Multivalent expression sheep listeria monocytogenes cervical cancer vaccine and preparation method thereof

Abstract

The invention discloses a multivalent expression sheep listeria vaccine for cervical cancer and a preparation method thereof, and the method comprises the following steps: on the basis of replacing sheep listeria LI [delta] ilo:: hly with hemolysin, constructing to obtain auxotrophic sheep listeria LI [delta] ilo:: hly [delta] dal [delta] dat; the fusion antigen is constructed by alternately arranging amino terminal and carboxyl terminal structural domains of four types of HPV6 / 11 / 16 / 18 E6 and E7 proteins and retaining an overlapping region of 16 amino acids at a connecting site; inserting the fusion antigen into a non-resistance plasmid pCW631 which carries a dal gene and replaces an Amp gene with an asd gene, so as to obtain a non-resistance plasmid pCW636; the resistance-free plasmid pCW636 is electrically transferred into the auxotroph sheep listeria monocytogenes LIdeltailo:: hly deltadal deltadat, and the sheep listeria monocytogenes cervical cancer vaccine is obtained. The cervical cancer vaccine provided by the invention can express the E6E7 protein in a multivalent manner, so that multiple protection on cervical cancer is realized.

Description

Technical Field

[0001] The present invention relates to the field of biological technologies, and particularly relates to a multivalent-expressing Listeria innocua cervical cancer vaccine for sheep and a preparation method thereof. Background Art

[0002] Cervical cancer is the most common gynecological malignant tumor. The high-incidence age of carcinoma in situ is 30 - 35 years old, and that of invasive carcinoma is 45 - 55 years old. In recent years, its incidence has shown a trend of getting younger. The widespread application 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. The traditional methods for treating cancer mainly include surgical resection, radiotherapy, chemotherapy, and therapeutic vaccines. Surgical resection, radiotherapy, and chemotherapy are prone to drawbacks such as metastasis and recurrence. In contrast, the advantage of vaccine therapy is that it causes less harm to the body and has low toxicity. Currently, the vaccines used for tumors are mainly traditional inactivated vaccines and subunit vaccines, which have disadvantages such as a long immune cycle and poor immune effect.

[0003] Vaccines based on bacteria as carriers have significant advantages in delivering exogenous antigens, including low production cost, high genetic stability, good safety, and high anti-tumor efficiency. The Listeria innocua balanced lethal system is a green vector system. Due to the high-copy number characteristics of plasmids, the Listeria innocua balanced lethal system may have a better immune effect. Existing Listeria-based cervical cancer vaccines are basically monovalent-expressing vaccines, and the protective power of monovalent-expressing vaccines is limited. There is a lack of a Listeria-based cervical cancer vaccine in the prior art that can multivalently express E6E7 proteins to better achieve multiple protection against cervical cancer.

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

[0005] The present invention provides a multivalent-expressing Listeria innocua cervical cancer vaccine for sheep and a preparation method thereof, aiming to solve the technical problems existing in the Listeria-based cervical cancer vaccine mentioned in the background art.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: In a first aspect of the present invention, a preparation method of a multivalent-expressing Listeria innocua cervical cancer vaccine for sheep is provided. The Listeria innocua cervical cancer vaccine for sheep is prepared by the following method: On the basis of replacing the hemolysin of Listeria innocua LIΔ ilo :: hly a nutritional auxotrophic Listeria innocua LIΔ ilo :: hly Δ dal Δ dat ; A fusion antigen was constructed by alternately arranging 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 overlapping region at the junction site; Insert the said fusion antigen into the carrier dal gene and use asd gene to replace Amp gene in the antibiotic-free plasmid pCW631 to obtain the antibiotic-free plasmid pCW636; Electroporate the said antibiotic-free plasmid pCW636 into the auxotrophic Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat to obtain the Listeria monocytogenes cervical cancer vaccine.

[0007] In an alternative embodiment of the first aspect of the present invention, on the basis of replacing the hemolysin in Listeria monocytogenes LIΔ ilo :: hly the auxotrophic Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat includes: Prepare cryopreserved Listeria monocytogenes with hemolysin replacement LIΔ ilo :: hly、 top10 Escherichia coli carrying the pCW619-LI dal plasmid and top10 Escherichia coli carrying the pCW619-LI dat plasmid; Resuscitate the cryopreserved Listeria monocytogenes with hemolysin replacement LIΔ ilo :: hly and prepare competent Listeria monocytogenes with hemolysin replacement; Resuscitate the cryopreserved top10 Escherichia coli carrying the said pCW619-LI dal plasmid and the cryopreserved top10 Escherichia coli carrying the said pCW619-LI dat plasmid, and extract the said pCW619-LI dal plasmid and the said pCW619-LI dat plasmid; Electroporate the said pCW619-LI dal plasmid and the said pCW619-LI dat plasmid into the competent Listeria monocytogenes with hemolysin replacement for homologous recombination in sequence to obtain the auxotrophic Listeria monocytogenes LIΔ ilo :: hly Δ dal Δdat 。

[0008] In an alternative embodiment of the first aspect of the present invention, replacing the lytic toxin of the cryopreserved Listeria innocua LIΔ ilo :: hly with resuscitation and preparation of competent lytic toxin-replaced Listeria innocua including: Replacing the lytic toxin of the cryopreserved Listeria innocua LIΔ ilo :: hly Melting at room temperature and culturing overnight with BHI broth to obtain resuscitated Listeria innocua; Culturing the resuscitated Listeria innocua in BHI broth containing sucrose for expansion and regularly measuring the OD600 value; When the OD600 value is between 0.4 - 0.7, adding penicillin G until the OD600 value shows a downward trend to obtain competent lytic toxin-replaced Listeria innocua.

