CN116676324B - System and method for constructing and releasing anti-tumor effector protein based on Kil protein - Google Patents
System and method for constructing and releasing anti-tumor effector protein based on Kil protein Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biology, and provides a system and a method for constructing and releasing anti-tumor effect proteins based on Kil proteins. The invention can activate immune system to kill tumor by expressing Kil protein in engineering bacteria and nanometer antibody therapeutic protein substance in bacteria. The data show that the engineering bacteria for constitutively expressing the Kil protein can reduce the activity of bacteria so as to improve the safety of bacterial treatment, but can ensure that the number of bacteria is at a certain level so as to achieve the aim of fully releasing therapeutic substances.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a system and a method for constructing and releasing anti-tumor effect protein based on Kil protein.
Background
Methods for treating tumors using live bacteria have been in clinical practice at the end of the 19 th century, which is considered to be the most primitive form of immunotherapy. Modern animal experimental researches show that bacteria show good safety and tumor treatment effect in a specific dosage. Part of bacteria have a tendency of growing and colonizing to the anaerobic environment inside the tumor, so that the tumor targeting is realized; the bacteria are used as complete prokaryotes, and have the capability of synthesizing complex biological macromolecules to enhance the tumor treatment effect; the bacteria mainly play an anti-tumor function through the cytotoxicity of the bacteria and activation of an immune system after the host is infected, so that the bacterial treatment has the potential of breaking through immune-free microenvironment and overcoming the adverse prognosis of tumors caused by genetics to become a universal treatment means. In 2002, the results of human clinical trials published in journal Journal of Clinical Oncology indicate that attenuated salmonella murine (attenuated Salmonella typhimurium), which is believed to have tumor targeting properties, can successfully colonize metastatic melanoma in a subset of patients within a safe dose range by intravenous injection, but fails to exhibit an anti-tumor effect. The clinical trial concludes that the inability to continue increasing bacterial doses within safe limits enhances intratumoral bacterial colonization as a major reason for the lack of bacterial anti-tumor effects as demonstrated in animal experiments. Therefore, it is currently the mainstream view that successful bacterial tumor treatment needs to improve targeting and antitumor ability of bacteria while compromising safety.
Synthetic biology is an interdisciplinary discipline that combines the disciplines of genetic engineering, systems biology, control, etc., which emphasizes the modular construction of genetic elements, devices and systems to achieve a target function in a target organism. The bacteria modified by genetic engineering or synthetic biology means are engineering bacteria. In 2016, the research of modifying bacteria to treat colon cancer mice with liver metastasis by using synthetic biology technology is reported for the first time, and the progress of the field of bacterial treatment of tumors is greatly promoted. In 2018, several papers for improving the therapeutic effect of bacterial tumor based on synthetic biology means are reported, which brings more possibility to the field. In the above research, a periodic bacterial lysis system based on flora induction is partially used to release protein with therapeutic function, so that good therapeutic effect is obtained in mice model of liver metastasis colon cancer and lymphoma. The periodic bacterial lysis system ensures the sustained release of bacterial synthetic products, so that the bacteria can enhance the anti-tumor effect by the direct cell perforation effect or the killing capacity of phagocytes and cytotoxic T cells (cytotoxic Tlymphocyte), maintain the bacteria in a lower number and obviously improve the safety of treatment. Bacteria with low toxicity and easy gene operation are used as vectors, and a complex biosynthesis system is used as a 'cell factory' to participate in tumor treatment, so that the targeting and killing ability of bacteria tumor or the immune response of a host is enhanced to treat the tumor, and the bacterial tumor has become a new development direction of bacterial tumor treatment.
However, there are still some problems in bacterial treatment of current tumors: 1) The bacterial self-secretion system is underdeveloped, and the generated protein active product is difficult to secrete outside the cell, so that the killing effect of the bacteria is limited. 2) It is desirable to limit the toxicity of bacteria to ensure therapeutic safety. Thus, in related studies for treating tumors by engineering bacteria, it is often required to release therapeutic substances synthesized in bacteria by a method of lysing the bacteria due to underdevelopment of bacterial secretion systems. Meanwhile, the bacterial quantity can be reduced by lysing bacteria, so that the safety of bacterial treatment is improved. This is also why the studies described above use periodic cleavage systems based on flora induction to release proteins. The system has the advantage of stably releasing the protein and reducing the overall bacterial load over a period of time to improve safety. However, for engineering bacteria using proteins as therapeutic substances, the actual release of therapeutic proteins from periodic lysis systems based on flora induction is low; and bacteria are easily cleared by the immune system at low points of periodic concussion, which significantly affects their long-acting colonization ability. There is a need for more optimal means to solve this problem.
