阅读说明：本技术 一种幽门螺杆菌口服疫苗 (Helicobacter pylori oral vaccine ) 是由 雷涵 岑黔鸿 高彤 任怡 于 2020-06-20 设计创作，主要内容包括：本发明涉及幽门螺杆菌口服疫苗领域,具体涉及一种融合表达幽门螺杆菌抗原基因与Aga2基因的重组酿酒酵母。本发明借助酿酒酵母表面展示系统,展示幽门螺杆菌的UreB或VacA蛋白,构建一种全新的针对幽门螺杆菌的口服疫苗。该口服疫苗能有效提高机体对幽门螺杆菌的免疫预防作用,具有良好的应用价值。(The invention relates to the field of helicobacter pylori oral vaccines, in particular to recombinant saccharomyces cerevisiae for fusion expression of a helicobacter pylori antigen gene and an Aga2 gene. The invention constructs a brand-new oral vaccine aiming at helicobacter pylori by means of a saccharomyces cerevisiae surface display system to display UreB or VacA protein of the helicobacter pylori. The oral vaccine can effectively improve the immunoprophylaxis of the organism to helicobacter pylori, and has good application value.)

1. A recombinant plasmid, characterized in that: the recombinant plasmid can fuse and express a helicobacter pylori antigen gene and an Aga2 gene to obtain a recombinant plasmid for expressing fusion protein;

the nucleotide sequence of the helicobacter pylori antigen gene is shown as any one of SEQ ID NO 1-2;

the peptide segment obtained by the helicobacter pylori antigen gene expression in the fusion protein is positioned at the C end of the peptide segment obtained by the Aga2 gene expression.

2. The recombinant plasmid of claim 1, wherein the recombinant plasmid is a backbone plasmid that is the pYD1 plasmid produced by Invitrogen.

3. The recombinant plasmid of claim 1, wherein said helicobacter pylori antigen gene is linked to a stop codon sequence.

4. A recombinant Saccharomyces cerevisiae, which is characterized in that: it is Saccharomyces cerevisiae containing the recombinant plasmid of any one of claims 1-2.

5. The recombinant Saccharomyces cerevisiae according to claim 4, wherein the Saccharomyces cerevisiae is S.

6. Use of the recombinant Saccharomyces cerevisiae according to claim 4 or 5 for the preparation of a helicobacter pylori vaccine.

7. Use according to claim 6, characterized in that: the vaccine is an oral vaccine.

8. A helicobacter pylori vaccine, characterized by: the recombinant saccharomyces cerevisiae of claim 4 or 5 is used as an active ingredient, and pharmaceutically acceptable auxiliary materials or auxiliary ingredients are added to prepare the preparation.

9. The vaccine of claim 8, wherein: the auxiliary component is enteric capsule.

10. A process for the preparation of the recombinant Saccharomyces cerevisiae as claimed in claim 4 or 5, comprising the steps of:

(1) constructing a recombinant plasmid capable of fusing and expressing a helicobacter pylori antigen gene and an Aga2 gene to obtain a recombinant plasmid for expressing a fusion protein;

(2) introducing the recombinant plasmid into competent saccharomyces cerevisiae to obtain the recombinant plasmid;

the nucleotide sequence of the helicobacter pylori antigen gene is shown as any one of SEQ ID NO 1-2.

Technical Field

The invention relates to the field of helicobacter pylori oral vaccines, in particular to a helicobacter pylori oral vaccine.