[0009] In an alternative embodiment of the first aspect of the present invention, resuscitating the cryopreserved top10 Escherichia coli carrying the pCW619-LI dal plasmid and the cryopreserved top10 Escherichia coli carrying the pCW619-LI dat plasmid and extracting the pCW619-LI dal plasmid and the pCW619-LI dat plasmid including: Resuscitating the cryopreserved top10 Escherichia coli carrying the pCW619-LI dal plasmid and the cryopreserved top10 Escherichia coli carrying the pCW619-LI dat plasmid by melting at room temperature and culturing overnight with BHI broth to obtain resuscitated dal plasmid Escherichia coli and resuscitated dat plasmid Escherichia coli; Using the centrifugal column type Tiangen plasmid mini-prep midiprep kit to extract plasmids from the resuscitated dal plasmid Escherichia coli and the resuscitated dat plasmid Escherichia coli to obtain the pCW619-LI dal plasmid and the pCW619-LI dat plasmid.

[0010] In an alternative embodiment of the first aspect of the present invention, inserting the fusion antigen into the antibiotic-free plasmid pCW631 carrying dal gene and replacing asd gene with Amp gene to obtain the antibiotic-free plasmid pCW636 including: Prepare Escherichia coli carrying the antibiotic-free plasmid pCW631 and Escherichia coli carrying the fusion antigen plasmid pCW635 for cryopreservation; After thawing the cryopreserved Escherichia coli carrying the antibiotic-free plasmid pCW631 and Escherichia coli carrying the plasmid pCW635 of the fusion antigen at room temperature, culture them overnight in LA broth to obtain Escherichia coli with the pCW631 plasmid and Escherichia coli with the pCW635 plasmid; Use the centrifugal column type Tiangen plasmid midiprep kit to extract plasmids from the Escherichia coli with the pCW631 plasmid and the Escherichia coli with the pCW635 plasmid to obtain the antibiotic-free pCW631 plasmid and the antigen pCW635 plasmid; Use high-fidelity enzyme and primer dal Vector to amplify the antibiotic-free pCW631 plasmid and recover it by gel electrophoresis to obtain a linearized vector; Use high-fidelity enzyme and primer HPV Insert to amplify the antigen pCW635 plasmid and recover it by gel electrophoresis to obtain an antigen insert fragment; Perform a metal bath reaction on the linearized vector and the antigen insert fragment according to a molar ratio of 1:2 to obtain an antigen-vector ligation product; Add the antigen-vector ligation product to competent Escherichia coli DH5αΔ asd for ligation product transformation; Extract the antibiotic-free plasmid pCW636 with positive antigen from the transformed competent Escherichia coli DH5αΔ asd ;

[0011] In an alternative embodiment of the first aspect of the present invention, before adding the antigen-vector ligation product to competent Escherichia coli DH5αΔ asd for ligation product transformation, it includes: Prepare cryopreserved Escherichia coli DH5αΔ asd with the asd gene knocked out; After thawing the Escherichia coli DH5αΔ asd at room temperature, culture it overnight in LB-DAP broth to obtain revived Escherichia coli DH5αΔ asd ; Use LB-DAP broth to expand the culture of the revived Escherichia coli DH5αΔ asd and regularly measure the OD600 value; When the OD600 value reaches 0.5, add a CaCl2 solution to obtain competent Escherichia coli DH5αΔ asd ;

[0012] In an alternative embodiment of the first aspect of the present invention, the fusion antigen comprises a promoter of the LLO hemolysin gene, an LLO signal peptide, an LLO predicted cleavage site, a GP33 protein gene, an antigen tag, and an E6E7 fusion gene, and the E6E7 fusion gene is an alternating arrangement of the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of four types of HPV6 / 11 / 16 / 18.

[0013] The second aspect of the present invention provides a multivalent-expressing Listeria monocytogenes subsp. innocua cervical cancer vaccine prepared by the preparation method according to any one of the first aspect of the present invention.

[0014] Beneficial effects: The present invention discloses a preparation method of a multivalent-expressing Listeria monocytogenes subsp. innocua cervical cancer vaccine, which is prepared by the following method: on the basis of replacing the hemolysin of Listeria monocytogenes subsp. innocua LIΔ ilo :: hly a nutritional-deficient Listeria monocytogenes subsp. innocua LIΔ ilo :: hly Δ dal Δ dat is constructed; a fusion antigen is constructed by alternately arranging 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 overlapping region at the ligation site; the fusion antigen is inserted into an antibiotic-free plasmid pCW631 carrying the dal gene and replacing the asd gene with the Amp gene to obtain an antibiotic-free plasmid pCW636; the antibiotic-free plasmid pCW636 is electrotransformed into the nutritional-deficient Listeria monocytogenes subsp. innocua LIΔ ilo :: hly Δ dal Δ dat to obtain the Listeria monocytogenes subsp. innocua cervical cancer vaccine. The cervical cancer vaccine of the present invention can multivalently express the E6E7 protein and achieve multiple protection against cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a step flowchart of the preparation method of a multivalent-expressing Listeria monocytogenes subsp. innocua cervical cancer vaccine of the present invention.

