CN108939064B - Cervical cancer therapeutic vaccine based on recombinant attenuated sheep listeria - Google Patents

Abstract

The invention relates to a cervical cancer therapeutic vaccine based on recombinant attenuated sheep listeria. The invention takes attenuated Listeria monocytogenes (Li for short) of sheep as a carrier, utilizes the unique characteristic that the Listeria monocytogenes can grow in host phagocyte cells and is a natural T cell immune activation adjuvant, effectively improves the specific immune response in a tumor microenvironment, and breaks the immune tolerance of an organism, thereby eliminating the persistent viral infection of the organism, achieving the good treatment effect of eliminating focus and inhibiting the development of tumors. The method has the advantages that the defect of low immunogenicity of nucleic acid and protein polypeptide vaccines is overcome, and the attenuated Listeria keeps the characteristics that the original strain can grow in cells and complete antigen presentation and has higher safety.

Description

Cervical cancer therapeutic vaccine based on recombinant attenuated sheep listeria

Technical Field

The invention belongs to the technical field of biology, and relates to a cervical cancer therapeutic vaccine based on recombinant attenuated sheep listeria.

Background

Cervical cancer poses a serious threat to the health of women worldwide and is a gynecological malignancy with mortality second to breast cancer. Research shows that HPV is a main risk factor of cervical cancer, and more than 90 percent of cervical cancer patients are accompanied by high-risk HPV infection, wherein 16 types and 18 types are the main types. 90% of the cancer types of cervical cancer are cervical squamous carcinoma, and the main pathogenic type is HPV16 type. The main carcinogenic factors of HPV are E6 and E7 proteins, and E6 destroys normal cell cycle regulation by inhibiting the activity of a tumor suppressor p53, so that cells are promoted to be immortalized; the E7 protein deprives pRb protein of its cancer-suppressing action by binding to and degrading it. The continuous high expression of E6 and E7 from the early stage of HPV infection and the expression only in the HPV infected tissues make them ideal targets for preparing cervical cancer therapeutic vaccines.

Compared with the traditional treatment means, such as surgical operation, radiotherapy and chemotherapy, the therapeutic vaccine has the characteristics of small damage and low toxicity. There are three main existing forms of therapeutic vaccines: protein polypeptide vaccines, viral (bacterial) vaccines and nucleic acid vaccines. The key point of success is to cause enough immune response, but the prior nucleic acid and protein polypeptide vaccines have low immunogenicity and obvious defects.

Disclosure of Invention

In view of the above, the present invention aims to provide a therapeutic vaccine for cervical cancer based on recombinant attenuated listeria ovis.

In order to achieve the purpose, the invention provides the following technical scheme:

the cervical cancer therapeutic vaccine based on the recombinant attenuated listeria ovis comprises the effective component of the recombinant attenuated listeria ovis which carries HPV16 type E6E7 fusion antigen epitope peptide genes.

Preferably, the attenuated Listeria ovis is obtained by completely knocking out two virulence genes, namely actA and plcB, of Listeria ovis (Li for short).

Preferably, the preparation method of the recombinant attenuated listeria ovis is obtained by taking the attenuated listeria ovis as a vector and carrying HPV16 type E6E7 fusion epitope peptide group.

Further preferably, the specific steps are as follows:

(1) synthesizing HPV16 type E6E7 fusion antigen epitope peptide gene fragments;

(2) constructing a targeting plasmid;

(3) preparing a competent cell Li delta actAplcB-lacZ;

(4) performing electric transformation on the competent cells prepared in the step (3) by using the targeting plasmid prepared in the step (2);

(5) and performing homologous recombination hybridization culture and screening verification on the Li delta actAplcB-lacZ and the targeting plasmid.

Still more preferably, the specific method of step (1) is: the HPV16 type E6 protein and E7 protein amino acid sequences are inquired from NCBI, then dominant T cell antigen epitopes are screened out and sequentially arranged through software (for example, CTLPred and NetMHC 4.0 software are used for analyzing possible CTL epitopes in the sequences, ProPred and NetMHCIIpan 3.1 software are used for analyzing possible Th epitopes in the sequences), then codon optimization is carried out according to Listeria preference codon tables to obtain optimized sequences, then HindIII enzyme digestion sites (AAGCTT) are added at the upstream, Xho I enzyme digestion sites (CTCGAG) are added at the downstream, and the designed sequences are finally directly obtained through synthesis. Wherein, the codon optimization method is realized by submitting the amino acid sequence to a website (such as http:// www.jcat.de /), and then selecting a sheep Listeria preferred codon optimization option; the synthesis method of the DNA sequence comprises the following steps: the DNA sequence was synthesized directly using a gene synthesizer model ABI 3900-Thermo Fisher Scientific et al for a total of 866bp, as shown in SEQ ID NO. 1.

Still more preferably, the specific method of step (2) is:

the fusion epitope peptide gene fragment obtained in the step (1) is subjected to double enzyme digestion by using restriction enzymes Hind III and Xho I, meanwhile, the plasmid pCW203 (patent CN103074361B) is subjected to enzyme digestion by using the same restriction enzymes, the fusion epitope peptide gene fragment is inserted into enzyme digestion sites of pCW203 Hind III and Xho I through technologies such as connection, transformation and the like, and the intermediate plasmid pCW203-E6E7 is obtained, and as shown in SEQ ID NO.2, the successful construction of the intermediate plasmid is verified through PCR verification, quality-improving particle verification and enzyme digestion verification. The fragment between the BamH I cleavage site and the Xho I cleavage site of the intermediate plasmid pCW203-E6E7 was excised and inserted between the BamH I cleavage site and the Xho I cleavage site of the recombinant plasmid pCW154 (patent CN103074361B) to obtain the targeting plasmid pCW154-E6E7, as shown in SEQ ID NO. 3.

Still more preferably, the specific method of step (3) is: li delta actAplcB-lacZ (patent CN103074361B) was cultured in BHI broth containing 0.5mol/L sucrose, and A was periodically measured₆₀₀When A is₆₀₀When the concentration is 0.4, adding penicillin G to ensure that the final concentration is 12.5 mu G/ml, and continuing culturing; when A is₆₀₀Centrifuging when the concentration is 0.7, washing, subpackaging and storing.

Still more preferably, the specific method of step (3) is: inoculation with Li.DELTA.actAplcB-lacZ (patent C)N103074361B) is cultured in 15ml of BHI liquid culture medium containing 0.5mol/L sucrose at 37 ℃ and 220rpm for 12-16 hours; the resulting bacterial liquid was transferred to 250ml of BHI broth containing 0.5mol/L sucrose, and A was periodically measured₆₀₀When A is₆₀₀When the concentration is 0.4, adding penicillin G to ensure that the final concentration is 12.5 mu G/ml, and continuing culturing; when A is₆₀₀When the concentration is 0.7, pouring the mixture into a 50mL centrifuge tube, centrifuging the mixture at 4 ℃ and 10000rpm for 5 minutes, and removing the supernatant; repeatedly washing the precipitate with 0.5mol/L ice-cold sucrose solution, adding 20ml 0.5mol/L sucrose solution each time, centrifuging at 4 deg.C 10000rpm for 10min, discarding the supernatant, and washing for 3 times; add 300. mu.l of resuspension of 0.5mol/L sucrose per tube, dispense pre-cooled sterile EP tubes at 50. mu.l/tube, store at-80 ℃.

Further preferably, the specific method of step (4) is: electrically transferring the targeting plasmid to a Li delta actAplcB-lacZ competent cell, and adding BHI broth to culture for 2 hours after the electric transfer is finished; the transformation liquid is coated on an erythromycin BHI agar plate, a single blue colony is screened out through blue-white spots, and plasmid extraction and PCR verification are carried out after pure culture.

Still more preferably, the specific method of step (4) is: taking out the prepared competent cells from a refrigerator at-80 deg.C, placing in an ice box, melting completely, slowly adding 5 μ l targeting plasmid into the competent cells Li Δ actAplcB-lacZ at 1/10 ratio with a precooling gun head at-20 deg.C, and making into ddH₂O negative control; after mild mixing, carrying out ice bath for 5 minutes, then completely transferring into an electric rotating cup, carrying out ice bath for 5 minutes, and carrying out electric rotation in an electric rotating instrument; after the electric transfer is finished, ice-bath is carried out for 5 minutes, 750 mu l of BHI broth preheated at 37 ℃ is added by using a gun head preheated at 37 ℃, the mixture is uniformly mixed and then is completely transferred to an EP tube, and the mixture is cultured for 2 hours at 150rpm by a shaking table at 30 ℃; and (3) coating the whole amount of the bacterial liquid on a BHI agar plate (BHI-Ery-X-gal, BEXI plate for short) containing 1-5 mug/ml of erythromycin and 20-60 mug/ml of X-gal by using a sterile L-shaped glass rod, culturing for 48-72 hours at 30 ℃, then, carrying out pure culture on the blue bacterial colony on the BEXI plate, culturing for 24 hours at 30 ℃, carrying out plasmid extraction and PCR verification.

More preferably, the electrotransfer conditions are: the voltage is 1500V, the duration is 5ms, and the inner diameter of the electric revolving cup is 1 mm.

More preferably, the specific method for extracting the plasmid is as follows: extracting plasmids according to the description of a plasmid extraction kit (the listeria is a gram-positive bacterium, lysozyme is required to be added before the Solution I is used, the final concentration of the lysozyme is 20mg/L, the lysozyme is added with the Solution I containing the lysozyme, then the bacteria are resuspended, the temperature is 37 ℃ in water bath for 45min), and finally electrophoresis is carried out after Elution by 30 mul of Elution Buffer; gel imaging is carried out, and whether a band exists or not is observed to judge whether the recombinant plasmid is successfully introduced, wherein the size of the plasmid is 9525 bp.

