CN110967482B - Kit for detecting infection of cryptobacter pyogenes of goats and detection method thereof - Google Patents
Kit for detecting infection of cryptobacter pyogenes of goats and detection method thereof Download PDFInfo
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Abstract
The invention provides an indirect ELISA kit for detecting goat occult pyogenes, which is characterized by comprising an ELISA plate coated with goat occult pyogenes recombinant hemolysin rPLO; the amino acid sequence of the rPLO is shown as SEQ ID NO. 1. The invention establishes an indirect ELISA kit capable of accurately detecting goat infected cryptococcus pyogenes by utilizing the goat cryptococcus pyogenes hemolysin recombinant protein rPLO. The kit has strong specificity, high sensitivity and good repeatability.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a goat cryptococcus pyogenes infection detection kit, a preparation method and a use method thereof.
Technical Field
The purulent occult bacillus is a symbiotic bacterium of the mucous membranes of economical livestock such as cattle, sheep, pigs and the like, mainly parasitizes in the respiratory tract, the digestive tract and the genitourinary tract of animals, and is a component of normal flora. The secretor pyogenes is a conditioned pathogen, can invade through skin and mucous membrane damages to cause adjacent tissue and organ infection, and can be inhaled into the lung to cause lung and chest pyogenic pneumonia, pyogenic pleurisy and the like.
The crypthecellobium pyogenes is one of the main pathogens causing goat body surface lymphadenitis and goat respiratory diseases (Zhang Su Hui et al, 2015). Clinically, sick sheep gradually lose weight and breathe quickly, and the phenomena of body surface lymphnoditis, abortion of pregnant ewes and the like are caused. The dissection shows the lung with the suppurative focus of soybean size, lung with purulent lesion of chest wall, etc. The pathological section shows myocardial fibrosis, serous-cellulosic degeneration in the lung and renal tubule lumen, lymph node steatosis, inflammatory cell infiltration of liver, and serous-purulent inflammation of spleen. The treatment of pulmonary infection is difficult due to low plasma concentration in the lung. The disease is widely spread in southwest areas, and causes serious economic loss to goat breeding in the areas.
Identified virulence factors of cryptococcus pyogenes are hemolysin (PLO), collagen-binding protein (CbpA), neuraminidase (Nan) NanH and NanP. PLO is the only exotoxin secreted by Cryptococcus pyogenes, and all isolates of Cryptococcus pyogenes produce PLO. Billington, et al 1997, cloned the Cryptococcus pyogenes plo gene and obtained its entire gene sequence. The plo open reading frame has 1605bp in total, encodes 534 amino acids and has a molecular weight of 57.9ku. The plo gene is located on a 2.7kb gene island, with the orf121 open reading frame upstream and the fstY, ffh genes downstream. PLO belongs to a member of the cholesterol-dependent cytolytic Family (CDC), such as the hemolysin of Streptococcus Pneumoniae (PLY), listeria monocytogenes (LLO), and the like, are members of the CDC Family. Such toxins exert their cytolytic activity by forming pores on eukaryotic cells. CDC affects a variety of physiological processes in the host including complement activation, up-regulation of cytokine levels, suppression of respiratory bursts and bactericidal activity of polymorphonuclear leukocytes and monocytes, and is cytotoxic to polymorphonuclear leukocytes and macrophages. PLO is also an important host-protective antigen (Jost et al, 1999). The DNA vaccine containing PLO gene and the eukaryotic expression plasmid containing IL-1 beta are combined to immunize, and the ability of the immunized mice to resist the infection of the cryptococcus pyogenes is enhanced (Huang et al, 2016). Furthermore, the laboratory demonstrated that both artificially infected mice and naturally infected sheep were able to produce antibodies against PLO only. The research results show that the PLO can be used for the development of cryptococcus pyogenes immune preparation products.
Yan Minghui (2016) prepared 2 monoclonal antibodies against PLO and confirmed that the critical regions of the antigen to which these 2 monoclonal antibodies were directed were amino acids 64 to 79 (i.e., VPVTKDQLKDDGTYTVF) and 58 to 75 (i.e., GESIENVLKDGT) of PLO. This is also shown in patent application No. CN 201510616098.8. In CN201510616098.8, a recombinant plasmid pET-30a (+) -PLO containing a PLO gene of Cryptococcus pyogenes constructed by Muxiangli et al (2013, proceedings of Chinese preventive veterinarian, 2013,35 (06): 477-480) was also used to induce expression of recombinant PLO without signal peptide, and the monoclonal antibody hybridoma culture cell supernatant or mouse ascites purified monoclonal antibody was detected by indirect ELISA method coated with the recombinant PLO. The application of this "epitope polypeptide of crypt pyogenes PLO in the preparation of antibody reagents for detecting anti-crypt pyogenes PLO protein" is mentioned in the claim of CN201510616098.8, which does not refer to the content of how to establish a specific, sensitive, and highly reproducible indirect ELISA kit of crypt pyogenes using recombinant PLO.
The cryptobacter pyogenes is a new pathogen of goats, and the harm to goats is gradually known. Accurate and convenient diagnosis methods are needed to be used as technical supports for effective prevention and control, and the ELISA method is one of the optional methods for detecting and diagnosing the infection of the goat cryptobacter pyogenes. At present, a cryptobacter pyogenes ELISA diagnostic kit suitable for the basement layer is lacked.
Disclosure of Invention
Therefore, the indirect ELISA kit capable of accurately detecting the goat infected cryptococcus pyogenes is established by utilizing the goat cryptococcus pyogenes hemolysin recombinant protein rPLO. The kit has strong specificity, high sensitivity and good repeatability.
