Oral hepatitis B vaccine and preparation method thereof

Abstract

The invention discloses an oral hepatitis B vaccine and a preparation method thereof. The preparation method comprises the following steps: preparing a milk exosome; construction and purification of hepatitis B surface antigen; and encapsulating the purified hepatitis B surface antigen into the milk exosome by adopting a membrane fusion method to obtain the oral hepatitis B vaccine. In the preparation method of the oral hepatitis B vaccine provided by the invention, the exosome is extracted and separated from fresh milk through a mechanical separation method and sucrose density gradient centrifugation and is used as an oral drug delivery carrier; secondly, inserting the hepatitis B virus surface antigen gene into engineered yeast, and expressing hepatitis B virus surface antigen protein; subsequently, mixing the purified hepatitis B surface antigen with cationic liposome, encapsulating the hepatitis B surface antigen into exosome by using a membrane fusion method, and carrying out characterization and activity test to obtain the oral hepatitis B vaccine. The oral hepatitis B vaccine prepared by the preparation method of the oral hepatitis B vaccine provided by the invention can tolerate the gastrointestinal tract environment in the body of an experimental mouse, can penetrate through the intestinal epithelial barrier, and finally stimulates the body to generate a hepatitis B antibody.

Description

Technical Field

[0001] The present invention relates to the technical field of hepatitis B vaccine, and in particular to an oral hepatitis B vaccine and a preparation method thereof. Background Art

[0002] Hepatitis B (HBV) is a global epidemic caused by the hepatitis B virus (HBV). According to the World Health Organization, nearly 1 million people die each year from complications such as liver failure, cirrhosis, and liver cancer caused by chronic HBV infection. Although large-scale vaccination of hepatitis B vaccine has significantly reduced the incidence rate, there are still some problems that limit its widespread application in developing countries: (1) The vaccine needs to be stored and transported under harsh cold chain conditions (2-8°C), resulting in high costs; (2) The main method of vaccination is intramuscular injection, which requires professional personnel to operate; (3) The use of aluminum hydroxide as an adjuvant may cause adverse reactions at the injection site, such as swelling and inflammation. Therefore, it is urgent to develop a new safe, low-cost, and convenient hepatitis B vaccine formulation.

[0003] Compared with intramuscular vaccines, oral vaccines have the advantages of convenient administration, low production cost, and no need for cold chain storage. However, oral vaccines face two major technical challenges: (1) It is necessary to overcome the complex digestive tract environment, such as gastric acid and digestive enzymes that may cause the vaccine to be degraded before reaching the intestine; (2) The antigen component of the vaccine is mainly protein, which is a large molecule that is difficult to effectively penetrate the intestinal epithelial barrier and cannot activate immune cells and stimulate antibody production. Summary of the invention

[0004] The present invention provides an oral hepatitis B vaccine, and further provides a method for preparing the oral hepatitis B vaccine. The oral hepatitis B vaccine prepared by the preparation method can tolerate the gastrointestinal environment in experimental mice, and can penetrate the intestinal epithelial barrier, and finally stimulate the body to produce hepatitis B antibodies. The oral hepatitis B vaccine provided by the present invention has the advantages of simple administration, safety and effectiveness in vivo, relatively low production cost, no need for refrigerated transportation and storage, etc., which helps to solve the problems in the prior art.

[0005] The first aspect of the present invention provides a method for preparing an oral hepatitis B vaccine, comprising the following steps:

[0006] Preparation of milk exosomes;

[0007] Construction and purification of hepatitis B surface antigen;

[0008] The purified hepatitis B surface antigen was encapsulated into milk exosomes by membrane fusion method to obtain an oral hepatitis B vaccine.

[0009] In one embodiment of the present invention, the membrane fusion method comprises the following steps:

[0010] The purified hepatitis B surface antigen is mixed with the cationic liposome and incubated to form a liposome-HBsAg complex;

[0011] The liposome-HBsAg complex is mixed with milk exosomes and incubated, and the cationic liposomes in the liposome-HBsAg complex undergo membrane fusion with the milk exosomes to obtain an oral hepatitis B vaccine.