Still more preferably, the specific method for PCR verification is: extracting all genes in bacteria by a boiling method to be used as a template, amplifying a target band HPV16E6E7, and carrying out PCR amplification by using sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers; the reaction cycle conditions were: 94 ℃ 3min → (94 ℃ 1min, 47 ℃ 30 sec, 72 ℃ 1min) × 30 cycles → 72 ℃ 10min → 4 ℃; the PCR results were observed electrophoretically, with the expected results: the fragment of HPV16E6E7 is about 852 bp.

The upstream primer HPV-f: 5'-CATCAAAAACGTACAGCAATG-3', as shown in SEQ ID NO. 4;

downstream primer HPV-r: 5'-TGGTTTTTGAGAACAAATTGG-3', as shown in SEQ ID NO. 5;

more preferably, the electrophoresis conditions are: 1% agarose gel; 90V, loading amount: 5 μ l of PCR product.

Further preferably, the specific method of step (5) is: continuously passaging sheep listeria carrying a targeting plasmid at 42 ℃ and 30 ℃, integrating a target fragment carried by the targeting plasmid into the listeria by utilizing a homologous recombination hybridization principle and a gene targeting technology, and screening out a suspicious strain by utilizing blue white spots and erythromycin sensitivity; then PCR screening and gene sequencing verification are carried out.

More preferably, the specific method of subculture is: and (3) streaking and inoculating a Li delta actAplcB-lacZ plate electrically transferred with a targeting plasmid into a BEXI plate, subculturing at 42 ℃, selecting a single blue colony to inoculate BHI broth, subculturing at 30 ℃, starting to transfer to the 3 rd generation of the broth, adding BHI into each generation of culture to dilute, respectively coating 100 mu l of diluted bacterium liquid on the BEXI and BXI (BHI agar plate containing 20-60 mu g/ml of X-gal) plates, and culturing for 24 hours at 30 ℃.

Even more preferably, subcultureThe specific method for cultivating the fish comprises the following steps: inoculating a BEXI plate with the Li delta actAplcB-lacZ streaked line electrically transferred with the targeting plasmid, subculturing at 42 ℃ for 2-3 generations (the culture time of each generation is 48 hours), selecting a single blue colony, inoculating 5ml of BHI broth, and subculturing at 30 ℃ and 200rpm for 6 generations (the culture time of each generation is 24 hours); starting at the 3 rd generation of the broth, 360. mu.l BHI broth (1:10 dilution) was added to 40. mu.l culture per generation and 1:10 dilutions were continued for several times until 10 was diluted⁶ Coating 10 times, respectively⁶Mu.l of the diluted bacterial solution was applied to BEXI and BXI plates and incubated at 30 ℃ for 24 hours; the positive colony Li Δ actAplcB-E6E7 grew as a white colony on BXI plates and did not grow on BEXI plates.

More preferably, the specific method of PCR screening is: and (3) amplifying the following three groups of gene fragments of the suspicious recombinant bacteria with correct screening results of the blue white spots and the erythromycin: anti-erythromycin gene, target antigen gene HPV16E6E7 and actA gene, and identifying corresponding vaccine strains.

More preferably, the specific method of PCR screening is: extracting the genome DNA of Li delta actAplcB-E6E7 by using a bacterial genome extraction kit as a template, and amplifying the following three groups of gene fragments: and identifying the recombinant bacteria Li delta actAplcB-E6E7 according to the amplification result of the anti-erythromycin gene, the target antigen gene HPV16E6E7 and the actA gene.

More preferably, the sequence shown in SEQ ID NO.6 and SEQ ID NO.7 of the erythromycin-resistant gene Ery is used as a primer for PCR amplification; the reaction cycle conditions were: 94 ℃ 3min → (94 ℃ 1min, 60 ℃ 30s, 72 ℃ 2min) × 30 cycles → 72 ℃ 10min → 4 ℃; the PCR results were observed electrophoretically, with the expected results: the Ery gene fragment is about 1471 bp.

An upstream primer Ery-f: 5'-GATAAGTCGACGATTCACAAAAAATAG-3', as shown in SEQ ID NO. 6;

downstream primer Ery-r: 5'-AAAACTAGTCCCGGG GCGAATTG-3', as shown in SEQ ID NO. 7.

More preferably, the Li actA gene takes sequences shown in SEQ ID NO.8 and SEQ ID NO.9 as primers for PCR amplification; the reaction cycle conditions were: 94 ℃ 3min → (94 ℃ 1min, 50 ℃ 30s, 72 ℃ 1min) × 30 cycles → 72 ℃ 10min → 4 ℃; the PCR results were observed electrophoretically, with the expected results: the Li actA gene fragment is about 600 bp.

An upstream primer Li-actA-f: 5'-GAAGCTAAAAGTGCAAATGTCCC-3', as shown in SEQ ID NO. 8;

downstream primer Li-actA-r: 5'-ATTTCTTTAATACTGCGTTTGGGG-3', as shown in SEQ ID NO. 9.

More preferably, the specific method for gene sequencing verification is as follows: identifying the correct corresponding vaccine strain by PCR, amplifying the target antigen gene HPV E6E7 by PCR with the genome DNA as a template, verifying the sequencing of the amplified product, and preserving the strain with the correct sequencing.

The invention has the beneficial effects that:

the cervical cancer therapeutic vaccine prepared by the invention carries HPV16 type E6E7 fusion gene, takes Listeria monocytogenes (Listeria ivanovii, Li for short) as a carrier, utilizes the unique characteristic that Listeria can grow in host phagocyte cells and is a natural T cell immune activation adjuvant, effectively improves the specific immune response in the tumor microenvironment, breaks the immune tolerance of an organism, thereby eliminating the continuous viral infection of the organism, and achieves the good treatment effect of eliminating focus and inhibiting the development of tumor. The method has the advantages that the defect of low immunogenicity of nucleic acid and protein polypeptide vaccines is overcome, and the attenuated Listeria keeps the characteristics that the original strain can grow in cells and complete antigen presentation and has higher safety.

The source bacteria of the invention is Li delta actAplcB-lacZ, the final attenuation is deletion attenuation of all genes of actA and plcB, the deleted genes are related to intercellular transfer of bacteria and escape of bacteria from secondary lysosomes, the virulence of the bacteria is reduced after deletion, compared with the attenuation mode of only deleting part of genes of hemolysin, the invention has better safety, and simultaneously, the deleted genes do not influence the antigen presentation of the bacteria in phagocytes. The inserted HPV gene is added with an E6 protein coding gene besides an E7 protein coding gene, and is formed by selecting antigen epitope peptide genes in the E6 and E7 protein coding genes and fusing the antigen epitope peptide genes, and the coding gene is optimized by a listeria codon and has uniqueness. The inserted HPV fusion antigen gene can improve the recognition of the vaccine bacteria to the tumor cells, attack more tumor cells and achieve better treatment effect. The screening of the positive bacteria is carried out by temperature, erythromycin and blue-white spot screening. The blue-white spot screening of the invention is identified by naked eyes, is simple and easy to implement and has high screening success rate. The HPV gene insertion site is at the downstream of the mpl gene, and the site does not influence the expression of other important proteins of bacteria and keeps the biological activity of the original strain as much as possible.

The method comprises the following specific steps:

1) pertinence: inducing specific cell factor for cervical cancer cell, targeting treating cancer tissue, and no damage to normal cell.

2) High efficiency: the vaccine strain has high immunocompetence and induces efficient cell factor expression. The Listeria strain has the function of efficiently activating the cellular immune function of an organism, so that the recombinant strain carrying the antigen can activate various anti-tumor immune mechanisms, activate specific tumor killer T cells and break the effective tumor killing mechanism of immune suppression reactivation.

3) Safety: the attenuated strain does not exist in vivo for a long time, does not cause infection, shows good tolerance in preclinical tests, is proved to be safe by other similar clinical tests, and can be well compatible with other tumor treatment means, such as cocktail-type immunosuppressants, surgeries, radiotherapy and chemotherapy.

4) And (3) stabilizing: the antigen gene carried by the vaccine strain prepared by the invention is integrated into the genome of the strain, and the antigen gene stably exists in the strain and cannot be lost.

5) The cost is low: the vaccine is low in production cost, rapid and economical, and can be produced on a large scale by using a standard bacterial fermentation technology without cell culture, cold chain transportation and storage.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a map of the structure of Li-targeted recombinant plasmids. A structural map of a Li targeting plasmid contains two Li homologous genes (Li mpl and Li orfBAldh), an ampicillin resistance gene (Amp), an erythromycin resistance gene (Ery), and a gene cassette (gene cassette): comprises a promoter phly, a HAepitope gene, a VSV-G epitope gene, a GP33 epitope gene, a GP61 epitope gene and a fusion protein HPV E6E7 gene.

FIG. 2 is a diagram showing the result of PCR identification electrophoresis after electrically converting Li Δ actAplcB-lacZ into a targeting plasmid. 1 is target fragment HPV16E6E7 amplified from bacteria after electrotransformation, and M is DNA ladder.

FIG. 3 is a diagram showing the result of identifying the electrophoresis of the upgraded grains after the targeting plasmid is electrically converted into Li delta actAplcB-lacZ. 1 is a plasmid extracted from the bacterium after electrotransformation, and M is DNA ladder.

FIG. 4 is a diagram showing the result of PCR identification electrophoresis of recombinant bacteria. The result of the PCR identification electrophoresis of Li delta actAplcB-E6E7 shows that + is a positive control, -is a negative control, and M is DL2000 DNA marker. A: 1 is an HPV16E6E7 fusion antigen gene amplified by taking a Li delta actAplcB-E6E7 genome as a template; b: 1 is an anti-erythromycin gene amplified by taking a Li delta actAplcB-E6E7 genome as a template; c: 1 is actA gene amplified using the Li delta actAplcB-E6E7 genome as a template. The PCR amplification result is in line with expectation, which indicates that the Li delta actAplcB-E6E7 recombinant vaccine strain is successfully constructed, and the HPV E6E7 gene segment in the targeting plasmid is integrated into the genome of the bacteria at a fixed point.