The purpose of the invention is realized by the following measures:
an indirect ELISA kit for detecting goat occult pyogenes, which is characterized by comprising an ELISA plate coated with goat occult pyogenes recombinant hemolysin rPLO; the amino acid sequence of the rPLO is shown as SEQ ID NO. 1.
The coating amount of the rPLO on a 96-well enzyme label plate is 0.2-1 mu g/well, and the coating condition is 37 ℃ for 2-4h.
The preparation method of the recombinant hemolysin rPLO comprises the following steps: the goat crypthecellus pyogenes PLO gene fragment is amplified through PCR, the rPLO gene fragment is cloned to pET28a, and the recombinant protein is expressed through a prokaryotic system.
Further, cloning the rPLO gene fragment into a recombinant vector of pET28a, transforming the rPLO gene fragment into escherichia coli BL21 (DE 3), and adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression; and (3) carrying out affinity purification on the expression product rPLO by using a 6 XHis tag and a nickel column to obtain a purified recombinant rPLO antigen.
The goat crypthecellus pyogenes PLO gene fragment is cloned into pET28a, IPTG induction expression is used in escherichia coli BL21 (DE 3), immunoblot analysis proves that goat serum immunized by the crypthecellus pyogenes, goat serum naturally infected by the crypthecellus pyogenes and mouse serum artificially infected by the crypthecellus pyogenes can identify the expression product, and the amino acid sequence of the recombinant hemolysin rPLO which can be identified by the crypthecellus pyogenes specific serum is shown in SEQ ID NO 1.
The rPLO obtained by the invention is efficiently expressed in the form of inclusion body, and can be expressed and purified in large quantity, so that the rPLO can meet the requirement of industrial production.
Furthermore, the preparation method of the ELISA plate coated with the Cryptococcus pyogenes rPLO comprises the following steps:
1) Diluting rPLO with 0.05mol/L sodium carbonate buffer solution with pH9.6, adding into an enzyme label plate, and coating amount is 0.2-1 μ g/hole;
2) Covering an enzyme label plate with a sealing plate film, and incubating for 2h at 37 ℃;
3) Discarding the liquid in the wells of the ELISA plate, adding a washing solution into the wells, standing for 1min, and repeating for 3-5 times. After discarding the liquid in the hole each time, patting the residual liquid on absorbent paper to be dry;
4) Blocking the ELISA plate by 0.01mol/L pH7.2 phosphate buffer solution containing 5% skimmed milk powder or 1-5% gelatin;
5) Placing the enzyme label plate at 37 ℃ for 2h, incubating, removing liquid in the hole, and patting dry on absorbent paper;
6) Adding phosphate buffer solution containing 5-20% of sucrose and having pH of 0.01mol/L into the wells of the ELISA plate, incubating at room temperature for 3-5h, removing the liquid in the wells, naturally drying, and vacuum-pumping and sealing with a packaging bag containing a desiccant.
Further, the kit comprises an enzyme-labeled antibody, wherein the enzyme-labeled antibody is horseradish peroxidase-labeled rabbit anti-goat IgG. And the enzyme-labeled antibody is subpackaged into 25 mL/bottle.
Further, the kit further comprises: sample diluent, washing solution, negative control serum, positive control serum, substrate developing solution A and B and stop solution.
Furthermore, the positive control serum is collected from goat serum containing PLO antibody after immunization of goat with Cryptobacterium pyogenes or rPLO.
Further, the sample diluent and the washing solution contain ProClin300.
The negative control serum is healthy goat serum which is not infected with cryptococcus pyogenes.
Specifically, the sample diluent, the washing solution, the substrate developing solutions A and B and the stop solution have the following formulas:
sample diluent: PBS:8g/L NaCl,0.2g/L KCl,3.58g/L Na 2 HPO 4 ·12H 2 O，0.272g/L KH 2 PO 4 1.0g/L BSA,1.0mL/L ProClin300, dissolved in deionized water. Subpackaging into 40 mL/bottle.
10-fold concentrated washing solution: 80g/L NaCl,2g/L KCl,35.8g/L Na 2 HPO 4 ·12H 2 O，2.72g/L KH 2 PO 4 10mL/L ProClin300, dissolved in deionized water. Subpackaging into 40 mL/bottle.
Substrate color development liquid A: 6.8g/L of sodium acetate, 0.8g/L of citric acid and 0.15ml/L of 30% hydrogen peroxide are dissolved in distilled water. Subpackaging in brown bottle, 15 mL/bottle.
Substrate color developing solution B: disodium EDTA 0.1g/L, citric acid 0.475g/L, glycerol 25mL/L, 3', 5' -Tetramethylbenzidine (TMB) powder 0.075g/L (0.15 g of TMB powder first dissolved in 3mL of DMSO), dissolved in distilled water. Subpackaging in brown bottle, 15 mL/bottle.
Stopping liquid: 108.7mL of concentrated sulfuric acid (98%) is added dropwise into 891.3mL of distilled water to obtain a 2mol/L sulfuric acid solution, and the solution is mixed uniformly. Subpackaging into 15 mL/bottle.
The invention also provides application of the kit in detecting the infection of the goat cryptobacter pyogenes.
