阅读说明：本技术 一种毕氏肠微孢子虫孢壁蛋白单克隆抗体的制备及其应用 (Preparation and application of monoclonal antibody of spore wall protein of nosema enteromorpha ) 是由 冯耀宇 蒙西南 肖立华 李娜 郭亚琼 于 2020-12-28 设计创作，主要内容包括：本发明公开了一种毕氏肠微孢子虫孢壁蛋白单克隆抗体的制备及其应用。基于重组蛋白EbSWP1制得的单克隆抗体对于检测毕氏肠微孢子虫具有良好的特异性,不与虾肝肠微孢子虫、兔脑炎微孢子虫、大肠杆菌等发生反应。本发明制备的单克隆抗体可与荧光素标记物偶联制得免疫荧光抗体,利用该抗体与环境、粪便中的毕氏肠微孢子虫做直接免疫荧光反应,可以快速、灵敏、准确地检验毕氏肠微孢子虫。与实验室最常用的PCR检测方法相比,本发明的免疫荧光检测方法操作简便,耗费时间短,且检测准确度高,以PCR结果为参考,本发明检测方法的准确率高于85％,该方法简便、快速、灵敏、准确的特点使其适用于基层检测应用。(The invention discloses a preparation method and application of a monoclonal antibody of spore wall protein of nosema peelii. The monoclonal antibody prepared based on the recombinant protein EbSWP1 has good specificity for detecting the Microsporidium Pediculus, and does not react with the shrimp liver intestine Microsporidium Pediculus, rabbit encephalitozoon Microsporidium Pekinensis, Escherichia coli and the like. The monoclonal antibody prepared by the invention can be coupled with a fluorescein marker to prepare an immunofluorescence antibody, and the antibody can be used for carrying out direct immunofluorescence reaction with the enterosporidium parvum in the environment and excrement, so that the enterosporidium parvum can be quickly, sensitively and accurately detected. Compared with the most common PCR detection method in laboratories, the immunofluorescence detection method disclosed by the invention is simple and convenient to operate, short in consumed time and high in detection accuracy, and the accuracy of the detection method is higher than 85% by taking a PCR result as a reference.)

1. The spore wall protein SWP1 of the nosema peelii is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 1.

2. A spore wall protein gene EbSWP1 of Microsporum peelii, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

3. A recombinant expression vector comprising the nucleotide sequence of the gene of claim 2.

4. A recombinant expression strain comprising the recombinant expression vector of claim 3.

5. The recombinant expression strain of claim 4, wherein the strain is E.coli.

6. A recombinant protein obtained by inducing expression of the recombinant expression strain of claim 4 or 5.

7. A monoclonal antibody produced from the recombinant protein of claim 6 as an immunogen.

8. An immunofluorescent antibody, wherein said immunofluorescent antibody is obtained by conjugating the monoclonal antibody of claim 7 to isothiocyanatofluorescein.

9. Use of the protein according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3, the recombinant expression strain according to claim 4, the recombinant protein according to claim 6, the monoclonal antibody according to claim 7 or the immunofluorescence antibody according to claim 8 for the preparation of a reagent or a kit for diagnosing or detecting an infection with Microsporum peelii.

10. A kit for diagnosing or detecting Microsporosis peelii infection comprising the monoclonal antibody of claim 7 or the immunofluorescent antibody of claim 8.

Technical Field

The invention belongs to the field of biotechnology. More particularly, relates to a spore wall protein monoclonal antibody of the nosema enteromorpha and application thereof in the detection of the nosema enteromorpha.

Background

Microsporidia (Microsporidia) is an obligate parasitic opportunistic unicellular pathogen in eukaryotic cells, can infect various mammals and birds, and seriously jeopardizes public health and safety. Among all microsporidian species, intestinal microsporidian (enterocytozobiumeusii) is the most common species of microsporidian disease in humans, susceptible to the population including aids patients, organ transplant patients, cancer patients, and even healthy children and the elderly, and can cause long-term fatal diarrhea in immunocompromised or hypo-immune patients and severe self-limiting diarrhea in immunocompromised patients. Due to the small size of microsporidia, there remains difficulty in the rapid clinical diagnosis of microsporidia infection.