FIG. 5 is a schematic diagram showing the structure of a foreign gene inserted into the genome of a recombinant strain Li Δ actAplcB-E6E7 and the genes before and after the insertion. From this figure, it was found that the insertion site of the foreign gene was deleted the actA and plcB genes from the recombinant bacterial genome. The inserted foreign gene expresses fusion protein under the action of self-contained promoter phly, and the fusion protein HAs secretion signal peptide, GP33 and GP61 epitope peptide tags and HA and VSV-G western blot detection tags.

FIG. 6 is a survival curve of C57BL/6 mice inoculated with the recombinant strain. C57BL/6 mice were vaccinated separately into the tail vein with different doses of Li Δ actAplcB-E6E7, i.e., 3.0X 10⁷CFU/only, 3.3X 10⁸CFU/only and 3.5X 10⁹CFU/3 inoculation dose, continuously observing survival condition of mice for 10 days, and drawing survival curve of each dose groupAnd calculating the survival rate of each group. Calculating the LD of Li delta actAplcB-E6E7 according to the modified Korotkoff method₅₀Is 4 x 10⁸CFU/only.

FIG. 7 is a graph of tumor volumes at different time points following caudal intravenous injection of recombinant Listeria ovis (Li Δ actAplcB-E6E7) immunotherapy in cervical cancer model C57BL/6 mice. C57BL/6 mice were inoculated with 1X 10 TC-1 cells subcutaneously on the right flank of day 0⁵One for each model, three immunotherapies were performed on the modeled mice on days 7, 14 and 21 after TC-1 cell inoculation, and the tumor volumes of each group of mice were recorded. In the figure, a is the tumor volume of each group of model mice on day 7 after TC-1 cell inoculation; b is the tumor volume at different time points after immunotherapy in each group of model mice. Wherein A is normal saline control group, B is Li carrier bacterium (Li delta actAplcB-lacZ) control group, and C is vaccine treatment group. There was no statistical difference in tumor volume between groups at the time of the first immunotherapy (day 7). After the second (day 14) and third tumor treatment (day 21), observation was made at day 42 (mice were sacrificed when the tumor length of the mice was 20 mm), no mice survived in both the normal saline control group and the vehicle bacteria control group, and the tumor size of the surviving mice in the treatment group was effectively controlled, wherein 1 mouse was completely cured.

FIG. 8 shows the survival of the cervical cancer model mice in each group after tail vein injection immunotherapy. Survival conditions of mice in each group after three times of immunotherapy on 7 days, 14 days and 21 days after TC-1 cell inoculation on cervical cancer model mice, wherein A is a normal saline control group, B is a Li carrier bacterium control group, and C is a vaccine treatment group. Survival analysis is carried out on the treatment group and each group, and the survival time of the cervical cancer model mouse is obviously prolonged by the treatment group compared with the treatment group by the normal saline (P is less than 0.05) and the survival time of the cervical cancer model mouse compared with the treatment group by the carrier control group (P is less than 0.05).

FIG. 9 is a photograph of a tumor of a 35d mouse which is a cervical cancer model. A is a normal saline control group; b is a Li carrier bacterium control group; and C is a vaccine treatment group.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1 preparation of the vaccine bacterium Li Δ actAplcB-E6E7

(1) HPV16E6E7 gene fragment synthesis

The method comprises the steps of inquiring HPV16 type E6 protein and E7 protein genes from NCBI, predicting dominant T cell epitope by online software analysis, selecting and combining fusion epitope peptide genes, carrying out corresponding optimization according to Listeria codons, adding HindIII enzyme cutting sites at the upstream and Xho I enzyme cutting sites at the downstream, and finally directly obtaining a DNA sequence (obtained in a form of clone plasmids pUC57-E6E7 provided by a company) through synthesis, wherein the fragment length is 866bp (shown as SEQ ID NO. 1).

(2) Construction of targeting plasmids

Construction of the intermediate plasmid pCW203-E6E7

Plasmids pUC57-E6E7 and pCW203 were extracted according to the plasmid instructions and eluted with 30. mu.L of Elution Buffer.

pUC57-E6E7 and pCW203 were double digested with HindIII and Xho I, 20. mu.L: pUC57-E6E7 or pCW203 < 1ng, HindIII 1. mu.L, Xho I1. mu.L, 10 XNEB Buffer 2. mu.L, ddH₂O make up the system to 20. mu.L. The plasmid is digested in water bath at 37 ℃ for 1h, 0.5 mu L of CIAP is added into the vector fragment, and dephosphorylation is carried out in water bath at 37 ℃ for 30 min. The enzyme digestion mixture is mixed with 6 XLoading Buffer, electrophoresis (1% agarose, 94V) is carried out, fragments with corresponding lengths are recovered through gel (pUC57-E6E7 recovers small fragments after enzyme digestion, namely fusion epitope peptide gene fragments with the length of 866 bp; pCW203 recovers large fragments after enzyme digestion, namely vector fragments with the length of about more than 10Kb), and the fragments are eluted by 30 mu L of precipitation Buffer.

Respectively taking fusion epitope peptide antigen gene fragments and carrier fragments according to a connection system: 50ng of vector, mol of insert to mol of vector fragment 5: 1, T4 Ligase 1. mu.L, 10 XLigase Buffer 5. mu.L, ddH₂O make up the system to 10. mu.l and attach for 1h at 22 ℃. The ligation product was gently mixed with competent cells of E.coli DH5 α at a volume of 1:10, ice-cooled for 30min, heat shocked at 42 ℃ for 45s, ice-cooled for 3min, and added with 500. mu.L of preheated SOB broth, mixed well, cultured at 37 ℃ and 180rpm for 1h, spread on LA plates (LB-Amp plates: LB plates containing 100. mu.g/mL Amp), and cultured at 37 ℃ for 16-20 h.

And (3) PCR screening: amplifying a target band HPV16E6E7 and a primer HPV-f/r (f: 5'-CATCAAAAACGTACAGCAATG-3', r: 5'-TGGTTTTTGAGAACAAATTGG-3') by using a bacterial DNA genome extracted by a boiling method as a template, wherein the reaction circulating conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 47 ℃ 30s, 72 ℃ 1min) × 30 cycles → 72 ℃ 10min → 4 ℃; electrophoretic Observation of PCR results (electrophoresis conditions: 1% agarose gel; 90V, loading: 5. mu.l PCR product), expected results: the fragment of HPV16E6E7 is about 852 bp.

Enzyme digestion verification: plasmids were extracted from positive bacteria screened and verified by PCR, digested with Hind III and Xho I according to the above digestion system, and the digested mixture was mixed with 6 × Loading Buffer for electrophoresis (1% agarose, 94V).

Sequencing and verifying: and extracting the plasmid which is expected by PCR and enzyme digestion verification, and sending the plasmid to a sequencing company for sequencing. Coli carrying the positive plasmid, which was completely correct after sequencing verification, was stored at-80 ℃.

(ii) construction of targeting plasmid

Plasmid pCW154 was extracted and eluted with 30. mu.L of Elution Buffer. Plasmid pCW154 and intermediate plasmid pCW203-E6E7 are respectively and uniformly mixed by restriction enzymes BamH I and Xho I according to the system of (2) (HindIII is replaced by BamH I), enzyme digestion and dephosphorylation are carried out, gel is recovered after electrophoresis, and small fragments after enzyme digestion of pCW203-E6E7, namely an insert fragment (931bp) and a pCW154 vector skeleton (long fragment after enzyme digestion, the length is about 8606 bp). And (3) uniformly mixing the systems according to the step (2), connecting, converting into escherichia coli DH5 alpha, coating the escherichia coli DH5 alpha on an LA plate, and performing PCR screening, BamHI and XhoI double-enzyme digestion verification and sequencing verification on a single growing colony, wherein the specific operation is the same as the step (2) (replacing HindIII enzyme with BamHI enzyme). The E.coli carrying the positive plasmid after sequencing verification was stored at-80 ℃. The structural map of the targeting plasmid is shown in FIG. 1.

(3) Preparation of Li delta actAplcB-lacZ competent cell for electrotransformation

Inoculating Li delta actAplcB-lacZ into 15ml BHI liquid culture medium containing 0.5mol/L sucrose, and culturing at 37 ℃ and 220rpm for 12-16 h. The resulting bacterial liquid was transferred to 250ml of BHI broth containing 0.5mol/L sucrose, and A was periodically measured₆₀₀When A is₆₀₀When the concentration is 0.4, penicillin G is added to the solution to a final concentration of 12.5. mu.g/ml, and the culture is continued. When A is₆₀₀When the concentration is 0.7, the mixture is poured into a 50mL centrifuge tube, centrifuged at 10000rpm at 4 ℃ for 5min, and the supernatant is discarded. The precipitate was washed repeatedly with 0.5mol/L ice-cold sucrose solution, 20ml of 0.5mol/L sucrose solution was added each time, centrifuged at 4 ℃ at 10000rpm for 10min, the supernatant was discarded, and washed 3 times. Add 300. mu.l of resuspension of 0.5mol/L sucrose per tube, dispense pre-cooled sterile EP tubes at 50. mu.l/tube, store at-80 ℃.

