阅读说明：本技术 斯氏艾美耳球虫mic1蛋白的应用 (Application of eimeria stutzeri MIC1 protein ) 是由 杨光友 魏闻芮 彭雪蓉 于 2019-11-18 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MIC1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MIC1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MIC1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 MIC1 protein as rabbit coccidiosis diagnosis antigen, and the like, wherein related experimental results show that the Eimeria sieboldii MIC1 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 MIC1 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.)

1. The application of the eimeria stuartii MIC1 protein as a diagnostic antigen of rabbit coccidiosis and/or the application in preparing the diagnostic antigen of rabbit coccidiosis.

2. Application of eimeria stutzeri MIC1 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. Application of eimeria stuartii MIC1 protein in preparation of 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 MIC1 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 stutzeri MIC1 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 stuartii MIC1 protein (Esmic1) as a diagnostic antigen of rabbit coccidiosis and application thereof in preparation of the diagnostic antigen of rabbit coccidiosis, so that Esmic1 has high specificity and sensitivity, and good immunogenicity and reactogenicity;

the invention also aims to provide application of EsMIC1 in preparation of a kit for detecting rabbit coccidiosis, so that an ELISA method established by EsMIC1 shows higher specificity and sensitivity and can be used for ELISA detection;

another purpose of the invention is to provide application of EMIC 1 in preparation of rabbit coccidiosis vaccines.

In this context, the eimeria stuartii MIC1 protein (espic 1) may be non-natural, e.g., synthetic or expressed from an artificial vector (often referred to in the art as the recombinant protein rlemic 1). 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.

MIC1 is a glycoprotein G-related protein comprising a TSP region of 5 repeating hydrophilic amino acids, a conserved transmembrane region (CTD), and a long terminal string of hydrophobic amino acids (CTR); no relevant studies on the MIC of rabbit coccidia represented by Eimeria siei are available.

The invention utilizes the real-time fluorescent quantitative PCR technology to analyze the relative transcription level of Eimeria stuartii MIC1 protein (Esmic1) in different development periods of an insect body, clones and expresses pronucleus to Esmic1, utilizes immunoblotting to verify the reactionogenicity of recombinant protein rEsmic1 and establishes an indirect ELISA method to evaluate the diagnostic value of the recombinant protein as a recombinant antigen to the liver type rabbit coccidiosis. The results showed that Esmic1 was expressed in the highest amount during the schizogenesis phase of Eimeria sieboldii, and the protein encoded by the ORF of the Emic 1 (711 bp in full length, SEQ ID NO: 1) gene had a molecular weight of about 25.89kDa (SEQ ID NO: 2). Immunoblotting showed that the recombinant protein rEsMIC1 could be recognized by Eimeria sieboldii positive serum, indicating that it has good reactogenicity. The indirect ELISA method established based on rEsMIC1 has 100% (48/48) sensitivity and 97.9% (47/48) specificity; in addition, rEsMIC1 detected rabbit serum antibodies at day 6, 8, and 10 after Eimeria sieboldii infection, and reached the highest detection rate at day 10 after infection, 62.5% (30/48). Therefore, Esmic1 is a candidate diagnostic antigen for rabbit coccidiosis caused by Eimeria sieboldii.

Based on the content, the invention provides the application of the eimeria stuartii MIC1 protein as a rabbit coccidiosis diagnosis antigen and the application in preparing the rabbit coccidiosis diagnosis antigen. Meanwhile, the invention also provides the application of the eimeria stutzeri MIC1 protein in the 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 the eimeria stuartii MIC1 protein in preparation of rabbit coccidiosis vaccines.