A use method of an indirect ELISA kit for detecting steinernema pyogenes of goats comprises the following steps:
1) And (3) taking the enzyme label plate (coated with rPLO) out of the kit, and diluting the serum sample to be detected by 50 times by using a sample diluent. Sucking 100 mu L of diluted serum into an enzyme label plate, and simultaneously setting two holes of positive control serum and negative control serum, wherein each hole is 100 mu L/hole;
2) Incubating at 37 deg.C for 30min;
3) Discarding liquid in the hole, adding 300 mu L/hole washing solution into the hole, standing for 1min, repeatedly washing for 3 times, and patting dry residual liquid in the hole on absorbent paper;
4) Adding 100 mu L of enzyme-labeled antibody into each hole;
5) Incubating at 37 ℃ for 30min;
6) Discarding liquid in the hole, adding 300 mu L/hole washing solution into the hole, standing for 1min, repeatedly washing for 3 times, and patting dry residual liquid in the hole on absorbent paper;
7) Adding 50 mu L of substrate color development liquid A and substrate color development liquid B into each hole, and slightly shaking and mixing uniformly to avoid overflowing of liquid in the holes;
8) Incubating at 37 ℃ for 15min;
9) Adding 50 mu L of stop solution into the hole;
10 OD of the test sample and the control are measured and recorded 450 The value is obtained.
The kit of the invention has the following judgment standards: average OD of negative control 450 Less than 0.4, positive controls with an average value greater than 1.0 are considered effective.
S is sample OD 450 Value, P is OD of positive control 450 And (4) average value. If the S/P value is more than 0.5, the sample is judged to be positive to the infection of the cryptobacter pyogenes. If the S/P value is less than or equal to 0.5 but greater than 0.4, the sample should be retested. If the test result is not changed, the sampling detection is carried out again after a certain time interval. And if the S/P value is less than 0.5 in the retest result, the sample is judged to be negative to the infection of the cryptobacter pyogenes. And (5) redoing if the experiment has no effect.
Advantageous effects
1. The invention provides a recombinant hemolysin rPLO which can be identified by goat specific serum immunized by goat cryptococcus pyogenes. The protein realizes high-efficiency expression in the form of inclusion body, can obtain at least 40mg of purified rPLO per 100mL of culture, has high yield, and can meet the requirement of industrialized production.
2. The indirect ELISA kit for detecting the infection of the goat cryptococcus pyogenes is established by using the cryptococcus pyogenes recombinant hemolysin rPLO and the use method thereof, and has the following effective effects: (1) The specificity is strong, and the infection of the cryptococcus pyogenes can be distinguished from the common goat pyogenic bacterial infection; (2) the sensitivity is high; (3) The method is simple to operate, is suitable for rapid diagnosis of the basic layer, and does not need to separate, culture and identify pathogenic bacteria according to the traditional method; (4) the detection time is short, and the detection is finished within 2 hours; (5) the preservation period is up to 1 year at the temperature of 2-8 ℃.
Drawings
FIG. 1 shows the double digestion of the recombinant plasmid pET28a-plo
M: marker;1: recombinant plasmid pET28a-plo
FIG. 2 is a PLO gene-induced expression analysis
M: a protein Marker;1: rPLO;2: no induction was performed.
FIG. 3 is the result of the recognition of rPLO by goat serum immunized with Cryptococcus pyogenes culture (immunoblot) M: a protein Marker;1: rPLO.
FIG. 4 is rPLO soluble expression analysis
M: a protein Marker;1: precipitating; 2: supernatant; 3: no induction was performed.
FIG. 5 shows the results of rPLO purification
M: a protein Marker;1: purified rPLO;2: dissolving the inclusion body; 3: and washing the inclusion bodies.
FIG. 6 shows the results of blocking experiments.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail. The experimental methods of the preferred embodiments, which do not indicate specific conditions, are generally performed according to conventional conditions, and the examples are given for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
EXAMPLE 1 preparation of rPLO
1. Construction of expression vector and recombinant Strain
A goat cryptococcus pyogenes PLO gene fragment is amplified through PCR, a target gene is cloned into pET28a, and a BL21 (DE 3) recombinant strain is constructed. The amino acid sequence of PLO cloned and expressed is shown in SEQ ID NO. 1, and the theoretical molecular weight of recombinant hemolysin rPLO is 33.5kD.
2. Inducible expression
Inoculating the recombinant engineering bacteria into 2mL liquid LB culture medium (containing 50mg/L kanamycin), shaking at 37 deg.C for overnight culture, transferring to fresh liquid LB culture medium (containing 50mg/L kanamycin) at 1%, and shaking at 37 deg.C for culture until the bacterial concentration reaches OD 600 If =0.6, IPTG was added at 0.1mmol/L, and the mixture was cultured at 37 ℃ under shaking at 180r/m for 3 hours. SDS-PAGE was used to analyze the expression induced by the recombinant protein.
As can be seen from FIG. 2, rPLO is clearly expressed.
3. Antiserum preparation
Preparing TSB culture medium, and autoclaving at 121 deg.C for 15min. And (3) selecting a single colony of the goat cryptococcus pyogenes, inoculating the single colony to a TSB culture medium, and shaking at 37 ℃ for 72h for culture. To the culture was added 0.1% formaldehyde solution and inactivated at 37 ℃ with shaking every 5h for 24h. Adding Freund's adjuvant for emulsification, and emulsifying agent prepared with Freund's complete adjuvant and emulsifying agent prepared with Freund's incomplete adjuvant for boosting immunity. The goat is immunized by injecting emulsifier into 5mL per muscle, 2 weeks are separated between each immunization, blood is collected before immunization and serum is separated, and the blood is collected 2 weeks after the last immunization and the serum is separated.
