance loading The hole L/.After sample-adding, after bromophenol blue enters separation gel, voltage can be increased to 90V, continue constant pressure electrophoresis until bromine Phenol indigo plant reaches gel sub.

4) it dyes

After electrophoresis, power supply is closed, gel slab is carefully taken out from electrophoresis tank, uses distilled water flushing.Then glue has been used Plate carefully takes out separation gel and is placed in plate, is subsequently poured into dyeing liquor, places it on the shaking table of horizontal shake and dyes 1-2h.

5) it decolourizes

After dyeing, dyeing liquor is poured into returnable bottle in case next time uses.It is subsequently poured into destainer, places it in water Decolourize 1h on the dynamic shaking table of yawing, after blue background purifies completely, gel is immersed to terminate in deionized water and is reacted.

6) observation and judgement of result

It is compared with the band of standard protein molecular weight Marker, observes and determine result.

7, the purifying of albumen is expressed

(1) Bacteria Culture

It takes to freeze and is inoculated in the LB liquid medium of 5mL in -40 DEG C of 5 μ L of strain, Kan concentration is 50 μ g/mL, and 37 DEG C are shaken Bed 200r/min, overnight.This bacterium solution is seeded in the LB culture medium of 200mL by 1:100, Kan concentration is 50 μ g/mL, 37 DEG C Shaking table culture induces 4h until the IPTG of final concentration of 1mmol/L is added in OD value when being 0.6~0.8.

(2) sample treatment

By 4 DEG C of bacterium solution, 6000r/min is centrifuged 20min, collects precipitating and is dissolved in 20mM phosphate buffer (pH=7.4), 4 DEG C, 6000r/min is centrifuged 20min, collects precipitating.Thallus is resuspended in the 20mM phosphate buffer (pH=7.4) of 20mL In, it mixes well rear ice-bath ultrasonic and is crushed, super 5s stops 5s, ultrasonic 45min.Later in 4 DEG C, 12000r/min is centrifuged 30min, It collects supernatant and crosses 0.45 μm of thick suspended matter of filter membrane removal, this liquid can be used as affinity chromatography loading sample.

(3) affinitive layer purification is carried out using Ni-NTA

A liquid: the 20mM phosphate buffer (pH=7.4) of the NaCl containing 0.5M

B liquid: the 20mM phosphate buffer (pH=7.4) of imidazoles containing 0.5M, 0.5M NaCl

1) column is filled: with the His Trap HP prepacked column of 3ml GE company

2) it balances: balancing pillar, flow velocity 1mL/min with the A liquid of 5 times of bed volumes.

3) loading: by sample loading, flow velocity 1mL/min collects efflux.

4) it washes column: washing pillar with the A liquid of 10 times of bed volumes, wash away unadsorbed albumen, flow velocity 1mL/min is collected Efflux.

5) it elutes: first with the 80mmol B liquid elution of 5 times of bed volumes, flow velocity 1mL/min, collection efflux, then with 4 The 250mmol B liquid elution of times bed volume, flow velocity 1mL/min collect efflux.

6) pillar is rinsed with the 250mmol B liquid of 5 times of bed volumes, trickle abandons it.

7) pillar is rinsed with the A liquid of 5 times of bed volumes, trickle abandons it.

8) column outlet is screwed, 20% ethyl alcohol of 3 times of bed volumes is added, 4 DEG C of refrigerators is placed in and saves.

9) a small amount of each step efflux is taken to carry out SDS-PAGE analysis respectively.

8, the testing result of expression product

(1) the SDS-PAGE analysis of expression product

SDS-PAGE analysis the result shows that, IPTG through 1mmol/L induces 4h, and genetic engineering bacterium obtains efficient expression, The specific protein band for generating 57kD, is consistent with the molecular mass of prediction, the result is shown in Figure 1.

(2) the SDS-PAGE analysis of protein purification figure is expressed

SDS-PAGE analysis the result shows that, flowing through can remove a part of foreign protein in peak, 80mmol B liquid can wash away largely Foreign protein, as a result PYK protein content is shown in Fig. 2 up to 95% in 250mmol B eluent.

