CN111575309A

CN111575309A - Construction method of genetic engineering lactic acid bacteria for expressing IL-21 and application of genetic engineering lactic acid bacteria in tumor immunotherapy

Info

Publication number: CN111575309A
Application number: CN201910118843.4A
Authority: CN
Inventors: 赵树立; 卓倩; 韩煜东
Original assignee: Nanjing First Hospital
Current assignee: Nanjing First Hospital
Priority date: 2019-02-18
Filing date: 2019-02-18
Publication date: 2020-08-25

Abstract

The invention belongs to the technical field of microbial gene resources, and discloses two sections of nucleotide sequences optimized by a human IL-21 codon and an expression system thereof in lactic acid bacteria. The method is characterized in that a food-grade lactobacillus intracellular expression plasmid vector pNZ8149-IL21 and a soluble expression plasmid vector pNZ8149-sIL21 are constructed, after the vectors are transferred into a lactobacillus NZ3900 strain through electricity, a food-grade inducer Nisin is used for inducing to obtain a gene engineering lactobacillus intracellular expression strain NZ3900-IL21 and a soluble expression strain NZ3900-sIL21 which have high expression of human IL-21, and pilot products NZ3900-IL21 lysate and NZ3900-sIL21 oral liquid are prepared by respectively expanding and producing according to the growth characteristics of the two strains. The two products can be directly orally taken by a tumor patient, can relieve gastrointestinal function toxicity caused by conventional treatment of the tumor patient, can enhance anti-tumor immune functions of CD8+ T cell activity of an organism, tumor microenvironment M1/M2 transformation and the like, has obvious inhibition effect on various tumors, and has important application value in tumor immunotherapy.

Description

Construction method of genetic engineering lactic acid bacteria for expressing IL-21 and application of genetic engineering lactic acid bacteria in tumor immunotherapy

Technical Field

The invention belongs to the technical field of microbial gene resources and biosynthesis, and particularly relates to construction of a pNZ8149-IL21 recombinant plasmid, a lactobacillus genetic engineering strain which is constructed by utilizing the plasmid and is used for heterogeneously expressing intracellularly and expressing human IL-21 in a soluble manner, and application of the lactobacillus genetic engineering strain in the aspect of antitumor immunity.

Background

Interleukin-21 (IL-21) is a cytokine discovered in recent years and involved in the proliferation, differentiation and effector processes of various immune cells, including NK cells, T cells, B cells, DC cells and the like, belongs to the type I cytokine family, and is mainly represented by CD4⁺Th2 cells synthesize and secrete proteins that play a key role in regulating innate and adaptive immune responses. With the research on the tumor immune gene therapy, the anti-tumor effect of IL-21 has been receiving attention in recent years. Studies suggest that IL-21 can induce and activate Th1 cells, enhance the immune function of body cells, generate a large amount of IFN-gamma, further promote the proliferation of NK cells and the CTL effect of CD8+ T cells, and achieve the purpose of inhibiting tumor growth. The effect of IL-21 in tumor immunotherapy is proved at present, but the short half-life (only 2-4 hours) and the generation of anti-drug antibodies and the like lead to poor drug-forming performance of IL-21 recombinant protein.

Lactococcus lactis (l.lactis) is one of the common bacteria in the gastrointestinal tract, participates in regulating the ecological balance of normal flora in the gastrointestinal tract of an organism, and has become a model strain (model LAB) widely applied in the industries of fermentation, food and health care products. Since l.lactis, which has no infectivity and pathogenicity and is modified to efficiently present foreign proteins to the mucosal immune system of a host and trigger immune response, has become a safe, effective, quality-controlled bacterial vaccine, gene therapy vector, etc. for which extensive research has been conducted in recent years. Previous studies have shown that oral administration of l.lactis lysate in combination with anti-CTLA-4 antibody can enhance the anti-tumor immune activity of the antibody, by suppressing the number of AOM/DSS-induced colorectal carcinoma tumors in mice, by reducing the survival rate of the animals, etc. Is a good immune enhancement adjuvant effect.

