Nucleic acid delivery carrier, delivery system and application thereof

Abstract

The invention discloses a nucleic acid delivery carrier, a delivery system and application thereof. According to the invention, a CpG sequence is inserted into multiple cloning sites of an eukaryotic expression vector, so that a delivery vector capable of efficiently delivering nucleic acid is constructed, and meanwhile, a nucleic acid delivery system consisting of the delivery vector and probiotics is constructed. On the basis of the delivery vector, nucleic acid to be delivered is inserted into the delivery vector and then transformed into probiotics to construct a recombinant bacterium preparation, namely the nucleic acid to be delivered can be delivered into a body in a manner of orally taking or injecting the recombinant bacterium preparation, so that the defects that only a recombinant expression vector is used for transforming recombinant bacteria, and the efficiency is high are overcome; and nucleic acid is difficult to efficiently deliver into a body. The nucleic acid delivery carrier or delivery system can be used for delivering the nucleic acid with the effect of treating or enhancing the body immunity into the body, that is, the nucleic acid delivery carrier or delivery system can be used for preparing drugs or immune preparations for treating diseases by delivering the nucleic acid, and is wide in application.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering. More specifically, it relates to a nucleic acid delivery vector, a delivery system and their applications. Background Art

[0002] Compared with traditional protein biologics, DNA biologics such as DNA vaccines, DNA drugs and immune regulatory related small nucleic acids (such as cytokines) do not require complex purification methods, and have the advantages of short production process, low cost, high stability and convenient storage and transportation, playing an important role in the fields of immune regulation and disease treatment. However, biological barriers such as endonucleases, mucolytic enzymes and ciliated epithelium existing in the mucosal system will hinder the uptake and transfection of naked DNA preparation plasmids by the body, and DNA biologics need to overcome multiple physiological barriers to reach the target. This makes DNA biologics face many difficulties and challenges in practical applications. To improve the stability and delivery efficiency of DNA biologics, a certain DNA delivery system is needed for delivery.

[0003] Ideally, the delivery vector of DNA biologics should be able to carry one or more DNA products through cell membranes and physical and chemical barriers, and exhibit targeted specificity and controllable drug release characteristics. At the same time, it is necessary to avoid the carrier itself having adverse toxicity and immunogenicity, or having harmful chemical and enzymatic degradation effects on the loaded DNA biologics. The delivery systems of DNA biologics mainly include: live bacterial vectors, bacterial ghosts, liposomes and microparticle sustained release systems, etc. Among them, the preparation operation process of bacterial ghosts is relatively cumbersome, and liposomes and microparticle sustained release systems generally have the problem of high preparation cost, and it is difficult to carry out large-scale industrial production. Considering the cost and benefit factors comprehensively, choosing a live bacterial vector as the delivery vector of DNA biologics has more advantages in medical and even livestock applications. For the administration method of the DNA delivery vector, compared with injection administration, the oral administration method has the advantages of lower cost and simpler operation.

[0004] At present, the live bacterial vectors applied in the biological field can be divided into two categories. One category is the in vivo flora (such as fecal bacteria) and the microorganisms (such as probiotics) screened and isolated therefrom. The other category is non-host intestinal native bacteria such as Escherichia coli Nissle 1917 (EcN), lactic acid bacteria, Salmonella and yeast that are suitable for genetic engineering modification. However, probiotics have problems of insufficient targeting and precision. Fecal microbiota transplantation has achieved certain therapeutic effects in the treatment of Clostridium difficile infection. However, it is also accompanied by risks such as great standardization difficulty, high effect heterogeneity and unclear treatment mechanism.

[0005] In addition, although EcN has been developed for delivering various drug protein molecules, the current research on the EcN delivery system mainly focuses on delivering the metabolites, proteins, and polypeptides of this engineered bacterium. For example, patients with phenylketonuria are unable to metabolize phenylalanine in their diet, and the accumulation of its toxicity will 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. That is, using EcN for protein molecule delivery mainly relies on the self-expression system of EcN cells to express the target protein or produce the corresponding metabolites. Limited by the number of administered EcN cells and the protein expression ability of this bacterium, the protein production is relatively low in this way of only relying on the self-expression system of the delivery bacterium to express proteins. There is no relevant report on the DNA delivery method that uses EcN to deliver DNA into cells and uses the body cells of human or animal receptors to express proteins. Moreover, it was found in the experiment that EcN cannot effectively deliver DNA into cells. When using EcN to carry plasmid vectors to deliver DNA, after the bacteria die, the plasmids in the bacteria are released, and a large amount of plasmid DNA will be degraded by the enzymes in the tissue fluid. Only a very small amount is randomly phagocytosed by the body tissue cells. Therefore, the proportion of DNA delivered into cells is very low and cannot achieve the delivery effect. Summary of the Invention

[0006] The present invention addresses the deficiencies in the above-mentioned prior art and provides a nucleic acid delivery vector, a delivery system, and their applications.

