阅读说明：本技术 一种筛选高表达外源蛋白的非整合减毒李斯特菌菌株的方法 (Method for screening non-integrated attenuated Listeria strain with high-expression foreign protein ) 是由 代楠 关剑 赵勇刚 于 2018-06-11 设计创作，主要内容包括：本公开涉及一种筛选高表达外源蛋白的非整合减毒李斯特菌菌株的方法。具体来说,本公开涉及一种检测由非整合的减毒李斯特菌所表达的外源蛋白的量的方法,一种筛选非整合的减毒李斯特菌的方法和依据上述筛选方法得到的非整合的减毒李斯特菌。本公开采用的方法耗时短、操作简洁、样品检测量大,能够满足同时生产大批量样品的同时,对生产的样品进行快速大量初筛的要求。即使非整合李斯特菌菌株间存在明显的表达差异,本公开的方法仍然可实现对非整合李斯特菌的快速大量筛选,进而获得表达效果最优的李斯特菌菌株,为非整合李斯特菌疫苗制备提供便利。(The present disclosure relates to a method of screening for non-integrating attenuated listeria strains that highly express foreign proteins. In particular, the present disclosure relates to a method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, a method of screening for a non-integrated attenuated listeria and non-integrated attenuated listeria obtained according to the screening method described above. The method has the advantages of short time consumption, simple operation and large sample detection amount, and can meet the requirement of rapidly and massively primary screening the produced samples while simultaneously producing a large number of samples. Even if obvious expression difference exists among the non-integrated listeria strains, the method disclosed by the invention can still realize rapid and large-scale screening of the non-integrated listeria strains, so that the listeria strains with optimal expression effects are obtained, and convenience is provided for preparation of non-integrated listeria vaccines.)

1. A method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria comprising the steps of:

(1) Mixing the listeria monocytogenes and a protein precipitator to obtain a mixed solution; uniformly mixing the mixed solution and precipitating;

(2) Collecting the foreign protein precipitate in the precipitate;

(3) Dissolving the exogenous protein precipitate by a protein loading buffer solution to obtain an exogenous protein dissolving solution;

(4) After the exogenous protein dissolving solution is denatured, spotting on a spotting membrane, and air-drying; adding sealing liquid for sealing;

(5) Incubating the sample application membrane with a substance capable of reacting with the foreign protein and having a detection label;

(6) Dripping a developing solution on the incubated sample application membrane, and developing by a developing device;

(7) The amount of the foreign protein expressed by the non-integrated attenuated listeria was detected by visualization.

2. The method of claim 1, wherein said non-integrating attenuated listeria comprises a recombinant nucleic acid molecule encoding an open reading frame for a recombinant polypeptide comprising a heterologous antigen fused to a derivative listeriolysin (LLO) polypeptide, said recombinant nucleic acid molecule further comprising a first promoter sequence; wherein

The derived listeriolysin (LLO) polypeptide is selected from a polypeptide which is obtained by substituting, repeating, deleting or adding one or more amino acids in an amino acid sequence shown as SEQ ID NO. 3 and has or partially has the activity of the listeriolysin (LLO) polypeptide shown as SEQ ID NO. 1.

3. The method of claim 2, wherein the amino acid sequence encoding said derived listeriolysin (LLO) polypeptide has a sequence that hybridizes to the amino acid sequence encoding a polypeptide as set forth in SEQ ID NO:1, has at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% identity compared to the amino acid sequence of the listeriolysin (LLO) polypeptide.

4. The method of claim 2 or 3, wherein said derived Listeriolysin (LLO) polypeptide is a polypeptide as set forth in SEQ ID NO 3.

5. The method of claim 2 or 3, wherein the heterologous antigen is selected from a tumor antigen or a non-tumor antigen; optionally, the non-tumor antigen is selected from OVA or a fragment having OVA function.

6. The method of claim 2, wherein said recombinant nucleic acid molecule further comprises a linker sequence linking a nucleotide sequence encoding said derived listeriolysin (LLO) polypeptide and a nucleotide sequence encoding said heterologous antigen; optionally, the heterologous antigen is selected from a tumor antigen or a non-tumor antigen.