Nisin, Nisin (Nisin), is a natural, highly effective, safe, biologically active antimicrobial peptide that has a strong inhibitory effect against many gram-positive bacteria, including food spoilage and pathogenic bacteria, and is currently the only bacteriocin in the world that is approved for use as a food additive. The recombinant gene is introduced into the system to prepare a preparation or a vaccine, the pNZ8149 vector, the NZ3900 strain and the inducer Nisin in the gene expression system are all food grade, the human recombinant IL-21 gene is introduced into NZ3900 probiotics through the system, and the active preparation is prepared through biological fermentation. The viable bacteria preparation can be directly orally taken, so that the complex and high-cost later extraction process of general genetic engineering bacteria is avoided, and a new idea is provided for producing safe and cheap biological products by genetic engineering. The invention provides a safe and nontoxic gene therapy vector, which provides theoretical basis and technical support for the feasibility study of later-stage probiotic immune preparations on tumor resistance.

Disclosure of Invention

In view of the pharmaceutical defect of the exogenous IL-21 recombinant protein, the aim of tumor immunotherapy can not be achieved by effectively utilizing the immune enhancing function of the exogenous IL-21 recombinant protein, and the invention mainly solves the problem that the IL-21 recombinant protein can be continuously and efficiently expressed in the body of a tumor patient by utilizing the IL-21 genetic engineering lactobacillus which is nontoxic, harmless and efficiently expressed in the body of the human body, thereby achieving the tumor immunotherapy effect.

In the first aspect of the invention, the pNZ8149-IL21 recombinant plasmid is characterized in that: the recombinant plasmid is named as pNZ8149-IL21, contains an optimized IL-21 gene sequence, can be stably replicated, and is expressed in a lactobacillus expression system in an intracellular manner. The plasmid is transformed into lactococcus lactis NZ3900 by an electrotransformation method to prepare an NZ3900-IL21 genetic engineering strain. The optimized IL-21 gene sequence has the sequence shown in SEQ ID NO: 1.

In the second aspect of the invention, the pNZ8149-sIL21 recombinant plasmid is characterized in that: the recombinant plasmid is named as pNZ8149-sIL21, contains lactobacillus signal peptide and optimized gene sequence of IL-21, can be stably replicated, and IL-21 is expressed in a lactobacillus expression system in a soluble manner. The plasmid is transformed into lactococcus lactis NZ3900 by an electrotransformation method to prepare an NZ3900-sIL21 genetic engineering strain. The optimized IL-21 gene sequence has the sequence shown in SEQ ID NO: 2.

In the third aspect of the invention, the invention provides an active lactobacillus preparation capable of directly orally expressing human IL-21, which is prepared by culturing NZ3900-sIL21 genetic engineering strains in a culture medium containing 8% brown sugar and 2% lactose and inducing with Nisin of 50ng/ml for 48 hours. The preparation contains active thallus no less than 10⁸cfu/ml. The microbial inoculum can be directly used for immunotherapy by oral administration, and has obvious inhibiting effect on digestive tract system tumors such as oral cancer, esophageal cancer, gastric cancer, intestinal cancer and the like without being limited by the stages and types of tumors.

The fourth aspect of the invention provides a lactobacillus lysate freeze-dried powder capable of directly orally expressing human IL-21, which is prepared by culturing NZ3900-IL21 genetic engineering strains in an M17 (0.5% lactose) culture solution and inducing with Nisin of 50ng/ml for 4 h. Collecting culture medium, centrifuging, collecting thallus, ultrasonic crushing and cracking (1,200bar, 4 deg.C, 2 min/time, 3 times), collecting lysate, adding freeze-drying protectant (12% of skimmed milk, 6% of trehalose and 2% of gel at mass ratio of 1: 1), and freeze-drying in a freeze-drying machine. The lactobacillus lysate freeze-dried powder can be diluted by normal saline and then orally taken to directly carry out immunotherapy, and has obvious inhibiting effect on digestive tract system tumors such as oral cancer, esophageal cancer, gastric cancer, intestinal cancer and the like without being limited by tumor stages and types.