[0007] The first object of the present invention is to provide a nucleic acid delivery vector.

[0008] The second object of the present invention is to provide a nucleic acid delivery system.

[0009] The third object of the present invention is to provide the application of the above-mentioned vector or the above-mentioned system in delivering nucleic acids.

[0010] The fourth object of the present invention is to provide the application of the above-mentioned vector or the above-mentioned system in preparing a product for delivering nucleic acids.

[0011] The fifth object of the present invention is to provide the application of the above-mentioned vector or the above-mentioned system in preparing a drug for treating diseases by delivering nucleic acids.

[0012] The sixth object of the present invention is to provide the application of the above-mentioned vector or the above-mentioned system in preparing an immunizing agent or an immunoadjuvant.

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

[0014] The eighth object of the present invention is to provide the application of the CpG sequence shown in SEQ ID NO.1 in improving the nucleic acid delivery efficiency or in preparing a product for promoting nucleic acid delivery.

[0015] The above object of the present invention is achieved by the following technical solutions:

[0016] CpG sequences refer to a class of oligodeoxyribonucleotides with non-methylated cytosine and guanine nucleotides (CpG) as the core, and these sequences can interact with Toll-like receptor 9 (in mammals) or Toll-like receptor 21 (in birds) of the body cells. During the screening of CpG sequences by the inventors of the present invention, a CpG sequence with strong binding ability to TLR9 / 21 was obtained, as shown in SEQ ID NO.1.

[0017] Aiming at the problem of low efficiency of using EcN to carry plasmid vectors to deliver DNA into body cells, the present invention discovers that by inserting the obtained CpG sequence into the multiple cloning site (MCS) of plasmid vector pVAX1 and simultaneously inserting the DNA to be delivered (represented by "X"), a recombinant vector pVAX1-X-CpG is constructed, and then it is transformed into EcN to construct a recombinant bacterial preparation. By means of oral administration or injection of the recombinant bacterial preparation, the DNA to be delivered is successfully delivered into body cells with high efficiency. That is, the present invention provides a nucleic acid delivery vector and a delivery system that can deliver nucleic acids into the body by injection or oral administration. Using the nucleic acid delivery vector and delivery system of the present invention, drugs or immunological preparations for treating diseases by delivering nucleic acids can be prepared. Therefore, the present invention requests protection for the nucleic acid delivery vector and system and their related applications.

[0018] The present invention provides a nucleic acid delivery vector, and 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 vector includes but is 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 plasmid vector pVAX1.

[0023] The present invention also provides a nucleic acid delivery system, and 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 EcN1917 and LH2018 strains.

[0027] Specifically, the probiotic Lactobacillus includes Lactiplantibacillus 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, and the method is as follows: inserting the nucleic acid to be delivered into the nucleic acid delivery vector and transforming 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 through a multiple cloning site.

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

[0032] Specifically, the recipients of the nucleic acid delivery vector or delivery system include, but are not limited to, poultry or livestock, or tissues or cells of poultry or livestock.

[0033] Specifically, the tissue includes the intestine.

[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] In view of the fact that the nucleic acid delivery vector or delivery system of the present invention can effectively deliver exogenous nucleic acid into body cells. Therefore, the present invention claims the application of the nucleic acid delivery vector or the nucleic acid delivery system in oral and / or injection delivery of nucleic acid.

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

[0039] Using the nucleic acid delivery vector or delivery system of the present invention, nucleic acids with therapeutic or immune-enhancing effects on the body can be delivered into the body, thereby achieving effects such as treating or enhancing the body's immunity. Therefore, the present invention also claims the use of the nucleic acid delivery vector or the nucleic acid delivery system in the preparation of drugs for treating diseases by delivering nucleic acids.

[0040] The present invention also claims the use of the nucleic acid delivery vector or the nucleic acid delivery system in the preparation of immunological agents or immune adjuvants.

[0041] In a specific embodiment of the present invention, the nucleic acid is delivered into the recipient by oral or injection using the nucleic acid delivery vector or the nucleic acid delivery system.

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

[0043] In a specific embodiment of the present invention, the present invention uses the nucleic acid delivery vector to transform probiotic Escherichia coli. In the absence of other exogenous DNA, the transformed recombinant bacteria also have the effect of stimulating the body's immunity and enhancing the body's immunity. That is, the present invention also provides an immunological agent, and the agent includes recombinant bacteria containing the nucleic acid delivery vector of the present invention.

[0044] Specifically, the recombinant bacteria are based on probiotic Escherichia coli as the starting strain.

[0045] Specifically, the immunological agent of the present invention also contains nucleic acids that can stimulate and enhance the body's immunity.

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

[0047] [[ID=2,4]]Specifically, the genes include 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 (Streptococcus pneumoniae common antigen gene).