[0016] Figure 2 is a complete flowchart of the preparation method of a multivalent-expressing Listeria monocytogenes subsp. innocua cervical cancer vaccine of the present invention.

[0017] Figure 3 is a schematic diagram of a fusion antigen of the present invention.

[0018] Figure 4 is a flowchart for evaluating the treatment effect of the present invention.

[0019] Figure 5 This is a graph showing the grouping of treatment effects and treatment strategies for the present invention.

[0020] Figure 6 This is a graph showing the change in tumor volume of a mouse bearing an HPV16 - type cervical cancer tumor for the present invention.

[0021] Figure 7 This is a survival curve graph of a mouse bearing an HPV16 - type cervical cancer tumor for the present invention.

[0022] Figure 8 This is a graph showing the change in tumor volume of a mouse bearing an HPV18 - type cervical cancer tumor for the present invention.

[0023] Figure 9 This is a survival curve graph of a mouse bearing an HPV18 - type cervical cancer tumor for the present invention. Detailed implementation manners

[0024] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the following further describes the present invention in detail with reference to the accompanying drawings and by way of examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] See Figure 1 and Figure 2 , the first aspect of the present invention provides a preparation method of a multivalent - expressing Listeria monocytogenes cervical cancer vaccine, including: S100. On the basis of replacing the hemolysin of Listeria monocytogenes LIΔ ilo :: hly , a nutritional - defective Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat is constructed; in the present invention, the hemolysin - replaced Listeria monocytogenes LIΔ ilo :: hly is a recombinant strain in which the Listeria monocytogenes hemolysin gene ( ilo ) is replaced with the Listeria ivanovii hemolysin gene ( hly ), and the nutritional - defective Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat means that on the basis of the hemolysin - replaced Listeria monocytogenes LIΔ ilo :: hly , the dal gene and the dat gene are knocked out.

[0026] In an optional implementation manner of the present invention, the replacing the hemolysin of Listeria monocytogenes LIΔ ilo ::hly Based on this, the auxotrophic Listeria monocytogenes LIΔ was constructed ilo :: hly Δ dal Δ dat including: S101. Prepare the hemolysin replacement Listeria monocytogenes LIΔ for cryopreservation ilo :: hly、 top10 Escherichia coli carrying the pCW619-LI dal plasmid and top10 Escherichia coli carrying the pCW619-LI dat plasmid; in this step, it can also be directly using the hemolysin replacement Listeria monocytogenes LIΔ constructed step by step ilo :: hly、 carrying the pCW619-LI dal plasmid top10 Escherichia coli and top10 Escherichia coli carrying the pCW619-LI dat plasmid, S102. Resuscitate the cryopreserved hemolysin replacement Listeria monocytogenes LIΔ ilo :: hly and prepare competent hemolysin replacement Listeria monocytogenes; in an alternative embodiment of the present invention, this step may include: resuscitating the cryopreserved hemolysin replacement Listeria monocytogenes LIΔ ilo :: hly Melting at room temperature and culturing overnight in BHI broth to obtain resuscitated Listeria monocytogenes; expanding the resuscitated Listeria monocytogenes in BHI broth containing sucrose and regularly measuring the OD600 value; when the OD600 value is between 0.4 - 0.7, adding penicillin G until the OD600 value shows a downward trend to obtain competent hemolysin replacement Listeria monocytogenes.

[0027] More specifically, this step may for example include: taking out the glycerol-preserved bacteria LIΔ from the -80°C refrigerator strain box ilo :: hly , melting at room temperature, inoculating into 100 μL to 5 mL BHI broth, and culturing overnight at 37°C and 200 rpm on a shaker, then using a sterile inoculation loop to pick one loop of the bacterial liquid and streaking it on the corresponding plate for temporary storage; taking the LIΔ preserved on the BHI plate ilo :: hlyInoculate the colony into 5 mL of BHI broth, and culture it in a shaker at 200 rpm for 16 - 18 h; Pipette 300 μL of the bacterial solution into 15 mL of BHI broth containing 0.5 mol / L sucrose, and culture it overnight in a shaker at 37 °C and 200 rpm; Transfer all of the overnight - cultured bacterial solution into 250 mL of BHI broth containing 0.5 mol / L sucrose, and culture it in a shaker at 37 °C and 200 rpm for scale - up culture. Measure the OD600 value every 1 h; When the OD600 value is between 0.4 - 0.7, add penicillin G with a final concentration of 12.5 g / L, and continue to culture it in a shaker until the OD600 value shows a slow or decreasing trend; Take 6 sterile 50 - mL centrifuge tubes and pre - cool them on ice. Aliquot the bacterial solution into the 50 - mL sterile centrifuge tubes, and centrifuge at 4 °C and 13000 rpm for 5 min; Discard the supernatant, add 20 mL of 0.5 mol / L sucrose solution, and gently pipette the centrifuge tube wall with a pipette gun to disperse the bacterial cell pellet. Centrifuge at 4 °C and 10000 rpm for 10 min; Discard the supernatant and repeat the washing once; Blot dry the supernatant, add 400 μL of 0.5 mol / L sucrose solution to the centrifuge tube and mix well. Aliquot it into sterile ep tubes at 50 μL / tube and store it at - 80 °C for later use.