Drawings

FIG. 1 is a graph of the alignment of partial sequencing results with expected sequences

FIG. 2 PCR identification of NZ3900-IL21 and NZ3900-sIL21 strains: (M.DNA Marker; 1.PNZ8149-IL21 positive plasmid; 2. blank control bacterium; 3. negative control; 4-10: PNZ8149-IL21 strain clone; 11-15: PNZ8149-sIL21 strain clone) (Marker has molecular weight of 100, 250, 500, 750, 1000, 1500, 2000bp from bottom to top respectively)

FIG. 3, SDS-PAGE identification of IL-21 protein expression of NZ3900-IL21 genetically engineered strain: wherein M: protein molecular weight; 1-4: nisin inducer 0, 30, 50 and 100ng/mL respectively induces for 4 h.

FIG. 4 Western Blot identification of IL-21 protein expression of NZ3900-IL21 genetically engineered strain: wherein M: protein molecular weight, lanes 1-4:

nisin inducer concentration

0, 30, 50 and 100ng/mL, induction for 4 h.

FIG. 5 is an ELISA identification of gene engineering strain sIL-21 protein expression of NZ3900-sIL 21: 1-4 are respectively: nisin inducer concentration of 0, 30, 50 and 100ng/mL, induction for 48 h.

FIG. 6 shows pilot products of NZ3900-sIL21 oral liquid and its control strain NZ3900 probiotic oral liquid preparation.

FIG. 7 shows pilot-scale products of lysates of NZ3900-IL21 genetically engineered bacteria and control strains NZ 3900.

FIG. 8 shows that NZ3900-sIL21 oral liquid and NZ3900-IL21 genetically engineered bacteria lysate enhance the food intake of mice: PBS blank control group; 2. a lyoprotectant control group; 3. oral liquid culture medium control group; NZ3900-sIL21 oral liquid group; NZ3900-IL21 lysate panel. P < 0.01vs PBS blank control group.

FIG. 9 shows that NZ3900-sIL21 oral liquid and NZ3900-IL21 genetically engineered bacteria lysate have no influence on the liver organ coefficients of mice. PBS blank control group; 2. a lyoprotectant control group; 3. oral liquid culture medium control group; NZ3900-sIL21 oral liquid group; NZ3900-IL21 lysate panel. P < 0.01vs PBS blank control group.

FIG. 10 shows that NZ3900-sIL21 oral liquid and NZ3900-IL21 genetically engineered bacteria lysate have no influence on mouse liver function serological indexes. Denotes p < 0.05vs PBS blank.

FIG. 11 NZ3900-sIL21 oral liquid and NZ3900-IL21 genetically engineered bacteria lysates inhibit tumor development in AOM/DSS mouse models. 1. Model PBS group; NZ3900 lysate control; NZ3900 oral control; NZ3900-IL21 lysate panel; NZ3900-sIL21 lactic acid bacteria oral liquid group.

FIG. 12 NZ3900-sIL21 oral liquid and NZ3900-IL21 genetically engineered bacteria lysates enhance the killing activity of immune cells in tumor tissues of AOM/DSS mouse models. Granzyme B is one of the killing active molecules of CD56+ NK cells and CD8+ T cells. 1. Model PBS group; NZ3900 lysate control; NZ3900 oral control; NZ3900-IL21 lysate panel; NZ3900-sIL21 lactic acid bacteria oral liquid group.

Detailed Description

The invention will be better understood from the following detailed description of the invention with reference to the drawings and specific examples, which, however, do not limit the scope of the invention.

The related culture medium and the preparation method thereof are as follows: GM medium (M17 broth + 0.5% lactose), GSGM medium (0.5M sucrose +25g/L glycine + M17 medium +5g/L glucose), GMMC recovery medium (M17 medium + 0.5% glucose +20mM MgC12+2mM CaC 12). Unless otherwise specified, the test methods and reagents in the examples are those conventionally used in the art and those commercially available.

Example 1: lactobacillus NZ3900 genetic engineering modification for expressing human IL-21 protein and preparation of oral liquid preparation and lysate freeze-dried powder thereof

1. Human IL-21 gene sequence optimization: according to the amino acid sequence (153 amino acids in total) of human IL-21 published by GeenBank, a signal peptide sequence of the first 29 amino acids is cut off, then the gene sequence is optimized after codons are optimized according to a lactic acid bacteria expression system, enzyme cutting sites of Nco I and Hind III are introduced into two ends of the sequence, and the optimized gene sequences are shown in a sequence table 1(SEQ ID NO: 1) and a sequence table 2(SEQ ID NO: 2). Then, a PAS (PCR-based amplified synthesis) method is adopted to design a full-length splicing primer, protective alkali is designed at two ends of the primer respectively, and a target gene sequence is synthesized.