(3) measurement of protein concentration

It is 416 μ g/mL with the PYK protein concentration of Pierce BCA protein quantification RNA isolation kit measurement after purification.

(2) coating of kit

(1) determination of ELIAS secondary antibody best effort concentration

The coating buffer of normal chicken serum and equivalent is mixed, by 1:2,1:4,1:8,1:16,1:32,1:64,1:128,1: 256, the dilution proportion of 1:512,1:1024,1:2048, coated elisa plate, 100 holes μ L/, each 1 column of concentration coating；Secondary antibody with The dilution proportion of 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,100 holes μ L/, each Concentration is coated with 1 row, determines the best effort concentration of ELIAS secondary antibody.

(2) determination of antigen PYK most preferably coating degree and best serum dilution

1) protein solution after purification is pressed to the dilution proportion of 1:10,1:20,1:40,1:80,1:160, packet with coating buffer By ELISA Plate, 100 holes μ L/, each 2 column of concentration coating.

2) it covers, 1-2min is vibrated on microoscillator, be placed in 37 DEG C of incubators and incubate 1h, be transferred to 4 DEG C overnight.

3) it is washed 3 times with cleaning solution, each 5min.Washing lotion is filled it up with when washing in hole, washing, which finishes, is absorbing water plank It is patted dry on paper.

4) it is closed with confining liquid, adds 100 holes μ L/, 37 DEG C of incubation 1h are washed 3 times.

5) by positive and negative serum respectively with 1:10,1:20,1:40,1:80 and 1:50,1:100,1:200,1:400 ratio Dilution does square matrix and titrates the determining best peridium concentration of expression product, 100 holes μ L/, and each concentration is coated with 1 row.37 DEG C of incubation 1h, Washing 3 times, drying.

6) ELIAS secondary antibody of 1:1000,100 holes μ L/, 37 DEG C of reaction 45min are added.Washing, drying.

7) 100 hole μ L/ of tmb substrate buffer, 37 DEG C of reaction 10min are added.

8) 100 μ L/ hole terminate liquids are added and terminate reaction, measure OD with microplate reader_450nmValue.

(3) determination of best serum dilution

Protein solution after purification is pressed to the dilution proportion of 1:160, coated elisa plate, 100 holes μ L/ with coating buffer.

Positive serum is divided into former times of dilution and 2 times dilute two kinds, then respectively with 1:50,1:100,1:200,1:400 Dilution proportion, 100 holes μ L/, each concentration are coated with 6 holes.

By negative serum respectively with the dilution proportion of 1:50,1:100,1:200,1:400,100 holes μ L/, each concentration packet By 1 row.

Coated recombinant protein under according to different conditions carries out ELISA measurement, to determine the optimum dilution degree of serum.

(4) operating procedure of ELISA

The operating procedure that ELISA is tested after above-mentioned condition determines is as follows:

96 hole elisa Plates, 100 holes μ L/, 37 DEG C of 1h are coated with after recombinant protein antigen is diluted to optium concentration with coating buffer 4 DEG C are added to stay overnight.Coating buffer is got rid of, with cleaning solution board-washing 3 times, each 5min.Then the confining liquid in 100 holes μ L/ is added in 37 DEG C 1h is closed, board-washing is taken out and is same as above.With serum dilution by after serum samples diluted to optimum dilution degree, 100 holes μ L/, every part of blood Final proof product set two repetitions.After 37 DEG C of reaction 1h, take out board-washing (ibid).Again plus ELIAS secondary antibody, 100 holes μ L/, 37 DEG C of reactions After 45min, take out board-washing (ibid).Then substrate solution is added, terminate liquid termination reaction is added after being protected from light 10min, stands I.e. in reading every hole absorbance value at 450nm in microplate reader, blank control wells are concurrently set.