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

[0049] In view of the fact that the present invention improves the nucleic acid delivery effect by inserting the CpG sequence shown in SEQ ID NO.1 into the plasmid vector. Therefore, the present invention also claims the use of the CpG sequence shown in SEQ ID NO.1 in improving the 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] The present 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 the present invention has the following advantages:

[0052] 1. High targeting specificity: The CpG sequence used in the present invention can highly specifically bind to TLR9 / TLR21 of body cells. By using probiotics to carry a recombinant vector containing the CpG sequence and the nucleic acid to be delivered into the body, after being recognized by body cells, the DNA can be delivered into the cells, and the body cells are used to express the delivered DNA, thereby exerting the efficacy of the delivered DNA. This process does not require the aid of special instruments or devices to transfect DNA into cells.

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

[0054] 3. Low cost and easy to scale up: The present invention uses intestinal probiotics for fermentation and culture, with low cost, easy-to-meet culture conditions, strong operability, and is very suitable for large-scale production. In addition to injection delivery, the nucleic acid delivery system of the present invention can also be delivered orally, which is not only simple to operate but also further reduces costs. This characteristic makes it not only applicable to the field of human medicine but also has significant advantages in large-scale applications in animal husbandry.

[0055] 4. Immune enhancement: After the nucleic acid delivery vector of the present invention is transformed into probiotic Escherichia coli, in the absence of other exogenous DNA, the transformed recombinant bacteria also have the effect of stimulating the body's immunity and enhancing the body's immunity. When the delivery vector also contains other DNAs with immune regulatory effects, the immune regulatory effect can be simultaneously exerted. Brief Description of the Drawings

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

[0057] Figure 2Detection results of the expression activity of Gluc 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 by oral administration (OR) or intracaudal vein injection (IM) at different times; all samples were tested 3 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments were indicated by "*", ***p < 0.001.

[0058] Figure 3 Detection results of the BSA antibody IgG titer in animals after immunizing animals with recombinant bacterial preparations of EcN, LH2018, BSA, EcN-CpG, LH2018-CpG, EcN-BSA-CpG, and LH2018-BSA-CpG by oral administration (OR) or intracaudal vein injection (IM); A in the figure is the detection result of the BSA antibody IgG titer in immunized mice; B in the figure is the detection result of the BSA antibody IgG titer in immunized chickens; all samples were tested 3 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments were indicated by "*", **p < 0.01, ***p < 0.001.

[0059] Figure 4 Detection results of the serum IgG and intestinal mucus IgA antibody titers in ducks immunized with recombinant bacterial preparations of EcN, LH2018, commercially available inactivated vaccine against duck infectious serositis (Vac), EcN-Porin-CpG, and LH2018-Porin-CpG; A in the figure is the detection result of the serum IgG antibody titer; B in the figure is the detection result of the intestinal mucus IgA antibody titer; all samples were tested 3 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments were indicated by "*", *p < 0.05, **p < 0.01, ***p < 0.001.

[0060] Figure 5 Detection results of the relative mRNA expression levels of IFN-γ and IL-4, the ratio of IFN-γ / IL-4, and the serum IgG titer in the spleens of ducklings immunized with PBS, Vac, EcN-CpG+Vac, and EcN-duIFNγ-CpG+Vac; A in the figure is the detection result of the relative expression level of IFN-γ in the spleen of ducklings; B in the figure is the detection result of the relative mRNA expression level of IL-4 in the spleen of ducklings; C in the figure is the ratio of IFN-γ / IL-4; D in the figure is the detection result of the serum IgG titer; all samples were tested 3 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments were indicated by "*", *p < 0.05, **p < 0.01.

[0061] Figure 6 Detection results of relative mRNA expression levels of IFN-γ and IL-4, ratio of IFN-γ / IL-4, and 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; A in the figure shows the detection results of relative IFN-γ expression level in the duckling intestine; B in the figure shows the detection results of relative IL-4 mRNA expression level in the duckling intestine; C in the figure shows the ratio of IFN-γ / IL-4; D in the figure shows the detection results of intestinal mucus IgA titer; all samples were tested 3 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments are indicated by "*", *p<0.05, **p<0.01, ***p<0.001.

[0062] Figure 7 Survival rates of ducklings immunized with PBS, Vac, EcN-CpG+Vac, LH2018-CpG+Vac, EcN-duIFNγ-CpG+Vac, and LH2018-duIFNγ-CpG+Vac, respectively, after infection with RA virulent strain GD1904.

