Multivalently expressed sheep Listeria cervical cancer vaccine and preparation method thereof

Abstract

The present invention discloses a multivalently expressed Listeria monocytogenes cervical cancer vaccine and a preparation method thereof, wherein the method comprises replacing the Listeria monocytogenes LIΔ with hemolysin. in :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. in :: hly Δ from the Δ given The fusion antigen was constructed by alternately arranging the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of the four HPV types 6 / 11 / 16 / 18 and retaining the overlapping region of 16 amino acids at the junction site; the fusion antigen was inserted into the carrier from the Gene combination asd Gene replacement Amp Gene non-resistance plasmid pCW631, obtain non-resistance plasmid pCW636; the non-resistance plasmid pCW636 is electroporated into the auxotrophic Listeria ovis LIΔ in :: hly Δ from the Δ given The cervical cancer vaccine of the present invention can multivalently express E6E7 proteins to achieve multiple protection against cervical cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to a multivalently expressed sheep Listeria cervical cancer vaccine and a preparation method thereof. Background Art

[0002] Cervical cancer is the most common gynecological malignancy. The peak age for in situ carcinoma is 30-35 years old, and for invasive carcinoma it is 45-55 years old. In recent years, the incidence has tended to be younger. The widespread use of cervical cytology screening in recent decades has enabled the early detection and treatment of cervical cancer and precancerous lesions, and the incidence and mortality of cervical cancer have decreased significantly. Traditional methods for treating cancer mainly include surgical resection, radiotherapy, chemotherapy, and therapeutic vaccines. Surgical resection, radiotherapy, and chemotherapy are prone to metastasis and recurrence. In comparison, vaccine therapy has the advantage of less damage to the body and low toxicity. Currently, the vaccines used for tumors are mainly traditional inactivated vaccines and subunit vaccines, which have disadvantages such as long immune cycles and poor immune effects.

[0003] Bacteria-based vaccines have significant advantages in delivering exogenous antigens, including low production costs, high genetic stability, good safety, and highly effective anti-tumor effects. The Listeria balanced lethal system is a green vector system. The Listeria balanced lethal system may have better immune effects due to the high copy characteristics of the plasmid. The existing Listeria-based cervical cancer vaccines are basically monovalent expression vaccines, and the protective effect of monovalent expression vaccines is limited. The existing technology lacks a Listeria cervical cancer vaccine that can polyvalently express E6E7 proteins to better achieve multiple protections against cervical cancer.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] The present invention provides a multivalently expressed sheep Listeria cervical cancer vaccine and a preparation method thereof, aiming to solve the technical problems existing in the Listeria cervical cancer vaccine mentioned in the background technology.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] The first aspect of the present invention provides a method for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine, wherein the Listeria monocytogenes cervical cancer vaccine is prepared by the following method:

[0008] Replacement of hemolysin in Listeria ovis LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat ;

[0009] The fusion antigen was constructed by alternating the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of four HPV types 6 / 11 / 16 / 18 and retaining a 16-amino acid overlapping region at the junction site.

[0010] The fusion antigen is inserted into the carrier dal Gene combination asd Gene replacement Amp gene from the resistance-free plasmid pCW631 to obtain the resistance-free plasmid pCW636;

[0011] The non-resistance plasmid pCW636 was electroporated into the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat In, obtain sheep Listeria cervical cancer vaccine.

[0012] In an optional embodiment of the first aspect of the present invention, the hemolysin replaces the sheep Listeria LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat include:

[0013] Prepare frozen stock of hemolysin-replacing Listeria ovis LIΔ ilo :: hly、 Carrying pCW619-LI dal coli carrying the top10 plasmids and pCW619-LI dat Top 10 E. coli plasmids;

[0014] Replace the hemolysin with Listeria ovis LIΔ for the frozen ilo :: hly Resuscitation is performed and competent hemolysin-replacing Listeria ovis is prepared;

[0015] The frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top10 E. coli of the plasmid was recovered and the pCW619-LI was extracted. dal Plasmids and the pCW619-LI dat plasmids;

[0016] The pCW619-LI dal Plasmids and the pCW619-LIdat The plasmids were sequentially electroporated into the competent hemolysin-replacing Listeria ovis for homologous recombination to obtain the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat .

[0017] In an optional embodiment of the first aspect of the present invention, the hemolysin is replaced with Listeria monocytogenes LIΔ ilo :: hly Resuscitation and preparation of competent hemolysin-replacing Listeria ovis include:

[0018] Replace the hemolysin with Listeria ovis LIΔ for the frozen ilo :: hly After thawing at room temperature, the cells were cultured overnight in BHI broth to obtain resuscitated Listeria ovis;

[0019] The revived Listeria ovis was expanded and cultured in BHI broth containing sucrose, and the OD600 value was measured regularly;

[0020] When the OD600 value is between 0.4 and 0.7, penicillin G is added until the OD600 value shows a downward trend, thereby obtaining competent hemolysin-replacing Listeria monocytogenes.

