Lactococcus lactis expression vector suitable for intraluminal induction of expression and use thereof
By constructing a Lactococcus lactis expression vector based on the Bifidobacterium ECX promoter and signal peptide, and using xylooligosaccharide inducers, the problem of low expression efficiency of genetically engineered probiotics in the intestinal lumen was solved, achieving safe and efficient exogenous gene expression and probiotic proliferation, which can be applied to disease treatment and health promotion.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHERN MEDICAL UNIVERSITY
- Filing Date
- 2024-07-09
- Publication Date
- 2026-06-19
Smart Images
Figure CN118773230B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical genetic engineering, specifically to a lactococcus expression vector suitable for in vitro expression and its application. Background Technology
[0002] In recent years, genetically engineered probiotics have attracted considerable attention. Due to their outstanding effects in improving gastrointestinal function and enhancing immunity, people have naturally considered using modern genetic engineering technology to express recombinant proteins or peptides with specific functions using probiotics as host bacteria for disease prevention and treatment. Some probiotics are used to express antimicrobial peptides such as nifedipine and pemphigoid to inhibit the growth of pathogenic bacteria and prevent bacterial infections; others are used to express anti-inflammatory factors (such as interleukin-10, IL-10) or mucosal protective factors (such as trefoil factor) for the treatment of gastritis and inflammatory bowel disease; still others are used to prepare oral vaccines for rotavirus, tetanus toxin C, and Salmonella; and some are used as vectors for gene therapy in the treatment of diseases such as malignant tumors. For example, some researchers have used genetically engineered Bifidobacterium to express endostatin for anti-tumor treatment. This research has been patented and is in clinical trials. Some researchers have used Bifidobacterium to express the cytosine deaminase (CD) gene as a vector, injecting it locally into solid tumors. The expressed and secreted CD can catalyze the low-toxicity chemotherapy drug 5-fluorocytosine (5-FC) to generate the more cytotoxic 5-fluorouracil (5-FU), thus enhancing the efficacy of chemotherapy while reducing its toxic side effects. Others have used recombinant lactic acid bacteria to express CD47 nanobodies to improve the body's active anti-tumor immunity. Probiotic-induced expression vectors are suitable for expressing both recombinant proteins that can be absorbed through the gastrointestinal epithelium to exert systemic effects and those that can directly function in the gastrointestinal tract. Antimicrobial peptides and trefoil factors can inhibit the growth of pathogenic bacteria and repair mucosal damage in the gastrointestinal tract; some enzymes and nutritional factors can promote the growth of probiotics, aid in food digestion and decomposition, or promote nutrient conversion and absorption; some recombinant proteins, such as intestinal peptide hormones, cytokines, and immune factors, can directly act on receptors on gastrointestinal epithelial cells, exerting biological effects through interactions with corresponding receptors. It is evident that probiotic inducible expression vectors have a very broad application prospect.
[0003] However, there is currently no suitable probiotic expression regulation system for inducing the expression of exogenous genes in the gastrointestinal tract. Existing gene expression regulation models in the field of genetic engineering, such as the lactose operon, tryptophan operon, temperature-sensitive operon, and nisin operon, are not suitable for genetically engineered probiotics, especially for inducing the expression of recombinant proteins in the intestinal lumen. The inducer used in the lactose operon, isopropyl-BD-thiopentanose glycoside (IPTG), is a synthetic glycoside that is toxic to humans and can only be used as an in vitro inducer, not an in vivo one. While tryptophan, the inducer of the tryptophan operon, can be taken orally, it is easily digested and broken down in the upper digestive tract, resulting in too small an amount reaching the intestines to reach the concentration needed to induce gene expression. The temperature-sensitive operon is even less suitable for in vivo application; controlling temperature in vivo is impractical. The nisin operon is only found in lactococci and streptococci, and nisin is an antimicrobial peptide that inhibits the growth of other bacteria. Therefore, the nisin operon is only suitable for inducing exogenous gene expression in Lactococcus lactis, and not suitable for regulating gene expression in other probiotics, thus having a limited scope of application.
[0004] The arabinose operon gene regulation expression system was used to express intestinal peptide hormones such as gastrin, immune factors such as interferon-α and thymosin-α, and anti-tumor drugs such as tumor suppressor in genetically engineered Bifidobacterium. Good therapeutic effects were achieved in both in vitro and animal experiments. However, the arabinose operon gene regulation expression system, derived from Escherichia coli, also has the following drawbacks: (1) high expression efficiency in genetically engineered E. coli, but low expression efficiency in genetically engineered probiotics; (2) expression efficiency is inhibited by glucose, which is both the main carbon source in in vitro culture media and the main digestive product of starch, and its concentration in the intestinal lumen is also high, thus inhibiting the expression level of the arabinose operon gene regulation expression system both in vitro and in vivo. Summary of the Invention
[0005] The purpose of this invention is to provide a lactococcus expression vector suitable for in vitro expression in the intestinal lumen. This expression vector can use lactococcus as the host bacterium and xylooligosaccharide or xylan as an inducer to induce the expression and secretion of exogenous target genes in the intestinal lumen or in vitro.
