CN109776662A - A kind of cocktail antigen for the detection of perlsucht allergy - Google Patents

Abstract

The present invention provides a kind of cocktail antigen for the detection of perlsucht allergy, includes ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX antigen；Wherein, the mass ratio of ESAT6 antigen, CFP10 antigen, FixB antigen and HSP antigen is 2:2:1.5:1.5.Cocktail antigen provided by the present invention can be used for preparing the detection antigen of perlsucht allergy.Cocktail antigen of the invention can substitute the ox type PPD and fowl type PPD for comparing that allergy uses, single intradermal allergy is carried out with this cocktail antigen, it can achieve the effect that compare allergy, two o'clock cropping, measurement skin depth, injection, 72 were as a child given an encore again and measure skin depth, calculate the sequence of maneuvers such as skin depth difference, it has been simplified to single-point operation and has been not necessarily to calculate, entire detection process is more easy.Meanwhile non-specific mycobacterial infections, the more special diagnosing ox tuberculosis such as can exclude fowl type mycobacteria.

Description

A kind of cocktail antigen for the detection of perlsucht allergy

Technical field

The invention belongs to animal epidemic detection technique fields, and in particular to it is a kind of for perlsucht allergy detection Cocktail antigen.

Background technique

Perlsucht is a kind of chronic debilitating Arbo infectious disease, is International Animal Health tissue (OIE) regulation The animal epidemic that must be notified to belongs to two class zoonosis in China, constitutes to cattle-raising, food safety and human health great It threatens.It according to statistics, is to be caused by Mycobacterium bovis, therefore grasp China's prapes status, quarantine and eliminate there are about 5% in people's tuberculosis Infecting infected cattle has important public health meaning.The quarantine of perlsucht can be classified as following three types at present: bacteriological detection is divided Sub- Biological Detection and immunology detection.Bacteriological detection and molecular biology for detection need to slaughter in sample collection process It slaughters an ox, is unsuitable for the requirement of poultry quarantine living.Immunological detection method includes Serologic detection and cellular immunity detection, due to branch Immune response caused by bacillus is based on cellular immunity, therefore most widely used at present is the intradermal allergy of tuberculin Detection method.

There are two ways to perlsucht tuberculin intradermal allergy, one is single intradermal allergy to test, It comprises the following steps that

1. preoperative medication: the cropping at one third on neck middle side part after ox is only numbered, with calliper to measure art portion center Rhicnosis thickness performs record.

2. injection: no matter large or small ox, the intracutaneous injection ox type PPD0.1mL at cropping.

3. observing response: determining result through 72h (± 4-6h) after intracutaneous injection.

4. result judgement: it is the positive that injection site skin depth difference, which is more than or equal to 4mm, and skin depth difference is greater than 2mm and is less than 4mm simultaneously It is suspicious；Suspicious animal is detected again after should being at least spaced 42 days, and result is the positive or suspicious, is judged to the positive.

And it is similar with single intradermal allergy method to compare allergy, comprises the following steps that

1. preoperative medication: (or neck unilateral side is at a distance of 15cm or more on two middle side part of neck at one third after ox is only numbered Two o'clock) cropping with calliper to measure art portion center rhicnosis thickness performs record.

2. injection: no matter large or small ox only, the intracutaneous injection ox type PPD0.1mL and fowl type PPD at two cropping points respectively 0.1mL。

3. observing response: determining result through 72h (± 4-6h) after intracutaneous injection.

4. result judgement, positive: ox type PPD reacting positive, ox type skin depth are 4mm or more thicker than fowl type skin depth；It is suspicious: ox type PPD is positive or suspicious, and the difference of ox type skin depth and fowl type skin depth is in 1-4mm；It is negative: ox type PPD reaction negative；Though or being ox type PPD is positive or suspicious but equal with fowl type skin depth or is less than fowl type skin depth.