[0021] In an optional embodiment of the first aspect of the present invention, the frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top10 E. coli of the plasmid was recovered and the pCW619-LI was extracted. dal Plasmids and the pCW619-LI dat Plasmids include:

[0022] The frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top 10 E. coli containing the plasmid were thawed at room temperature and cultured overnight in BHI broth to obtain recovery. dal Plasmids and E. coli recovery dat Plasmid E. coli;

[0023] The Tiangen Plasmid Miniprep Kit was used to recover the dal Plasmids in E. coli and the recovery dat Plasmid E. coli was extracted to obtain the pCW619-LI dal Plasmids and the pCW619-LIdat plasmid.

[0024] In an optional embodiment of the first aspect of the present invention, the fusion antigen is inserted into the carrier dal Gene combination asd Gene replacement Amp Gene-free plasmid pCW631, to obtain the resistance-free plasmid pCW636, including:

[0025] Prepare frozen Escherichia coli carrying the non-resistance plasmid pCW631 and the Escherichia coli carrying the fusion antigen plasmid pCW635;

[0026] Frozen E. coli carrying the non-resistant plasmid pCW631 and the plasmid pCW635 carrying the fusion antigen were thawed at room temperature and then cultured overnight in LA broth to obtain E. coli with the pCW631 plasmid and E. coli with the pCW635 plasmid;

[0027] The pCW631 plasmid E. coli and the pCW635 plasmid E. coli were extracted using a centrifugal column-type Tiangen plasmid mini-extraction kit to obtain the pCW631 non-resistance plasmid and the pCW635 antigen plasmid;

[0028] Use high-fidelity enzymes and primers dal Vector amplifies the pCW631 non-resistant plasmid and recovers it by gel agarose gel to obtain a linearized vector;

[0029] Amplify the pCW635 antigen plasmid using a high-fidelity enzyme and primer HPV Insert and recover the fragment by gel recovery to obtain the antigen insert fragment;

[0030] The linearized vector and the antigen insert are subjected to a metal bath reaction at a molar ratio of 1:2 to obtain an antigen-vector ligation product;

[0031] The antigen carrier ligation product was added to the competent Escherichia coli DH5αΔ asd Carry out the transformation of the ligation product;

[0032] From the competent Escherichia coli DH5αΔ asd The antigen-positive and non-antibiotic plasmid pCW636 was extracted from the culture medium.

[0033] In an optional embodiment of the first aspect of the present invention, the antigen carrier ligation product is added to the competent Escherichia coli DH5αΔ asd Before the transformation of the ligation product, the following steps are included:

[0034] Preparing cryopreserved knockouts asd Escherichia coli DH5αΔ asd ;

[0035] The Escherichia coli DH5αΔ asd After thawing at room temperature, culture overnight in LB-DAP broth to obtain resuscitated E. coli DH5αΔ asd ;

[0036] The resuscitation of Escherichia coli DH5αΔ asd Use LB-DAP broth for expansion culture and measure OD600 value regularly;

[0037] When the OD600 value reached 0.5, CaCl2 solution was added to obtain competent Escherichia coli DH5αΔ asd .

[0038] In an optional embodiment of the first aspect of the present invention, the fusion antigen includes the promoter of the LLO hemolysin gene, the LLO signal peptide, the LLO predicted cleavage site, the GP33 protein gene, the antigen tag and the E6E7 fusion gene, and the E6E7 fusion gene is the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of the four types of HPV6 / 11 / 16 / 18 arranged alternately.

[0039] The second aspect of the present invention provides a multivalently expressed Listeria monocytogenes cervical cancer vaccine, which is prepared by the preparation method described in any one of the first aspects of the present invention.

[0040] Beneficial effect: The present invention discloses a method for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine, which is prepared by the following method: replacing the hemolysin with the Listeria monocytogenes LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat The fusion antigen was constructed by alternately arranging the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of the four HPV types 6 / 11 / 16 / 18 and retaining the overlapping region of 16 amino acids at the junction site; the fusion antigen was inserted into the carrier dal Gene combination asd Gene replacement Amp Gene non-resistance plasmid pCW631, obtain non-resistance plasmid pCW636; the non-resistance plasmid pCW636 is electroporated into the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat The cervical cancer vaccine of the present invention can polyvalently express E6E7 proteins to achieve multiple protection against cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a flowchart of the steps of the preparation method of a multivalently expressed sheep Listeria cervical cancer vaccine of the present invention.

[0042] Figure 2 This is a complete schematic diagram of the process for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine of the present invention.

[0043] Figure 3 Schematic diagram of a fusion antigen of the present invention.

[0044] Figure 4 This is a flow chart of treatment effect evaluation of the present invention.

