Recombinant listeria monocytogenes membrane vesicle carrying foreign protein as well as preparation method and application of recombinant listeria monocytogenes membrane vesicle

Abstract

The invention relates to the technical field of biology, and discloses a recombinant listeria monocytogenes membrane vesicle carrying foreign protein and a preparation method and application thereof.LLO enriched on MVs is creatively used as a guide sequence, a fusion gene segment (phly-hly-foreign protein gene) is firstly designed and synthesized, and then the fusion gene segment (phly-hly-foreign protein gene) is synthesized; then subcloning the fusion gene segment phly-hly-foreign protein gene into a vector plasmid, so as to obtain a recombinant plasmid; replacing an antibiotic resistance gene in the recombinant plasmid with an asd gene to obtain an antibiotic-free expression recombinant plasmid; electrically transferring the recombinant plasmid into a listeria monocytogenes nutrition deletion strain LM [delta] daldat, and constructing to obtain a recombinant strain; and culturing the recombinant strain, collecting bacterial liquid, and extracting and purifying MVs. Compared with a wild strain LM, the MVs secreted by the recombinant strain have no obvious difference in shape structure and particle size, and the recombinant strain can successfully guide and express foreign proteins onto the LM MVs, so that the invention provides the application of the recombinant listeria monocytogenes membrane vesicles as vaccines, vaccine carriers, drug carriers or vaccine adjuvants in preparation of biological agents.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to a recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein, and a preparation method and application thereof. Background Art

[0002] Bacterial extracellular vesicles (BMVs) are lipid bilayer membrane nanostructures released by bacteria into the extracellular environment during their growth. Vesicles secreted by Gram-negative bacteria are called outer membrane vesicles (OMVs), while vesicles secreted by Gram-positive bacteria are called membrane vesicles (MVs). BMVs have a diameter between 20 and 200 nm and contain a variety of bacterial components, including lipids, proteins, nucleic acids, etc. They can mediate bacteria-bacteria and bacteria-host interactions and participate in a variety of biological activities such as bacterial pathogenicity, signal transduction, quorum sensing, and stress. The complex and diverse components on BMVs make them immunogenic and can effectively stimulate the body to produce an immune response. The nanoscale vesicle structure gives them the ability to carry exogenous antigens and encapsulate small molecule drugs. Therefore, BMVs are considered to be a promising vaccine carrier platform, drug delivery system, and protein delivery system.

[0003] Listeria is a genus of G + Non-spore-forming short rods are intracellular parasites. Currently, multiple species have been discovered, including Listeria monocytogenes (LM) and Listeria ivanovii (LI). LM and LI can produce a variety of virulence factors such as internalization and hemolysin, promote phagocytosis and escape from lysosomes, and stimulate both cellular and humoral immune responses. In 2013, Lee et al. first discovered that LM can also produce MVs, which also carry a large number of bacterial components, including the classic virulence factor Listeriolysin O (LLO).

[0004] Studies have found that LM MVs are similar to other BMVs, have good biosafety, high immunogenicity, and can be used as an immune adjuvant to regulate the body's humoral immune response. However, there are no reports on the use of LM MVs as vaccines, vaccine / drug delivery carriers, etc. Summary of the invention

[0005] In view of this, the present invention provides a recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein, and a preparation method and application thereof.

[0006] The specific technical solution of the invention is as follows:

[0007] A method for preparing recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins comprises the following steps:

[0008] Step 1, obtaining a fusion gene fragment comprising a hemolysin O gene promoter, a hemolysin O gene and a foreign protein gene: phly-hly-foreign protein gene;

[0009] Step 2, subcloning the fusion gene fragment phly-hly-foreign protein gene into the vector plasmid to obtain a recombinant plasmid;

[0010] Step 3, replacing the antibiotic resistance gene in the recombinant plasmid in step 2 with the asd gene to construct an antibiotic-free expression recombinant plasmid;

[0011] Step 4, electro-transferring the antibody-free expression recombinant plasmid obtained in step 3 into the Listeria monocytogenes nutrient-deficient strain LMΔdaldat to construct a recombinant strain;

[0012] Step 5: Cultivate the recombinant strain, collect the bacterial liquid, and extract and purify MVs.

[0013] The present invention provides a recombinant Listeria monocytogenes membrane vesicle carrying exogenous proteins, creatively using LLO enriched on MVs as a guide sequence. Compared with the wild strain LM, the shape structure and particle size of the MVs secreted by the recombinant strain LMΔdaldat::pCW630 have no obvious differences, and the recombinant strain LMΔdaldat::pCW630 can successfully guide the expression of exogenous proteins on LM MVs.