Currently, the microsporidian diagnosis methods commonly used in laboratories include PCR, conventional staining and electron microscopy. However, these methods are mainly used for the detection of Microsporosis Pediclosa Piercei in research laboratories and some government public health laboratories, and are rarely used in clinical diagnostic laboratories. The PCR-based detection method has high accuracy and has the outstanding advantages of further identifying the insect species and the genotype, but the PCR detection consumes higher time and cost. Conventional dyeing methods: KMnO₄Methyl violet method, acid-fast trichrome staining method, and staining with fluorescent stain Calcofluor M2RThe color method, Chromotrope 2R staining method, etc., which are simpler than the PCR method but have low specificity and cannot identify the species of microsporidian. The electron microscope is divided into a scanning electron microscope and a transmission electron microscope, is an extremely accurate identification method, can enable an experimenter to observe polar silks and other ultra-micro structures with species characteristics, is considered as a gold standard for identifying microsporidian species, is generally used for researches such as polypide invasion, cell positioning and the like, but is complex in operation, high in cost, long in time consumption, expensive in equipment, needs more professional operators, and is not suitable for conventional detection.

The immunofluorescence technique is also called as a fluorescence antibody technique, is a labeled immunity technique established on the basis of immunology, biochemistry and microscope techniques, has the advantages of convenient operation, visual image, convenient result evaluation and the like, is commonly used for clinical detection, and the key of the method is whether the method is suitable for primary detection of the nosema peelii. Although there are some immunoassay kits in the prior art, such as Bienusi-Glo of Waterborn, USA^TMThe kit can detect the Microsporum peelii by direct fluorescence immunoassay, and the antibody used by the kit is derived from an antigenic determinant outside the spore wall of the sporozoite, but the specificity is not strong, nonspecific staining can occur, and the price is high, so that the immunoassay method for the Microsporum peelii with stronger specificity is needed to be provided. Chinese patent CN 103728458A discloses the application of rabbit encephalitozoon cuniculi spore wall protein SWP1 in the preparation of a reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection, but no related hypoencephalitozoon cuniculi spore wall protein and related reports of the hypoencephalitozoon cuniculi spore wall protein in the detection of the hypoencephalitozoon cuniculi exist at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing an antibody and a method for rapidly and accurately detecting the Pythium bicolor in order to overcome the inconvenience of the existing Pythium bicolor detection method in basic detection application.

The first purpose of the invention is to provide a spore wall protein SWP1 of Microsporum peelii.

It is a second object of the present invention to provide a gene encoding said sporoderm protein SWP 1.

The third objective of the invention is to provide a recombinant expression vector.

The fourth purpose of the invention is to provide a recombinant expression strain.

The fifth object of the present invention is to provide a recombinant protein.

The sixth purpose of the invention is to provide a monoclonal antibody.

It is a seventh object of the present invention to provide an immunofluorescent antibody.

The eighth purpose of the invention is to provide the application of the protein, the gene, the recombinant expression vector, the recombinant expression strain, the recombinant protein, the monoclonal antibody and the immunofluorescence antibody in preparing a reagent or a kit for diagnosing or detecting the nosema peelii infection.

The ninth object of the present invention is to provide a kit for diagnosing or detecting Microsporidium peelii infection.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a spore wall protein SWP1 of the nosema peelii, and the amino acid sequence of the protein is shown in SEQ ID NO. 1.

The invention also provides a spore wall protein gene EbSWP1 of the nosema peelii, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

The invention also provides a recombinant expression vector containing the gene.

The invention also provides a recombinant expression strain containing the recombinant expression vector.

The invention also provides a recombinant protein, which is obtained by the induction expression of the recombinant expression vector or the recombinant expression strain.

The invention also provides a monoclonal antibody, which is prepared by taking the recombinant protein as an immunogen.

The invention also provides an immunofluorescence antibody, wherein the immunofluorescence antibody is obtained by coupling the monoclonal antibody with a fluorescein marker.

In addition, the invention also provides a kit for diagnosing or detecting the infection of the nosema peelii, which contains the monoclonal antibody or the immunofluorescence antibody.

Preferably, the recombinant expression strain is escherichia coli.

Preferably, the fluorescein label is fluorescein isothiocyanate (FITC, showing green under excitation light).

