阅读说明：本技术 人可溶性cd80融合基因转化乳酸菌及其应用 (Human soluble CD80 fusion gene transformed lactobacillus and application thereof ) 是由 曾位森 林子青 白杨 范宏英 孟晓静 于 2019-09-17 设计创作，主要内容包括：本发明公开了人可溶性CD80(hsCD80)融合基因转化乳酸菌,包括hsCD80基因或CTB-hsCD80融合基因转化乳酸菌,是将所述hsCD80基因或CTB-hsCD80融合基因插入到乳酸菌表达载体pLN并转化乳酸菌获得,还公开了该转化菌在制备具有肿瘤免疫治疗效果的口服活菌药物中的应用。本发明中hsCD80和CTB-hsCD80融合基因转化乳酸菌可制备成活菌悬液、乳制品、活菌菌粉、菌片或胶囊等活菌制剂,作为免疫治疗活菌药物口服后能在人体肠道内增殖和定植,应用于肿瘤、病毒感染或细胞内细菌感染、原发性和继发性免疫低下等疾病的预防与治疗。(The invention discloses a human soluble CD80(hsCD80) fusion gene transformed lactobacillus, which comprises an hsCD80 gene or CTB-hsCD80 fusion gene transformed lactobacillus, wherein the hsCD80 gene or CTB-hsCD80 fusion gene is inserted into a lactobacillus expression vector pLN and transformed lactobacillus to obtain the lactobacillus, and the invention also discloses the application of the transformed bacterium in the preparation of oral viable bacteria medicament with tumor immunotherapy effect. The fusion gene of the hsCD80 and the CTB-hsCD80 is transformed into lactic acid bacteria, which can be prepared into live bacteria preparations such as live bacteria suspension, dairy products, live bacteria powder, bacteria tablets or capsules, can be proliferated and colonized in human intestinal tracts after being taken as an immunotherapy live bacteria medicament, and is applied to the prevention and treatment of diseases such as tumors, virus infection or intracellular bacterial infection, primary and secondary low immunity and the like.)

1. The human soluble CD80 fusion gene transformed lactobacillus is characterized in that: the lactobacillus transformed by the hsCD80 gene or the CTB-hsCD80 fusion gene is obtained by inserting the hsCD80 gene or the CTB-hsCD80 fusion gene into a lactobacillus expression vector pLN and transforming lactobacillus, wherein the CTB-hsCD80 fusion gene is mainly formed by connecting an hsCD80 gene and a CTB gene, a cDNA sequence corresponding to a furin enzyme digestion polypeptide sequence is introduced between the hsCD80 gene and the CTB gene, and a secretory signal peptide SPK1 is introduced at the N end of an extracellular region structure domain of the hsCD80 gene.

2. The human soluble CD80 fusion gene transformed lactic acid bacterium of claim 1, wherein: the transformed lactic acid bacteria do not contain a drug-resistant gene, and can induce expression and secrete recombinant proteins hsCD80 and/or CTB-hsCD80 in intestinal lumens or in vitro.

3. The use of the human soluble CD80 fusion gene transformed lactic acid bacteria of claim 1 in the preparation of a live oral bacterial medicament with disease immunotherapy effect.

4. Use according to claim 3, characterized in that: the diseases are tumors, virus infection diseases, intracellular bacterial infection diseases, primary and secondary hypoimmunity diseases.

5. Use according to claim 4, characterized in that: the tumor is colorectal cancer or melanoma.

6. Use according to claim 3, characterized in that: the oral viable bacteria medicament is viable bacteria suspension, dairy product, viable bacteria powder, bacteria tablet or capsule.

Technical Field

The invention belongs to the technical field of human soluble CD80(hsCD80 for short) genes, and particularly relates to human soluble CD80 fusion gene transformed lactobacillus and application thereof.

Background

In recent years, immunotherapy targeting immune checkpoint (checkpoint) molecules is the hot spot of current anti-tumor research, and various immune checkpoint inhibitor drugs, such as PD-1 and PD-L1 humanized antibodies, have been approved for clinical application and have achieved good therapeutic effects.

