阅读说明：本技术 Cs/hpmcp纳米微粒在口服疫苗递送中的应用 (Application of CS/HPMCP nano particles in oral vaccine delivery ) 是由 肖书奇 李志伟 马志倩 李爽 于 2020-05-15 设计创作，主要内容包括：本发明公开了CS/HPMCP纳米微粒在口服疫苗递送中的应用,属于动物病毒学和动物基因工程技术领域。为了检测CS/HPMCP纳米微粒口服递送的免疫效力,本发明选取猪流行性腹泻病毒作为试验对象；本发明通过大肠杆菌表达系统表达COE蛋白,进一步通过离子交联方法制备了载有COE的CS/HPMCP纳米颗粒,并且评估了给小鼠口服载有COE的CS/HPMCP纳米微粒后,小鼠的体液免疫、细胞免疫和粘膜免疫；旨在为研发口服型纳米微粒佐剂提供有力的技术支持,同时为PEDV口服型亚单位疫苗的研究奠定前期基础。(The invention discloses application of CS/HPMCP nano particles in oral vaccine delivery, and belongs to the technical field of animal virology and animal genetic engineering. In order to detect the immune efficacy of oral delivery of CS/HPMCP nanoparticles, porcine epidemic diarrhea virus is selected as a test object; according to the invention, the COE protein is expressed by an escherichia coli expression system, the CS/HPMCP nano-particles loaded with the COE are further prepared by an ion crosslinking method, and the humoral immunity, the cellular immunity and the mucosal immunity of a mouse are evaluated after the CS/HPMCP nano-particles loaded with the COE are orally taken by the mouse; aims to provide powerful technical support for developing oral nanoparticle adjuvants and lay a preliminary foundation for researching PEDV oral subunit vaccines.)

The application of CS/HPMCP nanoparticles in oral vaccine delivery is characterized by comprising the following specific steps:

(1) constructing a target gene prokaryotic expression vector, and obtaining purified target protein through induced expression and purification;

(2) preparing CS/HPMCP/protein composite nanoparticles: the mass ratio of CS to HPMCP is 0.7: 1, the concentration of chitosan is 2mg/ml, the concentration of HPMCP is 4mg/ml, the concentration of acetic acid is 0.5mol/L, and the pH value of a chitosan solution is 5.5; under the condition that the rotating speed of a magnetic stirrer is 400rpm, the HPMCP solution and the target protein solution are mixed and then are dropwise added into the chitosan solution, and the stirring time is 10min, so that the CS/HPMCP/protein composite nano-particles are prepared.

2. Use of CS/HPMCP/protein composite nanoparticles according to claim 1 for the preparation of a vaccine.

3. The use of CS/HPMCP nanoparticles in oral vaccine delivery according to claim 1, wherein the step (1) of constructing a prokaryotic expression vector of PEDV COE, and performing induced expression and purification to obtain purified COE protein comprises the following steps:

amplifying a PEDV COE gene, wherein the primer sequence is as follows:

PEDV-SX-COE-F：5’-TCGGATCCGTTACTTTGCCATCATTTAATG-3’；SEQ ID NO.1；

PEDV-SX-COE-R：5’-TGCTCGAGAACGTCCGTGACACCTTC-3’；SEQ ID NO.2；

cloning the PEDV COE gene into a pET-32a prokaryotic expression vector by utilizing enzyme cutting sites BamHI and Xho I to obtain pET-32 a-COE;

thirdly, transforming the pET-32a-COE plasmid with correct sequencing into BL21(DE3) competent cells, adding IPTG with the final concentration of 1mmol/L, and carrying out induced expression for 3-4h to obtain the purified COE protein.

Application of CS/HPMCP/COE protein composite nanoparticles in preparation of vaccines for preventing porcine epidemic diarrhea.

Technical Field

The invention relates to the technical field of animal virology and animal genetic engineering, in particular to application of CS/HPMCP nano particles in oral vaccine delivery.