2. Sequencing and verifying the obtained optimized IL-21 gene sequence: the stored puc57 plasmid and the synthetic IL-21 (or IL-21 with signal peptide, i.e., sIL-21) gene sequence were double digested with Nco I and Hind III at 37 ℃ for 4 hours. Subsequently, the cleaved target band was recovered by gel electrophoresis using 1% (BioFlux gel recovery kit). Then, according to the instructions of the T4 DNA ligation kit, the DNA was ligated according to puc57 vector: IL-21 gene sequence was ligated at a molar concentration ratio of 1: 3 and reacted at 4 ℃ overnight. 2 mu.L of the ligation product was taken to transform competent E.coli DH5 alpha cells, then plated on LB solid medium plate containing 100. mu.g/ml ampicillin, cultured overnight at 37 ℃ for 12 hours, single colonies were randomly picked up to amplify bacteria in liquid LB medium containing 100. mu.g/ml ampicillin, plasmids were extracted, and DNA sequencing was performed for identification (see FIG. 1 for sequencing results).

Plasmid construction of PNZ8149-IL21 (or PNZ8149-sIL21) and electro-transformation and identification of NZ3900 lactic acid bacteria: amplifying strains with correct sequencing, extracting puc57-IL21(puc57-sIL21) plasmid and PNZ8149 plasmid by using a plasmid extraction kit, carrying out double enzyme digestion on NcoI and Hind III, collecting the enzyme digested IL-21 and PNZ8149 plasmids by using a gel recovery kit, and connecting by using a T4 DNA connection kit as above. Uniformly mixing 1 mu L of the ligation product and 40 mu L of NZ3900 competent cells, adding the mixture into an ice-precooled electric shock cup (0.2cm), and performing electroporation under the conditions of voltage of 2.0kV, electric pulse of 25 mu F and resistance of 200 omega; immediately adding 1.0mL of precooled GMMC recovery culture medium after pulse ending, placing the mixture on ice for 5min, transferring the mixture into a 1.5mL centrifuge tube, and culturing the mixture for 1 to 1.5 hours at the temperature of 30 ℃; uniformly spreading on GM culture plate containing 0.5% lactose, respectively spreading 10 μ L, 100 μ L, 900 μ L on the plate, standing at 30 deg.C for 40 hr after the bacterial solution is completely absorbed, and allowing transformant to appear. The transformants were picked and cultured overnight at 30 ℃ in 5mL of GM medium. Transformant plasmids were extracted and the plasmid upstream and downstream primers of pNZ 8149: 5-GCGTATCTATGGCTGTCA-3 and 5-TTCAATAATCCCTCCTCTC-3 are subjected to PCR identification and enzyme cutting identification by an enzyme cutting method. Finally, the size of the target gene is verified by agarose gel electrophoresis, the correct recombinant plasmid is identified and named as pNZ8149-IL21 (or pNZ8149-sIL21), and the correct genetic engineering strain is identified and named as NZ3900-IL21 (or NZ3900-sIL21) (the PCR identification result is shown in figure 2).

4, induced expression and identification of NZ3900-IL21 lactic acid bacteria genetic engineering strains: selecting correctly identified clone strain, inoculating into 5mL M17 (containing 0.5% lactose) culture solution, and culturing at 30 deg.C until thallus OD₆₀₀About 0.4; nisin of 50ng/mL was added to the remaining culture, and anaerobic culture was carried out at 30 ℃ for 4 hours to induce expression of the fusion protein. Finally, 1mL of the culture was removed at 10000r/mCentrifuging at room temperature for 2min in, resuspending the precipitate with protein electrophoresis sample buffer solution, ultrasonically crushing the resuspension solution, and collecting the supernatant; the expression of IL-21 was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Western Blot) (see FIGS. 3 and 4 for the results of the analyses).

