CN109776662B - Cocktail antigen for detecting bovine tuberculosis allergy - Google Patents

Abstract

The invention provides a cocktail antigen for detecting bovine tuberculosis allergy, which comprises an ESAT6 antigen, a CFP10 antigen, a FixB antigen and an HSPX antigen; wherein the mass ratio of the ESAT6 antigen, the CFP10 antigen, the FixB antigen and the HSP antigen is 2:2:1.5: 1.5. The cocktail antigen provided by the invention can be used for preparing a detection antigen for bovine tuberculosis allergy. The cocktail antigen can replace bovine PPD and avian PPD used for allergy comparison, the cocktail antigen is used for single intradermal allergy reaction, the effect of allergy comparison can be achieved, a series of operations of two-point shearing, skin thickness measurement, injection, returning to the field again after 72 hours, skin thickness difference calculation and the like are simplified into single-point operation without calculation, and the whole detection process is simpler and more convenient. Meanwhile, non-specific mycobacteria infection such as avian mycobacterium and the like can be eliminated, and bovine tuberculosis can be diagnosed more specifically.

Description

Cocktail antigen for detecting bovine tuberculosis allergy

Technical Field

The invention belongs to the technical field of animal epidemic disease detection, and particularly relates to a cocktail antigen for detecting bovine tuberculosis allergy.

Background

Bovine tuberculosis is a chronic consumable zoonosis infectious disease, is an animal epidemic disease which needs to be reported by international animal health Organization (OIE), belongs to two types of animal infectious diseases in China, and poses a great threat to cattle industry, food safety and human health. According to statistics, about 5 percent of human tuberculosis is caused by mycobacterium bovis, so that the method has important public health significance for mastering the current bovine tuberculosis situation in China and quarantining and eliminating infected cows. Quarantine of bovine tuberculosis can be currently classified into three categories: bacteriological testing, molecular biological testing, and immunological testing. Bacteriological and molecular biological detection methods require slaughtering of the cattle during sample collection and are not suitable for live animal quarantine requirements. The immunological detection method comprises serological detection and cellular immune detection, and the body immune response caused by mycobacteria is mainly cellular immune, so that the method is most widely applied to the detection of the tuberculin intradermal allergy at present.

There are two methods of intradermal allergy to bovine tuberculin, one being a single intradermal allergy test comprising the steps of:

preoperative treatment: after numbering cattle, shearing hair at the upper third of the middle part of the neck side, measuring the thickness of the central wrinkle of the operative part by a caliper, and recording.

Injection: bovine PPD was injected intradermally at the haircut site at 0.1mL for both large and small cattle.

Observation reaction: the results were judged 72h (+ -4-6 h) after intradermal injection.

Judging the result: the skin thickness difference of the injection part is more than or equal to 4mm and is positive, and the skin thickness difference is more than 2mm and less than 4mm and is suspicious; the suspicious animals should be detected at least after 42 days, and the result is positive or suspicious and positive.

The comparative allergy is similar to the single intradermal allergy method, and comprises the following steps:

preoperative treatment: after numbering cattle, shearing hair at one third of the middle part of two sides of the neck (or two points with a distance of more than 15cm on one side of the neck), measuring the thickness of the central wrinkle of the operative part by using a caliper, and recording.

Injection: no matter whether big or small cattle, 0.1mL of bovine PPD and 0.1mL of avian PPD were injected intradermally at two shearing points, respectively.

Observation reaction: the results were judged 72h (+ -4-6 h) after intradermal injection.

Judging the result, and determining the positivity: PPD reaction of cattle type is positive, and the thickness of the cattle type skin is more than 4mm thicker than that of the poultry type skin; and (3) suspicious: PPD of cattle type is positive or suspicious, and the difference between the cattle type skin thickness and the poultry type skin thickness is 1-4 mm; negative: bovine PPD reaction was negative; or is positive or suspicious for bovine PPD, but is equal to or less than the thickness of the poultry skin.