[0045] Figure 5 This is a diagram of the treatment effect of the present invention for the grouping of poor families and treatment strategies.

[0046] Figure 6 This is a graph showing changes in tumor volume in HPV16 cervical cancer-bearing mice according to the present invention.

[0047] Figure 7 This is a survival curve of HPV16 cervical cancer-bearing mice according to the present invention.

[0048] Figure 8 This is a graph showing changes in tumor volume in HPV18 cervical cancer-bearing mice according to the present invention.

[0049] Figure 9 This is a survival curve of HPV18 cervical cancer-bearing mice according to the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0051] See also Figure 1 and Figure 2 The first aspect of the present invention provides a method for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine, comprising:

[0052] S100, in which hemolysin replaces Listeria monocytogenes LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat In the present invention, the hemolysin replaces Listeria ovis LIΔ ilo :: hlyListeria monocytogenes hemolysin gene ( ilo ) was replaced by the Listeria monocytogenes hemolysin gene ( hly ) recombinant strain, the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat That is, the hemolysin replaces the ovis LIΔ ilo :: hly Based on the knockout dal Genes and dat Gene.

[0053] In an optional embodiment of the present invention, the hemolysin replaces the Listeria ovis LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat include:

[0054] S101. Prepare frozen hemolysin to replace Listeria ovis LIΔ ilo :: hly、 Carrying pCW619-LI dal coli carrying the top10 plasmids and pCW619-LI dat coli with plasmids; in this step, the hemolysin constructed step by step can also be used to directly replace the sheep Listeria LIΔ ilo :: hly、 Carrying pCW619-LI dal coli and carrying plasmid pCW619-LI dat Top 10 Plasmids for E. coli,

[0055] S102, replacing the frozen hemolysin with Listeria monocytogenes LIΔ ilo :: hly Resuscitation is performed to prepare competent hemolysin-substituted Listeria ovis; in an optional embodiment of the present invention, this step may include: freezing the hemolysin-substituted Listeria ovis LIΔ ilo :: hly After thawing at room temperature, the culture was cultured overnight in BHI broth to obtain revived Listeria ovis; the revived Listeria ovis was expanded and cultured in BHI broth containing sucrose, and the OD600 value was regularly measured; when the OD600 value was between 0.4 and 0.7, penicillin G was added until the OD600 value showed a downward trend, thereby obtaining competent hemolysin-replacing Listeria ovis.

[0056] More specifically, this step may include, for example, taking out the glycerol-stored bacterial LIΔ from the -80°C refrigerator bacterial box. ilo :: hly After thawing at room temperature, inoculate 100 μL to 5 mL of BHI broth and culture overnight in a shaker at 37°C and 200 rpm. Use a sterile inoculation loop to pick up a loop of bacterial liquid and streak it on the corresponding plate for temporary storage. Take the LIΔ ilo :: hly The colony was transferred to 5 mL of BHI broth and cultured on a shaker at 200 rpm for 16-18 h. 300 μL of bacterial solution was added to 15 mL of BHI broth containing 0.5 mol / L sucrose and cultured overnight on a shaker at 37°C and 200 rpm. All the overnight cultured bacterial solution was transferred to 250 mL of BHI broth containing 0.5 mol / L sucrose and cultured on a shaker at 37°C and 200 rpm. The OD600 value was measured every 1 h. When the OD600 value was between 0.4 and 0.7, penicillin G was added at a final concentration of 12.5 g / L and cultured on a shaker until the OD600 value reached 0. 00 value showed a slow or downward trend; take 6 50mL sterile centrifuge tubes and place them on ice for pre-cooling, divide the bacterial solution into 50mL sterile centrifuge tubes, and centrifuge at 4℃ and 13000rpm for 5min; discard the supernatant, add 20mL of 0.5mol / L sucrose solution, and gently blow the wall of the centrifuge tube with a pipette to disperse the bacterial precipitation, and centrifuge at 4℃ and 10000rpm for 10min; discard the supernatant and repeat the washing once; aspirate the supernatant, add 400μL of 0.5mol / L sucrose solution to the centrifuge tube, mix well, divide into sterile ep tubes at 50μL / tube, and store at -80℃ for use.

[0057] S103, the frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top10 E. coli of the plasmid was recovered and the pCW619-LI was extracted. dal Plasmids and the pCW619-LI dat In an optional embodiment of the present invention, this step may include: freezing the carrier pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top 10 E. coli containing the plasmid were thawed at room temperature and cultured overnight in BHI broth to obtain recovery. dal Plasmids and E. coli recovery dat Plasmid Escherichia coli; the recovery was performed using the Tiangen Plasmid Miniprep Kit dal Plasmids in E. coli and the recovery datPlasmid E. coli was extracted to obtain the pCW619-LI dal Plasmids and the pCW619-LI dat plasmid.

