阅读说明：本技术 一种巨型艾美耳球虫纳米亚单位疫苗及其制备方法和应用 (Eimeria maxima nano subunit vaccine and preparation method and application thereof ) 是由 李祥瑞 宋小凯 严若峰 徐立新 黄剑梅 于 2019-10-11 设计创作，主要内容包括：本发明涉及巨型艾美耳球虫(E.maxima)重组蛋白亚单位疫苗EmMIC3和纳米亚单位疫苗PLGA-EmMIC3,属于生物兽药技术领域。该纳米亚单位疫苗为用PLGA纳米材料包裹重组蛋白亚单位疫苗EmMIC3制成。经动物免疫保护性试验证实,上述重组蛋白亚单位疫苗EmMIC3和纳米亚单位疫苗PLGA-EmMIC3均能够有效抵抗巨型艾美耳球虫感染。本发明用纳米材料PLGA包裹重组蛋白EmMIC3形成了一个全新的疫苗形式,不同于单独EmMIC3重组蛋白,二者组分不同。并且EmMIC3重组蛋白与纳米材料PLGA包被以后,其免疫保护效果得到明显的提升,保护效果从一般提升到优秀。(The invention relates to an Eimeria maxima (E.maxima) recombinant protein subunit vaccine EmMIC3 and a nano subunit vaccine PLGA-EmMIC3, belonging to the technical field of biological veterinary medicines. The nanometer subunit vaccine is prepared by using PLGA nanometer material to wrap recombinant protein subunit vaccine EmMIC 3. Animal immune protective tests prove that the recombinant protein subunit vaccine EmMIC3 and the nano subunit vaccine PLGA-EmMIC3 can effectively resist the infection of the Eimeria maxima. According to the invention, the recombinant protein EmMIC3 is encapsulated by the nano material PLGA to form a brand-new vaccine form, which is different from the single EmMIC3 recombinant protein, and the two components are different. After the EmMIC3 recombinant protein and the nano material PLGA are coated, the immune protection effect is obviously improved, and the protection effect is generally improved to be excellent.)

1. The E.maxima nano subunit vaccine is a nano particle formed by PLGA coated recombinant protein EmMIC3, wherein the recombinant protein EmMIC3 is the E.maxima microline protein 3, and the amino acid sequence of the E.maxima nano subunit vaccine is shown in SEQ ID No. 1.

2. The E.maxima (E.maxima) nano subunit vaccine of claim 1, characterized in that the recombinant protein EmMIC3 is obtained by transferring E.maxima recombinant expression plasmid pET-32a (+) -MIC3 into Escherichia coli for expression, and purifying the expressed recombinant protein EmMIC3 by His protein purification column.

3. The eimeria maxima (e.maxima) nano subunit vaccine of claim 1, wherein the diameter of the eimeria maxima (e.maxima) nano subunit vaccine is 80nm to 330 nm.

4. The method of preparing the E.maxima (E.maxima) nano subunit vaccine of claim 1, characterized by comprising the steps of:

(1) Transforming E.coli BL21 competent cells by the E.maxima pET-32a (+) -MIC3 recombinant expression plasmid to obtain bacteria containing the pET-32a (+) -MIC3 recombinant expression plasmid;

(2) expressing and purifying the recombinant protein EmMIC3 of the Eimeria maxima;

(3) Preparing a nano subunit vaccine PLGA-EmMIC 3: preparing a dichloromethane solution of PLGA, wherein the mass volume concentration of PLGA is 4-5 g/100 mL; adding 5% PVA into a dichloromethane solution of PLGA, and uniformly mixing for 1-2min in a vortex manner; carrying out ultrasonic crushing for 3-5 min under an ice bath condition; dropwise adding the recombinant protein EmMIC3 solution prepared in the step (2) while swirling, and uniformly mixing for 1-2 min; carrying out ultrasonic crushing for 3-5 min under an ice bath condition to form milky primary emulsion; adding 5% PVA into the primary emulsion while swirling, and carrying out ultrasonic crushing for 3-5 min under an ice bath condition to form a double emulsion; stirring and volatilizing the double emulsion obtained after the ultrasonic treatment until the organic solvent is volatilized completely; centrifuging for 30-40 min at 28000-30000 r/min by using a refrigerated ultracentrifuge; after centrifugation is finished, respectively collecting supernatant and sediment; resuspending the precipitate obtained after ultracentrifugation by deionized water to obtain PLGA-coated recombinant protein suspension, placing the PLGA-coated recombinant protein suspension in a penicillin bottle, placing the penicillin bottle at the temperature of minus 80 ℃ for 1.5 to 2 hours, transferring the penicillin bottle into a vacuum freeze dryer, and carrying out freeze drying for 20 to 24 hours to obtain the E.maxima (E.maxima) nano subunit vaccine; wherein the volume of the 5% PVA added each time is 2-2.5 times of the volume of the dichloromethane solution of the PLGA; the ultrasonic breaking power is 40-50W, the ultrasonic is 3-5 s, and the interval is 5 s.