[0006] The objective of this invention is achieved through the following technical solution:
[0007] A lactococcus expression vector suitable for in vitro expression is proposed. It utilizes the promoter and secretory signal peptide of the exoxylanase ECX gene of Bifidobacterium to control the expression and secretion of exogenous genes. It can induce the expression and secretion of exogenous target genes in vitro or in the intestinal lumen using lactococcus as the host bacterium and xylooligosaccharide as the inducer.
[0008] Furthermore, the expression vector is constructed by using Bifidobacterium NCC2705 genomic DNA as a template, and PCR amplifying a DNA fragment including the ECX promoter PECX and the signal peptide SPecx using EXPF1 and EXPR1 primers; inserting the DNA fragment upstream of the multiple cloning site of the Lactococcus lactis expression vector pNZ8149, replacing the original nisin promoter (Pnisin) DNA sequence in the pNZ8149 vector, to obtain the pLXS expression vector;
[0009] The upstream primer ECXF1 sequence and the downstream primer ECXR1 nucleic acid sequence are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the nucleotide sequences of the ECX gene promoter and the secretory signal peptide DNA fragment are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
[0010] Furthermore, the method for constructing the expression vector is as follows:
[0011] (1) Based on the DNA base sequence of the promoter (Pecx) and secretory signal peptide (SPecx) of the extracellular xylanase ECX gene of Bifidobacterium longum NCC2705, a pair of primers were designed and synthesized. The endonuclease BamHⅠ restriction sequence was introduced at the 5' end of the upstream primer EXPF1, and the endonuclease NcoⅠ restriction sequence was introduced at the 5' end of the downstream primer EXPR1.
[0012] (2) Using the extracted Bifidobacterium longum NCC2705 genomic DNA as a template, the ECX promoter PECX and signal peptide SPecx DNA fragments were amplified by PCR using EXPF1 and EXPR1 primers, and the PECX-SPecx DNA fragments were purified and recovered.
[0013] (3) The recovered PECX-SPecx DNA fragment was digested with restriction enzymes BamHI and NcoI, and the expression vector pNZ8149 was digested with restriction enzymes PsuI and NcoI to remove the Pnisin DNA fragment in pNZ8149. The PECX-SPecx fragment and the large fragment of the pNZ8149 vector were recovered by gel electroporation. The PECX-SPecx fragment and the pNZ8149 vector digested fragment were mixed at a ratio of 5:1 and ligated with T4 DNA ligase. The mixture was then electroporated into Lactococcus lactis with the LacF gene missing. The pLXS recombinant expression vector was obtained by screening with a selective medium using lactose as the sole carbon source and then transformed into Lactococcus lactis.
[0014] (4) Extract plasmid DNA, and use restriction enzyme digestion and DNA sequencing to confirm that the PECX-SPecx fragment successfully replaced Pnisin and that the recombinant pLXS expression vector had the correct sequence. Extract pLXS plasmid DNA for later use.
[0015] Further, the ECX signal peptide mentioned in step (2) is Pediococcus K1 signal peptide, Lactococcus nonstructural protein signal peptide, or Bifidobacterium α-amylase signal peptide.
[0016] Furthermore, the Lactococcus lactis expression vector pNZ8149 is the nisin-induced expression system NICE, with gene expression controlled by the nisin promoter Pnisin, containing the LacF lactose auxotroph compensation gene, and not containing antibiotic resistance genes.
[0017] Furthermore, the selective medium is an EM medium with lactose as the sole carbon source, wherein the lactose content is 0.5%~2.0% (w / v).
[0018] Furthermore, the exogenous target gene is green fluorescent protein, long-acting glucagon-like peptide, and / or Exendin-4.
[0019] Furthermore, the electric shock conditions for the electro-conversion are: voltage 1.6kV~2.5kV, capacitance 20~25µF, and resistance 200~300Ω.
[0020] Furthermore, the engineered bacteria, which are cultured in vitro to ferment and transform into live bacterial preparations, can be taken orally with xylooligosaccharides or xylan as inducers. This can induce the expression and secretion of recombinant polypeptides or recombinant proteins in the intestinal lumen, thereby achieving the purpose of preventing and treating diseases.
[0021] Specifically, the ECXS signal peptide sequence in the pLXS expression vector can be replaced with the E. coli secretory signal peptide gⅢ sequence, and a foreign gene can be inserted at the NcoⅠ and XbaⅠ multiple cloning sites to obtain a recombinant expression vector carrying the foreign gene. Gene-transformed E. coli engineered bacteria can be obtained by conventional chemical transformation. Recombinant polypeptides or recombinant proteins can be induced to express and secrete in vitro or in the intestinal lumen with xylooligosaccharides. Finally, the gene-transformed E. coli can be prepared into a live bacterial preparation.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] (1) The *Lactococcus lactis* expression vector of the present invention, suitable for induction of exogenous gene expression in vitro and in the intestinal lumen, is suitable for inducing exogenous gene expression in vitro. The inducer xylooligosaccharide or xylan is present in high amounts in dietary fiber, cannot be digested and decomposed by the upper digestive tract and common bacteria, can only be partially decomposed and utilized by probiotics, and has the effect of stimulating the proliferation of probiotics. After oral administration, xylooligosaccharide or xylan is not easily digested and absorbed in the upper digestive tract, and can maintain a high concentration in the intestinal lumen, ensuring its efficiency in inducing exogenous gene expression, and has very high food safety.