[0014] Furthermore, the specific operation process in step 5 is as follows:

[0015] The recombinant strain was cultured in a culture medium. When the OD600 value of the bacterial solution reached 0.8 to 1.0, the bacterial solution was inoculated into the broth, shaken, centrifuged, and the supernatant was collected.

[0016] The supernatant is concentrated by ultrafiltration, the concentrate is centrifuged at high speed, and the precipitate is resuspended to obtain a crude MVs product;

[0017] The crude MVs extract was purified by density gradient centrifugation to obtain a purified MVs product.

[0018] Another object of the present invention is to provide recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins obtained by the above preparation method.

[0019] A recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein is prepared by the above-mentioned preparation method.

[0020] Another object of the present invention is to provide new applications of the membrane vesicles.

[0021] The above-mentioned recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins are used as vaccines, vaccine carriers, drug carriers or vaccine adjuvants in the preparation of biological preparations.

[0022] Furthermore, the biological agent is a vaccine, a drug or an immunomodulatory agent.

[0023] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0024] 1. The present invention provides a recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein, creatively using LLO enriched on MVs as a guide sequence, firstly designing and synthesizing a fusion gene fragment (phly-hly-exogenous protein gene), and then subcloning the fusion gene fragment phly-hly-exogenous protein gene into a vector plasmid to obtain a recombinant plasmid; then the antibiotic resistance gene in the recombinant plasmid is replaced to construct an antibiotic-free expression recombinant plasmid; the obtained antibiotic-free expression recombinant plasmid is electroporated into the Listeria monocytogenes nutritional deficiency strain LMΔdaldat to construct a recombinant strain; the recombinant strain is cultured, the bacterial liquid is collected, and the MVs are extracted and purified. Compared with the wild strain LM, there is no significant difference in the shape structure and particle size of the MVs secreted by the recombinant strain LMΔdaldat::pCW630, and the recombinant strain LMΔdaldat::pCW630 can successfully guide the expression of exogenous proteins into LMMVs. The application of recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins as vaccines, vaccine vectors, drug carriers or vaccine adjuvants in the preparation of biological preparations is proposed.

[0025] 2. The vesicles secreted by the recombinant strain obtained in the present invention showed a typical spherical structure under a transmission electron microscope, with an average particle size of 135.3 nm, and had no significant difference from the vesicles secreted by the wild strain.

[0026] 3. The plasmid transferred into the recombinant strain obtained by the present invention can be stably inherited, and the genetic characteristics of the constructed recombinant strain remain stable after 25 consecutive generations in vitro.

[0027] 4. The present invention is based on the balanced lethal system. By constructing an expression plasmid carrying the bacterial nutritional gene asd, the guide gene and the exogenous antigen fusion gene fragment, and not containing an antibiotic resistance gene, the expression plasmid is electroporated into the constructed nutritional deficiency strain to construct a recombinant strain, thereby guiding the exogenous antigen to the bacterial vesicle. The technical solution provided by the present invention can be extended to all exogenous protein antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Schematic diagram of the plasmid involved in preparing the recombinant strain LMΔdaldat::pCW630 of the present invention, wherein: Figure 1 a in is plasmid pCW627, Figure 1 b in the figure is plasmid pCW630.

[0029] Figure 2 Schematic diagram of the construction process of the recombinant strain LMΔdaldat::pCW630 of the present invention.

[0030] Figure 3 The colony PCR verification results of the recombinant strain LMΔdaldat::pCW630 prepared in the present invention.

[0031] Figure 4 This is the in vitro growth curve of the wild strain LM and the recombinant strain LMΔdaldat::pCW630 in the present invention.

[0032] Figure 5 This is an in vitro genetic stability test of the recombinant strain LMΔdaldat::pCW630 in the present invention.

[0033] Figure 6 These are transmission electron micrographs of the MVs secreted by the wild-type LM and the recombinant strain LMΔdaldat::pCW630 in the present invention, used to observe the morphology of the MVs.

[0034] Figure 7 This is a dynamic light scattering analysis of the MVs secreted by the wild-type LM and the recombinant strain LMΔdaldat::pCW630 in the present invention, used to characterize the particle size distribution of the MVs.