Background

Chitosan (CS) is a partially deacetylated form of chitin, formed by glucosamine and N-acetylglucosamine monomers linked by β - (1-4) glycosidic linkages, a linear polymer, exists in the exoskeletons of crustaceans, insect and fungal cell walls, the second most abundant polysaccharide in nature next to cellulose, chitosan and chitosan derivatives have been widely used in the fields of anti-tumor, wound dressing, cosmetics, food, tissue engineering and ophthalmology, due to their minimal toxicity, good biodegradability, excellent biocompatibility, and furthermore, due to the cationic properties of CS, mucosal adsorption properties, immunostimulatory properties, and the ability to instantaneously open epithelial tight junctions, it has also been used to deliver recombinant proteins via the mucosal route⁺And CD8⁺T cell immune response. Ion crosslinking is the most common method for preparing chitosan nanoparticles, and Tripolyphosphate (TPP) is the most common crosslinking agent. In addition, hypromellose phthalate (HPMCP) is an enteric biodegradable material approved by the Food and Drug Administration (FDA) for use in humans, and is another cross-linking agent. The substance is one of enteric coating excipients widely used in pharmaceutical industry, and is only in PH>And 5.5, the solution is completely dissolved. The pH of the gastrointestinal tract is known to be as follows: stomach (pH2.0-4.0), duodenum (pH5.5), jejunum (pH6.0) and ileum (pH 7.2-8.0). Thus, when HPMCP is used for oral delivery of proteins, it may reduce degradation of proteins in the stomach and increaseAbsorption of proteins in the intestinal tract. When HPMCP is used instead of TPP as a cross-linker, CS nanoparticles exhibit superior properties, such as increased mucoabsorbability and acid stability of the nanoparticles in the ileum. However, the nanoparticles are commonly used for oral delivery of insulin and heparin, and are rarely used for oral delivery of proteins or vaccines, and the factors affecting CS/HPMCP nanoparticle formation have not been systematically studied.

Porcine Epidemic Diarrheal (PED) is a Porcine susceptible acute highly-contagious intestinal disease caused by Porcine Epidemic Diarrheal Virus (PEDV). PEDV primarily infects small intestinal epithelial cells and causes villous atrophy, resulting in severe diarrhea, vomiting, dehydration, anorexia and high mortality in 7-10 day old piglets. The spike (S) protein of PEDV and the neutralizing epitope region (COE) of the S protein are major candidates for developing vaccines against PEDV subunits. Furthermore, PEDV-specific secretory iga (siga) antibodies in colostrum and milk produced by mucosal immunization are crucial for preventing PEDV invasion. Also, vaccines delivered by the mucosal route induce mucosal immunity more efficiently than traditional inactivated and attenuated PEDV vaccines that are immunized by intramuscular route or subcutaneous injection. However, oral vaccines for pregnant sows face several challenges, namely that the active ingredients in the vaccine may be degraded by gastric acid, pepsin and trypsin in the gastrointestinal tract, which would render the orally delivered vaccine unable to elicit an effective mucosal immune response against PEDV infection.

Therefore, providing CS/HPMCP nanoparticles for use in oral vaccine delivery is a problem that needs to be addressed by those skilled in the art.

Disclosure of Invention

In view of the above, the present invention provides the use of CS/HPMCP nanoparticles for oral vaccine delivery.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the CS/HPMCP nano particles in oral vaccine delivery comprises the following specific steps:

(1) constructing a target gene prokaryotic expression vector, and obtaining purified target protein through induced expression and purification;

(2) preparing CS/HPMCP/protein composite nanoparticles: the mass ratio of CS to HPMCP is 0.7: 1, the concentration of chitosan is 2mg/ml, the concentration of HPMCP is 4mg/ml, the concentration of acetic acid is 0.5mol/L, and the pH value of a chitosan solution is 5.5; under the condition that the rotating speed of a magnetic stirrer is 400rpm, the HPMCP solution and the target protein solution are mixed and then are dropwise added into the chitosan solution, and the stirring time is 10min, so that the CS/HPMCP/protein composite nano-particles are prepared.

Further, the CS/HPMCP/protein composite nano particle is applied to preparation of vaccines.

Further, a PEDV COE prokaryotic expression vector is constructed in the step (1), and purified COE protein is obtained through induced expression and purification, and the steps are as follows:

amplifying a PEDV COE gene, wherein the primer sequence is as follows:

PEDV-SX-COE-F：5’-TCGGATCCGTTACTTTGCCATCATTTAATG-3’；SEQ ID NO.1；

PEDV-SX-COE-R：5’-TGCTCGAGAACGTCCGTGACACCTTC-3’；SEQ ID NO.2；

cloning the PEDV COE gene into a pET-32a prokaryotic expression vector by utilizing enzyme cutting sites BamHI and XhoI to obtain pET-32 a-COE;

thirdly, transforming the pET-32a-COE plasmid with correct sequencing into BL21(DE3) competent cells, adding IPTG with the final concentration of 1mmol/L, and carrying out induced expression for 3-4h to obtain the purified COE protein.