4. Antibody recognition
The induced culture was centrifuged at 1 mL/min and 12000g/min for 1min, and 100. Mu.L of 2 Xloading buffer was added to the pellet, after which the cells were suspended in a boiling water bath for 10min, and subjected to SDS-PAGE for immunoblotting analysis. The primary antibody is antiserum prepared by immunizing the goat cryptococcus pyogenes, the antiserum is diluted by 1. The color developing agent is BCTP/NBT.
As can be seen in FIG. 3, rPLO was recognized by goat antiserum immunized against Cryptobacterium pyogenes of goats, producing a specific band consistent with the expected size.
5. Soluble expression analysis
Performing induced expression rPLO according to the method, centrifuging to collect thalli, adding 20mL PBS per 100mL culture to suspend thalli precipitation, and ultrasonically cracking bacterial suspension in ice-water bath (the ultrasonic power is 300W, the working time is 4s, the interval is 5s, and the ultrasonic cracking time is 15 min). 100 μ L of lysate was centrifuged at 12000g/min for 1min, and the pellet and supernatant were subjected to SDS-PAGE, respectively, to analyze the soluble expression of rPLO.
As can be seen in FIG. 4, rPLO was abundant in the induced culture pellet and no apparent rPLO was present in the supernatant, indicating that rPLO was expressed as inclusion bodies.
6. Recombinant protein purification
Dissolving the inclusion body by 8mol/L urea, eluting by a sample, washing the hybrid protein and imidazole solution, and purifying the recombinant protein by Ni-NTA affinity chromatography. The effect of recombinant protein purification was analyzed by SDS-PAGE, the purity was analyzed by gray-scanning the band of interest with Image Lab3.0, and the concentration of purified protein was determined using BCA kit.
As can be seen in FIG. 5, rPLO was purified from inclusion body lysates by Ni-NTA affinity purification to 40mg rPLO per 100mL of culture as determined by BCA kit assay and Image Lab3.0 analysis.
The inventors also performed the above-described experiments using the fragments shown in SEQ ID NO. 2 to SEQ ID NO. 6, which were not recognized by goat antiserum immunized with Cryptobacterium pyogenes, could not form inclusion bodies, and could not appear in the induced culture pellet.
Example 2 optimization of Indirect ELISA conditions
1. Determination of optimal coating concentration and optimal serum dilution
The extracted rPLO was diluted 1 (8.55 μ g/mL), 1 (4.28 μ g/mL), 1 (400 (2.14 μ g/mL) and 1 (800 μ g/mL) in carbonate buffer at pH9.6 and added to wells of a microplate separately at 100 μ L per well overnight at 4 ℃. The following day, wash plates 3 times with PBST, 200. Mu.L/well for 5min each. BSA 2% 200. Mu.L/well, 2h at 37 ℃ total blocking. After blocking, the plates were washed 3 times for 5min with PBST. Positive, negative sera were diluted in 1. An enzyme-labeled antibody labeled with horseradish peroxidase (HRP) is diluted by 1. Washed 4 times. The remaining steps were performed according to a conventional indirect ELISA procedure. In the specific embodiment, the ELISA plate used in each example is a 96-well ELISA plate.
And (4) judging a result: and (4) taking the antigen-antibody working concentration with the maximum P/N value as the optimal dilution in the titration result of the matrix method.
2. Coating temperature and time optimization
The enzyme label plate is coated with the determined rPLO concentration, the coating conditions are set to be 12h and 24h under the condition of 4 ℃, 1h, 2h, 3h and 4h under the condition of 37 ℃, and 6 groups are provided, wherein each group is repeated for two times. Serum was diluted at the determined optimal dilution, HRP-labeled enzyme-labeled antibody was diluted 1 10000, and the rest were tested according to a conventional indirect ELISA procedure. The group with the largest P/N value is taken as the optimal antigen coating condition.
3. Selection of the type of blocking agent and the blocking time
3.1 selection of blocking Agents
The ELISA plate was coated under the optimal conditions, 7 test groups were set using the non-blocking group as the control group, and blocking was performed using 10% fetal bovine serum, 5% skim milk powder, 1% gelatin, 5% gelatin, 0.1% BSA, 0.5% BSA, and 3% BSA as blocking agents, respectively. The blocking conditions were 37 ℃ for 1.5h. HRP enzyme-labeled antibody was diluted 1. The remainder were tested according to the conventional indirect ELISA procedure. And selecting the group with the OD value of the closed group lower than that of the non-closed group and the maximum P/N value as the optimal sealant.
3.2 selection of the blocking time
The ELISA plate was coated and blocked under optimal conditions for l h, 1.5h and 2h at 37 ℃. Serum was diluted at the determined optimal dilution, HRP enzyme-labeled antibody was diluted 1 10000, and the rest of the conventional indirect ELISA procedure was performed, with the group with the largest P/N value as the optimal blocking time.
4. Determination of optimal reaction time between serum and antigen
Coating and sealing the ELISA plate under the optimal condition, adding the serum diluted by the optimal dilution factor, and respectively acting for 0.5h, 1h, 1.5h and 2h at 37 ℃. HRP enzyme-labeled antibody was diluted 1 to 10000, and the rest was tested according to a conventional indirect ELISA procedure. The group with the largest P/N value was used as the optimal action time of the serum.
Determination of optimal working concentration and optimal action time of HRP-labeled antibody
5.1 Optimal working concentration of HRP-labeled antibody
According to the determined optimal condition test, the dilutions of HRP-labeled rabbit anti-goat IgG were set as: 1.