[0012] In the present invention, membrane fusion of cationic liposomes and milk exosomes occurs, so that the hepatitis B surface antigen is loaded inside the liposomes, so as to avoid the problem of the vaccine being degraded before reaching the intestine due to the complex environmental problems of the digestive tract. At the same time, milk exosomes can further avoid the problem that the hepatitis B surface antigen is difficult to effectively penetrate the intestinal epithelial barrier.

[0013] In one embodiment of the present invention, the purified HBsAg was mixed with cationic liposomes and incubated at room temperature for 30 min to form a liposome-HBsAg complex.

[0014] In one embodiment of the present invention, the liposome-HBsAg complex is mixed with milk exosomes and incubated at room temperature for 30 minutes to obtain an oral hepatitis B vaccine.

[0015] In one embodiment of the present invention, the preparation of milk exosomes comprises the following steps:

[0016] Fresh milk is separated by mechanical separation to obtain skimmed milk;

[0017] Skim milk was subjected to sucrose density gradient centrifugation to obtain milk exosomes.

[0018] In one embodiment of the present invention, skimmed milk is subjected to sucrose density gradient centrifugation comprising the following steps:

[0019] The skim milk was centrifuged at 100,000 g for 105 min, and the precipitate was resuspended in PBS to obtain a resuspension;

[0020] The resuspension was subjected to sucrose density gradient centrifugation, and the sucrose density gradient solutions used included sucrose solutions with mass concentrations of 2 mol / L, 1.65 mol / L, 1.3 mol / L, 0.95 mol / L and 0.6 mol / L from top to bottom.

[0021] In one embodiment of the present invention, the construction and purification of hepatitis B surface antigen comprises the following steps:

[0022] Obtaining the nucleic acid sequence of hepatitis B virus surface antigen protein from the NCBI database, codon-optimizing the nucleic acid sequence, and synthesizing the optimized nucleic acid fragment, the nucleic acid sequence of which is shown in SEQ ID NO.1;

[0023] The synthesized nucleic acid fragment was constructed into the pPIC9K expression vector by double enzyme digestion to obtain a recombinant expression plasmid;

[0024] The recombinant expression plasmid was transferred into Pichia pastoris, and purified by inducing expression to obtain purified hepatitis B surface antigen.

[0025] The second invention of the present invention provides an oral hepatitis B vaccine, which is prepared by the above preparation method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The oral hepatitis B vaccine preparation method provided by the present invention has the advantages of simplified process and high efficiency, significantly reducing the complexity and cost of production. At the same time, the method can be easily implemented in existing industrial production facilities without major transformation or new equipment, thereby accelerating the marketization process of the product.

[0028] 2. In the method for preparing an oral hepatitis B vaccine provided by the present invention, exosomes are extracted and separated from fresh milk by mechanical separation and sucrose density gradient centrifugation and used as an oral drug delivery carrier; secondly, the hepatitis B virus surface antigen gene is inserted into an engineered yeast to express the hepatitis B virus surface antigen protein; subsequently, the purified hepatitis B surface antigen and cationic liposomes are mixed, and the hepatitis B surface antigen is encapsulated into the exosomes by a membrane fusion method, and an oral hepatitis B vaccine is obtained after characterization and activity testing.

[0029] 3. The oral hepatitis B vaccine prepared by the oral hepatitis B vaccine preparation method provided by the present invention can tolerate the gastrointestinal environment in experimental mice, and can penetrate the intestinal epithelial barrier, and ultimately stimulate the body to produce hepatitis B antibodies.

[0030] 4. The oral hepatitis B vaccine provided by the present invention has the advantages of simple administration, safety and effectiveness in vivo, relatively low production cost, and no need for refrigerated transportation and storage, which helps to solve the problems in the prior art.

[0031] 5. The oral hepatitis B vaccine preparation method provided by the present invention has the advantages of simplified process and high efficiency, which significantly reduces the complexity and cost of production. At the same time, the method can be easily implemented in existing industrial production facilities without major transformation or new equipment, thereby accelerating the marketization process of the product.