5. The method of claim 4, wherein the E.maxima pET-32a (+) -MIC3 recombinant expression plasmid of step (1) is constructed according to the immunoprotection and binding ability of four kinds of E.maxima of the microwire proteins to chicken intestinal epithelial cells, Huangjing Longwu, Ph-Dai-Proc-thesis of Nanjing agriculture university, 2016.

6. the method of claim 4, wherein the step (2) of expressing the purified recombinant E.maxima protein EmMIC3 comprises: inoculating escherichia coli containing pET-32a (+) -MIC3 recombinant expression plasmid into LB liquid culture medium according to the volume ratio of 1: 80-120, culturing at 37 ℃ and 200r/min to OD₆₀₀And when the concentration is 0.4-0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.8-1 mmol/L for induction expression, and purifying the expressed recombinant protein EmMIC3 by a His protein purification column to obtain the purified recombinant protein EmMIC3 of the Eimeria maxima.

7. Use of the eimeria maxima (e.maxima) nano subunit vaccine of claim 1 in the preparation of a medicament for preventing infection by chicken eimeria maxima.

Technical Field

The invention relates to the technical field of biological veterinary drugs, and relates to an eimeria maxima nano subunit vaccine, a preparation method and an application thereof.

Background

Coccidiosis is a globally prevalent parasitic disease that seriously harms the poultry industry. The morbidity is 50-70%, the mortality is 20-30%, and can reach more than 80% in serious cases. The production efficiency of the tolerant chickens is greatly reduced, and the economic loss caused by each year exceeds 30 billion dollars. Currently, coccidiosis in chickens is mainly controlled by drugs. However, with the continuous emergence of drug-resistant strains, the prevention effect of the drug is obviously reduced, and the drug resistance of the coccidian and the drug residue of the coccidian resistant drug generated in the using process of the drug seriously affect the safety of animal-derived food. Efforts have therefore been made to find alternatives to anticoccidial drugs. Research finds that the immunoprophylaxis can solve the problem of drug residue, and is an ideal method for controlling coccidiosis. The current commercial coccidiosis resistant vaccines are all live vaccines, but the live vaccines have the defects of self, such as immune programs and using methods which are not easy to control; the production capacity is limited, the cost is high, the storage is difficult, and the requirements of the modern chicken industry are difficult to meet; in addition, most importantly, the live vaccine has the safety problems of possible strong toxicity, easy toxin dispersion and the like and the immune effect problem. With the rapid development of the field of molecular biology, new-generation vaccines such as subunit vaccines are emerging and increasingly play an important role in the control of livestock and poultry diseases. The subunit vaccine only contains an immunogen protein component of a pathogen which is necessary for generating protective immune response, and has the characteristics of no replication in chicken bodies, no pathogenicity to chicken, no toxin dispersion and the like. After the subunit vaccine is inoculated to the breeding hens, the resistance of the breeding hens can be obtained, and simultaneously, the ability of organisms to resist coccidium infection is enhanced. Therefore, subunit vaccines are of great research interest. However, currently, few subunit vaccines are commercially available.