[0024] (2) The *Lactococcus lactis* expression vector of the present invention, suitable for in vitro expression, shows very low expression of the exogenous gene without the addition of inducing agents such as xylooligosaccharides or xylan. Adding inducing agents such as xylooligosaccharides or xylan can efficiently induce the expression of the exogenous target gene in vitro or in the intestinal lumen.
[0025] (3) The lactococcus lactis expression vector of the present invention, which is suitable for in vitro induction of expression, can also use Gram-positive bacteria such as probiotics as host bacteria, as well as Gram-negative bacteria such as Escherichia coli as host bacteria; it is suitable for in vitro induction of exogenous gene expression and in vivo (intestinal lumen) induction of exogenous gene expression; it is suitable for expressing various cytokines, immune factors, special proteins and enzymes, polypeptide hormones, recombinant proteins, and antigen proteins; it can be used to prepare probiotic live bacteria drugs and oral vaccines; it can be used for compensatory treatment of nutritional deficiencies and genetic defects; treatment of infectious diseases; gene therapy of malignant tumors; and it can also be applied to the production of probiotic feed additives for poultry and livestock and disease prevention and control.
[0026] (4) The Lactococcus lactis expression vector of the present invention is suitable for induction expression in the intestinal lumen. The genetically engineered bacteria can be further processed into live bacterial suspensions, dairy products, live bacterial powders, bacterial tablets or capsules and other live bacterial preparations. After oral administration, they can proliferate and colonize in the human intestine. At the same time, consuming xylooligosaccharides or xylan can induce the expression of exogenous functional genes, thereby promoting health and preventing and treating diseases.
[0027] (5) The Lactococcus lactis expression vector of the present invention is suitable for induction expression in the intestinal lumen. In addition to the specific functions of the recombinant polypeptides or recombinant proteins expressed and secreted by the gene-transformed Lactococcus lactis, the Lactococcus lactis itself has the function of regulating the balance of gastrointestinal flora and improving gastrointestinal function. The inducer xylooligosaccharide or xylan can not only induce the expression of exogenous genes, but also has the beneficial effects of promoting the growth and proliferation of probiotics, lubricating the intestines and relieving constipation, and reducing lipids and weight loss. It has a synergistic effect with the gene-transformed engineered bacteria in promoting health and preventing and treating diseases. Attached Figure Description
[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments in the accompanying drawings, but this does not constitute any limitation on the present invention.
[0029] Figure 1 This is a schematic diagram of the construction of the exogenous gene inducible expression vector pLXS for Lactococcus lactis, and the recombinant expression vector containing the exogenous genes GFP, LGLP, and EX4.
[0030] Figure 2 The results of in vitro induction expression detection of GFP in Lactococcus lactis transformed with GFP in Example 1 are shown. A, B, and C are observation results under a regular light microscope, D and E are observation results under a fluorescence microscope, and F is the result of Western blot detection.
[0031] Figure 3 The results are from in vivo animal imaging observations after GFP transformation of Lactococcus lactis and induction of GFP expression in the intestinal lumen in Example 1.
[0032] Figure 4 The results of immunoassay for the in vitro expression of LGLP and EX4 peptides induced by LGLP and EX4 transformation of Lactococcus lactis in Example 2;
[0033] Figure 5 The results of LGLP and EX4 transformation of Lactococcus lactis expression supernatant in Example 2 showed that it promoted pancreatic islet cell proliferation and insulin secretion in vitro.
[0034] Figure 6 This refers to the results of oral treatment of obese diabetic (DB) mice with live Lactococcus lactis transformed by LGLP and EX4 in Example 2.
[0035] Figure 7 The results of immunohistochemical detection of pancreatic islet tissue after oral treatment of DB obese diabetic mice with live Lactococcus lactis transformed by LGLP and EX4 in Example 2 are shown. Detailed Implementation
[0036] The application method of the present invention is further illustrated below with reference to specific embodiments. The following embodiments and accompanying drawings are for illustrative purposes only and should not be construed as limiting the present invention.
[0037] Unless otherwise specified, the reagents used in the following examples are conventionally available commercially or biochemical reagents, and the methods and equipment used in the following examples are conventionally used in the art.
[0038] Example 1: Preparation of the exogenous gene inducible expression vector pLXS for Lactococcus lactis
[0039] (1) Based on the DNA base sequences of the promoter (Pecx) and secretory signal peptide (SPecx) of the exoxygnanase ECX gene of Bifidobacterium longum NCC2705, a pair of primers were designed and synthesized. An endonuclease BamHI restriction sequence was introduced at the 5' end of the upstream primer EXPF1, and an endonuclease NcoI restriction sequence was introduced at the 5' end of the downstream primer EXPR1. The nucleic acid sequences of the upstream primer ECXF1 and the downstream primer ECXR1 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide sequences of the ECX gene promoter and secretory signal peptide DNA fragment are shown in SEQ ID NO.3 and SEQ ID NO.4.