With the advancement of antibody engineering, it has been possible to express biologically active antibody components in bacteria, including single-chain variable regions (scfvs) of conventional antibodies and nanobodies (nanobodies) derived from heavy chain antibodies. The method lays a technical foundation for the combined use of bacterial treatment and immunotherapy. Nanobodies are variable region fragments of alpaca heavy chain only antibodies (camelid heavy-chain only antibodies, camelid HcAbs), i.e., single variable domains (variable domain of heavy chain of heavy-chain anti, VHH). Is the natural antigen binding fragment with the minimum molecular weight (15 kDa) discovered at present. Due to the small molecular weight, nanobodies have good tissue permeability. And can maintain biological activity for several weeks in 37 ℃ environment, and has good chemical stability. While nanobody drugs against adult acquired thrombopenia have been marketed in 2019, nanobodies often need to be used in combination with other monoclonal antibodies or as part of CAR-T or Fc-containing antibodies to exert an anti-tumor effect due to the lack of Fc-segments for neoplastic disease. As a document using anti-mouse CD47 nanobody suggests, the use of anti-mouse CD47 nanobody alone as a therapeutic substance in mice does not extend the course of treatment of tumors in mice engineered with bacteria, which are required to express therapeutic proteins inside the bacteria and to secrete them successfully, reducing bacterial toxicity to increase therapeutic safety.
The nanobody expressed by bacteria needs to be released outside the bacterial cell to function. However, bacterial surface transport systems are inherently difficult to secrete recombinant proteins expressed by foreign genes outside the cell. Direct cleavage of bacterial release proteins by toxic proteins is an effective approach to solve this problem.
In the current research of modifying bacteria to treat tumors by using synthetic biology ideas, the most classical lysis method is a synchronous lysis release system based on an AHL flora induction system published in the journal of Nature of 2016 (doi: 10.1038/aperture 18930). The principle is as follows: N-Acyl-homoserine lactones (N-Acyl-Homoserine Lactones, AHL) are small molecules synthesized by the LuxI protein and freely diffuse among bacteria, and an AHL-LuxR complex formed after the small molecules are combined with the LuxR protein which is expressed in bacteria can be used for inducible activation of the LuxI promoter, so that the expression of LuxI, phi X174E short peptides and target proteins is activated. The phi X174E gene derived from phage phi X174 (phiX 174) can code a short peptide containing 91 amino acids, and the short peptide can directly inhibit the formation of lipid I (lipid I) by inhibiting the activity of the translocation enzyme Mray of escherichia coli, thereby inhibiting the formation of the cell wall of the escherichia coli to cause the death and the lysis of bacteria. The expression quantity of LuxI is increased, AHL production is increased, positive feedback can continue to activate the expression of phi X174E short peptide and target protein, and the bacterial flora almost maintains a synchronous state due to free diffusion of AHL. Finally, under the action of phi X174E short peptide, most bacteria die synchronously, and the target protein is successfully released. However, due to local concentration differences, there are always non-dead bacteria. When the average concentration of AHL decreases with liquid exchange, the process is restarted with the non-killed bacteria. This process has been shown to work effectively in animals.
Although the above-described synchronized lysis system achieves the aim of reducing bacterial toxicity and successful release of the product, it also has certain drawbacks: (1) The synchronous lysis system can cause that bacteria are easily cleared by an immune system when the vibration is low, and the field planting efficiency is reduced. (2) Repeated lysis of bacteria to low populations can result in a substantial decrease in the overall yield of bacterial proteins due to repeated reductions in the number of bacterial populations.
In the literature, the in vivo imaging system (in vivo imaging system, IVIS) of small animals was used to detect the presence of self-harboring bioluminescent bacteria in mice. The conclusion shows that the control group bacteria without the synchronous lysis system maintained a higher bioluminescence signal after growth to the plateau phase, whereas the experimental group bacteria with the synchronous lysis system showed periodic fluorescence signal changes as described above. By extracting image data results in original research animal experiments, MATLAB is used for plotting the change condition of in-vivo biological fluorescence signals. Since the original literature has already described: the expression of the target protein is synchronous with the growth state of bacteria, and almost consistent. Thus, a Logistic curve describing the number of colonies can be used to model the change in fluorescence of bacteria in the absence of the lysis system. The results are shown in FIG. 1, wherein the abscissa in FIG. 1 represents time (hours) and the ordinate represents the intensity of the bioluminescence signal. The lower line of the graph is the biofluorescence signal (noted) from bacteria with their own bioluminescence and flora induction release system in mice, extracted from Jeff Hasty and reported by Nature. The upper line is a bacterial growth curve (noted) without lysis system fitted to the IVIS signal in the original article using Logistic equation with the maximum environmental capacity of the experimental group set to K. The shaded portions are respectively: the decrease in fluorescence signal when K was decreased from 1/2K in the (upper) control group, and the actual decrease in fluorescence signal in the (lower) experimental group. As can be seen from FIG. 1, when the number of bacteria in the control group reaches 1/2K by using external factors at the end of the log phase, the fluorescence signal of the bacteria is reduced, i.e., the amount of protein product released will be much higher than that of the synchronous lysis release system. Simulation results show that the release amount of the target product can be obviously increased by cracking bacteria in the stage close to the platform through an asynchronous cracking means. In summary, there is an urgent need to design a new lysis system to improve release of the target bacterial product and reduce toxicity of the engineering bacteria.