(4) Electrotransformation of targeting plasmid into Li delta actAplcB-lacZ

Taking out the prepared competent cells from a refrigerator at-80 deg.C, placing in an ice box, melting completely, slowly adding 5 μ l targeting plasmid into the competent cells Li Δ actAplcB-lacZ at 1/10 ratio with a precooling gun head at-20 deg.C, and making into ddH₂And (4) O negative control. After mild mixing, the mixture is iced for 5min, then the mixture is completely transferred into an electric rotating cup, the ice-cooling is carried out for 5min, and the electric rotating is carried out in an electric rotating instrument (the electric rotating condition is that the voltage is 1500V, the duration is 5ms, and the inner diameter of the electric rotating cup is 1 mm). After the completion of the electrotransfer, the mixture was ice-cooled for 5min, 750. mu.l of BHI broth preheated at 37 ℃ was added to the flask with the tip preheated at 37 ℃, the mixture was mixed well and transferred to an EP tube, and the mixture was cultured for 2 hours at 150rpm with a shaker at 30 ℃. And (3) coating the bacterial liquid on a BHI agar plate (BHI-Ery-X-gal, BEXI plate) containing 1-5 mug/ml of erythromycin and 20-60 mug/ml of X-gal by using a sterile L-shaped glass rod in a full amount, culturing at 30 ℃ for 48-72 hours, wherein the positive bacteria are blue colonies, performing pure culture on the BEXI plate, culturing at 30 ℃ for 24 hours, and performing PCR verification and quality-improved particle verification, wherein the steps are shown in a figure 2 and a figure 3.

And (3) PCR verification: extracting all genes of bacteria by a boiling method as a template, amplifying a target band E6E7 and a primer HPV-f/r (f: 5'-CATCAAAAACGTACAGCAATG-3', r: 5'-TGGTTTTTGAGAACAAATTGG-3'), wherein the reaction circulation conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 47 ℃ 30s, 72 ℃ 1min) × 30 cycles → 72 ℃ 10min → 4 ℃; electrophoretic Observation of PCR results (electrophoresis conditions: 1% agarose gel; 90V, loading: 5. mu.l PCR product), expected results: the fragment of HPV16E6E7 is about 852 bp. As shown in FIG. 2, it was suggested that the targeting plasmid had been transferred into the bacterium.

Extracting plasmid according to the plasmid extraction kit (Listeria is gram-positive bacteria, lysozyme is required to be added before Solution I is used, the final concentration of lysozyme is 20mg/L, the bacteria are resuspended after the Solution I containing lysozyme is added, water bath at 37 ℃ is carried out for 45min), and finally electrophoresis is carried out after Elution by 30 mul of Elution Buffer. Gel imaging is carried out, and whether a band exists or not is observed to judge whether the recombinant plasmid is successfully introduced, wherein the size of the plasmid is 9525 bp. As shown in FIG. 3, the plasmid extraction was successful, and the length was as expected, indicating that the targeting plasmid was successfully transferred.

(5) Homologous recombination and screening of Li delta actAplcB-lacZ and targeting plasmid

Homologous recombination hybridization culture of Li delta actAplcB-lacZ and targeting plasmid

Li delta actAplcB-lacZ streaked BEXI plate with electrically transferred targeting plasmid was subcultured at 42 deg.c for 2-3 passages (48 hr per passage), and a single blue colony was inoculated into 5ml BHI broth and subcultured at 30 deg.c and 200rpm for 6 passages (24 hr per passage). At the beginning of the 3 rd passage of the broth, 40. mu.l of the culture was diluted 1:10 with 360. mu.l of BHI and 10 serial dilutions were made⁶ Coating 10 times, respectively⁶Mu.l of the diluted bacterial solution was plated on BEXI and BXI (BHI agar plates containing X-gal 20-60. mu.g/ml) and cultured at 30 ℃ for 24 hours. The positive colony Li Δ actAplcB-E6E7 grew as a white colony on BXI plates and did not grow on BEXI plates.

② the Li delta actAplcB-E6E7 identified by PCR

The bacterial genome extraction kit extracts the genome DNA of Li delta actAplcB-E6E7 as a template, and carries out amplification of the following three groups of gene fragments: and identifying the recombinant bacteria Li delta actAplcB-E6E7 according to the amplification result of the anti-erythromycin gene, the target antigen gene cassette and the actA gene. As shown in FIG. 4, the sequence from left to right is A, B, C, wherein 1 in A is HPV16E6E7 amplified by taking Li delta actAplcB-E6E7 genomic DNA as a template, the primers and the reaction conditions are shown in (2) first, 1 in B is an amplified target band anti-erythromycin gene Ery, about 1471bp, the primers Ery-f/r (f: 5'-GATAAGTCGACGATTCACAAAAAATAG-3', r: 5'-AAAACTAGTCCCGGG GCGAATTG-3'), and the reaction cycle conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 60 ℃ 30s, 72 ℃ 2min) × 30 cycles → 72 ℃ 10min → 4 ℃; 1 in C is amplified Li actA gene, about 600bp, a primer Li-actA-f/r (f: 5'-GAAGCTAAAAGTGCAAATGTC CC-3', r: 5'-ATTTCTTTAATACTGCGTTTGGGG-3'), and the reaction circulation conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 55 ℃ 30s, 72 ℃ 1min) × 30 cycles → 72 ℃ 10min → 4 ℃; . The Li delta actAplcB-E6E7 carries an E6E7 fusion gene and does not carry an anti-erythromycin gene, and the actA gene is knocked out through PCR identification. The identification result is consistent with the expectation, and Li delta actAplcB-E6E7 is successfully prepared.

Third Gene sequencing verification

After PCR identification, using bacterial genome DNA as a template, carrying out PCR amplification on a target antigen gene HPV16E6E7, carrying out sequencing verification on an amplification product, and carrying out strain preservation on a strain with correct sequencing. The structural schematic diagram of the foreign gene inserted into the recombinant bacterial genome and the genes before and after the insertion site is shown in FIG. 5.

(6) Candidate strain LD of Li delta actAplcB-E6E7 vaccine₅₀Measurement of (2)

After thawing the Li.DELTA.actaaplcb-E6E 7 strain stored at-20 ℃ in a water bath at 37 ℃, 10. mu.l of each strain was inoculated into 5ml of BHI broth and cultured overnight at 200rpm at 37 ℃. On the next day, 40. mu.l of the once-recovered bacterial suspension was taken and cultured in 20ml of BHI broth at 37 ℃ and 200rpm overnight for two recoveries.

Inoculating 17.5ml of each of the twice recovered bacterial solutions to 350ml of BHI broth, culturing at 37 ℃ and 200rpm by shaking until A is obtained₆₀₀When the value is between 0.3 and 0.7, determining A at the moment according to the growth curve of Listeria₆₀₀The corresponding bacterial quantity is calculated, and concentration or dilution treatment is carried out according to the predicted bacterial inoculation dose: taking a certain amount of bacterial liquid for centrifugation, carrying out 13000rpm for 2min, carrying out re-centrifugation after re-suspension by using normal saline, then re-suspending the bacterial liquid to the bacterial infection dosage by using the normal saline, and placing the bacterial liquid on ice for standby.

After 6-8 weeks old C57BL/6 female mice are adaptively fed for 3 days, the mice are randomly divided into 3 groups, 7 mice in each group are inoculated with 3.0 multiplied by 10 tail veins respectively⁷CFU/only, 3.3X 10⁸CFU/only and 3.5X 10⁹CFU/only; continuously observing for 10 days, counting the death and survival conditions of the mice, and drawing survival curves of each dose group, as shown in figure 6; calculating LD by using improved fitting method₅₀And at 0.1 × LD₅₀As the optimal subsequent immunization dose.

Example 2: evaluation of therapeutic effect of vaccine strain on cervical cancer

(1) TC-1 tumor cell culture and cervical cancer model establishment

Using RPMI1640 Medium containing 10% fetal calf serum, 1% penicillin and streptomycin at 37 deg.C with 5% CO₂Culturing TC-1 cells in an incubator until the density reaches about 80%, rinsing the cells with Hank's solution for 2-3 times, digesting with pancreatin, counting by using PBS (phosphate buffer solution) for heavy suspension, and preparing the cells into the concentration of 1 × 10⁶The cell suspension of the mice is 100 mu l, and the cell suspension is injected into the right flank of the C57BL/6 female mouse with the age of 6-8 weeks, so that a mouse HPV infection tumor model is established, and the tumor can be formed in a macroscopic view after about 7 days. Tumor volume according to formula 1/2 × (a × b)²) Calculate (a, b are the longest and shortest diameters of the tumor, respectively). Statistical analysis of the data was performed using SPSS software. The mean value of the two samples is compared by t test, P<A difference of 0.05 is statistically significant. As shown in FIG. 7a, the tumor volume was uniform in the groups on day 7, and there was no statistical difference between the groups.

(2) Immunotherapy of modeled mice

The experiment establishes a negative control group: injecting Normal Saline (NS) with the same volume as that of the treatment group on 7 th, 14 th and 21 th days of the model building respectively; control group of Li-carrier bacteria: injecting 0.1 × LD on 7, 14 and 21 days of molding respectively₅₀Li Δ actAplcB-lacZ (injection dose 2X 10)⁷CFU/r), treatment group: injecting 0.1 × LD on 7, 14 and 21 days of molding respectively₅₀Li Δ actAplcB-E6E7 (injection dose 4X 10)⁷CFU/only).