(5) determination of ELISA yin and yang attribute critical value

The 45 parts of progress indirect ELISA detections of chicken serum for taking laboratory to save.Every part of sample repeats two holes, as a result takes it flat Mean value calculates the average value (X) and standard variance (SD) of sample OD450nm value, according to principle of statistics, the OD450nm of sample When average value (X)+3SD of value > negative sample OD450nm value, the positive can be judged in 99.9% level.

(6) specific test

It is positive that chicken virus mycoplasma positive serum, chicken sramana positive serum, white diarrhea are detected respectively with the ELISA method of foundation The positive serum of serum, Mycoplasma bovis and mycoplasma ovine pneumoniae, and verified whether cross reaction.

(7) sensitivity tests

Chicken Mycoplasma synoviae standard positive serum is done into 1:40,1:80,1:160,1:320,1:640,1:1280,1: 2560, eight dilutions of 1:5120, remaining condition are tested by optimum reaction condition.

(8) repetitive test

18 parts of chicken serums are chosen, use twice test by coated ELISA Plate, 3 repetitions of each progress, calculates its coefficient of variation.

(9) comparative test and the detection to other animals

Have detected the positive and feminine gender blood of 93 portions of chickens altogether with above-mentioned ELISA operation sequence and the yin and yang attribute critical value determined Clearly, and with the result of U.S. IDEXX chicken Mycoplasma synoviae antibody test it compares.

(3) result

(1) determination of ELIAS secondary antibody best effort concentration

From table 1 it follows that when ELIAS secondary antibody 1:800 dilutes, the OD of serum₄₅₀Value is close to 1, it is thus determined that recombination egg The best effort concentration of white ELIAS secondary antibody is 1:1000.

The determination of 1. ELIAS secondary antibody best effort concentration of table

(2) antigen is most preferably coated with condition and determines

From Table 2, it can be seen that recombinant protein 37 DEG C of 1h then 4 DEG C of overnight coatings under the conditions of, positive serum and yin The 0D of property serum₄₅₀Value difference is maximum, illustrates that coating effect is preferable, it is thus determined that the optimum condition of recombinant protein is 1:160.

2. recombinant protein of table is most preferably coated with the determination of condition

(3) definitive result (square matrix titration) of best serum dilution and ELIAS secondary antibody

The extension rate that serum is worked as in square matrix titration display is 1:400, when the dilution of antigen is 1:160, positive serum OD₄₅₀Value can reach 2.729, and the OD of negative serum₄₅₀Value is 0.138, yin and yang attribute serum OD₄₅₀Value difference is maximum, therefore selects 1:400 is best serum diluting multiple, and the optimum diluting multiple of ELIAS secondary antibody is 1:1000 (the results are shown in Table 3).

The OD of 3. square matrix of table titration₄₅₀It is worth result

(4) definitive result of ELISA yin and yang attribute critical value

Indirect ELISA detection is carried out by 45 parts of chicken negative serums.The X that averages is 0.144, and standard variance SD is 0.050, determine the critical point X+3SD=0.144+3 × 0.050=0.294 of yin and yang attribute.That is the OD of sample to be tested₄₅₀Value is greater than It is the positive when 0.294, is less than or equal to 0.294 for negative (the results are shown in Table 4).

The ELISAOD of 4.45 parts of chicken negative serums of table₄₅₀Value

(5) specificity experiments result

Using established ELISA condition, ELISA reaction plate is coated with recombinant protein, with chicken virus mycoplasma positive serum, chicken The positive serum progress ELISA intersection examination of sramana's positive serum, white diarrhea positive serum, Mycoplasma bovis and mycoplasma ovine pneumoniae Test, the results showed that foundation using recombinant protein as antigen examine chicken Mycoplasma synoviae serum antibody ELISA method specificity compared with Good (the results are shown in Table 5).

5. specific test result of table

(6) sensitivity tests result

PYK recombinant protein is coated with by best peridium concentration, and chicken Mycoplasma synoviae positive serum is 1:40,1: 80, eight dilutions of 1:160,1:320,1:640,1:1280,1:2560,1:5120, remaining condition by optimum reaction conditions into Row ELISA test.As a result difficult by visual color variation after being developed the color by ELISA when positive serum is diluted to 1:2560 The negative, positive findings with judgement, but microplate reader still can detect.(the results are shown in Table 6).