[0040] (2) Using the extracted Bifidobacterium longum NCC2705 genomic DNA as a template, the ECX promoter PECX and signal peptide SPecx DNA fragments were amplified by PCR using EXPF1 and EXPR1 primers, and the PECX-SPecx DNA fragments were purified and recovered.
[0041] (3) The recovered PECX-SPecx DNA fragment was digested with restriction enzymes BamHI and NcoI, and the expression vector pNZ8149 was digested with restriction enzymes PsuI and NcoI to remove the Pnisin DNA fragment in pNZ8149. The PECX-SPecx fragment and the large fragment of the pNZ8149 vector were recovered by gel electroporation. The PECX-SPecx fragment and the pNZ8149 vector digested fragment were mixed at a ratio of 5:1 and ligated with T4 DNA ligase. The mixture was then electroporated into Lactococcus lactis with the LacF gene missing. The pLXS recombinant expression vector was obtained by screening with a selective medium using lactose as the sole carbon source and then transformed into Lactococcus lactis.
[0042] (4) Extract plasmid DNA, and use restriction enzyme digestion and DNA sequencing to confirm that the PECX-SPecx fragment successfully replaced Pnisin and that the recombinant pLXS expression vector had the correct sequence. Extract pLXS plasmid DNA for later use.
[0043] Example 2: Preparation, identification, and expression regulation of Lactococcus lactis transformed with GFP gene
[0044] 1. Construction of pLXS-GFP recombinant expression vector
[0045] (1) Design and synthesize a pair of primers, introducing an NcoI restriction site at the 5' end of the upstream primer GFPF1 and an XbaI restriction site at the 5' end of the downstream primer GFPR1. The base sequences of primers GFPF1 and GFPR1 are shown in the sequence listing (SEQ ID NO:8 and SEQ ID NO:9);
[0046] (2) Using reporter gene vector pEGFP-N1 (Clontech) DNA as a template, and GFPF1 and GFPR1 as primers, the cDNA fragment of the GFP gene was amplified by PCR, and the cDNA fragment was purified and recovered.
[0047] (3) The GFP gene cDNA fragment and the pLXS Lactococcus lactis expression vector DNA were digested with restriction enzymes NcoI and XbaI. The GFP gene fragment and the pLXS vector fragment were separated by agarose gel electrophoresis. The GFP gene and vector DNA fragments were recovered and purified using a gel DNA recovery kit. The GFP gene and vector DNA fragments were mixed in a certain ratio, and ligation buffer and T4 DNA ligase were added. The mixture was ligated at 16°C for 6-12 hours to form the pLXS-GFP recombinant expression vector.
[0048] The gene sequence of GFP is shown in SEQ ID NO:5.
[0049] 2. Preparation and identification of *Lactococcus lactis* transformed with GFP gene
[0050] (1) Lactococcus lactis was transformed by electroporation. 1-3 μg of ligation product was added to 100 μl of competent cells of Lactococcus lactis NZ3900 (provided by BioVector Vector Culture Collection Center) with LacF gene deletion. After mixing, the mixture was transferred to an electroporation cuvette with a width of 2 mm. The cuvette was placed in the electroporation tank and the electroporation conditions were set as follows: 2.5 kV, capacitance 25 µF, resistance 200 Ω.
[0051] (2) Culture and screening of *Lactococcus lactis* colonies transformed with the GFP gene. After electroporation, 200-400 µL of EM medium containing 20 mM MgCl2, 2 mM CaCl2, and 0.5% lactose was added to suspend and rinse the bacteria. The bacteria were then spread onto EM agarose plates containing 0.5% (w / v) lactose and 0.04% bromocresol purple and incubated at 30°C for 48 h. Single yellow colonies were picked and inoculated into 5 mL of EM liquid medium containing 0.5% lactose for amplification and culture for 24 h.
[0052] (3) Sequencing identification of *Lactococcus lactis* transformed with the GFP gene. Collect 5-10 mL of bacterial culture, add lysozyme to a final concentration of 10 mg / mL, digest at 37°C for 30 min, and extract plasmid using a standard plasmid extraction kit. Send the extracted plasmid to a DNA sequencing company for sequencing identification of *Lactococcus lactis* transformed with the GFP gene using universal sequencing primers upstream of the pNZ8149 vector.
[0053] (4) Select the GFP-transformed Lactococcus lactis that were correctly identified by sequencing and expand the culture in EM medium. Add glycerol to the final concentration of 25%, dispense and store in a -20℃ freezer to obtain the original strain of GFP-transformed Lactococcus lactis.
