Oral recombinant lactic acid bacteria formulations for targeted intestinal delivery of antimicrobial peptides and IL-22

Abstract

The invention discloses a preparation method of a recombinant lactic acid bacteria preparation for expressing antibacterial peptide and interleukin 22 (IL-22) and application of a composite microecological preparation composed of the antibacterial peptide and the interleukin 22 in prevention and treatment of animal bacterial infection diseases. The probiotic preparation provided by the invention comprises lactic acid bacteria, antibacterial peptide and interleukin, the recombinant lactic acid bacteria preparation can be taken orally without generating toxicity to an organism, and the recombinant lactic acid bacteria preparation can be colonized in intestinal tracts and secrete the antibacterial peptide and IL-22. The antibacterial peptide secreted by the recombinant lactic acid bacteria can punch bacterial cell membranes to cause bacterial death, does not cause generation of drug-resistant bacteria, and can reduce or replace the use of antibiotics; iL-22 secreted by the recombinant lactic acid bacteria can promote the repair of damaged tissues of intestinal tracts, activate the immune system of the organism and enhance the resistance; the probiotic effect of lactic acid bacteria can regulate the balance of intestinal flora of the organism, promote nutrient absorption and enhance the immunity of the organism. The preparation provided by the invention has the advantages of innovativeness, good safety, no toxic or side effect, simple administration mode, remarkable effect and the like, can effectively reduce the use of antibiotics, and provides a new solution for preventing and treating bacterial diseases in animal husbandry.

Description

Technical Field

[0001] The present invention relates to the fields of probiotics and biomedicine, and specifically to the preparation of a composite probiotic preparation of Lactococcus lactis expressing antimicrobial peptide (LL-37) and interleukin 22 (IL-22), as well as the application of the composite probiotic preparation in treating bacterial diseases in animals. Background Art

[0002] Lactic acid bacteria are widely distributed in nature and the human body. They are a group of Gram-positive bacteria that ferment sugars and produce lactic acid. Lactic acid bacteria thrive in acidic environments and can colonize and proliferate in the host's gastrointestinal tract, regulating the balance of intestinal flora and improving the intestinal microecological environment. They also possess probiotic effects such as anti-tumor, anti-inflammatory, and anti-allergic properties, as well as physiological benefits such as immunomodulation. Compared to other active carriers (such as attenuated Escherichia coli, Salmonella, and viruses), lactic acid bacteria are non-immunogenic and do not stimulate specific immune responses. Therefore, using lactic acid bacteria as genetically engineered receptors, and applying genetic engineering techniques to purposefully and selectively develop novel probiotic preparations, can better leverage their probiotic properties and immune effects. Lactic acid bacteria's beneficial properties for the host and their strong tolerance to the acidic gastrointestinal environment make them promising vehicles for targeted protein drug delivery. LL-37 is the only member of the cathelicidins antimicrobial peptide family in the human body. It has a wide range of anti-pathogenic activities and can effectively kill a variety of pathogens, especially Escherichia coli. LL-37 directly forms holes in the bacterial cell membrane, destroying the integrity of the cell membrane, thereby effectively preventing the development of bacterial resistance. IL-22 is a CD4 + A cytokine secreted by a subset of T cells. IL-22 plays a key balancing role between regulating immune responses and promoting tissue repair, and can promote the healing process of tissue damage by promoting cell proliferation, enhancing the regenerative capacity of epithelial cells, and regulating inflammatory responses. Therefore, the microecological preparation of the present invention simultaneously expresses and secretes LL-37 and IL-22 in the intestine, which can achieve the dual effect of killing pathogens while repairing damaged intestinal epithelium. Currently, LL-37 is mainly obtained by in vitro purification of Escherichia coli and yeast systems, and has the disadvantages of low expression purity, large side effects, and high cost. There are no reports on the in vitro expression of IL-22. The present invention aims to design a food-grade composite microecological preparation that secretes LL-37 and IL-22 in the intestine through oral colonization. Summary of the Invention

[0003] The purpose of the present invention is to provide a new means to replace antibiotics for the prevention and treatment of bacterial diseases. It provides a lactic acid bacteria composite microecological preparation that can colonize the body's intestinal tract and express and secrete LL-37 and IL-22 in the intestine. The preparation simultaneously exerts the probiotic effect of lactic acid bacteria, the bactericidal effect of LL-37 and the tissue repair effect of IL-22, and has a significant preventive and therapeutic effect on bacterial infectious diseases.