33 molding mice were randomly divided into 3 groups of 11 mice each. The first immunotherapy was performed on day 7 after TC-1 cell injection, and the bacterial solution was prepared according to example 1(6), and the amount of the tail vein inoculated with the bacterial solution was 100. mu.l, and the tail vein of the NS control group was inoculated with NS of the same volume. A second booster treatment was given on day 14 and a third booster treatment was given on day 21, at the same method and dose as the first. Mice body weight, tumor growth and survival were observed and recorded every 2 days (mice were sacrificed when tumors reached 20mm in length) and according to the formula 1/2 × (a × b)²) Tumor volumes were calculated (a, b are the longest and shortest diameters of the tumor, respectively). Survival comparisons Log rank test, P<A difference of 0.05 is statistically significant. The results are shown in FIGS. 7b, 8, and 9 for the booster immunotherapyAfter 42 days, compared with the Li carrier bacterium control group and the normal saline group, the tumor size of the treatment group is effectively controlled, and 1 mouse in the treatment group is cured (figure 7). The survival rate of the mice in the treatment group is obviously improved compared with the mice in the Li carrier bacterium control group (P is less than 0.05) and the negative control group (P is less than 0.05). The vaccine strain prepared by experiments has effective control and treatment effects on cervical cancer. (FIG. 8, FIG. 9)

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing Songyue Biotech Co., Ltd

<120> therapeutic vaccine for cervical cancer based on recombinant attenuated listeria ovis

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 866

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aagctttgca tcaaaaacgt acagcaatgt tccaagatcc acaagaatct ggtcgtaaat 60

taccacaatt atgtacagaa ttacaaacaa caattcatga tattatttta gaatgtgttt 120

attgtaaaca acaattatta cgtcgtgaag tttatgattt cgcattccgt gatttatgta 180

ttgtttatcg tgatggtaac ccatatgcag tttgtgataa atgtttaaaa ttctattcta 240

aaatttctga atatcgtcat catggtgata caccaacatt acatgaatat atgttagatt 300

tacaaccaga aacaacagat ttatatggtt atggtcaatt aaacgattct tctgaagaag 360

aagatgaaat tgatggtcca gcaggtcaag cagaaccaga tcgtgcacat tataacattg 420

ttacattctg ttgtaaatgt gattctacat tagataaatg tttaaaattc tattctaaaa 480

tttctgaata tcgtcattat tgttattctg tttatggtac aacattagaa caacaatata 540

acaaaccatt atgtgattta ttaattcgtt gtattaactg tcaaaaacca ttatgtccag 600

aagaaaaaca acgtcattta gataaaaaac aacgtttcca taacattcgt ggtcgttgga 660

caggtcgttg tatgtcttgt tgtcgttctt ctcgtacacg tcgtgaaaca caattacatt 720

ataacattgt tacattctgt tgtaaatgtg attctacatt acgtttatgt gttcaatcta 780

cacatgttga tattcgtaca ttagaagatt tattaatggg tacattaggt attgtttgtc 840

caatttgttc tcaaaaacca ctcgag 866

<210> 2

<211> 13913

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgcggccgcc agtgtgatgg atatctgcag aattaattcg gctttctaga gtgactttta 60