The main reason why malignant tumors can evade immune surveillance is that both malignant tumor cells and immune cells express some immune co-suppression molecules (co-suppression molecules), also called immune checkpoint molecules, to generate immune suppression and evade immune surveillance of the body. This is also a major obstacle to breakthrough for tumor therapy. B7-H1(CD274) and B7-DC (CD273) are two important immunosuppressive molecules, both of which are homologous molecules of CD80(B7.1), and can be combined with receptor programmed death factor 1 (PD-1) on T cells to inhibit generation of antitumor immunity and inhibit killing of tumors by effector T cells (Te). Thus, both are also referred to as PD-1 ligand 1(PD-L1) and ligand 2(PD-L2), respectively. PD-L1 is widely expressed in various malignant tumor cells and is up-regulated by cytokines such as interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). PD-L2 is mainly expressed in DC, macrophage, mast cell, etc. Both PD-L1 and PD-L2 bind to the receptors PD-1 or CD80 on T cells. On one hand, the activation of T cells is inhibited, and the apoptosis of the T cells is induced; on the other hand, the method inhibits the cytotoxic effect of Te cells on tumor cells. In recent years, targeted therapy of immunosuppressive molecules such as PD-1 and its ligands PD-L1, PD-L2, and CTLA-4 has become a hotspot of research on antitumor therapy. The humanized antibodies or inhibitors of PD-1, PD-L1, PD-L2 and CTLA-4 have favorable curative effect in clinical treatment tests of malignant tumors such as breast cancer, colon cancer and lung cancer, the humanized antibodies of PD-1, namely nivolumab and pembrolizumab and the humanized antibodies of PD-L1, namely atezolizumab, have been approved by FDA in Japan, America, Australia and other countries, and are applied to the treatment of malignant tumors.

In addition to PD-1, immune cells, upon activation, express a number of checkpoint molecules, such as cytotoxic T cell antigen 4(CTLA-4), TIM3, LAG-3, and the like. These checkpoint molecules prevent over-and autoimmunity from occurring during the course of the immune response through negative feedback regulation mechanisms. CTLA-4 is a negative regulatory receptor for CD80, occurring primarily in activated T cells, regulatory T cells (tregs), and memory T cells (Tm). The combination of CD80 and CTLA-4 inhibits the activation of helper T cells (Th) and Tm cells, stimulates the proliferation of Treg cells and induces the generation of immune tolerance. The CTLA-4 antibody ipilimumab was the first checkpoint antibody drug approved for the market, primarily for metastatic melanoma treatment. TIM3 and LAG-3 are expressed predominantly in activated CD4⁺And CD8⁺T cells, inhibiting the activation of Th1 and the killing effect of Te. Inhibitors or antibodies to TIM3 and LAG-3 may also enhance the killing of Te against tumor cells.

However, immune checkpoint inhibitors have less clinical applicability. Only less than one third of tumor patients have definite curative effect after being treated by the PD-1 antibody, and most tumor patients have poor or ineffective curative effect after being taken. This is because checkpoint inhibitors act primarily by blocking immunosuppressive pathways, increasing the anti-tumor effect of already activated immune cells of the body, while having less efficacy in stimulating active anti-tumor immunity. For example, the PD-1 antibody can only improve the immune effect of Te on tumor cells, and the PD-L1 antibody only has better curative effect on PD-L1 positive tumors. However, immune cells and malignant tumors usually express a plurality of different immunosuppressive molecules, and the expression level of the immunosuppressive molecules of different individuals is greatly different, and not all patients can generate more anti-tumor Te cells by themselves. Therefore, in order to improve the anti-tumor effect of the immunotherapy drug, the anti-tumor active immunity of the organism must be firstly stimulated, or the active immunity drug and the checkpoint inhibitor are jointly used, so that the better effect can be obtained.