[0004] In order to achieve the above objectives, the first aspect of the present invention provides a method for constructing a recombinant Lactococcus lactis preparation expressing LL-37 and IL-22, and an identification experiment of the expression and secretion of LL-37 and IL-22 by recombinant lactic acid bacteria; The second aspect of the present invention provides an experiment on the colonization time and safety evaluation of a recombinant lactic acid bacteria preparation expressing LL-37 and IL-22 in the intestinal tract of animals; The third aspect of the present invention provides an experiment on the inhibition of the growth of multiple bacteria in vitro by a composite probiotic expressing LL-37 and IL-22; The fourth aspect of the present invention provides an experiment to evaluate the effect of a probiotic preparation expressing LL-37 and IL-22 in treating and preventing Escherichia coli disease in chickens.

[0005] Through the above scheme, the lactic acid bacteria preparation provided by the present invention can colonize and secrete LL-37 and IL-22 in the intestinal tract of animals, playing the multiple roles of lactic acid bacteria in regulating the balance of intestinal flora, antimicrobial peptides in killing bacteria, and IL-22 in repairing damaged intestinal epithelium. The lactic acid bacteria, expression vectors, and inducers involved in the present invention are all food grade and will not cause damage to the body. The recombinant lactic acid bacteria preparation provided by the present invention can achieve targeted delivery of LL-37 and IL-22 in the intestinal tract of animals, achieve an excellent combination of probiotics, antimicrobial peptides, and IL-22, and can effectively prevent and treat diseases caused by bacterial infections in animals. Therefore, the present invention is particularly conducive to breaking the current dilemma of abuse of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 The results of gel electrophoresis identification of LL-37 and IL-22 gene fragments in recombinant lactic acid bacteria are shown in Figure 2. Figure 2 Western Blot identification of recombinant lactic acid bacteria expressing LL-37 and IL-22; Figure 3 is the colonization time of recombinant lactic acid bacteria in the mouse intestine; Figure 4 The results of HE staining of organ tissue sections in acute toxicology experiments in mice; Figure 5 Observation of inhibition zone for in vitro antibacterial experiment of recombinant lactic acid bacteria; Figure 6fecal morphology changes in chickens treated with recombinant lactic acid bacteria for Escherichia coli infection; Figure 7 HE staining of tissue sections of chickens infected with Escherichia coli treated with recombinant lactic acid bacteria; Figure 8 To detect the expression level of inflammatory factors in chickens infected with Escherichia coli by recombinant lactic acid bacteria; Figure 9 HE staining of tissue sections to show that recombinant lactic acid bacteria can prevent Escherichia coli disease in chickens; Figure 10 Detection of expression levels of inflammatory factors in recombinant lactic acid bacteria to prevent Escherichia coli disease in chickens. DETAILED DESCRIPTION

[0007] The present invention is described below through specific embodiments. In the following examples, the Lactococcus lactis strain (NZ3900) and vector (pNZ8149) were obtained from the Shaanxi Stem Cell Engineering Technology Research Center; the gene sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd.; Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., and chicks were purchased from the Lvxin Hatchery in Deyang, Sichuan; and pathogenic Escherichia coli was isolated from the feces of sick chickens at a farm.

[0008] In the first aspect of the present invention, a recombinant lactic acid bacteria probiotic preparation expressing LL-37 and IL-22 was constructed by gene editing. Using the NCBI database, the LL-37 and IL-22 sequences were designed. A restriction enzyme site (Xba1) matching the pNZ8149 promoter was added to the 5' end, and a 3×FLAG sequence followed by a restriction enzyme site (Kpn1) matching the pNZ8149 promoter was added to the 3' end. The gene fragments were assembled into the pNZ8149 vector via double T4 ligation to form a recombinant vector. The recombinant vector was then transformed into the NZ3900 strain by electroporation, and positive colonies were screened on Elliker plates to obtain the recombinant lactic acid bacteria strain. The recombinant lactic acid bacteria plasmid was extracted and identified, as shown in the following example. Figure 1 Therefore, using the extracted plasmid as a template, PCR amplification with LL-37 and IL-22 specific primers was possible, and gel electrophoresis identified gene bands that matched the sizes of LL-37 and IL-22. Sequencing confirmed that the gene sequence was completely correct, and the gene sequence is as follows: LL-37-3×Flag (5'→3'): ctgctgggtgatttcttccggaaatctaaagagaagattggcaaagagtttaaaagaattgtccagagaatcaaggattttttgcggaatcttgtacccaggacagagtccGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAG IL-22-3×Flag (5'→3'): .