Disclosure of Invention
In order to solve the problems, the invention provides a system and a method for constructing and releasing anti-tumor effector protein based on Kil protein. The invention establishes a system for continuously releasing protein substances by bacteria based on Kil proteins, and the system can ensure that the bacteria stably release protein therapeutic substances simultaneously expressed in the bacteria when the population quantity is high. The Kil protein is a bacteriocin release protein derived from E.coli (bacteriocin release protein, BRP). Under the condition of natural low expression quantity, the bacterial outer membrane can be destroyed, and substances in the periplasmic space can be released. The immune system is activated to kill tumors by expressing Kil proteins in engineering bacteria and simultaneously expressing therapeutic effector proteins (such as CD47 nanobodies and PD-L1 nanobodies) in bacteria. The Kil protein can mediate bacterial death after expression, so as to avoid continuous mass amplification of bacteria and increase safety. In animal experiments, the system shows remarkable treatment effect on tumors and remarkably improves treatment safety.
The technical scheme of the invention is as follows:
a system for releasing anti-tumor effector protein constructed based on a Kil protein, comprising a Kil protein expression module and a therapeutic effector protein module, wherein the Kil protein expression module comprises a promoter, an RBS, a Kil protein sequence and a terminator for expressing the Kil protein, and the therapeutic effector protein module comprises the promoter, the RBS, the therapeutic effector protein sequence and the terminator for expressing the therapeutic effector protein.
The nucleotide sequence of the Kil protein sequence is shown as SEQ ID No. 1.
The therapeutic effector protein sequence is a CD47 nanobody sequence and/or a PD-L1 nanobody sequence.
The promoter for expressing the Kil protein is any one or a combination of several of J23109, J23114 and J23115.
The nucleotide sequence of the promoter J23109 for expressing the Kil protein is shown as SEQ ID No.2, the nucleotide sequence of the promoter J23114 for expressing the Kil protein is shown as SEQ ID No.3, and the nucleotide sequence of the promoter J23115 for expressing the Kil protein is shown as SEQ ID No. 4.
The nucleotide sequence of RBS of the Kil protein expression Module is AAGAAGGAA.
The nucleotide sequence of the terminator of the Kil protein expression module is shown as SEQ ID No. 5.
The nucleotide sequences of the system are respectively shown as SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15 and SEQ ID No. 16.
The application of the system in preparing anti-tumor products.
The system for releasing the anti-tumor effect protein constructed based on the Kil protein comprises the following construction method: the Kil proteins are expressed in bacteria by means of plasmid vectors and/or insert genomes based on molecular biological methods, and the therapeutic effector proteins are simultaneously expressed in bacteria by means of plasmid vectors and/or insert genomes.
It should be noted that the Kil protein expression module in the system of the present invention further includes a Kil protein expression module formed based on a mutant Kil protein sequence.
The beneficial effects of the invention are as follows:
the system for releasing the anti-tumor effector protein based on the Kil protein construction comprises a Kil protein expression module and a therapeutic effector protein module, wherein the Kil protein expression module comprises a promoter, an RBS, a Kil protein sequence and a terminator for expressing the Kil protein, and the therapeutic effector protein module comprises the promoter, the RBS, the therapeutic effector protein sequence and the terminator for expressing the therapeutic effector protein. The invention establishes a system for continuously releasing protein substances by bacteria based on Kil proteins, and the system can ensure that the bacteria stably release protein therapeutic substances simultaneously expressed in the bacteria when the population quantity is high. The invention can activate immune system to kill tumor by expressing Kil protein in engineering bacteria and nanometer antibody therapeutic protein substance in bacteria. The data show that the engineering bacteria for constitutively expressing the Kil protein can reduce the activity of bacteria so as to improve the safety of bacterial treatment, but can ensure that the number of bacteria is at a certain level so as to achieve the aim of fully releasing therapeutic substances.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a graph of the variation in fluorescence of bacteria in the absence of the lysis system using a Logistic curve describing the number of colonies;
FIG. 2 shows a graph comparing nanobody leakage for different systems;
FIG. 3 shows a strain containing a Kil protein cleavage SystemE. coli MG1655A nanobody leakage pattern in (a);
FIG. 4A shows a strain containing a Kil protein cleavage SystemE. coli MG1655And strains without Kil protein cleavage SystemE. coli MG1655OD of (d) 600 A value comparison graph;
FIG. 4B shows a strain containing a Kil protein cleavage SystemE. coli MG1655And strains without Kil protein cleavage SystemE. coli MG1655A comparison of bacterial colony formation numbers;
FIG. 5 shows a comparison of survival curves of mice after injection of different strains into a highly tumor-bearing mouse model.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
A Kil protein expression module is constructed by molecular cloning, and comprises a promoter, RBS, a Kil protein sequence and a terminator for expressing the Kil protein, wherein the sequence of each element is shown in Table 1.
