CN114672502A - Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application - Google Patents

Abstract

The present invention relates to a plasmid comprising: a gene encoding a temperature sensitive element; a manipulation unit that is located downstream of the gene encoding a temperature sensitive element and is capable of being regulated by the temperature sensitive element; a foreign gene expressing a protein of interest located downstream of the manipulation unit. The plasmid of the present invention can regulate the expression of a target protein by the manipulation unit regulated by the temperature sensitive element, which is inactivated when the temperature is increased to a given temperature so that the manipulation unit initiates the expression of the target protein, and which inhibits the manipulation unit from initiating the expression of the target protein when the temperature is lower than the given temperature. The invention further provides a construction method of a temperature-controlled cell-free synthesis system through the plasmid, and the expression of target protein in the cell-free system is regulated and controlled through temperature.

Description

Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application

Technical Field

The invention belongs to the field of biological reaction systems, and particularly relates to a construction method of a temperature-controlled cell-free reaction system, a plasmid used by the method and application of the temperature-controlled cell-free reaction system.

Background

Bioprocess control is an important issue in the fields of bio-manufacturing and genetic engineering. The protein synthesis and the space-time activation and regulation of the biological function of a specific site have important significance in the fields of biotechnology, medicine, industrial production and the like. The control method commonly used at present is to regulate gene expression by utilizing the chemical effect of molecules. However, this method also has some disadvantages, including instability of chemical effect, low spatial accuracy and other adverse side reactions that may be brought about by chemical inducers. The use of physical signals such as light and heat to control biological processes is an ideal method. Compared with the poor permeability of optical signals to organisms, the temperature is taken as a unique physical signal, and the overall and local temperature can be accurately and effectively controlled. Temperature regulated expression systems rely on a powerful and finely regulated promoter, avoiding the use of special media, toxic or expensive chemical inducers.

Currently, temperature regulated expression has been successfully used for the production of many recombinant proteins and peptides. Temperature is a very well-defined signal that can be spatially directed by means of external triggers such as focused ultrasound, infrared light and magnetic particle hyperthermia). An ideal thermal biological switch that is responsive to temperature changes should have a sensitive switching effect to sharp temperature changes. Heat shock proteins have been rapidly identified as the most compelling example of known selective and inducible transcriptional regulation in eukaryotic cells. The lipid-mediated changes in conformation at different temperatures result in different enzymatic properties of the membrane-associated protein, which confers the membrane-associated protein with temperature sensing properties. Temperature-sensitive RNA molecules, known as "RNA thermometers," can be designed to have a flexible switching response temperature with high sensitivity. In addition, the transcriptional repressor provides on-off control of the threshold for bacterial gene expression in the biomedical related 32-46 ℃ range.

However, the above-described intracellular regulatory mechanisms in response to temperature changes face some inherent difficulties. Temperature is an important factor affecting enzyme activity and complex cellular metabolism, and temperature changes may result in changes in the intensity and metabolic flux of cells, thereby adversely affecting the target pathway. Furthermore, interactions between different synthetic pathways in vivo may limit effective control of protein expression. Therefore, a more efficient method is being sought to avoid unnecessary interference of metabolic pathways with temperature control pathways.

The cell-free protein synthesis (CFPS) system is a platform for the in vitro synthesis of proteins that should have been produced and are difficult to produce in a living organism. CFPS takes exogenous DNA or mRNA as a template, and inorganic salt, reaction substances and energy substrates are supplemented in the system. The synthesis of proteins can be carried out without the cellular environment by the action of various enzymes provided by the cell extract. Compared with a cell system, the CFPS can avoid the interference of intracellular metabolic pathways on target metabolic pathways at different temperatures, and improve the specificity of temperature regulation pathways. In addition, cell-free systems are characterized by their simplicity and time efficiency, as an open synthesis platform, without membrane constraints. They require only the addition of certain substances to the biosynthetic reaction which can be completed within a few hours, so that the factors influencing the bonds of the control elements in CFPS can be rapidly explored and screened. In addition, previous studies have shown that cell-free synthesis systems controlled by physical signals are encapsulated in liposomes to mimic population communication of living cells, and that such responsive artificial cells have the potential to deliver drugs.

Disclosure of Invention

The present invention establishes a genetic loop in response to temperature in a CFPS system, thereby controlling protein synthesis by temperature in vitro (fig. 1). Phage lambda repression of thermosensitive variants cI857 (abbreviated as cI in the present invention) was used to construct temperature controlled cell free protein synthesis (hereinafter referred to as tcCFPS) system and to accomplish reconstitution of plasmid and optimization of cell extracts and redox conditions. The present invention also establishes artificial cells that can control protein synthesis by temperature.