[0058] More specifically, this step may include, for example, taking out the bacteria carrying pCW619-LI from the -80°C freezer box. dal coli carrying the top10 plasmids and pCW619-LI dat After the top 10 E. coli carrying the plasmid were thawed at room temperature, 50 μL of each was added to 5 mL of LA broth, and cultured overnight in a shaker at 37°C and 200 rpm. A sterile inoculation loop was used to streak a loop of bacterial liquid onto the corresponding plate for temporary storage. dal and pCW619-LI dat The top 10 E. coli strains harboring the plasmid were each inoculated into 5 mL of LA broth and cultured overnight on a shaker. Plasmids were extracted according to the instructions of the Tiangen Plasmid Miniprep Midi Kit (spin column format). After elution with sterile ultrapure water, the purity and concentration were determined using a Nanodrop instrument and stored at -20°C until use.

[0059] S104, the pCW619-LI dal Plasmids and the pCW619-LI dat The plasmids were sequentially electroporated into the competent hemolysin-replacing Listeria ovis for homologous recombination to obtain the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat .

[0060] Specifically, this step may include, for example, storing the competent LIΔ ilo :: hly and plasmid pCW619-LI stored at -20°C dal , put it on ice and slowly melt it, at the same time, pre-cool the tip of the pipette and pre-heat 750 μL of BHI broth containing 1 mol / L sucrose at 37°C; take 5 μL of plasmid pCW619-LI dal To 50 μL LIΔ ilo :: hlyIn the competent state, gently tap the bottom of the tube to mix, then place it on ice and let it stand for 5 minutes; aspirate the competent state and plasmid mixture in the EP tube into the gap in the middle of the electroporation cup, gently tap the table to eliminate the bubbles, and let it stand on ice for 5 minutes; electroporate at 1500V voltage for 5ms and immediately place it on ice for 5 minutes; add 750μL of preheated BHI broth containing 1mol / L sucrose to the electroporation cup, mix it thoroughly with the competent state and plasmid mixture, transfer it to a sterile EP tube, and place it on a shaker at 37℃ and 200rpm for incubation for 2-3 hours; spread it on a D-BE3 plate preheated in a 37℃ constant temperature incubator and incubate it at 37℃ for 24h-48h.

[0061] The single colony grown on the plate was streaked onto a new D-BE3 plate and cultured at 37°C for 24-48 hours. The newly grown single colony on the plate was used as a template and the primers LI specific for Listeria monocytogenes and E53-LI specific for the plasmid backbone were used to generate the plasmid. dal Perform PCR verification. Load 5 μL of the PCR reaction product onto an agarose gel for electrophoresis at 90 V for 30 min.

[0062] The positive colonies screened were streaked onto D-BE3 plates and cultured in a 42°C constant temperature incubator for 48 h before subculturing. A single colony on the third generation plate was used as a template to generate the homologous LI dal PCR was performed using the same primers to verify whether gene exchange had occurred in the colonies; positive bacteria with gene exchange were picked and inoculated into D-BHI broth, cultured in a shaker at 30°C and 200 rpm for 24 h, and then subcultured and continued to be cultured; after 6 consecutive generations, 100 μL of the 6th generation bacterial solution was taken and diluted 10-fold to 10-7; the D-BHI plate was preheated in a 37°C constant temperature incubator, and 800 μL of each dilution of bacterial solution was taken in a small amount and multiple times in a biosafety cabinet and spread on the D-BHI plate, and cultured in a 37°C constant temperature incubator for 24-48 h.

[0063] Resistance screening: Single colonies grown on D-BHI plates were streaked onto D-BE3 and D-BHI plates, and cultured in a 37°C incubator for 48 h.

[0064] PCR screening: Single colonies that grew on D-BHI plates but not on D-BE3 plates were selected as templates and PCR was performed using LI dal PCR was performed with the primers to screen dal LIΔ gene knockout ilo :: hly .

[0065] Sequencing: The colonies that were positive in both resistance screening and PCR amplification were sequenced with homologous LI dal PCR amplification was performed using high-fidelity enzymes and the PCR products were sent for sequencing.

[0066] The bacteria that are correct after resistance screening, PCR screening and sequencing verification are the ones that have knocked out LI. dal LIΔ of gene ilo :: hly , named LIΔ ilo :: hly Δ dal .

[0067] LIΔ was prepared in the same manner ilo :: hly Δ dal Competent, then pCW619-LI dat The plasmid was electroporated into competent cells, and then homologous recombination and screening were performed. The primers for PCR screening were homologous LI dat and LI dat The bacteria that are correct after resistance screening, PCR screening and sequencing verification are the ones that have knocked out LI dat LIΔ of gene ilo :: hly Δ dal Δ dat , named LIΔ ilo :: hly ΔΔdd.