[0035] Figure 8 The Western blot analysis of the secretion of MVs by the wild-type LM and the recombinant strain LMΔdaldat::pCW630 in the present invention is shown. Figure 8 In a, the primary antibody was rabbit anti-LLO polyclonal antibody, and the secondary antibody was goat anti-rabbit IgG antibody; Figure 8 In b, the primary antibody is mouse anti-OVA monoclonal antibody, and the secondary antibody is goat anti-mouse IgG antibody. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] The following examples disclose in detail the method and application of directing exogenous proteins into the membrane vesicles of Listeria monocytogenes.

[0039] In the following examples, the exogenous protein used is ovalbumin OVA. In other embodiments, the exogenous protein is not limited to OVA protein, and can be other exogenous proteins, such as HPV16 E6 protein, HPV16 E7 protein, influenza virus hemagglutinin protein, etc.

[0040] Example 1

[0041] Construct the non-antibody plasmid pCW630, such as Figure 2 shown.

[0042] 1.1 Construction of plasmid pCW627

[0043] After the design of the phly-hly-ova fusion gene was completed, the company conducted full gene synthesis and subcloning construction to obtain the pCW627 plasmid, and the plasmid pCW627 was electroporated into the competent state of the nutrient-deficient Escherichia coli DH5αΔasd (Construction of a balanced lethal system of Listeria expressing cervical cancer antigen gene and its biological characteristics, Journal of Sichuan University (Medical Edition), November 2023, 52(6):1159-1166) to construct the recombinant strain DH5αΔasd::pCW627. The sequence of the fusion gene fragment phly-hly-ova is as shown in SEQ ID NO:1, and the sequence of the plasmid pCW627 is as shown in SEQ ID NO:2, and the schematic diagram is shown in Figure 1 a in.

[0044] 1.2 Construction of plasmid pCW628

[0045] The EryR gene in pCW627 was replaced with the phly-LM dal gene to obtain pCW628, whose sequence is shown in SEQ ID NO:3.

[0046] 1.2.1 Extraction of plasmids pCW627 and pCW-gfp-LM dal

[0047] Escherichia coli DH5αΔasd::pCW627 stored at -20°C and the strain LMΔdd:dal (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, Ou Q, Liu T, Huang H, Tang T, Wang C. A Listeria ivanovii balanced-lethal system may be a promising antigen carrier for vaccine construction. Microb Biotechnol. 2022 Nov; 15(11): 2831-2844.) stored at -20°C were streaked and inoculated into 5 mL of LB (Luria-Bertani) solid medium (hereinafter referred to as LA plate) supplemented with 100 μg / mL ampicillin (Ampicillin, Amp), and cultured overnight at 37°C. A single colony was inoculated into 5 mL of LB liquid medium (hereinafter referred to as LA broth) supplemented with 100 μg / mL Amp, and incubated overnight at 37°C at 200 rpm. According to the instructions of the plasmid extraction kit, plasmids pCW-gfp-LM dal and pCW627 were extracted respectively and eluted with an appropriate amount of sterile ddH2O. The plasmid concentration and purity were determined using Nanodrop 2000.

[0048] 1.2.2 Preparation of Vector628 fragment

[0049] Plasmid pCW627 was used to prepare vector fragment Vector 628. 10 μg of plasmid pCW627 was double-digested with restriction endonucleases SalⅠ and SpeⅠ, and the two fragments were separated by agarose gel electrophoresis (1.5% agarose gel, 90V voltage), and the large fragment (7907 bp) was purified and recovered using a gel recovery kit, i.e., vector fragment Vector 628. Nanodrop 2000 was used to determine the concentration and purity of the recovered fragment. The sequence of vector fragment Vector 628 is shown in SEQ ID NO: 4.

[0050] 1.2.3 Preparation of insert fragment phly-LM dal

[0051] Primers phly-LM dal-F / R are used to amplify the insert fragment phly-LM dal from the plasmid pCW-gfp-LM dal, and the amplification can be performed under conventional PCR conditions. After the amplification, the fragments are separated by agarose gel electrophoresis (1.5% agarose gel, 90V voltage), and the insert fragment phly-LM dal (1381bp) is purified and recovered using a gel recovery kit. The sequence of primer phly-LM dal-F is as shown in SEQ ID NO:5, the sequence of phly-LM dal-R is as shown in SEQ ID NO:6, and the sequence of the insert fragment phly-LM dal is as shown in SEQ ID NO:7.

[0052] 1.2.4 Connection conversion

[0053] Use a seamless ligation kit to connect the vector fragment Vector628 and the insert fragment phly-LM dal. For specific operations, see the instructions.