CD80 is an important immune co-stimulatory molecule (CSMs) that plays an important role in immunity against tumors and viral infections. Because of the immunosuppressive mechanism of malignant tumor, an immune co-stimulatory molecule is required to participate in the mechanism to stimulate the body to generate effective anti-tumor immune response. CD80 is the most important immune co-stimulatory molecule, first found in activated B lymphocytes, designated B7.1. Later studies found that CD80 was widely distributed among Antigen Presenting Cells (APC), Dendritic Cells (DC), T cells, B cells and myeloid cells. Human CD80 is a type i transmembrane protein consisting of 288 amino acid residues (aa), with the gene located in the long arm of chromosome 3 (3q 3.1). Wherein 1-34aa is a signal peptide, 35-242 is an extracellular structure, and comprises two immunoglobulin-like structural regions. 243-263aa is a transmembrane helix structure, 264-288 is an intracellular structure region. Mouse CD80 consists of 306 aa, has high homology with human CD80 amino acid sequence, and has similar structural characteristics and biological effect.

CD80 is known to have two major receptors, one is CD28 and the other is CTLA-4. CD28 is constitutively expressed on T cells. CD80 stimulates the proliferation and activation of naive T cells by binding to CD28, differentiating them into class I helper T cells (Th1), and secreting interleukin 2(IL-2), promoting cellular immunity. The binding of CD80-CD28 in turn also stimulates the secretion of IL-6 by APC cells, further stimulating T cell proliferation and activation. CTLA-4 is a negative regulatory receptor, mainly found in Te, Th1, Treg and Tm cells. The combination of CD80 and CTLA-4 inhibits the activation of Th and Tm cells, stimulates the proliferation of Treg cells and induces the generation of immune tolerance.

CD80 has the effect of activating and modulating Tm, Te and Treg. After the CD80 gene is knocked out, the immunity of the mice is obviously reduced. After lymphocyte choriomeningitis virus (LCMV) stimulation, the numbers of Th1, Treg, Tm and Te cells in peripheral blood and spleen were all reduced.

The soluble CD80(sCD80 for short) only containing the extracellular domain can activate cellular immune response and prevent the action of immunosuppressive molecules, shows very good anti-tumor effect and is expected to become a novel immunotherapy medicament with high efficiency and wide application range. CD80 has been used mainly as an immunoadjuvant molecule for preparing tumor vaccines or pathogenic microorganism vaccines by transfecting cells or coexpressing with antigens. Human soluble CD80(hsCD80) gene is transfected into human melanoma cell, and can overcome the immunosuppression caused by PD-L1, induce immune response and inhibit the growth and metastasis of human melanoma ectopic tumor inoculated to subcutaneous mouse.

Recent studies have shown that soluble CD80(sCD80) in mice can significantly enhance the anti-tumor immune response of peripheral blood lymphocytes in mice and humans. After sCD80-Fc is injected, the number of activated lymphocytes, the number of effector lymphocytes and the secretion of IFN-gamma of tumor-bearing mice are obviously increased, and the survival time is greatly prolonged. sCD80 can exert anti-tumor effects through multiple pathways: enhancing specific anti-tumor immune response through interaction with CD28 on a T cell membrane; ② by reducing the expression of PD-L1 and competitive combination with PD-L1, the combination of immunosuppressive molecules PD-L1 and PD-L2 and receptor PD-1 is blocked, and the anti-tumor immune killing effect of Te is enhanced; and the CD80 can be combined with other activated check point molecules such as CTLA-4, TIM3 and LAG-3, and the like to overcome immune tolerance and stimulate anti-tumor immune response. CD80 stimulates the differentiation of T lymphocytes from CD28 knockout mice. The comparative research results show that the anti-tumor curative effect of the soluble CD80 is obviously better than that of anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies, the application range is wider, the anti-tumor drug is suitable for treating various malignant tumors, and the anti-tumor drug shows a very good anti-tumor therapeutic prospect.

However, the existing immunotherapy drugs have the inherent disadvantages of high production cost and inconvenient administration besides small application range, and limit the clinical popularization and application of the existing immunotherapy drugs. The traditional biological medicines (including antibodies) can not be produced by separating and purifying proteins, and must be administered by injection, so that the two inherent disadvantages of high production cost and inconvenient administration exist. If the conventional production method is adopted to prepare the sCD80, the problems of high production cost and inconvenience in medication are still existed, which are difficult to overcome. Therefore, there is a need to develop new expression production modes and administration routes of sCD 80.