[0063] Figure 8 Detection results of mRNA expression levels of tight junction protein components occludin, claudin, and peripheral membrane protein, and mRNA expression levels of cytokines in the ceca of geese immunized with recombinant bacterial preparations of PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG, respectively; A in the figure shows the detection results of mRNA expression levels of tight junction protein components occludin, claudin, and peripheral membrane protein in the goose ceca; B in the figure shows the detection results of mRNA expression levels of cytokines IL2, IL6, IL10, and IL12b in the goose ceca; C in the figure shows the detection results of mRNA expression levels of cytokines IFN-α, IFN-β, IFN-γ, and IFN-κ in the goose ceca; all samples were tested 5 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments are indicated by "*", *p<0.05, **p<0.01, ***p<0.001.

[0064] Figure 9Detection results of total serum 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, respectively, after Salmonella challenge; A in the figure shows the detection results of total serum IgY; B in the figure shows the detection results of anti-Salmonella IgY titers; all samples were tested 5 times, and the results were recorded as mean ± standard deviation; significant differences between different treatments are indicated by "*", **p < 0.01, ***p < 0.001. Detailed implementation mode

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

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

[0067] EcN in the embodiments of the present invention is Escherichia coli Nissle 1917 strain, and LH2018 is a probiotic Escherichia coli strain screened from chicken intestines, which can colonize in avian intestines for more than 190 h. All the strains used are stored in the Microbiology Laboratory of the College of Life Sciences, South China Agricultural University.

[0068] Example 1 Delivery of Gluc in animals

[0069] In this example, Gluc is used as the reporter gene, and the delivery of Gluc in mouse spleen cells is used to illustrate the construction, usage method, and delivery effect of the nucleic acid delivery vector described in the present invention. Among them, the schematic diagram of the construction of the nucleic acid delivery vector (recombinant vector containing CpG sequence and DNA to be delivered) is as Figure 1 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 respectively recombined onto the vector (recombined into the multiple cloning site of the vector) using a seamless cloning kit (Beyotime, #D7010S), to obtain 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 onto the pVAX1-CpG vector using a seamless cloning kit (Beyotime, #D7010S), to obtain the pVAX1-Gluc-CpG recombinant plasmid containing the Gluc gene sequence. The pVAX1-Gluc recombinant vector and the pVAX1-Gluc-CpG recombinant vector were respectively transformed into EcN and LH2018 strains by heat shock transformation, to obtain the probiotic recombinant strains EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG and LH2018-Gluc-CpG. The obtained probiotic recombinant strains were propagated using LB medium, cultured in a shaker at 37 °C for 12 h, and the cells were collected by centrifugation, washed and resuspended with PBS solution 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 will not be expressed in bacteria. If the recombinant vector is successfully delivered into the mouse body and expressed after the mouse is orally administered or injected with the recombinant probiotic preparation containing the recombinant vector, Gluc can be detected in the mouse spleen cells (because spleen cells are rich in B cells and macrophages, and these cells 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 orally administered at a dose of 1×10 7 CFU / mouse, or at a dose of 1×10 5Mice (BALB / c mice) were immunized by injecting doses of CFU / mouse; the negative controls were oral or injected with the original EcN or LH2018 without any carrier. After oral or injection immunization, the mice were euthanized at 8 h, 12 h, and 24 h after immunization, and the spleens of the mice were aseptically removed and prepared into cell suspensions (the method is shown in Wang Lianrong et al., 2010), and then Gluc was detected by the method of Maguire et al. (2009). That is, 100 μL of Gauss luciferase reporter gene cell lysate (Beyotime, #RG135M) was added to the mouse spleen cell suspension, and after pipetting and mixing, it was shaken on a shaker at medium speed for 15 min to obtain a cell lysate sample; the Gauss luciferase detection substrate coelenterazine and the Gauss luciferase detection buffer were mixed at a ratio of 1:10 to prepare a Gauss luciferase detection working solution; 10 μL of the cell lysate sample was added to a 96-well plate pre-added with 100 μL of the Gauss luciferase detection buffer, and the Gauss luciferase detection working solution was added at a dose of 10 μL per well, mixed, and immediately continuously monitored three times using a multifunctional microplate reader.

[0075] In the present invention, after immunizing mice with different recombinant bacterial preparations of EcN, LH2018, EcN-Gluc, LH2018-Gluc, EcN-Gluc-CpG, and LH2018-Gluc-CpG by oral (OR) or injection (IM) at different times, the detection results of the expression activity of Gluc in mouse spleen cells are as Figure 2 shown. The results showed that compared with the control, the expression activity of Gluc in the spleen cells of the mice in the OR-EcN-Gluc-CpG, OR-LH2018-Gluc-CpG, IM-EcN-Gluc-CpG, or IM-LH2018-Gluc-CpG group was significantly increased (p < 0.001), indicating that DNA was successfully delivered into the mice and expressed using the method described in the present invention. In addition, Figure 2 it also showed that compared with injection, delivering DNA by oral administration of recombinant bacterial preparations could cross the intestinal tissue barrier and be expressed in the peripheral immune organ (spleen).

