阅读说明：本技术 斯氏艾美耳球虫sag4蛋白的应用 (Application of Eimeria siei SAG4 protein ) 是由 杨光友 陶圆圆 彭雪蓉 于 2019-11-22 设计创作，主要内容包括：本发明涉及生物技术领域,公开了斯氏艾美耳球虫SAG4蛋白作为兔球虫病诊断抗原等一系列相关应用,相关实验结果显示,斯氏艾美耳球虫SAG4蛋白能被斯氏艾美耳球虫阳性血清识别,具有良好的免疫原性和反应原性；同时在间接ELISA方法中表现出极高敏感性和特异性,种种结果证明斯氏艾美耳球虫SAG4蛋白可以作为兔球虫病的诊断抗原,以及应用到相关疫苗和检测试剂盒中。(The invention relates to the technical field of biology, and discloses a series of related applications of Eimeria sieboldii SAG4 protein as rabbit coccidiosis diagnosis antigen, and the like, wherein related experimental results show that the Eimeria sieboldii SAG4 protein can be identified by Eimeria sieboldii positive serum, and has good immunogenicity and reactogenicity; meanwhile, the protein shows extremely high sensitivity and specificity in an indirect ELISA method, and various results prove that the Eimeria sieboldii SAG4 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.)

1. The application of Eimeria siei SAG4 protein as a diagnostic antigen of rabbit coccidiosis and/or in the preparation of the diagnostic antigen of rabbit coccidiosis.

2. Application of Eimeria siei SAG4 protein in preparation of a kit for diagnosing rabbit coccidiosis.

3. The use according to claim 2, wherein the kit is an ELISA kit.

4. The use according to claim 3, wherein the ELISA kit is a kit based on an ELISA indirect method.

5. The application of Eimeria siei SAG4 protein in preparing vaccine for rabbit coccidiosis.

6. An ELISA kit for diagnosing rabbit coccidiosis, which is characterized by comprising a solid phase carrier coated with Eimeria siei SAG4 protein.

7. The ELISA kit of claim 6, further comprising one or more of an enzyme-labeled secondary antibody, a washing solution, a developing solution, a blocking solution, a diluting solution and a stop solution.

Technical Field

The invention relates to the technical field of biology, in particular to application of Eimeria sieboldii SAG4 protein.

Background

Rabbit coccidiosis (rabbitcoccidiosis) is a protozoal disease caused by various coccidia of the genus eimeria parasitizing in the intestine and liver of rabbits, is a common and multiple disease of rabbits, and is one of the major diseases seriously harming rabbits. Eimeria stiedai (Eimeria stiedai), a rabbit coccidium most virulent, attacks primarily the liver and bile duct epithelial cells of rabbits, causing cirrhosis and cholestasis, causing severe hepatic rabbit coccidiosis. The diseased rabbits are mainly characterized clinically by diarrhea, growth and development retardation, weakness and emaciation, and can cause death when seriously infected. Because liver type rabbit coccidiosis has high morbidity and mortality, Eimeria sieboldii is regarded as one of the most harmful pathogens in domestic rabbit raising industry, and the development of domestic rabbit raising industry is severely restricted.

The rabbit coccidiosis is distributed worldwide, causing serious economic loss to rabbit industry in many countries, and the liver type rabbit coccidiosis is the most pathogenic and prevalent. At present, the diagnosis of the disease needs to be performed by a cesarean section of a diseased rabbit, and an effective prenatal diagnosis method is not available; eimeria sieboldii oocysts have been studied and used as natural diagnostic antigens for diagnosis, however, the oocysts as the diagnostic antigens have a series of problems that antigen standard products are difficult to collect and prepare, sources and dosage are difficult to determine, and popularization and application are difficult.

In view of the strong pathogenicity of Eimeria stipitis and the severity of its harm to the rabbit industry, the diagnosis of diseased rabbits using reliable diagnostic techniques is the basis for effective prevention and control. However, the current methods for prenatal diagnosis of the disease are still lack of reports, so that the establishment of a serological diagnosis method for accurately diagnosing the hepatic rabbit coccidiosis has important significance for preventing and treating the disease.

Disclosure of Invention

In view of the above, the invention aims to provide Eimeria sieversii SAG4 protein (ESAG 4) as a diagnostic antigen of rabbit coccidiosis and an application thereof in preparation of the diagnostic antigen of rabbit coccidiosis, so that ESAG 4 has high specificity and sensitivity, and good immunogenicity and reactogenicity;

the invention also aims to provide application of EsSAG4 in preparation of a kit for detecting rabbit coccidiosis, so that an ELISA method established by the EsSAG4 shows higher specificity and sensitivity and can be used for ELISA detection;

another object of the invention is to provide the use of ESAG 4 in the preparation of a rabbit coccidiosis vaccine.