The gastrointestinal tract is the simplest and most convenient and economic bioreactor for generating and transforming biological medicines, and probiotics such as lactic acid bacteria and the like are good carriers for oral administration of immunotherapy medicines. The gastrointestinal tract of the human body is a complex and dynamic ' micro-ecological world ' and is also a high-efficiency ' factory ' for biochemical metabolism '. It is estimated that the total number of bacteria in the intestine of an adult exceeds 10¹⁴The total amount of biochemical metabolism corresponds to the total amount of metabolism of the liver. If the "factory" of the gastrointestinal tract could be effectively utilized as a bioreactor for drug production and transformation, the advantages of economy, convenience and high efficiency would no doubt be provided, and a new approach for disease prevention and treatment would be provided.

Lactic acid bacteria and bifidobacteria are the most suitable and safer host bacteria for the intestinal bioreactor. The probiotics such as lactobacillus and bifidobacterium grow in oral cavity, vagina and digestive tract of human body for a long time, especially anoxic parts such as lower digestive tract (ileum, colon) and the like, and the human body even takes the probiotics as a part of the human body, so that immunological rejection reaction is not generated basically. The probiotics have the functions of improving the intestinal micro-ecological environment, inhibiting the growth of pathogenic bacteria, promoting nutrient absorption, decomposing toxic products, regulating immunity, preventing tumorigenesis and the like. The lactobacillus and the bifidobacterium have long residence time and large quantity in the lower digestive tract, have accumulation effect, are beneficial to the expression, absorption and utilization of peptide drugs, and are very suitable to be used as carriers for the expression and administration of the polypeptide drugs in the intestinal tract.

In recent years, the use of genetically engineered probiotics for oral vaccine preparation or gene therapy has become a research hotspot. The probiotics can be used for expressing rotavirus antigens, tetanus toxin C and other antigens to prepare oral vaccines or expressing Cytosine Deaminase (CD) genes to carry out gene therapy of malignant tumors. The use of probiotics such as lactobacillus to express immune factors such as interferon, thymosin and the like obtains positive curative effect in the treatment research of animal models with diseases such as virus infection, malignant tumor and the like. Phase II clinical trials have been completed with studies on the treatment of enteritis with lactococcus expressing interleukin 10 (IL-10).

The lactobacillus is used for expressing human soluble CD80(hsCD80 for short), although the two problems of high production cost and inconvenient medication can be overcome. But are limited to the treatment of gastrointestinal malignancies. In order to make the lactobacillus-expressed human sCD80 exert the systemic anti-tumor immunity effect, the problem that the macromolecule sCD80 expressed by lactobacillus in intestinal tract passes through the intestinal mucosa barrier and enters the blood circulation must be solved.

The expression of the fusion with cholera toxin subunit B (CTB) is an effective way to promote the crossing of intestinal mucosa barrier by macromolecular drugs. CTB is a regulatory subunit recognized and bound by cholera toxin and intestinal epithelial cells. Cholera toxin consists of 1 a subunit (CTA) and 5B subunits (CTB). CTB binds specifically to the receptor on intestinal epithelial cells, ganglioside M1(GM1), without enterotoxicity. Intestinal epithelial cells are rich in GM 1. Fusion expression with CTB is an effective method for recombinant proteins to cross the intestinal mucosal barrier.

Researches show that the efficiency of GFP and IFN-alpha absorbed through intestinal mucosa and entering blood circulation after the tobacco leaves are orally taken can be remarkably improved by carrying out fusion expression on Green Fluorescent Protein (GFP) or IFN-alpha and CTB in the tobacco. A certain amount of GFP can be detected in tissues such as submucosa, inferior vena cava, liver, spleen and the like. Adding a special protease enzyme digestion sequence between the GFP and the CTB, carrying out enzyme digestion after the fusion protein enters the intestinal epithelial cells, and dissociating the GFP and the CTB. CTB was detected only in the epithelial cells, while GFP was detected only in the tissues of inferior vena cava, liver, spleen, etc. The fusion expression of the target protein and CTB is expected to break through the intestinal mucosa barrier.

Disclosure of Invention

The invention aims to provide human soluble CD80(hsCD80 for short) fusion gene transformed lactobacillus, which can be prepared into oral dosage forms, does not carry antibiotics or drug resistance genes, can be induced and expressed in vivo or in vitro, particularly in intestinal tracts by an inducer Nisin (lactoglobulin), and is used for preventing and treating various diseases such as tumors and the like.