Compare allergy due to additionally having used fowl type PPD to be detected, the spy of allergy can be dramatically increased The opposite sex, can more accurate diagnosing ox tuberculosis.But it is excessively cumbersome due to comparing allergy, almost it is difficult to promote and answers With.

Summary of the invention

The object of the present invention is to provide a kind of cocktail antigens for the detection of perlsucht allergy, can be used for diagnosing Perlsucht.Cocktail antigen of the invention can distinguish fowl type PPD, ox type PPD, and can exclude the non-spies such as fowl type mycobacteria The influence of anisotropic mycobacterial infections.

Cocktail antigen used in the present invention includes ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX anti- It is former；Wherein, the mass ratio of ESAT6 antigen, CFP10 antigen, FixB antigen and HSP antigen is 2:2:1.5:1.5.

Cocktail antigen provided by the present invention can be used for preparing the detection antigen of perlsucht allergy.

Another aspect of the present invention provides a kind of detection antigen of perlsucht allergy, is resisted by above-mentioned cocktail Original preparation；

A kind of detection antigen of perlsucht allergy, wherein ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX The final concentration of antigen is respectively 40 μ g/mL, 40 μ g/mL, 30 μ g/mL, 30 μ g/mL.

Another aspect of the present invention provides a kind of perlsucht allergy diagnostic reagent, wherein including above-mentioned chicken tail Wine antigen and/or detection antigen.

Cocktail antigen of the invention can substitute the ox type PPD and fowl type PPD for comparing that allergy uses, with this chicken tail Wine antigen carries out single intradermal allergy, it can achieve the effect that compare allergy, by two o'clock cropping, measurement skin depth, Injection, 72 were as a child given an encore again to be measured skin depth, calculates the sequence of maneuvers such as skin depth difference, has been simplified to single-point operation and without meter It calculates, entire detection process is more easy.Meanwhile the non-specific mycobacterial infections such as can exclude fowl type mycobacteria, more Special diagnosing ox tuberculosis.

Detailed description of the invention

Fig. 1: the not antidiastole effect picture of synantigen

Fig. 2: the antidiastole effect picture of different antigen combinations

Fig. 3: ESAT6 concentration curve；

Fig. 4: CFP10 curve graph；

Fig. 5: FixB concentration curve；

Fig. 6: HSPX concentration curve；

Fig. 7: protein electrophoresis figure；

Fig. 8: new single site skin test and the time-consuming comparison figure for comparing skin test；

Fig. 9: new single site skin test figure compared with conventional method excludes fowl type mycobacterial infections.

Specific embodiment

The present invention is described in detail with reference to the accompanying drawing.

Embodiment 1: the screening of allergy cocktail antigen

1, the screening of antigen component

ESAT6, CFP10 are mixed and are referred to as EC as a kind of antigen, concentration is identified according to one pack system concentration.By EC, Tetra- kinds of antigen diluents of FixB, MPB83 and HSPX are the concentration of 10 μ g/mL of final concentration, use natural infection ox, avian tuberculosis mycobacterium sense It contaminates 3 groups of animals such as ox and healthy ox and carries out allergy Skin-test.

In the average almost consistent situation of skin depth difference of four kinds of antigen detection avian tuberculosis mycobacterium infection animals and healthy ox Under, the average skin depth that the average skin depth difference that EC antigen detects natural infected animal is greater than MPB83, FixB, HSPX antigen is poor.That is EC When as antidiastole antigen, the skin depth difference of natural infected animal and nonspecific infection animal is much larger than other antigens, table The antidiastole effect of bright EC is better than other components (Fig. 1).

It using EC as indispensable component, is combined with by other each components, forms EC/FixB (being abbreviated as ECF), EC/ 3 kinds of MPB83 (being abbreviated as ECM), EC/HSPX (being abbreviated as ECH) etc. combinations, while being acted on and being passed with PPDB/PPDA (being abbreviated as BA) The control of system method.The result shows that ECF and ECH combination is combined better than ECP.It combines EC with FixB and HSPX simultaneously, forms EC/ FixB/HSPX (is abbreviated as ECFH), and compared with ECF and ECH and BA.As a result think ECFH better than ECF and ECH.It is thus determined that Using ESAT6/CFP10/FixB/HSPX combination as cocktail antigen component (Fig. 2).