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a spore wall protein SWP1 of Microsporum peelii, a coding gene and a recombinant protein EbSWP1 thereof, and a monoclonal antibody of the spore wall protein of the Microsporum peelii prepared by utilizing the recombinant protein EbSWP1 has good specificity and does not react with shrimp liver intestinal microsporidian, rabbit encephalitozoon microsporidian, Escherichia coli and the like. The monoclonal antibody can be coupled with a fluorescein marker to prepare an immunofluorescence antibody. The antibody is used for carrying out direct immunofluorescence reaction with the enteron pycnidia in the environment and excrement, so that the enteron pycnidia infection can be quickly, sensitively and accurately detected. The immunofluorescence antibody is used for detecting the Pediococcus peelii infection, the time for completing the whole operation is only 2 hours, the time is far less than that of the most common PCR detection method in a laboratory, the accuracy is high, when the antibody is used for detecting a cattle manure sample, the PCR result is taken as the reference, the accuracy is more than 85 percent, and the antibody has the characteristics of simplicity, convenience, rapidness, sensitivity and accuracy, so that the antibody can be suitable for detecting the Pediococcus peelii infection at the basic level.

Drawings

FIG. 1 is a diagram showing the results of recombinant protein detection.

FIG. 2 is a schematic diagram showing the microscopic examination result of the fluorescence immunoassay of Microsporozoite peelii using FITC-conjugated spore wall protein monoclonal antibody of Microsporozoite peelii.

FIG. 3 shows Bienusi-Glo^TM(existing foreign commercial reagents) staining results.

FIG. 4 shows the result of staining with the fluorescent stain Calcofluor M2R.

FIG. 5 shows Chromotrope 2R staining results (requiring a professional to identify microscopic results).

FIG. 6 shows the results of the direct immunofluorescence reaction with non-specific microorganisms.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation of recombinant protein SWP1 of spore wall protein of Microsporidium peelii

1. Clone transformation and protein induction expression of spore wall protein SWP1 gene

The inventor previously found a spore wall protein SWP1 of Microsporidium peelii, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene EbSWP1 of which is shown in SEQ ID NO. 2. The gene EbSWP1 full length coding the spore wall protein SWP1 of the Microsporidium peelii was amplified by Polymerase Chain Reaction (PCR), and the used primer was synthesized by Biotechnology Inc. (Shanghai, China) and has the sequence:

SWP1-F 5'-CGGGATCCTATCAAGAAACAAAAAGATATATTG-3' (underlined as BamH I restriction enzyme site);

SWP1-R 5'-TTGTCGACATTAAATTTTTCAAGATGGTTTC-3' (underlined is a Sal I restriction enzyme site);

the reaction system is shown in Table 1

TABLE 1

The PCR cycle consisted of a 98 ℃ cycle lasting 30 s; 35cycles of 98 ℃ for 10s, 55 ℃ for 30s and 72 ℃ for 30 s; a cycle at 72 ℃ for 5 minutes. PCR Using Phusion^TMHigh fidelity DNA polymerizationEnzyme (cat 530L; Thermo, Waltham, MA, USA) amplification. The PCR product was purified using TaKaRaMiniBEST DNA fragment purification kit (cat No. 9761; TaKaRa, Tokyo, Japan).

The recombinant EbSWP1-pCold I vector was transformed into E.coli BL21(DE3) competent cells and cultured in LB medium supplemented with 100. mu.g/mL ampicillin. Expression of the recombinant protein EbSWP1 was induced by adding 1mM isopropylthio- β -galactoside (IPTG) to the culture and cultured at 15 ℃ at 180rpm for 12 h.

2. Detection of recombinant proteins

Expression results were checked using SDS-PAGE and Western blot analysis of anti-His-tag antibody.

BL21(DE3) cultures were collected by centrifugation and lysed by sonication on ice, the lysates centrifuged and the resulting supernatants filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane filter (Millipore, Billerica, MA, USA), the filtrate was loaded onto a column containing Ni-NTA His-bind resin at room temperature, the recombinant protein EbSWP1 was eluted from the resin with 250mM imidazole buffer and analyzed using SDS-PAGE and blot.