[0054] Thaw E. coli TOP10 competent cells on ice, add 5 μL of ligation product, mix gently with fingertips, and let stand on ice for 30 minutes. Heat shock in 42℃ water bath for 45 seconds, quickly transfer to ice, and let stand for 2 minutes. Add 700 μL LB broth and resuscitate at 37℃ 200rpm for 1 hour. After the recovery, centrifuge at 12000rpm for 2 minutes, discard about 600 μL of supernatant, and resuspend the bacterial pellet. According to the experimental needs, aspirate different volumes of resuspended bacterial solution and spread it on LA plates and culture overnight at 37℃.

[0055] 1.2.5 Screening

[0056] Pick a single colony grown on the LA plate and add it to 20 μL ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. Primers pCW628-SX-F / R are used for colony PCR screening. The sequence of pCW628-SX-F is as shown in SEQ ID NO: 8, and the sequence of pCW628-SX-R is as shown in SEQ ID NO: 9.

[0057] 1.2.6 Sequencing

[0058] The correct single colony verified by colony PCR was selected and inoculated into LA broth, and the plasmid was extracted and sent to a sequencing company for sequencing. The plasmid with the correct sequencing was named pCW628, and the plasmid pCW628 was electroporated into the competent cell of the nutrient-deficient Escherichia coli DH5αΔasd to construct the recombinant strain DH5αΔasd::pCW628.

[0059] 1.3 Construction of the anti-antibody plasmid pCW630

[0060] The asd gene was used to replace the AmpR gene in pCW628 to obtain pCW630, whose sequence is shown in SEQ ID NO:10.

[0061] 1.3.1 Bacterial strain recovery

[0062] The Escherichia coli DH5αΔasd (Construction of the balanced lethal system of Listeria expressing cervical cancer antigen gene and its biological characteristics, Journal of Sichuan University (Medical Edition), November 2023, 52(6):1159-1166) stored at -20°C was streaked and inoculated into a solid culture medium supplemented with 50 μg / mL diaminopimelic acid (DAP) (hereinafter referred to as DAP-LB plate), and cultured at 37°C overnight; the Escherichia coli DH5αΔasd::pCW628 stored at -20°C was inoculated into a 5mLLA plate and cultured at 37°C overnight.

[0063] 1.3.2 Preparation of competent E. coli DH5αΔasd

[0064] Pick a single colony and inoculate it into 5mL LB liquid culture medium (hereinafter referred to as DAP-LB broth) supplemented with 50μg / mL DAP, and shake at 37℃200rpm overnight. Pipette 1mL of fresh bacterial solution and inoculate it into 50mL DAP-LB broth, shake at 37℃200rpm until the OD600 value is about 0.5 to collect the bacteria. Transfer the bacterial solution to a 50mL centrifuge tube, ice bath for 30min, centrifuge at 4℃8000rpm for 3min, and discard the supernatant. Add 10mL pre-cooled 0.1mol / L CaCl2 to resuspend, centrifuge at 4℃6000rpm for 3min, and discard the supernatant. Resuspend with 1mL pre-cooled 0.1mol / L CaCl2, divide into 50μL / tube and store at -80℃.

[0065] 1.3.3 Preparation of vector fragment Vector 630 and insert fragment asd

[0066] Plasmid pCW628 was extracted, and the vector fragment and the insert fragment were prepared with reference to Examples 1.2.2 and 1.2.3. Primers Vector630-F / Vector630-R were used to amplify the vector fragment Vector 630 from plasmid pCW628, and primers asd-F / asd-R were used to amplify the insert fragment asd from plasmid pYA3342 (Construction of Listeria Balanced Lethal System Expressing Cervical Cancer Antigen Gene and Study on Its Biological Characteristics, Journal of Sichuan University (Medical Edition)). The sequence of primer Vector630-F is as SEQ ID NO: 11, the sequence of Vector630-R is as SEQ ID NO: 12, the vector fragment Vector 630 is as SEQ ID NO: 13, the sequence of primer asd-F is as SEQ ID NO: 14, the sequence of asd-R is as SEQ ID NO: 15, and the sequence of insert fragment asd is as SEQ ID NO: 16.

[0067] 1.3.4 Connection conversion

[0068] Referring to Example 1.2.4, the vector fragment Vector 630 and the insert fragment asd were ligated and transformed, and the ligation product was transformed into DH5αΔasd competent cells, spread on LB plates and cultured at 37°C overnight.