7. the method of claim 6, wherein the linker sequence comprises a nucleotide sequence encoding the sequence set forth in SEQ ID NO 16; optionally, the linker sequence comprises one, two, or more than three repeats of the sequence shown in SEQ ID NO 16.

8. The method of claim 6 or 7, wherein said listeriolysin (LLO) polypeptide-derived nucleotide sequence is linked to a linker sequence comprising said heterologous antigen encodes an amino acid sequence set forth in SEQ ID NO. 14 or SEQ ID NO. 15.

9. The method of any one of claims 1 to 8, wherein the promoter sequence is selected from the group consisting of a sequence encoding the Phly gene; optionally, the recombinant nucleic acid molecule further comprises a tag sequence for detection or a gene encoding a metabolite; preferably, the metabolite is selected from the group consisting of secondary metabolites.

10. The method of any one of claims 1-9, wherein the protein precipitating agent is selected from a TCA/acetone solution.

11. the method according to any one of claims 1 to 10, wherein, after the foreign protein precipitate is obtained, before the foreign protein precipitate is dissolved, impurity components in the foreign protein precipitate are removed; optionally, the impurity component is selected from TCA and/or acetone.

12. A method of screening for non-integrated attenuated listeria comprising comparing the amount of a foreign protein expressed by a non-integrated attenuated listeria detected by the method of any one of claims 1-11, and screening for non-integrated attenuated listeria having a higher level of expression of the foreign protein.

13. The method of screening for non-integrated attenuated listeria of claim 12, wherein the non-integrated attenuated listeria is screened.

Technical Field

The present disclosure relates generally to the field of biotechnology. In particular, the present disclosure provides a method of screening for strains. More specifically, the present disclosure provides a method of screening for non-integrating attenuated listeria strains that highly express a foreign protein.

Background

Listeria monocytogenes (Lm) is an important food-borne pathogenic bacterium, and may cause serious Listeria diseases for the elderly, children, pregnant women, and immunosuppressed people [1 ]. Listeria, a gram-positive intracellular parasite, can survive and multiply in epithelial and phagocytic cells. Due to its unique infection process, listeria can induce inflammatory response and activate the combination of MHC class I and class II antigen presentation pathways at the same time, making listeria a vaccine vector with great application prospects [2-4 ].

At present, there are two main international approaches for constructing tumor vaccines by using attenuated LM strains as vectors: integrative and non-integrative listeria vaccines [5 ]. The integrative listeria vaccine usually uses recombinant plasmid to integrate exogenous gene into listeria genome at fixed point by homologous recombination technology, has the advantages of stable exogenous protein expression, obvious activation of organism specific immune response and the like in application, but has the defects of long vaccine construction period, complicated integrative construction and screening process and the like, thereby prolonging the vaccine preparation period and being not beneficial to clinical personalized treatment. In contrast, non-integrated listeria vaccines are constructed only by plasmid electrotransformation, and have simple and rapid process and short vaccine preparation period, and can secrete and express foreign proteins and effectively activate specific immune response of organisms [6-8 ]. However, the inventors found that due to the random number of plasmids electrically transformed into Listeria, significant expression differences exist among single colonies in the same culture plate after screening by using antibiotics, and finally, the treatment effects of vaccines in different batches are different. Therefore, the search and establishment of a technology capable of rapidly screening the Listeria strains with high expression foreign genes in large quantity lays a foundation for the application of the non-integrated Listeria vaccine.