2, the concentration screening of 4 kinds of antigen

It is 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 40 μ that EC, FixB, HSPX3 kind antigen protein are done gradient dilution respectively G/mL, 50 μ g/mL, dilute again after freeze-drying, respectively as allergy skin test antigen, use natural infection ox, fowl point 3 groups of animals such as branch bacillus infection ox and healthy ox are tested.

The result shows that 2 groups of animals such as avian tuberculosis mycobacterium infected cattle and healthy ox are when all antigen concentrations rise, OD value Without significant change.And natural infection ox, when 4 kinds of albumen are as antigen, skin depth difference can with the concentration of albumen rise and on It rises, i.e., the skin depth difference difference between natural infection ox and other 2 groups of oxen can be increased as antigen concentration rises, but EC is in concentration After reaching 40 μ g/mL, skin depth difference no longer obviously rise (see Fig. 3-4), FixB and HSPX after concentration reaches 30 μ g/mL, Skin depth difference no longer obviously rises (Fig. 5-6), it was demonstrated that the optimal use of optimal use concentration 40 the μ g/mL, FixB and HSPX of EC are dense Degree is 30 μ g/mL

Embodiment 2: cocktail antigen of the invention is prepared according to determining antigen combination and concentration

One, preparation method

(1) test material

1. key instrument

Constant temperature oscillator: its woods Bell's instrument manufacturing Co., Ltd, Haimen City

Decolorization swinging table: QILINBEIER company

Constant current constant voltage electrophoresis apparatus: Beijing company, Jun Yi instrument plant

Ultrasonic cell disruptor: the new biological Co., Ltd of sesame science and technology in Ningbo

The universal half-dried transfer electrophoresis tank of Trans-SD: Beijing Kai Yuanxinrui Instrument Ltd.

Ice machine: Anting Scientific Instrument Factory, Shanghai

Ultraviolet-uisible spectrophotometer: SHIMADZU

Trace dna protein assay: Nanodrop

Freeze drier: Toshiba

2. recombinant plasmid

Recombinating pET-28a-HSPX, pET-28a-FixB, pET-28a-ESAT6 and pET-28a-CFP10 is inventor Building.

3. main agents

Glacial acetic acid, isopropanol, Tween-20, hydrochloric acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, Coomassie brilliant blue, benzyl sulphur Acyl fluorides, isopropylthiogalactoside, acrylamide, methene-acrylamide, ammonium persulfate, PEG 20000, PBS buffering Liquid etc. is purchased from Sinopharm Chemical Reagent Co., Ltd., lauryl sodium sulfate, sodium chloride, potassium chloride, Coomassie brilliant blue, methyl Sulfuryl fluoride (PMSF), isopropylthiogalactoside (IPTG) etc. are purchased from Shanghai Sheng Gong bioengineering Co., Ltd

4, main agents configure

(1) coomassie brilliant blue R_250 dyeing liquor: weighing 1g coomassie brilliant blue R_250, measures 250ml isopropanol, is placed in 1L It is stirred evenly in beaker, 100ml glacial acetic acid is added, is settled to 1L, after filter paper is filtered to remove particulate matter, room temperature preservation.

(2) coomassie brilliant blue R_250 destainer: measuring glacial acetic acid 100ml, and dehydrated alcohol 50ml adds deionized water to mix Afterwards, 1L, room temperature preservation are settled to.

(3) 30%Acrylamide (acrylamide): acrylamide 290g is weighed, methene-acrylamide 10g is in 1L beaker In, about 600mL deionized water is added, is settled to 1L after stirring and dissolving, 0.45 μm of membrane filtration decontamination is placed in 4 in brown bottle DEG C save.