5.2 Optimal working time of HRP (horse radish peroxidase) -labeled antibody
According to the determined optimal working concentration of the HRP-labeled antibody, the test is divided into 4 groups of 0.5h, 1h, 1.5h and 2h at 37 ℃ according to the action time. The rest of the procedures were performed according to the conventional indirect ELISA procedure, and the group with the largest P/N value was used as the optimal action time of the HRP-labeled antibody.
Selection of the color development time of TMB
Performing test according to the determined optimal conditions, setting the color development time to be 5min, 10min, 15min and 20min at 37 ℃, and performing other operations according to a conventional indirect ELISA program, wherein the group with the maximum P/N value is used as the optimal color development time of the substrate.
The experimental results are as follows:
(1) Determination of optimal coating concentration of antigen and optimal dilution of serum
The optimal antigen coating concentration and the optimal serum dilution were screened, and it was found that the OD values decreased with the increase of the positive serum dilution when the antigen sample was diluted 80, 160, 320 and 640 times, respectively. Positive serum OD 320-fold (2.14. Mu.g/mL) rPLO and 50-fold serum dilutions 450 The value is more than 1.00, the P/N ratio is maximum, so the optimal coating concentration of the antigen is 2.14 mu g/mL, and the coating amount of the antigen can be determined to be 0.2 mu g-1 mu g/hole; the optimal dilution of serum was 1The results are shown in Table 1.
TABLE 1 determination of optimal dilution of antigen and serum
(2) Optimization of coating conditions
The OD value of the positive serum increases along with the prolonging of the coating time under the condition of 37 ℃, the coating time is 2-4h at 37 ℃, and the P/N value is in a peak value interval. Therefore, the antigen coating conditions can be set to 37 ℃ for 2-4h. The results are shown in Table 2.
TABLE 2 coating Condition optimization test results
(3) Selection of type of blocking agent and blocking time
The P/N value of the sealant is obviously higher than that of other sealants by using 5% of skimmed milk powder or 1-5% of gelatin as the sealant. The results are shown in Table 3. 5% skimmed milk powder or gelatin is used as a sealant, and when the sealant is sealed for 2h, the P/N value is the maximum and is 6.690. Thus, the optimal blocking time for antigen coated plates was 2h. The results are shown in Table 4.
TABLE 3 selection of classes of blocking agents
TABLE 4 selection of blocking time
(4) Selection of serum action time
As can be seen from Table 5, the OD value of the positive serum increased and the P/N value increased with the increase of the serum action time within 0.5-1.5h, but the difference was not obvious.
TABLE 5 selection of serum action time
(5) Selection of dilution times and incubation times of HRP-labeled antibodies
As can be seen from table 6, the OD values of the positive and negative sera decreased with increasing dilution of the HRP-labeled antibody. When the HRP-labeled antibody was 64000 fold diluted, the P value was 1.581, the N value was 0.295, and the P/N maximum was 5.359. Thus, the optimal dilution factor for HRP-labeled antibody was 64000 fold.
TABLE 6 selection of dilution factor for HRP-labeled antibodies
The HRP-labeled antibody was diluted 64000 times with the HRP-labeled antibody for the optimal reaction time test, and the results are shown in Table 7. When the reaction time of the HRP-labeled antibody is within 0.5-2h, the P/N value is in a descending trend along with the time. The results show that the optimal working time of the HRP-labeled antibody is 0.5h at 37 ℃.
TABLE 7 selection of reaction time for HRP-labeled secondary antibodies
(6) Selection of TMB development time
As can be seen from Table 8, when the color was developed for 15min, the P value was 1.517, the N value was 0.318, and the P/N ratio was the largest and was 4.770. The optimal color development time of TMB was 15min.
TABLE 8 selection of TMB development time
Example 3 Indirect ELISA method Main technical index test
1. Specificity test
1.1 Cross-over test
Respectively preparing oil emulsion antigens with culture bacteria liquid of Escherichia coli, staphylococcus aureus, streptococcus and Corynebacterium pseudotuberculosis, and respectively immunizing goat. Separating serum, detecting by established indirect ELISA method, and establishing positive serum control and negative serum control of goat cryptococcus pyogenes. And judging whether the serum of the bacteria is positive to the goat cryptococcus pyogenes antibody according to the S/P value.
Detection effectiveness and cross reaction judgment standard: average OD of negative controls 450 Less than 0.4, positive controls with an average value greater than 1.0 are considered effective. S is sample OD 450 Value, P is OD of positive control 450 Average value. If the S/P value is greater than 0.5, the sample should be judged as positive for cross-reaction. If the S/P value is less than or equal to 0.5 but greater than 0.4, the sample should be retested. If the S/P value is less than 0.4, the cross reaction is negative.
1.2 blocking assay
Taking goat cryptococcus pyogenes positive serum for dilution by a multiple ratio, wherein the dilution is from 1 to 1, and the number of the positive serum is 6, dividing each dilution into two parts, and adding rPLO protein with the optimal dilution multiple in the same volume into one part, wherein the rPLO protein is a blocking group; the other portion was added with an equal volume of coating buffer, which is a blocking control group. The treated serum was incubated at 37 ℃ for 1.5h and centrifuged at 10000rpm for 20min. Negative sera were also diluted 2-fold, from 1. Experiments were performed under the determined optimal reaction conditions and the blocking rate was calculated. The blocking rate was calculated as follows:
blocking rate = (OD value of blocking control-OD value after blocking) ÷ OD value of blocking control × 100%.
The detection results of the cross test are shown in Table 9, the S/P values of the antiserum of escherichia coli, staphylococcus aureus, streptococcus and corynebacterium pseudotuberculosis are all less than 0.4, namely the rPLO has no cross reaction with the serum. The indirect ELISA method established by the test has better specificity.