[0069] 1.3.5 Screening

[0070] Positive clones were screened with reference to Example 1.2.5. Primers asd-SX-F / R were used for colony PCR screening. The sequence of asd-SX-F is shown in SEQ ID NO: 17, and the sequence of asd-SX-R is shown in SEQ ID NO: 18.

[0071] 1.3.6 Sequencing

[0072] Select a single colony that has been verified by colony PCR and inoculated into LB broth, extract the plasmid and send it to a sequencing company for sequencing. The plasmid that has been sequenced correctly is named pCW630, and the plasmid pCW630 is electroporated into the competent cell of the nutrient-deficient Escherichia coli DH5αΔasd to construct the recombinant strain DH5αΔasd::pCW630. See the schematic diagram of the pCW630 plasmid for details. Figure 1 b in.

[0073] Example 2

[0074] Construction of LMΔdaldat::pCW630

[0075] 2.1 Preparation of LMΔdaldat competent cells

[0076] 2.1.1 Bacterial strain recovery

[0077] Take the LMΔdaldat strain (Construction of the balanced lethal system of Listeria expressing cervical cancer antigen gene and its biological characteristics, Journal of Sichuan University (Medical Edition), November 2023, 52(6):1159-1166) stored at -20°C and streak it onto a Brain-Heart Infusion Broth (BHI) plate (hereinafter referred to as D-BHI plate) supplemented with 200 μg / mL D-alanine, and culture it at 37°C for 24 hours. Pick a single colony and inoculate it onto another D-BHI plate and culture it at 37°C for 24 hours.

[0078] 2.1.2 Preparation of competent cells

[0079] Pick 3 to 4 single colonies on the above plate and inoculate them into 15 mL of BHI liquid culture medium (hereinafter referred to as D-BHI broth) (containing 0.5 mol / L sucrose) with 200 μg / mL D-alanine, and shake at 37°C, 200 rpm overnight. The next morning, inoculate the aforementioned 15 mL of fresh bacterial solution into 250 mL of D-BHI broth (containing 0.5 mol / L sucrose), and shake at 37°C, 200 rpm. Adjust to zero with D-BHI broth (containing 0.5 mol / L sucrose), and monitor the OD600 value of the bacterial solution regularly. When the OD600 value reaches 0.4, add penicillin G solution (final concentration is 12.5 μg / mL) and continue to shake. When the OD600 value of the bacterial solution reaches 0.7 or begins to decrease, collect the bacteria. Divide the bacterial solution into 50 mL centrifuge tubes, centrifuge at 4°C, 13000 rpm for 5 minutes, and discard the supernatant. Add 20mL of pre-cooled 0.5mol / L sucrose solution to resuspend the bacterial pellet, centrifuge at 4℃10000rpm for 10min, discard the supernatant, and repeat this step once. Resuspend the bacterial pellet with 300μL of 0.5mol / L sucrose solution, divide into 50μL / tube and store at -80℃. Collect the bacterial cells on ice throughout the process.

[0080] 2.2 Preparation of plasmid pCW630

[0081] Pick out E. coli DH5αΔasd::pCW630 stored at -20℃ and streak it onto LB plate and culture it at 37℃ overnight to recover the strain. Pick out a single colony and inoculate it into 5mL LB broth and shake it at 37℃ 200rpm overnight. Extract plasmid pCW630 according to the instructions of the plasmid extraction kit and elute it with 35μL sterile ddH2O. Use Nanodrop 2000 to determine the plasmid concentration and purity.

[0082] 2.3 Electroporation

[0083] Precool 10μL and 200μL pipette tips and electroporation cups at -20℃, and preheat 1000μL pipette tips and BHI broth (containing 1mol / L sucrose) at 37℃. Thaw LM competent cells and plasmid pCW630 on ice. Pipette 5μL plasmid solution dropwise and add to the corresponding 50μL competent cells, mix gently with fingertips, and place on ice for 5min. Transfer the above mixture to the electroporation cup and place on ice for 5min. Electroporate at 1500V for 5ms on the electroporator, remove the electroporation cup, and place on ice for 5min. Add 700μL BHI broth (containing 1mol / L sucrose) to the electroporation cup, mix the bacteria, and then aspirate into a 1.5mL EP tube. Resuscitate at 37℃ and 200rpm for 2h. After the recovery, centrifuge at 12000rpm for 2min, discard about 600μL supernatant, and resuspend the bacterial pellet. Spread all the resuspended bacterial solution onto a BHI plate and culture in a 37°C incubator for 48 to 72 hours.

