A nucleic acid delivery vector, delivery system and use thereof

Abstract

The application discloses a nucleic acid delivery carrier, a delivery system and application thereof. The nucleic acid delivery carrier is constructed by inserting a CpG sequence into a multi-cloning site of a eukaryotic expression carrier, and the nucleic acid delivery system is constructed by the delivery carrier and probiotics. Based on the delivery carrier, the nucleic acid to be delivered is inserted into the delivery carrier and then transformed into probiotics to construct a recombinant bacteria preparation. The nucleic acid to be delivered can be delivered into the body by orally taking or injecting the recombinant bacteria preparation, which overcomes the deficiency that the nucleic acid is difficult to be efficiently delivered into the body by only using the recombinant expression carrier to transform the recombinant bacteria. The nucleic acid delivery carrier or the delivery system can be used to deliver the nucleic acid with therapeutic or immune effect into the body, and the nucleic acid delivery carrier or the delivery system can be used to prepare a medicine or an immune preparation for treating diseases by delivering the nucleic acid, and has wide application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. More specifically, it relates to a nucleic acid delivery vector, delivery system, and its applications. Background Technology

[0002] Compared to traditional protein biologics, DNA vaccines, DNA drugs, and small nucleic acid molecules related to immune regulation (such as cytokines) are DNA biopharmaceuticals that do not require complex purification methods. They offer advantages such as shorter production processes, lower costs, higher stability, and ease of storage and transportation, playing a crucial role in immunomodulation and disease treatment. However, biological barriers present in the mucosal system, such as endonucleases, mucolytic enzymes, and ciliated epithelium, can hinder the uptake and transfection of naked DNA plasmids by the body. DNA biopharmaceuticals must overcome multiple physiological obstacles to reach their target sites. This presents numerous difficulties and challenges for the practical application of DNA biopharmaceuticals. To improve the stability and delivery efficiency of DNA biopharmaceuticals, specific DNA delivery systems are necessary.

[0003] Ideally, a delivery carrier for DNA biopharmaceuticals should be able to carry one or more DNA products across cell membranes and physicochemical barriers, exhibiting targeted specificity and controllable drug release characteristics. Simultaneously, it should avoid adverse toxicity and immunogenicity of the carrier itself, or harmful chemical and enzymatic degradation of the loaded DNA biopharmaceutical. Delivery systems for DNA biopharmaceuticals mainly include live bacterial carriers, bacterial shadowing agents, liposomes, and microparticle sustained-release systems. Among these, the preparation process of bacterial shadowing agents is relatively cumbersome, while liposomes and microparticle sustained-release systems generally suffer from high preparation costs, making large-scale industrial production difficult. Considering both cost and benefit factors, live bacterial carriers are more advantageous as delivery carriers for DNA biopharmaceuticals in medical and even animal husbandry applications. Regarding the administration method of DNA delivery carriers, oral administration offers advantages over injection administration in terms of lower cost and ease of operation.

[0004] Currently, live microbial vectors used in the field of biology can be divided into two categories: one is in vivo flora (such as fecal microbiota) and microorganisms isolated from them (such as probiotics); the other is non-host intestinal native bacteria such as Escherichia coli Nissle 1917 (EcN), lactic acid bacteria, Salmonella, and yeast, which are suitable for engineering modification. However, probiotics have problems with insufficient targeting and precision. Fecal microbiota transplantation has achieved certain therapeutic effects in treating Clostridium difficile infection, but it is also accompanied by risks such as difficulty in standardization, high heterogeneity of effects, and unclear treatment mechanisms.

[0005] Furthermore, while EcN has been developed for delivering various drug protein molecules, current research on EcN delivery systems mainly focuses on delivering metabolites, proteins, and peptides from engineered bacteria. For example, patients with phenylketonuria (PKU) are unable to metabolize phenylalanine from their diet, and the accumulation of its toxicity can affect brain development. Isabella VM et al. modified EcN to express phenylalanine-metabolizing enzymes, converting the toxic metabolite phenylalanine into trans-cinnamic acid and phenylpyruvic acid. In other words, protein molecule delivery using EcN primarily relies on the EcN cell's own expression system to express the target protein or produce corresponding metabolites. Limited by the number of EcN cells used and the protein expression capacity of the bacteria, this method, which relies solely on the delivery bacteria's own expression system, results in relatively low protein yields. As for DNA delivery methods using EcN to deliver DNA into cells and then using human or animal recipient cells to express proteins, no relevant reports have been found. Furthermore, the experiment revealed that EcN cannot effectively deliver DNA into cells. When using EcN to carry plasmid vectors to deliver DNA, the plasmids are released after the bacteria die. A large amount of plasmid DNA is degraded by enzymes in the tissue fluid, and only a very small amount is randomly phagocytosed by the body's tissue cells. Therefore, the proportion of DNA delivered into cells is very low, failing to achieve the desired delivery effect. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention provides a nucleic acid delivery vector, delivery system, and its applications.

[0007] The first objective of this invention is to provide a nucleic acid delivery vector.

[0008] A second objective of this invention is to provide a nucleic acid delivery system.

[0009] A third object of the present invention is to provide the application of the vector, or the system, in the delivery of nucleic acids.

[0010] A fourth object of the present invention is to provide the use of the carrier, or the system, in the preparation of products that deliver nucleic acids.

[0011] A fifth object of the present invention is to provide the use of the carrier, or the system, in the preparation of a medicament for treating diseases by delivering nucleic acids.

[0012] A sixth object of the present invention is to provide the use of the carrier or the system in the preparation of immunomodulators or immunoadjuvants.

[0013] The seventh object of the present invention is to provide an immunomodulator.

[0014] An eighth object of the present invention is to provide the use of the CpG sequence shown in SEQ ID NO.1 in improving nucleic acid delivery efficiency or in the preparation of products for promoting nucleic acid delivery.