[0009] The second aspect of the present invention provides an identification experiment of recombinant lactic acid bacteria expressing and secreting LL-37 and IL-22 The recombinant strain was induced to express the exogenous protein by using an appropriate concentration of Nisin, and Western Blot detection was performed using a FLAG-specific antibody, such as Figure 2 As shown, recombinant lactic acid bacteria can express LL-37 and IL-22 proteins. The optimal induction dose of Nisin is 10-100 ng / mL, the induction temperature is 30℃, and the induction time is 3 h-4 h.

[0010] The third aspect of the present invention provides a method for colonization time of recombinant lactic acid bacteria in mouse intestine. The present invention provides an experiment to explore the colonization time of recombinant probiotic preparation in mice intestine, which is specifically implemented as follows: 12 6-8 week old Kunming mice were selected, 8 in the experimental group and 4 in the control group. 8 The mice were gavaged with a dose of 100 CFU / 100 g, and the control group was gavaged with an equal volume of PBS once a day for 14 consecutive days. The intestinal contents of the experimental and control group mice were then collected for 5 consecutive days. After the fecal genome was extracted, PCR amplification was performed using FLAG-specific primers, such as Figure 3 As shown in Figure 3, the recombinant probiotic preparation can colonize the mouse intestine for more than 5 days.

[0011] The fourth aspect of the present invention is to evaluate the safety of recombinant lactic acid bacteria expressing LL-37 and IL-22 in animals. In order to evaluate the safety of recombinant probiotic preparations, the present invention provides an acute toxicity experiment on mice, which is specifically implemented as follows: 12 6-8 week old Kunming mice were selected and divided into 2 groups. The experimental group was treated with 10 10 CFU / 100g dose (normal feeding concentration is 10 8 CFU / 100g) were administered orally to mice, while the control group was administered orally with an equal volume of PBS once a day for 7 consecutive days. Organs were collected for HE staining of tissue sections, and serum was collected for biochemical index detection. Figure 4 As shown in Table 1, a 100-fold dose of recombinant lactic acid bacteria did not cause histological changes in the heart, liver, spleen, and intestines of mice. As shown in Table 1, a 100-fold dose of recombinant lactic acid bacteria did not cause changes in biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), lactate dehydrogenase (LDH), urea (UREA), creatinine (CREA), and creatine kinase (CK). These results demonstrate that the recombinant lactic acid bacteria provided by the present invention do not cause toxic damage to animals and pose no safety risks in clinical use.

[0012] Table 1 Control 1 Control 2 Control 3 Control 4 Experiment 1 Experiment 2 Experiment 3 Experiment 4 ALT 57.5 45.2 52.6 60.4 47.5 55.6 42.8 55.9 AST 178 164.9 152.3 182.6 156.5 168.7 156.3 189.7 AST / ALT 3.1 3.6 2.9 3.0 3.2 3 3.7 3.4 TP 58.7 61.1 56.1 61.7 57.6 54.4 54.1 54.2 ALB 36.5 34.6 34.8 36.9 35.1 31.7 34.5 31.5 UREA 29.7 20.6 28.8 28.8 29.5 25.1 33 28.4 CK 6.38 8.86 7.99 8.99 7.07 6.54 5.97 8.65 LDH 970.3 1094.5 1012.3 1035.5 1070.8 1128 995.6 1282.5 .

[0013] The fifth aspect of the present invention is to evaluate the in vitro antibacterial effect of the recombinant lactic acid bacteria preparation. The present invention provides an in vitro antibacterial experiment of a recombinant lactic acid bacteria preparation against Escherichia coli, Salmonella and Staphylococcus aureus. The specific implementation steps are as follows: 10 6 100 μL of Escherichia coli, Salmonella and Staphylococcus aureus liquid with CFU / mL was evenly spread with a spreading rod. Four wells were punched in each culture medium with an 8 mm puncher. No substance was added to one well as a negative control. The remaining three wells were added with equal volumes of sensitive antibiotics (as a positive control), supernatant of recombinant lactic acid bacteria expressing LL-37, and supernatant of recombinant lactic acid bacteria expressing IL-22. Cultured in a 37°C biochemical incubator for 16 h and the results were observed. Figure 5 As shown, obvious inhibition zones were formed around the two recombinant lactic acid bacteria preparations, and the diameters of the inhibition zones were basically consistent with those formed by the antibiotics.