TABLE 9 Cross test
Remarking: "-" indicates negative, and "+" indicates positive.
The blocking test results are shown in table 10 and fig. 6. When the serum dilution times are fixed, the OD value of the blocking group is obviously lower than that of the blocking control group, and the blocking rate is more than 60 percent, which indicates that the rPLO can block the reaction of positive serum and the positive serum. The result shows that the indirect ELISA method established by the test has good specificity.
TABLE 10 blocking test
2. Repeatability test
2.1 in-Board repeat test
Taking 3 parts of serum, carrying out tests on the same ELISA plate according to determined conditions, and carrying out detection on each part of serum by 3 times in a repeated way for 6 times. The in-plate coefficient of variation was calculated for each serum.
2.2 repeat test between plates
3 portions of serum are taken and respectively tested on 4 enzyme label plates according to the determined conditions, and each portion of serum is repeated for 4 times. The interplate coefficient of variation was calculated for each serum.
The in-plate reproducibility test results are shown in Table 11. The in-plate variation coefficients of the three serum samples are respectively 8.11%, 8.07% and 4.91%, and are all less than 10%, and the indirect ELISA method established by the test has good in-plate repeatability.
TABLE 11 in-Board repeatability test results
Remarking: coefficient of variation = standard deviation/mean × 100%.
The results of the plate-to-plate reproducibility tests are shown in Table 12. The coefficients of variation between plates for the three serum samples were 8.18%, 3.14% and 5.16%, respectively, all less than 10%. The indirect ELISA method established by the test has good repeatability between plates.
TABLE 12 results of the repeatability test between the plates
Remarking: coefficient of variation = standard deviation/mean × 100%.
3. Sensitivity test
Diluting the occult pyogenes positive serum by 2 times, wherein the dilution is total to 7 dilutions from 1 to 200 to 1. S/N>2 is judged to be positive, S is the OD of the sample 450 Value, N is OD of negative control 450 Average value.
As can be seen from table 13, the OD value of the goat cryptococcus pyogenes positive serum decreased with the increase of dilution factor, and when the serum was diluted 8000 times, the OD value was 0.401,0.401/0.157=2.554 > 2, and the goat cryptococcus pyogenes positive serum was judged to be positive. When the serum was diluted 16000 times, the OD value was 0.262,0.262/0.157=1.669 <2, and it was judged to be negative. The highest serum dilution of which the detection result is positive by the goat cryptococcus pyogenes antibody is 1.
TABLE 13 sensitivity test results
4. Shelf life
Adding 0.01mol/LpH7.4 phosphate buffer solution containing 5% sucrose into the wells of the ELISA plate, incubating at room temperature for 3h, discarding the liquid in the wells, naturally drying, placing the ELISA plate into a packaging bag containing a drying agent, vacuumizing, sealing, and storing at 2-8 deg.C. And taking out the kit at regular intervals, detecting standard positive serum, standard negative serum and quality control serum, and evaluating main technical indexes of the kit, such as specificity, sensitivity and the like.
The test shows that the kit can be placed for 15 months at the temperature of 2-8 ℃ and the effective period is as long as 1 year.
EXAMPLE 4 preparation and Assembly of the kits
1. The preparation method of the ELISA plate coated with the cryptococcus pyogenes rPLO comprises the following steps:
1) Diluting rPLO prepared in example 1 with 0.05mol/L sodium carbonate buffer solution with pH9.6, adding into 96-well enzyme label plate, and coating amount is 0.2-1 μ g/well;
2) Covering an enzyme label plate with a sealing plate film, and incubating at 37 ℃ for 2-4h;
3) Discarding liquid in the holes of the enzyme label plate, adding a washing solution into the holes, standing for 3min, repeating for 3-5 times, and after discarding the liquid in the holes each time, patting the residual liquid on absorbent paper to be dry;
4) Blocking the ELISA plate by 0.01mol/L pH7.2 phosphate buffer solution containing 5% skimmed milk powder or 1-5% gelatin;
5) Placing the enzyme label plate at 37 ℃ for 2h, incubating, removing liquid in the hole, and patting on absorbent paper to dry;
6) Adding phosphate buffer solution containing 5-20% of sucrose and having pH of 0.01mol/L into the wells of the ELISA plate, incubating at room temperature for 3-5h, discarding the liquid in the wells, naturally drying, and vacuum-pumping and sealing with a packaging bag containing desiccant.
2. The enzyme-labeled antibody is rabbit anti-goat IgG labeled by horseradish peroxidase. The enzyme-labeled antibody is subpackaged into 25 mL/bottle.
3. The positive control serum is collected from goat serum containing PLO antibody after immunizing goat with Cryptococcus pyogenes or rPLO. The positive control serum was diluted with the sample diluent and split into 1 mL/tube.
4. The negative control serum is healthy goat serum without infecting cryptococcus pyogenes. The negative control serum was diluted with the sample diluent and dispensed into 1 mL/tube.
5. Preparation of a sample diluent: PBS:8g/L NaCl,0.2g/L KCl,3.58g/L Na 2 HPO 4 ·12H 2 O，0.272g/L KH 2 PO 4 1.0g/L BSA,1.0mL/L ProClin300, dissolved in deionized water. The mixture is subpackaged into 40 mL/bottle.
Preparation of 6.10-fold concentrated washing solution: 80g/L NaCl,2g/L KCl,35.8g/L Na 2 HPO 4 ·12H 2 O，2.72g/L KH 2 PO 4 10mL/L ProClin300, dissolved in deionized water. Subpackaging into 40 mL/bottle.