Specifically, the following technical scheme is provided:

1. a plasmid, comprising:

a gene encoding a temperature sensitive element;

a manipulation unit that is located downstream of the gene encoding a temperature sensitive element and is capable of being regulated by the temperature sensitive element;

a foreign gene expressing a protein of interest located downstream of the manipulation unit.

2. The plasmid according to item 1, wherein the manipulation unit that can be regulated by the temperature sensitive element means that when the temperature is increased to a given temperature, the temperature sensitive element is inactivated so that the manipulation unit initiates expression of the target protein; and the temperature sensitive element inhibits the manipulation unit from initiating the expression of the target protein when the temperature is lower than a given temperature, preferably 37-40 ℃.

3. The plasmid according to item 1 or 2, wherein the temperature sensitive element and the operator are used in combination, and the temperature sensitive element and the operator are selected from any one of bacteriophage lambda-repressed cI-pR-pL promoter, TpA protein-TpA promoter, TetR thermo-sensitive variant-tet operator of E.coli repressor protein, LacI (Ts) -lacO site + LacI promoter, or E.coli heat shock protein-pHSP promoter, and preferably, the temperature sensitive element and the operator are selected from bacteriophage lambda-repressed cI-pR-pL promoter.

4. The plasmid according to any one of items 1 to 3, wherein the initial plasmid used for constructing the plasmid is selected from any one of pSB3K3, pET series, pGEX series, pMAL series, pQE-1, pQE-60, pGS-21a, pCDFDuet-1, pRSFDuet-1 or pBluescriptII series, preferably pSB3K 3.

5. The plasmid according to any one of claims 1 to 4, wherein the target protein is selected from any one or more of a fluorescent protein, a vaccine protein, an antibody protein, a biocatalytic enzyme, a membrane protein, a polypeptide, a cytokine protein, a hormone protein, and a complement protein.

6. The plasmid according to claim 5, wherein the fluorescent protein is any one or more selected from red fluorescent protein, green fluorescent protein, orange fluorescent protein and yellow fluorescent protein.

7. Use of the plasmid according to any one of items 1-6 in a cell-free reaction system for synthesizing a protein of interest.

8. A liposomal drug delivery system comprising:

the plasmid according to any one of claims 1 to 4, wherein the protein of interest is a pharmaceutical protein;

cell-free extracts of prokaryotic or eukaryotic cells; and temperature sensitive proteins

9. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract;

-mixing the cell extract with a plasmid comprising a gene encoding a temperature sensitive element and a manipulation unit capable of being regulated by the temperature sensitive element located downstream of the gene encoding the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell-free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the target protein in a reaction system at a given temperature.

10. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract mixture of a cell extract comprising a temperature sensitive element and a cell extract not comprising a temperature sensitive element;

-mixing the cell extract mixture with a plasmid comprising a manipulation unit capable of being manipulated by the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the protein of interest in a reaction system at a given temperature.

11. The method of item 10, wherein the volume ratio of the temperature sensitive element-containing cell extract to the temperature sensitive element-free cell extract is 1:2 to 2: 1.

12. The method of any one of claims 9 to 11, wherein the cell extract is a cell extract from escherichia coli, archaebacteria, malt cells, yeast cells, rabbit reticulocytes, tobacco leaf cells, insect cells, or chinese hamster ovary cells.

13. The method according to any one of items 9 to 12, wherein the temperature sensitive element and the operator are used in combination, and the temperature sensitive element and the operator are selected from any one of bacteriophage lambda-repressed cl-pL promoter, TlpA protein-TlpA promoter, e.coli repressor TetR temperature sensitive variant-tet operator, e.coli repressor LacI (ts) -lacO site + LacI promoter, or e.coli heat shock protein-pHSP promoter, preferably the temperature sensitive element and the operator are selected from bacteriophage lambda-repressed cl-pR-pL promoter.

14. The method according to any one of items 9 to 13, wherein the energy source substance is one or more selected from sucrose, maltose, glucose-6-phosphate, fructose-1, 6-diphosphate, phosphoglycerate, phosphocreatine, adenosine triphosphate, acetyl phosphate, glutamate, polyphosphate, and phosphoenolpyruvate.

15. The method according to any one of claims 9 to 14, wherein the amino acid mixture is one or more selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, arginine, and histidine.