[0054] 3. Detection of GFP gene-transformed Lactococcus lactis GFP-induced expression in vitro and in the intestinal lumen
[0055] (1) In vitro induction and determination of GFP expression in *Lactococcus lactis* transformed with the GFP gene. *Lactococcus lactis* transformed with the GFP gene were cultured and multiplied in EM medium in vitro. When the bacterial density reached OD600 of 0.6-1.2, xylooligosaccharide at a final concentration of 0.1-2.0% was added to induce expression for 6-12 hours. The bacterial suspension was spread on a glass slide, dried in air over a flame, and observed under a fluorescence microscope to see if the engineered bacteria emitted green fluorescence due to GFP expression. The culture supernatant and bacterial cells were collected by centrifugation, and the expression level of GFP was detected by Western blotting with a GFP antibody.
[0056] (2) Detection of GFP-induced expression in the intestine of live *Lactococcus lactis* transformed with the GFP gene after oral administration. *Lactococcus lactis* transformed with the GFP gene was cultured and multiplied in EM medium in vitro. When the bacterial density reached OD600 of 1.2–1.5, the cells were collected by centrifugation. The cells were resuspended in physiological saline and xylooligosaccharide (1.0–10.0% concentration) to achieve a viable cell rate of approximately 10%. 9 A bacterial suspension of CFU / ml was fed to experimental mice. Four hours later, the mice were euthanized by cervical dislocation, and the entire gastrointestinal tract was isolated. Green fluorescence due to GFP expression could be observed under in vivo imaging.
[0057] Figure 2 This is the result of detecting the in vitro induced expression of GFP in *Lactococcus lactis* transformed with GFP, such as... Figure 2 As shown, GFP transformation of Lactococcus lactis causes bromocresol purple to turn yellow, and the transformed bacterial colonies and their surrounding area turn yellow. Figure 2 -A); 2-B shows the morphology of the transformed bacteria under a phase-contrast microscope; 2-C shows the Gram staining observation of the transformed bacteria; 2-D: fluorescence microscopy observation results of the transformed bacteria before induction; after 3 hours of induction with xylooligosaccharides, the GFP transformed bacteria were observed under a fluorescence microscope, and each bacterium emitted bright green fluorescence ( Figure 2 -E); before induction with an inducer, only weak fluorescence was observed in the GFP-transformed bacteria. After 6 hours of induction with different final concentrations of xylooligosaccharides, GFP expression could be detected by Western blotting using an anti-GFP antibody. Within the xylooligosaccharide range of 0.2%–2.0%, the expression level of GFP was directly proportional to the concentration of the xylooligosaccharide inducer (2–F).
[0058] Mice were fed a mixture of GFP-transformed live lactic acid bacteria and 10.0% xylooligosaccharide. Six hours later, the mice were sacrificed, and the entire gastrointestinal tract was isolated and placed in a clean petri dish for observation using a small animal in vivo imaging system. Figure 3 This image shows in vivo animal imaging results after GFP transformation of lactic acid bacteria and GFP expression in the intestinal lumen. Figure 3 As shown, bright green fluorescence was observed in the mouse intestine; without the inducer, live GFP-transformed bacteria fed alone to mice only showed very weak fluorescence in the cecum; while live empty vector-transformed bacteria fed to mice with the inducer showed no fluorescence in the intestine. This indicates that the inducer xylooligosaccharide can induce gene transformation of Lactococcus lactis to express recombinant GFP protein in the intestinal lumen.
[0059] Example 3: Preparation of live lactic acid bacteria transformed with LGLP and EX4 genes and their application in the treatment of diabetes.
[0060] 1. Construction of pLXS-LGLP and pLXS-EX4 recombinant expression vectors
[0061] (1) Based on the amino acid sequence of the mature human GLP-1 polypeptide and the codon preference of Lactococcus lactis, the second amino acid in the GLP-1 amino acid sequence was modified to glycine (Gly) or valine (Val) to avoid inactivation by endopeptidase IV (DPP4), thus obtaining the long-acting GLP-1 (LGLP) gene. The long-acting GLP-1 (LGLP) cDNA sequence was chemically synthesized from the whole gene. Based on the amino acid sequence of the American venom lizard Exendin-4 (EX4) polypeptide and the codon preference of Lactococcus lactis, the EX4 gene cDNA sequence was designed. An endonuclease NcoI restriction site was introduced at the 5' end of the LGLP and EX4 cDNAs, and an endonuclease XbaI restriction site was introduced at the 3' end. The LGLP and EX4 cDNA sequences were synthesized by chemical synthesis of the whole gene. The cDNA sequences of the LGLP and EX4 genes are shown in the sequence listing (SEQ ID NO:6 and SEQ ID NO:7).
[0062] (2) The cDNA fragments of the LGLP and EX4 genes, as well as the DNA of the pLXS Lactococcus lactis expression vector, were digested with restriction enzymes NcoI and XbaI. The LGLP and EX4 gene fragments and the pLXS vector fragment were separated by agarose gel electrophoresis. The LGLP, EX4 and vector digested fragments were recovered and purified using a gel DNA recovery kit. The LGLP and EX4 digested fragments were mixed with the vector digested fragments in a certain ratio, and ligation buffer and T4 DNA ligase were added. The mixture was ligated at 16°C for 6-12 hours to form the pLXS-LGLP and pLXS-EX4 recombinant expression vectors.