6. sensitivity tests result of table

(7) repetitive test result

18 parts of chicken serums are taken, detection 3 times is respectively repeated with the coated ELISA Plate of two batches, calculates standard deviation.As a result it repeats to try It tests the middle coefficient of variation and is up to 7.82%, minimum 2.46%, 18 parts of serum coefficient of variation are all smaller, have preferable repeat Property.

(8) comparative test result testing result

1) ELISA method that application ID EXX box is tested and established detects 93 parts of chicken serum samples, detection knot Fruit is as shown in table 8, and feminine gender detection coincidence rate is 97.8%, and using IDEXX testing inspection result as reference, PYK-ELISA is tested Detect the sample number of result (same sample is feminine gender through two methods detection) consistent with its divided by test sample sum, i.e., Coincidence rate must be detected.The detection coincidence rate of PYK-ELISA test and IDEXX are 97.8%.

The testing result of table 7.IDEXX and ELISA detection chicken serum

2) ELISA method that application ID EXX is tested and established detects 91 parts of chicken serum samples, application ID EXX The ELISA method that box is tested and established detects 91 parts of chicken serum samples, and testing result is as shown in table 9, positive inspection Coincidence rate is 98.9% out, and using IDEXX testing inspection result as reference, it is consistent with its that PYK-ELISA is tested detection result The sample number of (same sample is the positive through two methods detection) is divided by test sample sum to get detection coincidence rate.PYK- The detection coincidence rate of ELISA test and IDEXX are 98.9%.

The testing result of table 8.IDEXX and ELISA detection chicken serum

The major advantage of the present invention compared with the existing technology is as follows:

(1) high specificity and sensibility are high.

(2) testing result is read with ELISA microplate reader, and as a result objective, just, visually observing so as to avoid people may go out Existing error, there is a situation where fail to judge or misjudge.

(3) repeatability is high, same sample repeated detection, as a result unanimously；Or it is detected with the antigen of different batches Product, as a result unanimously, this is the basic demand for developing kit.

(4) broad spectrum activity is strong, chicken Mycoplasma synoviae other kinds submitted in the pyk gene that the present invention selects and GeneBank Gene homology reach 100%, with the PYK albumen of expression, more parts of chicken serums are detected by indirect ELISA, detect As a result high with synchronous IDEXX kit test result coincidence rate, illustrate that the present invention can be used for examining for chicken Mycoplasma synoviae It is disconnected.

(5) recombination bacillus coli of all express express target protein PYK of the present invention, property non-hazardous to people can use fermentation Tank large-scale production realizes quality control, guarantees the uniform quality of ELISA kit produced.

(6) operation of the present invention is simple, is not only suitable for the investigation of field pandemic disease, is also applied for laboratory example and examines It is disconnected.Sequence:

SEQ ID No.1

>pyk gene(1431bp)