The Western blotting (Western blotting) technology is the most widely used experimental technology in the present protein expression and analysis research, combines the traditional high-resolution sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) electrophoresis with the immunodetection technology with high sensitivity and strong specificity, analyzes the expression of the target protein most effectively, and plays an important role in molecular biology. A sample is separated after SDS-PAGE gel electrophoresis, proteins in the gel are transferred to a chemically inert macromolecule transfer membrane by an electroblotting method, then a primary antibody (primary antibody) specific to a target protein is subjected to an immune binding reaction with the target protein on the transfer membrane, a secondary antibody (secondary antibody) marked (such as HRP) is combined with the primary antibody, and qualitative and semi-quantitative analysis of the target protein is completed by detecting a secondary antibody marker, so that the method has the advantages of high sensitivity, strong specificity and the like [9-10 ]. Dot blotting (Dot blotting) is a method of detecting antibody immunological binding directly on a membrane. The method omits the step of electrophoretic separation, and belongs to the simplified Western blot, northern blot and the like which can be used for detecting DNA, RNA and protein. In addition, the method allows quantification of protein concentration by direct measurement of radioactivity counts for isotopically labeled probes or indirect autoradiography and photometric density values for enzymatically labeled probes [11 ]. The method has no electrophoretic separation step, so that the size of the protein in the sample cannot be detected, but the method is suitable for coarse screening of the sample due to simple and quick experimental process and capability of simultaneously detecting a large number of samples, and is widely applied to the fields of rapid detection of food, screening and evaluation of antibody effectiveness and the like [12-13 ]. In protein expression detection, the Western blot technology has complicated experimental process and long experimental time consumption, and in Listeria expression detection, the Western blot experiment needs large sample preparation amount, complex preparation operation and small sample preparation amount in a single time, and is not beneficial to rapid coarse screening of mass samples.

Because the above problems still exist in the existing schemes, there is a need to provide a method for rapidly screening non-integrated attenuated listeria strains with high expression of foreign proteins, so as to solve the problem that the listeria strains may have differential expression among the colonies generated in the process of preparing the listeria strains.

Disclosure of Invention

Problems to be solved by the invention

Based on the defects in the prior art, the method for rapidly screening the non-integrated attenuated Listeria strain with high expression foreign protein is provided, the problem of differential expression among colonies is well solved, and a foundation is laid for the clinical application of the Listeria vaccine.

Means for solving the problems

In one embodiment, the present disclosure provides a method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria comprising the steps of:

(1) Mixing the listeria monocytogenes and a protein precipitator to obtain a mixed solution; uniformly mixing the mixed solution and precipitating;

(2) Collecting the foreign protein precipitate in the precipitate;

(3) Dissolving the exogenous protein precipitate by a protein loading buffer solution to obtain an exogenous protein dissolving solution;

(4) After the exogenous protein dissolving solution is denatured, spotting on a spotting membrane, and air-drying; adding sealing liquid for sealing;

(5) Incubating the sample application membrane with a substance capable of reacting with the foreign protein and having a detection label;

(6) Dripping a developing solution on the incubated sample application membrane, and developing by a developing device;

(7) The amount of the foreign protein expressed by the non-integrated attenuated listeria was detected by visualization.

In one embodiment, the present disclosure provides a method of detecting the amount of a foreign protein expressed by a non-integrating attenuated listeria, wherein said non-integrating attenuated listeria comprises a recombinant nucleic acid molecule encoding an open reading frame for a recombinant polypeptide comprising a heterologous antigen fused to a derivative listeriolysin (LLO) polypeptide, said recombinant nucleic acid molecule further comprising a first promoter sequence; wherein, the derived listeriolysin (LLO) polypeptide is selected from the polypeptide which is obtained by substituting, repeating, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3 and has or partially has the activity of the listeriolysin (LLO) polypeptide shown in SEQ ID NO. 1. Optionally, the derived listeriolysin (LLO) polypeptide is a polypeptide as shown in SEQ ID No. 3.

In one embodiment, the present disclosure provides a method for detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, wherein said non-integrated attenuated listeria comprises a recombinant nucleic acid molecule comprising an amino acid sequence encoding said derived listeriolysin (LLO) polypeptide and a nucleic acid sequence encoding a polypeptide as set forth in SEQ ID NO:1, has at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% identity compared to the amino acid sequence of the listeriolysin (LLO) polypeptide. Optionally, the derived listeriolysin (LLO) polypeptide is a polypeptide as shown in SEQ ID No. 3.

In one embodiment, the present disclosure provides a method for detecting the amount of a heterologous protein expressed by a non-integrating attenuated listeria, wherein said non-integrating attenuated listeria comprises a recombinant nucleic acid molecule encoding an open reading frame for a recombinant polypeptide comprising a heterologous antigen fused to a derivative listeriolysin (LLO) polypeptide, wherein said heterologous antigen is selected from a tumor antigen or a non-tumor antigen; optionally, the non-tumor antigen is selected from OVA or a fragment having OVA function.