[0084] 2.4 Colony PCR

[0085] Pick a single colony on the BHI plate and add it to 20 μL ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The screening primers are asd-SX-F / R and OVA-SX-F / R. The asd-SX-F sequence is such as SEQ ID NO:17, the asd-SX-R sequence is such as SEQ ID NO:18, the OVA-SX-F sequence is such as SEQ ID NO:19, and the OVA-SX-R sequence is such as SEQ ID NO:20. Amplify according to conventional PCR conditions. After amplification, analyze by agarose gel electrophoresis (1% agarose gel, 90V voltage).

[0086] 2.5 Strain verification and preservation

[0087] The recombinant strains screened by colony PCR were verified using primers HomoLMdal-F / R, HomoLMdat-F / R, asd-SX-F / R and OVA-SX-F / R (HomoLMdal-F sequence such as SEQ ID NO:21, HomoLMdal-R sequence such as SEQ ID NO:22, HomoLMdat-F sequence such as SEQ ID NO:23, HomoLMdat-R sequence such as SEQ ID NO:24, asd-SX-F sequence such as SEQ ID NO:17, asd-SX-R sequence such as SEQ ID NO:18, OVA-SX-F sequence such as SEQ ID NO:19, OVA-SX-R sequence such as SEQ ID NO:20). The verification results are shown in the table below. Figure 3 a in Figure 3 b in Figure 3 The c in Figure 3The verified recombinant strain was saved in time and named LMΔdaldat::pCW630. Figure 3 In the figure, M is a 250 bp DNA Marker; in a, 1 and 2 are respectively amplified by primers HomoLMdal-F / R for LM and LMΔdaldat::pCW630; in b, 1 and 2 are respectively amplified by primers HomoLMdat-F / R for LM and LMΔdaldat::pCW630; in c, LMΔdaldat::pCW630 is amplified by primers asd-SX-F / R; in d, LMΔdaldat::pCW630 is amplified by primers asd-SX-F / R.

[0088] Example 3

[0089] LMΔdaldat::pCW630 growth curve assay

[0090] Pick the recombinant strain LMΔdaldat::pCW630 stored at -20℃ and streak it onto a BHI plate and culture it at 37℃ for 24h. Pick a single colony and inoculate it into 5mL BHI broth, and shake it at 37℃ and 200rpm overnight. Take an appropriate amount of bacterial liquid and inoculate it into 50mL BHI broth, adjust the absorbance value OD600 at 600nm to 0.06, shake it at 37℃ and 200rpm, measure OD600 every 1h, and draw a growth curve based on OD600. The growth curve of LM and LMΔdaldat::pCW630 is shown in Figure 2. Figure 4 As shown in the figure, the growth trends of LMΔdaldat::pCW630 and LM are basically the same. LM and LMΔdaldat::pCW630 entered the logarithmic growth phase after 5 hours of culture and entered the growth plateau phase after 10 hours.

[0091] Example 4

[0092] Genetic stability of LMΔdaldat::pCW630 in vitro

[0093] Pick the recombinant strain LMΔdaldat::pCW630 stored at -20℃, streak it onto a BHI plate and culture it at 37℃ for 24h. Pick a single colony and inoculate it into 5mL BHI broth, and shake it at 37℃ and 200rpm overnight. Take 10μL of fresh bacterial BHI broth culture and inoculate it into 5mL BHI broth, shake it at 37℃ and 180rpm for 16h to obtain the first generation culture. Take 10μL of the first generation culture and inoculate it into 5mL BHI broth and continue to culture it. As mentioned above, pass it through generations continuously for 25 generations. The in vitro genetic stability of the 1st, 5th, 10th, 15th, 20th, and 25th generations of bacterial cultures was detected by PCR, and the primers were HomoLMdal-F / R, HomoLMdat-F / R, HomoLIdal-F / R, HomoLIdat-F / R, LMdal-SX-F / R and asd-SX-F / R. Figure 5 As shown, the genetic characteristics of the constructed recombinant strain LMΔdaldat::pCW630 are very stable in in vitro continuous passage. Figure 5 where M is a 250 bp DNA marker; the 1st, 5th, 10th, 15th, 20th and 25th generations of LMΔdaldat::pCW630 were amplified using primers HomoLMdal-F / R, HomoLMdat-F / R, asd-SX-F / R and OVA-SX-F / R, respectively.

[0094] Example 5

[0095] Extraction, purification and characterization of MVs

[0096] 5.1 Preparation of large volume culture supernatant

[0097] 5.1.1 Bacterial strain recovery

[0098] The recombinant strain LMΔdaldat::pCW630 stored at -20°C was streaked onto a BHI plate and cultured at 37°C for 24 h. A single colony was picked and inoculated onto another BHI plate and cultured in a 37°C incubator for 24 h.

