CN117867022A - Recombinant adenovirus for expressing helicobacter pylori protein, and preparation method and application thereof - Google Patents
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Abstract
The invention provides a recombinant adenovirus for expressing helicobacter pylori protein, a preparation method and application thereof, wherein the method comprises the following steps: inserting helicobacter pylori genes into a shuttle plasmid vector through enzyme cutting sites KpnI and HindIII to obtain a recombinant adenovirus shuttle plasmid; the recombinant adenovirus shuttle plasmid and adenovirus skeleton plasmid are co-transferred into competent cells for recombination to obtain recombinant plasmids; linearizing the recombinant plasmid with restriction enzyme PacI and transfecting into cells to package recombinant adenovirus, thereby obtaining recombinant adenovirus expressing helicobacter pylori protein. The recombinant adenovirus expressing helicobacter pylori protein may be used as new carrier vaccine or new non-toxic adjuvant vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a recombinant adenovirus for expressing helicobacter pylori protein, a preparation method and application thereof.
Background
Helicobacter pylori (Helicobacter Pylori, HP) is a helical, microaerophilic gram-negative bacterium that colonizes the gastrointestinal tract, particularly the stomach, of the human body. HP can survive in gastric acid environment because it encodes urease, producing ammonia and CO 2 Microenvironment, and thus is not affected by gastric acid. The prevalence of helicobacter pylori infection varies widely from country to country, around 50% worldwide. If not treated with the relevant therapy, HP infection may persist for a lifetime. The pathogen has been demonstrated to induce peptic ulcers, gastritis, gastric proliferative polyps, gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric cancer. Furthermore, according to the conclusion of the World Health Organization (WHO) international cancer research institute working group in 1994, HP has been considered a class I (established) carcinogen for humans. The current global new cases of gastric cancer are about 110 ten thousand per year, the 4 th gastric cancer is discharged in the cause of malignant tumor death, and about 80 ten thousand people die from gastric cancer per year. The occurrence of gastric cancer is affected by a variety of factors including helicobacter pylori infection, family history and genetic predisposition of gastric cancer, age, environment, lifestyle, diet, smoking, drinking, etc., wherein at least 90% of gastric cancer is associated with helicobacter pylori infection. The infection rate of helicobacter pylori in China is about 60 percent, and the infection rate of 8-9 hundred million people is about national, wherein the incidence rate of gastric cancer is about 1 percent, and the gastric cancer is the cancer with the incidence rate of the second place and is the second next to lung cancer. China is a high-incidence country of gastric cancer, and more than half of new gastric cancers are in China worldwide.
Current traditional triple regimens of PPI (proton pump inhibitor) plus two antibiotics treat HP with gradually decreasing eradication rates, now eradication rates below or 80%. The fifth chinese guideline for helicobacter pylori treatment commonly recommended four-way regimen includes bismuth, PPI and two antibiotics. However, the worldwide drug resistance problem layer is endless, and researches report that HP has different degrees of drug resistance to tetracycline, amoxicillin, clarithromycin, levofloxacin and metronidazole, and the radical cure rate of the first-line scheme is reduced to 75%. Meanwhile, the addition of new auxiliary drugs such as bismuth agents or other probiotics and prokinetic drugs does not play a great role, but rather increases the economic burden of patients, reduces the compliance of the patients, and gradually increases the influence of long-term periods on intestinal flora and adverse reactions. In addition, triple quadruple therapy, although curing HP infection, is easy to relapse. The co-meal among friends is an important cause of reinfection, while the separate meals are not easy to reinfect. Helicobacter pylori can be isolated from gastrointestinal secretions, saliva, gums and faeces of patients infected with helicobacter pylori in the prior art, indicating that gastric-oral, oral-oral and faecal-oral transmission are possible important transmission routes. The poor economic conditions are risk factors for H.pylori infection, as the poor economic conditions are associated with more crowded living conditions which favour the in-home transmission of H.pylori, and the need for designing and manufacturing H.pylori protective and therapeutic vaccines is now increasing.
Helicobacter pylori has not yet been marketed as a commercial vaccine. Despite the great efforts of researchers, the production of effective helicobacter pylori vaccines remains a complex task. Since helicobacter pylori is well-adapted to the intragastric environment and is able to evade immune system mechanisms leading to persistent infections, current attempts to develop efficient vaccines have failed. And most of researches on helicobacter pylori vaccines are limited to animal experiments at present and cannot be used for clinical experiments due to antigen degradation, potential toxicity of vaccine adjuvants and the like. The development of a novel vector vaccine or a novel non-toxic adjuvant vaccine is therefore a great focus in the development of helicobacter pylori vaccines at the present time.