(4) 10% ammonium persulfates: weighing 1g ammonium persulfate, and stirring and dissolving after 10ml deionized water is added, and 4 DEG C save 2 weeks Left and right.

(5) 10%SDS solution: weighing 10g SDS in 100mL beaker, and 75mL deionized water is added to dissolve by heating in 68 DEG C, Enriching hydrochloric acid tune pH value is settled to 100mL, room temperature preservation to 7.2.

(6) 1M Tris-HCl (pH6.8): weighing 121.1g Tris in 1L beaker, and 800mL deionized water is added, stirs Dissolution is mixed, is 6.8 with concentrated hydrochloric acid tune pH value, is settled to 1L.After 121 DEG C of high pressure sterilizations, room temperature preservation.

(7) 1.5M Tris-HCl (pH8.8): weighing 181.7g Tris in 1L beaker, and 800mL deionized water is added, Dissolution is sufficiently stirred, is 8.8 with concentrated hydrochloric acid tune pH value, is settled to 1L.After 121 DEG C of high pressure sterilizations, room temperature preservation.

(8) 5 × SDS-PAGE electrophoretic buffers: weighing Tris 15.1g, Glycine 94g, SDS 5g in 1L beaker, 1L, room temperature preservation are settled to after deionized water stirring and dissolving is added.

(9) 1M IPTG solution: 2.38g IPTG is weighed in 10mL centrifuge tube, appropriate deionization is added, after completely dissolution It is settled to 10mL, with 0.22 μm of filter filtration sterilization, 1mL/ parts is dispensed, is stored in -20 DEG C.

(10) Tris 5.81g, glycine 2.93g, SDS transferring film buffer (Transfer buffer): are weighed 0.375g after adding deionized water dissolving, adds 200mL methanol and sufficiently dissolves, be settled to 1L, room temperature storage is spare.

(2) preparation method

The inducing expression of 1.ESAT6, CFP10, FixB and HSPX albumen

(1) building is contained into pET-28a-ESAT6, pET-28a-CFP10, pET-28a-FixB and pET-28a-HSPX Bacterial strain is successively inoculated in the LB liquid medium containing kanamycins (Kan+) resistance, and 37 DEG C, 200r/min shaken cultivation 12h.

(2) pET-28a-ESAT6, pET-28a-CFP10, pET-28a-HSPX and the pET-28a- being incubated overnight are taken respectively Each 50 μ L of FixB bacterium solution is successively inoculated in the fresh LB liquid medium of 50mL (Kan+40 μ g/mL), 37 with 1:1000 ratio DEG C, when 200r/min cultivates about 0.6 2-3h to OD600nm, respectively take 1mL bacterium solution as control is not induced, 4 DEG C save backup.

(3) addition IPTG to final concentration of 1mmol/L, 37 DEG C, 200r/min, pET-28a-ESAT6, pET-28a- CFP10, pET-28a-FixB and pET-28a-HSPX Fiber differentiation collect thallus after inducing 4h.

(4) 1mL bacterium solution is taken out respectively, and 12000r/min is centrifuged 2min, discards supernatant, and collects thallus.

2. protein purification

(1) bacterium is collected: taking bacterium solution after inducing expression, 4 DEG C are centrifuged 10min with 8000r/min, culture medium are discarded, with suitable It measures PBS buffer solution and bacterial sediment is resuspended, 4 DEG C are centrifuged 10min with 8000r/min, discard supernatant, and collect bacterial sediment.

(2) cellular lysate: with 15mL combination/washing buffer (20mM Tris-HCl, 500mM NaCl, 10mM imidazoles, PH 8.0) thallus is resuspended, ultrasonication 30min, 5s/ times, is spaced 5s, 400W on ice.4 DEG C are centrifuged 10min with 8000r/min, Retain supernatant.