mu.L of the purified protein was added to 10. mu.L of the protein loading buffer, followed by incubation in a metal bath preheated to 100 ℃ for 10 minutes to perform polyacrylamide gel electrophoresis. After completion of electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for transfer, after completion of membrane transfer, blocked with 5% skim milk powder for 2 hours, the mouse anti-his tag monoclonal antibody was diluted with 5% skim milk powder in an appropriate ratio, and then the primary antibody was incubated at 4 degrees overnight, the primary antibody was recovered the next day, and TBST was added and washed 3 times, 5 minutes each, the HRP-labeled goat anti-mouse was diluted with 5% skim milk powder in an appropriate ratio, and the PVDF membrane was incubated at room temperature for 1 hour. After completion of incubation, the secondary antibody was recovered, and TBST was added and washed 3 times for 5 minutes each, and finally, 1mL of TMB color developing solution was added to develop the PVDF membrane. As shown in fig. 1, a new protein band appeared after induction, consistent with the expected size; the result of Western blot analysis shows that a single protein band appears, and the size of the single protein band is consistent with that expected, which indicates that the recombinant protein EbSWP1 is successfully obtained.

EXAMPLE 2 preparation of monoclonal antibody against spore wall protein of Microsporum peelii

1. Three 5-week-old female BALB/c mice were immunized five times at 2-week intervals (subcutaneous injections) with a first vaccination of 1:1 emulsion of 200. mu.g of SWP1 recombinant protein per 1mL, and subsequent immunizations with Freund's incomplete adjuvant. The level of immunity in the mice was monitored by indirect ELISA. Before the fusion, mice were injected intraperitoneally with 100. mu.g of SWP1 protein dissolved in 100. mu.L PBS as a shock immunization, and 3 days later splenocytes were collected and fused with SP2/0 cells.

2. Feeder cells were prepared (0.5 to 1 day in advance)

(1) Preparing 2 balb/c mice with the age of 6 weeks, and fasting for 12h in advance;

(2) killing after neck breaking, and soaking in 75% alcohol to wet hair;

(3) the abdominal skin was cut open and sterile injected with 10mL of incomplete DMEM;

(4) lightly press and shake the mouse;

(5) extracting ascites;

(6) repeating the steps of 2-5, centrifuging at 1000rpm for 5min, adding 10mL HAT, and counting;

(7) diluting to 4 × 5 × 10⁴Per mL;

(8) laying 5 plates per 100 μ L of well;

3. SP2/0 cell Collection

(1) Performing amplification culture before fusion, preparing 10 cells of 25cm²Each flash contains 5mL of culture solution;

(2) washing with incomplete culture medium twice, blowing off, centrifuging at 1000rpm for 5min, and counting;

(3)10mL of incomplete DMEM is subjected to resuspension and counting;

4. spleen cell preparation

(1) Preparing 3 plates, wherein each plate is filled with 10mL of incomplete culture medium;

(2) carrying out eyeball blood collection on the immunized mouse, and killing the mouse after neck breaking; soaking for 3-5 min at 75%;

(3) cutting the abdominal skin, cutting the peritoneum, and aseptically taking out the spleen;

(4) removing the spleen with forceps from the first dish;

(5) washing several times in a second dish;

(6) grinding with a needle tube core in a third plate;

(7) sieving the grinded spleen cell suspension with a disposable 200-mesh cell sieve, and counting;

5. cell fusion

(1) Splenocytes were mixed with SP2/0 cells: SP2/0: splenocytes 1: 5;

(2) supplementing incomplete culture medium to 30mL, and centrifuging at 1000rpm for 5 min;

(3) the supernatant is sucked up, and the tube bottom is knocked to loosen the cells;

(4) placing the centrifuge tube containing cells in a 37 ℃ water bath, adding 1mL of 37 ℃ preheated 50% PEG-1500, adding while rotating, adding after 45s, and standing for 10 s;

(5) adding 30mL of serum-free DMEM while rotating, wherein the adding speed is gradually increased, wherein the adding speed is 1mL at 1min, 2mL at 2min and 3mL at 3 min;

(6) water bath at 37 deg.C for 10 min;

(7) centrifuging at 1000rpm for 5min, discarding supernatant, blowing gently with 10mL pipette for several times to disperse large cell mass, and adding HAT to 50 mL;

(8) packaging into 96-well plates with each well being 100 mL;

6. monoclonal cell screening

(1) After 5 days of fusion, half of the wells from which cells grew were subjected to fluid exchange;

(2) after 7 days of fusion, the wells in which the cells grew were completely changed, and 50. mu.L of each well was extracted the next day for ELISA detection;

(3) performing amplification culture on ELISA positive holes, and screening out single clones by using a limiting dilution method;