TABLE 1 sequence of elements of Kil protein expression Module
Further, through a high-throughput screening platform, the Kil protein expression module sequence after further optimization is obtained through screening, and is shown as SEQ ID No.6, specifically:
GGCAGTAAAAAGACGTAAACTTTCCCAGAATCCTGCCGATATTATCCCACAAAATTTGTCACACAAGGAAGCTGAATGGGGAGCAGCATGCGCAAACGCTTTTTTGTGGGCATTTTTGCGATTAACCTGCTGGTGGGCTGCCAGGCGAACTATATTCCGGATGTGCAGGGCGGCACCATTGCGCCAAGCAGCAGCAGCAAACTGACCGGCATTGCGGTGCAGTAAATAGCCAATTATTGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCTTAC。
further, a therapeutic effector protein module-CD 47 nanobody expression system is constructed, which comprises a J23100 promoter, a RBS, a CD47 nanobody sequence of pelB signal peptide and a terminator, wherein the sequence of the CD47 nanobody expression system is shown as SEQ ID No.7, and specifically comprises the following components:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGGAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCATTATTTTTAAAATTAACGATATGGGCTGGTATCGCCAGGCGCCGGGCAAACGCCGCGAATGGGTGGCGGCGAGCACCGGCGGCGATGAAGCGATTTATCGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGCGAAAAACAGCGTGTTTCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGGTGATTAGCACCGATCGCGATGGCACCGAATGGCGCCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG。
another therapeutic effector protein module-PD-L1 nanobody expression system is also constructed, comprising a J23100 promoter, a RBS, a CD47 of pelB signal peptide, a PD-L1 nanobody sequence and a terminator; the PD-L1 nanometer antibody expression system has a sequence shown in SEQ ID No.8, and is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTTCAGCTGGTTGAAACCGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGCGTCTGGTTTCACCTTCTCTATGCACGCGATGACCTGGTACCGTCAGGCGCCGGGTAAACAGCGTGAACTGGTTGCGGTTATCACCTCTCACGGTGACCGTGCGAACTACACCGACTCTGTTCGTGGTCGTTTCACCATCTCTCGTGACAACACCAAAAACATGGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTACTGCAACGTTCCGCGTTACGACTCTTGGGGTCAGGGTACCCAGGTTACCGTTTCTTCTGGTGGTCTGCCGGAAACCGGTGGTCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG。
the following describes the above technical scheme in detail with reference to specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-CD47-Double Terminator-J23109-RBS-Kil-Terminator, and the sequence of the system constructed based on the Kil protein for releasing anti-tumor effect protein is shown as SEQ ID No. 9.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGGAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCATTATTTTTAAAATTAACGATATGGGCTGGTATCGCCAGGCGCCGGGCAAACGCCGCGAATGGGTGGCGGCGAGCACCGGCGGCGATGAAGCGATTTATCGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGCGAAAAACAGCGTGTTTCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGGTGATTAGCACCGATCGCGATGGCACCGAATGGCGCCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 2
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-CD47-Double Terminator-J23114-RBS-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 10.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGGAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCATTATTTTTAAAATTAACGATATGGGCTGGTATCGCCAGGCGCCGGGCAAACGCCGCGAATGGGTGGCGGCGAGCACCGGCGGCGATGAAGCGATTTATCGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGCGAAAAACAGCGTGTTTCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGGTGATTAGCACCGATCGCGATGGCACCGAATGGCGCCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 3
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-CD47-Double Terminator-J23115-RBS-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 11.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGGAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCATTATTTTTAAAATTAACGATATGGGCTGGTATCGCCAGGCGCCGGGCAAACGCCGCGAATGGGTGGCGGCGAGCACCGGCGGCGATGAAGCGATTTATCGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGCGAAAAACAGCGTGTTTCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGGTGATTAGCACCGATCGCGATGGCACCGAATGGCGCCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTATAGCTAGCTCAGCCCTTGGTACAATGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 4
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-CD47-Double Terminator-Optimized-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 12.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGGAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCATTATTTTTAAAATTAACGATATGGGCTGGTATCGCCAGGCGCCGGGCAAACGCCGCGAATGGGTGGCGGCGAGCACCGGCGGCGATGAAGCGATTTATCGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGCGAAAAACAGCGTGTTTCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCACCGCGGTGATTAGCACCGATCGCGATGGCACCGAATGGCGCCGCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCGGCAGTAAAAAGACGTAAACTTTCCCAGAATCCTGCCGATATTATCCCACAAAATTTGTCACACAAGGAAGCTGAATGGGGAGCAGCATGCGCAAACGCTTTTTTGTGGGCATTTTTGCGATTAACCTGCTGGTGGGCTGCCAGGCGAACTATATTCCGGATGTGCAGGGCGGCACCATTGCGCCAAGCAGCAGCAGCAAACTGACCGGCATTGCGGTGCAGTAAATAGCCAATTATTGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCTTAC。