Background

Helicobacter pylori (h. pylori) is a novel gram-negative bacterium that spirals or sigmoids on the surface of gastric mucosal epithelial cells. The length of the material is 2.5-4.0 um, and the width is 0.5-1.0 um. One end of the thallus is provided with 4-7 flagella which provide power for the H.pyrori planting process and have the anchoring function in the planting process, so that the thallus is tightly attached to the gastric mucosa to prevent displacement. The pyhori is an obligate microaerophilic bacterium, has strict requirements on the growth environment and grows in 5 to 8 percent of oxygen environment. After being infected by h.pyrori, it attaches to gastric epithelial cells, colonizes and proliferates in vivo, causing the occurrence of active gastritis, changes the physiological function of the stomach, causes gastric acid hypersecretion, resulting in chronic gastritis, acute gastritis, duodenal ulcer and peptic ulcer. Meanwhile, it destroys mucosa secreting acid, causing the risk of diseases such as atrophic gastritis, gastric mucosa-associated lymphoma and gastric cancer, and is the main pathogenic bacterium of digestive system diseases. The National Institute of Health (NIH) has established that most recurrent peptic ulcers are caused by infection with helicobacter pylori, which the World Health Organization (WHO) lists in a list of carcinogens.

Currently, triple therapies (proton pump inhibitors, clarithromycin + amoxicillin, omeprazole) are mainly used clinically to treat diseases associated with h. Antibiotics play an important role in eradicating h.pyri, but the clinical problem that comes with it is that antibiotics also kill other probiotics in the gut, affecting the stability of the gut flora. In addition, with the rapid rise of antibiotic resistance, the dosage of antibiotics in clinical treatment schemes is increasing continuously, the curative effect is not satisfactory, and the problem of bacterial resistance is occurring continuously. It is noteworthy that h.pyri is susceptible to repeated infections, which makes antibiotics contradictory in clinical treatment regimens, i.e. increased amounts of antibiotics do not increase the effectiveness of clinical healing. The World Health Organization (WHO) published a list of 12 resistant bacteria and bacterial families that pose the greatest threat to human health, with helicobacter pylori (resistant to clarithromycin) ranking 6 th. Thirdly, the use of antibiotics has many adverse reactions such as allergy and the like. Amazing that about 70 million people who die globally each year directly or indirectly from antibiotic resistance and the number of them increases year by year, the body injuries and adverse reactions caused thereby are immeasurable.

The Saccharomyces cerevisiae (s. cerevisiae) surface display system displays foreign proteins on the cell surface using the a-lectin receptor of Saccharomyces cerevisiae. The lectin receptor consists of two subunits encoded by the AGA1 and AGA2 genes. The Aga1 protein (Aga1p,725 amino acids) is covalently linked to the extracellular matrix of the beta-glucan yeast cell wall after secretion by the cell. The Aga2 protein (Aga2p,69 amino acids) binds to Aga1p through two disulfide bonds and remains attached to the cells after secretion by contact with Aga1 p. Commercial pYD1 (Invitrogen, USA) is a 5.0kb Saccharomyces cerevisiae compatible expression plasmid that allows the fusion of the gene of interest to AGA 2. The N-terminal part of Aga2p needs to be linked to Aga1p, while the protein of interest can be fused to the C-terminus, presented on the cell surface of s.cerevisiae. Saccharomyces cerevisiae can display high molecular weight proteins, and can effectively fold, glycosylate and form disulfide bonds on expressed exogenous eukaryotic proteins. Wei Q utilizes the Saccharomyces cerevisiae surface display system to display the metal regulated protein MerR and demonstrates its usefulness for bioabsorption and bioremediation of environmental mercury pollutants. Karbaanowicz T utilizes the Saccharomyces cerevisiae surface display system to display neurotoxin as a technique for producing recombinant toxins, revealing the potential application of the Saccharomyces cerevisiae-based surface display system in industry.

Which has many advantages over other display systems. First, saccharomyces cerevisiae has recognized safety (GRAS) and has been used in large quantities in the food and pharmaceutical industries. Secondly, the cell wall surface component of the saccharomyces cerevisiae EBY100 contains adjuvants beta-1, 3-D-glucan and mannan, and can enhance the immune response of organisms to antigens. Thirdly, the saccharomyces cerevisiae molecular display system has typical eukaryotic specificity, post-translational modification mechanisms, and is capable of expressing many functional proteins required for post-translational modification. And thirdly, the culture method of the saccharomyces cerevisiae is simple, low in cost and short in growth period, and can be applied to industrial production in a large scale.