[0076] Example 2 Delivery of the gene encoding BSA protein in animals

[0077] Bovine serum albumin (BSA) is the most abundant protein in plasma, accounting for approximately 60% of the total protein. It has the property of binding various hydrophobic ligands such as fatty acids and tryptophan, and has been used as a model protein in many different biophysical, biochemical, and physicochemical studies. In this example, a recombinant bacterium carrying the gene encoding BSA protein was constructed. After immunizing recipient animals (mice and chickens) orally or by injection with the recombinant strain, the BSA antibody titer in the animal body was detected to explore whether the BSA gene carried by pVAX1-BSA-CpG could be expressed in recipient animal cells. If the BSA gene was successfully delivered into the animal body (mice and chickens) and expressed, the BSA antibody (IgG) titer in the serum could be detected in vivo.

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

[0079] Using the pVAX1-CpG constructed in the example as a vector, the coding DNA sequence of BSA (bovine Genebank ID: 280717) was inserted into the MCS of the vector using the same seamless cloning kit to obtain the pVAX1-BSA-CpG plasmid containing the BSA sequence. The constructed recombinant plasmid was heat-shock transformed into EcN and LH2018 according to the aforementioned method (1 in Example 1) to prepare EcN-BSA-CpG and LH2018-BSA-CpG recombinant bacterium preparations. pVAX1-CpG was heat-shock transformed into EcN and LH2018 in the same way to prepare EcN-CpG and LH2018-CpG recombinant bacterium preparations as controls.

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

[0081] Mice were immunized with EcN, LH2018, BSA, EcN-CpG, LH2018-CpG, EcN-BSA-CpG, and LH2018-BSA-CpG recombinant bacterium preparations respectively. The administration method for the BSA group was injection after emulsification with Freund's adjuvant, and the dose was 50 μg / animal; the administration methods for the other groups were continuous oral administration (OR) for 3 days (or tail vein injection (IM) for 1 day), and the oral dose was 1×10 7 CFU / animal (the injection dose was 1×10 5 CFU / animal); immunization was carried out twice with an interval of 7 days; 7 days after the second immunization, ELISA kits (Shanghai Enzyme-linked Biology) were used to detect the BSA antibody (IgG) titer in the serum, and the results were as Figure 3As shown in A of []. The results showed that compared with the control, significant BSA antibodies were detected in mice orally and injectably administered with EcN-BSA-CpG or LH2018-BSA-CpG (OR and IM) recombinant bacterial preparations, indicating that BSA was successfully delivered into the 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, respectively. The administration method for the BSA group was injection after emulsification with Freund's adjuvant, with a dose of 100 μg per chicken; the administration method for the remaining groups was continuous oral administration for 3 days, with a dose of 1×10 8 CFU per chicken; immunization was performed twice, with an interval of 7 days; 7 days after the second immunization, ELISA kits (Shanghai Enzyme-linked Biology) were used to detect the BSA antibody (IgG) titer in the serum, and the results were as Figure 3 shown in B of []. The results showed that compared with the control, significant BSA antibodies were detected in chickens orally administered with EcN-BSA-CpG or LH2018-BSA-CpG recombinant bacterial preparations, indicating that BSA was successfully delivered into the chickens and expressed.

[0084] Example 3 Delivery of the gene encoding Porin protein in animals

[0085] Riemerella anatipestifer (RA) is a Gram-negative bacterium that can cause avian Riemerella anatipestifer disease. It can cause severe perihepatitis, pericarditis, and peritonitis in poultry such as ducks and geese, and is one of the pathogenic bacteria that cause significant economic losses in poultry production and breeding, especially ducklings are most susceptible to infection. Outer membrane porin protein (Porin) is a membrane channel protein derived from RA, which has high immunogenicity and can also induce host immune responses, participate in bacterial drug resistance, and complement activation. Research has shown that the Porin gene is highly conserved, can be used for the identification of strains, is an important antigen component of the bacterial body, and can provide cross-immune protection for various types of strains. Porin protein has good immunogenicity, providing a certain experimental basis for the "antigen-antibody" reaction.

[0086] The present invention constructs a recombinant bacterial preparation carrying the Porin gene. The recombinant bacterial preparation is used to immunize ducks orally. By detecting the Porin antibody titer in the animal body, it can be explored whether the Porin gene carried by the recombinant bacterial preparation can be successfully delivered into the ducks and expressed. The duck genome does not have the Porin gene and does not express the Porin protein. After ducks orally take the recombinant bacterial preparation carrying the Porin gene, if the Porin antibody (IgG and IgA) titer is detected in the body, it indicates that Porin has been successfully delivered into the animal body (ducks) 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 the 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 to obtain the pVAX1-Porin-CpG plasmid containing the Porin gene sequence. According to the aforementioned method (1 in Example 1), the constructed recombinant plasmid was heat-shock transformed into EcN and LH2018, and EcN-Porin-CpG and LH2018-Porin-CpG recombinant bacterial preparations were prepared.