[0014] The sixth aspect of the present invention is to evaluate the effect of the recombinant lactic acid bacteria preparation on the treatment of Escherichia coli disease The present invention also provides a method for treating chicken diseases induced by pathogenic Escherichia coli by using recombinant lactic acid bacteria, wherein the oral dose of pathogenic Escherichia coli is 10 8 CFU / 100g body weight, and after 24 hours, the chicks showed symptoms such as depression, loss of appetite, and diarrhea, indicating that the chick disease model was successfully constructed. 8 CFU / 100g body weight, once a day, for 7 consecutive days. Integrating the comprehensive evaluation of the fecal morphology changes of chicks, HE staining of organ tissue sections, and the expression levels of inflammatory factors, the recombinant lactic acid bacteria has a significant therapeutic effect on the treatment of pathogenic Escherichia coli disease. The specific implementation is as follows: 24 3-day-old three-yellow chickens were selected, and after 7 days of adaptive feeding, they were randomly divided into 3 groups (control group, model group and treatment group). The control group was not treated and was raised normally. The chicks in the model group and treatment group were subjected to an Escherichia coli-induced infection model. The treatment group was gavaged with recombinant lactic acid bacteria suspended in PBS, and the model group was gavaged with an equal volume of PBS. Figure 6 As shown in the figure, the feces of the chicks in the model group were yellow-white and watery. The feces of the chicks in the treatment group were close to those in the control group in color and state. Figure 7 The HE staining results of the cut edges of the chick organs show that the heart and liver tissues of the model group were accompanied by a large number of diffuse bleeding spots, and the small intestinal villi were severely broken. The abnormal bleeding spots in the heart and liver tissues of the treatment group disappeared, and the intestinal villi returned to normal and were similar to the tissue structure of the control group. The changes in inflammatory factors were detected by fluorescent quantitative PCR. The results are shown in the figure. Figure 8As shown, the expression levels of inflammatory factors TNF-α, IL-6, and IL-10 in the liver and small intestine tissues of chicks in the treatment group were significantly different from those in the model group and approached those in the control group. Therefore, the recombinant lactic acid bacteria provided by the present invention have a good effect in treating diseases caused by bacterial infections and can replace the use of antibiotics.

[0015] Evaluation of the effect of the probiotic preparation provided by the seventh aspect of the present invention on preventing Escherichia coli disease in chicks The present invention provides an evaluation of the effect of a recombinant lactic acid bacteria system on preventing pathogenic Escherichia coli infection in chicks, characterized in that the chicks are orally administered with 10% recombinant lactic acid bacteria provided by the present invention. 8 CFU / 100g body weight, once a day, for 14 consecutive days, and then gavage the chicks for 10 8 The recombinant lactic acid bacteria were infected with pathogenic Escherichia coli at a concentration of 100 CFU / 100 g body weight. After 24 hours, the test was performed. Combined with the HE staining of organ tissue sections and the expression levels of inflammatory factors, the comprehensive evaluation showed that the recombinant lactic acid bacteria had a significant preventive effect on the infection of pathogenic Escherichia coli. The specific implementation was as follows: 24 three-day-old three-yellow chickens were selected and adaptively fed for 7 days. They were then randomly divided into three groups (control group, infection group and prevention group). The control group was not treated and was raised normally. The prevention group was gavage-fed with 10 CFU / 100 g of PBS-suspended 10 8 CFU / 100g body weight of recombinant lactic acid bacteria, once a day, for 14 consecutive days, while the infection group was gavaged with the same volume of PBS, once a day, for 14 consecutive days. 8 CFU / 100g body weight of pathogenic Escherichia coli, and the test was conducted after 24 hours of feeding. Figure 9 As shown in the figure, HE staining of liver and intestinal tissue sections showed that there were multiple diffuse hemorrhagic lesions in the liver and spleen of the infection group, while the degree of liver lesions in the prevention group was low and close to that of the control group; the intestinal villi of the infection group were severely broken, while the integrity of the intestinal villi in the prevention group was close to that of the control group. Figure 10 As shown, the expression levels of inflammatory factors TNF-α, IL-6, and IL-10 in the liver and intestinal tissues of chicks in the prevention group were significantly different from those in the infection group and approached those in the control group. Therefore, it can be concluded that the recombinant lactic acid bacteria provided by the present invention have a good effect in preventing diseases caused by bacterial infection.