[0028] S103. Resuscitate the top10 Escherichia coli carrying the pCW619 - LI dal plasmid and the top10 Escherichia coli carrying the pCW619 - LI dat plasmid, and extract the pCW619 - LI dal plasmid and the pCW619 - LI dat plasmid; In an alternative embodiment of the present invention, this step may include: Resuscitate the top10 Escherichia coli carrying the pCW619 - LI dal plasmid and the top10 Escherichia coli carrying the pCW619 - LI dat plasmid. After thawing at room temperature, culture them overnight in BHI broth to obtain the resuscitated dal plasmid Escherichia coli and the resuscitated dat plasmid Escherichia coli; Use the centrifugal column - type Tiangen plasmid miniprep mid - volume kit to extract plasmids from the resuscitated dal plasmid Escherichia coli and the resuscitated dat plasmid Escherichia coli to obtain the pCW619 - LI dal plasmid and the pCW619 - LI dat plasmid.

[0029] More specifically, this step may include, for example: Take out the top10 Escherichia coli carrying the pCW619 - LI dal plasmid and the top10 Escherichia coli carrying the pCW619 - LIdat Top10 Escherichia coli containing the plasmid was thawed at room temperature. Then, 50 μL of each was inoculated into 5 mL of LA broth and cultured overnight at 37 °C and 200 rpm on a shaker. After that, one loop of the bacterial solution was picked with a sterile inoculation loop and streaked on the corresponding plate for temporary storage. The revived Top10 Escherichia coli carrying pCW619-LI dal and pCW619-LI dat plasmid were each inoculated into 5 mL of LA broth and cultured overnight on a shaker. Plasmid extraction was carried out according to the instructions of the Tiangen Midiprep Plasmid Kit (centrifugal column type). After elution with sterile ultrapure water, the purity and concentration were measured with a nanodrop instrument and stored at -20 °C for later use.

[0030] S104. The pCW619-LI dal plasmid and the pCW619-LI dat plasmid were sequentially electrotransformed into the competent hemolysin-replacing Listeria monocytogenes for homologous recombination to obtain the auxotrophic Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat .

[0031] Specifically, this step may include, for example: thawing the competent LIΔ ilo :: hly stored in an -80 °C refrigerator and the plasmid pCW619-LI dal stored at -20 °C slowly on ice. At the same time, the pipette tips were pre-cooled and 750 μL of BHI broth containing 1 mol / L sucrose was preheated at 37 °C; 5 μL of the plasmid pCW619-LI dal was added to 50 μL of LIΔ ilo :: hly competent cells. After gently flicking the bottom of the tube to mix, it was placed on ice and left to stand for 5 min; the mixture of competent cells and plasmid in the ep tube was aspirated into the gap in the middle of the electroporation cuvette, and the table was gently tapped to remove the bubbles, and then left to stand on ice for 5 min; after electroporating at 1500 V for 5 ms, it was immediately placed on ice for 5 min; the preheated 750 μL of BHI broth containing 1 mol / L sucrose was added to the electroporation cuvette, mixed well with the mixture of competent cells and plasmid, transferred to a sterile EP tube, and cultured at 37 °C and 200 rpm on a shaker for 2 - 3 h; it was spread on a D-BE3 plate preheated in a 37 °C incubator and cultured at 37 °C for 24 h - 48 h.

[0032] The single colonies grown on the plate were streaked and inoculated on a new D-BE3 plate and cultured at 37 °C for 24 h - 48 h. The newly grown single colonies on the plate were taken as templates, and with the Listeria monocytogenes-specific primer LI and the plasmid backbone-specific primer E53-LI dalPerform PCR verification. Take 5 μL of the PCR reaction product for agarose gel electrophoresis, set the voltage at 90 V, and the running time at 30 min.

[0033] Inoculate the selected positive colonies by streaking on a D - BE3 plate, culture in a constant temperature incubator at 42 °C for 48 h and then sub - culture; take the single colonies on the 3rd - generation plate as templates, and perform PCR with homologous LI dal primers to verify whether gene exchange has occurred in the colonies; pick the positive bacteria with gene exchange and inoculate them into D - BHI broth, place them on a shaker at 30 °C and 200 rpm for 24 h and then sub - culture; after continuous culturing for 6 generations, take 100 μL of the 6th - generation bacterial liquid, serially dilute it 10 - fold continuously to 10 - 7; pre - heat the D - BHI plate in a constant temperature incubator at 37 °C, and in a biosafety cabinet, take 800 μL of the bacterial liquid of each dilution in small amounts and apply it to the D - BHI plate multiple times, and culture it in a constant temperature incubator at 37 °C for 24 h - 48 h.

[0034] Resistance screening: Streak the single colonies grown on the D - BHI plate onto the D - BE3 and D - BHI plates respectively, and culture in a constant temperature incubator at 37 °C for 48 h.