[0015] The above-mentioned objective of this invention is achieved through the following technical solution:

[0016] CpG sequences refer to a class of oligodeoxyribonucleotides with unmethylated cytosine and guanine nucleotides (CpG) as their core, which can interact with Toll-like receptor 9 (mammalians) or Toll-like receptor 21 (avian) in organismal cells. During the screening of CpG sequences, the inventors of this invention obtained a CpG sequence with strong binding ability to TLR9 / 21, as shown in SEQ ID NO.1.

[0017] To address the problem of low efficiency in delivering DNA into cells using EcN-carrying plasmid vectors, this invention discovers a method to construct a recombinant vector pVAX1-X-CpG by inserting the obtained CpG sequence into the multiple cloning site (MCS) of the plasmid vector pVAX1, along with the DNA to be delivered (denoted by "X"). This vector is then transformed into EcN to create a recombinant bacterial preparation. The DNA to be delivered is successfully and efficiently delivered into cells via oral or injectable administration of this recombinant bacterial preparation. In other words, this invention provides a nucleic acid delivery vector and delivery system capable of delivering nucleic acids into the body via injection or oral administration. Using the nucleic acid delivery vector and delivery system described in this invention, drugs or immunotherapeutic agents that treat diseases by delivering nucleic acids can be prepared. Therefore, this invention seeks protection for the nucleic acid delivery vector and system and their related applications.

[0018] The present invention provides a nucleic acid delivery vector, wherein the vector is a recombinant vector containing the CpG sequence shown in SEQ ID NO.1.

[0019] Specifically, the vector contains a eukaryotic promoter.

[0020] Specifically, the plasmid vector used to construct the recombinant vector is a eukaryotic expression plasmid vector.

[0021] Specifically, the eukaryotic expression plasmid vectors include, but are not limited to, pVAX1, pVAX2, pcDNA3.1, or pFAR4.

[0022] In a specific embodiment of the present invention, the plasmid vector used to construct the nucleic acid delivery vector is pVAX1, which is constructed by inserting the CpG sequence into the multiple cloning site of the plasmid vector pVAX1.

[0023] The present invention also provides a nucleic acid delivery system, wherein the system contains the nucleic acid delivery vector and probiotics that can be transformed by the nucleic acid delivery vector.

[0024] Optionally, the probiotics are intestinal probiotics.

[0025] Specifically, the probiotics include, but are not limited to, probiotic Escherichia coli, Lactobacillus, and Bacillus.

[0026] Specifically, the probiotic Escherichia coli includes strains EcN1917 and LH2018.

[0027] Specifically, the probiotic lactic acid bacteria include Lactobacillus plantarum.

[0028] Specifically, the probiotic Bacillus includes probiotic Bacillus subtilis.

[0029] The present invention also provides a method for delivering nucleic acid using the nucleic acid delivery vector or delivery system, the method comprising: inserting the nucleic acid to be delivered into the nucleic acid delivery vector and transforming it into probiotics to prepare a recombinant bacterial preparation, and delivering the obtained recombinant bacterial preparation into a recipient by oral administration or injection.

[0030] Specifically, the nucleic acid to be delivered is inserted into the vector via a multiple cloning site.

[0031] Specifically, the nucleic acid to be delivered is DNA.

[0032] Specifically, the recipient of the nucleic acid delivery vector or delivery system includes, but is not limited to, poultry or livestock, or the tissues or cells of poultry or livestock.

[0033] Specifically, the tissue includes the intestines.

[0034] Specifically, the cells include immune cells.

[0035] More specifically, the immune cells include dendritic cells (DCs), macrophages, and lymphocytes.

[0036] More specifically, the lymphocytes are B cells.

[0037] Given that the nucleic acid delivery vector or delivery system described in this invention can effectively deliver exogenous nucleic acids into the body's cells, this invention seeks protection for the use of the nucleic acid delivery vector or the nucleic acid delivery system in oral and / or injectable delivery of nucleic acids.

[0038] The present invention also claims protection for the use of the nucleic acid delivery vector or the nucleic acid delivery system in the preparation of products for oral and / or injectable delivery of nucleic acids.

[0039] Using the nucleic acid delivery vector or delivery system described in this invention, nucleic acids with therapeutic or immune-enhancing effects can be delivered into the body, thereby achieving therapeutic or immune-enhancing effects. Therefore, this invention also claims protection for the use of the nucleic acid delivery vector or the nucleic acid delivery system in the preparation of medicaments for treating diseases by delivering nucleic acids.

[0040] The present invention also claims protection for the use of the nucleic acid delivery vector or the nucleic acid delivery system in the preparation of immunomodulators or immunoadjuvants.

[0041] In a specific embodiment of the present invention, the nucleic acid delivery vector or the nucleic acid delivery system is used to deliver nucleic acid into the receptor via oral or injectable means.

[0042] Specifically, the recipient of the nucleic acid delivery vector or delivery system includes poultry or livestock, or the tissues or cells of poultry or livestock.

[0043] In a specific embodiment of the present invention, the present invention utilizes the nucleic acid delivery vector to transform probiotic Escherichia coli. Even without other exogenous DNA, the resulting recombinant bacteria also have the effect of stimulating the body's immunity and enhancing its immunity. That is, the present invention also provides an immunomodulatory agent comprising recombinant bacteria containing the nucleic acid delivery vector of the present invention.

[0044] Specifically, the recombinant bacteria are derived from probiotic Escherichia coli strains.

[0045] Specifically, the immune preparations described in this invention also contain nucleic acids that stimulate and enhance the body's immunity.

[0046] Specifically, the nucleic acid is a gene sequence.

