阅读说明：本技术 一种堆型艾美耳球虫纳米亚单位疫苗及其制备方法和应用 (Eimeria acervulina nano subunit vaccine and preparation method and application thereof ) 是由 李祥瑞 严若峰 宋小凯 徐立新 黄剑梅 于 2019-10-11 设计创作，主要内容包括：本发明公开了一种堆型艾美耳球虫纳米亚单位疫苗及其制备方法和应用。一种堆型艾美耳球虫纳米亚单位疫苗,所述的堆型艾美耳球虫纳米亚单位疫苗是由PLGA包裹重组蛋白EaMIC3形成的纳米粒子,所述的重组蛋白EaMIC3为堆型艾美耳球虫微线蛋白3,其氨基酸序列如SEQ ID NO.1所示。本发明将EaMIC3重组蛋白包被于纳米材料PLGA形成了一个全新的疫苗形式,得到免疫保护效果较高的堆型艾美耳球虫PLGA纳米亚单位疫苗。(The invention discloses an Eimeria acervulina nano subunit vaccine and a preparation method and application thereof. The eimeria acervulina nano subunit vaccine is a nano particle formed by PLGA (poly (lactic-co-glycolic acid)) encapsulated recombinant protein EaMIC3, wherein the recombinant protein EaMIC3 is Eimeria acervulina microwire protein 3, and the amino acid sequence of the eimeria acervulina nano subunit vaccine is shown as SEQ ID No. 1. According to the invention, EaMIC3 recombinant protein is coated on a nano material PLGA to form a brand-new vaccine form, so that the Eimeria acervulina PLGA nano subunit vaccine with a higher immune protection effect is obtained.)

1. The eimeria acervulina nano subunit vaccine is characterized in that the eimeria acervulina nano subunit vaccine is a nano particle formed by PLGA (poly-lactic-co-glycolic acid) coated recombinant protein EaMIC3, the recombinant protein EaMIC3 is eimeria acervulina microwire protein 3, and the amino acid sequence of the eimeria acervulina nano subunit vaccine is shown as SEQ ID No. 1.

2. The eimeria acervulina nano subunit vaccine as claimed in claim 1, characterized in that the recombinant protein EaMIC3 is obtained by transferring E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF into Escherichia coli for expression, and purifying the expressed recombinant protein EaMIC3 by His protein purification column.

3. The eimeria acervulina nano-subunit vaccine of claim 1, wherein the eimeria acervulina nano-subunit vaccine particle size is 143nm to 186 nm.

4. The method for preparing the eimeria acervulina nano subunit vaccine as claimed in claim 1, which comprises the following steps:

(1) E.coli BL21 competent cells are transformed by E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF to obtain Escherichia coli containing pET-32a-EaMIC3 ORF recombinant expression plasmid;

(2) Expressing and purifying Eimeria acervulina recombinant protein EaMIC 3;

(3) Preparing a nano subunit vaccine PLGA-EaMIC 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 EaMIC3 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 suspension into a vacuum freeze dryer, and carrying out freeze drying for 20 to 24 hours to obtain the eimeria acervulina 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.

5. The method according to claim 4, wherein the E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF described in step (1) is constructed as described in Zhang Zhen, role of chicken Eimeria acervulina microwire protein in invasion site specificity.

6. The method of claim 4, wherein the step (2) of expressing the purified Eimeria acervulina recombinant protein EaMIC3 is performed by: inoculating escherichia coli containing pET-32a-EaMIC3 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 EaMIC3 by a His (His-protein) purification column to obtain the purified Eimeria acervulina recombinant protein EaMIC 3.

7. The preparation method according to claim 4, wherein the ultrasonic breaking power in step (3) is 40-50W per time, the ultrasonic is 3-5 s per time, and the interval is 5s per time.

8. The use of the eimeria acervulina nano-subunit vaccine of claim 1 in the preparation of a medicament for the prevention of eimeria acervulina infection in a chicken.

Technical Field

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

Background

Coccidiosis in chickens is an intestinal disease caused by protozoan parasites of the genus eimeria and is one of the most important poultry diseases in the world. The morbidity of the coccidiosis in the chicken is 50-70%, the mortality rate is 20-30%, and the mortality rate is up to 80% in serious cases. Coccidiosis in chickens can cause extensive damage to the intestinal tracts of the chickens, infected chickens lose weight, the feed conversion rate is reduced, the laying rate of the laying hens is reduced, and the economic loss caused to the poultry industry every year exceeds $ 30 billion. At present, the main method for controlling the coccidiosis of the chicken is to add an accurately metered anti-coccidiosis drug into the feed. However, the long-term use of the anticoccidial drug leads to the continuous emergence of drug-resistant strains, so that the effect of the drug on preventing and treating the coccidiosis in chickens is obviously reduced. In recent years, the widespread emergence of antimicrobial drug resistance has raised concerns about the safety of antimicrobial drugs and their potential impact on human health, animal health and the environment. In addition, the european veterinary consortium issued a document claiming to strengthen the regulatory requirements of anticoccidial drugs in 2016, indicating its position for strengthening the regulatory requirements of anticoccidial drugs. The residual anticoccidial drug generated in the using process of the anticoccidial drug seriously affects the safety of animal-derived food. The demand of people on a method for preventing and treating the chicken coccidiosis without drug residues is increasingly urgent. Research finds that immunoprophylaxis can solve the problem of drug residue. The immune prevention method of the coccidia live vaccine and subunit vaccine with the anti-coccidia effect is considered as an ideal method for controlling coccidiosis. The current commercially available coccidian-resistant vaccines are all coccidian live vaccines, and although the coccidian live vaccines have excellent effect of controlling coccidiosis, the coccidian live vaccines have the safety problems and the immune effect problems of possible strong toxicity, easy toxin dissipation and the like due to limited production amount, difficult storage, difficult control of a using method and high cost, so that the requirements of modern chicken raising industry are difficult to meet. In recent years, the field of molecular biology has been rapidly developed, and the emergence of subunit vaccines has been promoted. 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, and the subunit vaccine has the advantages of safe use, stable property, convenient transportation, low production cost and the like, so the subunit vaccine has great research significance. In addition, subunit vaccines play an increasingly important role in the control of livestock and poultry epidemic diseases. However, currently, few subunit vaccines are commercially available. The continuous development of nanotechnology makes it possible to design nanoparticles of different composition, size, shape and surface characteristics, and also creates 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 addition, nanoparticles are changing the diagnosis of diseases and 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 acervulina (e.acervulina) is one of the most widespread and most harmful species.