Some virus vaccines constructed by using a saccharomyces cerevisiae surface display system currently exist, for example, patent application No. 201510405035.8 discloses a vaccine for yeast display of grass carp hemorrhagic disease injection and a preparation method thereof, and the vaccine is prepared by displaying GCRV-VP7 protein on the surface of a saccharomyces cerevisiae cell EBY100 delta Mnn9 after glycosylation gene knockout. However, no report of helicobacter pylori oral vaccine constructed by a yeast surface display system is found at present.

The invention firstly applies the saccharomyces cerevisiae surface display technology to construct the helicobacter pylori oral vaccine, and inspects the immune protection efficiency of a saccharomyces cerevisiae surface display platform as a helicobacter pylori oral vaccine delivery carrier by constructing an animal model infected by helicobacter pylori. Further, feasible strategies and methods are provided for developing the saccharomyces cerevisiae surface display platform-based bacterial oral vaccine.

Disclosure of Invention

In order to solve the problems, the invention provides a recombinant saccharomyces cerevisiae and application thereof.

The technical scheme of the invention comprises the following steps:

a recombinant plasmid, which can fuse and express a helicobacter pylori antigen gene and an Aga2 gene to obtain a recombinant plasmid for expressing fusion protein;

the nucleotide sequence of the helicobacter pylori antigen gene is shown as any one of SEQ ID NO 1-2;

the peptide segment obtained by the helicobacter pylori antigen gene expression in the fusion protein is positioned at the C end of the peptide segment obtained by the Aga2 gene expression.

The recombinant plasmid was constructed from pYD1 plasmid produced by Invitrogen as a backbone plasmid, as described above.

As the aforementioned recombinant plasmid, the helicobacter pylori antigen gene is linked with a stop codon sequence.

A recombinant Saccharomyces cerevisiae, which is a Saccharomyces cerevisiae containing any one of the recombinant plasmids.

Recombinant s.cerevisiae EBY100 as described previously, said s.cerevisiae.

The application of the recombinant saccharomyces cerevisiae in preparing the helicobacter pylori vaccine.

The vaccine is an oral vaccine for the aforementioned use.

A helicobacter pylori vaccine is a preparation prepared by taking any one of the recombinant saccharomyces cerevisiae as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

As for the vaccine, the auxiliary component is enteric-coated capsules.

A method for preparing the recombinant saccharomyces cerevisiae comprises the following steps:

(1) constructing a recombinant plasmid capable of fusing and expressing a helicobacter pylori antigen gene and an Aga2 gene to obtain a recombinant plasmid for expressing a fusion protein;

(2) introducing the recombinant plasmid into competent saccharomyces cerevisiae to obtain the recombinant plasmid;

the nucleotide sequence of the helicobacter pylori antigen gene is shown as any one of SEQ ID NO 1-2.

The invention can effectively prevent the infection of the helicobacter pylori and has very good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

Note: lane represents the Lane of electrophoresis.

FIG. 1; a cultured h.

FIG. 2: PCR electrophoretogram of UreB and VacA genes

Lane 1 DNA marker (DL 2,000); lane 2: UreB gene is 1710 bp;

lane 1 DNA marker (DL 2,000); lane2 VacA gene is 780 bp.

FIG. 3: plasmid pYD1 after double digestion, Lane 1: DNA marker (DL5,000); lane2 plasmid pYD1 after double digestion is 5009 bp.

FIG. 4: double-enzyme digestion identification map of recombinant plasmids pYD1-UreB and pYD 1-VacA.

Lane 1 DNA marker (DL5,000); lane2, pYD1-UreB is subjected to double enzyme digestion by Nhe I/EcoR I;

lane 1 DNA marker (DL5,000); lane 2: pYD1-VacA was digested simultaneously with Nhe I/EcoR I.

FIG. 5: PCR identification electrophoretogram of recombinant Saccharomyces cerevisiae EBY100/pYD1, S.cerevisiae EBY100/pYD1-UreB and S.cerevisiae EBY100/pYD1-VacA

Lane 1 DNA marker (DL 2,000); lane2 PCR result of forward and reverse primer amplification of pYD1 with S.cerevisiae EBY100/pYD1 genome DNA as template.