16. The method of any one of items 9 to 15, wherein the oxidizing substance is oxidized glutathione (GSSG) and the reducing substance is reduced Glutathione (GSH), and the molar ratio of GSSG to GSH is 2:1 to 4: 1.

17. The method of any of claims 9-16, wherein the given temperature is 37-40 ℃.

Effects of the invention

The invention establishes a cell-free expression system for synthesizing temperature control protein, and can realize the synthesis of the temperature control protein according to specific temperature by adding the plasmid with the temperature sensitive element prepared by the invention. By introducing the temperature sensitive element, the temperature control approach is prevented from being interfered by an unnecessary metabolic approach in the temperature regulation and expression process in the prior art, so that the temperature control cell-free expression system can better realize temperature control without generating other additional influences.

The present invention uses engineered plasmids as a means of introducing a temperature sensitive element replication template. Bringing these genetic elements together into a reaction system via engineered plasmids has proven to be a cost-effective and efficient approach.

The invention further optimizes the conditions of the cell-free expression system, optimizes the redox characteristics by adding different amounts of oxidizing substances and reducing substances, and can obtain the maximum temperature control induced expression multiple of 3.40 times. By changing the types and the proportion of cell extracts in the cell-free expression system, the temperature-controlled induced expression fold of 4.10 times can be obtained at most. By analyzing the reaction kinetics, the regulation and control of the cI protein on the pR-pL operon-promoter at different temperatures can be more clearly revealed, and a theoretical basis is provided for the possible application of the synthesis and the transmission of the therapeutic protein in the future.

The invention successfully constructs the liposome which is activated at a specific temperature and has the protein expression function, and the liposome is constructed by adopting a water-in-oil emulsion transfer method on the basis of 1-palmitoyl-2 oleyl-sn-glycerol-3-phosphorylcholine (POPC) and cholesterol. In order to ensure sufficient reaction time in the artificial cells to obtain a certain amount of fluorescence expression, liposomes were prepared at reaction temperatures exceeding 12 h. From the flow cytometry results, it was shown that the tcCFPS encapsulated liposomes were responsive to temperature control. The feasibility of the response model of the artificial cells to the temperature signals is proved.

The plasmid with the temperature sensitive element constructed by the invention can also control protein synthesis in cells.

Drawings

FIG. 1 is a temperature controlled cell free protein synthesis system (tcCFPS) established in the present invention;

FIG. 2 is a model of the tcCFPS regulatory mechanism using the temperature sensitive circuit cI-pR-pL;

FIG. 3A is a diagram of the structure of plasmid pcI;

FIG. 3B is a diagram of the structure of plasmid p.DELTA.cI;

FIG. 4 is a graph showing the comparison of expression of plasmid pcI at different temperatures in a cell system;

FIG. 5 is a graph showing the comparison of expression of plasmid pcI at different temperatures in a cell-free system;

FIG. 6 is a graph showing a comparison of GFP expression at different temperature molar ratios of GSSG to GSH in a cell-free system;

FIG. 7 is a graph of the average fluorescence and ratio of GFP at different temperatures when different volume fractions of a mixed cell extract of cI-BS cell extracts were added to a cell-free system;

FIG. 8 is a graph of the concentration of cI-mRNA in tcCFPS at different temperatures;

FIG. 9 is a graph of the concentration of GFP-mRNA in tcCFPS at different temperatures;

FIG. 10 is an artificial cell model containing tcCFPS;

FIG. 11 is a graph comparing the expression of GFP in liposomes at different temperatures;

FIG. 12A is a confocal image of Liss RhodPE fluorescence in liposomes at 30 ℃;

FIG. 12B is a confocal image of Liss RhodPE fluorescence in liposomes at 37 ℃;

FIG. 12C is a confocal image of Liss RhodPE fluorescence and GFP fluorescence in liposomes at 30 ℃;

FIG. 12D is a confocal image of Liss RhodPE fluorescence and GFP fluorescence in liposomes at 37 ℃.

Detailed Description

It should be noted that certain terms are used throughout the description and following claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the invention is to be determined by the claims appended hereto.

The present invention in a first aspect relates to: a plasmid.

In a specific embodiment, there is provided a plasmid comprising: a gene encoding a temperature sensitive element; a manipulation unit that is located downstream of the gene encoding a temperature sensitive element and is capable of being regulated by the temperature sensitive element; a foreign gene expressing a protein of interest located downstream of the manipulation unit.