tgttgaggca ttaacatttg ttaacgacga taaagggaca gcaggactag aataaagcta 120

taaagcaagc atataatatt gcgtttcatc tttagaagcg aatttcgcca atattataat 180

tatcaaaaga gaggggtggc aaacggtatt tggcattatt aggttaaaaa atgtagaagg 240

agagtgaaac ccatgaaaaa aataatgcta gtttttatta cacttatatt agttagtcta 300

ccaattgcgc aacaaactga agcaaaggat gcatcggatc catatccata tgatgttcca 360

gattatgcat cattgaattc aatgagaagt gaaagaccac aagctttgca tcaaaaacgt 420

acagcaatgt tccaagatcc acaagaatct ggtcgtaaat taccacaatt atgtacagaa 480

ttacaaacaa caattcatga tattatttta gaatgtgttt attgtaaaca acaattatta 540

cgtcgtgaag tttatgattt cgcattccgt gatttatgta ttgtttatcg tgatggtaac 600

ccatatgcag tttgtgataa atgtttaaaa ttctattcta aaatttctga atatcgtcat 660

catggtgata caccaacatt acatgaatat atgttagatt tacaaccaga aacaacagat 720

ttatatggtt atggtcaatt aaacgattct tctgaagaag aagatgaaat tgatggtcca 780

gcaggtcaag cagaaccaga tcgtgcacat tataacattg ttacattctg ttgtaaatgt 840

gattctacat tagataaatg tttaaaattc tattctaaaa tttctgaata tcgtcattat 900

tgttattctg tttatggtac aacattagaa caacaatata acaaaccatt atgtgattta 960

ttaattcgtt gtattaactg tcaaaaacca ttatgtccag aagaaaaaca acgtcattta 1020

gataaaaaac aacgtttcca taacattcgt ggtcgttgga caggtcgttg tatgtcttgt 1080

tgtcgttctt ctcgtacacg tcgtgaaaca caattacatt ataacattgt tacattctgt 1140

tgtaaatgtg attctacatt acgtttatgt gttcaatcta cacatgttga tattcgtaca 1200

ttagaagatt tattaatggg tacattaggt attgtttgtc caatttgttc tcaaaaacca 1260

ctcgagtata cagatattga aatgaataga ttaggaaaat gataagtcga cgattcacaa 1320

aaaataggca cacgaaaaac aagttaaggg atgcagttta tgcatccctt aacttactta 1380

ttaaataatt tatagctatt gacaagagat aagaattgtt caaagctaat attgtttaaa 1440

tcgtcaattc ctgcatgttt taaggaattg ttaaattgat tttttgtaaa tattttcttg 1500

tattctttgt taacccattt cagaacgaaa taattatact tttgtttatc tttgtgtgat 1560

attcttgatt tttttctact taatctgata agtgagctat tcactttagg tttaggatga 1620

aaatattctc ttggaaccat acttaatata gaaatatcaa cttctgccat taaaagtaat 1680

gccaatgagc gttttgtatt taataatctt ttagcaaacc cgtattccac gattaaataa 1740

atctcattag ctatactatc aagaacaatt ttgcgtatta tatccgtact tatgttataa 1800

ggtatattac catatatttt ataggattgg tttttaggaa atttaaactg caatatatcc 1860

ttgtttaaaa cttggaaatt atcgtgatca acaagtttat tttctgtagt tttgcataat 1920

ttatggtcta tttcaatggc agttacgaaa ttacacctct ttactaattc aagggtaaaa 1980

tggccttttc ctgagccgat ttcaaagata ttatcatgtt catttaatct tatatttgtc 2040

attattttat ctatattatg ttttgaagta ataaagtttt gactgtgttt tatatttttc 2100

tcgttcatta taaccctctt taatttggtt atatgaattt tgcttattaa cgattcatta 2160

taaccactta ttttttgttt ggttgataat gaactgtgct gattacaaaa atactaaaaa 2220

tgcccatatt ttttcctcct tataaaatta gtataattat agcacgagct ctgataaata 2280

tgaacatgat gagtgatcgt taaatttata ctgcaatcgg atgcgattat tgaataaaag 2340

atatgagaga tttatctaat ttcttttttc ttgtaaaaaa agaaagttct taaaggtttt 2400

atagttttgg tcgtagagca cacggtttaa cgacttaatt acgaagtaaa taagtctagt 2460

gtgttagact ttatgaaatc tttatacgtt tatatatatt tattatccgg aggtgtagca 2520

tgtctcattc aattttgagg gttgccagag ttaaaggatc aagtaataca aacgggatac 2580

aaagacataa tcaaagagag aataaaaact ataataataa agacataaat catgaggaaa 2640

catataaaaa ttatgatttg attaacgcac aaaatataaa gtataaagat aaaattgatg 2700

aaacgattga tgagaattat tcagggaaac gtaaaattcg gtcagatgca attcgccccg 2760

ggactagtgc ggccgctaat aatcttgcgc ttcgatgaca acagctgtac cagatgcagt 2820

gaccattagc attccgttat cagctccaag cacttcataa tcaatatcaa caccgataac 2880

ggcatttgcg ccaatatctt tcgcacgttg ttccatttca cgaattgctt cctcgcgagc 2940

attaataagt tcatcttcat agccttgcga acggcccccg aagaaatttc gaagtccagc 3000

cccaatatct ttcataaagt taacgccagt gatgacttcg ccgaaaacga tttttttata 3060

ttcgataatt tgtttgcctt caatatttgg tgaagttgtt acaatcatga gttatcccta 3120

cagtttttct tttatcatac ctcttagtac tttttctagt caaaggatat ccggttattt 3180

cgtacgattt cgcgcttttt ctatataaga aatagcatct ggaactttac aagctgtatt 3240

tccaaggttt acatgaactt tcccgactga tttcgcggct tccatcgctt tttcgtgcaa 3300

atcgtctttg taaattccac aagtgataat aaaaccgttc attgaatagc gggttctatt 3360

ggtttctgtt tgcagcgttt ctttcgcgcg ttgaagtaat gtctctgctt caggcaaatt 3420

gttaatatgt tcgctgtaaa tctgccagcc catggattta gttaaatcat aatcactttc 3480

aatccatgtt ttggcgaaaa ggagtgcatc atcacgtttg cttacaatcg atgctagtcc 3540

aaaagttagt tgtgaccatg tttcgcgaaa agcgatattc catttttcga tttgttcggt 3600

ggttattttg tttggattaa ttgctagcaa accgagataa atcaagtcac tattatttga 3660

ttcaatcagt tttacggcga gttcgtgatt ttttgtcagt ttttctcggc ggatgatttt 3720

ctttaaatca ccaattttta gtccgtaaag atctaatgaa tccggacaac cgtgattacg 3780

aaaaattttg atcgtattgg ggttttctaa ggcttgtagc tcggtgtcaa gttggtcaaa 3840

agtaatcata cgcgtcactc ctctcgaata aagtaagtat aacaaaaaaa gcatgcgaag 3900

gcgcatgctt taggatttaa gaatattagt ctatttgttt cattgcgtcg tctagctaga 3960

attaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 4020

ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 4080

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4140

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4200

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4260

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4320

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4380

tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc 4440

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 4500

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 4560

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 4620

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4680

gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc 4740

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4800

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 4860

gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4920

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 4980

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5040

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5100

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5160

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5220

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5280

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5340

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5400

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5460

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5520

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5580

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5640

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5700

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5760

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5820

ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5880

gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5940

gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 6000

acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 6060

gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 6120

cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 6180

cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 6240

caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac 6300

agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct 6360

ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc acttgatgcc 6420

tccgtgtaag ggggaatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat 6480

gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa 6540

acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg 6600

cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat 6660

ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa 6720

accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca 6780

cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag 6840

ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc 6900

cgacgatgat aagctgtcaa acatgagaat taattcccga ttatgtcttt tgcgcactcg 6960

gcttaaacca gttttcgctg gtgcgaaaaa agagtgtctt gtgacaccta aattcaaaat 7020

ctatcggtca gatttatacc gatttgattt tatatattct tgaataacat acgccgagtt 7080

atcacataaa agcgggaacc aatcatcaaa tttaaacttc attgcataat ccattaaact 7140

cttaaattct acgattcctt gttcatcaat aaactcaatc atttctttaa ttaatttata 7200

tctatctgtt gttgttttct ttaataattc atcaacatct acaccgccat aaactatcat 7260

atcttctttt tgatatttaa atttattagg atcgtccatg tgaagcatat atctcacaag 7320