Compared with the allergy, the avian PPD is additionally used for detection, so that the specificity of the allergy can be obviously increased, and the tuberculosis of the cattle can be more accurately diagnosed. But the comparative allergic reaction is too complicated, so the popularization and the application are difficult.

Disclosure of Invention

The invention aims to provide a cocktail antigen for detecting bovine tuberculosis allergy, which can be used for diagnosing bovine tuberculosis. The cocktail antigen can distinguish avian PPD and bovine PPD, and can eliminate the influence of non-specific mycobacteria infection such as avian mycobacteria.

The cocktail antigen used in the invention comprises ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX antigen; wherein the mass ratio of the ESAT6 antigen, the CFP10 antigen, the FixB antigen and the HSP antigen is 2:2:1.5: 1.5.

The cocktail antigen provided by the invention can be used for preparing a detection antigen of bovine tuberculosis allergy.

In another aspect, the invention provides a detection antigen for bovine tuberculosis allergy, which is prepared from the cocktail antigen;

the detection antigen for the bovine tuberculosis allergy has the final concentrations of 40 mug/mL, 30 mug/mL of ESAT6 antigen, 40 mug/mL of CFP10 antigen, FixB antigen and HSPX antigen.

In another aspect, the invention provides a diagnostic reagent for bovine tuberculosis allergy, which comprises the cocktail antigen and/or the detection antigen.

The cocktail antigen can replace bovine PPD and avian PPD used for allergy comparison, the cocktail antigen is used for single intradermal allergy reaction, the effect of allergy comparison can be achieved, a series of operations of two-point shearing, skin thickness measurement, injection, returning to the field again after 72 hours, skin thickness difference calculation and the like are simplified into single-point operation without calculation, and the whole detection process is simpler and more convenient. Meanwhile, non-specific mycobacteria infection such as avian mycobacterium and the like can be eliminated, and bovine tuberculosis can be diagnosed more specifically.

Drawings

FIG. 1: differential diagnostic effect map of different antigens

FIG. 2: differential diagnostic effect map of different antigen combinations

FIG. 3: ESAT6 concentration profile;

FIG. 4: CFP10 graph;

FIG. 5: FixB concentration profile;

FIG. 6: HSPX concentration profile;

FIG. 7: protein electrophoretogram;

FIG. 8: time consumption comparison graphs of the novel single-point skin test and the comparative skin test;

FIG. 9: a comparison of the novel single-point skin test with the traditional method for excluding avian mycobacterial infection.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

Example 1: screening of cocktail antigens for allergy

1. Screening of antigenic Components

The ESAT6, CFP10 is mixed as an antigen called EC, and the concentration is marked by single component concentration. Four antigens, EC, FixB, MPB83 and HSPX, were diluted to a final concentration of 10. mu.g/mL, and 3 groups of animals, such as naturally infected cattle, Mycobacterium avium infected cattle and healthy cattle, were used for allergy skin tests.

Under the condition that the average skin thickness difference values of four antigen detection avian mycobacterium infected animals and healthy cattle are almost consistent, the average skin thickness difference of EC antigen detection naturally infected animals is larger than that of MPB83, FixB and HSPX antigens. That is, when EC is used as an antigen for differential diagnosis, the difference between the skin thickness of naturally infected animals and that of non-specifically infected animals is much larger than that of other antigens, which indicates that the differential diagnosis effect of EC is better than that of other components (FIG. 1).

EC is taken as an essential component, and is combined with other components to form 3 combinations such as EC/FixB (ECF), EC/MPB83 (ECM), EC/HSPX (ECH) and the like, and the combination is compared with a PPDB/PPDA (BA) action traditional method. The results indicate that the ECF and ECH combinations are superior to the ECP combinations. EC was combined simultaneously with FixB and HSPX to form EC/FixB/HSPX (abbreviated as ECFH) and compared to ECF and ECH and BA. The results suggest that ECFH outperformed ECF and ECH. The ESAT6/CFP10/FixB/HSPX combination was therefore identified as a cocktail antigen component (FIG. 2).