example 5
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-PD-L1-Double Terminator-J23109-RBS-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 13.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTTCAGCTGGTTGAAACCGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGCGTCTGGTTTCACCTTCTCTATGCACGCGATGACCTGGTACCGTCAGGCGCCGGGTAAACAGCGTGAACTGGTTGCGGTTATCACCTCTCACGGTGACCGTGCGAACTACACCGACTCTGTTCGTGGTCGTTTCACCATCTCTCGTGACAACACCAAAAACATGGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTACTGCAACGTTCCGCGTTACGACTCTTGGGGTCAGGGTACCCAGGTTACCGTTTCTTCTGGTGGTCTGCCGGAAACCGGTGGTCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 6
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-PD-L1-Double Terminator-J23114-RBS-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 14.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTTCAGCTGGTTGAAACCGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGCGTCTGGTTTCACCTTCTCTATGCACGCGATGACCTGGTACCGTCAGGCGCCGGGTAAACAGCGTGAACTGGTTGCGGTTATCACCTCTCACGGTGACCGTGCGAACTACACCGACTCTGTTCGTGGTCGTTTCACCATCTCTCGTGACAACACCAAAAACATGGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTACTGCAACGTTCCGCGTTACGACTCTTGGGGTCAGGGTACCCAGGTTACCGTTTCTTCTGGTGGTCTGCCGGAAACCGGTGGTCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 7
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-PD-L1-Double Terminator-J23115-RBS-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 15.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTTCAGCTGGTTGAAACCGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGCGTCTGGTTTCACCTTCTCTATGCACGCGATGACCTGGTACCGTCAGGCGCCGGGTAAACAGCGTGAACTGGTTGCGGTTATCACCTCTCACGGTGACCGTGCGAACTACACCGACTCTGTTCGTGGTCGTTTCACCATCTCTCGTGACAACACCAAAAACATGGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTACTGCAACGTTCCGCGTTACGACTCTTGGGGTCAGGGTACCCAGGTTACCGTTTCTTCTGGTGGTCTGCCGGAAACCGGTGGTCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCTTTATAGCTAGCTCAGCCCTTGGTACAATGCTAGCCTGTATTGATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACATATGAGGAAAAGATTTTTTGTGGGAATATTCGCGATAAACCTCCTTGTTGGATGTCAGGCTAACTATATACCTGATGTTCAGGGAGGGACCATCGCACCATCCTCCTCTTCTAAACTGACGGGGATCGCGGTTCAGTAGTACTAGAGTCACACTACCTAGAGCCAGGCATCAAATGGCTCACCTTCGGGTGGGCCTTTCTGCG。
example 8
The embodiment provides a system for releasing anti-tumor effect protein based on Kil protein, which specifically comprises the following steps: J23100-pel-PD-L1-Double Terminator-Optimized-Kil-Terminator, the sequence of the system is shown in SEQ ID No. 16.
The sequence is specifically as follows:
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGAGCGATTGATGAAGTACCTGCTGCCGACCGCCGCCGCCGGCCTGCTGCTGCTGGCCGCCCAGCCGGCCATGGCCATGGCGCAGGTTCAGCTGGTTGAAACCGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGCGTCTGGTTTCACCTTCTCTATGCACGCGATGACCTGGTACCGTCAGGCGCCGGGTAAACAGCGTGAACTGGTTGCGGTTATCACCTCTCACGGTGACCGTGCGAACTACACCGACTCTGTTCGTGGTCGTTTCACCATCTCTCGTGACAACACCAAAAACATGGTTTACCTGCAGATGAACTCTCTGAAACCGGAAGACACCGCGGTTTACTACTGCAACGTTCCGCGTTACGACTCTTGGGGTCAGGGTACCCAGGTTACCGTTTCTTCTGGTGGTCTGCCGGAAACCGGTGGTCACCACCACCACCACCACTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACATGGATCCATGGTTAGCCCTCCCACACATAACCAGGAGGTCAGATTATTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGATAGATTGCTAAGCTAGTATTTCAGCGAATTCATCGGCTTCATCAGAAGATCAGCTTCTGTGCCGGCAGTAAAAAGACGTAAACTTTCCCAGAATCCTGCCGATATTATCCCACAAAATTTGTCACACAAGGAAGCTGAATGGGGAGCAGCATGCGCAAACGCTTTTTTGTGGGCATTTTTGCGATTAACCTGCTGGTGGGCTGCCAGGCGAACTATATTCCGGATGTGCAGGGCGGCACCATTGCGCCAAGCAGCAGCAGCAAACTGACCGGCATTGCGGTGCAGTAAATAGCCAATTATTGAAGGCCTCCCTAACGGGGGGCCTTTTTTTGTTTCTGGTCTCCCTTAC。
experimental example
1. In vitro experiments for releasing nanobodies based on Kil protein cleavage System
1.1 evaluation of nanobody Release Capacity based on Kil protein cleavage System
To explore the ability of nanobodies to release based on Kil protein cleavage System at different time and different promoters, kit containing constitutively expressed anti-CD 47 nanobody modules and containing different constitutive promotersE. coli DH5aEngineering bacteria are continuously cultured. Control of bacterial Access OD at initiation 600 The value is 0.02, the corresponding strain is continuously cultured for 72 hours, sampling is carried out for 1 time every 24 hours, sample preparation is reserved, and the content of the nano antibody in the supernatant after the culture medium is centrifuged is detected through Western Blot by using a 6X Histag antibody so as to judge the leakage condition of the nano antibody under different Kil protein cleavage system structures. As a Control, the J23100-pel-CD47-Double Terminator plasmid (CD 47 Control) without Kil protein cleavage system was used.