In recent years, the development of nanotechnology has made it possible to design nanoparticles of different composition, size, shape and surface characteristics, and also to create opportunities for their application in the medical field. Since the size of the nanoparticles is similar to that of cellular components, they can enter living cells through endocytic mechanisms, especially pinocytosis. The nano particles are widely applied as vaccine carrier transportation tools and immunopotentiators, can improve the stability of antigens, enhance the presentation and immunogenicity of the antigens, and can target the presentation and slow release of the antigens. In fact, nanoparticles are changing the diagnosis of disease, as well as providing bioactive substances for disease prevention and treatment. Poly (lactic-co-glycolic acid) (PLGA) has good biocompatibility and biodegradability, is a nano material approved by the U.S. FDA and European drug administration for clinical treatment, and has shown good immune enhancement effect in HIV DNA vaccine. At present, no application research report of the PLGA nano subunit vaccine in chicken coccidia is found.

There are 7 internationally recognized coccidiosis pathogens of chicken, eimeria tenella (e.tenella), eimeria necatrix (e.necatrix), eimeria acervulina (e.acervulina), eimeria maxima (e.maxima), eimeria brunetti (e.brunetti), eimeria praecox (e.praecox) and eimeria mitis (e.mitis), respectively. Of these, eimeria maxima (e.maxima) is one of the most widely distributed and most harmful species. The e.tenella microwire protein 3 (etcic 3) has been reported as a key molecule determining e.tenella parasitism in the cecum, however, the key molecule determining e.maxima parasitism has not been reported.

Disclosure of Invention

The invention aims to provide a nano subunit vaccine PLGA-EmMIC3 capable of preventing E.maxima chicken coccidium infection.

The invention also aims to provide a preparation method of the nano subunit vaccine.

The purpose of the invention can be realized by the following technical scheme:

The E.maxima nano subunit vaccine is a nano particle formed by PLGA packaging recombinant protein EmMIC3, wherein the recombinant protein EmMIC3 is E.maxima microlin protein 3, and the amino acid sequence of the E.maxima nano subunit vaccine is shown in SEQ ID No. 1.

The recombinant protein EmMIC3 is preferably obtained by transferring E.maxima recombinant expression plasmid pET-32a (+) -MIC3 into escherichia coli for expression and purifying the expressed recombinant protein EmMIC3 by a His protein purification column.

The particle size of the E.maxima nano subunit vaccine is preferably 80nm-330 nm.

The preparation method of the E.maxima nano subunit vaccine comprises the following steps:

(1) Transforming E.coli BL21 competent cells by the E.maxima pET-32a (+) -MIC3 recombinant expression plasmid to obtain bacteria containing the pET-32a (+) -MIC3 recombinant expression plasmid;

(2) Expressing and purifying the recombinant protein EmMIC3 of the Eimeria maxima;

(3) Preparing a nano subunit vaccine PLGA-EmMIC 3: preparing a dichloromethane solution of PLGA, wherein the mass volume concentration of PLGA is 4-5 g/100 mL; adding 5% PVA into a dichloromethane solution of PLGA, and uniformly mixing for 1-2min in a vortex manner; carrying out ultrasonic crushing for 3-5 min under an ice bath condition; dropwise adding the recombinant protein EmMIC3 solution prepared in the step (2) while swirling, and uniformly mixing for 1-2 min; carrying out ultrasonic crushing for 3-5 min under an ice bath condition to form milky primary emulsion; adding 5% PVA into the primary emulsion while swirling, and carrying out ultrasonic crushing for 3-5 min under an ice bath condition to form a double emulsion; stirring and volatilizing the double emulsion obtained after the ultrasonic treatment until the organic solvent is volatilized completely; centrifuging for 30-40 min at 28000-30000 r/min by using a refrigerated ultracentrifuge; after centrifugation is finished, respectively collecting supernatant and sediment; resuspending the precipitate obtained after ultracentrifugation by deionized water to obtain PLGA-coated recombinant protein suspension, placing the PLGA-coated recombinant protein suspension in a penicillin bottle, placing the penicillin bottle at the temperature of minus 80 ℃ for 1.5 to 2 hours, transferring the penicillin bottle into a vacuum freeze dryer, and carrying out freeze drying for 20 to 24 hours to obtain the E.maxima (E.maxima) nano subunit vaccine; wherein the volume of the 5% PVA added each time is 2-2.5 times of the volume of the dichloromethane solution of the PLGA; the ultrasonic breaking power is 40-50W, the ultrasonic is 3-5 s, and the interval is 5 s.