[0063] Figure 1This is a schematic diagram showing the probiotic exogenous gene inducible expression vector pLXS in Examples 1-2, as well as GFP, LGLP and EX4, which are used to construct recombinant expression vectors pLXS-GFP, pLXS-LGLP and pLXS-EX4 carrying exogenous genes.
[0064] 2. Preparation and identification of *Lactococcus lactis* transformed with LGLP and EX4 genes
[0065] (1) Lactococcus lactis was transformed by electroporation. 1-3 μg of pLXS-LGLP and pLXS-EX4 ligation products were added to 100 μL of competent cells of Lactococcus lactis with LacF gene deletion (BioVector vector culture center), respectively. After mixing, the mixture was transferred to an electroporation cuvette with a width of 2 mm. The cuvette was placed in the electroporation tank and the electroporation conditions were set as follows: 2.5 kV, capacitance 25 µF, resistance 200 Ω.
[0066] (2) Culture and screening of *Lactococcus lactis* colonies transformed with LGLP and EX4 genes. After electroporation, 200-400 µL of EM medium containing 20 mM MgCl2, 2 mM CaCl2, and 0.5% lactose was added to suspend and rinse the bacteria. The bacteria were then spread onto EM agarose plates containing 0.5% (w / v) lactose and 0.04% bromocresol purple and incubated at 30°C for 48 h. Single colonies were picked and amplified in 5 mL of EM liquid medium containing 0.5% lactose for 24 h.
[0067] (3) Sequencing identification of *Lactococcus lactis* transformed with LGLP and EX4 genes. Collect 5-10 mL of bacterial culture, add lysozyme to a final concentration of 10 mg / mL, digest at 37°C for 30 min, and extract plasmids using a standard plasmid extraction kit. Send the extracted plasmids to a DNA sequencing company for sequencing identification of *Lactococcus lactis* transformed with LGLP and EX4 genes using universal sequencing primers downstream of the pNZ8149 vector.
[0068] (4) The LGLP and EX4 transformed lactic acid bacteria that were correctly identified by sequencing were expanded in EM medium, and glycerol was added to a final concentration of 25%. After being dispensed, they were stored in a -20℃ freezer to obtain the original strain of Lactococcus lactis transformed by LGLP and EX4 genes.
[0069] 3. Experiments on the in vitro promotion of pancreatic islet cell proliferation and insulin secretion by Lactococcus lactis expression products transformed with LGLP and EX4 genes.
[0070] (1) INS-1 cells (rat pancreatic islet cells) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 0.5 mM pyruvate and 5 μM β-mercaptoethanol in a CO2 incubator at 37°C.
[0071] (2) INS-1 cells were seeded at 5 x 10³ cells / well into 96-well culture plates and cultured until the logarithmic growth phase;
[0072] (3) Lactococcus lactis transformed with empty vectors pLXS, GLP-8G, and EX4 genes at a ratio of 1:100 was inoculated into RPMI1640 basal medium and cultured at 30℃ until the OD600 reached 0.8-1.0. Then, xylooligosaccharide was added to a final concentration of 0.1-2.0% to induce expression, and the culture was continued for 6 hours. The supernatant of Lactococcus lactis transformed with pLXS, GLP-8G, and EX4 genes was taken, filtered through a 0.22μm filter to remove bacteria, and the pH was adjusted to 7.3 with Tis-NaCl. The supernatant was added to INS-1 cells in 96-well culture plates, and an equal volume of RPMI1640 was added as a blank control group. Four replicates were set up for each group, and the culture was continued for 24 hours.
[0073] (4) Cell proliferation (survival) assay: Add 10 μl of CCK-8 solution to each well containing cultured cells, continue incubation for 2 hours, and then measure the absorbance at 450 nm using an ELISA reader. With the average OD450 nm of the blank control group as 100%, calculate the cell survival rate (percentage relative to the blank control group).
[0074] (5) Determination of insulin expression and secretion levels in cell culture supernatant: Using an insulin ELISA kit, according to the kit's instructions, add cell culture supernatant of each group to a microplate, cover with a membrane, incubate for 2 hours, wash 3 times with washing buffer, add enzyme-labeled secondary antibody and incubate for 40 minutes, repeat the above operation, wash 3 times, add dimethylbenzidine (TMB) chromogenic substrate, add stop solution, and measure the absorbance (OD) value at 450 nm using an ELISA reader; at the same time, measure the absorbance (OD) value using insulin standard, plot a standard curve, and calculate the corresponding insulin level of each cell culture supernatant sample.
[0075] 4. Animal experiments on the treatment of obesity and diabetes with live Lactococcus lactis transformed with LGLP and EX4 genes.
[0076] (1) Culture of LGLP and EX4 gene-transformed bacteria. LGLP and EX4-transformed lactococci (obtained in Example 1) were revived in MRS medium. After the bacteria reached a certain concentration, they were inoculated into MRS medium at a certain ratio (1:100-500). When the bacterial density reached OD600 of 1.0-1.5, the bacteria were collected.