[0032] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the oral hepatitis B vaccine process of the present invention;

[0034] Figure 2 This is a characterization result diagram of milk exosomes in an embodiment of the present invention; wherein, Figure 2 (A) is the transmission electron microscopy characterization result of milk exosomes. Figure 2 (B) is the NTA characterization result of milk exosomes. Figure 2 (C) is the result of flow cytometry characterization of milk exosomes (CD9, CD63);

[0035] Figure 3 The figure shows the expression and purification of hepatitis B surface antigen and the characterization results of hepatitis B-exosome vaccine; Figure 3 (A) is the characterization result of HBsAg gene PCR product, Figure 3 (B) is the result of immunoblotting of HBsAg protein. Figure 3 (C) is the transmission electron microscopy characterization result of cationic liposome-HBsAg protein-exosome;

[0036] Figure 4 This is the result of the hepatitis B surface antigen expression test (cell experiment) and the hepatitis B antibody titer test of mice oral vaccine; Figure 4 (A) is the result of immunoblotting characterization of hepatitis B antibody in cell experiments. Figure 4 (B) is the result of in vivo hepatitis B antibody enzyme-linked immunosorbent assay. DETAILED DESCRIPTION

[0037] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0038] Oral vaccines currently face two major technical challenges: (1) The need to overcome the complex digestive tract environment, such as gastric acid and digestive enzymes that may cause the vaccine to be degraded before reaching the intestines; (2) The antigenic components of the vaccine are mainly proteins, which are large molecules that are difficult to effectively penetrate the intestinal epithelial barrier and cannot activate immune cells and stimulate antibody production.

[0039] Therefore, a new vaccine delivery vehicle is needed that can protect the structure and function of the vaccine while stimulating an immune response. Oral strategies for hepatitis B vaccines based on nanomaterials have shown potential. Early studies have attempted to use chitosan nanoparticles or silica as delivery materials, but the development of these materials has been limited by their low bioavailability and metabolic rate and potential side effects.

[0040] With the rise of emerging nanomedicine oral technology, exosomes have received increasing attention in the field of drug delivery. Exosomes are lipid bilayer membrane vesicles with a diameter of about 30-150 nanometers, which are secreted by almost all cells and widely present in a variety of body fluids. They are natural carriers of information exchange. Exosomes are not only numerous and easy to enrich, but also have the characteristics of high biocompatibility, low immunogenicity and good cellular uptake. Studies have shown that drugs packaged in exosomes can effectively avoid enzymatic degradation and have low immunogenicity and good safety. Milk is rich in exosomes, which can be used as a delivery vehicle for oral vaccines, and compared with cell culture, milk is easier to obtain and has a low cost. Studies have confirmed that milk exosomes can survive in gastric acid and intestinal degradation environments, and have the ability to cross the gastrointestinal barrier, making them a potential oral delivery tool.

[0041] Yeast is an important system for recombinant protein expression. It has the advantages of fast growth, easy genetic manipulation and large-scale culture. It also has the post-translational processing ability of eukaryotic cells and can produce recombinant proteins with biological activity. As a rapidly developing host for foreign protein expression, Pichia pastoris can efficiently produce foreign gene products, the expression vector is stable and inherited, and it is suitable for high-density and high-yield industrial fermentation. In addition, it can perform many typical protein post-translational modifications of eukaryotic organisms, such as glycosylation.

[0042] In the present invention, exosomes extracted from milk are used to encapsulate hepatitis B surface antigen (HBsAg) induced to be expressed in engineered yeast, thereby preparing an oral hepatitis B vaccine. This oral vaccine can tolerate the gastrointestinal environment in experimental mice, penetrate the intestinal epithelial barrier, and effectively stimulate the production of hepatitis B antibodies.

[0043] In the present invention, HBsAg is the abbreviation of Hepatitis B surface antigen. In the present invention, liposome-HBsAg complex and liposome-Hepatitis B surface antigen protein complex refer to the same substance.

[0044] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art in practical applications according to the present invention still belong to the scope of protection of the present invention.

[0045] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0046] Example

[0047] This embodiment provides an oral hepatitis B vaccine, which is prepared by the following steps:

[0048] Step a (purification and characterization of milk exosomes): mechanical separation was used to separate fat from fresh milk, and sucrose density gradient centrifugation was performed to obtain milk exosomes.