The specific operation steps are as follows:
a. single colonies were picked from LB plates and inoculated into 3mL liquid LB medium containing 100. Mu.g/L ampicillin and cultured overnight at 37℃and 220 rpm.
b. Inoculating overnight cultured bacterial culture solution into 3mL liquid LB medium containing 100 μg/L ampicillin, and calculating to obtain initial OD of the culture medium 600 After 0.02, the cells were incubated at 37℃and 220rpm for a period of time according to the experimental protocol.
c. 1mL of the medium was centrifuged at 12000rpm for 1 minute.
d. 77. Mu.L of the centrifuged supernatant was placed in a 0.2 mL PCR tube, 23. Mu.L of a protein loading buffer was added, and the mixture was subjected to a treatment at 95℃for 5 minutes in a PCR instrument to complete protein preparation.
e. Western immunoblotting experiments were performed on the above samples, and nanobodies were detected using a mouse anti-6 XHistag monoclonal antibody to determine the nanobody content in the culture supernatant.
The results are shown in fig. 2, where nanobody leakage exhibited a significant time dependence. The most efficient promoter of J23115 showed a significantly deeper band at 48 hours relative to the other groups, representing a significant leakage of nanobodies. Since the three promoters were ranked from strong to weak in intensity as J23115, J23114, J23109, respectively, this is consistent with theoretical predictions. Both the J23114 and J23115 promoters showed a greater amount of leakage at 72 hours relative to the control group. Based on the above data, J23115 was finally selected as a promoter for Kil protein expression for subsequent animal experiments.
Based on the above experiments, it can be seen that: j23115 promoter-initiated, kil protein-based cleavage System (J23115-RBS-Kil-Terminator) essentially meets the nanobody release requirements. Next, the J23115-RBS-Kil-Terminator system was assembled into the J23100-pel-PD-L1-Double Terminator plasmid structure as well, and the anti-CD 47 nanobody and the anti-PD-L1 nanobody containing the J23115-RBS-Kil-Terminator system were transferred intoE. coli MG1655Verifying whether Kil proteins are capable of being in candidate target strainsE. coli MG1655The same cleavage function is exhibited.
By controlling bacterial access at the startOD 600 The corresponding strain was cultured for 72 hours at a value of 0.02. Culture medium was taken at 72 hours, supernatant was centrifuged and sampled, and nanobodies in supernatant were detected by Western Blot to observe leakage of the structured nanobodies. The control group is a CD47 and PD-L1 nanobody constitutive expression vector without Kil protein cleavage system.
The results are shown in FIG. 3, whereE. coli MG1655The Kil protein cleavage system exerts excellent effects: for the strain containing J23100-pel-CD47-Double Terminator, the control group (FIG. 3, lane 2, control) containing no J23115-RBS-Kil-Terminator showed almost no nanobody leakage from the supernatant, but the experimental group (FIG. 3, lane 3, J23115 Kil) containing J23115-RBS-Kil-Terminator showed significant supernatant nanobody leakage. For the strain containing J23100-pel-PD-L1-Double Terminator, the control group (FIG. 3, lane 4, control) supernatant containing no J23115-RBS-Kil-Terminator showed almost no nanobody leakage, but the experimental group (FIG. 3, lane 5, J23115 Kil) containing J23115-RBS-Kil-Terminator showed significant supernatant nanobody leakage; among them, lane 1 in FIG. 3 is a 15 kilodaltons marker band.