(4) after two times of continuous screening, performing indirect immunofluorescence experiment by using the obtained monoclonal cell culture solution supernatant, detecting antibody effect, and determining strains;

example 3 conjugation of monoclonal antibodies to spore wall protein FITC of Microsporum peelii

(1) Preparing a protein sample to be crosslinked dissolved in 0.1M sodium carbonate buffer (pH 9) with the concentration being more than or equal to 2 mg/mL;

(2) dissolving FITC in anhydrous DMSO to obtain a solution with a concentration of 1mg/mL (FITC solution is prepared before labeling experiment and protected from light);

(3) for 1mL of protein solution, 50. mu.L of FITC solution is added, and the protein solution is gently stirred while adding 5. mu.L of FITC solution at a time;

(4) after the required FITC is added, incubating the reaction solution at 4 ℃ for 8h in a dark place;

(5) addition of NH₄Cl to a final concentration of 50mM, and terminating the reaction at 4 ℃ for 2 h;

(6) adding xylene green to a concentration of 0.1% and glycerol to a concentration of 5%;

(7) desalting by using a desalting centrifugal column;

(8) the conjugate was stored at 4 ℃ in the dark and 0.1% preservative was added.

Example 4 detection of Microsporozoan peelii

The immunofluorescent antibodies prepared in example 3 were used to detect samples collected from cattle and monkeys, respectively, and Bienusi-Glo was used to detect the same^TMThe detection kit (the existing foreign commercialized reagent), the Calcofluor M2R staining method, the Chromotrope 2R staining method and the nested PCR method are used for detecting the same batch of samples, and the detection results of the Pythium pisorum obtained by the method are analyzed and compared.

1. Direct fluorescence immunoassay

(1) Coating the sample on a high-adsorption glass slide in a thin and uniform manner, and waiting for natural drying;

(2) after the slide is fully dried, flame fixing is carried out for 3 times, and each time lasts for 1 s;

(3) 1% BSA-PBS, incubation in a 37 ℃ wet box for 30 min;

(4) pouring or sucking away 1% BSA-PBS on the slide, adding the antibody diluted by 5 times, and incubating for 1h at 37 ℃ in a wet box;

(5) washing the slide with PBS for 3 times, and placing the slide on a shaking table for 3-5 min each time;

(6) airing the liquid on the glass slide until the liquid is nearly dry, and dropwise adding the anti-quenching sealing tablet;

(7) nail polish was applied around the coverslip in a circle.

(8) Observing as soon as possible, if the observation can not be immediately carried out, and storing the mixture in a light-proof wet box at 4 ℃.

The direct fluorescence immunoassay result is shown in figure 2, wherein the green part is directly immunofluorescent-stained nosema enteromorpha, and the length and width are about 0.8-1.5 μm. The background is clean, and no non-specific staining exists, which indicates that the prepared immunofluorescence antibody has good specificity. (Bar 2 μm)

2、Bienusi-Glo^TMKit detection

(1) Coating the sample on a high-adsorption glass slide in a thin and uniform manner, and waiting for natural drying;

(2) after the slide is fully dried, flame fixing is carried out for 3 times, and each time lasts for 1 s;

(3) adding Bienusi-Glo (1 x) dropwise, and incubating for 1h at 37 ℃ in a wet box;

(4) washing the slide with PBS for 3 times, and placing the slide on a shaking table for 3-5 min each time;

(5) airing the liquid on the glass slide until the liquid is nearly dry, and dropwise adding the anti-quenching sealing tablet;

(6) coating a circle of nail polish around the cover glass;

(7) observing as soon as possible, if the observation can not be immediately carried out, and storing the mixture in a light-proof wet box at 4 ℃.

Bienusi-Glo^TMThe detection result of the kit is shown in figure 3, and the result shows that the kit has non-specific staining, the length and width of the stained substance in the figure is more than 2 mu m and is not the Microsporosis pythaea.

3. Calcofluor M2R staining method

(1) Taking a small amount of sample suspension, thinly coating a layer on a glass slide, naturally airing, and fixing by using 4% paraformaldehyde;

(2)1:1, dropwise adding a proper amount of Calcofluor M2R dye and 10% KOH solution;

(3) pouring off the solution after 1min, dripping the anti-quenching tablet and sealing;

the detection result of the Calcofluor M2R staining method is shown in figure 4, and it can be seen from the figure that all fungi are stained, and Microsporophores Pediclosae of enteron cannot be distinguished, so the method is not suitable for detecting environmental samples.