(3) pillar balances: balance pillar to room temperature takes lower bottom cap, removes top cap, surplus liquid is allowed to flow down, and clamps Pillar places it vertically, at the top of pillar upward.Pillar, flow control are washed with combination/washing buffer of twice of column volume In 0.5-1mL/min.

(4) it washes column: the supernatant obtained in step (2) being added to above resin (with 0.45 μm of membrane filtration before loading), is received Collect the liquid flowed through.If desired, the liquid flowed through is rejoined pillar, so that albumen maximum capacity is incorporated on pillar.

(5) column is washed: with combination/washing buffer of twice of column volume (if inclusion body, with containing for twice column volume 8mol/L urea washes buffer) pillar is washed, collect the liquid flowed through.

(6) destination protein elutes: with the elution buffer of twice of column volume (if inclusion body, with containing for twice column volume 8mol/L urea elution buffer) albumen of elution band His label from resin.Step is repeated twice, in different Guan Zhongshou Collect each component.The protein eluted is analyzed with SDS-PAGE.

(7) resin cleans: if it is observed that back pressure increases or resin significantly pollutes, with the 0.5M of 15 times of column volumes NaOH cleans filter cylinder.The time of contact of permission is 30 minutes, and correspondingly adjusts flow rate.It is rebalanced with the 1xPBS of 10 volumes, Pillar is stored in the NaOH of 20-30% ethyl alcohol or 10-100mM.

(8) dialyse: bag filter is boiled twice with bag filter treatment fluid, each 10min, then is boiled twice with ultrapure water, often Protein solution after purification is put into bag filter by secondary 10min, and both ends are clipped with clip, is successively being contained 4M, 3M, 2M and is being free of Renaturation in the renaturation solution of urea keeps 4 DEG C of low temperature environments, and every 4-6h replaces a solution until bag filter two sides concentration is consistent.

(9) it measures concentration: measuring protein concentration using trace dna protein assay.

The SDS-PAGE electrophoresis of 3.ESAT6, CFP10, HSPX and FixB albumen

12%SDS-PAGE electrophoresis is carried out to the destination protein of elution.

(1) board-washing loading board: first being cleaned glass plate with pure water before glue, then is rinsed for several times with MilliQ grades of water.Then will Glass plate is fixed on bracket, is filled ultrapure water between the groove of two plates, is checked whether leakage, and moisture is vacated, and drying is stand-by.

(2) glue: 12% separation gel and 5% concentration glue are prepared, is shown in Table 1, is uniformly mixed after sequentially adding each component.

1 SDS-PAGE separation gel of table and concentration glue configuration scheme

(3) encapsulating: each component mix after, separation gel is slowly injected into plastic plate, avoid generate bubble, about 8mL or so, Add dehydrated alcohol fluid-tight, 37 DEG C of incubation 30min to complete solidification.Dehydrated alcohol is outwelled, filter paper blots residual liquid.Fill concentration Glue plugs comb, 37 DEG C of incubation 30min to complete solidification.

(4) sample treatment: isometric 2 × Protein Loading Buffer being added into protein sample, after mixing, 100 DEG C are boiled 5min.

(5) electrophoresis: 15 μ L sample loadings, 80V constant pressure electrophoresis 30min protein concentrate sample, then 120V electrophoresis 1h are loaded.

(6) dyeing-decolorzing: the gel after electrophoresis is put into the staining plate containing appropriate coomassie brilliant blue staining liquid, 50r/ Min dyes at least 1h.After the completion of dyeing, in decolorization swinging table, 50r/min, Coomassie brilliant blue destainer is decolourized repeatedly to gel It is fully transparent.

(7) electrophoretic band of purifying protein it is single, clear (Fig. 7).

4. prepared by finished product

By ESAT6, CFP10, FixB and HSPX albumen of purifying, it is concentrated into 0.7mg/mL's using PEG 20000 Concentration mixes according to the ratio of 2:2:1.5:1.5, is distributed into 5mL/ bottles, saves after freeze-drying in 4 DEG C.