In this context, the eimeria stuartii SAG4 protein (EsSAG4) may be non-natural, e.g., synthetic or expressed from an artificial vector (often referred to in the art as the recombinant protein rsesag 4). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance.

SAGs are located on the surface of cell membranes of protozoa and are a type of surface protein related to Glycosyl Phosphatidylinositol (GPI), and related research results of SAGs in Toxoplasma gondii (Toxoplasma gondii), Plasmodium (Plasmodium), neospora caninum (Neosporinum) and Sarcocystis neurona (Sarcocystis neurona) are reported in the literature, and no related research on SAG gene family of Eimeria leprae is seen at present.

The invention clones and expresses prokaryotic cells of EsSAG4, verifies the reactogenicity of recombinant protein rEsSAG4 by immunoblotting, and establishes an indirect ELISA method to evaluate the diagnostic value of the recombinant protein rEsSAG4 as a recombinant antigen to the liver rabbit coccidiosis. The result showed that the protein encoded by the ORF of the ESAG 4 (full length 444bp, SEQ ID NO: 1) gene had a molecular weight of about 16.17kDa (SEQ ID NO: 2). Immunoblotting showed that the recombinant protein rESAG 4 could be recognized by Eimeria sieboldii positive serum, indicating that it has good reactogenicity. The indirect ELISA method established based on rsesag 4 had a sensitivity of 97.92% (47/48) and a specificity of 100% (48/48). Therefore, ESAG 4 can be used as a candidate diagnostic antigen for rabbit coccidiosis caused by Eimeria sieboldii.

Based on the content, the invention provides the application of the Eimeria siei SAG4 protein as a diagnostic antigen of rabbit coccidiosis and the application in preparing the diagnostic antigen of rabbit coccidiosis. Meanwhile, the invention also provides application of Eimeria sieboldii SAG4 protein in preparation of a kit for diagnosing rabbit coccidiosis; among them, the kit is preferably an ELISA kit, and more specifically, the ELISA kit is a kit based on an ELISA indirect method. In addition, the invention also provides application of Eimeria stewartii SAG4 protein in preparation of rabbit coccidiosis vaccines.

According to the application of Eimeria sieboldii SAG4 protein in preparation of the kit, the invention provides an ELISA kit for diagnosing rabbit coccidiosis, which comprises the Eimeria sieboldii coated with the ELISA kitA solid phase carrier of SAG4 protein (ESAG 4). In a specific embodiment of the invention, the solid phase carrier can be selected from a 96-well culture plate or a similar solid phase carrier, the ESAG 4 is coated at a concentration of 1.45 mug/well, and the carrier can be coated with a coating solution consisting of 0.39g Na₂CO₃，35mMNaHCO₃And adjusting the pH value to 9.6 to obtain the product, wherein the concentration of the NaCl is 0.2M.

After the core components of the kit are determined, the ELISA kit further comprises one or more than two of enzyme-labeled secondary antibody, washing solution, developing solution, confining solution, diluent and stop solution.

The enzyme-labeled secondary antibody is preferably goat anti-rabbit IgG labeled with HRP, in the specific embodiment of the invention, the enzyme-labeled secondary antibody is a product purchased from biological engineering Limited company of Bausch & Wuhan, and the dilution ratio of the enzyme-labeled secondary antibody is 1: 3000A;

the washing solution is preferably PBS-T washing solution, in the specific embodiment of the invention, the PBS-T washing solution is composed of 0.01M PBS + 0.05% Tween-20; the color development liquid is preferably TMB color development liquid;

the blocking fluid is preferably skim milk, which in a specific embodiment of the invention is 5% diluted in 0.01M PBS solution.

The stop solution is preferably a sulfuric acid solution, and the concentration is preferably 2 mol/L; the preparation method comprises slowly dripping 21.7mL of 98% concentrated sulfuric acid into 178mL of deionized water, cooling to room temperature, and storing at 4 ℃;

the diluent is preferably 0.01M PBS; the preparation method comprises 8g of NaCl, 0.2g of KCl and 1.42g of Na₂HPO₄，0.27gKH₂PO₄Dissolving in 800mL deionized water, dissolving to 1L, sterilizing, and storing at room temperature.