In the context of this specification, a "plasmid" is defined as a double-stranded DNA molecule in a closed loop form, which is a DNA molecule other than a chromosome (or a karyopoiesis) in an organism such as a bacterium, a yeast, or an actinomycete, and which is present in the cytoplasm of the cell (except for a yeast, and the plasmid for a yeast is present in the nucleus of the cell), has an autonomous replication ability, and can maintain a constant copy number in a daughter cell and express genetic information carried therein. The plasmid is not necessary for the growth and propagation of bacteria, and can be automatically lost or eliminated by artificial treatment, such as high temperature, ultraviolet ray, etc. The genetic information carried by the plasmid can endow the host bacteria with certain biological characters, and is beneficial to the survival of the bacteria under specific environmental conditions. Like the bacterial genome, plasmids also belong to circular double-stranded DNA (covalently closed circular DNA, cccDNA). The present invention uses engineered plasmids as a means of introducing both protein replication templates (along with promoters responsive to temperature sensitive proteins) and temperature sensitive proteins. Bringing these genetic elements together into a reaction system via a plasmid has proven to be a cost-effective and efficient method.

In the context of the present specification, "operator unit" refers to a promoter or an operator in the field of bioengineering, or a tandem promoter-operator.

In the context of this specification, a "promoter" is defined as a DNA sequence recognized, bound and transcribed by RNA polymerase, which contains conserved sequences required for specific binding of RNA polymerase and transcription initiation, most of which is located upstream of the transcription initiation point of a structural gene, and is not transcribed per se, in accordance with the general definition of the field of bioengineering. However, some promoters, such as tRNA promoters, are located downstream of the transcription start site and these DNA sequences can be transcribed. The nature of the promoter was originally identified by mutations that increase or decrease the transcription rate of the gene. Promoters are generally located upstream of the transcription start site.

In the context of the present specification, "operon" is defined in accordance with the general definition of the field of bioengineering, and refers to a group of key nucleotide sequences, including an Operator gene (Operator), a common promoter, and one or more structural genes used as a motif for the production of messenger rna (mrna). An operon is typically composed of more than 2 coding sequences in tandem with promoter, operator, and other regulatory sequences in a genome. The promoter sequence is a specific DNA sequence that RNA polymerase binds to and initiates transcription. Within certain regions of the promoter sequences of many prokaryotic genes, there are usually some similar sequences, called consensus sequences, in the-10 and-35 regions upstream of the transcription start site. The consensus sequence for E.coli and some bacterial promoters is TATAAT, also known as the Pribnow box (PribnowBox), in the-10 region and TTGACA in the-35 region. Any base mutation or variation in these consensus sequences affects the binding of RNA polymerase to the promoter sequence and initiation of transcription. Thus, the degree of identity with the consensus sequence determines the magnitude of transcriptional activity of the promoter sequence. The operator sequence is the binding site for the prokaryotic repressor protein. When the operator sequence binds to the repressor protein, it blocks the binding of RNA polymerase to the promoter sequence, or prevents the RNA polymerase from moving forward along the DNA, repressing transcription, mediating negative regulation. Also, a specific DNA sequence in the prokaryotic operon regulatory sequences binds to an activator protein, which activates transcription and mediates positive regulation.

In a specific embodiment, the manipulation unit capable of being manipulated by the temperature sensitive element is a unit in which the temperature sensitive element is inactivated when the temperature is increased to a given temperature, so that the manipulation unit initiates expression of a target protein; and said temperature sensitive element inhibits the manipulation unit from initiating expression of the target protein when the temperature is lower than a given temperature, preferably the given temperature is 37-40 deg.C, and may be, for example, 37 deg.C, 37.5 deg.C, 38 deg.C, 38.5 deg.C, 39 deg.C, 39.5 deg.C, 40 deg.C.

In the present invention, the gene encoding the temperature sensitive element is used in combination with the operator, for example, when the temperature sensitive element is phage lambda repression cI, the operator is pR-pL promoter.

The bacteriophage lambda repression cI refers to cI857 described in Tunable thermal biolistics for in vivo control of microbial therapeutics, which is named cI38 for short in the present invention, i.e., the expression of "bacteriophage lambda repression cI", "cI 857", "cI 38" and "cI" in the present invention has the same meaning, and bacteriophage lambda repressor cI is a well-known temperature sensitive variant acting on a tandem pR-pL operator-promoter, which can effectively regulate a gene cloned downstream of the promoter.

In other embodiments of the present invention, the temperature sensitive element and the operator may be any one of a TlpA protein-TlpA promoter, a TetR thermo-sensitive variant-tet operator of E.coli repressor protein, LacI (Ts) -lacO site + LacI promoter, or a E.coli heat shock protein-pHSP promoter.