[0099] 5.1.2 Preparation of large volume culture supernatant

[0100] Pick a single colony and inoculate it into 5mL BHI broth, shake at 37℃200rpm overnight. Take 250μL of fresh bacterial solution and inoculate it into 25mL BHI broth, shake at 37℃200rpm for 16h. After adjusting the OD600 value of the above bacterial solution to about 1.0, take 10mL of bacterial solution and inoculate it into 1L BHI broth, shake at 37℃200rpm for 16h. Centrifuge at 4℃12000g for 10min and collect the culture supernatant.

[0101] 5.2 MVs extraction and purification

[0102] 5.2.1 Ultrafiltration concentration-ultracentrifugation

[0103] The culture supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was ultrafiltered and concentrated to 35 mL through a 500 KDa cutoff module. The concentrate was ultracentrifuged at 140,000 g for 6 h at 4°C, and the supernatant was discarded. The precipitate was resuspended in 500 μL PBS solution, which was the crude MVs extract.

[0104] 5.2.2 Density gradient centrifugation purification

[0105] In a 4mL centrifuge tube, iodixanol solutions with concentrations of 45%, 35%, 30%, 25%, 20%, 15%, and 10% were layered in sequence (from bottom to top), and the crude MVs extract was layered on the top layer. The samples were ultracentrifuged at 180000g at 4°C for 4h (with the smallest acceleration), and the samples were collected in layers after the centrifugation. 20μL of sample was taken from each layer, and an appropriate amount of SDS-PAGE protein loading buffer was added. The protein was denatured by boiling for 10min, and the density gradient centrifugation effect was observed by SDS-PAGE gel electrophoresis (5% concentrated gel, 10% separation gel) and Coomassie brilliant blue staining.

[0106] 5.2.3 Washing and desalting

[0107] According to the results of Coomassie Brilliant Blue staining, the liquid layer without flagella was combined, the volume was supplemented with PBS solution, and ultra-high-speed centrifugation was performed again at 140000g at 4°C for 6 hours, and the supernatant was discarded. The precipitate was resuspended in 50 μL PBS solution to obtain the purified MVs product.

[0108] 5.3 MVs characterization

[0109] 5.3.1 BCA method for protein concentration measurement

[0110] According to the instructions of the BCA protein assay kit, the protein concentration of the MVs purified product was determined for subsequent experiments.

[0111] 5.3.2TEM characterization of MVs morphology

[0112] The MVs suspension was dropped onto a 400-mesh copper grid and stained using 2% phosphotungstic acid negative staining. The morphological characteristics of the MVs were characterized by observation under a transmission electron microscope (80 KV). Figure 6 As shown, MVs (indicated by red arrows) showed typical spherical structures under TEM with a clean background, and the shape, structure, and size of MVs secreted by LMΔdaldat::pCW630 (indicated by red arrows) were not significantly different from those of LM.

[0113] 5.3.3 Dynamic light scattering analysis to characterize MVs particle size characteristics

[0114] The MVs suspension was diluted to 1 mL with PBS solution at a ratio of 1:20, added to a cuvette and measured by a particle size analyzer (Malvern Zetasizer Nano ZS). The average particle size of MVs was reported as Z-average, and the particle size distribution characteristics of MVs were presented as Intensity PSD. Figure 7 The average particle size of MVs secreted by LM was 129.3 nm, and the average particle size of MVs secreted by LMΔdaldat::pCW630 was 135.3 nm, and the particle size distribution of the two was basically the same.

[0115] Example 6

[0116] Detection of the fusion protein LLO-OVA on MVs

[0117] 6.1 MVs extraction and purification

[0118] MVs were extracted and purified according to Examples 5.1 and 5.2, and the protein concentration of the purified MVs product was determined according to the instructions of the BCA protein assay kit.