2. Bacterial Activity verification
Continuous lysis of bacteria in animals to release nanobodies requires that the bacteria themselves remain viable, whereas expression of Kil proteins affects bacterial growth. In order to explore the influence of the Kil protein cleavage system on the activity of engineering bacteria, the OD of the engineering bacteria liquid culture medium containing the Kil protein cleavage system after 24 hours of culture is measured 600 Values and bacterial colony generation numbers are used for representing the survival condition of engineering bacteria. According to the above experiments, the J23115 constitutive promoter was used to express the Kil protein and simultaneously constitutively express anti-CD 47 nmE. coli MG1655Engineering bacteria (J23100-pel-CD 47-Double Terminator-J23115-RBS-Kil-Terminator) as experimental group only constitutively expresses anti-CD 47 nmE. coli MG1655The activity of engineering bacteria (J23100-pel-CD 47-Double Terminator) was verified as a control group.
The specific experimental steps are as follows:
a. single colonies were picked from LB plates and inoculated into 3mL liquid LB medium containing 100. Mu.g/L ampicillin and cultured overnight at 37℃and 220 rpm.
b. The bacterial culture solution cultured overnight was inoculated into 3mL of liquid LB medium containing 100. Mu.g/L ampicillin, and the initial OD of the medium was calculated 600 After 0.02, the cells were cultured at 37℃and 220rpm for 24 hours.
c. OD of bacterial culture after 24 hours of culture was measured in NanoDrop One using a cuvette with an optical path of 1cm 600 。
d. Diluting the bacterial culture solution 10 6 After doubling, 100. Mu.L was uniformly spread on a plate. Counts were taken after overnight incubation.
The results show that the OD measured by the engineering bacteria of the experimental group at 24 hours 600 The value is lower than that of the control group, but the good growth state is still maintained, and the OD thereof is kept 600 The value was about 5.0 (fig. 4A). The bacterial colony generation number shows that the activity of the engineering bacteria of the experimental group is lower than that of the control group at 24 hours, but the better survival activity is still maintained, and the number of the living bacteria (namely the bacterial number in FIG. 4B) is 2.2x10 8 CFU/mL (also referred to as 22X 10) 7 CFU/mL, as in fig. 4B). The experimental result shows that the engineering bacteria for constitutive expression of Kil protein can reduce the activity of bacteria to improve the safety of bacterial treatment, but can ensure that the bacterial quantity is at a certain level so as to achieve the aim of fully releasing therapeutic substances.
3. Animal experiment for releasing nanobody based on Kil protein system
Since no therapeutic module is foundE. coli MG1655Tumor regression was induced, so that mice with high tumor were used to subcutaneously transplant lymphoma model mice (i.e., 2000mm in size was formed 3 Left and right giant KA202 cell line subcutaneous tumor mice) verifies the therapeutic effect of the therapeutic module after intratumoral injection of the engineering bacteria. To verify the effect of nanobody-Kil protein cleavage systems individually in mice, we injected 100ul 1 x 10 intratumorally every 4 days for high tumor bearing mice after random grouping and confirming no statistical difference in tumor bearing size (p=0.474) among groups 9 CFU/mlE. coli MG1655The original strain (original strain), the engineering bacterium containing the anti-CD 47 nanobody and the Kil protein cleavage system (CD 47-Kil), the engineering bacterium containing the anti-PD-L1 nanobody and the Kil system (PD-L1-Kil), PBS buffer (PBS control), and the tumor growth and the survival of the mice were continuously observed within 20 days (FIG. 5).
The specific operation is as follows:
a. mixing DMEM culture medium and IMDM culture medium in a biosafety cabinet at a ratio of 1:1; fetal bovine serum was added to give a volume ratio of 10% in the final medium. And adding Penicillin Streptomycin diabody according to the proportion of 100:1 and adding sterile beta-mercaptoethanol according to the proportion of 500:1.7.
b. The cryopreserved KA202 lymphoma cell line (containing Feeder) was removed from the liquid nitrogen and quickly transferred to a 37℃water bath for rapid thawing.
c. Adding appropriate amount of cell line into six-hole plate containing above culture medium, wherein the usage amount of each hole culture medium is about 2.5 mL, and CO is 7.5% at 37deg.C 2 Culturing under the condition. Subculturing is performed according to the cell condition.
d. The cell culture broth was transferred to a 50 mL BD tube and thoroughly mixed. mu.L of the cell culture solution and 10. Mu.L of the phenol blue solution were each taken and thoroughly mixed in a 1.5 mL EP tube. mu.L of the mixture was added to a cell counting plate and counted under a microscope.
e. The 50 mL BD tube was centrifuged at 300 Xg for 5 minutes. After discarding the supernatant, it was resuspended using 20 mL DMEM medium.
f. Again, the 50 mL BD tube was centrifuged at 300×g for 5 minutes. After discarding the supernatant, the suspension was resuspended in an appropriate amount of DMEM medium to give a final resuspension with a viable cell count of 1X 10 7 /mL。
g. KA202 lymphoma cell line was subcutaneously injected with an insulin needle in C57BL/6 mice after 100. Mu.L of the cell line was resuspended in DMEM medium.