[0047] Specifically, the genes include the Porin gene, duIFNγ gene, goIFNγ gene, poGP5 gene (porcine reproductive and respiratory syndrome virus GP5 gene), poPCV3 gene (porcine circovirus type 3 gene), and splafB gene (streptococcal universal antigen gene).

[0048] Optionally, the immunomodulator is an oral or injectable immunomodulator.

[0049] Given that the present invention improves the delivery efficiency of nucleic acids by inserting the CpG sequence shown in SEQ ID NO.1 into a plasmid vector, the present invention also claims protection for the use of the CpG sequence shown in SEQ ID NO.1 in improving nucleic acid delivery efficiency or in the preparation of products for promoting nucleic acid delivery.

[0050] The present invention has the following beneficial effects:

[0051] This invention constructs a delivery vector capable of efficiently delivering nucleic acids by inserting a CpG sequence into the multiple cloning site of a eukaryotic expression vector, and simultaneously constructs a nucleic acid delivery system composed of the delivery vector and probiotics. The nucleic acid delivery vector or delivery system constructed by this invention has the following advantages:

[0052] 1. High targeting specificity: The CpG sequence used in this invention can bind to TLR9 / TLR21 of the body's cells with high specificity. Probiotics carry a recombinant vector containing the CpG sequence and the nucleic acid to be delivered into the body. After being recognized by the body's cells, the DNA can be delivered into the cells. The body's cells express the delivered DNA, thereby exerting the effect of the delivered DNA. This process does not require the use of special instruments or devices to transfect DNA into the cells.

[0053] 2. Highly efficient delivery and expression: The nucleic acid delivery vector of the present invention contains corresponding expression elements of humans or recipient animals, which can be used to express the target protein efficiently using recipient animal cells, thereby achieving the purpose of efficiently delivering DNA biological products and more effectively exerting the immune effect of DNA products.

[0054] 3. Low cost and easy to scale: This invention uses intestinal probiotics for fermentation culture, which is inexpensive, easy to meet the culture conditions, and highly operable, making it very suitable for large-scale production. Furthermore, the nucleic acid delivery system described in this invention can be delivered orally in addition to injection, which is not only simple to operate but also further reduces costs. This characteristic makes it not only suitable for human medicine but also has significant advantages for large-scale application in animal husbandry.

[0055] 4. Enhanced Immunity: After the nucleic acid delivery vector of the present invention is transformed into probiotic Escherichia coli, the recombinant bacteria obtained by transformation, without the presence of other exogenous DNA, also have the effect of stimulating the body's immunity and enhancing the body's immunity. When the delivery vector also contains other immunomodulatory DNA, it can exert immunomodulatory effects at the same time. Attached Figure Description

[0056] Figure 1 This is a schematic diagram illustrating the construction of the nucleic acid delivery vector (a recombinant vector containing a CpG sequence and the DNA to be delivered) according to the present invention.

[0057] Figure 2This study investigated the expression activity of Glucose in mouse spleen cells after immunization with recombinant bacterial preparations of EcN, LH2018, EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG, and LH2018-Gluc-CpG at different time points following oral (OR) or intravenous (IM) administration. All samples were tested three times, and results are recorded as mean ± standard deviation. Significant differences between different treatments were indicated by "*", with ***p<0.001.

[0058] Figure 3 The results of BSA antibody IgG titers in animals were obtained after oral (OR) or intravenous (IM) administration of recombinant bacterial preparations of EcN, LH2018, BSA, EcN-CpG, LH2018-CpG, EcN-BSA-CpG, and LH2018-BSA-CpG. Figure A shows the BSA antibody IgG titer in immunized mice; Figure B shows the BSA antibody IgG titer in immunized chickens. All samples were tested three times, and results are recorded as mean ± standard deviation. Significant differences between treatments are indicated by "*", **p<0.01, ***p<0.001.

[0059] Figure 4 The results of serum IgG and intestinal mucus IgA antibody titers in ducks immunized with EcN, LH2018, commercially available duck inactivated vaccine (Vac), EcN-Porin-CpG, and LH2018-Porin-CpG recombinant bacterial preparations are shown in the figure. Figure A represents the serum IgG antibody titer test result; Figure B represents the intestinal mucus IgA antibody titer test result. All samples were tested three times, and the results are recorded as mean ± standard deviation. Significant differences between different treatments are indicated by "*", *p<0.05, **p<0.01, ***p<0.001.

[0060] Figure 5 The relative expression levels of IFN-γ and IL-4 mRNA, the IFN-γ / IL-4 ratio, and serum IgG titers in the spleens of ducklings immunized with PBS, Vac, EcN-CpG+Vac, and EcN-duIFNγ-CpG+Vac, respectively, were measured. Figure A shows the relative expression level of IFN-γ in the duckling spleen; Figure B shows the relative expression level of IL-4 mRNA in the duckling spleen; Figure C shows the IFN-γ / IL-4 ratio; and Figure D shows the serum IgG titer. All samples were tested three times, and the results are recorded as mean ± standard deviation. Significant differences between different treatments are indicated by "*", *p<0.05, **p<0.01.

[0061] Figure 6 The relative expression levels of IFN-γ and IL-4 mRNA, the IFN-γ / IL-4 ratio, and the intestinal mucus IgA titer in the intestines of ducklings immunized with PBS, Vac, EcN-CpG+Vac, LH2018-CpG+Vac, EcN-duIFNγ-CpG+Vac, and LH2018-duIFNγ-CpG+Vac, respectively, were measured. Figure A shows the relative expression level of IFN-γ in the duckling intestines; Figure B shows the relative expression level of IL-4 mRNA in the duckling intestines; Figure C shows the IFN-γ / IL-4 ratio; and Figure D shows the intestinal mucus IgA titer. All samples were tested three times, and the results are recorded as mean ± standard deviation. Significant differences between different treatments are indicated by "*", where *p<0.05, **p<0.01, and ***p<0.001.