[0035] PCR screening: Select the single colonies that grow on the D - BHI plate but not on the D - BE3 plate as templates, and perform PCR with LI dal primers to screen out dal the LI gene - knocked - out LIΔ ilo :: hly .

[0036] Sequencing: For the colonies that are positive in both resistance screening and PCR amplification, perform PCR amplification with high - fidelity enzyme using homologous LI dal as primers, and send the PCR products for sequencing.

[0037] The bacteria that are verified correctly through resistance screening, PCR screening and sequencing are the LIΔ dal with the LI gene knocked out, ilo :: hly , name it LIΔ ilo :: hly Δ dal .

[0038] Prepare the competent cells of LIΔ ilo :: hly Δ dal in the same way, then electro - transform the pCW619 - LI dat plasmid into the competent cells, and then perform homologous recombination and screening. The primers for PCR screening are homologous LI dat and LI dat; The bacteria that have passed the resistance screening, PCR screening, and sequencing verification are correct, and they are the ones with the LI dat gene knocked out, namely LIΔ ilo :: hly Δ dal Δ dat , which is named LIΔ ilo :: hly ΔΔdd.

[0039] S200. A fusion antigen was constructed by alternately arranging 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 overlapping region at the junction site; see Figure 3 , in the present invention, the fusion antigen includes the promoter of the LLO hemolysin gene, the LLO signal peptide, the predicted cleavage site of LLO, the GP33 protein gene (the H-2Db-restricted epitope of the glycoprotein GP33 of lymphocytic choriomeningitis virus (LCMV), containing residues 33 to 41, and its amino acid sequence is KAVYNFATM), an antigen tag (residues 98 to 106 of the hemagglutinin molecule of human influenza virus (YPYDVPDYA)), 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 four types of HPV6 / 11 / 16 / 18 arranged alternately.

[0040] S300. Insert the fusion antigen into the antibiotic-free plasmid pCW631 carrying dal gene and replace asd gene with Amp gene to obtain the antibiotic-free plasmid pCW636; in an alternative embodiment of the present invention, this step may include: S301. Prepare frozen Escherichia coli carrying the antibiotic-free plasmid pCW631 and Escherichia coli carrying the plasmid pCW635 of the fusion antigen.

[0041] S302. After thawing the frozen Escherichia coli carrying the antibiotic-free plasmid pCW631 and the Escherichia coli carrying the plasmid pCW635 of the fusion antigen at room temperature, culture them overnight in LA broth to obtain Escherichia coli with the pCW631 plasmid and Escherichia coli with the pCW635 plasmid; specifically, this step may include taking out the Escherichia coli carrying pCW635 and pCW631 from the -80°C refrigerator freezer, thawing at room temperature, inoculating 50 μL into 5 mL LA broth; culturing overnight at 37°C and 200 rpm in a shaker, and then using a sterile inoculation loop to pick one loop of the bacterial liquid and streak it on the corresponding plate for temporary storage.

[0042] S303. Use the centrifugal column type Tiangen plasmid mini-prep midiprep kit to extract plasmids from the pCW631 plasmid Escherichia coli and the pCW635 plasmid Escherichia coli, obtaining the pCW631 antibiotic-free plasmid and the pCW635 antigen plasmid. The plasmid extraction process in this step can refer to the pCW619-LI plasmid extraction steps, which will not be elaborated in detail in the present invention. dal The plasmid extraction steps will not be elaborated in detail in the present invention.

[0043] S304. Use high-fidelity enzyme and primers dal Vector to amplify the pCW631 antibiotic-free plasmid and recover the gel, obtaining a linearized vector. Specifically, this step may include: using high-fidelity enzyme and primers dal Vector to amplify the vector plasmid pCW631, prepare a 0.5% agarose gel, load all PCR reaction products for agarose gel electrophoresis, set the voltage to 90V, and run for 40 minutes; after electrophoresis, quickly cut the required band under ultraviolet light and place it in a clean EP tube; weigh the agarose gel block and perform gel recovery according to the omega Gel Extraction Kit instructions. After eluting with sterile ultrapure water, measure its purity and concentration with a nanodrop instrument and store it at -20°C for later use.

[0044] S305. Use high-fidelity enzyme and primers HPV Insert to amplify the pCW635 antigen plasmid and recover the gel, obtaining an antigen insertion fragment. The specific process of this step can refer to step S304.

[0045] S306. Perform a metal bath reaction on the linearized vector and the antigen insertion fragment at a molar ratio of 1:2 to obtain an antigen-vector ligation product; specifically, this step may include: prepare the reaction system on ice, where the molar ratio of the linearized vector to the insertion fragment should be 1:2, and calculate the addition amount according to the molar ratio and plasmid concentration. After the system is prepared, immediately place it in a 50°C metal bath for reaction for 15 minutes. After the reaction, place it on ice and immediately perform transformation.

[0046] S307. Add the antigen-vector ligation product to competent Escherichia coli DH5αΔ asd for ligation product transformation; in an optional embodiment of the present invention, before this step, it is necessary to first prepare competent Escherichia coli DH5αΔ asd , and the steps to prepare competent Escherichia coli DH5αΔ asd may include: prepare frozen Escherichia coli DH5αΔ asd from which the asd gene has been knocked out; thaw the Escherichia coli DH5αΔ asd at room temperature and culture it overnight with LB-DAP broth to obtain revived Escherichia coli DH5αΔasd ; The revived Escherichia coli DH5αΔ asd was cultured in LB-DAP broth for amplification, and the OD600 value was measured regularly; when the OD600 value reached 0.5, a CaCl2 solution was added to obtain competent Escherichia coli DH5αΔ asd .