Disclosure of Invention

The invention aims to provide an Eimeria acervulina nano subunit vaccine.

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

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

the eimeria acervulina nano subunit vaccine is a nano particle formed by PLGA (poly (lactic-co-glycolic acid)) encapsulated recombinant protein EaMIC3, wherein the recombinant protein EaMIC3 is Eimeria acervulina microwire protein 3, and the amino acid sequence of the eimeria acervulina nano subunit vaccine is shown as SEQ ID No. 1.

The recombinant protein EaMIC3 is preferably obtained by transferring E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF into escherichia coli for expression and purifying the expressed recombinant protein EaMIC3 by a His protein purification column.

The particle size of the eimeria acervulina nano subunit vaccine is 143nm-186 nm.

The preparation method of the eimeria acervulina nano subunit vaccine comprises the following steps:

(1) e.coli BL21 competent cells are transformed by E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF to obtain Escherichia coli containing pET-32a-EaMIC3 ORF recombinant expression plasmid;

(2) Expressing and purifying Eimeria acervulina recombinant protein EaMIC 3;

(3) Preparing a nano subunit vaccine PLGA-EaMIC 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 EaMIC3 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 suspension into a vacuum freeze dryer, and carrying out freeze drying for 20 to 24 hours to obtain the eimeria acervulina 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 construction method of the E.acervulina recombinant expression plasmid pET-32a-EaMIC3 ORF described in the step (1) is preferably Zhang Zheng super, and the role of chicken Eimeria acervulina microwire protein in invasion site specificity.

The method for expressing and purifying Eimeria acervulina recombinant protein EaMIC3 in the step (2) is optimized: inoculating escherichia coli containing pET-32a-EaMIC3 ORF 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 EaMIC3 by a His (His-protein) purification column to obtain the purified Eimeria acervulina recombinant protein EaMIC 3.

In the step (3), the optimal ultrasonic crushing power is 40-50W, the ultrasonic treatment is carried out for 3-5 s, and the interval is 5 s.

The invention relates to application of an Eimeria acervulina nano subunit vaccine in preparing a medicament for preventing infection of chicken Eimeria acervulina.

The invention has the following advantages and effects:

(1) At present, no research and report of the eimeria acervulina PLGA nano subunit vaccine exists, and the invention fills the blank of the research of the eimeria acervulina PLGA nano subunit vaccine. (2) Eimeria acervulina microwin 3(EaMIC3) is present on the top of Eimeria acervulina sporozoites and can be combined with chicken duodenal epithelial cells, and furthermore, antiserum of EaMIC3 can remarkably inhibit the invasion of Eimeria acervulina into host cells, indicating that EaMIC3 is related to the sporozoite invasion process. According to the invention, the EaMIC3 recombinant protein is coated on the nano material PLGA to form a brand-new vaccine form, and after the EaMIC3 recombinant protein and the nano material PLGA are coated, the coccidiosis resistance index (ACI) is improved (from 173.76 to 176.6), which indicates that the immune protection effect of the Eimeria acervulina is enhanced. (3) The invention improves the reported embedding process of the nano material, the concentration of PVA is improved to 5% from 1% reported, and the embedding rate of the nano vaccine is obviously improved.

Drawings

FIG. 1SDS-PAGE analysis of purified EaMIC3 fusion protein.

m: protein Mark (kDa); 1: purified EaMIC3 fusion protein

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

Detailed Description

Base material:

1. sporulation of oocysts: eimeria acervulina sporulated oocysts, belonging to Jiangsu strain heap type, were rejuvenated and sporulated in chickens every 3 months with sporulation rates above 80% (Sowa, Lizhou Qing. Chicken coccidiosis science [ M ]. Beijing: Chinese agriculture university Press, 1998.).

2. Experimental animals: the 0-day-old cymbidium white chicks are purchased from a Shuangli hatching poultry farm in Anhai county, raised in a strictly sterile and coccidian-free environment from the time of shelling to the end of an experiment, and fed with food and water freely.

3. Strain: e.coli BL21 strain of recombinant expression plasmid E.acervulina pET-32a-EaMIC3 ORF (Zhang Zheng super, role of chicken Eimeria acervulina microwire protein in invasion site specificity, Ph academic paper 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).