According to the technical scheme, the Eimeria sieboldii SAG4 protein is used as a series of related applications such as rabbit coccidiosis diagnosis antigen, and related experimental results show that the Eimeria sieboldii SAG4 protein can be identified by Eimeria sieboldii positive serum, and has good immunogenicity and reactogenicity; meanwhile, the protein shows extremely high sensitivity and specificity in an indirect ELISA method, and various results prove that the Eimeria sieboldii SAG4 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.

Drawings

FIG. 1 shows the expression purification of rEsSAG4 and immunoblot results; wherein, lane M: a protein standard substance marker; lane 1: induction of rEsSAG4 (unpurified) expressed from E.coli BL21(DE3) with IPTG; lane 2: purified rEsSAG4(8 μ g); lane 3: eresag 4 recognized by eimeria siella-positive sera; lane 5: eresag 4 recognized by eimeria siella-negative sera; lanes 5-7: rESAG 4 recognized by rabbit E-Coccidium-positive sera; lanes 8-10: rESAG 4 recognized by rabbit sarcoptidosis positive serum;

FIG. 2 shows the detection of E.stuartii positive and negative serum samples using an established indirect ELISA method; where the horizontal line represents the Cut-Off value of the ELISA method (Cut-Off 0.447), 48 eimeria siella positive and negative serum samples were detected, respectively, with significant variability between the data (x indicates P < 0.01).

Detailed Description

The invention discloses application of Eimeria sibirica SAG4 protein, and a person skilled in the art can use the content for reference and appropriately modify the process parameters to realize the application. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the embodiments described herein without departing from the spirit and scope of the invention.

The invention carries out reverse transcription to synthesize cDNA by extracting Eimeria sporulated oocyst total RNA of Eimeria spelundii and taking OligodT (18) as a reverse transcription primer, and amplifies an Eimeria spelundii SAG4 protein coding sequence from the cDNA. After T cloning, the amplified product is introduced into an expression vector in a way of enzyme digestion connection, and prokaryotic expression is carried out by escherichia coli to obtain recombinant rEsSAG 4.

In experiments of a specific embodiment, all experimental animals were treated strictly according to the "animal protection law of the people's republic of china" (draft published on 9/18 th in 2009). All procedures were performed according to the rules of animal care and use of the animal ethics committee of the university of Sichuan agriculture (China, Yaan) (approval No.: 2015-028).

Eimeria sieboldii species used in the present invention were isolated from the liver and gall bladder of naturally infected New Zealand rabbits, sporulated in 2.5% potassium dichromate solution at 28 ℃ and then stored at 4 ℃; 48 30-day-old coccidiosis-free young rabbits were bred by animal parasitosis research center of Sichuan university of agriculture and strictly raised in coccidiosis-free environment, during which boiled drinking water and 80 ℃ roasted rabbit feed were provided for feeding, and anticoccidial drugs diclazuril and decoquinate were alternately used while regular spray-burning of rabbit cages to prevent coccidiosis contamination.

48 parts of Eimeria sieboldii negative rabbit serum in the test is collected from 48 coccidian-free young rabbits, the coccidian-free young rabbits check excrement by a saturated saline floating method every day in the period of 30-35 days of age, and the coccidian oocysts are negative after 5 days of continuous excrement check; negative sera were used to determine Cut-Off values and specificity of indirect ELISA. 48 parts of Eimeria sieboldii positive rabbit serum were collected from artificially infected coccidian sporulated oocysts (8X 10)⁴One/one) to 30 days and the liver was confirmed by necropsy to show significant symptoms in 48 experimental rabbits; positive sera were used to determine the sensitivity of the indirect ELISA method. 3 parts of rabbit sarcoptidosis (sarcoptidosis cabeii) positive serum were collected from New Zealand rabbits artificially infected with rabbit sarcoptidosis, 3 parts of rabbit coccidiosis positive serum were collected from New Zealand rabbits naturally infected with and having detected coccidiosis etiologically; rabbit sarcoptidosis and rabbit enterococcidia positive sera were used for immunoblotting to verify the cross-reactivity of the recombinant protein with the rest of the rabbit parasites. All serum samples were stored at-20 ℃ until use.

All data are expressed as mean ± standard deviation (s.d.) and all correlation analyses were performed using GraphPad Prism version 5.0(GraphPad Software). The analysis of intra-and inter-batch variability was done using IBM SPSS statistics 22.0(SPSSSOFware), and a P value less than 0.05 was judged as a significant data difference.

The application of the Eimeria siei SAG4 protein provided by the invention is further explained below.