[0047] Specifically, this step may include: thawing DH5αΔ asd competent cells on ice, adding 5 μL of the ligation product to 100 μL of the competent cells, gently flicking the bottom of the tube to mix evenly, and placing it on ice for 30 min; after heat shock in a metal bath at 42 °C for 45 s, immediately cooling it on ice for 2 min; adding 900 μL of LB broth to the tube, culturing it in a shaker at 37 °C and 200 rpm for 1 h, spreading the broth in the tube on an LB plate in a biosafety cabinet, and culturing it overnight in a 37 °C incubator.

[0048] S308. Extract the antigen-positive antibiotic-free plasmid pCW636 from the transformed competent Escherichia coli DH5αΔ asd . Specifically, this step may include: picking a monoclonal colony on the plate and inoculating it into 5 mL of LB broth, culturing it overnight in a shaker at 37 °C and 200 rpm, extracting the plasmid according to the aforementioned related method, using the extracted plasmid as a template, and using asd primers, dal gene primers, and HPV-seq primers for PCR amplification; sending the plasmid with positive PCR amplification for sequencing verification, and the plasmid with correct verification is the antibiotic-free plasmid containing the target antigen fragment, asd nutritional gene, and dal nutritional gene, which is named pCW636.

[0049] S400. Electrotransform the antibiotic-free plasmid pCW636 into the auxotrophic Listeria monocytogenes of sheep LIΔ ilo :: hly Δ dal Δ dat to obtain the cervical cancer vaccine of Listeria monocytogenes of sheep. Specifically, this step may include: taking out the glycerol-preserved strains LIΔ ilo :: hly ΔΔdd and LMΔΔdd from the -80 °C refrigerator freezer box, melting them at room temperature, inoculating 100 μL into 5 mL of BHI broth, culturing them overnight in a shaker at 37 °C and 200 rpm, and then using a sterile inoculation loop to pick one loop of the bacterial liquid and streak it on the corresponding plate for temporary storage. Extract the constructed plasmid pCW636 and the plasmid pCW631 constructed by our research group in the early stage. Prepare LIΔ ilo :: hlyFor competent cells of ΔΔdd and LMΔΔdd, D-alanine needs to be added to the medium to enable the strains to obtain nutrients for growth. Electrotransform plasmid pCW636 into LIΔ ilo :: hly In competent cells of ΔΔdd and LMΔΔdd, electrotransform pCW631 into LIΔ ilo :: hly In competent cells of ΔΔdd.

[0050] Product verification: Use the single colonies grown on the electrotransformation plate with HPV-seq primers, asd gene primers, dal gene primers and Listeria-specific primer LI for PCR amplification verification, and sequence the PCR amplification products for verification.

[0051] The primer sequence table used in the preparation method of the multivalent-expressing Listeria monocytogenes cervical cancer vaccine of the present invention is shown in Table 1 below: Table 1 Primer sequence table

[0052] The plasmid sequence table used in the preparation method of the multivalent-expressing Listeria monocytogenes cervical cancer vaccine of the present invention is shown in Table 2 below: Table 2 Plasmid sequence table

[0053] To better illustrate the therapeutic effect of the Listeria monocytogenes cervical cancer therapeutic vaccine of the present invention, the following experimental models were constructed for verification in the present invention.

[0054] 1. Establish a mouse model bearing HPV16 and 18 cervical cancer tumors Recovery of TC-1 cells and U14-E6 cells: Preheat the water bath to 37 °C in advance; take out the cryopreservation tube containing the TC-1 cell suspension from the liquid nitrogen tank, quickly shake it constantly in the 37 °C water bath until it completely melts, then aspirate all the cell suspension into a 15 mL centrifuge tube containing 9 mL of 1640 complete medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of 1640 complete medium to resuspend, transfer it to a T25 culture flask, supplement with 5 mL of 1640 complete medium, shake well and culture overnight in a 37 °C, 5% CO2 cell culture incubator. U14-E6 cells are recovered using DMEM complete medium, and other operations are the same as those for TC-1 cells.

[0055] 2. Subculture of TC-1 cells and U14-E6 cells: Take out the T25 culture flask containing TC-1 cells, discard the supernatant, add 3 mL of PBS, gently shake, then discard the PBS, and repeat the rinsing with PBS once; add 1 mL of trypsin containing EDTA, place it in a 37°C incubator for digestion for 1 min, observe the cell digestion under a microscope, when most of the cells become round and detached, quickly take it back to the biosafety cabinet, add 2 mL of 1640 complete medium to terminate digestion, gently blow down all the cells with a pipette tip; aspirate the liquid into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend with 1 mL of 1640 complete medium. Divide the cell suspension into new T25 flasks at a ratio of 1:2, add 6 mL of 1640 complete medium, shake well, and culture overnight in a 37°C, 5% CO2 cell incubator. U14-E6 cells are subcultured using DMEM complete medium, and other operations are the same as those for TC-1 cells.