7. Preparation of substrate color development solution A: 6.8g/L of sodium acetate, 0.8g/L of citric acid and 0.15ml/L of 30% hydrogen peroxide are dissolved in distilled water. Subpackaging in brown bottle, 15 mL/bottle.
8. Preparation of a substrate color developing solution B: disodium EDTA 0.1g/L, citric acid 0.475g/L, glycerol 25mL/L, 3', 5' -Tetramethylbenzidine (TMB) powder 0.075g/L (0.15 g of TMB powder first dissolved in 3mL of DMSO), dissolved in distilled water. Subpackaging in brown bottle, 15 mL/bottle.
9. Preparation of a stop solution: 108.7mL of concentrated sulfuric acid (98%) is added dropwise into 891.3mL of distilled water to obtain a 2mol/L sulfuric acid solution, and the solution is mixed uniformly. Subpackaging into 15 mL/bottle.
10. Kit assembly
According to each component prepared by the preparation method, 2 enzyme label plates coated and sealed are configured according to each kit, and 1 bottle of each sample diluent, 10-time concentrated cleaning solution, negative control serum, positive control serum, substrate developing solution A, substrate developing solution B, enzyme labeled antibody and stop solution and 1 part of operation instruction are assembled.
EXAMPLE 5 method of Using the kit
The use method of the kit comprises the following steps:
1) And (3) taking the ELISA plate coated with rPLO out of the kit, diluting the serum sample to be detected by 50 times with a sample diluent, and sucking 100 mu L of mixed solution into the ELISA plate. Setting two holes of positive control serum and negative control serum at the same time, 100 mu L/hole, and diluting the negative serum and the positive serum by 50 times according to the serum to be seen by using a sample diluent;
2) Incubating at 37 ℃ for 30min;
3) Discarding liquid in the hole, adding 300 mu L/hole washing solution into the hole, standing for 3min, repeatedly washing for 3 times, and patting dry residual liquid in the hole on absorbent paper;
4) Adding 100 mu L of enzyme-labeled antibody into each hole;
5) Incubating at 37 ℃ for 30min;
6) Discarding liquid in the hole, adding 300 mu L/hole washing solution into the hole, standing for 3min, repeatedly washing for 3 times, and patting dry residual liquid in the hole on absorbent paper;
7) Adding 50 mu L of substrate color development liquid A and substrate color development liquid B into each hole, and slightly shaking and mixing uniformly to avoid overflowing of liquid in the holes;
8) Incubating at 37 ℃ for 15min;
9) Adding 50 mu L of stop solution into the hole;
10 OD of the test sample and the control are measured and recorded 450 The value is obtained.
The kit of the invention has the following judgment standards: average OD of negative control 450 Less than 0.4, positive controls with an average greater than 1.0 are effective in the test orientation.
S is sample OD 450 Value, P is OD of positive control 450 Average value. If the S/P value is more than 0.5, the sample is judged to be positive to the infection of the cryptobacter pyogenes. If the S/P value is less than or equal to 0.5 but greater than 0.4, the sample should be retested. If the test result is not changed, re-sampling and detecting are carried out after a certain time interval, and the re-detection S/P value is less than 0.5, the sample is judged to be negative to the infection of the cryptobacter pyogenes. If the experiment is invalid, the experiment should be redone.
SEQUENCE LISTING
<110> institute of zootechnics in Chongqing City
<120> indirect ELISA method for detecting goat cryptobacter pyogenes infection
<160>
<210> 1
<211> 245
<212> protein
<213> Artificial (Artificial sequence)
<400> 1
TDGLSAPRAS ISPMDKVDLK SAQETNETSV DKYIRGLKYD PSGVLAVKGE SIENVPVTKD 60
QLKDGTYTVF KHERKSFNNL RSDISAFDAN NAHVYPGALV LANKDLAKGS PTSIGIARAP 120
QTVSVDLPGL VDGKNKVVIN NPTKSSVTQG MNGLLDGWIQ RNSKYPDHAA KISYDETMVT 180
SKRQLEAKLG LGFEKVSAKL NVDFDAIHKR ERQVAIASFK QIYYTASVDT PTSPHSVFGP 240
NVTAQ 245
<210> 2
<211> 253
<212> protein
<213> Artificial (Artificial sequence)
<400> 2
DLKDRGVNNK NPLGYISSVS YGRQIFVKLE TTSTSNDVQA AFSGLFKAKF GNLSTEFKTK 60
YADILNKTRA TVYVVGGSAR GGVEVATGNI DALKKIIKEE STFSTKVPAV PVSYAVNFLK 120
DNQLAAVRSS GDYIETTATT YKSGEITFRH GGGYVAKFRL KWDEISYDPQ GKEIRTPKTW 180
SGNWVGRTAG FRETIQLPAN ARNIHVEAGE ATGLAWDPWW TVINKKNLPL VPHREIVLKG 240
TTLNPWVEEN VKP 253
<210> 3
<211> 125
<212> protein
<213> Artificial (Artificial sequence)
<400> 3
TDGLSAPRAS ISPMDKVDLK SAQETNETSV DKYIRGLKYD PSGVLAVKGE SIENVPVTKD 60
QLKDGTYTVF KHERKSFNNL RSDISAFDAN NAHVYPGALV LANKDLAKGS PTSIGIARAP 120
QTVSV 125
<210> 4
<211>120
<212> protein
<213> Artificial (Artificial sequence)
<400>4
DLPGLVDGKN KVVINNPTKS SVTQGMNGLL DGWIQRNSKY PDHAAKISYD ETMVTSKRQL 60
EAKLGLGFEK VSAKLNVDFD AIHKRERQVA IASFKQIYYT ASVDTPTSPH SVFGPNVTAQ 120
<210> 5
<211>120
<212> protein
<213> Artificial (Artificial sequence)
<400>5
DLKDRGVNNK NPLGYISSVS YGRQIFVKLE TTSTSNDVQA AFSGLFKAKF GNLSTEFKTK 60
YADILNKTRA TVYVVGGSAR GGVEVATGNI DALKKIIKEE STFSTKVPAV PVSYAVNFLK 120
DNQL 124
<210> 6
<211>120
<212> protein
<213> Artificial (Artificial sequence)
<400>6
AAVRSSGDYI ETTATTYKSG EITFRHGGGY VAKFRLKWDE ISYDPQGKEI RTPKTWSGNW 60
VGRTAGFRET IQLPANARNI HVEAGEATGL AWDPWWTVIN KKNLPLVPHR EIVLKGTTLN 120
PWVEENVKP 129
Claims (10)
1. An indirect ELISA kit for detecting goat occult pyogenes infection, which is characterized by comprising an ELISA plate coated with goat occult pyogenes recombinant hemolysin rPLO; the amino acid sequence of the rPLO is shown as SEQ ID NO. 1.