In one embodiment, the present disclosure provides a method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, wherein said non-integrated attenuated listeria comprises a recombinant nucleic acid molecule further comprising a linker sequence linking a nucleotide sequence encoding said derived listeriolysin (LLO) polypeptide and a nucleotide sequence encoding said heterologous antigen; wherein the heterologous antigen is selected from a tumor antigen or a non-tumor antigen. Optionally, the linker sequence comprises a nucleotide sequence encoding the sequence shown as SEQ ID NO 16; optionally, the linker sequence comprises one, two, or more than three repeats of the sequence shown in SEQ ID NO 16. Optionally, linked to the nucleotide sequence of the derived listeriolysin (LLO) polypeptide, the amino acid sequence encoded by the linker sequence comprising the heterologous antigen is set forth in SEQ ID No. 14 or SEQ ID No. 15.

In one embodiment, the present disclosure provides a method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, wherein said non-integrated attenuated listeria comprises a recombinant nucleic acid molecule comprising a promoter sequence selected from the group consisting of a sequence encoding the Phly gene; optionally, the recombinant nucleic acid molecule further comprises a tag sequence for detection or a gene encoding a metabolite; preferably, the metabolite is selected from the group consisting of secondary metabolites.

In one embodiment, the present disclosure provides a method of detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, wherein the protein precipitating agent is selected from a TCA/acetone solution.

In one embodiment, the present disclosure provides a method for detecting the amount of a foreign protein expressed by a non-integrated attenuated listeria, wherein, after obtaining a precipitate of the foreign protein, the foreign protein precipitate is removed of impurity components prior to dissolving the foreign protein precipitate; optionally, the impurity component is selected from TCA and/or acetone.

In another embodiment, the present disclosure provides a method for screening non-integrated attenuated listeria, wherein the amount of the foreign protein expressed by the non-integrated attenuated listeria detected by the aforementioned detection method is compared to screen non-integrated attenuated listeria with higher expression level of the foreign protein.

ADVANTAGEOUS EFFECTS OF INVENTION

The plasmid pAM401 adopted by the method is very stable in the process of multiple passages of Listeria, and the phenomenon of plasmid loss or mutation is not found after 10-20 passages, so that the plasmid pAM can be safely used for vaccine construction. The expression transcription of the antigen gene selects a promoter Phly carried by Listeria LLO, the promoter is stable and efficient, and the gene which is constructed by transcription and translation and is used for coding the heterologous antigen can be well started. Meanwhile, with the signal peptide sequence of the LLO, after the vaccine infected cell escapes out of the lysosome, the expressed protein is secreted into the bacterial extracellular plasma to induce the cellular immune response. Optionally, a protein detection tag, such as Flag or His tag, is added to the plasmid for detection of protein expression secretion.

Optionally, the vector construction method adopted by the disclosure and the vector obtained by the method can be free from the influence of enzyme cutting sites on the heterologous antigen, and are convenient to operate, high in insertion efficiency and accurate in insertion.

Optionally, in the technical scheme adopted by the disclosure, in the design optimization of the antigen peptide, the expression characteristics of listeria are met according to the codon preference of escherichia coli as an optimization standard, and the antigen peptide optimally designed by the method can be expressed in listeria and has good stability. The listeria carried heterologous antigen can be efficiently secreted into host cells to fully activate specific tumor immune response, and theoretically obtain better treatment effect.

Optionally, the technical scheme adopted by the disclosure greatly improves the expression of the non-integrated listeria tumor vaccine antigen peptide, so that the antigen peptide has a more prominent effect on anti-tumor immune response.

The present disclosure employs a method for rapid screening of non-integrated transfected listeria strains with high expression of foreign genes. Compared with the Western blot technology used in the conventional protein expression detection, the method disclosed by the invention is short in time consumption, simple in operation and large in sample detection amount, and can meet the requirement of rapidly and massively primary screening the produced samples while simultaneously producing a large number of samples.

Optionally, even if there is an obvious expression difference between the non-integrated listeria strains, the method disclosed by the present disclosure can still realize rapid mass screening of the non-integrated listeria strains, thereby obtaining the listeria strains with the optimal expression effect, and providing convenience for the preparation of non-integrated listeria vaccines.