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

[0090] Ducklings (3 - 5 days old) were immunized with EcN, LH2018, a commercially available inactivated vaccine against duck plague (Commercial Inactive Vaccine, Vac, Qilu Animal Health Products Co., Ltd., Shandong, China), EcN-Porin-CpG and LH2018-Porin-CpG recombinant bacterial preparations respectively. Vac was administered according to the instructions; the administration method for the other groups was continuous oral administration for 3 days, with a dose of 1×10 8 CFU / duck; immunization was carried out twice, with an interval of 7 days; at 7 days, 14 days and 21 days after the second immunization, ELISA kits (Shanghai Enzyme-linked Biology) were used to detect the IgG titer in the serum and the IgA titer in the intestinal mucus. The results are as Figure 4 shown. The results showed that IgG was detected in the duck serum and IgA was detected in the intestinal mucosa after orally administering EcN-Porin-CpG or LH2018-Porin-CpG recombinant bacterial preparations, indicating that the Porin gene was successfully delivered into the ducks and expressed. The IgA antibody titer in the intestinal mucus after administering LH2018-Porin-CpG was significantly higher than that after administering Vac or EcN-Porin-CpG. The possible reason is that LH2018 can colonize in the intestine for more than 190 h, and the intestinal mucosal immune effect is better.

[0091] Immune Regulatory Function of the DNA Encoding Gene of duIFNγ in Ducklings

[0092] In this example, a recombinant bacterial preparation carrying the coding sequence of duck duIFNγ (Genebank ID: AF087134) was constructed. This recombinant bacterial preparation was used as an oral adjuvant for the vaccine and co-immunized with Vac in ducklings. By detecting the relative expression levels of IFN-γ and interleukin-4 (IL-4) mRNA in the spleens and intestines of ducklings and calculating their ratios, measuring the serum IgG and intestinal mucus IgA antibody titers, and conducting a challenge experiment with the virulent strain GD1904 of RA, the immune adjuvant effects of the EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG recombinant bacterial preparations in delivering IFNγ were explored.

[0093] 1. Preparation of EcN-duIFNγ-CpG and LH2018-duIFNγ-CpG Recombinant Bacterial Preparations

[0094] Using pVAX1-CpG as the vector, the duIFNγ-encoding DNA sequence was inserted into the MCS of the vector using a seamless cloning kit to obtain the recombinant plasmid pVAX1-duIFNγ-CpG containing the duIFNγ-encoding sequence. The constructed recombinant plasmid was heat-shock transformed 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-shock transformed into EcN and LH2018 in the same way, and EcN-CpG and LH2018-CpG recombinant bacterial preparations were prepared as controls.

[0095] 2. Detection of the Relative Expression Levels of IFN-γ and IL-4 mRNA in the Spleens of Ducklings

[0096] Ducklings were immunized with PBS, Vac, EcN-CpG+Vac, and EcN-duIFNγ-CpG+Vac, respectively. Vac was administered according to the instructions; the administration methods of the remaining groups were continuous oral administration for 3 days, with a dose of 1×10 8 CFU per feather; immunization was carried out twice, with an interval of 7 days; a challenge experiment was conducted using the virulent strain GD1904 of RA. Ducklings were subcutaneously injected with 1 mL of the virulent strain GD1904 of RA (7.5×10 6 CFU / mL) on the 35th day; at 3 days after the second immunization and 7 days after the challenge, qRT-PCR was used to detect the relative expression levels of IFN-γ and IL-4 mRNA in the spleens of ducklings in each group and calculate the ratio of IFN-γ / IL-4 in the spleen. The results are as follows in turn Figure 5as shown in A - C. The results showed that at 3 days after the second immunization and 7 days after virus challenge, the IFN - γ expression level when Vac was used in combination with EcN - duIFNγ - CpG was significantly higher than that when Vac was used alone ( Figure 5 in A), indicating that EcN - duIFNγ - CpG as a vaccine adjuvant could enhance the immune response of the body. At 7 days after virus 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 in A), indicating that EcN - duIFNγ - CpG delivering the duIFNγ - encoding gene DNA could effectively enhance the immune response of the body. The ratio of IFN - γ / IL - 4 in the EcN - duIFNγ - CpG + Vac group was higher than that in the EcN - CpG + Vac ( Figure 5 in C), indicating that by delivering the duIFNγ gene, EcN - duIFNγ - CpG could be used as a good vaccine adjuvant to regulate the type of immune response, making the immune response more inclined to Th1 - type cell immunity and contributing to the clearance of the infective agent.