2. Concentration screening of 4 antigens

EC, FixB and HSPX3 antigen proteins are respectively diluted into 10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL and 50 mu g/mL in a gradient way, and are re-diluted after freeze-drying, and are respectively used as allergic skin test antigens to carry out tests by using 3 groups of animals such as naturally infected cattle, avian mycobacterium infected cattle, healthy cattle and the like.

The results show that the OD value of 2 groups of animals such as the avian mycobacterium infected cattle, the healthy cattle and the like has no obvious change when all the antigen concentrations rise. When naturally infected cattle and 4 proteins are used as antigens, the skin thickness difference of the naturally infected cattle and 4 proteins is increased along with the increase of the concentration of the proteins, namely, the skin thickness difference between the naturally infected cattle and other cattle in 2 groups is increased along with the increase of the concentration of the antigens, but the skin thickness difference of the EC is not obviously increased after the concentration of the EC reaches 40 mu g/mL (shown in figures 3-4), and the skin thickness difference of the FixB and HSPX is not obviously increased after the concentration of the FixB and HSPX reaches 30 mu g/mL (shown in figures 5-6), so that the optimal use concentration of the EC is 40 mu g/mL, and the optimal use concentration of the FixB and HSPX is 30 mu g/mL

Example 2: preparation of cocktail antigens of the invention based on defined antigen combinations and concentrations

First, preparation method

(I) test materials

1. Main instrument

A constant temperature oscillator: marigold instruments manufacturing Ltd

And (3) decoloring and shaking table: QILINBEIER Corp

Flow-stabilizing voltage-stabilizing electrophoresis apparatus: beijing Junyi Instrument factory Co Ltd

Ultrasonic cell crusher: ningbo Xinzhi scientific and biological Co., Ltd

Trans-SD general type semi-dry transfer electrophoresis tank: beijing Kaiyun instruments Co Ltd

An ice maker: shanghai' an pavilion scientific instrument factory

Ultraviolet-visible spectrophotometer: SHIMADZU

Micro nucleic acid protein determinator: nanodrop

Freeze-drying the instrument: toshiba

2. Recombinant plasmid

Recombinant pET-28a-HSPX, pET-28a-FixB, pET-28a-ESAT6 and pET-28a-CFP10 were constructed by the inventors.

3. Primary reagent

Glacial acetic acid, isopropanol, Tween-20, hydrochloric acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, Coomassie brilliant blue, phenylmethylsulfonyl fluoride, isopropylthiogalactoside, acrylamide, methylene-acrylamide, ammonium persulfate, polyethylene glycol 20000, PBS buffer and the like from national drug group chemical reagents Co., Ltd, and sodium dodecyl sulfate, sodium chloride, potassium chloride, Coomassie brilliant blue, methylsulfonyl fluoride (PMSF), Isopropylthiogalactoside (IPTG) and the like from Shanghai Biotech engineering Co., Ltd

4. Primary reagent configuration

(1) Coomassie brilliant blue R-250 staining solution: weighing 1g of Coomassie brilliant blue R-250, measuring 250ml of isopropanol, putting the mixture into a 1L beaker, uniformly stirring, adding 100ml of glacial acetic acid, fixing the volume to 1L, filtering by using filter paper to remove particulate matters, and storing at room temperature.

(2) Coomassie brilliant blue R-250 destaining solution: measuring 100ml of glacial acetic acid and 50ml of absolute ethyl alcohol, adding deionized water, uniformly mixing, fixing the volume to 1L, and storing at room temperature.

(3) 30% Acrylamide (Acrylamide): weighing 290g of acrylamide and 10g of methylene-acrylamide in a 1L beaker, adding about 600mL of deionized water, stirring to dissolve, fixing the volume to 1L, filtering with a 0.45 mu m filter membrane to remove impurities, and storing in a brown bottle at 4 ℃.