[0062] Figure 7 The survival rate of ducklings immunized with PBS, Vac, EcN-CpG+Vac, LH2018-CpG+Vac, EcN-duIFNγ-CpG+Vac and LH2018-duIFNγ-CpG+Vac after infection with the virulent RA strain GD1904 was determined.

[0063] Figure 8 The mRNA expression levels of tight junction protein components (dense protein 1, closure protein, and peripheral membrane protein) and cytokine expression levels in the cecum of geese immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, respectively, were detected. Figure A shows the mRNA expression levels of these components in the goose cecum; Figure B shows the mRNA expression levels of cytokines IL2, IL6, IL10, and IL12b; Figure C shows the mRNA expression levels of cytokines IFN-α, IFN-β, IFN-γ, and IFN-κ. All samples were tested five times, and results are recorded as mean ± standard deviation. Significant differences between treatments are indicated by *, where *p < 0.05, **p < 0.01, and ***p < 0.001.

[0064] Figure 9The results of serum total IgY and anti-Salmonella IgY titers in geese immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG after Salmonella challenge were shown in the figure. Figure A shows the serum total IgY detection results, and Figure B shows the anti-Salmonella IgY titer detection results. All samples were tested 5 times, and the results are recorded as mean ± standard deviation. Significant differences between different treatments are indicated by "*", **p<0.01, ***p<0.001. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] In this embodiment of the invention, EcN is Escherichia coli Nissle 1917 strain, and LH2018 is a probiotic Escherichia coli strain screened from chicken intestines. This strain can colonize poultry intestines for more than 190 hours. All strains used are preserved in the microbiology laboratory of the College of Life Sciences, South China Agricultural University.

[0068] Example 1: Delivery of Glucose in Animals

[0069] This embodiment uses Gluc as a reporter gene and illustrates the construction, usage, and delivery effect of the nucleic acid delivery vector described in this invention by demonstrating the delivery of Gluc in mouse spleen cells. A schematic diagram of the construction of the nucleic acid delivery vector (a recombinant vector containing a CpG sequence and the DNA to be delivered) is shown below. Figure 1 As shown.

[0070] 1. Preparation of EcN-Gluc-CpG and LH2018-Gluc-CpG recombinant bacterial preparations

[0071] Using pVAX1 as a vector, the CpG sequence (shown in SEQ ID NO.1) and the Gluc gene sequence were recombined into the vector (recombined into the multiple cloning site of the vector) using a seamless cloning kit (Beyotime, #D7010S), resulting in the pVAX1-CpG recombinant vector containing the CpG sequence and the pVAX1-Gluc recombinant vector containing the Gluc gene sequence. Then, using pVAX1-CpG as a vector, the gene sequence encoding Gluc (Gluc, shown in SEQ ID NO.2) was recombined into the pVAX1-CpG vector using a seamless cloning kit (Beyotime, #D7010S), resulting in the pVAX1-Gluc-CpG recombinant plasmid containing the Gluc gene sequence. The pVAX1-Gluc and pVAX1-Gluc-CpG recombinant vectors were transformed into EcN and LH2018 strains, respectively, via heat shock transformation to obtain probiotic recombinant strains EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG, and LH2018-Gluc-CpG. The obtained probiotic recombinant strains were propagated using LB medium and cultured in a shaker at 37°C for 12 h. The bacterial cells were collected by centrifugation, washed with PBS, and resuspended to obtain the corresponding recombinant bacterial preparations.

[0072] 2. Detection of Gluc expression in mouse spleen cells

[0073] The pVAX1-Gluc-CpG recombinant vector is driven by a eukaryotic promoter and is not expressed in bacteria. Mice are given a recombinant probiotic preparation containing this recombinant vector orally or by injection. If the recombinant vector is successfully delivered into the mice and expressed, Gluc can be detected in the mouse spleen cells (because spleen cells are rich in B cells and macrophages, which highly express TLR9 and can be efficiently recognized by CpG).

[0074] The recombinant bacterial preparations of EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG, and LH2018-Gluc-CpG were respectively administered at 1×10⁻⁶. 7 Administer orally at a dose of CFU / animal, or at a dose of 1×10 5Mice (BALB / c mice) were immunized with CFU / mouse injections; oral or injectable raw EcN or LH2018 without any carriers served as negative controls. Mice were euthanized at 8h, 12h, and 24h post-immunization, and their spleens were aseptically harvested to prepare cell suspensions (see Wang et al., 2010). Glucose levels were then measured using the method described by Maguire et al. (2009). Add 100 μL of Gaussian luciferase reporter gene cell lysis buffer (Beyotime, #RG135M) to mouse spleen cell suspension, mix well by pipetting, and then lyse on a shaker at a moderate speed for 15 min to obtain cell lysis samples. Prepare Gaussian luciferase detection working solution by mixing the Gaussian luciferase detection substrate coelenterin and Gaussian luciferase detection buffer at a ratio of 1:10. Take 10 μL of cell lysis sample and add it to a 96-well plate pre-filled with 100 μL of Gaussian luciferase detection buffer. Add 10 μL of Gaussian luciferase detection working solution to each well, mix well, and immediately monitor three times continuously using a multi-functional microplate reader.

[0075] This invention, through oral (OR) or intravenous (IM) administration, analyzed the expression activity of Glucose in mouse spleen cells after immunizing mice with recombinant bacterial preparations of EcN, LH2018, EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG, and LH2018-Gluc-CpG at different time points. The results are as follows: Figure 2 As shown in the figure. The results showed that, compared with the control, the expression activity of Gluc in spleen cells of mice in the OR-EcN-Gluc-CpG, OR-LH2018-Gluc-CpG, IM-EcN-Gluc-CpG, or IM-LH2018-Gluc-CpG groups was significantly increased (p<0.001), indicating that the method described in this invention successfully delivered DNA into mice and achieved expression. Furthermore, Figure 2 This also demonstrates that, compared to injection, delivery of DNA via oral recombinant bacterial preparations can cross the intestinal tissue barrier and enter the peripheral immune organ (spleen) for expression.