[0068] S200, a fusion antigen is constructed by alternating the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of four types of HPV6 / 11 / 16 / 18 and retaining a 16-amino acid overlap region at the junction site; see Figure 3 In the present invention, the fusion antigen includes the LLO hemolysin gene promoter, LLO signal peptide, LLO predicted cleavage site, GP33 protein gene (H-2Db restricted epitope of lymphocytic choriomeningitis virus (LCMV) glycoprotein GP33, including residues 33 to 41, and its amino acid sequence is KAVYNFATM), an antigen tag (derived from residues 98 to 106 of the human influenza virus hemagglutinin molecule (YPYDVPDYA)) and the E6E7 fusion gene, wherein the E6E7 fusion gene is the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of four types of HPV6 / 11 / 16 / 18 arranged alternately.

[0069] S300, inserting the fusion antigen into the carrier dal Gene combination asd Gene replacement Amp Gene-free plasmid pCW631 to obtain the resistance-free plasmid pCW636; in an optional embodiment of the present invention, this step may include:

[0070] S301. Prepare frozen Escherichia coli carrying the non-resistance plasmid pCW631 and the Escherichia coli carrying the fusion antigen plasmid pCW635.

[0071] S302. Frozen E. coli carrying the non-resistance plasmid pCW631 and the plasmid pCW635 carrying the fusion antigen are thawed at room temperature and cultured overnight in LA broth to obtain E. coli carrying the pCW631 plasmid and E. coli carrying the pCW635 plasmid. Specifically, this step may include removing the E. coli carrying pCW635 and pCW631 from a -80°C freezer, thawing them at room temperature, and adding 50 μL of the E. coli to 5 mL of LA broth; culturing them overnight in a shaker at 37°C and 200 rpm, and then using a sterile inoculating loop to streak a loopful of the bacterial solution onto corresponding plates for temporary storage.

[0072] S303, using the centrifugal column type Tiangen plasmid mini-extraction kit to extract the pCW631 plasmid E. coli and the pCW635 plasmid E. coli to obtain the pCW631 non-antibiotic plasmid and the pCW635 antigen plasmid. The plasmid extraction process of this step can refer to pCW619-LI dal The steps of plasmid extraction are not described in detail herein.

[0073] S304, use high-fidelity enzymes and primers dal Vector amplification of the pCW631 non-resistant plasmid and gel recovery to obtain a linearized vector. Specifically, this step may include: using a high-fidelity enzyme and primers dal Vector amplification vector plasmid pCW631 was prepared on a 0.5% agarose gel. All PCR reaction products were loaded onto the gel for electrophoresis at 90 V for 40 min. After electrophoresis, the desired band was quickly excised under UV light and placed in a clean EP tube. The agarose gel block was weighed and recovered according to the instructions of the Omega Gel Extraction Kit. After elution with sterile ultrapure water, the purity and concentration were determined using a nanodrop instrument. The gel was then stored at -20°C for later use.

[0074] S305: Amplify the pCW635 antigen plasmid using a high-fidelity enzyme and primer HPV Insert and recover the fragment by gel gel to obtain the antigen insert fragment. The specific process of this step can be referred to step S304.

[0075] S306. The linearized vector and the antigen insert are reacted in a metal bath at a molar ratio of 1:2 to obtain an antigen-vector ligated product. Specifically, this step may include preparing the reaction system on ice, wherein the molar ratio of the linearized vector to the insert is 1:2, and the amount added is calculated based on the molar ratio and plasmid concentration. After the system is prepared, it is immediately placed in a 50°C metal bath for reaction for 15 minutes. After the reaction is completed, the reaction is placed on ice and immediately transformed.

[0076] S307, adding the antigen carrier ligation product into competent Escherichia coli DH5αΔ asd In an optional embodiment of the present invention, before this step, it is necessary to prepare competent Escherichia coli DH5αΔ asd , prepare competent E. coli DH5αΔ asd Steps may include: preparing cryopreserved knockout asd Escherichia coli DH5αΔ asd ; The Escherichia coli DH5αΔ asd After thawing at room temperature, culture overnight in LB-DAP broth to obtain resuscitated E. coli DH5αΔ asd ; The resuscitation of Escherichia coli DH5αΔ asd Use LB-DAP broth to expand the culture and regularly measure the OD600 value; when the OD600 value reaches 0.5, add CaCl2 solution to obtain competent Escherichia coli DH5αΔ asd .

[0077] Specifically, this step may include: thawing DH5αΔ on ice asd Competent medium: 5 μL of ligation product was added to 100 μL of competent medium, and the tube bottom was gently flicked to mix. The tube was placed on ice for 30 min. After heat shock in a 42°C metal bath for 45 s, the tube was immediately placed on ice for 2 min. 900 μL of LB broth was added to the tube, and the tube was placed on a shaker at 37°C and 200 rpm for 1 h. The broth in the tube was spread on an LB plate in a biosafety cabinet and cultured in a 37°C constant temperature incubator overnight.