(4) 10% ammonium persulfate: ammonium persulfate of 1g is weighed, added with deionized water of 10ml, stirred and dissolved, and stored for about 2 weeks at 4 ℃.

(5) 10% SDS solution: weighing 10g SDS into a 100mL beaker, adding 75mL deionized water, heating and dissolving at 68 ℃, adding concentrated hydrochloric acid to adjust the pH value to 7.2, fixing the volume to 100mL, and storing at room temperature.

(6)1M Tris-HCl (pH6.8): 121.1g of Tris is weighed in a 1L beaker, 800mL of deionized water is added, the mixture is stirred and dissolved, the pH value is adjusted to 6.8 by concentrated hydrochloric acid, and the volume is adjusted to 1L. Sterilizing at 121 deg.C under high pressure, and storing at room temperature.

(7)1.5M Tris-HCl (pH8.8): 181.7g Tris is weighed into a 1L beaker, 800mL deionized water is added, the mixture is fully stirred and dissolved, the pH value is adjusted to 8.8 by concentrated hydrochloric acid, and the volume is adjusted to 1L. Sterilizing at 121 deg.C under high pressure, and storing at room temperature.

(8)5 XSDS-PAGE running buffer: weighing 15.1g of Tris, 94g of Glycine and 5g of SDS into a 1L beaker, adding deionized water, stirring and dissolving, then fixing the volume to 1L, and storing at room temperature.

(9)1M IPTG solution: weighing 2.38g IPTG into a 10mL centrifuge tube, adding a proper amount of deionized water, fully dissolving, metering to 10mL, filtering and sterilizing by using a 0.22 mu m filter, subpackaging by 1 mL/part, and storing at-20 ℃.

(10) Transfer buffer (Transfer buffer): weighing Tris 5.81g, glycine 2.93g and SDS 0.375g, adding deionized water for dissolving, then adding 200mL of methanol for fully dissolving, fixing the volume to 1L, and storing at room temperature for later use.

(II) preparation method

Inducible expression of ESAT6, CFP10, FixB and HSPX proteins

(1) The constructed strains containing pET-28a-ESAT6, pET-28a-CFP10, pET-28a-FixB and pET-28a-HSPX are sequentially inoculated into LB liquid culture medium containing kanamycin (Kan +) resistance and are cultured for 12 hours at 37 ℃ under the condition of 200r/min with shaking.

(2) 50 mu L of overnight-cultured bacterial solutions of pET-28a-ESAT6, pET-28a-CFP10, pET-28a-HSPX and pET-28a-FixB are respectively taken and sequentially inoculated into 50mL of fresh LB liquid culture medium (Kan +40 mu g/mL) according to the proportion of 1:1000, and when the culture is carried out for 2-3h at 37 ℃ and 200r/min until the OD600nm is about 0.6, 1mL of bacterial solutions are respectively taken as non-induced control and stored for standby at 4 ℃.

(3) Adding IPTG to the final concentration of 1mmol/L, performing induction culture at 37 ℃ and 200r/min on pET-28a-ESAT6, pET-28a-CFP10, pET-28a-FixB and pET-28a-HSPX, and collecting thalli after induction for 4 h.

(4) Respectively taking out 1mL of bacterial liquid, centrifuging at 12000r/min for 2min, discarding supernatant, and collecting thalli.

2. Protein purification

(1) And (3) bacteria collection: taking the bacteria liquid after induction expression, centrifuging for 10min at 8000r/min at 4 ℃, discarding the culture medium, re-suspending the thallus precipitate with a proper amount of PBS buffer solution, centrifuging for 10min at 8000r/min at 4 ℃, discarding the supernatant, and collecting the thallus precipitate.