[0077] (2) Preparation of live bacterial suspension. Once the transformed bacteria reach a certain density, centrifuge at 5000g-10000g for 5-10 minutes to collect the bacteria. Add an appropriate amount of physiological saline and xylooligosaccharide (the inducing agent) to a final concentration of 10.0% to resuspend the bacteria, achieving a viability rate of approximately 10%. 9CFU / ml bacterial suspension. Store at 4-8°C.
[0078] (3) Preparation of freeze-dried live bacterial preparations. After the transformed bacteria are cultured to a certain density, the bacteria are collected by centrifugation at 5000g-10000g for 5-10 minutes. The bacteria are resuspended and mixed with a freeze protection solution (15% skim milk powder, 5% glycerol, 0.9% NaCl) in a certain proportion, dispersed evenly, and pre-frozen in an ultra-low temperature freezer until completely frozen. Then, the bacteria are placed in a freeze dryer and dried under a vacuum of 10.0-12.00 Pa until the moisture content of the bacterial powder is >3%. After freeze-drying, the bacterial powder is taken out, dispensed into sterile sealed containers, and stored at -20℃ for later use.
[0079] (4) Preparation of spray-dried enteric-coated live bacteria preparation. After the transformed bacteria are cultured to a certain density, they are collected by centrifugation at 5000g-10000g for 5-10 minutes. Enteric encapsulation material and a protectant are added for resuspension. The mixture is then spray-dried into enteric-coated live bacteria powder under specific drying conditions: 100-120℃, fan frequency 50Hz, and liquid flow rate 20r / s. The powder is stored in a refrigerator at 4-8℃ for later use.
[0080] (5) Determination of bacterial activity of live Lactococcus lactis powder. Weigh 0.1 g of powder, resuspend in 1.0 mL MRS medium, and dilute in a gradient of 1:100, 1:1000, and 1:1000. Spread 0.1 mL of each solution onto MRS solid medium plates and incubate at 37 °C for 48 h in an anaerobic incubator. Calculate the cfu / g of the powder.
[0081] (6) Oral administration of live Lactococcus lactis to treat obese diabetic mice. DB homozygous obese diabetic mice (BKS) were used... leptr- / - Mice were randomly divided into 5 groups, with 6-8 mice in each group: a blank control group (BC) orally administered sterile resuspension; a negative control group orally administered pLXS-transformed Lactobacillus lactis (Vector group); experimental groups orally administered pLXS-LGLP-transformed bacteria (LGLP group) and pLXS-EX4-transformed bacteria (EX4 group), respectively; and a positive control group intraperitoneally injected with EX4 polypeptide (EX4-IP group). A group of age-matched normal mice was also included as a control (NC). Mice in each group fed the transforming bacteria were given 0.1 ml (approximately 6 × 10⁻⁶) of the transforming bacteria. 9 CFU (Chronic Fumarate) enteric-coated live bacteria powder. Administered once daily for 4 weeks. EX4-IP group mice received an intraperitoneal injection of 100 ng once daily for 4 weeks. Food intake and body weight were measured every other day, fasting blood glucose was measured weekly, and an oral glucose tolerance test (OGTT) was performed every two weeks. After 4 weeks, mice were fasted for 12 hours, their eyeballs were enucleated to collect peripheral blood, and they were euthanized by cervical dislocation. The liver, pancreas, intestines, and intestinal contents were collected. Figure 5Peripheral blood glucose, total triglycerides (TRIG), and total cholesterol (CHOL) levels were measured using a micro-volume fully automated biochemical analyzer. Pancreatic islet structure was analyzed using immunohistochemistry. Pancreatic islet cell proliferation in mice was detected using BrdU in vivo tracing (DNA replication) combined with immunofluorescence. Liver tissue sections were stained with Oil Red O to assess the improvement of fatty liver.
[0082] Figure 4 Immunoassay results for the in vitro induced expression of LGLP and EX4 peptides in lactic acid bacteria transformed with LGLP and EX4. Figure 4 As shown, after 0-36 hours of induction with xylooligosaccharide at a final concentration of 0.5% for LGLP and EX4 transformed lactic acid bacteria, the protein in the supernatant culture was detected by Western blotting using anti-GLP-1 and anti-Exendin-4 antibodies. Lane 1: Empty vector transformed bacteria (Vector) induced with xylooligosaccharide for 24 hours; Lanes 2-6: LGLP and EX4 transformed lactic acid bacteria induced with xylooligosaccharide for 0-36 hours. The LGLP peptide in the supernatant of LGLP transformed lactic acid bacteria reached its peak 12-24 hours after xylooligosaccharide induction; the EX4 peptide in EX4 transformed lactic acid bacteria reached its peak 36 hours after xylooligosaccharide induction.