In a specific embodiment, the target protein is selected from one or more of fluorescent protein, vaccine protein, antibody protein, biocatalytic enzyme, membrane protein, polypeptide, cytokine protein, hormone protein, and complement protein, and there is no particular limitation on the "target protein" related to the present invention in terms of molecular weight, chemical properties, and physical properties.

In the context of the present specification, "vaccine protein" complies with the general definition in the biotechnology field, covering both subunit and polypeptide vaccines. At present, DNA recombination technology makes it possible to obtain large quantities of pure antigen molecules. Compared with vaccines prepared by using pathogens as raw materials, the vaccine has revolutionary change in technology, so that the quality is easier to control, and the price is higher. From the standpoint of efficacy, some subunit vaccines, such as acellular pertussis, HBsAg, etc., are highly immunogenic at low doses; while others have lower immunity and require a stronger adjuvant than aluminium salts. Peptide vaccines are typically manufactured by chemical synthesis techniques. Its advantages are simple components and easy quality control. However, as the molecular weight and structural complexity of the immunogen decreases, the immunogenicity also decreases significantly. Thus, these vaccines typically require special structural designs, special delivery systems, or adjuvants.

In the context of the present specification, "biocatalytic enzyme" complies with the general definition in the field of biotechnology and may also be referred to simply as "enzyme". The chemical nature of an enzyme is that of a protein (with the exception of a few RNAs), and therefore it also has a primary, secondary, tertiary, or even quaternary structure. According to their molecular composition, they can be classified into simple enzymes and conjugated enzymes. The enzyme protein in the combined enzyme is a protein part, the cofactor is a non-protein part, and the enzyme protein and the cofactor have catalytic activity only when the enzyme protein and the cofactor are combined into a whole enzyme. Enzymes are a very important class of biocatalysts (biochatalysts). Due to the action of enzymes, chemical reactions in organisms can be efficiently and specifically carried out under extremely mild conditions. Many enzymes can also be used as drugs, such as asparaginase.

In the context of the present specification, "membrane proteins" conform to the general definition in the field of biotechnology, i.e. refer to proteins comprised by biological membranes. Membrane proteins can be divided into three main groups: the extrinsic membrane proteins or peripheral membrane proteins, the intrinsic membrane proteins or integral membrane proteins, and the lipocalins. The membrane protein includes glycoprotein, carrier protein and enzyme. Usually, some saccharides are linked outside the membrane protein, and these saccharides are equivalent to transmitting signals into cells through the change of molecular structures of the saccharides themselves. The function of membrane proteins is multifaceted. Membrane proteins can act as "carriers" to transport substances into and out of cells. Some membrane proteins are specific receptors for hormones or other chemicals, such as receptors on thyroid cells that receive thyrotropin from the pituitary gland. Various enzymes are also present on the membrane surface, which allow specific chemical reactions to take place on the membrane, such as endoplasmic reticulum membrane, which catalyses the synthesis of phospholipids, etc. The recognition function of the cell is also determined by the proteins on the membrane surface. These proteins are often surface antigens. Surface antigens bind to specific antibodies, such as the human cell surface with a protein antigen HLA, a dimer that is very diverse. Membrane proteins play a very important role in many vital activities of an organism, such as proliferation and differentiation of cells, energy conversion, signal transduction, and material transport. It is estimated that about 60% of drug targets are membrane proteins.

In the context of the present specification, "polypeptide" may be used interchangeably with "polypeptide drug" and encompasses both endogenous and exogenous polypeptides; the former is intrinsic polypeptide of human body, such as enkephalin, thymosin, pancreatic polypeptide, etc.; the latter is selected from snake venom, sialic acid, bee venom, frog venom, scorpion venom, hirudin, derivatives of Amorphophallus rivieri and cecropin secreted by fly, which has antitumor, antiviral, and antibacterial activities.

In the context of the present specification, "antibody" is in accordance with the general definition in the field of biotechnology, i.e. antibody (antibody) refers to a protein with protective effects produced by the body as a result of stimulation by an antigen. It (immunoglobulins are not just antibodies) is a large Y-shaped protein secreted by plasma cells (effector B-cells), used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, etc., and found only in body fluids such as blood of vertebrates, and on the cell membrane surface of B-cells.