(2) And (3) cracking thalli: the cells were resuspended in 15mL binding/washing buffer (20mM Tris-HCl, 500mM NaCl, 10mM imidazole, pH 8.0) and sonicated on ice for 30min at 5 s/time intervals of 5s, 400W. Centrifuging at 8000r/min for 10min at 4 deg.C, and collecting supernatant.

(3) Column equilibration: the column is equilibrated to room temperature, the bottom cap is removed, the top cap is removed, excess liquid is allowed to flow down, the column is clamped so that it is placed vertically, with the top of the column facing up. The column was washed with two column volumes of binding/washing buffer, the flow rate was controlled between 0.5-1 mL/min.

(4) And (3) column washing: the supernatant obtained in step (2) was added on top of the resin (filtered through a 0.45 μm filter before loading) and the liquid was collected. If necessary, the flow through liquid is re-fed to the column to maximize the ability of the protein to bind to the column.

(5) And (3) column washing: the column is washed with two column volumes of binding/washing buffer (in the case of inclusion bodies, two column volumes of washing buffer containing 8mol/L urea) and the liquid that flows through is collected.

(6) Eluting the target protein: the His-tagged proteins were eluted from the resin using two column volumes of elution buffer (two column volumes of elution buffer containing 8mol/L urea if inclusion bodies). The procedure was repeated twice and the fractions were collected in different tubes. The eluted proteins were analyzed by SDS-PAGE.

(7) Resin cleaning: if an increase in back pressure or significant contamination of the resin is observed, the cartridge is washed with 15 column volumes of 0.5M NaOH. The allowed contact time was 30 minutes and the flow rate was adjusted accordingly. The column was re-equilibrated with 10 volumes of 1xPBS and stored in 20-30% ethanol or 10-100mM NaOH.

(8) And (3) dialysis: boiling the dialysis bag with dialysis bag treatment solution twice, each time for 10min, boiling with ultrapure water twice, each time for 10min, filling the purified protein solution into the dialysis bag, clamping two ends with clips, renaturing in renaturation solution containing 4M, 3M, 2M and no urea in sequence, maintaining 4 deg.C low temperature environment, and replacing the solution every 4-6h until the concentrations of two sides of the dialysis bag are consistent.

(9) And (3) measuring the concentration: protein concentration was determined using a micro nucleic acid protein analyzer.

SDS-PAGE electrophoresis of ESAT6, CFP10, HSPX and FixB proteins

The eluted target protein was subjected to 12% SDS-PAGE.

(1) Washing and plate loading: before the glue is prepared, the glass plate is cleaned by pure water and then washed by MilliQ grade water for several times. And then fixing the glass plate on a bracket, filling ultrapure water between the grooves of the two plates, checking whether leakage occurs, emptying water, and drying for later use.

(2) Preparing glue: 12% of separation glue and 5% of concentrated glue are prepared, shown in table 1, and the components are added in sequence and mixed uniformly.

TABLE 1 SDS-PAGE separation gel and concentrated gel formulation system

(3) Glue pouring: after the components are mixed uniformly, the separation gel is slowly injected into the gel preparation plate to avoid generating bubbles, about 8mL, absolute ethyl alcohol is added for liquid sealing, and the mixture is incubated at 37 ℃ for 30min until the mixture is completely solidified. The absolute ethanol was decanted off and the residual liquid was blotted with filter paper. Injecting concentrated gel, inserting a comb, and incubating at 37 deg.C for 30min until completely coagulating.

(4) Sample treatment: to the Protein sample, an equal volume of 2 XProtein Loading Buffer was added, mixed well and boiled at 100 ℃ for 5 min.

(5) Sample adding electrophoresis: mu.L of the sample was loaded, and the protein sample was concentrated by 80V constant voltage electrophoresis for 30min and then subjected to 120V electrophoresis for 1 h.

(6) Dyeing and decoloring: and placing the gel after electrophoresis into a staining dish containing a proper amount of Coomassie brilliant blue staining solution, and staining for at least 1h at 50 r/min. After dyeing is finished, repeatedly decoloring the Coomassie brilliant blue decoloring solution in a decoloring shaker at 50r/min until the gel is completely transparent.