acctttcaca cttcctgcaa tctgcggaat agtcgcattc aattcttctg ttattatttt 7380

tatctgttca taagatttat taccctcata catcactaga atatgataat gctctttttt 7440

catcctacct tctgtatcag tatccctatc atgtaatgga gcactacaaa ttgaatgtgt 7500

aactctttta aatactctaa ccactcggct ttgctgattc tggatataaa acaaatgtcc 7560

aattacgtcc tcttgaattt ttcttgtttt cagtttcttt tattacattt tcgctcatga 7620

tataataacg gtgctaatac acttaacaaa atttagtcat agataggcag catgccagtg 7680

ctgtctatct ttttttgttt aaaatgcacc gtattcctcc tttgcatatt tttttattag 7740

aataccggtt gcatctgatt tgctaatatt atatttttct ttgattctat ttaatatctc 7800

attttcttct gttgtaagtc ttaaagtaac agcaactttt ttctcttctt ttctatctac 7860

aactatcact gtacctccca acatctgttt ttttcacttt aacataaaaa acaacctttt 7920

aacattaaaa acccaatatt tatttatttg tttggacaat ggacaatgga cacctagggg 7980

ggaggtcgta gtacccccct atgttttctc ccctaaataa ccccaaaaat ctaagaaaaa 8040

aagacctcaa aaaggtcttt aattaacatc tcaaatttcg catttattcc aatttccttt 8100

ttgcgtgtga tgcgctgcgt ccattaaaaa tcctagagct ttgcaaccga aagttaatag 8160

ctgtcgctac tactttcgct tacgctctaa gtatatttta aggactgtca cacgcaaaaa 8220

gttttctcgg cataaaagta cctctacatc tctaaatcgt ctgtacgctg tttctcacgc 8280

tttctatcga tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc 8340

atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc 8400

ctgactgcgt tagcaattta actgtgataa actaccgcat taaagctagc tttaaggcta 8460

aatgccgaat ggttggcacc taccgcattg gcaaccgtgg cagaagaggg cgcatccgtt 8520

ttggcgaaaa agagtaaaac ggcgaggatg agtgcacagc cagagcccag ccagaaaaca 8580

aactgattat tgatggtgaa catgatgccg acaatcgagg cacacagcgc ccagccaaca 8640

cagccaaaca tccgcgcgcg accaaattcg aaattactgc gacggctgac tttctcaata 8700

aatgcctcta ctgctggcgc accggcgtta aaacaaaagc ctagataaat accaccaaca 8760

atcgatccta ctaaaatgtt gtattgtaac agtggcccga agataaaaat aaagaacggc 8820

gcaaacatca ctaacatgcc ggtaataatc cacagcaggt atttgcgcag cccgagtttg 8880

tcagaaagca gaccaaacag cggttggaat aatagcgaga acagagaaat agcggcaaaa 8940

ataatacccg tatcactttt gctgatatgg ttgatgtcat gtagccaaat cgggaaaaac 9000

gggaagtagg ctcccatgat aaaaaagtaa aagaaaaaga ataaaccgaa catccaaaag 9060

tttgtgtttt ttaaatagta cataatggat ttccttacgc gaaatacggg cagacatggc 9120

ctgcccggtt attattattt ttgacaccag accaactggt aatggtagcg accggcgctc 9180

agctggaatt aattccgccg atactgacgg gctccaggag tcgtcgccac caatccccat 9240

atggaaaccg tcgatattca gccatgtgcc ttcttccgcg tgcagcagat ggcgatggct 9300

ggtttccatc agttgctgtt gactgtagcg gctgatgttg aactggaagt cgccgcgcca 9360

ctggtgtggg ccataattca attcgcgcgt cccgcagcgc agaccgtttt cgctcgggaa 9420

gacgtacggg gtatacatgt ctgacaatgg cagatcccag cggtcaaaac aggcggcagt 9480

aaggcggtcg ggatagtttt cttgcggccc taatccgagc cagtttaccc gctctgctac 9540

ctgcgccagc tggcagttca ggccaatccg cgccggatgc ggtgtatcgc tcgccacttc 9600

aacatcaacg gtaatcgcca tttgaccact accatcaatc cggtaggttt tccggctgat 9660

aaataaggtt ttcccctgat gctgccacgc gtgagcggtc gtaatcagca ccgcatcagc 9720

aagtgtatct gccgtgcact gcaacaacgc tgcttcggcc tggtaatggc ccgccgcctt 9780

ccagcgttcg acccaggcgt tagggtcaat gcgggtcgct tcacttacgc caatgtcgtt 9840

atccagcggt gcacgggtga actgatcgcg cagcggcgtc agcagttgtt ttttatcgcc 9900

aatccacatc tgtgaaagaa agcctgactg gcggttaaat tgccaacgct tattacccag 9960

ctcgatgcaa aaatccattt cgctggtggt cagatgcggg atggcgtggg acgcggcggg 10020

gagcgtcaca ctgaggtttt ccgccagacg ccactgctgc caggcgctga tgtgcccggc 10080

ttctgaccat gcggtcgcgt tcggttgcac tacgcgtact gtgagccaga gttgcccggc 10140

gctctccggc tgcggtagtt caggcagttc aatcaactgt ttaccttgtg gagcgacatc 10200

cagaggcact tcaccgcttg ccagcggctt accatccagc gccaccatcc agtgcaggag 10260

ctcgttatcg ctatgacgga acaggtattc gctggtcact tcgatggttt gcccggataa 10320

acggaactgg aaaaactgct gctggtgttt tgcttccgtc agcgctggat gcggcgtgcg 10380

gtcggcaaag accagaccgt tcatacagaa ctggcgatcg ttcggcgtat cgccaaaatc 10440

accgccgtaa gccgaccacg ggttgccgtt ttcatcatat ttaatcagcg actgatccac 10500

ccagtcccag acgaagccgc cctgtaaacg gggatactga cgaaacgcct gccagtattt 10560

agcgaaaccg ccaagactgt tacccatcgc gtgggcgtat tcgcaaagga tcagcgggcg 10620

cgtctctcca ggtagcgaaa gccatttttt gatggaccat ttcggcacag ccgggaaggg 10680

ctggtcttca tccacgcgcg cgtacatcgg gcaaataata tcggtggccg tggtgtcggc 10740

tccgccgcct tcatactgca ccgggcggga aggatcgaca gatttgatcc agcgatacag 10800

cgcgtcgtga ttagcgccgt ggcctgattc attccccagc gaccagatga tcacactcgg 10860

gtgattacga tcgcgctgca ccattcgcgt tacgcgttcg ctcatcgccg gtagccagcg 10920

cggatcatcg gtcagacgat tcattggcac catgccgtgg gtttcaatat tggcttcatc 10980

caccacatac aggccgtagc ggtcgcacag cgtgtaccac agcggatggt tcggataatg 11040

cgaacagcgc acggcgttaa agttgttctg cttcatcagc aggatatcct gcaccatcgt 11100

ctgctcatcc atgacctgac catgcagagg atgatgctcg tgacggttaa cgcctcgaat 11160

cagcaacggc ttgccgttca gcagcagcag accattttca atccgcacct cgcggaaacc 11220

gacatcgcag gcttctgctt caatcagcgt gccgtcggcg gtgtgcagtt caaccaccgc 11280

acgatagaga ttcgggattt cggcgctcca cagtttcggg ttttcgacgt tcagacgtag 11340

tgtgacgcga tcggcataac caccacgctc atcgataatt tcaccgccga aaggcgcggt 11400

gccgctggcg acctgcgttt caccctgcca taaagaaact gttacccgta ggtagtcacg 11460

caactcgccg cacatctgaa cttcagcctc cagtacagcg cggctgaaat catcattaaa 11520

gcgagtggca acatggaaat cgctgatttg tgtagtcggt ttatgcagca acgagacgtc 11580

acggaaaatg ccgctcatcc gccacatatc ctgatcttcc agataactgc cgtcactcca 11640

acgcagcacc atcaccgcga ggcggttttc tccggcgcgt aaaaatgcgc tcaggtcaaa 11700

ttcagacggc aaacgactgt cctggccgta accgacccag cgcccgttgc accacagatg 11760

aaacgccgag ttaacgccat caaaaataat tcgcgtctgg ccttcctgta gccagctttc 11820

atcaacatta aatgtgagcg agtaacaacc cgtcggattc tccgtgggaa caaacggcgg 11880

attgaccgta atgggatagg ttacgttggt gtagatgggc gcatcgtaac cgtgcatctg 11940

ccagtttgag gggacgacga cagtatcggc ctcaggaaga tcgcactcca gccagctttc 12000

cggcaccgct tctggtgccg gaaaccaggc aaagcgccat tcgccattca ggctgcgcaa 12060

ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 12120

atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 12180

aacgacggga tccataaaaa ctagcattat ttttttcatg ggtttcactc tccttctaca 12240

ttttttaacc taataatgcc aaataccgtt tgccacccct ctcttttgat aattataata 12300

ttggcgaaat tcgcttctaa agatgaaacg caatattata tgcttgcttt atagctttat 12360

tctagtcctg ctgtcccttt atcgtcgtta acaaatgtta atgcctcaac ataaaagtca 12420

ctctagtaca tttttatctc atttttataa tcctgataga gataatactt atttgccggg 12480

ttttgctaat gcgaaaataa caggagcaaa gtatttcaat caatcggtga ctgattaccg 12540

agaagggaaa tttgacacag cgttttataa attaggccta gcaatccatt attatacgga 12600

tattagtcaa cctatgcacg ccaataattt taccgcaata tcataccctc caggctacca 12660

ctgtgcatat gaaaattacg tagataccat taaacacaat tatcaagcaa cggaagacat 12720

ggtagcaaaa agattttgct cagatgacgt gaaagactgg ctctatgaaa atgcgaaaag 12780

ggcgaaagcg gactacccga aaatagtcaa tgcgaaaact aaaaaatcat atttagtagg 12840

aaattctgaa tggaaaaagg atacagtgga acctactgga gctagactaa gagattcaca 12900

gcaaactttg gcaggttttt tagaattttg gtctaaaaaa acaaatgaat aacaatattt 12960

aggaatacat tcttatccac tcgttagcgg gtggatatat tttatgggga ggaagtaagc 13020

caaatgtata taaaagggag gttaatcttt ttctttgtag tgttagtaat cgcgttatgt 13080

tccgtattaa tattgcttat aataaaaata agcgtatgga aagatgaacc atttcattta 13140

agcgatgcaa aggagattga gtgtcttgga agttgtgaaa taaaaaatac taatcaaaaa 13200

atccatttct tctccataaa agagaatttg tttgaggaaa agggtgatat agctggaatt 13260

ttaaatgaag acgaacaaaa agttgctgat aagtccattt tcattgttat tttggatgat 13320

gaaaaaggga ttgcgaacga agagtaaagc ggtttgttag gtagtcttga atagttaaaa 13380

aacaatatag gaggaacatc tatgaaaatg cttaaaaagg gtaccgcagt tctatttgta 13440

atgatcatgg cagttatgtt agtcgcgtgt ggggataaag aagaaacgaa aacttacaca 13500

ctttcccaaa atggtgttga ttctaaatta acatacacat ataagggaga caaagttact 13560

aaacaaacag ctgaaaatac aatgtcatat gcttcgttag gtgtggcttc aaaagaagac 13620

gctgaaaaaa tgcttaaagc aacaagcgat aaattccaag gtattgatgg tttaaaagaa 13680

aagattgaat ataaagatga taaagctatt gaaacactag aagtagatta cactaaaatc 13740

tcttcggaag atatgaacaa aatccctggt atgtcttcaa atggtgatac ttctaaaggg 13800

attagcatgg aggaatctgc aaaaatgcta gaatcccaag gctataaaga agtttcaaaa 13860

taaatttgca ataaaaaaga agtatccgat tcatttggat acttcttttt tat 13913

<210> 3

<211> 9525

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaattcccga ttatgtcttt tgcgcactcg gcttaaacca gttttcgctg gtgcgaaaaa 60

agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt 120

tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa 180

tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat 240

aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc 300

atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg 360

atcgtccatg tgaagcatat atctcacaag acctttcaca cttcctgcaa tctgcggaat 420

agtcgcattc aattcttctg ttattatttt tatctgttca taagatttat taccctcata 480

catcactaga atatgataat gctctttttt catcctacct tctgtatcag tatccctatc 540

atgtaatgga gcactacaaa ttgaatgtgt aactctttta aatactctaa ccactcggct 600

ttgctgattc tggatataaa acaaatgtcc aattacgtcc tcttgaattt ttcttgtttt 660

cagtttcttt tattacattt tcgctcatga tataataacg gtgctaatac acttaacaaa 720

atttagtcat agataggcag catgccagtg ctgtctatct ttttttgttt aaaatgcacc 780

gtattcctcc tttgcatatt tttttattag aataccggtt gcatctgatt tgctaatatt 840

atatttttct ttgattctat ttaatatctc attttcttct gttgtaagtc ttaaagtaac 900

agcaactttt ttctcttctt ttctatctac aactatcact gtacctccca acatctgttt 960

ttttcacttt aacataaaaa acaacctttt aacattaaaa acccaatatt tatttatttg 1020

tttggacaat ggacaatgga cacctagggg ggaggtcgta gtacccccct atgttttctc 1080

ccctaaataa ccccaaaaat ctaagaaaaa aagacctcaa aaaggtcttt aattaacatc 1140

tcaaatttcg catttattcc aatttccttt ttgcgtgtga tgcgctgcgt ccattaaaaa 1200

tcctagagct ttgcaaccga aagttaatag ctgtcgctac tactttcgct tacgctctaa 1260

gtatatttta aggactgtca cacgcaaaaa gttttctcgg cataaaagta cctctacatc 1320

tctaaatcgt ctgtacgctg tttctcacgc tttctatcga tcccgcaaga ggcccggcag 1380

taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat 1440

gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa 1500

actaccgcat taaagcttgt cgacgattca caaaaaatag gcacacgaaa aacaagttaa 1560

gggatgcagt ttatgcatcc cttaacttac ttattaaata atttatagct attgacaaga 1620

gataagaatt gttcaaagct aatattgttt aaatcgtcaa ttcctgcatg ttttaaggaa 1680

ttgttaaatt gattttttgt aaatattttc ttgtattctt tgttaaccca tttcagaacg 1740

aaataattat acttttgttt atctttgtgt gatattcttg atttttttct acttaatctg 1800

ataagtgagc tattcacttt aggtttagga tgaaaatatt ctcttggaac catacttaat 1860

atagaaatat caacttctgc cattaaaagt aatgccaatg agcgttttgt atttaataat 1920

cttttagcaa acccgtattc cacgattaaa taaatctcat tagctatact atcaagaaca 1980

attttgcgta ttatatccgt acttatgtta taaggtatat taccatatat tttataggat 2040

tggtttttag gaaatttaaa ctgcaatata tccttgttta aaacttggaa attatcgtga 2100

tcaacaagtt tattttctgt agttttgcat aatttatggt ctatttcaat ggcagttacg 2160

aaattacacc tctttactaa ttcaagggta aaatggcctt ttcctgagcc gatttcaaag 2220

atattatcat gttcatttaa tcttatattt gtcattattt tatctatatt atgttttgaa 2280

gtaataaagt tttgactgtg ttttatattt ttctcgttca ttataaccct ctttaatttg 2340

gttatatgaa ttttgcttat taacgattca ttataaccac ttattttttg tttggttgat 2400

aatgaactgt gctgattaca aaaatactaa aaatgcccat attttttcct ccttataaaa 2460

ttagtataat tatagcacga gctctgataa atatgaacat gatgagtgat cgttaaattt 2520

atactgcaat cggatgcgat tattgaataa aagatatgag agatttatct aatttctttt 2580

ttcttgtaaa aaaagaaagt tcttaaaggt tttatagttt tggtcgtaga gcacacggtt 2640

taacgactta attacgaagt aaataagtct agtgtgttag actttatgaa atctttatac 2700

gtttatatat atttattatc cggaggtgta gcatgtctca ttcaattttg agggttgcca 2760

gagttaaagg atcaagtaat acaaacggga tacaaagaca taatcaaaga gagaataaaa 2820

actataataa taaagacata aatcatgagg aaacatataa aaattatgat ttgattaacg 2880

cacaaaatat aaagtataaa gataaaattg atgaaacgat tgatgagaat tattcaggga 2940

aacgtaaaat tcggtcagat gcaattcgcc ccgggactag tttcgttatg gcttaaattg 3000

caataacgct ttttgggatg gtcaggaaat tctttatgga gatggagata agaagaatta 3060

caaaccattt tcatgtgcaa aaaatatcgt cgctcatgaa ttaacgcatg ctgtcattca 3120

acactcagca ggtttagaat acgaagggca agcaggtgca ttaaatgaat cattcgcgga 3180

tgtctttagt tattttatta ctccagacaa ttggttaata ggtgaggatg tctgtttaca 3240

tggaatgaac agcaggagag taagaagttt aaaagaacca gataaataca accaagcggc 3300

acacatgaat gagtatgagt caatgccgat tacagaagaa tatgattggg gaggggttca 3360

tttcaatagc ggaataccta acaaagctgc ttataatacc atcacgaaag ttggtaggga 3420

acaggcagaa caactatatt ttcgagcatt aaagtattac ttaacgaaaa aatcccaatt 3480

tgtcgatgct aagaatgcgc ttcaacaggc ggcgagagac ttatacagcg aagaagtggc 3540

taaaaaagta ggagaagcat gggaagaagt tggcgttaga taagggatgt cctccaagcc 3600

aaattaacaa atgttaaaga aaagcggttt ctcccaatgc tattcttgaa gcaattcatg 3660

aattgttttc ctaaattagc taatagggag gttttaacaa gagtcaaaaa ctctaaacag 3720

cctgaattgt atactgttct tacgcaggta ttagagagcg gccgccagtg tgatggatat 3780

ctgcagaatt aattcggctt tctagagtga cttttatgtt gaggcattaa catttgttaa 3840

cgacgataaa gggacagcag gactagaata aagctataaa gcaagcatat aatattgcgt 3900

ttcatcttta gaagcgaatt tcgccaatat tataattatc aaaagagagg ggtggcaaac 3960

ggtatttggc attattaggt taaaaaatgt agaaggagag tgaaacccat gaaaaaaata 4020

atgctagttt ttattacact tatattagtt agtctaccaa ttgcgcaaca aactgaagca 4080

aaggatgcat cggatcttaa agctgtttat aattttgcta ctatgaagga tccatatcca 4140

tatgatgttc cagattatgc atcattgaat tcaatgagaa gtgaaagacc acaagctttg 4200

catcaaaaac gtacagcaat gttccaagat ccacaagaat ctggtcgtaa attaccacaa 4260

ttatgtacag aattacaaac aacaattcat gatattattt tagaatgtgt ttattgtaaa 4320

caacaattat tacgtcgtga agtttatgat ttcgcattcc gtgatttatg tattgtttat 4380

cgtgatggta acccatatgc agtttgtgat aaatgtttaa aattctattc taaaatttct 4440

gaatatcgtc atcatggtga tacaccaaca ttacatgaat atatgttaga tttacaacca 4500

gaaacaacag atttatatgg ttatggtcaa ttaaacgatt cttctgaaga agaagatgaa 4560

attgatggtc cagcaggtca agcagaacca gatcgtgcac attataacat tgttacattc 4620

tgttgtaaat gtgattctac attagataaa tgtttaaaat tctattctaa aatttctgaa 4680

tatcgtcatt attgttattc tgtttatggt acaacattag aacaacaata taacaaacca 4740

ttatgtgatt tattaattcg ttgtattaac tgtcaaaaac cattatgtcc agaagaaaaa 4800

caacgtcatt tagataaaaa acaacgtttc cataacattc gtggtcgttg gacaggtcgt 4860

tgtatgtctt gttgtcgttc ttctcgtaca cgtcgtgaaa cacaattaca ttataacatt 4920

gttacattct gttgtaaatg tgattctaca ttacgtttat gtgttcaatc tacacatgtt 4980

gatattcgta cattagaaga tttattaatg ggtacattag gtattgtttg tccaatttgt 5040

tctcaaaaac cactcgaggg attaaatgga ccagatattt ataaaggagt ttatcaattt 5100

aaaagtgttg aatttgatgt cgagtataca gatattgaaa tgaatagatt aggaaaatga 5160

taagtcggcg gccgcacaaa aaacggaaat cagttagtaa aactggtttc cgttttttat 5220

taatagtctt gagcctcaat aacaacagca gtgccagatg ctgtaaccat taacatacca 5280

ttatccgctc caagtacttc ataatcgata tccacaccaa ttacggcatt tgctccgata 5340

tctttggcgc gctgttccat ctctttaata gcttcctcac gtgcgttaat taactcgtct 5400

tcataacctt gtgatcgtcc accgaaaaag tttctgaggc cagctccaat gtctttcata 5460

aaattaacac cagtaattac ttctccgaaa acgatttttt tatattcgat aatttgcttg 5520

ccttcaatat ttggtgaggt agttacaatc attattaaat ccctccagtt ttcttttatc 5580

atacctctaa gttgtttttt tagtcaaagg atagctggtt attttttacg ggcgtgggct 5640

ttttcgatat aagtaatcgc atctgggact ttgcaagcag tttttcctaa atctacatat 5700

actttaccta ttgattctgc tgcttccaat gcttttgtgt aaagttcctc tttataaatt 5760

ccgcaagcga tgatataact attcatagag taacgagttc tgttcgtttc agattgtaaa 5820

gattcttttg cgagttccag taattcctcg gcttcaggca aagtcgcgat gtgttctgtg 5880

aaaatttgcc agcccattga tttagttaaa tcagcatcac ttttaatcca tttcctagca 5940

aaggtgagtg cgtcatcacg tttacttaca acggaggcta gcccaaaagt taacatcgtc 6000

caagcatctc gaaaagctat attccatttc tcgatttgtg aaacagctac ttttttaggg 6060

ttcacggcaa gtaaaccaag gtaaattaag tcgctattat tagactcgat taattggagt 6120

gctaattcgt gatttttatt caatttttca cggcgaataa ttttctttaa atcaccaatc 6180

tttaatctct agaacttttt taagtgtatc tgcggagttt ttcatttgtt ctttttcttt 6240

gtcatttaag ttcatttcaa caatatggcg aacgccttga cggttaacga ctgctggtgc 6300

acctatataa atatcgttca taccgtaatg gccatctaaa taaacagaaa gtggcaaaat 6360

cgcattttcg ttatttagaa ttgcttttgt aatacgagca agagctgcag caacgccgta 6420

gaatgtagcg ccttttttat taataatttc ataagctgca tcacgaacac ttacgaaaat 6480

agtatccatt gcaccttgtt catcttcgct aatccattca gtaattggaa ggccgccgac 6540

agttgtgtgg ctccatgctg ggaattcttg aagacgaaag ggcctcgtga tacgcctatt 6600

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 6660

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 6720

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 6780

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 6840

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 6900

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 6960

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 7020

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 7080

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 7140

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 7200

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 7260

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc 7320

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 7380

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 7440

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 7500

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 7560

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 7620

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 7680

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 7740

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 7800

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 7860

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 7920

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 7980

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 8040

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 8100

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 8160

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 8220

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 8280

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 8340

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 8400

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 8460

tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 8520

tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 8580

gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 8640

cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 8700

gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 8760

tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 8820

cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg 8880

tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc 8940

tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc 9000

tgtttggtca cttgatgcct ccgtgtaagg gggaatttct gttcatgggg gtaatgatac 9060

cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 9120

tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 9180

ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 9240

agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 9300

gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 9360

tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 9420

ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg 9480

gccaggaccc aacgctgccc gacgatgata agctgtcaaa catga 9525

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

catcaaaaac gtacagcaat g 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggtttttga gaacaaattg g 21

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gataagtcga cgattcacaa aaaatag 27

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaaactagtc ccggggcgaa ttg 23

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaagctaaaa gtgcaaatgt ccc 23

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atttctttaa tactgcgttt gggg 24

Claims (3)

1. The cervical cancer therapeutic vaccine based on the recombinant attenuated listeria ovis is characterized in that the effective component of the vaccine is the recombinant attenuated listeria ovis carrying HPV16 type E6E7 fusion antigen epitope peptide gene;

the attenuated sheep listeria is obtained by completely knocking out two virulence genes namely actA and plcB from the sheep listeria;

the preparation method of the recombinant attenuated sheep listeria is obtained by taking the attenuated sheep listeria as a carrier and carrying HPV16 type E6E7 fusion antigen epitope peptide; the method comprises the following specific steps:

(1) synthesizing HPV16 type E6E7 fusion antigen epitope peptide gene fragments;

(2) constructing a targeting plasmid;

(3) preparing a competent cell Li delta actAplcB-lacZ;

(4) performing electric transformation on the competent cells prepared in the step (3) by using the targeting plasmid prepared in the step (2);

(5) performing homologous recombination hybridization culture and screening verification on the Li delta actAplcB-lacZ and the targeting plasmid;

the specific method of the step (1) is as follows: inquiring HPV16 type E6 protein and E7 protein amino acid sequences from NCBI, screening dominant T cell epitope through software, sequentially arranging, carrying out corresponding optimization according to Listeria codon to obtain an optimized sequence, adding HindIII enzyme cutting site at the upstream, adding Xho I enzyme cutting site at the downstream, and finally directly obtaining the designed sequence through synthesis, wherein 866bp is shown in SEQ ID NO. 1;

the specific method of the step (2) is as follows: carrying out double enzyme digestion on the fusion epitope peptide gene fragment obtained in the step (1) by using restriction enzymes Hind III and Xho I, simultaneously carrying out enzyme digestion on the plasmid pCW203 by using the same restriction enzymes, inserting the fusion epitope peptide gene fragment into enzyme digestion sites of pCW203 Hind III and Xho I through technologies such as connection, transformation and the like to obtain an intermediate plasmid pCW203-E6E7, and verifying the successful construction of the intermediate plasmid through PCR verification, quality-improving particle verification and enzyme digestion verification as shown in SEQ ID NO. 2; cutting off a fragment between the BamH I enzyme cutting site and the Xho I enzyme cutting site of the intermediate plasmid pCW203-E6E7, and inserting the fragment between the BamH I enzyme cutting site and the Xho I enzyme cutting site of the recombinant plasmid pCW154 to obtain a targeting plasmid pCW154-E6E7 shown in SEQ ID No. 3;

the specific method of the step (3) is as follows: li delta actAplcB-lacZ was cultured in BHI broth containing 0.5mol/L sucrose, and A was periodically measured₆₀₀When A is₆₀₀When the concentration is 0.4, adding penicillin G to ensure that the final concentration is 12.5 mu G/ml, and continuing culturing; when A is₆₀₀Centrifuging when the concentration is 0.7, washing, subpackaging and storing.

2. The vaccine of claim 1, wherein the specific method of step (4) is: electrically transferring the targeting plasmid to a Li delta actAplcB-lacZ competent cell, and adding BHI broth to culture for 2 hours after the electric transfer is finished; the transformation liquid is coated on an erythromycin BHI agar plate, a single blue colony is screened out through blue-white spots, and plasmid extraction and PCR verification are carried out after pure culture.

3. The vaccine of claim 1, wherein the specific method of step (5) is: continuously passaging sheep listeria carrying a targeting plasmid at 42 ℃ and 30 ℃, integrating a target fragment carried by the targeting plasmid into the listeria by utilizing a homologous recombination hybridization principle and a gene targeting technology, and screening out a suspicious strain by utilizing blue white spots and erythromycin sensitivity; then PCR screening and gene sequencing verification are carried out.

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