[0083] Figure 5 The results showed that LGLP and EX4-transformed *Lactococcus lactis* expression supernatant promoted pancreatic islet cell proliferation and insulin secretion in vitro. Figure 5 As shown, recombinant LGLP and EX4 peptides expressed and secreted by *Lactococcus lactis* transformed with LGLP and EX4 can promote pancreatic islet cell proliferation and insulin secretion in vitro. Treatment of rat INS-1 islet cells with *Lactococcus lactis* culture supernatant at concentrations of 60 ng and 100 ng for 48 hours significantly promoted INS-1 islet cell proliferation, with a cell proliferation rate (percentage) exceeding that of cells treated with empty vector-transformed bacteria (negative control cells) by 100%. Treatment of rat INS-1 islet cells with *Lactococcus lactis* culture supernatant at concentrations of 60 ng and 100 ng for 24 hours significantly promoted insulin secretion, with the 100 ng concentration showing a better effect. Only the 100 ng concentration of *Lactococcus lactis* culture supernatant significantly promoted insulin secretion; the 20 ng and 60 ng concentrations of *EX4* culture supernatant had no significant effect on insulin secretion. Therefore, high concentrations of recombinant LGLP and EX4 peptides significantly promote insulin secretion from cultured islet cells in vitro.
[0084] Figure 6 Results of oral administration of live lactic acid bacteria transformed from LGLP and EX4 to obese diabetic (DB) mice. Figure 6As shown, oral treatment with LGLP and EX4-transformed live Lactococcus lactis, along with an inducer, significantly reduced fasting blood glucose and improved glucose tolerance in obese diabetic mice. After 2 weeks of treatment with LGLP-transformed Lactococcus lactis, fasting blood glucose decreased significantly; after 3 weeks of treatment with EX4-transformed Lactococcus lactis and EX4 peptide intraperitoneal injection (EX4-IP), oral blood glucose levels decreased significantly. The hypoglycemic effect of LGLP-transformed Lactococcus lactis was superior to that of EX4-transformed Lactococcus lactis and also superior to the EX4 peptide intraperitoneal injection group (EX4-IP). Oral treatment with LGLP and EX4-transformed live Lactococcus lactis significantly improved glucose tolerance in diabetic mice. After 4 weeks of treatment with LGLP-transformed Lactococcus lactis, blood glucose concentration decreased significantly 60 minutes after oral glucose administration, and returned to near normal levels by 90 minutes. Compared with the empty vector-transformed bacteria treatment group, EX4-transformed Lactococcus lactis and EX4 peptide intraperitoneal injection (EX4-IP) also showed a better effect in improving oral glucose tolerance. The LGLP-transformed bacteria were more effective than the EX4-transformed Lactococcus lactis group in improving oral glucose tolerance, and also more effective than the EX4 peptide intraperitoneal injection group.
[0085] Figure 7 This is the immunohistochemical result of pancreatic islet tissue in DB obese diabetic mice after oral treatment with live Lactococcus lactis transformed by LGLP and EX4. Figure 7 As shown, oral administration of live Lactococcus lactis transformed by LGLP and EX4 significantly restored the structure and function of the pancreatic islets in obese diabetic mice. Immunohistochemical analysis of islet tissue revealed that pancreatic islet function was significantly impaired in obese diabetic (DB) mice, with smaller islet volume and lower insulin secretion. After 4 weeks of oral treatment with live Lactococcus lactis transformed by LGLP and EX4 plus an inducer, pancreatic islet function significantly recovered, islet volume significantly increased, and insulin secretion increased. BrdU immunofluorescence assays tracing pancreatic islet cell proliferation (DNA replication) showed that oral treatment with live Lactococcus lactis transformed by LGLP and EX4 plus an inducer for 4 weeks significantly stimulated islet cell (β-cell) proliferation, significantly increased islet volume, and increased insulin secretion (green fluorescence).
[0086] The above embodiments are some implementations of the present invention and are not limited to the present invention. For those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A Lactococcus lactis expression vector suitable for intraluminal induction of expression, characterized in that, By utilizing the promoter and secretory signal peptide of the extracellular exoxylanase ECX gene of Bifidobacterium to control the expression and secretion of exogenous genes, the expression and secretion of exogenous target genes can be induced in the intestinal lumen or in vitro using Lactococcus lactis as the host bacterium and xylooligosaccharide as the inducer. The method for constructing the expression vector is as follows: using Bifidobacterium NCC2705 genomic DNA as a template, the DNA sequence fragment including the promoter and secretory signal peptide of the ECX gene is amplified by PCR using upstream primer ECXF1 and downstream primer ECXR1; the fragment is inserted upstream of the multiple cloning site of the Lactococcus lactis expression vector pNZ8149, replacing the original nisin promoter Pnisin DNA sequence in the pNZ8149 vector, to obtain the pLXS expression vector; The nucleic acid sequences of the upstream primer ECXF1 and the downstream primer ECXR1 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the nucleotide sequences of the ECX gene promoter and the secretory signal peptide DNA fragment are shown in SEQ ID NO:3 and SEQ ID NO:
4.
2. The *Lactococcus lactis* expression vector suitable for in vitro expression as described in claim 1, characterized in that, The exogenous target gene is green fluorescent protein, long-acting glucagon-like peptide, and / or Exendin-4.