[0078] S308, from the transformed competent Escherichia coli DH5αΔ asd The antigen-positive non-resistant plasmid pCW636 was extracted from the plate. Specifically, this step may include: picking a monoclonal colony on the plate and inoculating it into 5 mL of LB broth, placing it on a shaker at 37°C and 200 rpm for overnight culture, extracting the plasmid according to the aforementioned related methods, using the extracted plasmid as a template, and asd Primers, dalGene primers and HPV-seq primers are used for PCR amplification; the plasmids that are positive for PCR amplification are sent for sequencing verification, and the correct plasmids are those that contain the target antigen fragments, asd Nutritional genes and dal The nutritional gene-free plasmid was named pCW636.

[0079] S400, electrotransforming the non-resistant plasmid pCW636 into the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat Specifically, this step may include: taking out the glycerol-stored strain LIΔ from a -80°C freezer box; ilo :: hly After thawing at room temperature, 100 μL of ΔΔdd and LMΔΔdd were added to 5 mL of BHI broth and cultured overnight on a shaker at 37°C and 200 rpm. A sterile inoculating loop was then used to streak a loopful of the bacterial solution onto the corresponding plate for temporary storage. The constructed plasmid pCW636 and the plasmid pCW631 previously constructed by our research group were extracted. ilo :: hly ΔΔdd and LMΔΔdd competent cells require the addition of D-alanine to the culture medium to allow the strain to obtain nutrients and grow. ilo :: hly In the ΔΔdd and LMΔΔdd competent cells, pCW631 was electroporated into LIΔ ilo :: hly ΔΔdd competent state.

[0080] Product verification: Single colonies grown on the electroporated plate were detected by using HPV-seq primers, asd Gene primers, dal Gene primers and Listeria-specific primer LI were used for PCR amplification verification, and the PCR amplification products were sequenced for verification.

[0081] The primer sequences used in the preparation method of the multivalently expressed Listeria monocytogenes cervical cancer vaccine of the present invention are shown in Table 1 below:

[0082] Table 1 Primer sequence list

[0083]

[0084] The plasmid sequence list used in the preparation process of the multivalently expressed sheep Listeria cervical cancer vaccine of the present invention is shown in Table 2 below:

[0085] Table 2 Plasmid sequence list

[0086]

[0087] In order to better illustrate the therapeutic effect of the Listeria monocytogenes cervical cancer therapeutic vaccine of the present invention, the present invention constructed the following experimental model for verification.

[0088] 1. Establishment of HPV16 and HPV18 cervical cancer tumor-bearing mouse models

[0089] To resuscitate TC-1 and U14-E6 cells: Preheat a water bath to 37°C in advance. Remove the cryovial containing TC-1 cell suspension from the liquid nitrogen tank and rapidly shake it in a 37°C water bath until completely thawed. Transfer the entire cell suspension into a 15-mL centrifuge tube containing 9 mL of 1640 complete medium. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in 1 mL of 1640 complete medium. Transfer the tube to a T25 culture flask, add 5 mL of 1640 complete medium, shake well, and culture overnight in a 37°C, 5% CO2 cell culture incubator. Resuscitate U14-E6 cells using DMEM complete medium. All other procedures are the same as for TC-1 cells.

[0090] 2. Passaging TC-1 and U14-E6 cells: Remove the T25 flask containing TC-1 cells, discard the supernatant, add 3 mL of PBS, gently shake, discard the PBS, and rinse once with PBS. Add 1 mL of trypsin containing EDTA and incubate at 37°C for 1 minute. Observe the cell digestion under a microscope. When most cells become round and detach, quickly return to the biosafety cabinet and add 2 mL of 1640 complete medium to terminate the digestion. Gently dislodge all cells with a pipette. Aspirate the liquid into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend in 1 mL of 1640 complete medium. Aliquot the cell suspension into a new T25 flask at a 1:2 ratio. Add 6 mL of 1640 complete medium, shake well, and culture overnight in a 37°C, 5% CO2 cell culture incubator. U14-E6 cells are passaged using DMEM complete medium. Other procedures are the same as for TC-1 cells.

[0091] 2. Establishment of HPV16 and 18 cervical cancer tumor-bearing mouse models

[0092] Take TC-1 cells and U14-E6 cells at a density of about 80% at passage 3-4, discard the supernatant, add 3 mL of PBS, shake gently, discard the PBS, and rinse with PBS again once; add 1 mL of trypsin containing EDTA, place in a 37°C incubator for digestion for 1 minute, add 2 mL of complete medium to terminate digestion, and gently blow off all cells with a pipette tip; aspirate the liquid into a 15 mL centrifuge tube, place at 1000 rpm for 5 minutes, discard the supernatant, and add 1 mL of PBS to resuspend the cells. Count the cells and adjust the cell concentration to 1×10 with PBS. 6 100 μL of TC-1 cell and U14-E6 cell suspensions were subcutaneously inoculated into the right abdomen of C57BL / 6 mice, and the tumor formation in the mice was observed.