Inducing soluble expression and identification of NZ3900-sIL21 lactic acid bacteria genetic engineering strain: selecting a clone strain with correct identification, inoculating the clone strain in a 8% brown sugar and 2% lactose culture medium, carrying out anaerobic fermentation at 30 ℃ until OD600 is about 0.4, adding Nisin with the concentration of 50ng/ml, and continuing to induce for 48h to induce the expression of the fusion protein. Finally, 1mL of the culture is taken out, centrifuged at 10000r/min at room temperature for 2min, and the culture supernatant is collected. Finally, the secretion expression of IL-21 in the culture medium was measured by ELISA (see FIG. 5 for the results of the identification). The strain with the highest expression level of the supernatant and the culture conditions were subjected to pilot scale-up, and the culture was bottled as an oral liquid for future use (see fig. 6).

6, preparing NZ3900-IL21 freeze-dried powder of the genetically engineered lactobacillus lysate: amplification culture and induction expression of NZ3900-IL21 were carried out in the manner described above for induction expression of NZ3900-IL21 by lactic acid bacteria. Centrifuging, collecting the bacterial pellet, and processing according to 10⁹After bottling the dosage of cpu/ml/bottle, carrying out ultrasonication and lysis (1200bar, 4 ℃,2 min/time, 3 times), finally adding 1ml of freeze-drying protective agent (12% of skim milk, 6% of trehalose and 2% of gel in mass fraction ratio respectively) into each bottle according to the mass ratio of 1: 1, and finally putting into a freeze dryer for freeze-drying (see figure 7).

Example 2: toxicity study of NZ3900-IL21 lysate and NZ3900-sIL21 oral liquid on 4-week gavage in mice

SPF grade 4 week old Balb/c mice 100 (18-22g, half male and half female) were purchased from the laboratory animal center, Yangzhou university. The male and female animals are raised in cages and are raised in the animal experiment center of the first hospital in Nanjing under the conditions that the temperature is 23 +/-2 ℃ and the humidity is 55 +/-5%. After a one-week quarantine period, 20 animals (male and female halves) were divided into 5 groups, namely a blank PBS control group, a lyoprotectant control group (i.e., 12% skim milk + 6% trehalose + 2% gelatin), a NZ3900-IL21 lysate lyophilized powder group (each vial of lyophilized powder dissolved in 1ml PBS), a NZ3900-sIL21 oral liquid group, and an oral liquid medium group (8% brown sugar + 2% lactose). The administration is carried out by gavage at a dose of 0.2 ml/time/day for 28 consecutive days. The mental state, physical signs and excretions of the mice were observed every day, and the death time and number were recorded. Body weight and food consumption were weighed once a week. 12 hours after the last dose, the eyeball was bled and sacrificed for systematic dissection, and the heart, liver, spleen, lung, kidney and thymus were weighed and organ indices calculated. The collected blood samples were subjected to measurement of biochemical indices of blood, and the above organs were preserved in formalin solution for subsequent histopathological examination.

As a result:

1. general observations were: compared with a blank control group, all mice of the administration group have no death occurrence, no adverse reaction, and no difference of all behaviors from normal mice. Some mice have red and swollen eyes and slow movement during grasping due to operation reasons, but can recover automatically after free movement.

2. Body weight and food intake: although the body weight of the mice in each group showed stable increase, although the food intake of the NZ3900-IL21 lysate lyophilized powder group and the NZ3900-sIL21 oral liquid group was significantly higher than that of the PBS blank control group, the lyophilized protectant control group and the oral liquid medium control group (p < 0.01) (FIG. 8), the body weight of the mice in 5 groups was not statistically different.

3. Effect on mouse organ index: compared with the PBS blank control group, the liver index of the oral liquid culture medium control group is obviously increased (p is less than 0.05). The other dose groups were not significantly different from the blank control group. This is probably due to the sugar content in the culture medium of the oral liquid, and the sugar content in the NZ3900-sIL21 oral liquid is metabolized by the fermentation of lactic acid bacteria, so it has no abnormal toxicity to the body (FIG. 9).