Embodiment 3: the application of allergy cocktail antigen

Cocktail antigen prepared by the present invention is used as perlsucht allergy antigen, the specific steps are as follows:

1, antigen is prepared:

By ESAT6, CFP10, FixB and HSPX albumen of purifying, it is concentrated into 0.7mg/ respectively using PEG 20000 The concentration of mL is mixed and made into cocktail antigen of the invention according to the ratio of 2:2:1.5:1.5, is distributed into 1mL/ bottles, after freeze-drying It is saved in 4 DEG C.

2, allergy is tested

Take 5mLPBS before use, be added into the cocktail antigen being lyophilized, used after mixing, make ESAT6, CFP10, The final concentration of FixB and HSPX is respectively 40 μ g/mL, 40 μ g/mL, 30 μ g/mL, 30 μ g/mL.

Allergy test is carried out according to following step:

1. preoperative medication: the cropping at one third on neck middle side part after ox is only numbered, with calliper to measure art portion center Rhicnosis thickness performs record.

2. injection: no matter large or small ox, respectively in neck side intracutaneous injection cocktail antigen 0.1mL.

3. observing response: determining result through 72h (± 4-6h) after intracutaneous injection.

4. result judgement: it is the positive that injection site skin depth difference, which is more than or equal to 4mm, and skin depth difference is greater than 2mm and is less than 4mm simultaneously Suspicious, it is feminine gender that skin depth difference, which is less than 2mm,；Suspicious animal is detected again after should being at least spaced 42 days, and result is positive or suspicious , it is judged to the positive.

Using cocktail antigen, single intradermal allergy is carried out, (cocktail antigen single-point Skin-test, referred to as newly Type single-point skin test), that is, it can reach while (comparing Skin-test, referred to as using the comparison allergy of ox type PPD and fowl type PPD To compare skin test) effect.To the healthy ox of 5 natural infection oxen, 5 avian tuberculosis mycobacterium infected cattles and 5, while being detected, As a result new single site skin test and compare skin test and have 10 positives, 5 feminine genders, the coincidence rate of the two is 100%.It is shown in Table 2.

Table 2: new single site skin test and the comparative test for comparing skin test

The simple neck allergy of the perlsucht (letter of new single site skin test and traditional perlsucht quarantine method, that is, traditional Referred to as traditional single-point skin test) it compares, it is possible to authenticate fowl type mycobacterial infections reduce the nonspecific interference of test.To 5 fowl The detection of mycobacterial infections Niu Jinhang perlsucht, traditional single-point skin test is positive findings, and fowl type mycobacteria elimination factor is 0%；And new single site skin test is negative findings, fowl type mycobacteria elimination factor is 100% (Fig. 8).

New single site skin test skin test compared with compares, and cropping, injection, measures skin depth again, calculates measurement skin depth Deng can save the time (Fig. 9) of about half.Correspondingly, its workload also reduces half, experiment process is simplified, is reduced Test operation.

Claims (5)

1. a kind of cocktail antigen, which is characterized in that the cocktail antigen include ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX antigen；Wherein, the mass ratio of ESAT6 antigen, CFP10 antigen, FixB antigen and HSP antigen is 2:2: 1.5:1.5.

2. application of the cocktail antigen described in claim 1 in the detection antigen of preparation perlsucht allergy.

3. a kind of detection antigen of perlsucht allergy, which is characterized in that the detection antigen is by claim 1 institute The cocktail antigen preparation stated.

4. a kind of detection antigen of perlsucht allergy, which is characterized in that ESAT6 antigen in the detection antigen, The final concentration of CFP10 antigen, FixB antigen and HSPX antigen is respectively 40 μ g/mL, 40 μ g/mL, 30 μ g/mL, 30 μ g/mL.

5. a kind of perlsucht allergy detects antigen, which is characterized in that include claim 1 in the detection antigen The cocktail antigen and/or detection antigen as claimed in claim 3.