Drawings

FIG. 1 shows the differences in the expression levels of proteins expressed by single colonies formed by the different strains described above when preparing non-integrated Listeria strains. Wherein M represents a 250KDa protein ladder standard reference substance; shown in lanes 1-5 are Lm10403 S.DELTA.acta (pAM 401-LLO), respectively_1-28-LLO_22-523-(G4S)₂-E7peptide-LLO_524-529His) supernatant protein pellet concentrates on colonies No. 1-5 on the plate.

FIG. 2 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-267-PstI-LLO_524-529his) differences in protein expression between different single colonies. Wherein, sequences No. 1-8 show LM10403 S.DELTA.acta (pAM 401-LLO)_1-28-LLO_22-267-PstI-LLO_524-529His) plate 1-8 colony culture 1ml supernatant protein precipitate concentrate.

FIG. 3 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-267-PstI-LLO_524-529His) differences in protein expression between different single colonies. Wherein, sequences No. 1-8 show LM10403 S.DELTA.acta (pAM 401-LLO)_1-28-LLO_22-523-(G4S)₂-OVA₂₈-LLO_524-529His) plate 1-8 colony culture 1ml supernatant protein precipitate concentrate.

FIG. 4 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-523-(G4S)₂-NY-ESO-1-LLO_524-529His) differences in protein expression between different single colonies. Wherein, sequences No. 1-8 show LM10403 S.DELTA.acta (pAM 401-LLO)_1-28-LLO_22-523-(G4S)₂-NY-ESO-1-LLO_524-529His) plate 1-8 colony culture 1ml supernatant protein precipitate concentrate.

FIG. 5 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-523-(G4S)₂-OVA₂₈-LLO_524-529His) expression of the protein expressed by bacteria No. 1 to 6.

FIG. 6 shows that LM10403 S.DELTA.acta (pAM 401-LLO) was detected by Western blot method_1-28-LLO_22-523-(G4S)₂-OVA₂₈-LLO_524-529His) expression of the protein expressed by bacterium No. 2, No. 4, and No. 6.

FIG. 7 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-267-(G4S)₂-E7-LLO_524-529His) expression of the protein expressed by bacteria Nos. 1 to 8.

FIG. 8 shows LM10403S Δ actA (pAM 401-LLO) detection using the method of the present disclosure_1-28-LLO_22-267-(G4S)₂-E7-LLO_524-529His) expression of a protein expressed by a progeny strain of bacterium No. 1.

FIG. 9 shows the effect of LLO full length and conventional LLO signal peptide on foreign protein expression.

FIG. 10 shows the effect of different sizes of antigenic peptides and the presence or absence of the G4S sequence on the expression vector.

FIG. 11 shows the results of tumor curves obtained by subcutaneous inoculation of mice with EG7-OVA to generate tumors, followed by treatment with tumor vaccines.

FIG. 12 shows the test results of tumor generation by mice subcutaneously inoculated with EG7-OVA and treated with tumor vaccine (T test analysis).

Figure 13 shows the activation of Lm OVA vaccine on specific immune responses in mice by ELISPOT.

FIG. 14 shows tetramer experimental validation OVA₂₈tumor vaccines are specifically immunoreactive in mice.

FIG. 15 shows the detection of OVA₂₈Effective dose of tumor vaccine and activation of tumor-specific immune response in mice (tumor size growth curve results).

FIG. 16 shows the detection of OVA₂₈Effective dose of tumor vaccine and activation of tumor-specific immune response in mice (ELISPOT test results).

FIG. 17 shows OVA₂₈The tumor vaccine was compared with OVA-integrated Listeria vaccine for efficacy (tumor size growth curve results).

FIG. 18 shows OVA₂₈The tumor vaccine was compared with OVA-integrated Listeria vaccine for efficacy (ELISPOT test results after 7 days).

FIG. 19 shows OVA₂₈The tumor vaccine was compared with OVA-integrated Listeria vaccine for efficacy (ELISPOT test results after 12 days).

FIG. 20 shows OVA₂₈The tumor vaccine was the validation result (plate-coated result) of the non-integrative OVA listeria vaccine.

FIG. 21 shows OVA₂₈the tumor vaccine is the verification result of the non-integrative OVA listeria vaccine (the identification result of agarose gel electrophoresis).