[0076] Example 2: Delivery of the gene encoding the BSA protein in an animal.

[0077] Bovine serum albumin (BSA) is the most abundant protein in blood plasma, accounting for approximately 60% of total protein. It possesses the ability to bind various hydrophobic ligands, including fatty acids and tryptophan, and has been used as a model protein in many different biophysical, biochemical, and physicochemical studies. In this embodiment, a recombinant bacterium carrying a gene encoding the BSA protein was constructed. Recipient animals (mice and chickens) were immunized orally or by injection with the recombinant strain. The BSA antibody titer in the animals was then detected to investigate whether the BSA gene carried by pVAX1-BSA-CpG could be expressed in the recipient animal cells. If the BSA gene was successfully delivered to and expressed in the animals (mice and chickens), the BSA antibody (IgG) titer could be detected in the serum.

[0078] 1. Preparation of EcN-BSA-CpG and LH2018-BSA-CpG recombinant bacterial preparations

[0079] Using the pVAX1-CpG constructed in the examples as a vector, the BSA-encoding DNA sequence (Bovine Genebank ID: 280717) was inserted into the MCS of the vector using the same seamless cloning kit, resulting in the pVAX1-BSA-CpG plasmid containing the BSA sequence. The constructed recombinant plasmid was heat-shocked into EcN and LH2018 according to the aforementioned method (1 in Example 1) to prepare EcN-BSA-CpG and LH2018-BSA-CpG recombinant bacterial preparations. The pVAX1-CpG was heat-shocked into EcN and LH2018 using the same method to prepare EcN-CpG and LH2018-CpG recombinant bacterial preparations as controls.

[0080] 2. Detection of BSA antibody IgG titer in mice

[0081] Mice were immunized with recombinant bacterial preparations of EcN, LH2018, BSA, EcN-CpG, LH2018-CpG, EcN-BSA-CpG, and LH2018-BSA-CpG. The BSA group was administered via injection after emulsification with Freund's adjuvant at a dose of 50 μg per mouse; the other groups were administered via oral (OR) for 3 consecutive days (or via tail vein injection (IM) for 1 day), with an oral dose of 1 × 10⁻⁶. 7 CFU / animal (injection dose is 1×10) 5 CFU / animal); two immunizations were performed, 7 days apart; 7 days after the second immunization, the serum BSA antibody (IgG) titer was detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology), and the results were as follows. Figure 3As shown in Figure A. The results showed that, compared with the control, mice orally and by injection of recombinant bacterial preparations of EcN-BSA-CpG or LH2018-BSA-CpG (OR and IM) showed significant BSA antibodies in vivo, indicating that BSA was successfully delivered to mice and expressed.

[0082] 3. Detection of BSA antibody IgG titer in chickens

[0083] Chickens were immunized with recombinant strains of EcN, LH2018, BSA, EcN-CpG, LH2018-CpG, EcN-BSA-CpG, and LH2018-BSA-CpG. The BSA group was administered via injection after emulsification with Freund's adjuvant at a dose of 100 μg per bird; the other groups were administered orally for 3 consecutive days at a dose of 1 × 10⁻⁶. 8 CFU / feather; two immunizations, 7 days apart; 7 days after the second immunization, the serum BSA antibody (IgG) titer was detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology). The results are as follows. Figure 3 As shown in B in the figure. The results showed that, compared with the control, chickens orally administered EcN-BSA-CpG or LH2018-BSA-CpG recombinant bacterial preparations showed significant BSA antibodies in vivo, indicating that BSA was successfully delivered into the chickens and expressed.

[0084] Example 3: Delivery of the gene encoding the Porin protein in an animal.

[0085] *Riemerella anatipestifer* (RA) is a Gram-negative bacterium that causes anatipestifer infection in birds, including ducks and geese. It causes severe perihepatitis, pericarditis, and peritonitis, making it a major cause of economic losses in poultry farming, especially in ducklings. Outer membrane porin protein (Porin) is a membrane channel protein derived from RA. It possesses high immunogenicity and can induce host immune responses, participate in bacterial resistance, and activate complement. Studies have shown that the Porin gene is highly conserved and can be used for strain identification. It is an important antigenic component of the bacterial cell and can provide cross-immune protection for various strains. The good immunogenicity of Porin protein provides some experimental evidence for antigen-antibody reactions.

[0086] This invention constructs a recombinant bacterial preparation carrying the Porin gene. By orally immunizing ducks with this preparation, the success of the Porin gene delivery and expression in the duck can be investigated by detecting the Porin antibody titer in the animal. Since the duck genome does not contain the Porin gene and does not express Porin protein, the detection of Porin antibody (IgG and IgA) titers after oral administration of the recombinant bacterial preparation indicates that Porin has been successfully delivered to the animal (duck) and expressed.

[0087] 1. Preparation of EcN-Porin-CpG and LH2018-Porin-CpG recombinant bacterial preparations

[0088] Using the pVAX1-CpG constructed in Example 1 as a vector, the gene sequence encoding the Porin protein (Uniprot NO: E4TDA8) was inserted into the MCS of the vector using the same seamless cloning kit, resulting in the pVAX1-Porin-CpG plasmid containing the Porin gene sequence. The constructed recombinant plasmid was then heat-shocked into EcN and LH2018 according to the aforementioned method (1 in Example 1), and recombinant bacterial preparations of EcN-Porin-CpG and LH2018-Porin-CpG were prepared.