Disclosure of Invention
In order to solve the technical problems, the invention provides a recombinant adenovirus expressing helicobacter pylori protein, a preparation method and application thereof, and the recombinant adenovirus expressing helicobacter pylori protein can be used as a novel vector vaccine or a novel nontoxic adjuvant vaccine.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for producing a recombinant adenovirus expressing helicobacter pylori protein, the method comprising:
inserting helicobacter pylori genes into a shuttle plasmid vector through enzyme cutting sites KpnI and HindIII to obtain a recombinant adenovirus shuttle plasmid; wherein, the helicobacter pylori gene HP-Me is a gene fragment consisting of the combined dominant antigen epitope of five genes of NapA, ureA, ureB, hpaA, lpp;
the recombinant adenovirus shuttle plasmid and adenovirus skeleton plasmid are co-transferred into competent cells for recombination to obtain recombinant plasmids;
linearizing the recombinant plasmid with restriction enzyme PacI and transfecting into cells to package recombinant adenovirus, thereby obtaining recombinant adenovirus expressing helicobacter pylori protein.
Further, the coding gene sequence of the helicobacter pylori gene HP-Me is shown as SEQ ID NO: 1.
Further, the mass ratio of the recombinant adenovirus shuttle plasmid to the adenovirus backbone plasmid when co-transferred into competent cells is 9-11:1.
in a second aspect of the invention, the recombinant adenovirus expressing helicobacter pylori protein obtained by the method.
In a third aspect of the invention, there is provided the use of said recombinant adenovirus expressing helicobacter pylori protein in the preparation of a helicobacter pylori protective vaccine or a therapeutic vaccine.
In a third aspect of the invention, there is provided a protective or therapeutic vaccine against helicobacter pylori comprising the recombinant adenovirus expressing helicobacter pylori protein and an adjuvant.
Further, the vaccine is in a dosage form selected from one of a liquid preparation, a freeze-dried preparation, a capsule preparation, a tablet and a pill.
The invention can be used as vaccine to treat mucosal infection such as intramuscular injection, subcutaneous injection, nasal cavity, oral cavity, etc.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the recombinant adenovirus for expressing the helicobacter pylori protein is obtained by integrating the dominant helicobacter pylori antigen into an adenovirus genome for the first time, and the host is delivered by the recombinant adenovirus to induce the host to generate strong humoral and cellular immune response aiming at the helicobacter pylori antigen, so that the recombinant adenovirus can be used as a helicobacter pylori protective vaccine or a therapeutic vaccine, and can also be used for functional research of the helicobacter pylori protein and production of the helicobacter pylori protein by using cells as bioreactors. The gene fragment formed by combining dominant antigen epitopes of five genes of which the dominant antigen gene HP-Me is NapA, ureA, ureB, hpaA, lpp is creatively selected for the first time, and has good immunogenicity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows construction of a completely novel H.pylori gene-carrying adenovirus fragment by cloning H.pylori genes into a shuttle plasmid by engineering the shuttle plasmid fragment of the adenovirus packaging system.
FIG. 2 shows the content of specific IgG in serum after immunization of mice with recombinant adenovirus expressing H.pylori protein. Wherein, PBS group is a negative control group, ad5-wt is adenovirus skeleton plasmid Ad5 with wild type deleted E1 gene, ad5-HP is recombinant adenovirus expressing helicobacter pylori protein constructed in the embodiment of the invention, red bar graph is before immunization of mice, and blue bar graph is after immunization of mice;
FIG. 3 shows the content of specific IgA in gastric mucosa after immunization of mice with recombinant adenovirus expressing helicobacter pylori protein.
FIG. 4 shows the number of H.pylori colonies in the stomach of mice after immunization of mice with recombinant adenovirus expressing H.pylori protein.
FIG. 5 shows the condition of intragastric inflammation in mice after immunization with recombinant adenovirus expressing helicobacter pylori protein.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The effects of the present application will be described in detail with reference to examples and experimental data. Unless otherwise specified, the molecular cloning methods, protein expression purification methods, cell culture methods, various detection methods, and the like mentioned in the following schemes are all conventional experimental methods, and can be obtained by querying documents; the relevant reagents used may be purchased from the corresponding reagent suppliers.
EXAMPLE 1 construction of recombinant adenoviruses expressing helicobacter pylori proteins
1. Construction of recombinant adenovirus shuttle plasmid
As shown in FIG. 1, the HP-Me fragment (the coding gene sequence is shown as SEQ ID NO: 1) H.pylori gene fragment was ligated with a shuttle plasmid vector (purchased from Addgene, cat. 16403) through cleavage sites KpnI and HindIII to obtain a recombinant adenovirus shuttle plasmid.
2. Packaging of recombinant adenoviruses
The recombinant adenovirus shuttle plasmid obtained above was transformed into BJ5183 competent cells together with adenovirus backbone plasmid Ad5 (purchased from Addgene, cat# 16400) lacking the E1 gene for recombination. Colonies were picked from the plates and colonies containing successful recombination were identified. And (5) performing amplification culture on the positive colonies and extracting recombinant plasmids. Recombinant plasmids were linearized with the restriction enzyme PacI and transfected into HEK293 or 911 cells to package recombinant adenovirus. Cells were harvested 7-10 days after transfection with cell scraping. After centrifugation, the cells were resuspended in PBS. Cells were frozen in liquid nitrogen and lysed in a 37 ℃ water bath with vigorous shaking. This procedure was repeated a total of 4 times and after the end it was stored at-20 ℃.