[0049] Step b (Purification and characterization of hepatitis B surface antigen): Obtain the nucleic acid sequence of hepatitis B virus surface antigen protein from the NCBI database, optimize the codons, and entrust Nanjing GenScript to synthesize the fragments. The nucleotide sequence is shown in SEQ ID NO.1, and the protein amino sequence is shown in SEQ ID NO.2. The synthesized fragments are constructed into the pPIC9K expression vector by double enzyme digestion to obtain a recombinant expression plasmid; the recombinant expression plasmid is transferred into Pichia pastoris, and purified by induction expression to obtain purified hepatitis B surface antigen;

[0050] Step c (preparation of oral hepatitis B vaccine): incubate the purified hepatitis B surface antigen protein and cationic liposomes at room temperature for 30 minutes to form a liposome-HBsAg complex; further mix the liposome-HBsAg complex with the milk exosomes obtained in step a, incubate at room temperature for 30 minutes, and allow the cationic liposomes and exosomes to fuse membranes. The hepatitis B surface antigen is encapsulated into the exosomes by the membrane fusion method to form an oral hepatitis B vaccine containing the hepatitis B surface antigen-exosome carrier.

[0051] In this embodiment, step a specifically includes: centrifuging skim milk at 100,000 g (Beckman, USA) for 105 minutes, and then resuspending the precipitate in 1 mL PBS to obtain a resuspension; then placing the resuspension on top of a discontinuous density gradient sucrose medium (7 ml volume per corner) composed of 2 mol / L, 1.65 mol / L, 1.3 mol / L, 0.95 mol / L and 0.6 mol / L, and centrifuging at 100,000 g for 20 h at 4°C; the milk exosomes are separated by centrifugal force between the fractions with sucrose density of 1.3 mol / L and 1.65 mol / L, and the exosomes are separated from the impurities; the collected milk exosome precipitate is further centrifuged at 100,000 g, 4°C for 105 min, and the milk exosomes are stored at -80°C for later use.

[0052] In step (a), the morphology of milk exosomes was characterized by transmission electron microscopy after sucrose density gradient centrifugation, which showed a typical lipid bilayer membrane teacup-shaped vesicle structure ( Figure 2 A), and the particle size of exosomes was measured by nanoparticle tracking (NTA), which was distributed in the range of 30-180nm ( Figure 2B). Further, in this step, the molecular markers (CD63, CD9) on the surface of the purified milk exosome membrane were characterized by flow cytometry. The milk exosomes were labeled with fluorescent antibodies of CD9 and CD63, respectively. The experimental group used anti-CD9 labeled with Alexa Fluor 488 and anti-CD63 labeled with 488 to label the milk exosomes, respectively, and the control group used IgG labeled with Alexa Fluor 488 to label the milk exosomes. Compared with the control group, the fluorescence signal of the experimental group was significantly shifted ( Figure 2 C), indicating that CD9 and CD63 characteristic proteins on the surface of milk exosomes were successfully identified by flow cytometry. Based on the above results, it was determined that milk exosomes were successfully extracted from milk.

[0053] In this embodiment, step (b) specifically includes the following steps:

[0054] First, double-ended primers were added to amplify the HBs-Ag gene fragment by PCR technology, with pre-denaturation at 95°C for 8 minutes; denaturation at 95°C for 35 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 50 seconds, for a total of 35 cycles; finally, extension at 72°C for 5 minutes and storage at 4°C. The PCR product was recovered by gel electrophoresis for the target gene ( Figure 3 A). The pPIC9K expression vector was double-digested with XhoI and NotI, and the double-digested fragments were recovered. The target gene and the pPIC9K expression vector fragment were then connected at a molar ratio of 3:1 at 4°C overnight. The target gene was constructed into the double-digested pPIC9K expression vector by DNA recombinase, transformed into competent cells DH5α, plated on low-salt LB plates, cultured at 37°C for 16-24h, single colonies were picked, plasmids were extracted, and pPIC9K-HBs-Ag plasmids were obtained. 200μL of Pichia pastoris was mixed with 20μL of pPIC9K-HBs-Ag plasmid, ice-bathed for 5min, and then 1mL of culture medium was added and incubated at 37°C for 30min. Then 100μL of bacterial solution was spread on a solid culture medium plate and inverted at 37°C for 2 days. Pick a single clone, inoculate the Pichia pastoris with the HBs-Ag gene into 3L of culture medium, culture at 37°C, 250rpm for about 16-18h, until the bacterial OD 600 = 0.6, add 1mM IPTG, culture at 18°C, 250rpm for about 24h, and induce the expression of the recombinant protein at low temperature. Subsequently, collect the bacterial liquid, centrifuge at 4°C 12000rpm / min for 20min to harvest the Pichia pastoris, add 50mL of lysis buffer to lyse the precipitate, and then filter through a 0.45μm filter, and use AKTA protein purifier for affinity chromatography. The purified HBsAg protein is identified by Western Blot, and the cellulose membrane is incubated with anti-HBsAg to determine the molecular weight of the purified protein to be 25Kda ( Figure 3 B), HBsAg protein was successfully purified.