[0089] 2. Detection of Porin antibody (IgG and IgA) titers in ducklings

[0090] Ducklings (3-5 days old) were immunized with EcN, LH2018, commercially available inactivated duck disease vaccine (Vac, Shandong Qilu Animal Health Products Co., Ltd., China), EcN-Porin-CpG, and LH2018-Porin-CpG recombinant bacterial preparations, respectively. Vac was administered according to the instructions; the other groups were administered orally for 3 consecutive days at a dose of 1×10⁻⁶. 8 CFU / feather; immunized twice, 7 days apart; serum IgG and intestinal mucus IgA titers were detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology) at 7, 14, and 21 days after the second immunization. Results are as follows: Figure 4 As shown in the figure. The results showed that IgG was detected in duck serum and IgA was detected in intestinal mucosa after oral administration of EcN-Porin-CpG or LH2018-Porin-CpG recombinant bacterial preparations, indicating that the Porin gene was successfully delivered into the duck and expressed. The IgA antibody titer in intestinal mucosa after administration of LH2018-Porin-CpG was significantly higher than that after administration of Vac or EcN-Porin-CpG, possibly because LH2018 can colonize the intestine for more than 190 hours, resulting in better intestinal mucosal immunity.

[0091] Example 4: Immunomodulatory function of duIFNγ encoding gene DNA in ducklings

[0092] This embodiment constructed a recombinant bacterial preparation carrying the coding sequence of duck duIFNγ (Genebank ID: AF087134). This recombinant bacterial preparation was used as an oral adjuvant for the vaccine and co-immunized ducklings with Vac. The relative expression levels of IFN-γ and interleukin-4 (IL-4) mRNA in the spleen and intestine of ducklings were detected and their ratios were calculated. Serum IgG and intestinal mucus IgA antibody titers were measured, and a challenge experiment with the virulent RA strain GD1904 was conducted to explore the immunizing adjuvant effect of the EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG recombinant bacterial preparations in delivering IFNγ.

[0093] 1. Preparation of recombinant bacterial preparations of EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG

[0094] Using pVAX1-CpG as a vector, the duIFNγ-coding DNA sequence was inserted into the MCS of the vector using a seamless cloning kit to obtain the pVAX1-duIFNγ-CpG recombinant plasmid containing the duIFNγ-coding sequence. The constructed recombinant plasmid was heat-shocked into EcN and LH2018 according to the aforementioned method (1 in Example 1), and EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG recombinant bacterial preparations were prepared. pVAX1-CpG was heat-shocked into EcN and LH2018 using the same method, and EcN-CpG and LH2018-CpG recombinant bacterial preparations were prepared as controls.

[0095] 2. Detection of relative expression levels of IFN-γ and IL-4 mRNA in duckling spleen

[0096] Ducklings were immunized with PBS, Vac, EcN-CpG+Vac, and EcN-duIFNγ-CpG+Vac, respectively. Vac was administered according to the instructions; the other groups were administered orally for 3 consecutive days at a dose of 1×10⁻⁶. 8 CFU / feather; two immunizations, 7 days apart; challenge experiment using virulent RA strain GD1904, ducklings were subcutaneously injected with 1 mL of virulent RA strain GD1904 (7.5 × 10⁻⁶) on day 35. 6 CFU / mL); At 3 days after the second immunization and 7 days after challenge, qRT-PCR was used to detect the relative expression levels of IFN-γ and IL-4 mRNA in the spleen of ducklings in each group, and the IFN-γ / IL-4 ratio in the spleen was calculated. The results are as follows: Figure 5As shown in A to C. The results showed that 3 days after the second immunization and 7 days after challenge, the IFN-γ expression level of Vac combined with EcN-duIFNγ-CpG was significantly higher than that of Vac alone. Figure 5 The result (A) indicates that EcN-duIFNγ-CpG, as a vaccine adjuvant, can enhance the body's immune response. Seven days after challenge, the IFN-γ expression level in the EcN-duIFNγ-CpG+Vac group was significantly higher than that in the EcN-CpG+Vac group (…). Figure 5 The results (A) indicate that EcN-duIFNγ-CpG delivery of duIFNγ encoding gene DNA can effectively enhance the body's immune response. The IFN-γ / IL-4 ratio in the EcN-duIFNγ-CpG+Vac group was higher than that in the EcN-CpG+Vac group. Figure 5 The C in the figure indicates that EcN-duIFNγ-CpG, by delivering the duIFNγ gene, can act as a good vaccine adjuvant to modulate the type of immune response, making the immune response more inclined to Th1-type cellular immunity, which helps to clear infectious agents.

[0097] 3. Determination of serum IgG titer in ducklings

[0098] Ducklings were immunized with PBS, Vac, EcN-CpG+Vac, and EcN-duIFNγ-CpG+Vac, respectively, and challenged with the virus. The immunization and challenge methods were as described previously (Example 4, 2). Serum IgG titers were detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology) at 7 days, 14 days, and 7 days after the second immunization, respectively. The results are as follows: Figure 5 As shown in D in the figure. The results showed that at 14 days after the second immunization and 7 days after challenge, the serum IgG titer of Vac combined with EcN-duIFNγ-CpG was significantly higher than that of Vac alone. Figure 5 (D in the middle).