Further, the CS/HPMCP/COE protein composite nano-particles are applied to preparation of a vaccine for preventing porcine epidemic diarrhea.

The porcine epidemic diarrhea virus is only a specific example, the CS/HPMCP nano particles can also embed other proteins, and the obtained composite particles are used in the fields of vaccines, pharmacy and the like.

The CS/HPMCP nano particles can be applied to vaccine development, and vaccines can be delivered through oral administration, nasal drops, genital tract and other mucosal routes.

According to the technical scheme, compared with the prior art, the invention discloses the application of CS/HPMCP nanoparticles in oral vaccine delivery, and the pH-sensitive CS/HPMCP nanoparticles can be used for oral delivery of vaccines; in order to test the immune efficacy of the oral delivery of the nanoparticles, the invention selects porcine epidemic diarrhea virus as a test object; according to the invention, the COE protein is expressed by an escherichia coli expression system, the CS/HPMCP nano-particles loaded with the COE are further prepared by an ion crosslinking method, and the humoral immunity, the cellular immunity and the mucosal immunity of a mouse are evaluated after the CS/HPMCP nano-particles loaded with the COE are orally taken by the mouse; aims to provide powerful technical support for developing oral nanoparticle adjuvants and lay a preliminary foundation for researching PEDV oral subunit vaccines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the amplification of a COE target gene according to the present invention;

wherein, M: DNA molecular weight standard DL 8000; 1: a COE target gene;

FIG. 2 is a diagram showing the double digestion results of the target fragment and the vector of the present invention;

wherein, M: DNA molecular weight standard DL 8000; 1: the target gene enzyme digestion product; 2: carrying out enzyme digestion on the carrier;

FIG. 3 is a diagram showing PCR identification of recombinant plasmid liquid of the present invention;

wherein, M: DNA molecular weight standard DL 8000; 1-8: identifying the bacterial liquid; -: negative control; +: a positive control;

FIG. 4 is a drawing showing SDS-PAGE according to the present invention to identify purified proteins;

wherein, M: a protein Marker; 1, purified protein;

FIG. 5 is a diagram showing the western-blot identification of purified proteins according to the present invention;

wherein, M: a protein Marker; 1, purified protein;

FIG. 6 is an SDS-PAGE graph showing the identification of proteins released from CS/HPMCP/COE protein composite nanoparticles according to the present invention;

wherein, M: a protein Marker; 1, protein released from CS/HPMCP/COE protein composite nano particles; 2: CS/HPMCP unloaded nanoparticles; 3: a positive control; 4: CS/HPMCP/COE protein composite nanoparticles which are not treated by SDS;

FIG. 7 is a diagram of a western-blot for identifying proteins released by CS/HPMCP/COE protein composite nanoparticles according to the present invention;

wherein, M: a protein Marker; 1, protein released from CS/HPMCP/COE protein composite nano particles; 2: CS/HPMCP unloaded nanoparticles; 3: a positive control; 4: CS/HPMCP/COE protein composite nanoparticles which are not treated by SDS;

FIG. 8 is a graph showing the detection of anti-COE IgG antibody levels in serum by indirect ELISA according to the present invention;

FIG. 9 is a graph showing the measurement of anti-COE IgA antibody levels in serum by indirect ELISA according to the present invention;

FIG. 10 is a graph showing the results of the digestive tract secretory sIgA assay of the present invention;

FIG. 11 is a diagram showing the results of detecting the secretion of IL-4 in the supernatant by the ELISA kit of the present invention;

FIG. 12 is a diagram showing the effect of detecting lymphocyte proliferation by the MTT method of the present invention;

FIG. 13 is a diagram showing the results of detecting IFN-. gamma.secretion in the supernatant by the ELISA kit of the present invention;

FIG. 14 is a diagram showing the detection of relative expression of IFN-. gamma.mRNA by fluorescent quantitative PCR according to the present invention;

wherein, in fig. 8-14, x: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: the difference was not significant.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.