In the context of the present specification, "cytokine" is in accordance with the general definition in the field of biotechnology, i.e. a class of small molecule proteins with a wide range of biological activities, which are synthesized and secreted by immune cells (e.g. monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.). Cytokines generally modulate immune responses by binding to corresponding receptors to regulate cell growth, differentiation, and effects. Cytokines (CK) are low molecular weight soluble proteins that are produced by a variety of cells induced by immunogens, mitogens or other stimulants and have a variety of functions of regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells and damaged tissue repair. Specifically, it can be classified into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, and the like.

In the context of this specification, "complement" is defined generally in the biotechnology field, meaning a serum protein present in human and vertebrate serum and tissue fluids that is thermolabile, enzymatically active after activation, and mediates immune and inflammatory responses. It can be activated by antigen-antibody complexes or microorganisms, leading to lysis or phagocytosis by pathogenic microorganisms.

In still another embodiment, the fluorescent protein is any one or more selected from the group consisting of red fluorescent protein, green fluorescent protein, orange fluorescent protein, and yellow fluorescent protein.

In the context of the present specification, a "fluorescent protein" conforms to the general definition in the field of biotechnology and is a marker molecule often used in biological research. The earliest Green Fluorescent Protein (GFP) was found in the jellyfish of a academy name Aequorea victoria in 1962 by Nomura et al, and a second GFP was isolated from a marine coral. Wherein the jellyfish GFP is a monomer protein consisting of 238 amino acids, the molecular weight is about 27KD, GFP fluorescence is mainly generated in the presence of oxygen, the amide of glycine at the 67 th position in the molecule is subjected to nucleophilic attack on the carboxyl of serine at the 65 th position to form an imidazolyl group at the 5 th position, and after the alpha tyrosine bond of tyrosine at the 66 th position is subjected to dehydrogenation reaction, an aromatic group is combined with the imidazolyl group, so that a p-carboxybenzoic acid azolidone chromophore is formed in a GFP molecule to emit fluorescence. After the understanding of this principle, GFP has been widely used in biological studies, and various manufacturers such as Promega, Stratagene (including orange protein preparation technology from hong Kong university), Clontech (now Takara) and the like have produced corresponding products. In particular, green fluorescent protein GFP, the sequence of which is listed below, is used in a particular embodiment of the invention.

In a specific embodiment, the present invention constructs a pcI plasmid having a DNA portion sequence set forth in SEQ ID No.7 of the sequence Listing, which comprises, in order, a cI gene sequence, a pR operon sequence, a pL operon sequence, and a GFP gene sequence.

In another specific embodiment, the present invention constructs a p Δ cI plasmid having a DNA portion sequence as set forth in SEQ ID No.8 of the sequence listing, which in turn comprises a pR operon sequence, a pL operon sequence, and a GFP gene sequence.

The present invention in a second aspect relates to: the application of the plasmid in a cell-free protein synthesis system.

In a specific embodiment, there is provided the use of the above plasmid in a cell-free protein synthesis system for synthesizing a target protein, wherein a temperature sensitive element as a cofactor is further added to the cell-free protein synthesis system.

In a more specific embodiment, there is provided the use of the above plasmid for synthesizing a target protein in a cell-free protein synthesis system based on an E.coli extract, wherein a temperature sensitive element bacteriophage lambda repressing cI is further added as a cofactor in the cell-free protein synthesis system.

Here, the temperature-sensitive element is introduced by providing a culture containing a plasmid expressing the temperature-sensitive element.

The present invention in a third aspect relates to: various products based on the above applications.

In one embodiment, a liposomal drug delivery system is provided, comprising the plasmid described above; wherein the protein of interest is a pharmaceutical protein; cell-free extracts of prokaryotic or eukaryotic cells; and a temperature sensitive protein; and the drug delivery system needs to be stored at low temperature before being administered to a human body to prevent contamination.

The present invention in a fourth aspect relates to: a method for constructing a temperature-controlled cell-free reaction system.

In one embodiment, a method for constructing a temperature-controlled cell-free reaction system is provided, which comprises the following steps:

-providing a cell extract;

-mixing the cell extract with a plasmid comprising a gene encoding a temperature sensitive element and a manipulation unit capable of being regulated by the temperature sensitive element located downstream of the gene encoding the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell-free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the protein of interest in a reaction system at a given temperature.

The invention further provides another construction method of the temperature-controlled cell-free reaction system, which comprises the following steps:

-providing a cell extract mixture of a cell extract comprising a temperature sensitive element and a cell extract not comprising a temperature sensitive element;

-mixing the cell extract mixture with a plasmid comprising a manipulation unit capable of being manipulated by the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the protein of interest in a reaction system at a given temperature.