[0099] 4. Detection of relative expression levels of IFN-γ and IL-4 mRNA in the intestines of ducklings

[0100] Ducklings were immunized with PBS, Vac, EcN-CpG+Vac, LH2018-CpG+Vac, EcN-duIFNγ-CpG+Vac, and LH2018-duIFNγ-CpG+Vac, respectively, and challenged with the virus. The immunization and challenge methods were as described previously (Example 4, 2). At 3 days after the second immunization and 7 days after challenge, the relative mRNA expression levels of IFN-γ and IL-4 in the intestines of each group of ducklings were detected by qRT-PCR, and the IFN-γ / IL-4 ratio in the intestines was calculated. The results are as follows: Figure 5As shown in A to C. The results showed that IFN-γ expression levels were significantly higher in the group using Vac in combination with EcN-duIFNγ-CpG or LH2018-duIFNγ-CpG than in the group using Vac alone, both 3 days after the second immunization and 7 days after challenge. Figure 6 (A) indicates that EcN-duIFNγ-CpG, as a vaccine adjuvant, can enhance the body's immune response. At 3 days post-secondary immunization and 7 days post-challenge, the IFN-γ expression level in the EcN-duIFNγ-CpG+Vac group was significantly higher than that in the EcN-CpG+Vac group; the IFN-γ expression level in the LH2018-duIFNγ-CpG+Vac group was significantly higher than that in the LH2018-CpG+Vac group (A). Figure 6 A) indicates that EcN-duIFNγ-CpG or LH2018-duIFNγ-CpG delivering duIFNγ-encoding gene DNA can effectively enhance the body's immune response.

[0101] The IL-4 expression levels in the EcN-duIFNγ-CpG+Vac group or the LH2018-duIFNγ-CpG+Vac group were significantly lower than those in the Vac group. Figure 6 (B in the text). Furthermore, 3 days after the second immunization and 7 days after the challenge, the IFN-γ / IL-4 levels of EcN-duIFNγ-CpG or LH2018-duIFNγ-CpG combined with Vac were significantly higher than those in the Vac group ( Figure 6 B) indicates that EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG deliver the duIFNγ gene and can effectively regulate the type of immune response as a vaccine adjuvant, making the immune response more inclined to Th1-type cellular immunity, which helps to clear infectious agents.

[0102] Another Figure 6 As shown in C, the IFN-γ / IL-4 ratio in the EcN-duIFNγ-CpG+Vac group was higher than that in the EcN-CpG+Vac group; the IFN-γ / IL-4 ratio in the LH2018-duIFNγ-CpG+Vac group was higher than that in the LH2018-CpG+Vac group. This indicates that the duIFNγ gene delivered by EcN-duIFNγ-CpG, as a vaccine adjuvant, can regulate the type of immune response, making the immune response more inclined towards Th1-type cellular immunity, which helps to clear infectious agents.

[0103] 5. Determination of IgA titer in duckling intestinal mucus

[0104] Ducklings were immunized with PBS, Vac, EcN-CpG+Vac, LH2018-CpG+Vac, EcN-duIFNγ-CpG+Vac, and LH2018-duIFNγ-CpG+Vac, respectively, and challenged with the virus. The immunization and challenge methods were as described previously (Example 4, 2). Serum IgA titers were detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology) at 7 days, 14 days, and 7 days after the second immunization, respectively. The results are as follows: Figure 6 As shown in D in the diagram.

[0105] The results showed that at 7 days and 14 days after the second immunization and at 7 days after challenge, the intestinal mucus IgA titers in both the EcN-duIFNγ-CpG+Vac group and the LH2018-duIFNγ-CpG+Vac group were significantly higher than those in the Vac alone group. Figure 6 (D in the middle).

[0106] 6. Survival rate detection of ducklings challenged with the virulent RA strain GD1904

[0107] The virulent RA strain GD1904 was used for challenge experiments, and the challenge method was as described previously (Example 4, step 2). The survival rate was recorded 7 days after challenge, and the results are as follows: Figure 7 As shown in the figure. The results showed that the combined use of EcN-duIFNγ-CpG or LH2018-duIFNγ-CpG as vaccine adjuvants with Vac could improve the survival rate of ducklings, and the survival rate of ducklings using LH2018-duIFNγ-CpG as a vaccine adjuvant was higher than that of ducklings using EcN-duIFNγ-CpG.

[0108] The above results indicate that, compared with EcN-CpG and LH2018-CpG, EcN-duIFNγ-CpG and LH2018-goIFNγ-CpG, by delivering the duIFNγ gene, can be used as oral adjuvants for commercial duck disease vaccines, significantly enhancing the Vac immunization effect, regulating the type of immune response, making the immune response more inclined towards Th1-type cellular immunity, and contributing to the clearance of infectious agents.

[0109] Furthermore, since LH2018 has a better colonization effect in the gut than EcN, the duIFNγ gene adjuvant delivered by the LH2018-duIFNγ-CpG recombinant bacterial preparation is more effective than EcN-duIFNγ-CpG.

[0110] Example 5: Immunoprotective effect of goIFNγ encoding gene DNA against Salmonella in geese

[0111] This embodiment constructs a recombinant bacterial preparation carrying the coding sequence of goose goIFNγ (Genebank ID: AY524421.1). This recombinant bacterial preparation is used as an oral adjuvant for the vaccine and co-immunized goslings with Vac. The intestinal tissue damage score can be obtained by detecting the mRNA relative expression levels of tight junction protein components and cecal cytokines in the goose cecum, measuring serum total IgY, and measuring the anti-Salmonella IgY titer after challenge. The anti-Salmonella immunoprotective effect of IFNγ delivered by the EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations can be explored.

[0112] 1. Preparation of recombinant bacterial preparations of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG

[0113] Using pVAX1-CpG as a vector, the goIFNγ-coding DNA sequence was inserted into the MCS of the vector using a seamless cloning kit (Beyotime, #D7010S) to obtain the pVAX1-goIFNγ-CpG recombinant plasmid containing the goIFNγ-coding sequence. The constructed recombinant plasmid was heat-shocked into EcN and LH2018 according to the aforementioned method (Example 1, 1), and EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations were prepared. pVAX1-CpG was heat-shocked into EcN and LH2018 using the same method, and EcN-CpG and LH2018-CpG recombinant bacterial preparations were prepared as controls.