Preferably, the construction method of the E.maxima pET-32a (+) -MIC3 recombinant expression plasmid in the step (1) refers to the immunoprotection of four microline proteins of the Eimeria maxima and the binding capacity with chicken intestinal epithelial cells, Huanglongtiao, Ph-Dai of Nanjing agriculture university, 2016.

the preferable method for expressing and purifying the recombinant protein EmMIC3 of the Eimeria maxima in the step (2) is as follows: inoculating escherichia coli containing pET-32a (+) -MIC3 recombinant expression plasmid into LB liquid culture medium according to the volume ratio of 1: 80-120, culturing at 37 ℃ and 200r/min to OD₆₀₀And when the concentration is 0.4-0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.8-1 mmol/L for induction expression, and purifying the expressed recombinant protein EmMIC3 by a His protein purification column to obtain the purified recombinant protein EmMIC3 of the Eimeria maxima.

The invention relates to application of E.maxima (E.maxima) nano subunit vaccine in preparing a medicine for preventing chicken E.maxima infection.

the invention has the following advantages and effects:

(1) At present, the research and the report of the PLGA nano subunit vaccine of the Eimeria maxima are not seen, and the invention fills the blank of the research of the PLGA nano subunit vaccine of the Eimeria maxima. (2) According to the invention, the recombinant protein EmMIC3 is encapsulated by the nano material PLGA to form a brand-new vaccine form, which is different from the single EmMIC3 recombinant protein, and the two components are different. After the EMMIC3 recombinant protein and the nano material PLGA are coated, the immune protection effect is obviously improved, and the protection effect is improved from good (ACI 167.03) to excellent (ACI 184). (3) The invention improves the reported embedding process of the nano material, the concentration of PVA is improved to 5% from 1% of the reported concentration, and the embedding rate of the nano vaccine is remarkably improved (from 44.7% -82.7% to 90.09%).

Drawings

FIG. 1SDS-PAGE analysis of EmMIC3 fusion proteins.

M: protein marker (kDa); 1: pET-32a before induction; 2: pET-32a after 5h of induction; 3: pET-32a-EmMIC3 after purification; 4: pET-32a-EmMIC3 before induction; 5: pET-32a-EmMIC3 was obtained 5h after induction.

FIG. 2 scanning electron microscope results of nanometer subunit vaccine PLGA-EmMIC 3.

Detailed Description

Base material:

1. Sporulation of oocysts: the Jiangsu strain Eimeria maxima sporulate oocysts, and the sporulation rate is more than 80 percent after chicken rejuvenation and sporulation every 3 months (Sowa, Lizhou Qing. chicken coccidiosis science [ M ]. Beijing: China agricultural university Press, 1998.).

2. Experimental animals: the 0-day-old cymbidium white chicks were purchased from a Shuangli hatchery in Anhai county, raised from the time of emergence to the end of the experiment in a strictly sterile, coccidian-free environment, and were fed and drunk freely.

3. Strain: e.coli BL21 strain of recombinant expression plasmid E.maxima pET-32a (+) -MIC3 (Huanglongzhu, immunoprotection of four kinds of E.maxima microline protein and binding capacity with chicken intestinal epithelial cells. doctor academic thesis of Nanjing university of agriculture, 2016) was transformed.

4. Tool enzyme and reagent: protein molecular weight Marker was purchased from Thermo Fisher Scientific; HIS fusion protein purification kit (GE, USA), polyacrylamide, N' -methylene bisacryloyl, and Coomassie brilliant blue were purchased from Shanghai chemical reagent subpackaging factories; poly (glycolide-co-glycolide) (PLGA, Poly (D, L-lactide-co-glycolide) lactides: glycolide 65:35, Mw 40000-75000), polyvinyl alcohol (PVA, Poly (vinyl alcohol), Mw 31000-50000) from Sigma Aldrich; the other reagents are domestic analytical purifiers.

5. The main apparatus comprises: refrigerated tabletop centrifuge (Eppendorf centrifuge 5417R); ultraviolet-visible spectrophotometer (Bio-Rad); air bath shaking table (THZ, Experimental facilities of Taicano city, Jiangsu); an electrophoresis apparatus (DYY-11B, six instruments factories of Beijing); refrigerated ultracentrifuge (Beckman Coulter, usa); a vacuum freeze dryer (labbonco, usa); scanning electron microscope (Japanese JEOL JSM-IT 100).