(7) The electrophoretic band of the purified protein was single and clear (FIG. 7).

4. Preparation of finished product

The purified ESAT6, CFP10, FixB and HSPX proteins were concentrated to a concentration of 0.7mg/mL using polyethylene glycol 20000, mixed at a ratio of 2:2:1.5:1.5, and split-filled into 5 mL/bottle, lyophilized and stored at 4 ℃.

Example 3: use of cocktail antigens for allergy

The cocktail antigen prepared by the invention is used as an antigen for bovine tuberculosis allergy, and comprises the following specific steps:

1. preparing an antigen:

the purified ESAT6, CFP10, FixB and HSPX proteins are respectively concentrated to the concentration of 0.7mg/mL by using polyethylene glycol 20000, mixed according to the ratio of 2:2:1.5:1.5 to prepare the cocktail antigen of the invention, subpackaged into 1 mL/bottle, freeze-dried and stored at 4 ℃.

2. Allergy test

Before use, 5mL of PBS is added into the lyophilized cocktail antigen, and the mixture is mixed uniformly so that the final concentrations of ESAT6, CFP10, FixB and HSPX are respectively 40 mug/mL, 30 mug/mL and 30 mug/mL.

Allergy tests were carried out as follows:

preoperative treatment: after numbering cattle, shearing hair at the upper third of the middle part of the neck side, measuring the thickness of the central wrinkle of the operative part by a caliper, and recording.

Injection: no matter the cattle or the calves, 0.1mL of cocktail antigen was injected into the neck and the side of the neck.

Observation reaction: the results were judged 72h (+ -4-6 h) after intradermal injection.

Judging the result: the skin thickness difference of the injection part is more than or equal to 4mm, the skin thickness difference is more than 2mm and less than 4mm, the injection part is suspicious, and the skin thickness difference is less than 2mm, the injection part is negative; the suspicious animals should be detected at least after 42 days, and the result is positive or suspicious and positive.

By using the cocktail antigen, a single intradermal allergy (cocktail antigen single spot skin test, abbreviated as novel single spot skin test) is performed, and the effect of a comparative allergy (comparative skin test, abbreviated as comparative skin test) using both bovine PPD and avian PPD can be achieved. The detection is carried out on 5 naturally infected cows, 5 avian mycobacteria infected cows and 5 healthy cows simultaneously, the novel single-point skin test and the comparative skin test of the result have 10 positive heads and 5 negative heads, and the coincidence rate of the two is 100%. See table 2.

Table 2: comparative testing of novel single point skin test and comparative skin test

Compared with the traditional method for quarantine of bovine tuberculosis, namely the traditional simple cervical allergy of bovine tuberculosis (referred to as the traditional single-point skin test for short), the novel single-point skin test can identify the avian mycobacterium infection and reduce the nonspecific interference of the test. The tuberculosis of the cattle infected by 5 avian mycobacteria is detected, the traditional single-point skin test is a positive result, and the elimination rate of the avian mycobacteria is 0%; the new single-point skin test is a negative result, and the avian mycobacterium exclusion rate is 100% (fig. 8).

The new single point skin test can save about half the time compared with the comparative skin test in shearing, measuring skin thickness, injecting, measuring skin thickness again, calculating, etc. (fig. 9). Correspondingly, the workload is reduced by half, the test flow is simplified, and the test operation is reduced.

Claims (2)

1. A cocktail antigen comprising ESAT6 antigen, CFP10 antigen, FixB antigen and HSPX antigen; wherein the mass ratio of the ESAT6 antigen, the CFP10 antigen, the FixB antigen and the HSP antigen is 2:2:1.5: 1.5.

2. use of the cocktail antigen of claim 1 in the preparation of a test antigen for tuberculosis allergy in cattle.

Images