In a specific embodiment, the temperature sensitive element-containing Cell extract can be prepared by expressing a temperature sensitive element-containing plasmid in a Cell to an amount, and then preparing the Cell extract according to the method of Wen et al (A Cell-Free Biosensor for Detecting plasmid Sensing Molecules in P. Aeroginosa-induced reactivity samples. ACS Synth. biol.2017,6(12), 2293-2301.).

In another specific embodiment, the cell extract containing the temperature sensitive element may also be a cell extract containing no temperature sensitive element to which the temperature sensitive element is directly added.

The cell extract not containing a temperature sensitive element means a cell extract in which a plasmid containing a temperature sensitive element is not expressed in a cell and a temperature sensitive element is not added, and is also prepared according to the method of Wen et al.

In a preferred embodiment, the present invention constitutes a temperature-controlled cell-free reaction system by mixing an extract of E.coli BL21Star (DE3) expressing the cI repressor protein with an extract of cells of E.coli BL21Star (DE3) not containing the cI repressor protein in various proportions and adding the mixture to the cell-free system together with the plasmid p.DELTA.cI.

In a specific embodiment, the cell extract comprising a temperature sensitive element has a cI content of 7.2 ug/mL.

In a further preferred embodiment, the volume ratio of the cell extract comprising the temperature sensitive element to the cell extract not comprising the temperature sensitive element is 1:2-2:1, e.g. may be 1:2, 1:1.5, 1:1, 1.5:1, 2:1, preferably the volume fraction of the cell extract comprising the cI is 0.67, i.e. the volume ratio of the cell extract not comprising the temperature sensitive element is 2: 1.

In a specific embodiment, the cell is escherichia coli, archaebacteria, malt cells, yeast cells, rabbit reticulocytes, tobacco leaf cells, insect cells, or chinese hamster ovary cells.

In yet another embodiment, the cellular extract is a cellular extract from escherichia coli, archaebacteria, malt cells, yeast cells, rabbit reticulocyte, tobacco leaf cells, insect cells, or chinese hamster ovary cells.

In a further embodiment, said temperature sensitive element and said operator unit are used in combination, said temperature sensitive element and said operator unit being selected from any one of the bacteriophage lambda-repressing cI-pR-pL promoter, Tlpa protein-Tlpa promoter, TetR thermo-sensitive variant-tet operator of E.coli repressor protein, LacI (Ts) -lacO site + LacI promoter or E.coli heat shock protein-pHSP promoter, preferably said temperature sensitive element and said operator unit are selected from the bacteriophage lambda-repressing cI-pR-pL promoter.

In yet another embodiment, the energy source substance may be sucrose, maltose, glucose-6-phosphate, fructose-1, 6-diphosphate, phosphoglycerate, phosphocreatine, adenosine triphosphate, acetyl phosphate, glutamate, polyphosphate, and/or phosphoenolpyruvate.

In another embodiment, the amino acid mixture comprises glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, arginine, and histidine. And the transcription and translation auxiliary substance is selected from: one or more of potassium glutamate, ammonium glutamate, potassium oxalate, magnesium glutamate, oxidative glutathione, reductive glutathione, iodoacetamide, putrescine, spermidine, NAD, NADH, ATP, CTP, GTP, UTP, AMP, CMP, GMP, UMP, CoA, tRNA and folinic acid.

In one embodiment of the present invention, an oxidizing substance and a reducing substance are added to the temperature-controlled cell-free reaction system to make the temperature control effect better.

Preferably, the oxidized species is oxidized glutathione (GSSG) and the reduced species is reduced Glutathione (GSH).

Glutathione (GSH), the most abundant multifunctional antioxidant in the cell, has been demonstrated to be a natural scavenger of free radical detoxification and active electrophiles. In addition, glutathione disulfide (GSSG) is an endogenous peptide, the oxidized form of GSH. The two redox regulators were added to the pcI plasmid containing tcCFPS in different ratios to obtain different redox conditions.

In a preferred embodiment, the molar ratio of GSSG to GSH is from 2:1 to 4:1, for example, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4: 1.

In one embodiment, the given temperature is 37-40 deg.C, and may be, for example, 37 deg.C, 37.5 deg.C, 38 deg.C, 38.5 deg.C, 39 deg.C, 39.5 deg.C, 40 deg.C.