h. The growth of the tumor of the mice is observed, and the tumor body is about 2000mm to be measured by using a vernier caliper 3 At the time of intratumoral injection into mice 100. Mu.L of 1X 10 9 CFU/ml corresponding bacteria or buffer. The primary culture supernatant was discarded by centrifugation for each bacterial injection and resuspended in PBS buffer. Dynamic movementThe experiments were divided into PBS buffer control group (PBS control in FIG. 5),E. coli MG1655Control group (original strain in FIG. 5), CD47-Kil experimental group, PD-L1-Kil experimental group. There were at least 4 mice per group, with specific numbers given in the risk table below (risk table, table 2).
TABLE 2 Risk Table of mice after injection of different strains into highly tumor bearing mouse models
i. Subcutaneous tumor size was measured every 4 days using vernier calipers and survival was observed. The experiment was ended on day 20.
As shown in FIG. 5, mice injected with the original strain all died within 48 hours after injection, whereas the CD47-Kil, PD-L1-Kil, PBS control group did not die within 48 hours. Thus indicating that it may be bacteremia infection death caused by the bacteria themselves. The strain containing the Kil protein lysis system does not show the situation, which indicates that the function of the Kil protein lysis bacterial outer membrane plays a part in attenuation and obviously increases the treatment safety.
At day 14, all mice in the intratumoral PBS buffer group died. At the end of the experiment, 1 mouse still survived in the CD47-Kil group, and 2 mice still survived in the PD-L1-Kil group. Statistical analysis was performed using Log-rank method, and there was a significant statistical difference (p < 0.001) between CD47-Kil and PD-L1-Kil engineered bacteria containing nanobody-Kil proteolytic system and PBS control group and original strain. This demonstrates that engineered bacterial systems containing Kil proteins and therapeutic effector proteins have significant therapeutic effects.
In this experiment, complete tumor disappearance occurred on day 4 after inoculation with engineering bacteria in the CD47-Kil group 1 mice (i.e., the 1 mice that survived the CD47-Kil group). Whereas 3 mice surviving on day 20 (i.e., 1 mouse surviving the CD47-Kil group and 2 mice surviving the PD-L1-Kil group) all exhibited significant tumor shrinkage. While in the other groups (i.e., PBS buffer control group and PBS buffer control groupE. coli MG1655Control group), all ofNo tumor shrinkage was observed.
As reported in the prior art, the use of anti-mouse CD47 nanobodies alone as therapeutic substances in mice does not extend the survival time of mice (Proc. Natl. Acad. Sci. USA, 113, 19, vol. 19, 2646-2654, [ Proceedings of the National Academy of Sciences, 113 (19) E2646-E2654], literature ID doi: 10.1073/pnas.1604268113.). This study demonstrates that better therapeutic results were obtained with the combination of bacteria with anti-murine CD47 nanobodies (CD 47-Kil panel).
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention.
Claims (8)
1. The system for releasing the anti-tumor effector protein constructed based on the Kil protein is characterized by comprising a Kil protein expression module and a therapeutic effector protein module, wherein the Kil protein expression module comprises a promoter, an RBS, a Kil protein sequence and a terminator for expressing the Kil protein, and the therapeutic effector protein module comprises the promoter, the RBS, the therapeutic effector protein sequence and the terminator for expressing the therapeutic effector protein;
the therapeutic effector protein sequence is a CD47 nanobody sequence and/or a PD-L1 nanobody sequence;
the promoter for expressing the Kil protein is any one of J23109, J23114 and J23115.
2. The system of claim 1, wherein the nucleotide sequence of the Kil protein sequence is set forth in SEQ ID No. 1.
3. The system of claim 1, wherein the nucleotide sequence of the promoter J23109 for expressing the Kil protein is shown in SEQ ID No.2, the nucleotide sequence of the promoter J23114 for expressing the Kil protein is shown in SEQ ID No.3, and the nucleotide sequence of the promoter J23115 for expressing the Kil protein is shown in SEQ ID No. 4.
4. The system of claim 1, wherein the nucleotide sequence of the RBS of the Kil protein expression module is AAGAAGGAA.
5. The system of claim 1, wherein the terminator of said Kil protein expression module has the nucleotide sequence shown in SEQ ID No. 5.
6. The system according to claim 1, wherein the nucleotide sequence of the system is shown in any one of SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, SEQ ID No.14, SEQ ID No. 15.
7. Use of a system according to any one of claims 1-6 for the preparation of an anti-tumor product.
8. The method of construction of the system according to any one of claims 1 to 6, wherein the Kil protein expression module is expressed in bacteria by means of a plasmid vector and/or an insert genome based on molecular biology methods and the therapeutic effector protein module is expressed in bacteria simultaneously by means of a plasmid vector and/or an insert genome.
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