[0093] 3. Immunotherapy

[0094] Mouse grouping: After establishing HPV16 and 18 cervical cancer tumor-bearing mouse models, the two types of mice were divided into 5 groups for immunotherapy, namely PBS group, vector group, PD-1 group, vaccine group, and vaccine-PD-1 combined immune group, with 10 mice in each group.

[0095] Immunotherapy: C57BL / 6 mice were immunized with the vaccine strain and the vector strain at a dose of 0.1LD50 on the 7th, 14th, and 21st days, respectively, through the tail vein. At the same time, mice in the PD-1 group and the combined immunization group were intraperitoneally injected with PD-1 monoclonal antibody at a dose of 200µg / mouse on the 9th, 12th, and 17th days, respectively. The specific process can be found in Figure 4 and Figure 5 .

[0096] 4. Determination of treatment effect

[0097] Determination of mouse tumor volume: Starting from the 7th day, the length and width of the mouse tumor were measured and recorded every 1 day with a vernier caliper, and the tumor volume of the mouse was calculated using the formula (V = 1 / 2 length × width 2 ).

[0098] Calculate the median survival time: when mice die and the tumor volume reaches 2000mm 3 Or the tumor disappeared and remained stable for 7 days, which was considered to have reached the observation endpoint. The death and cure status of mice were recorded. When the last mouse reached the observation endpoint, the median survival time of mice in each group was sorted and calculated.

[0099] The therapeutic effect on HPV16 cervical cancer-bearing mice: The day of tumor cell inoculation was defined as day 0. From day 7 onwards, the tumor volume was measured every 1 day. The changes in tumor volume of HPV16 cervical cancer-bearing mice were as follows: Figure 6The tumor volume of the vaccine group and the combined immunization group was slower than that of the PBS group and the vector strain group, and the tumor volume was smaller. Figure 7 As shown, the tumor cure rate of the vaccine group was 30%, and the median survival time was 50 days; the tumor cure rate of the combined immunization group was 20%, and the median survival time was 41 days; compared with the PBS group, vector group and PD-1 group, the median survival time of the vaccine group and combined immunization group was significantly prolonged, and the differences were statistically significant.

[0100] The therapeutic effect on HPV18 cervical cancer-bearing mice: The changes in tumor volume in HPV18 cervical cancer-bearing mice are as follows: Figure 8 Compared with the PBS group and the vector strain mice, the tumor volume of the tumor-bearing mice in the vaccine group, PD-1 group and combined immunization group increased at a slower rate and was smaller. Figure 9 As shown, the tumor cure rate of the vaccine group was 40%, and the median survival time was 56 days; the tumor cure rate of the PD-1 monoclonal antibody group was 90%, and the tumor cure rate of the combined immunization group was 80%; compared with the vector strain group and PBS group, the median survival time of the vaccine group, PD-1 monoclonal antibody group and combined immunization group was significantly prolonged, and the differences were statistically significant.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine, characterized in that: include: Replacement of hemolysin in Listeria ovis LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat ; The fusion antigen is constructed by alternating the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of the four HPV types 6 / 11 / 16 / 18 and retaining a 16-amino acid overlapping region at the junction site; the gene sequence of the fusion antigen includes the promoter of the LLO hemolysin gene, the LLO signal peptide, the LLO predicted cleavage site, the GP33 protein gene, the antigen tag, and the E6E7 fusion gene. The E6E7 fusion gene is an alternating arrangement of the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of the four HPV types 6 / 11 / 16 / 18. The arrangement order of the E6E7 fusion gene is: 16E6N-16E7N-16E6C-16E7C-18E6N-18E7N-18E6C-18E7C-6E6N-6E7N-6E6C-6E7C-11E6N-11E7N-11E6C-11E7C; Insert the gene sequence of the fusion antigen into the dal Gene combination asd Gene replacement Amp The nucleotide sequence of the non-resistance plasmid pCW631 is shown in SEQ ID: 27, and the nucleotide sequence of the non-resistance plasmid pCW636 is shown in SEQ ID: 29; The non-resistance plasmid pCW636 was electroporated into the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat In, obtain sheep Listeria cervical cancer vaccine.