[0114] 2. mRNA expression levels of tight junction protein components CLDN1, OCLN, and ZO-1 in goose cecum

[0115] Geese were immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, respectively, orally for 3 consecutive days at a dose of 1×10⁻⁶. 8 CFU / feather; two immunizations, 7 days apart; 7 days after the second immunization, oral administration of virulent strain Salmonella (10) 11 CFU / feather was administered once daily for 4 consecutive days. The mRNA expression levels of the tight junction protein components Claudin 1 (CLDN1), Occludin (OCLN), and Zonulaoccludens-1 (ZO-1) in the goose cecum were then measured. The results are as follows: Figure 8As shown in Figure A. The results showed that the tight junction protein components OCLN and ZO-1 in the EcN-goIFNγ-CpG group were significantly higher than those in the EcN-CpG group; the tight junction protein components CLDN1, OCLN, and ZO-1 in the LH2018-goIFNγ-CpG group were significantly higher than those in the LH2018-CpG group, indicating that the recombinant bacterial preparations of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG significantly enhanced the intestinal barrier function by successfully delivering the goIFNγ encoding gene.

[0116] 3. Detection of mRNA expression levels of goose cecal cytokines

[0117] Geese were immunized with Salmonella using recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, respectively. After 4 days of continuous challenge, the mRNA expression levels of cecal cytokines in the geese were measured. The results are as follows: Figure 8 As shown in B and C in the figure. The immunization and challenge methods are the same as above (2 in Example 5). The results showed that IL2, IL6, IL10, IL12b, IFN-α, IFN-β, IFN-γ, and IFN-κ were all significantly upregulated in the EcN-goIFNγ-CpG group compared to the EcN-CpG group; IL2, IL6, IL10, IL12b, IFN-α, IFN-β, IFN-γ, and IFN-κ were all significantly upregulated in the LH2018-goIFNγ-CpG group compared to the LH2018-CpG group, indicating that the recombinant bacterial preparations of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG significantly upregulated cellular immunity by delivering the goIFNγ encoding gene.

[0118] 4. Goose intestinal lesion score

[0119] Geese were immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG and challenged with Salmonella. After 4 days of continuous challenge, the intestinal lesion score of the geese was calculated (intestinal lesion score (healthy = 0; low lesion = 1; moderate lesion = 2; severe lesion = 3; tissue necrosis = 4)). The immunization and challenge methods were the same as above (2 in Example 5). The results are shown in Table 1. Oral administration of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG to geese reduced the lesion score to 1 and 0, respectively, indicating that the recombinant bacterial preparations of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG can effectively protect the integrity of intestinal tissue and strengthen the immune barrier by delivering the goIFNγ encoding gene, and the recombinant bacterial preparation of LH2018-goIFNγ-CpG has a better protective effect on the intestine.

[0120] Table 1. Goose intestinal lesion scoring

[0121]

[0122] Geese were immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG and challenged with Salmonella. After 4 days of continuous challenge, the intestinal lesion scores of the geese were calculated (healthy = 0; low lesion = 1; moderate lesion = 2; severe lesion = 3; tissue necrosis = 4).

[0123] 5. Determination of total serum IgY in geese

[0124] Geese were immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, respectively, using the same immunization method as in Example 5, 2. Serum total IgY was analyzed by DNA dot blot at 7 days after the first and second immunizations, respectively. The results are as follows... Figure 9 As shown in A in the figure. The results showed no significant difference in serum total IgY among the groups.

[0125] 6. Determination of Salmonella IgY resistance in geese

[0126] Geese were immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, and then challenged with Salmonella, respectively. The immunization and challenge methods were the same as in Example 5, section 2. Anti-Salmonella IgY titers were detected using ELISA kits (Shanghai Enzyme-Linked Biotechnology) on days 7 and 14 post-challenge. The results are as follows: Figure 9As shown in Figure B. The results showed that 7 days after challenge, the anti-Salmonella IgY titers in both the EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG groups were low, but significantly increased after 14 days of challenge. This indicates that in the early stages of challenge, EcN and LH2018 significantly improved the intestinal barrier by delivering the goIFNγ gene, preventing the challenged strain from crossing the intestine, thus resulting in low antibody levels in the blood. As the challenge time increased, a small amount of the strain entered the bloodstream, inducing strong antibody immunity. This suggests that the recombinant strains EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG can effectively antagonize Salmonella and maintain infection control by delivering the goIFNγ encoding gene.

[0127] These results demonstrate that both EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG can successfully deliver the goIFNγ encoding gene DNA in geese. Compared with EcN-CpG and LH2018-CpG, EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG significantly upregulate cellular immunity, inhibit intestinal lesions, strengthen the immune barrier, and exert potent mucosal and systemic immune protection against Salmonella.

[0128] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid delivery vector, characterized in that, The vector is a recombinant vector containing the CpG sequence shown in SEQ ID NO.1; the plasmid vector used to construct the recombinant vector is a eukaryotic expression plasmid vector, and the recombinant vector is constructed by inserting the CpG sequence into the multiple cloning site of the eukaryotic expression plasmid vector.

2. A nucleic acid delivery system, characterized in that, The system contains the carrier of claim 1 and probiotics that can be transformed by the carrier of claim 1.

3. The system according to claim 2, characterized in that, The probiotics mentioned are intestinal probiotics.

4. The use of the carrier of claim 1, or the system of claim 2 or 3, in the preparation of products for oral and / or injectable delivery of nucleic acids.

5. The use of the carrier of claim 1, or the system of claim 2 or 3, in the preparation of a medicament for treating diseases by delivering nucleic acids.

6. The use of the carrier of claim 1, or the system of claim 2 or 3, in the preparation of immunomodulators or immunoadjuvants.

7. An immunomodulatory agent, characterized in that, The formulation comprises recombinant bacteria containing the vector of claim 1.

8. The use of the CpG sequence shown in SEQ ID NO.1 in the preparation of products for facilitating nucleic acid delivery.

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