According to the application of the eimeria stuartii MIC1 protein in the preparation of the kit, the invention provides an ELISA kit for diagnosing rabbit coccidiosis, which comprises a solid phase carrier coated with the eimeria stuartii MIC1 protein (EsMIC 1). 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 Esmic1 coating concentration is 1.15 mu g/well, and the carrier can be coated by a coating solution, wherein the coating solution is 0.39g of Na ₂CO ₃，35mMNaHCO ₃And 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, and in the specific embodiment of the invention, the PBS-T washing solution consists of 0.01MPBS + 0.05% Tween-20; the color development liquid is preferably TMB color development liquid;

the confining liquid is preferably skimmed milk, in a specific embodiment of the invention, the skimmed milk is 5% in concentration, diluted in a 0.01MPBS 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 Na ₂HPO ₄，0.27gKH ₂PO ₄Dissolving in 800mL deionized water, dissolving to 1L, sterilizing, and storing at room temperature.

According to the technical scheme, the Eimeria sieboldii MIC1 protein is used as a series of relevant applications such as rabbit coccidiosis diagnosis antigen, and relevant experimental results show that the Eimeria sieboldii MIC1 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 MIC1 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.

Drawings

FIG. 1 shows the results of the expression purification of rEsMIC 1; wherein, lane M: a protein standard substance marker; lane 1: induction of rEsMIC1 expressed by E.coli BL21(DE3) with IPTG (unpurified); lane 2: rEsMIC1 after purification with His-tagged protein purification column;

FIG. 2 shows the relative expression of Esmic1 at different developmental stages of Eimeria sieboldii; wherein the data are presented as mean plus standard deviation, with significant differences in the expression level of esic 1 at the non-sporulated oocyst stage from all other developmental stages (. lambda.denotes P < 0.05);

FIG. 3 shows an immunoblot analysis of rEsMIC 1; wherein, lane M: a protein standard substance marker; lane 1: eresmic 1 for eimeria stuartii positive serum identification; lane 2: rEsMIC1 for Eimeria sienna negative serum identification; lanes 3-5: rEsMIC1 recognized by rabbit sarcoptic mite positive serum; lanes 6-8: rEsMIC1 recognized by rabbit coccidia positive serum;

FIG. 4 shows the detection of E.stuartii positive and negative serum samples using an established indirect ELISA method; wherein, the horizontal line represents the Cut-Off value of the ELISA method (Cut-Off is 0.309), 48 eimeria siella positive and negative serum samples were detected, respectively, with significant difference between the data (×) representing P < 0.01;

FIG. 5 shows serum samples measured at days 0, 6, 8, and 10 after Eimeria sieboldii infection using an established indirect ELISA method; in this case, the horizontal line represents the Cut-Off value of the ELISA method (Cut-Off 0.309), and 48 sera were detected on days 0, 6, 8, and 10 after eimeria siella infection, respectively (P < 0.01).

Detailed Description

The invention discloses application of eimeria stutzeri MIC1 protein, and a person skilled in the art can use the content for reference and appropriately modify 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 amplifies the protein coding sequence of Eimeria siella MIC1 from cDNA by extracting Eimeria siella sporulated oocyst total RNA and reverse transcribing to cDNA. After T cloning, the amplified product is introduced into an expression vector in an enzyme digestion connection mode, and prokaryotic expression is carried out by using escherichia coli to obtain recombinant rEsMIC 1.

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 a naturally infected New Zealand rabbit, sporulated in 2.5% potassium dichromate solution at 28 ℃ and stored at 4 ℃; eimeria sieboldii non-sporulated oocysts, merozoites and gametophytes for transcript level analysis were collected and stored in liquid nitrogen as reported (JING J, LIUC, ZHU S X, et al. 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 in the present invention testThe coccidian negative rabbit serum is collected from 48 coccidian-free young rabbits, the coccidian-free young rabbits check excrement by a saturated saline floating method every day during the age of 30-35 days, 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 25 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. 48 sera from E.sieboldii at days 6, 8, and 10 after artificial infection were collected from experimental rabbits at the corresponding times and used to evaluate the early diagnostic value of the recombinant protein. 3 parts of rabbit sarcoptidosis scabies (Sarcoptes scabbiei) positive serum was collected from a New Zealand rabbit artificially infected with rabbit sarcoptidosis scabies, 3 parts of rabbit coccidiosis positive serum was collected from a New Zealand rabbit naturally infected with and etiologically detected for coccidiosis; 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 use of the E.stuartii MIC1 protein provided by the present invention is further described below.