[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 then challenged with the virus. The immunization and challenge methods were as described above (2 in Example 4). At 7 days and 14 days after the second immunization and 7 days after virus challenge, ELISA kits (Shanghai Enzyme - linked Biology) were used to detect the IgG titer in the serum. The results were as Figure 5 shown in D. The results showed that at 14 days after the second immunization and 7 days after virus challenge, the serum IgG titer when Vac was used in combination with EcN - duIFNγ - CpG was significantly higher than that when Vac was used alone ( Figure 5 in D).

[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 then challenged with the virus. The immunization and challenge methods were as described above (2 in Example 4). At 3 days after the second immunization and 7 days after virus challenge, qRT - PCR was used to detect the relative expression levels of IFN - γ and IL - 4 mRNA in the intestines of each group of ducklings and calculate the ratio of IFN - γ / IL - 4 in the intestines. The results were successively as Figure 5as shown in A - C. The results showed that on day 3 after the second immunization and day 7 after virus challenge, the IFN - γ expression levels when Vac was used in combination with EcN - duIFNγ - CpG or LH2018 - duIFNγ - CpG were significantly higher than those in the group using Vac alone ( Figure 6 in A), indicating that EcN - duIFNγ - CpG as a vaccine adjuvant could enhance the immune response of the body. On day 3 after the second immunization and day 7 after virus 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 ( Figure 6 in A), indicating that EcN - duIFNγ - CpG or LH2018 - duIFNγ - CpG delivering the duIFNγ - encoding gene DNA could effectively enhance the immune response of the body.

[0101] The IL - 4 expression levels in both the EcN - duIFNγ - CpG + Vac group and the LH2018 - duIFNγ - CpG + Vac group were significantly lower than those in the Vac group ( Figure 6 in B). And on day 3 after the second immunization and day 7 after virus challenge, the IFN - γ / IL - 4 ratios when EcN - duIFNγ - CpG or LH2018 - duIFNγ - CpG was used in combination with Vac were higher than those in the Vac group ( Figure 6 in B), indicating that EcN - duIFNγ - CpG and LH2018 - duIFNγ - CpG delivering the duIFNγ gene could effectively regulate the type of immune response as vaccine adjuvants, making the immune response more inclined to Th1 - type cellular immunity and contributing to the clearance of infective substances.

[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, indicating that the duIFNγ gene delivered by EcN - duIFNγ - CpG as a vaccine adjuvant could regulate the type of immune response, making the immune response more inclined to Th1 - type cellular immunity and contributing to the clearance of infective substances.

[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. The immunization and challenge methods were as described above (2 in Example 4). The titers of IgA in serum were detected using ELISA kits (Shanghai Enzyme-linked Biology) at 7 days and 14 days after the second immunization and 7 days after challenge. The results were as Figure 6 shown in D of

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

[0106] 6. Detection of the survival rate in the challenge experiment of ducklings with the RA virulent strain GD1904

[0107] A challenge experiment was carried out using the RA virulent strain GD1904. The challenge method was as described above (2 in Example 4). The survival rate at 7 days after challenge was recorded. The results were as Figure 7 shown. The results showed that both EcN-duIFNγ-CpG or LH2018-duIFNγ-CpG used as vaccine adjuvants in combination with Vac could improve the survival rate of ducklings, and the use of LH2018-duIFNγ-CpG as a vaccine adjuvant had a higher survival rate of ducklings than EcN-duIFNγ-CpG.

[0108] The above results indicated that compared with EcN-CpG and LH2018-CpG, EcN-duIFNγ-CpG and LH2018-goIFNγ-CpG could be used as oral adjuvants for commercial duck plague vaccines by delivering the duIFNγ gene, significantly enhancing the immune effect of Vac, regulating the type of immune response, making the immune response more inclined to Th1-type cell immunity, and contributing to the clearance of infective substances.

[0109] In addition, since LH2018 had a better intestinal colonization effect than EcN, the immune adjuvant effect of the duIFNγ gene delivered by the LH2018-duIFNγ-CpG recombinant bacterium preparation was stronger than that of EcN-duIFNγ-CpG.

[0110] Example 5 Immune protection of the goIFNγ-encoding gene DNA against Salmonella in geese

[0111] In this example, a recombinant bacterial preparation carrying the coding sequence of goose goIFNγ (Genebank ID: AY524421.1) was constructed. This recombinant bacterial preparation was used as an oral adjuvant for the vaccine and co-immunized with Vac in goslings. The relative mRNA expression levels of tight junction protein components and cecal cytokines in the goose cecum were detected, the intestinal tissue damage was scored, the total serum IgY was measured, and the IgY titer against Salmonella was measured after challenge to explore the anti-Salmonella immunoprotective effect of the EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations in delivering IFNγ.