[0055] The nucleotide sequence of the fragment synthesized in step (b) is as follows:

[0056] ATGGAGAGCACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTCTAGACTCGTGGTGGACTTCTCCAATTTTCTAGGGGGAGCACCCACGTGTCCTGGCCAAAATTCGCAGTCCCCAAC CTCCAATCACTCACCAACCTCTTGTCCTCCAACTTGTCCTGGCTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTACTTCCAGGAACAT CAACTACCAGCACGGGACCATGCAGAACCTGCACGATTCCTGCTCAAGGAACCTCTATGTTTCCCTCTTGTTGCTGTACAAAACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTC TCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGGTATACATTTGA

[0057] The amino sequence of the fragment protein synthesized in step (b) is shown as SEQ ID NO.2:

[0058] MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTLPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPNMESTTSGFLGPLLVLQAGFFLLTRIL Tip

[0059] The double-ended primer sequences in step (b) are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] In this embodiment, step (c) also includes characterizing the obtained oral hepatitis B vaccine, which specifically includes the following steps: detecting the morphology of the oral hepatitis B vaccine by electron microscopy, using 3% phosphotungstic acid staining solution to negatively stain the oral hepatitis B vaccine of the hepatitis B surface antigen-exosome carrier for 10 seconds, and observing the successful encapsulation of the hepatitis B surface antigen by milk exosomes by transmission electron microscopy imaging ( Figure 3 C).

[0064] In this example, the oral vaccine obtained in step (c) is further subjected to cell experiments and animal experiments to verify that the oral vaccine obtained in the present invention can successfully enter the test immune cells and express anti-hepatitis B viral antibodies. The specific steps are as follows:

[0065] Step (d) (cell experiment): The anti-hepatitis B virus antibodies in the cell culture medium supernatant were quantified by protein immunoblotting. First, 0.5 mg / mL oral vaccine was used to act on human lymphocyte B cells for 48 hours, and then the cell supernatant was collected, and the supernatant was ultrafiltered using a 30 kDa ultrafiltration tube to concentrate the protein and determine the protein concentration. Then, protein immunoblotting analysis was performed, and the protein was electrophoresed at 0 V constant voltage for 70 minutes. After the end, the gel was transferred to a PVDF membrane and electrophoresed at 120 V constant voltage. Then, 5% 2X protein loading buffer was added to the PVDF membrane and heated at 95 ° C for 5 minutes. 10 ug of protein sample was added to 8% SDS-PAGE gel, blocked with 9% skim milk powder, and incubated with anti-HBsAg antibody. Finally, HRP-labeled secondary antibody was added for incubation and color development. Figure 4 A shows that compared with the exosome empty group, the hepatitis B vaccine experimental group successfully detected HBsAg antibodies with a molecular weight of approximately 144kDa.

[0066] Step e (animal experiment): The experimental mice were divided into two groups: an oral saline control group (5 mice), an oral empty exosome carrier control group (5 mice) and an oral hepatitis B vaccine test group (5 mice). The experimental group was fed with hepatitis B vaccine orally on the 1st, 15th and 36th days, respectively, while the control group was also fed with the same dose of saline or empty exosome carrier orally. The physiological indicators of the mice were monitored daily. At different time points, the enzyme-linked immunosorbent assay method was used to determine the titer of hepatitis B antibodies in the serum of the two groups of mice. The specific process was to first rinse and activate the 96-well ELISA plate for detecting hepatitis B antibodies with buffer, then add the mouse serum sample and incubate at room temperature for 1 hour, then wash with washing buffer 3 times, 5 minutes each time, then add the HRP-labeled reagent A in the detection kit to the 96-well plate, incubate at room temperature for 30 minutes, then wash with washing buffer 3 times, 5 minutes each time, and finally add the color developing reagent for color development. The group with hepatitis B antibodies will show blue. The results are as follows Figure 4 B shows that hepatitis B antibodies were detected in the serum of mice injected with the hepatitis B vaccine, while almost no hepatitis B antibodies were produced in the PBS group and the exosome empty group, indicating that the oral hepatitis B vaccine prepared in this experiment can successfully induce the production of hepatitis B antibodies in mice.