The invention also aims to provide the application of the human soluble CD80 fusion gene transformed lactobacillus in preparing oral viable bacteria medicaments with tumor immunotherapy effects.

The first object of the present invention can be achieved by the following technical solutions: the human soluble CD80 fusion gene transformed lactobacillus comprises an hsCD80 gene or CTB-hsCD80 fusion gene transformed lactobacillus, which is obtained by inserting the hsCD80 gene or CTB-hsCD80 fusion gene into a lactobacillus expression vector pLN and transforming lactobacillus, wherein the CTB-hsCD80 fusion gene is mainly formed by connecting an hsCD80 gene and a CTB gene, a cDNA sequence corresponding to a furin enzyme digestion polypeptide sequence is introduced between the hsCD80 gene and the CTB gene, and a secretory signal peptide SPK1 is introduced at the N end of an extracellular region structure of the hsCD80 gene.

Optionally, the transformed lactic acid bacteria do not contain a drug resistance gene, which can induce expression and secretion of recombinant proteins hsCD80 and/or CTB-hsCD80 in the intestinal lumen or in vitro.

The hsCD80 or CTB-hsCD80 fusion gene transformed lactobacillus can be expressed in intestinal tract, and can directly act in the intestinal tract to prevent and treat intestinal tumor.

The CTB-hsCD80 fusion gene transformed lactic acid bacteria expressed CTB-hsCD80 fusion protein can be endocytosed by intestinal epithelial cells through the endocytosis mediated by a CTB receptor, and is specifically digested by furin, and free hsCD80 with immunological activity is released to the outside of cells or blood circulation, so that the intestinal mucosal barrier is broken through, lymphocyte activation is stimulated, the cellular immunity level of an organism is improved, and the immunosuppressive pathways of PD-1/PD-L1 and the like are blocked.

The preparation method of the human soluble CD80 fusion gene transformed lactobacillus can comprise the following steps:

(1) firstly, synthesizing hsCD80 and CTB-hsCD80 fusion genes, and introducing a cDNA sequence corresponding to a furin enzyme digestion polypeptide sequence between an hsCD80 gene and a CTB gene in a CTB-hsCD80 fusion gene;

(2) inserting the hsCD80 and/or CTB-hsCD80 fusion gene into a lactobacillus expression vector pLN, transforming the recombinant hsCD80 and CTB-hsCD80 fusion gene expression vector into LacF gene-deleted lactobacillus by an electrotransformation method, and screening the hsCD80 and CTB-hsCD80 fusion gene transformed lactobacillus by adopting a selective medium taking lactose as a unique carbon source.

In the preparation method of the human soluble CD80 fusion gene transformed lactobacillus:

optionally, the lactobacillus expression vector pLN in step (2) is a plasmid vector containing a LacF nutrition screening gene, containing no antibiotic resistance gene, and capable of inducing the expression of a target gene in vitro or in the intestinal lumen by using an inducer nisin (lactocin).

Optionally, the lactic acid bacteria in step (3) are lactic acid bacteria of the genus Lactobacillus such as lactococcus lactis (lactococcus lactis), Lactobacillus Acidophilus (Lactobacillus Acidophilus), or Lactobacillus plantarum (Lactobacillus plantarum).

Further, the preparation method of the human soluble CD80 fusion gene transformed lactobacillus comprises the following steps:

(1) firstly, synthesizing an SPK1-hsCD80 and an SPK1-CTB-hsCD80 fusion gene, and introducing a cDNA sequence corresponding to a furin enzyme digestion polypeptide sequence between an hsCD80 gene and a CTB gene in the CTB-hsCD80 fusion gene;

(2) the fusion genes of SPK1-hsCD80 and SPK1-CTB-hsCD80 are inserted into a lactobacillus expression vector pLN to obtain recombinant plasmids pLN-SPK1-hsCD80 and pLN-SPK1-CTB-hsCD 80.