atgtataaagaaaccgataaaagaacaaaattagtagctaccatcggaccttcaagtgataattatgaaatgttaaa aaaattagttcaagcaggtgtaacttgcgtgagagctaattttagtcacggatcatacgaagaacaaaaaaataaat ttaacttagcaaaacaagtttcaaaagaattaaaccttcctctttcattaatgctagataccaaaggacctgaaatt cgtgttggtaaaatgaaagatggtgttcaacttattaagcaaggaacaatgcttgatattttaaccactgaagaagc atataaaaacctagaaggaaattcagaacaaatttcagtgtcatatgatatgtcacttgacttgcaagttaatgatt cagtacttttagatgatggaaaactatcaacaaaagttgtaaaagtttcaaaaggtcttgtacaagtttatgtgcaa aacaatcacaaacttaaaacaaataaaagaattaaccttccaggagttgactttagccttccatttttaagtcctaa agatgttgaagacgttaaatttggagttcaaaatggaattagctatgtagcagcttcttttgttaactctgctgaaa acgtaaaacaacttagaaaagttttagtagaaaatggcggagagcacattgaaattatttctaaaattgaatcaact ttaggaattaaaaatattgaccaaatcatcgaagcttcagatggaataatggtggctagaggtgacctaggacttga agttccttactacgaagttccatactaccaaaaaatgattattagaaaatgtcgtgaagcaggtaaaccagttatcg ttgcaactcaaatgcttgattcaatggaaaactcacctcatccaacaagagcagaagtaactgatgtttattttgca gtagaacttggagcagattcaaccatgctttcaggagaatcagcaaatggtggatttcctctagaagctgttcaaac catgacaaaaatttctaagcgtgcagaaagagaattctattcaaaaatctactacccagtacatttagaaaaaatta aagaaaaactaggttcagatcttcgttcattaattgcatacgaaattgctaaaaaaactcaaggcaacgaatacaaa tttgcaattgttttatctagaactggaaaacttcttaaaaaggtagctaactatcgtccaaatactacaatcgtagg tattttagatgatgaaaaactaacaaattcatttggtatttattcaagtgttttcacctcattagattcaaaagaat tattttcagaaattaaaaaagaccattcaaaagctattttagctctaaaaccttttgaatattctaaaggtgataaa ttccttgtagttgaaaatgattctattaaagaatatacagttcaa

SEQ ID No.2

>PYK protein(477AA)

MYKETDKRTKLVATIGPSSDNYEMLKKLVQAGVTCVRANFSHGSYEEQKNKFNLAKQVSKELNLPLSLM LDTKGPEIRVGKMKDGVQLIKQGTMLDILTTEEAYKNLEGNSEQISVSYDMSLDLQVNDSVLLDDGKLSTKVVKVSK GLVQVYVQNNHKLKTNKRINLPGVDFSLPFLSPKDVEDVKFGVQNGISYVAASFVNSAENVKQLRKVLVENGGEHIE IISKIESTLGIKNIDQIIEASDGIMVARGDLGLEVPYYEVPYYQKMIIRKCREAGKPVIVATQMLDSMENSPHPTRA EVTDVYFAVELGADSTMLSGESANGGFPLEAVQTMTKISKRAEREFYSKIYYPVHLEKIKEKLGSDLRSLIAYEIAK KTQGNEYKFAIVLSRTGKLLKKVANYRPNTTIVGILDDEKLTNSFGIYSSVFTSLDSKELFSEIKKDHSKAILALKP FEYSKGDKFLVVENDSIKEYTVQ

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. It is all within the contents of the present invention and principle, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

Sequence table

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

<120>a kind of kit and preparation method for detecting chicken Mycoplasma synoviae antibody