[0056] 2. Establishment of HPV16 and 18 cervical cancer xenograft mouse models Take TC-1 cells and U14-E6 cells at about 80% density in the 3rd - 4th generation, discard the supernatant, add 3 mL of PBS, gently shake, then discard the PBS, and repeat the rinsing with PBS once; add 1 mL of trypsin containing EDTA, place it in a 37°C incubator for digestion for 1 min, add 2 mL of complete medium to terminate digestion, gently blow down all the cells with a pipette tip; aspirate the liquid into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells with 1 mL of PBS. Count the cells and adjust the cell concentration to 1×10 6 cells / mL with PBS; inject 100 μL of the TC-1 cell and U14-E6 cell suspensions subcutaneously into the right abdomen of C57BL / 6 mice, and observe the tumor formation in the mice.

[0057] 3. Immunotherapy Grouping of mice: After establishing the HPV16 and 18 cervical cancer xenograft mouse models, the two types of mice are respectively divided into 5 groups: PBS group, vector group, PD-1 group, vaccine group, and vaccine-PD-1 combination immunotherapy group for immunotherapy, with 10 mice in each group.

[0058] Immunotherapy: According to the prime-boost-final boost method, the vaccine strain and vector strain are respectively immunized into C57BL / 6 mice via the tail vein at a dose of 0.1 LD50 on the 7th, 14th, and 21st days; meanwhile, the mice in the PD-1 group and the combination immunotherapy group are intraperitoneally injected with PD-1 monoclonal antibody at a dose of 200 μg / mouse on the 9th, 12th, and 17th days. The specific procedure can be seen in Figure 4 and Figure 5 .

[0059] 4. Determination of treatment effect Measuring the tumor volume of mice: From the 7th day, the length and width of the tumors in mice were measured and recorded every 1 day using vernier calipers, and the tumor volume of mice was calculated using the formula (V = 1 / 2 length × width 2 ).

[0060] Calculating the median survival time: When mouse death occurred, the tumor volume reached 2000 mm 3 or the tumor disappearance state was stable for 7 days, it was regarded as reaching the observation endpoint, and the death and cure conditions of the mice were recorded. When the last mouse reached the observation endpoint, the median survival time of the mice in each group was sorted out and calculated.

[0061] Therapeutic effect on mice bearing HPV16 - type cervical cancer: Taking the day of inoculating tumor cells as the 0th day, from the 7th day, the tumor volume was measured once every 1 day. The changes in the tumor volume of mice bearing HPV16 - type cervical cancer were as Figure 6 . Compared with the PBS group and the vector - strain mice, the tumor volume of the tumor - bearing mice in the vaccine group and the combined immunotherapy group increased at a slower rate and was smaller. The survival curve of mice bearing HPV16 - type cervical cancer was as Figure 7 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 immunotherapy group was 20%, and the median survival time was 41 days; compared with the PBS group, the vector group and the PD - 1 group, the median survival time of the vaccine group and the combined immunotherapy group was significantly prolonged, and the differences were statistically significant.

[0062] Therapeutic effect on mice bearing HPV18 - type cervical cancer: The changes in the tumor volume of mice bearing HPV18 - type cervical cancer were as Figure 8 , compared with the PBS group and the vector - strain mice, the tumor volume of the tumor - bearing mice in the vaccine group, the PD - 1 group and the combined immunotherapy group increased at a slower rate and was smaller. The survival curve of mice bearing HPV18 - type cervical cancer was as Figure 9 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 immunotherapy group was 80%; compared with the vector - strain group and the PBS group, the median survival time of the vaccine group, the PD - 1 monoclonal antibody group and the combined immunotherapy group was significantly prolonged, and the differences were statistically significant.

[0063] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a multivalent expressed Listeria monocytogenes cervical cancer vaccine for sheep, characterized in that, Comprising: On the basis of replacing the hemolysin of Listeria ivanovii LIΔ ilo :: hly , the auxotrophic Listeria ivanovii LIΔ ilo :: hly Δ dal Δ dat ; A fusion antigen was constructed by alternately arranging 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 overlapping region at the ligation site; Insert the fusion antigen into a kanamycin-free plasmid pCW631 carrying dal gene and replace asd gene with Amp gene to obtain the kanamycin-free plasmid pCW636; Electroporate the antibiotic-free plasmid pCW636 into the auxotrophic Listeria ivanovii LIΔ ilo :: hly Δ dal Δ dat to obtain the Listeria ivanovii cervical cancer vaccine.

2. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine according to claim 1, characterized in that, On the basis of replacing the hemolysin of Listeria ivanovii LIΔ ilo :: hly , a auxotrophic Listeria ivanovii LIΔ ilo :: hly Δ dal Δ dat is constructed, including: Hemolysin to be cryopreserved replaces Listeria ivanovii LIΔ ilo :: hly, Top10 Escherichia coli carrying pCW619-LI dal plasmid and Top10 Escherichia coli carrying pCW619-LI dat plasmid; Replace the cryopreserved hemolysin with Listeria ivanovii LIΔ ilo :: hly Resuscitate it to prepare competent hemolysin-replaced Listeria ivanovii Thaw the cryopreserved Top10 Escherichia coli carrying the pCW619-LI dal plasmid and the cryopreserved Top10 Escherichia coli carrying the pCW619-LI dat plasmid, resuscitate them, and extract the pCW619-LI dal plasmid and the pCW619-LI dat plasmid; The pCW619-LI dal plasmid and the pCW619-LI dat plasmid were successively electrotransformed into the competent hemolysin-replaced Listeria monocytogenes for homologous recombination to obtain auxotrophic Listeria monocytogenes LIΔ ilo :: hly Δ dal Δ dat .

3. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine for sheep according to claim 2, characterized in that, Replacing the cryopreserved hemolysin with Listeria ivanovii LIΔ ilo :: hly Performing resuscitation and preparing competent hemolysin-replaced Listeria ivanovii, including: Replace the cryopreserved hemolysin with Listeria ivanovii LIΔ ilo :: hly After thawing at room temperature, culture it overnight in BHI broth to obtain revived Listeria ivanovii; The revived Listeria monocytogenes was cultured in BHI broth containing sucrose for expansion, and the OD600 value was measured regularly; When the OD600 value was between 0.4 and 0.7, penicillin G was added until the OD600 value showed a downward trend to obtain the competent hemolysin-replaced Listeria monocytogenes.

4. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine according to claim 3, characterized in that, The cryopreserved top10 Escherichia coli carrying the pCW619-LI dal plasmid and the cryopreserved top10 Escherichia coli carrying the pCW619-LI dat plasmid are resuscitated, and the pCW619-LI dal plasmid and the pCW619-LI dat plasmid are obtained by extraction, including: The cryopreserved top10 Escherichia coli carrying the pCW619-LI dal plasmid and the cryopreserved top10 Escherichia coli carrying the pCW619-LI dat plasmid were thawed at room temperature and cultured overnight in BHI broth to obtain the revived dal plasmid Escherichia coli and the revived dat plasmid Escherichia coli; Use the Tiangen Plasmid Mini Kit (Midiprep) to perform plasmid extraction on the revived dal plasmid Escherichia coli and the revived dat plasmid Escherichia coli to obtain the pCW619-LI dal plasmid and the pCW619-LI dat plasmid.

5. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine according to claim 4, characterized in that, Inserting the fusion antigen into a drug-free plasmid pCW631 carrying dal gene and replacing asd gene with Amp gene to obtain the drug-free plasmid pCW636 includes: Prepare Escherichia coli carrying the antibiotic-free plasmid pCW631 and Escherichia coli carrying the plasmid pCW635 of the fusion antigen for cryopreservation; The cryopreserved Escherichia coli carrying the antibiotic-free plasmid pCW631 and the Escherichia coli carrying the plasmid pCW635 of the fusion antigen were thawed at room temperature and cultured overnight in LA broth to obtain Escherichia coli with the pCW631 plasmid and Escherichia coli with the pCW635 plasmid; The pCW631 plasmid Escherichia coli and the pCW635 plasmid Escherichia coli were subjected to plasmid extraction using the centrifugal column type Tiangen plasmid miniprep midiprep kit to obtain the pCW631 antibiotic-free plasmid and the pCW635 antigen plasmid; Use high-fidelity enzymes and primers dal Amplify the pCW631 antibiotic-free plasmid using Vector and recover it by gel electrophoresis to obtain a linearized vector; The pCW635 antigen plasmid was amplified using a high-fidelity enzyme and primers HPV Insert and recovered by gel electrophoresis to obtain an antigen insertion fragment; The linearized vector and the antigen insertion fragment were subjected to a metal bath reaction at a molar ratio of 1:2 to obtain an antigen-vector ligation product; Add the antigen carrier ligation product to competent Escherichia coli DH5αΔ asd for ligation product transformation; Extract the antigen-positive antibiotic-free plasmid pCW636 from the transformed competent Escherichia coli DH5αΔ asd .

6. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine according to claim 5, characterized in that, Adding the antigen carrier ligation product to competent Escherichia coli DH5αΔ asd before the transformation of the ligation product includes: Escherichia coli DH5αΔ asd gene ready for cryopreservation; asd ​ For the Escherichia coli DH5αΔ asd After thawing at room temperature, culture it overnight with LB-DAP broth to obtain the revived Escherichia coli DH5αΔ asd ; For the revived Escherichia coli DH5αΔ asd Perform scale-up culture using LB-DAP broth and regularly measure the OD600 value; Add CaCl2 solution when the OD600 value reaches 0.5 to obtain competent Escherichia coli DH5αΔ asd .

7. The preparation method of the multivalent expressed Listeria monocytogenes cervical cancer vaccine according to claim 1, characterized in that, The fusion antigen includes the promoter of the LLO hemolysin gene, the LLO signal peptide, the predicted cleavage site of LLO, the GP33 protein gene, an antigen tag, and the E6E7 fusion gene, and the E6E7 fusion gene is an alternating arrangement of the amino-terminal and carboxyl-terminal domains of the E6 and E7 proteins of four types of HPV6 / 11 / 16 / 18.

8. A multivalent expressed Listeria monocytogenes cervical cancer vaccine for sheep, characterized in that, Prepared by the preparation method according to any one of claims 1-7.

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