2. The method for preparing the multivalently expressed Listeria monocytogenes cervical cancer vaccine according to claim 1, characterized in that: The hemolysin replacement in Listeria ovis LIΔ ilo :: hly Based on this, the auxotrophic Listeria ovis LIΔ was constructed. ilo :: hly Δ dal Δ dat include: Prepare frozen stock of hemolysin-replacing Listeria ovis LIΔ ilo :: hly, Carrying pCW619-LI dal coli carrying the top10 plasmids and pCW619-LI dat Top 10 E. coli plasmids; Replace the hemolysin with Listeria ovis LIΔ for the frozen ilo :: hly Resuscitation is performed and competent hemolysin-replacing Listeria ovis is prepared; The frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top10 E. coli of the plasmid was recovered and the pCW619-LI was extracted. dal Plasmids and the pCW619-LI dat plasmids; The pCW619-LI dal Plasmids and the pCW619-LI dat The plasmids were sequentially electroporated into the competent hemolysin-replacing Listeria ovis for homologous recombination to obtain the auxotrophic Listeria ovis LIΔ ilo :: hly Δ dal Δ dat .

3. The method for preparing the multivalently expressed Listeria monocytogenes cervical cancer vaccine according to claim 2, characterized in that: The hemolysin is replaced with Listeria ovis LIΔ ilo :: hly Resuscitation and preparation of competent hemolysin-replacing Listeria ovis include: Replace the hemolysin with Listeria ovis LIΔ for the frozen ilo :: hly After thawing at room temperature, the cells were cultured overnight in BHI broth to obtain resuscitated Listeria ovis; The revived Listeria ovis was expanded and cultured in BHI broth containing sucrose, and the OD600 value was measured regularly; When the OD600 value is between 0.4 and 0.7, penicillin G is added until the OD600 value shows a downward trend, thereby obtaining competent hemolysin-replacing Listeria monocytogenes.

4. The method for preparing the multivalently expressed Listeria monocytogenes cervical cancer vaccine according to claim 3, characterized in that: The frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top10 E. coli of the plasmid was recovered and the pCW619-LI was extracted. dal Plasmids and the pCW619-LI dat Plasmids include: The frozen cells carrying the pCW619-LI dal coli and the frozen pCW619-LI carrying the plasmid dat The top 10 E. coli containing the plasmid were thawed at room temperature and cultured overnight in BHI broth to obtain recovery. dal Plasmids and E. coli recovery dat Plasmid E. coli; The Tiangen Plasmid Miniprep Kit was used to recover the dal Plasmids in E. coli and the recovery dat Plasmid E. coli was extracted to obtain the pCW619-LI dal Plasmids and the pCW619-LI dat plasmid.

5. The method for preparing the multivalently expressed Listeria monocytogenes cervical cancer vaccine according to claim 4, characterized in that: The gene sequence of the fusion antigen is inserted into the dal Gene combination asd Gene replacement Amp Gene-free plasmid pCW631, to obtain the resistance-free plasmid pCW636, including: Prepare frozen Escherichia coli carrying the non-resistance plasmid pCW631 and the Escherichia coli carrying the fusion antigen plasmid pCW635; Frozen E. coli carrying the non-resistant plasmid pCW631 and the plasmid pCW635 carrying the fusion antigen were thawed at room temperature and then cultured overnight in LA broth to obtain E. coli with the pCW631 plasmid and E. coli with the pCW635 plasmid; The pCW631 plasmid E. coli and the pCW635 plasmid E. coli were extracted using a centrifugal column-type Tiangen plasmid mini-extraction kit to obtain the pCW631 non-resistance plasmid and the pCW635 antigen plasmid; Use high-fidelity enzymes and primers dal Vector amplifies the pCW631 non-resistant plasmid and recovers it by gel agarose gel to obtain a linearized vector; Amplify the pCW635 antigen plasmid using a high-fidelity enzyme and primer HPV Insert and recover the fragment by gel recovery to obtain the antigen insert fragment; The linearized vector and the antigen insert are subjected to a metal bath reaction at a molar ratio of 1:2 to obtain an antigen-vector ligation product; The antigen carrier ligation product was added to the competent Escherichia coli DH5αΔ asd Carry out the transformation of the ligation product; From the competent Escherichia coli DH5αΔ asd The antigen-positive and non-antibiotic plasmid pCW636 was extracted from the culture medium.

6. The method for preparing the multivalently expressed Listeria monocytogenes cervical cancer vaccine according to claim 5, characterized in that: The antigen carrier ligation product is added to the competent Escherichia coli DH5αΔ asd Before the transformation of the ligation product, the following steps are included: Preparing cryopreserved knockouts asd Escherichia coli DH5αΔ asd ; The Escherichia coli DH5αΔ asd After thawing at room temperature, culture overnight in LB-DAP broth to obtain resuscitated E. coli DH5αΔ asd ; The resuscitation of Escherichia coli DH5αΔ asd Use LB-DAP broth for expansion culture and measure OD600 value regularly; When the OD600 value reached 0.5, CaCl2 solution was added to obtain competent Escherichia coli DH5αΔ asd .

7. A multivalently expressed Listeria monocytogenes cervical cancer vaccine, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6.

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