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1948

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

ncstngrtca ncatgtataa agaaaccgat aaaagaacaa aattagtagc taccatcgga 60

ccttcaagtg ataattatga aatgttaaaa aaattagttc aagcaggtgt aacttgcgtg 120

agagctaatt ttagtcacgg atcatacgaa gaacaaaaaa ataaatttaa cttagcaaaa 180

caagtttcaa aagaattaaa ccttcctctt tcattaatgc tagataccaa aggacctgaa 240

attcgtgttg gtaaaatgaa agatggtgtt caacttatta agcaaggaac aatgcttgat 300

attttaacca ctgaagaagc atataaaaac ctagaaggaa attcagaaca aatttcagtg 360

tcatatgata tgtcacttga cttgcaagtt aatgattcag tacttttaga tgatggaaaa 420

ctatcaacaa aagttgtaaa agtttcaaaa ggtcttgtac aagtttatgt gcaaaacaat 480

cacaaactta aaacaaataa aagaattaac cttccaggag ttgactttag ccttccattt 540

ttaagtccta aagatgttga agacgttaaa tttggagttc aaaatggaat tagctatgta 600

gcagcttctt ttgttaactc tgctgaaaac gtaaaacaac ttagaaaagt tttagtagaa 660

aatggcggag agcacattga aattatttct aaaattgaat caactttagg aattaaaaat 720

attgaccaaa tcatcgaagc ttcagatgga ataatggtgg ctagaggtga cctaggactt 780

gaagttcctt actacgaagt tccatactac caaaaaatga ttattagaaa atgtcgtgaa 840

gcaggtaaac cagttatcgt tgcaactcaa atgcttgatt caatggaaaa ctcacctcat 900

ccaacaagag cagaagtaac tgatgtttat tttgcagtag aacttggagc agattcaacc 960

atgctttcag gagaatcagc aaatggtgga tttcctctag aagctgttca aaccatgaca 1020

aaaatttcta agcgtgcaga aagagaattc tattcaaaaa tctactaccc agtacattta 1080

gaaaaaatta aagaaaaact aggttcagat cttcgttcat taattgcata cgaaattgct 1140

aaaaaaactc aaggcaacga atacaaattt gcaattgttt tatctagaac tggaaaactt 1200

cttaaaaagg tagctaacta tcgtccaaat actacaatcg taggtatttt agatgatgaa 1260

aaactaacaa attcatttgg tatttattca agtgttttca cctcattaga ttcaaaagaa 1320

ttattttcag aaattaaaaa agaccattca aaagctattt tagctctaaa accttttgaa 1380

tattctaaag gtgataaatt ccttgtagtt gaaaatgatt ctattaaaga atatacagtt 1440

caartcanct yryshrsysr ghrysaahry rrrssnyrty sysanayahr ysargasnhr 1500

syryrnyssn yshsnaysna ryssnrrtsh rysyrrgayy styssyanys nyhrtshrhr 1560

ayryssnysn rnraryrstr snasnsrass yysrhrysaa ysarysyana yransnsnsy 1620

syshrsnysr gsnryashrr hrryssasay shyansnyry raaarhasnr asnaysnrgy 1680

saasnyysry srhryyssns narsytaarg ysyaryryra ryryrnystr gysysrgayy 1740

sraaahrnts rtsnrrsrhr rgaahrsayr haayasrhrt ryrasnyyhr aanhrthrys 1800

rysrgarghy rrysyryrra sysysysyrs rgrayraysy shrnysnyry shaarrghry 1860

ysysysaasn yrrgrsnhrh rayssyshrs nrhyyrrrah hrrsryshry sysssrysaa 1920

ysrhyrrysy syshaasnsr ysyrhran 1948

Claims (6)

1. a kind of kit for detecting chicken Mycoplasma synoviae antibody, which is characterized in that including chicken Mycoplasma synoviae PYK albumen Elisa plate, ELIAS secondary antibody, Sample dilution, cleaning solution, confining liquid, TMB, terminate liquid, positive control and negative control blood Clearly.

2. a kind of kit for detecting chicken Mycoplasma synoviae antibody according to claim 1, which is characterized in that described ELIAS secondary antibody is the rabbit-anti chicken monoclonal antibody of horseradish peroxidase-labeled；The positive control is natural infection chicken bursa synovialis The chicken serum of mycoplasma；The negative control is normal chicken serum；NaCl containing 8.0g, 0.22g in every liter of the cleaning solution KH₂PO₄, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O, 0.5mL Tween-20；The confining liquid is washing containing 5% horse serum Liquid is washed, Sample dilution is the cleaning solution of the BSA containing 5%；The terminate liquid is the sulfuric acid solution of 2M.

3. a kind of kit for detecting chicken Mycoplasma synoviae antibody according to claim 1, which is characterized in that above-mentioned dose The preparation method that PYK albumen is recombinated used in box, it is characterized in that the gene sequence of artificial synthesized chicken Mycoplasma synoviae PYK albumen It is classified as template amplification and obtains sequence shown in SEQ ID No.1, said gene is imported into prokaryotic expression carrier PET-30a (+) In, obtain recombinant expression carrier；Competent escherichia coli cell DH5 α is converted with recombinant expression carrier, extracts recombinant plasmid, warp PCR and digestion identification, filter out positive colony PET-30a-pyk；Above-mentioned recombinant plasmid transformed to BL21 (DE3) competence is thin After born of the same parents' inducing expression, albumen is expressed in SDS-PAGE electrophoretic analysis, then takes Ni column affinity chromatography method, is carried out to destination protein pure Change, then detects protein purification as a result, and analyzing the antigenicity of PYK albumen with SDS-PAGE and Western blot respectively.