4. Chromotrope 2R staining method

(1) Taking a small amount of sample suspension, thinly coating a layer on a glass slide, naturally airing, and fixing by flame;

(2) soaking in anhydrous methanol for 5 minutes;

(3) placing the dye vat filled with Chromotrope dye in a water bath kettle at 37 ℃, and inserting the glass slide into the dye vat to soak for 90 min;

(4) rinsing with 90% acidic alcohol (995.5mL of 90% ethanol +4.5mL of glacial acetic acid) for 10 seconds;

(5) rinsing with 95% alcohol for 10 s;

(6) soaking in 95% alcohol for 5 min;

(7) soaking in 100% ethanol for 5 min;

(8) soaking in 100% ethanol for 5 min;

(9) soaking for 10 minutes by using dimethylbenzene or dimethylbenzene substitute;

(10) soaking with xylene or xylene substitute for 10 min;

(11) and (5) sealing by using a neutral gum sealing agent.

The detection result of the Chromotrope 2R staining method is shown in figure 5, the Microsporosis Pectinatus Pilat is oval with the size of about 0.8-1.5 mu m, the middle or one end is pink, and in actual operation, the Microsporosis Pectinatus Pilat after Chromotrope 2R staining is not easy to observe and needs to be identified by a professional under a microscope.

5. Nested PCR assay

The samples collected in cattle and monkeys were identified using a nested PCR method based on Internal Transcribed Spacers (ITS) using primer pairs:

Eb-ITS-F1：GATGGTCATAGGGATGAAGAGCTT

Eb-ITS-R1：TATGCTTAAGTCCAGGGAG

Eb-ITS-F2：AGGGATGAAGAGCTTCGGCTCTG

Eb-ITS-R2：AGTGATCCTGTATTAGGGATATT

the enzyme used in nested PCR was DreamTaq PCR Master Mix (2X), and the PCR reaction system is shown in Table 2

TABLE 2

PCR Primary Mix	n＝1		Secondary Mix	n＝1
					Water	19.5		Water	18
Fermentas Mix(2X)	25		Fermentas Mix(2X)	25
					BSA(10mg/ml)	2		F2(10μM)	2.5
F1(10μM)	1.25		R2(10μM)	2.5
					R1(10μM)	1.25		Total	48
Total	49		Primary PCR Product	2
					DNA	1

The nested PCR procedure was as follows:

a first round: 5min at 94 ℃; 35cycles at 94 ℃ for 45s,55 ℃ for 45s, and 68 ℃ for 1 min; 68 ℃ for 7min

And a second round: 5min at 94 ℃; 35cycles at 94 ℃ for 45s,55 ℃ for 45s, and 68 ℃ for 1 min; 68 ℃ for 7min

And respectively calculating the positive rates of the direct immunofluorescence, Chromorope 2R and nested PCR detection samples, comparing the direct immunofluorescence detection result with the Chromorope 2R and nested PCR detection results, and calculating the direct immunofluorescence sensitivity, wherein the direct immunofluorescence sensitivity is shown in a table 3.

TABLE 3

As shown in Table 3, the direct immunofluorescence analysis using the immunofluorescence antibody of the present invention has an accuracy rate higher than 100% compared with the detection result of Chromotrope 2R, and has an accuracy rate higher than 85% compared with the nested PCR detection result.

Example 5 specific detection of immunofluorescent antibodies

The immunofluorescence antibody prepared in the embodiment 3 is used for direct immunofluorescence analysis of the prawn enterohepatic microsporidian, the rabbit encephalitozoon microsporidian and the escherichia coli respectively to observe whether the antibody can be dyed by the immunofluorescence antibody, and the steps of the direct immunofluorescence analysis are the same as those in the embodiment 4.

The specific detection result is shown in figure 6, the left graph A is the direct immunofluorescence result of shrimp liver intestine microsporidian, the left graph B is the direct immunofluorescence result of rabbit brain inflammation microsporidian, the left graph C is the direct immunofluorescence result of escherichia coli, the corresponding right graph is the microorganism shot under a differential interference microscope (DIC), the result shows that non-specific staining does not exist, and the obtained immunofluorescence antibody is good in specificity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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