[0067] In summary, in the method for preparing an oral hepatitis B vaccine provided by the present invention, exosomes are extracted and separated from fresh milk by mechanical separation and sucrose density gradient centrifugation, and used as an oral drug delivery carrier; secondly, the hepatitis B virus surface antigen gene is inserted into the engineered yeast to express the hepatitis B virus surface antigen protein; then, the purified hepatitis B surface antigen and cationic liposome are mixed, and the hepatitis B surface antigen is encapsulated into the exosomes by the membrane fusion method, and the oral hepatitis B vaccine is obtained after characterization and activity testing. The oral hepatitis B vaccine can tolerate the gastrointestinal environment in experimental mice, and can penetrate the intestinal epithelial barrier, and finally stimulate the body to produce hepatitis B antibodies. The oral vaccine of the invention has the advantages of simple administration, safety and effectiveness in vivo, relatively low production cost, and no need for refrigerated transportation and storage, which helps to solve the problems in the prior art.

[0068] In addition, the oral hepatitis B vaccine preparation method provided by the present invention has the advantages of simplified process and high efficiency, significantly reducing the complexity and cost of production. At the same time, the method can be easily implemented in existing industrial production facilities without major modifications or new equipment, thereby accelerating the marketization process of the product.

[0069] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A method for preparing an oral hepatitis B vaccine, characterized in that: The following steps are involved: Preparation of milk exosomes; Construction and purification of hepatitis B surface antigen; The purified hepatitis B surface antigen was encapsulated into milk exosomes by membrane fusion method to obtain an oral hepatitis B vaccine.

2. The method for preparing an oral hepatitis B vaccine according to claim 1, characterized in that: The membrane fusion method comprises the following steps: The purified hepatitis B surface antigen is mixed with the cationic liposome and incubated to form a liposome-HBsAg complex; The liposome-HBsAg complex is mixed with milk exosomes and incubated, and the cationic liposomes in the liposome-HBsAg complex undergo membrane fusion with the milk exosomes to obtain an oral hepatitis B vaccine.

3. The method for preparing an oral hepatitis B vaccine according to claim 2, characterized in that: The purified HBsAg was mixed with cationic liposomes and incubated at room temperature for 30 minutes to form a liposome-HBsAg complex.

4. The method for preparing an oral hepatitis B vaccine according to claim 3, characterized in that: The liposome-HBsAg complex was mixed with milk exosomes and incubated at room temperature for 30 minutes to obtain an oral hepatitis B vaccine.

5. The method for preparing an oral hepatitis B vaccine according to claim 2, characterized in that: The preparation of milk exosomes includes the following steps: Fresh milk is separated by mechanical separation to obtain skimmed milk; Skim milk was subjected to sucrose density gradient centrifugation to obtain milk exosomes.

6. The method for preparing an oral hepatitis B vaccine according to claim 5, characterized in that: Sucrose density gradient centrifugation of skim milk includes the following steps: The skim milk was centrifuged at 100,000 g for 105 min, and the precipitate was resuspended in PBS to obtain a resuspension; The resuspension was subjected to sucrose density gradient centrifugation, and the sucrose density gradient solutions used included sucrose solutions with mass concentrations of 2 mol / L, 1.65 mol / L, 1.3 mol / L, 0.95 mol / L and 0.6 mol / L from top to bottom.

7. The method for preparing an oral hepatitis B vaccine according to any one of claims 1 to 6, characterized in that: The construction and purification of hepatitis B surface antigen includes the following steps: Obtaining the nucleic acid sequence of hepatitis B virus surface antigen protein from the NCBI database, codon-optimizing the nucleic acid sequence, and synthesizing the optimized nucleic acid fragment, the nucleic acid sequence of which is shown in SEQ ID NO.1; The synthesized nucleic acid fragment was constructed into the pPIC9K expression vector by double enzyme digestion to obtain a recombinant expression plasmid; The recombinant expression plasmid was transferred into Pichia pastoris, and purified by inducing expression to obtain purified hepatitis B surface antigen.

8. An oral hepatitis B vaccine, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 7.

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