(3) The recombinant plasmids pLN-SPK1-hsCD80 and pLN-SPK1-CTB-hsCD80 are transformed into lactobacillus with lacF gene deletion by a conventional electrotransformation method, and a selective medium taking lactose as a unique carbon source is adopted to screen out the hsCD80 gene or the CTB-hsCD80 fusion gene transformed lactobacillus.

Namely, the preparation method of the transformed lactic acid bacteria comprises the following steps: firstly, synthesizing hsCD80 and CTB-hsCD80 fusion genes, and optimizing codons to enable the fusion genes to be suitable for lactobacillus expression; introducing an amino acid enzyme digestion sequence which is unique to furin between the hsCD80 and the CTB gene; inserting the fusion gene of the hsCD80 and the CTB-hsCD80 into a broad host expression vector pLN of gram-positive bacteria, connecting the upstream with a gene sequence of SPK1 signal peptide (Pediococcus K1 signal peptide), and controlling the expression by a lactoglobulin promoter (Pnisin); then, the expression vector of the hsCD80 or CTB-hsCD80 fusion gene is transformed into lactobacillus with lacF gene deletion by an electrotransformation method, and the selection culture medium taking lactose as a unique carbon source is adopted to screen the hsCD80 and CTB-hsCD80 transformed lactobacillus.

In the preparation method of the transformed lactic acid bacteria:

alternatively, the lactic acid bacterium host bacterium is lactococcus lactis (lactococcus lactis) in which the lacF gene is deleted.

Alternatively, the method for cloning the fusion gene of SPK1-hsCD80 or SPK1-CTB-hsCD80 into the lactobacillus expression vector pLN is a conventional gene recombination and cloning method.

Optionally, the lactobacillus expression vector pLN is derived from nisin induced expression system (NICE), contains LacF nutrition screening gene, and does not contain antibiotic resistance gene (nisin induced expression system (NICE) is currently a more general expression vector, and the commercially available vector is pNICE).

Optionally, the shock condition at the time of electrotransformation is: the voltage is 1.6kV-2.5kV, the capacitance is 25 muF, and the resistance is 200 omega.

Optionally, the selection medium is an EM medium containing only lactose as a sole carbon source, and the mass volume percentage of the lactose is 0.5% -2.0%.

Further, the preparation method of the transformed lactic acid bacteria comprises the following steps:

synthesis of the hsCD80 fusion genes SPK1-hsCD80 and SPK1-CTB-hsCD 80:

(1.1) optimizing codons according to the SPK1 signal peptide amino acid sequence to obtain a cDNA sequence of the SPK1 gene, wherein the sequence table is shown as SEQ ID NO: 1 is shown in the specification;

(1.2) according to the gene sequence and the functional region structure of the hsCD80, optimizing the codon to obtain the cDNA sequence of the hsCD80 gene only containing the extracellular region structure of the hsCD80, wherein the sequence table is shown as SEQ ID NO: 2 is shown in the specification;

(1.3) according to the CTB gene sequence, optimizing the codon to obtain the CTB gene cDNA sequence, wherein the sequence table is shown as SEQ ID NO: 3 is shown in the specification;

(1.4) connecting the cDNA sequences of the SPK1 and the hsCD80 gene to form a fusion gene, and adding restriction endonuclease digestion sequences of NcoI and XbaI to the 5 'end and the 3' end of the fusion gene respectively to synthesize the SPK1-hsCD80 gene;

(1.5) connecting cDNA sequences of the SPK1, CTB and hsCD80 genes to form a fusion gene, introducing a cDNA sequence corresponding to a furin enzyme digestion polypeptide sequence between the CTB and the hsCD80 genes, and adding restriction endonuclease digestion sequences NcoI and XbaI to the 5 'end and the 3' end of the fusion gene respectively to synthesize the SPK1-CTB-hsCD80 fusion gene;

construction of transformants of the fusion Gene of hsCD80 and CTB-hsCD80

(2.1) fusing the gene of the SPK1-hsCD80 and the gene of the SPK1-CTB-hsCD80 in a lactobacillus expression vector pLN to obtain a connector;

(2.2) adopting an electric shock transformation method to transform the connector into lactococcus lactis with LacF gene deletion, and adopting a selective medium EM screening with lactose as a unique carbon source to obtain the hsCD80 gene or CTB-hsCD80 fusion gene transformed lactobacillus after electric shock.