[0119] 6.2 Western blot analysis of the fusion protein LLO-OVA on MVs

[0120] Take 30μLMVs sample, add 7.5μL SDS-PAGE protein loading buffer (5×), boil for 10min to denature the protein. Prepare SDS-PAGE gel (5% concentrated gel, 10% separation gel, 1.5mm), load the sample, electrophoresis at constant voltage 80V for 30min, and then transfer to 120V for 1h. After electrophoresis, transfer the protein band to PVDF membrane, and the transfer condition is constant current 200mA on ice for 1.5h. After the transfer, use 5% skim milk powder to block at room temperature for 2h, wash once with TBST, and then incubate with antibodies. The primary antibodies are Rabbit Anti-Listeriolysin (LLO) antibody (Abcam) and Ovalbumin Monoclonal antibody (Wuhan Sanying), incubated overnight at 4℃; the secondary antibodies are goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) (Biyuntian), incubated at room temperature for 1h. After color development, the images were obtained using the Chemidox XRS gel imaging system (Bio-Rad). The results of WB detection using LLO as the target are shown in Figure 8 a in the figure, the results of WB detection using OVA as the target are shown in Figure 8 b. Figure 8It can be seen that the constructed recombinant strain LMΔdaldat::pCW630 can use LLO to guide the expression of exogenous protein OVA to secreted MVs, where Figure 8 Middle M: ​​Protein Marker; 1: MVs secreted by LM; 2: MVs secreted by LMΔdaldat::pCW630.

[0121] This embodiment provides a recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein, creatively uses LLO enriched on MVs as a guide sequence, designs and synthesizes a fusion gene fragment (phly-hly-exogenous protein gene) that fuses the hemolysin gene promoter, the hemolysin gene and the exogenous protein gene, and the hemolysin encoding gene hly is connected to the exogenous protein encoding gene through a flexible linker, and the hemolysin promoter phly is used to start the expression of the fusion protein LLO-exogenous protein. In the embodiment, the synthesized fusion gene fragment phly-hly-ova is subcloned into the expression plasmid pCW627, and pCW627 carries the phly-hly-ova fusion gene fragment. Then, the two resistance genes EryR and AmpR on the plasmid pCW627 are replaced with LM dal and asd, respectively, to construct the non-resistance expression plasmid pCW630. The non-antibody expression plasmid pCW630 was electroporated into the nutritional deficiency strain LMΔdaldat to construct a stable recombinant strain LMΔdaldat::pCW630. The recombinant strain MVs were obtained using the LM MVs extraction (ultrafiltration concentration-ultracentrifugation) and purification (density gradient centrifugation) protocols we established and optimized. Compared with the wild-type LM, the shape structure and particle size of the MVs secreted by the recombinant strain LMΔdaldat::pCW630 were not significantly different, and the recombinant strain LMΔdaldat::pCW630 could successfully guide the expression of exogenous protein OVA on LM MVs. The application of recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins as vaccines, vaccine carriers, drug carriers or vaccine adjuvants in the preparation of biological preparations was proposed.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins, characterized in that: The following steps are involved: Step 1, obtaining a fusion gene fragment comprising a hemolysin O gene promoter, a hemolysin O gene and a foreign protein gene: phly-hly-foreign protein gene; Step 2, subcloning the fusion gene fragment phly-hly-foreign protein gene into the vector plasmid to obtain a recombinant plasmid; Step 3, replacing the antibiotic resistance gene in the recombinant plasmid in step 2 with the asd gene to construct an antibiotic-free expression recombinant plasmid; Step 4, electro-transferring the antibody-free expression recombinant plasmid obtained in step 3 into the Listeria monocytogenes nutrient-deficient strain LMΔdaldat to construct a recombinant strain; Step 5: Cultivate the recombinant strain, collect the bacterial liquid, and extract and purify MVs.

2. The method for preparing recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins according to claim 1, characterized in that: The fusion gene fragment contains the hemolysin O gene promoter and the hemolysin O gene, and the foreign protein gene is fused downstream of the hemolysin O gene.

3. The method for preparing recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins according to claim 1, characterized in that: The specific operation process in step 5 is as follows: The recombinant strain was cultured in a culture medium. When the OD600 value of the bacterial solution reached 0.8 to 1.0, the bacterial solution was inoculated into the broth, shaken, centrifuged, and the supernatant was collected. The supernatant is concentrated by ultrafiltration, the concentrate is centrifuged at high speed, and the precipitate is resuspended to obtain a crude MVs product; The crude MVs extract was purified by density gradient centrifugation to obtain a purified MVs product.

4. A recombinant Listeria monocytogenes membrane vesicle carrying exogenous protein, characterized in that: The method is described in any one of claims 1 to 3.

5. Use of the recombinant Listeria monocytogenes membrane vesicles carrying exogenous proteins as claimed in claim 4 as vaccines, vaccine carriers, drug carriers or vaccine adjuvants in the preparation of biological preparations.

6. The use according to claim 5, characterized in that: The biological preparation is a vaccine, a drug or an immunomodulatory preparation.

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