3. Amplification of recombinant adenoviruses
293 cells were plated in cell culture dishes at a confluency of 70% and the virus-containing supernatant described above was added at a volume ratio of 30-50%. The cells were observed 2-3 days after infection, and generally a clear cell lysis or lesion phenomenon was seen. Viruses are collected when 50% of the cells leave the float, typically 3-5 days after infection. If the virus titer is insufficient, the above procedure may be repeated to amplify the virus.
4. Detection of specific antibody titer in mouse serum and gastric mucosa after recombinant adenovirus immunization
Helicobacter pylori protein is coated on ELISA plates to detect gastric tissue homogenates obtained from recombinant adenovirus immunized mice and helicobacter pylori specific antibodies in serum. FIG. 2 shows the serum specific IgG titer, 10% after immunization with recombinant adenovirus expressing helicobacter pylori protein constructed in the example of the invention 5 The method comprises the steps of carrying out a first treatment on the surface of the FIG. 3 shows the specific IgA titer in stomach tissue of 2 after immunization with recombinant adenovirus expressing helicobacter pylori protein constructed in the example of the invention 8 。
Example 2 use of live recombinant adenovirus as helicobacter pylori vaccine
SPF-grade BALB/c mice at 6 weeks of age were divided into three groups: control group 1, control group 2 and experimental group, 7 mice per group. Mouse nasal drip PBS in control group 1 and mouse nasal drip in control group 2 was infected with wild-type adenovirus. Mice in the experimental group were infected with recombinant adenovirus by nasal drops. The above procedure was repeated three weeks after infection as booster immunization. Two weeks after booster immunization, mice were infected with H.pylori. Mice were lavaged 4 times with helicobacter pylori over a period of one week. Four weeks after the completion of the stomach lavage, mice were bled and euthanized. The stomachs of mice were homogenized and serially diluted in broth culture and plated on Columbia blood agar plates for 5 days.
The results are shown in FIG. 4, and it is clear from FIG. 4 that the helicobacter pylori load in the stomach of mice immunized with the recombinant adenovirus is significantly reduced as compared with the control group.
As shown in fig. 5, the results of the detection of the inflammation in the stomach of the mice by HE staining the stomach tissue of the mice are shown in fig. 5, and compared with the control group, the lymphocyte infiltration level and the gastritis level in the stomach of the mice immunized by the recombinant adenovirus are obviously reduced.
In summary, it is known that recombinant adenoviruses expressing helicobacter pylori proteins induce in the host body a strong humoral and cellular immune response against helicobacter pylori antigens by the delivery host, and can be used as protective or therapeutic vaccines for helicobacter pylori, and also can be used for functional studies of helicobacter pylori proteins and for producing helicobacter pylori proteins by using cells as bioreactors.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. A method for producing a recombinant adenovirus expressing helicobacter pylori protein, the method comprising:
inserting helicobacter pylori genes into a shuttle plasmid vector through enzyme cutting sites KpnI and HindIII to obtain a recombinant adenovirus shuttle plasmid; wherein the helicobacter pylori gene is HP-Me, and a gene fragment consisting of the combined dominant antigen epitope of five genes of NapA, ureA, ureB, hpaA, lpp;
the recombinant adenovirus shuttle plasmid and adenovirus skeleton plasmid are co-transferred into competent cells for recombination to obtain recombinant plasmids;
linearizing the recombinant plasmid with restriction enzyme PacI and transfecting into cells to package recombinant adenovirus, thereby obtaining recombinant adenovirus expressing helicobacter pylori protein.
2. The method for preparing recombinant adenovirus expressing helicobacter pylori protein according to claim 1, wherein the nucleotide sequence of the helicobacter pylori gene is shown in SEQ ID NO: 1.
3. The method for preparing recombinant adenovirus expressing helicobacter pylori protein according to claim 1, wherein the mass ratio of the recombinant adenovirus shuttle plasmid to the adenovirus backbone plasmid when co-transferred into competent cells is 9-11:1.
4. a recombinant adenovirus expressing helicobacter pylori protein prepared by the method of any one of claims 1 to 3.
5. Use of the recombinant adenovirus expressing helicobacter pylori protein according to claim 4 for the preparation of a helicobacter pylori protective vaccine or a therapeutic vaccine.
6. A protective or therapeutic helicobacter pylori vaccine, characterized in that the vaccine comprises the recombinant adenovirus expressing helicobacter pylori protein according to claim 4 and an adjuvant.
7. The protective or therapeutic vaccine for helicobacter pylori according to claim 6, characterized in that the vaccine is in a dosage form selected from one of a liquid formulation, a lyophilized formulation, a capsule formulation, a tablet and a pill.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311706974.7A CN117867022A (en) | 2023-12-12 | 2023-12-12 | Recombinant adenovirus for expressing helicobacter pylori protein, and preparation method and application thereof |
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| CN202311706974.7A CN117867022A (en) | 2023-12-12 | 2023-12-12 | Recombinant adenovirus for expressing helicobacter pylori protein, and preparation method and application thereof |
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