4. a kind of kit for detecting chicken Mycoplasma synoviae antibody according to claim 3, which is characterized in that chicken synovia The preparation of capsule mycoplasma PYK albumen, may comprise steps of:

1) amplifying target genes: using artificial synthesized chicken Mycoplasma synoviae PYK protein gene sequence as template, pass through PCR method Its pyk gene is expanded, clones to obtain target gene, upstream primer adds BamH I restriction enzyme site, and downstream primer adds Xhol I restriction enzyme site, primer are as follows:

Pyk upstream region of gene primer: 5 '-GGCTTGGATCCATGTATAAAGAAACCG-3 '

Pyk downstream of gene primer: 5 '-GCCCTCGAGTTGAACTGTATATTC-3 '

2) double digestion is carried out with BamH I, Xhol I after the recycling of Pyk gene and PET-30a (+) plasmid extract, recycles purpose piece Section links 12-16 hours at 16 DEG C in the ratio of T4DNA connection enzyme effect decline target gene and carrier 5:1 in molar ratio, Conversion is extracted plasmid by state cell DH5 α, obtains positive restructuring expression plasmid PET- after BamH I, Xhol digestion identification are correct 30a-pyk

3) it then will extract plasmid PET-30a-pyk and convert BL21 (DE3) competent cell, the positive plasmid bacterium of acquisition is in 37 DEG C Culture is added IPTG to final concentration of 1mmol/L and carries out inducing expression, collect inducing expression when A600 value reaches 0.6-0.8 4 hours thallus afterwards, ultrasonic disruption take precipitating to carry out SDS-PAGE electrophoretic analysis after centrifugation.

4) affinitive layer purification is carried out using Ni-NTA, destination protein after purification carries out SDS-PAGE analysis, the results showed that The IPTG of 1mmol/L induces 4h, and genetic engineering bacterium obtains efficient expression, generates the specific protein band of about 57KD, and pre- The molecular mass of survey is consistent；

The antigenicity of elisa assay purifying protein.

5. a kind of kit for detecting chicken Mycoplasma synoviae antibody according to claim 1 or 3, which is characterized in that adopt With the elisa plate of the following steps preparation chicken Mycoplasma synoviae PYK albumen: making coating buffer with carbonate buffer solution, use Above-mentioned coating buffer will dilute 2.6 μ g/mL, be added in ELISA reaction plate by 100 holes μ L/, and 37 DEG C are closed 1 hour, and 4 DEG C were coated with Night pats dry, and closes 1 hour with 37 DEG C of 5% horse serum, is washed, patted dry with aluminium film vacuum seal with the PBST containing 0.05% tween Close preservation, spare.

6. a kind of kit for detecting chicken Mycoplasma synoviae antibody according to claim 1, which is characterized in that use ELISA method detects chicken Mycoplasma synoviae serum antibody, specific steps are as follows:

(1) it closes: confining liquid, every hole 100 μ L, 37 DEG C of closing 1h being added into enzyme mark version micropore；

(2) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(3) it is loaded: to be detected blood serum sample 100 μ L of the addition after 1:200 times of sample diluting liquid dilution into enzyme mark version micropore, 37 DEG C of closing 1h.

(4) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(5) add ELIAS secondary antibody: every hole, which is added, has diluted enzyme labelled antibody working solution 100 μ L, 37 DEG C of incubation 45min；

(6) it washs: pouring out the liquid in hole, 300 μ L of cleaning solution is added in every hole, washs 3 times and pats dry；

(7) TMB developing solution is added in every hole, and every 100 μ L of hole, 37 DEG C are protected from light incubation 10min；

(8) the every 100 μ L of hole of terminate liquid is added in every hole, reads OD in microplate reader immediately₄₅₀。