The second object of the present invention can be achieved by the following technical solutions: the application of the human soluble CD80 fusion gene transformed lactobacillus in preparing oral viable bacteria medicament with disease immunotherapy effect.

Optionally, the disease is a tumor, a virus-infected disease, an intracellular bacterial-infected disease, a primary and a secondary hypoimmunity disease.

Optionally, the tumor is colorectal cancer or melanoma.

Optionally, the oral viable bacteria medicament is a viable bacteria suspension, a dairy product, a viable bacteria powder, a bacteria tablet or a capsule.

Compared with the prior art, the invention has the following advantages:

(1) the invention has the characteristics that the fusion gene of human hsCD80 and CTB-hsCD80 is transformed into lactobacillus, and does not carry antibiotic or drug resistance genes;

(2) in the invention, the fusion genes of hsCD80 and CTB-hsCD80 in the lactobacillus transformed by the fusion genes of hsCD80 and CTB-hsCD80 can be induced and expressed by an inducer Nisin in vitro or in vivo, particularly in intestinal tracts;

(3) according to the invention, the fusion gene of the hsCD80 and the CTB-hsCD80 is transformed into lactobacillus to be expressed in intestinal tracts, and the lactobacillus can directly act in the intestinal tracts to prevent and treat intestinal tumors;

(4) according to the invention, CTB-hsCD80 fusion gene transformed lactic acid bacteria expressed CTB-hsCD80 fusion protein can be endocytosed by intestinal epithelial cells through the endocytosis mediated by CTB receptors, and is specifically digested by furin, and free hsCD80 with immunological activity is released to the outside of cells or in blood circulation, so that the intestinal mucosa barrier is broken through, lymphocyte activation is stimulated, the cellular immunity level of an organism is improved, and the effects of immunosuppressive pathways such as PD-1/PD-L1 and the like are blocked;

(5) the fusion gene of the hsCD80 and the CTB-hsCD80 is transformed into the lactic acid bacteria, and the lactic acid bacteria can be prepared into live bacteria preparations such as live bacteria suspension, dairy products, live bacteria powder, bacteria tablets or capsules, can be proliferated and colonized in intestinal tracts of human bodies after being orally taken, and is applied to the prevention and treatment of diseases such as tumors, virus infection or intracellular bacterial infection, primary and secondary low immunity and the like.

Drawings

FIG. 1 is a schematic diagram showing the construction of a lactic acid bacteria expression vector of the CTB-hsCD80 fusion gene in example 1;

FIG. 2 is a graph comparing the significant inhibition of the growth of subcutaneous colon cancer transplants in CT-26 mice by live bacteria of the transformed lactic acid bacteria, HsCD80 and CTB-hsCD80, in example 2, and A1 is an anatomical photograph; a2 is a tumor size statistical chart;

FIG. 3 is a statistical chart showing that the living bacteria of hsCD80 and CTB-hsCD80 transformed lactic acid bacteria in example 2 significantly stimulate the differentiation and maturation of spleen lymphocytes of mice with CT-26 tumor cells orally, A1 is the proportion of spleen lymphocyte subtypes, and A2 is the proportion of spleen activated lymphocytes;

FIG. 4 is a graph comparing the significant inhibition of the growth of melanoma transplantable tumors in B16F10 mice by oral administration of live bacteria of the hsCD80 and CTB-hsCD80 transformed lactic acid bacteria of example 3, A1 is an anatomical photograph; a2 is a tumor size statistical chart;

FIG. 5 is a statistical graph showing that live bacteria of hsCD80 and CTB-hsCD80 transformed lactic acid bacteria in example 3 significantly stimulate the differentiation and maturation of splenic lymphocytes of B16F10 tumor-bearing mice orally;

FIG. 6 is a graph comparing the significant inhibition of the growth of primary colon cancer in APC mice by live bacteria of hsCD80 and CTB-hsCD80 transformed lactic acid bacteria orally in example 4, A1 is a dissected photograph; a2 is a statistical chart.

Detailed Description

The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are raw reagent raw materials which are conventionally commercially available or commercially available, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.