2. The kit of claim 1, wherein the amount of rPLO coating on the microplate is 0.2-1 μ g/well.
3. The kit of claim 1, wherein the rPLO is prepared by a method comprising the steps of:
the goat cryptococcus pyogenes PLO gene fragment is amplified through PCR, the rPLO gene fragment is cloned to pET28a, and the recombinant protein is expressed through a prokaryotic system.
4. The kit of claim 3, wherein the recombinant vector is transformed into Escherichia coli BL21, and IPTG is added for induction expression; and (3) carrying out affinity purification on the expression product rPLO by using a 6 XHis tag and a nickel column to obtain a purified recombinant rPLO antigen.
5. The kit of any one of claims 1-4, wherein the rPLO-coated microplate is prepared by a method comprising the steps of:
(1) Diluting the purified rPLO with 0.05mol/L sodium carbonate buffer solution with pH9.6, and adding into the hole of an enzyme label plate, wherein the coating amount is 0.2-1 mu g/hole;
(2) Attaching an enzyme label plate to a plate sealing membrane, and incubating at 37 ℃ for 2-4h;
(3) Discarding liquid in the pores of the enzyme label plate, adding a washing solution into the pores, standing for 1min, repeating for 3 times, discarding the liquid in the plate after each washing, and patting the residual liquid in the pores on absorbent paper after the washing solution is discarded after the last washing;
(4) Blocking the enzyme label plate by 0.01mol/L phosphate buffer solution with pH7.2 containing 5% skimmed milk powder or 1-5% gelatin, incubating at 37 deg.C for 2h, removing blocking solution, washing for 3 times, patting on absorbent paper, and air drying;
(5) Vacuum-sealing with a packaging bag containing desiccant, and storing at 2-8 deg.C.
6. The kit of any one of claims 1 to 4, wherein the kit comprises an enzyme-labeled antibody which is horseradish peroxidase-labeled rabbit anti-goat IgG.
7. The kit according to any one of claims 1 to 4, wherein the kit comprises a substrate color developing solution A and a substrate color developing solution B, and the substrate color developing solution A and the substrate color developing solution B mainly comprise hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine, respectively.
8. The kit of any one of claims 1 to 4, wherein the kit comprises a positive control serum obtained by inactivating Cryptobacterium pyogenes in goats or by collecting goat serum containing PLO antibodies after immunization of goats with rPLO.
9. The kit according to any one of claims 1 to 4, wherein the kit comprises a sample diluent, a washing solution, a negative control serum, a positive control serum, a substrate developing solution A, a substrate developing solution B and a stop solution; the sample diluent contains ProClin300.
10. The method of using the kit of any one of claims 1 to 9, comprising the steps of:
(1) Diluting a serum sample to be detected by 50 times by using a sample diluent, taking 100 mu L of diluted serum to a hole of an enzyme label plate, and simultaneously setting two holes of positive control serum and negative control serum, wherein each hole is 100 mu L;
(2) Incubating at 37 ℃ for 30min;
(3) Discarding liquid in the pores of the enzyme label plate, adding 300 mu L of washing liquid into each pore, standing for 1min, repeating for 3 times, discarding the liquid in the plate after washing each time, and patting the residual liquid in the pores on absorbent paper;
(4) Adding 100 mu L of enzyme-labeled antibody into each hole;
(5) Incubating at 37 ℃ for 30min;
(6) Repeating the step (3);
(7) Adding 50 mu L of substrate color developing solution A and substrate color developing solution B into each hole, and slightly shaking and uniformly mixing;
(8) Incubating at 37 ℃ for 15min;
(9) Adding 50 mu L of stop solution into each hole;
(10) Measuring OD450 readings by using a microplate reader;
(11) And judging whether the test is established or not, and calculating the S/P ratio to judge whether the sample to be detected is positive or negative.
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| 孟祥莉 ; 母晓宇 ; 刘晓丹 ; 徐凝 ; 王璞 ; 高明春 ; 张文龙 ; 王君伟 ; .化脓隐秘杆菌溶血素蛋白的原核表达及其溶血活性鉴定.中国预防兽医学报.(第06期), * |
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