Lane 1 DNA marker (DL5,000); lane2, PCR result of positive and negative primer amplification of pYD1 by using S.cerevisiae EBY100/pYD1-UreB genome DNA as a template; lane 3, PCR result of UreB positive and negative primer amplification by using S.cerevisiae EBY100/pYD1-UreB genome DNA as template;

lane 1 DNA marker (DL 2,000); lane2, PCR result of positive and negative primer amplification of pYD1 by taking S.cerevisiae EBY100/pYD1-VacA genomic DNA as a template; lane 3, PCR result of positive and negative primer amplification of VacA using S.cerevisiae EBY100/pYD1-VacA genomic DNA as template.

FIG. 6: pattern diagram for constructing helicobacter pylori by saccharomyces cerevisiae surface display

A. Schematic diagram of a saccharomyces cerevisiae surface display system displaying the UreB protein.

B. Schematic diagram of a surface display system for Saccharomyces cerevisiae displaying VacA protein.

FIG. 7: western blot detection of specific expression of UreB and VacA proteins

Western blot analysis of EBY100/pYD 1-UreB: lane 1: a protein standard; lane2 EBY100/pYD1 lysate; lane 3: EBY100/pYD1-UreB lysate;

western blot analysis of EBY100/pYD 1-VacA. Lane 1: a protein standard; lane 2: EBY100/pYD1 lysate; lane 3: EBY100/pYD1-VacA lysate.

FIG. 8: immunofluorescence analysis results of EBY100/pYD1-UreB, A. negative control EBY100/pYD 1; EBY100/pYD 1-UreB. (magnification: 400X).

FIG. 9: immunofluorescence analysis results of EBY100/pYD1-VacA, A. negative control EBY100/pYD 1; EBY100/pYD 1-VacA. (magnification: 400X).

FIG. 10: flow cytometry analysis of EBY100/pYD1-UreB, A. negative control EBY100/pYD 1; EBY100/pYD1-UreB, (count: 10,000 cells).

FIG. 11: flow cytometry analysis of EBY100/pYD1-VacA results, A. negative control EBY100/pYD 1; EBY100/pYD1-VacA, (count: 10,000 cells).

FIG. 12: oral immunization schedule.

FIG. 13: ELISA was used to determine anti-UreB specific serum IgG titers, and the EBY100/pYD1-UreB and EBY100/pYD1-UreB + EBY100/pYD1-VacA groups were statistically significant (p <0.05) compared to the PBS and EBY100/pYD1 groups.

FIG. 14: ELISA was used to determine anti-VacA specific serum IgG titers, and the EBY100/pYD1-VacA and EBY100/pYD1-UreB + EBY100/pYD1-VacA groups were statistically significant (p <0.05) compared to the PBS and EBY100/pYD1 groups.

FIG. 15: ELISA detected anti-UreB specific secretory IgA, and EBY100/pYD1-UreB group and EBY100/pYD1-UreB + EBY100/pYD1-VacA group were statistically significant (p <0.05) compared to PBS group and EBY100/pYD1 group.

FIG. 16: ELISA detected anti-VacA specific secretory IgA, and EBY100/pYD1-VacA and EBY100/pYD1-UreB + EBY100/pYD1-VacA had statistical significance (p <0.05) compared to PBS and EBY100/pYD1 groups.

FIG. 17: quantitative analysis of the colonization of pylori SS1 in the stomach of mice, EBY100/pYD1-UreB, EBY100/pYD1-VacA and EBY100/pYD1-UreB + EBY100/pYD1-VacA were statistically significant (p <0.05) compared to PBS and EBY100/pYD 1.

FIG. 18: the results of the urease assay were statistically significant in the EBY100/pYD1-UreB, EBY100/pYD1-VacA and EBY100/pYD1-UreB + EBY100/pYD1-VacA groups (p <0.05) compared to the PBS group and EBY100/pYD1 group.

Detailed Description