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

[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-shock transformed into EcN and LH2018 according to the aforementioned method (1 in Example 1), and EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations were prepared. pVAX1-CpG was heat-shock transformed into EcN and LH2018 in the same way, 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 the goose cecum

[0115] Geese were immunized with PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG, and LH2018-goIFNγ-CpG recombinant bacterial preparations, respectively. The administration method was continuous oral administration for 3 days, and the dose was 1×10 8 CFU / bird; immunization was carried out twice with an interval of 7 days; 7 days after the second immunization, the virulent Salmonella strain (10 11 CFU / bird) was administered by gavage once a day. After continuous challenge for 4 days, the mRNA expression levels of tight junction protein components claudin 1 (CLDN1), occludin (OCLN), and zonula occludens-1 (ZO-1) in the goose cecum were detected. The results were as follows Figure 8As shown in A in []. 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 strengthened the intestinal barrier function by successfully delivering the goIFNγ-encoding gene.

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

[0117] Geese were immunized with PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations respectively and challenged with Salmonella. After continuous challenge for 4 days, the mRNA expression levels of cytokines in goose ceca were detected. The results were successively as Figure 8 shown in B and C in []. The immunization and challenge methods were 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 in the EcN-goIFNγ-CpG group were significantly up-regulated compared with those in the EcN-CpG group; IL2, IL6, IL10, IL12b, IFN-α, IFN-β, IFN-γ and IFN-κ in the LH2018-goIFNγ-CpG group were significantly up-regulated compared with those in the LH2018-CpG group, indicating that the recombinant bacterial preparations of EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG delivered the goIFNγ-encoding gene and significantly up-regulated the cellular immune level.

[0118] 4. Goose intestinal lesion score

[0119] Goose was immunized with PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations respectively, and challenged with Salmonella. After continuous challenge for 4 days, the intestinal lesions of geese were scored (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 could reduce the lesion score to 1 and 0 respectively, indicating that EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations could effectively protect the integrity of intestinal tissue and strengthen the immune barrier by delivering the goIFNγ-encoding gene, and the LH2018-goIFNγ-CpG recombinant bacterial preparation had a better intestinal protection effect.

[0120] Table 1 Intestinal lesion score of geese

[0121]

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

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

[0124] Goose was immunized with PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations respectively, and the immunization method was the same as above (2 in Example 5). Total serum IgY was analyzed by DNA dot blot at 7 days after the first immunization and 7 days after the second immunization respectively. The results are shown in Figure 9 A as shown. The results showed that there was no significant difference in total serum IgY among groups.

[0125] 6. Determination of anti-Salmonella IgY of geese

[0126] Goose was immunized with PBS, EcN-CpG, LH2018-CpG, EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant bacterial preparations respectively, and challenged with Salmonella. The immunization and challenge methods were the same as above (2 in Example 5). The anti-Salmonella IgY titer was detected using ELISA kits (Shanghai Enzyme-linked Biology) at 7 days and 14 days after challenge respectively. The results are shown in Figure 9As shown in B in []. The results showed that the IgY titers against Salmonella in the EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG groups were both low 7 days after challenge, but significantly increased 14 days after challenge. This indicates that in the early stage of challenge, EcN and LH2018 significantly improved the intestinal barrier by delivering the goIFNγ gene, preventing the challenged strains from crossing the intestine. Therefore, the antibody level in the blood was not high. As the challenge time extended, a small amount of the strains entered the blood, inducing a strong antibody immune level, suggesting that the EcN-goIFNγ-CpG and LH2018-goIFNγ-CpG recombinant strains can effectively antagonize Salmonella infection by delivering the goIFNγ-encoding gene.

[0127] The above results indicate 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 can significantly up-regulate the cellular immune level, inhibit intestinal lesions, strengthen the immune barrier, and exert a strong 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in 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.

2. The carrier according to claim 1, wherein The vector contains a eukaryotic promoter.

3. A nucleic acid delivery system, characterized in that, The system contains the vector described in claim 1 and probiotics that can be transformed by the vector described in claim 1.

4. The system according to claim 3, wherein The probiotics are intestinal probiotics.

5. Use of the vector according to claim 1 or 2, or the system according to claim 3 or 4 in oral and / or injectable delivery of nucleic acids.

6. Use of the vector according to claim 1 or 2, or the system according to claim 3 or 4 in the preparation of a product for oral and / or injectable delivery of nucleic acids.

7. Use of the vector according to claim 1 or 2, or the system according to claim 3 or 4 in the preparation of a drug for treating diseases by delivering nucleic acids.

8. Use of the vector according to claim 1 or 2, or the system according to claim 3 or 4 in the preparation of an immunopreparation or an immunoadjuvant.

9. An immune preparation, characterized in that, The preparation includes recombinant bacteria containing the vector described in claim 1 or 2.

10. Use of the CpG sequence shown in SEQ ID NO.1 in improving nucleic acid delivery efficiency or in the preparation of a product for promoting nucleic acid delivery.

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