[0016] The above describes in detail the construction method, animal safety evaluation, antibacterial testing, and evaluation of the preventive effect of the recombinant lactic acid bacteria preparation expressing LL-37 and IL-22 of the present invention. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, the recombinant lactic acid bacteria preparation of the present invention can be subjected to various simple modifications, or the probiotic preparation of the present invention can be amplified for production. These simple modifications and amplification production should be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. The present invention provides a composite microecological preparation that can effectively prevent and treat bacterial infectious diseases in animals, characterized in that: The preparation can express active antimicrobial peptide (LL-37) and interleukin 22 (IL-22) in the animal intestine, thereby effectively preventing and treating bacterial infectious diseases in animals.

2. The microecological preparation according to claim 1, Its characteristics are as follows: (1) The bacteria, inducers, and expression vectors used in this preparation are all food grade and can be taken orally directly; (2) The expression vector used in this preparation is a lactic acid bacteria promoter vector and has no resistance screening marker, so it will not cause any pollution to the environment or animals; (3) The LL-37 and IL-22 expressed by this preparation are conserved sequences and are suitable for multiple species; (4) The LL-37 and IL-22 expressed by this preparation have fusion expression tag sequences at the ends.

3. The probiotic preparation according to claim 1 or 2, wherein the bacterial species is Lactococcus lactis (NZ3900).

4. The probiotic preparation according to claim 1 or 2, wherein the inducer is Nisin, which is a food additive.

5. The probiotic preparation according to claim 1, 2 or 4, wherein the induction concentration of the inducer Nisin is 10-100 ng / mL.

6. The probiotic preparation according to claim 1 or 2, wherein the expression vector for driving the expression of LL-37 and IL-22 is a Lactococcus lactis promoter vector (pNZ8149) and is free of exogenous resistance contamination.

7. The probiotic preparation according to claim 1 or 2, wherein the LL-37 can perforate the cell walls of sensitive bacteria, causing death, wherein the bacteria include Escherichia coli, Salmonella, Staphylococcus aureus, etc.

8. The proecological preparation according to claim 1 or 2, wherein the IL-22 sequence is a conserved sequence and is applicable to multiple species, including mice, dogs, cats, chickens, ducks, pigs, cattle, sheep, etc.

9. The proecological preparation according to claim 1 or 2, wherein LL-37 and IL-22 are fused to the terminal ends with an expression tag sequence, wherein the tag sequence is a 3×FLAG sequence.

10. The probiotic preparation according to claim 1 or 2, through an intestinal colonization experiment in mice, proves that the probiotic preparation can colonize in mice for more than 5 days.

11. The use according to claim 10, wherein 8 The mice were gavaged with the proecological preparation provided by the present invention at a dose of CFU / 100 g body weight / day for 7 consecutive days. The feeding of the proecological preparation of the present invention was stopped on the 8th day. At the same time, the mouse feces were collected for 5 consecutive days, and the Flag tag in the fecal genome was identified using PCR amplification technology, which proved that the proecological preparation provided by the present invention can colonize in the mouse intestine for more than 5 days.

12. The probiotic preparation according to claim 1 or 2 is proven to have good safety through acute toxicity experiments in mice.

13. The use according to claim 12, wherein mice were orally gavaged at a dose 100 times the normal dose for 14 consecutive days, and the mice were in good mental state, no deaths occurred, and the morphology of various organs was normal after autopsy. There were no significant differences in serum alanine aminotransferase, aspartate aminotransferase, total protein, albumin, lactate dehydrogenase, urea, and creatine kinase compared with the control group.

14. The probiotic preparation according to claim 1 or 2 has achieved remarkable results in the treatment of Escherichia coli infection in chickens.

15. The use according to claim 14, wherein the Escherichia coli-induced chicken infection model is treated with the probiotic preparation according to claim 1 or 2, and the dosage of the composite probiotic preparation is 108 CFU / 100g body weight / day.

16. The use according to claims 14 and 15, wherein the proecological preparation significantly improves the fecal characteristics of infected chicks in the treatment of Escherichia coli disease in chicks. The chicks in the treatment group gain weight faster, have significantly reduced inflammatory symptoms of organs, and effectively alleviate liver and kidney damage.

17. The probiotic preparation according to claim 1 or 2 has achieved good results in preventing Escherichia coli in chickens.

18. The use according to claim 17, wherein chicks are gavaged with the probiotic preparation according to claim 1 or 2, and then infected with Escherichia coli 7 days later. The test results show that compared with the control group, the chicks gavaged with the probiotic preparation have significantly lower organ inflammatory symptoms and liver and kidney function damage.

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