FIG. 22 shows OVA₂₈The tumor vaccine is the verification result of the non-integrative OVA listeria vaccine (the identification result of agarose gel electrophoresis).

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.

The term "TCA/acetone solution" in this disclosure refers to a mixture of TCA and acetone.

The term "NY-ESO-1 antigen" in this disclosure refers to the New York esophageal squamous carcinoma antigen 1.

The term "E7 polypeptide" in the present disclosure refers to an E7 polypeptide derived from Human Papilloma Virus (HPV).

The term "OVA" in the present disclosure refers to chicken egg albumin (Ovalbumin), also known as chicken egg albumin, consisting of 386 amino acids, having a molecular weight of about 45 kD.

The term "Phyy" in this disclosure is a promoter encoding the LLO (lysin) gene.

The term "vaccine" in the present disclosure is an immune preparation for preventing diseases, which is prepared from pathogenic microorganisms (such as bacteria, etc.) and metabolites thereof by artificial attenuation, inactivation, or using transgenosis, etc.

The term "substitution, repetition, deletion or addition of one or more amino acids" in the present disclosure includes "conservative mutation". The term "conservative mutation" in the present disclosure refers to a conservative mutation that can normally maintain the function of a protein. A representative example of conservative mutations is conservative substitutions. Conservative substitution refers to, for example, a mutation in which Phe, Trp, Tyr are substituted for each other when the substitution site is an aromatic amino acid; a mutation wherein Leu, Ile and Val are substituted with each other when the substitution site is a hydrophobic amino acid; a mutation wherein Gln and Asn are substituted with each other in the case of a polar amino acid; a mutation wherein Lys, Arg and His are substituted with each other in the case of a basic amino acid; a mutation wherein Asp and Glu are substituted with each other in the case of an acidic amino acid; in the case of an amino acid having a hydroxyl group, the amino acid is substituted for Ser or Thr. Examples of the substitution regarded as conservative substitution include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Glu, Gln, His or Arg, substitution of Met with Met, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe, Tyr, His, Val or Phe, and substitution of Met with Phe or Phe. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.

"methods in general Biology in the art" in the present disclosure can be referred to corresponding methods described in publications such as "Current Protocols in Molecular Biology, Wiley publication", "Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory publication", and the like.

Technical scheme

In the technical scheme of the disclosure, the meanings represented by the numbers of the nucleotide and amino acid sequence table in the specification are as follows:

SEQ ID NO. 1 shows the nucleotide sequence of wild-type Listeriolysin LLO (LLO)₅₂₉)

SEQ ID NO:2 shows the amino acid sequence (LLO) of wild-type Listeriolysin LLO₅₂₉)

SEQ ID NO 3 shows a nucleotide sequence (LLO) of recombinant listeriolysin LLO₅₄₀)

SEQ ID NO. 4 shows an amino acid sequence (LLO) of recombinant listeriolysin LLO₅₄₀)

SEQ ID NO. 5 shows LLO₂₈Amino acid sequence of (1)

SEQ ID NO 6 shows LLO₂₈Nucleotide sequence of (A)

SEQ ID NO 7 shows OVA₂₈Nucleotide sequence of (A)

SEQ ID NO 8 shows OVA₂₈Amino acid sequence of (1)

SEQ ID NO 9 shows OVA₈Amino acid sequence of (1)

SEQ ID NO 10 shows OVA₈nucleotide sequence of (A)

11 shows the 5' homologous nucleotide sequence

12 shows the 3' homologous nucleotide sequence

SEQ ID NO 13 shows the amino acid sequence of the linker sequence

SEQ ID NO. 14 shows OVA₈amino acid sequence linked to a linker sequence

SEQ ID NO 15 shows OVA₂₈Amino acid sequence linked to a linker sequence

16 is the amino acid sequence of the E7 polypeptide

17 shows the nucleotide sequence of the E7 polypeptide

18 is the amino acid sequence of NY-ESO-1 polypeptide shown in SEQ ID NO

SEQ ID NO 19 shows the nucleotide sequence of NY-ESO-1 polypeptide

20 shows LLO_22-267Amino acid sequence of (1)

Shown in SEQ ID NO 21 is LLO_22-267Nucleotide sequence of (A)