4. Effect on biochemical indices of mouse blood: compared with a PBS blank control group, the glutamic-pyruvic transaminase and the glutamic-oxalacetic transaminase of the oral liquid culture medium control group are increased (P is less than 0.05), and the triglyceride is obviously increased (P is less than 0.05). The indexes of the rest groups have no significant difference compared with the PBS blank control group (figure 10).

5. And (3) histopathological detection: except for the mild fatty liver in the oral liquid culture medium control group, no organic abnormality was found in other organs.

Example 3: tumor inhibition effect of NZ3900-IL21 lysate and NZ3900-sIL21 lactobacillus oral liquid preparation in mouse AOM/DSS colorectal tumor model

SPF grade 4 week old Balb/c mice 60 (18-22g, female) were purchased from the laboratory animal center, Yangzhou university. The male and female animals are raised in cages and are raised in the animal experiment center of the first hospital in Nanjing under the conditions that the temperature is 23 +/-2 ℃ and the humidity is 55 +/-5%. 10 mice were left as normal controls, and the remaining mice were modeled using AOM/DSS in combination to induce precancerous lesions. The mice are modeled after 10mg/kg of AOM is intraperitoneally injected in a single dose on the 1 st day of the beginning of the experiment, and then 3% DSS aqueous solution is continuously and freely drunk for 5 days on the 5 th, 26 th and 47 th days, and the aqueous solution provided by a laboratory is regularly drunk at other time periods, so that the inflammatory adenoma canceration animal model is established. During the molding period, 5 mice died, and at the end of the molding on day 52, the remaining 45 mice were divided into 5 groups on average (i.e., model PBS group, NZ3900 lysate control group, NZ3900-IL21 lysate group, NZ3900 oral liquid control group, and NZ3900-sIL21 lactic acid bacteria oral liquid group), and 9 mice were each administered by gavage at 0.2 ml/time/2 days for 30 days.

As a result:

1. number of colorectal adenomas in each group: the number of tumor foci in the colorectal (p < 0.05) was not statistically different between NZ3900-IL21 lysate group and NZ3900-sIL21 lactic acid bacteria oral liquid group (p > 0.05) compared to model PBS group (FIG. 11).

2. Anti-tumor immune effects in colorectal tumor tissues: analysis of the results by immunohistochemical staining detection of CD8, CD56, and granzyme B in tumor tissues of each group indicated that positive staining for CD8, CD56, and granzyme B was significantly stronger in tumor tissues of NZ3900-IL21 lysate group and NZ3900-sIL21 lactobacillus oral liquid group than in model PBS control group (fig. 12). The results show that the gene engineering strain lysate and the oral liquid can effectively enhance the number and the activity of anti-tumor immune cells in mouse colorectal tissues after gastric lavage, thereby inhibiting the occurrence and the development of tumors.

Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

1. A recombinant plasmid vector, characterized in that the vector is pNZ8149-sIL21, which contains human interleukin 21(IL-21) suitable for soluble expression in a lactic acid bacteria host, and has the nucleotide sequence shown below:

2. a recombinant plasmid vector, characterized in that the vector is pNZ8149-IL21, which contains human interleukin 21(IL-21) suitable for intracellular expression in a lactic acid bacteria host, and has the nucleotide sequence shown below:

3. the vector of claim 1, which is genetically engineered lactic acid strain NZ3900-sIL21, characterized in that the vector is transformed with food grade probiotic strain NZ 3900.

4. The strain of claim 3, which produces NZ3900-sIL21 oral liquid, wherein the oral liquid is prepared by fermenting food-grade raw materials (8% brown sugar + 2% lactose) in a culture system and performing induction by using a food-grade inducer Nisin. The oral liquid can be used for immunotherapy and adjuvant therapy of digestive tract tumor.

5. The vector of claim 2, which is genetically engineered lactic acid strain NZ3900-IL21, characterized in that the vector is transformed with a food grade probiotic strain NZ 3900.

6. The strain of claim 5, which produces NZ3900-IL21 lysate characterized in that the lysate is prepared by induction with a food grade inducer Nisin and the lysate contains higher levels of IL-21. The lysate can be diluted and orally taken to be used for immunotherapy and adjuvant therapy of digestive tract tumors.