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1 plasmid construction

The phage lambda repressor cI is a well-known temperature-sensitive variant that acts on the tandem pR-pL operator-promoter, which effectively regulates the gene cloned downstream of the promoter. When the temperature is below 37 ℃ (usually 28-32 ℃), it will inhibit gene expression, while when the host RNA polymerase raises the temperature above 37 ℃, the mutated suppressor will be inactivated and transcribed, the regulatory mechanism is shown in fig. 2.

All plasmids used in the present invention were constructed according to standard molecular biology techniques.

In the present invention, pSB3K3 plasmid (Addgene #78636) was used as a backbone, and GFP (green fluorescent protein) was selected as a reporter protein (synthesized by GENEWIZ). The fluorescent reporter gene was cloned by Gibson assembly under the control of pL/pR promoter, and the expression repressor cI and the gene containing the pR-pL operational promoter and the temperature control loop for GFP fluorescent reporter protein were ligated to the plasmid backbone of pSB3K3 to give plasmid pSB3K3-cI-GFP (referred to herein as the pcI plasmid), as shown in FIG. 3A. A plasmid for knocking out the cI protein gene was constructed in the same manner, and designated pSB3K 3-GFP. DELTA.cI (hereinafter referred to as p. DELTA.cI) as a negative control, as shown in FIG. 3B.

Plasmid DNA was purified using the OMEGA plasmid Mini Kit and Qiagen plasmid Maxi Kit. Plasmid sequences were verified by the Tianyi biotechnology.

The sequence information of the promoter, RBS and part of the gene during plasmid preparation is shown in Table 1.

TABLE 1 promoter, RBS and partial Gene sequence information

The DNA partial sequence of pcI plasmid is shown in SEQ ID No.7 of the sequence table, and the sequence sequentially comprises a cI gene sequence, a pR operon sequence, a pL operon sequence and a GFP gene sequence.

The partial DNA sequence of the p.DELTA.cI plasmid is shown in SEQ ID No.8 of the sequence Listing, which comprises a pR operon sequence, a pL operon sequence and a GFP gene sequence in this order.

Example 2: expression of plasmids in cell systems

The pcI plasmid and the p.DELTA.cI plasmid were transferred into E.coli BL21 Star (DE3) and cultured at 30 ℃ and 37 ℃ for 12 hours, respectively. The OD600 and fluorescence intensity of the bacterial solution were then determined. As shown in FIG. 4, the GFP levels of the pcI plasmid were all higher than 30 ℃ at 37 ℃ to confirm that the constructed pcI plasmid can activate the expression of the target protein at the high temperatures expected in the cells.

Example 3: expression of plasmids in cell-free systems

The protein synthesis control capacity of tcCFPS was tested by adding pcI plasmid directly to the cell-free system and initiating the reaction at different temperatures.

Preparation of cell-free extract (BS) of Escherichia coli BL21 Star (DE3)

After overnight culture with E.coli, 200mL of 2 XYT-P medium was inoculated into a 1-L flask at a dilution of 1: 20. The culture was added to a 4l fermenter and incubated at 37 ℃ and 500rpm for 3.5 hours. The cell microspheres were washed twice with 100mL of ice-cold S30A buffer (14mM mg-glutamic acid, 60mM k-glutamic acid, 50mM Tris, pH 7.7) and then centrifuged at 8000 Xg 4 ℃ for 15 minutes. The cell microspheres were then resuspended in 30ml of frozen S30A buffer, transferred to a pre-weighed 50ml Falcon conical tube and centrifuged at 4 ℃ at 10000 Xg for 10 minutes. Finally, the tubes were weighed and flash frozen in nitrogen prior to storage at-80 ℃.

The next day, the frozen cell microspheres were thawed in ice and then resuspended in 1ml of S30A buffer per gram of cell microspheres. The cell suspension was disrupted by a high pressure disrupter for 2 times at 15000-. After centrifugation, the supernatant was carefully transferred to a new tube, and 3. mu.L of 1M DTT was added per 1ml of lysate. Shaking was carried out at 37 ℃ and 120 rpm for 80 minutes, and the remaining mRNA was digested with endogenous nuclease. The extract was centrifuged at 12000 Xg for 10 minutes at 4 ℃ and the supernatant was transferred to a 6-8kDa MWCO dialysis bag and dialyzed against 1L of S30B buffer (14mM Mg-glutamate,60mM Kglutamate,. about.5 mM Tris, pH 8.2) at 4 ℃ for 3 hours. Centrifuge at 12000 Xg for 10 min at 4 ℃. The supernatant was collected to give a cell-free extract of E.coli BL21 Star (DE 3).

Expression in cell-free systems

Adding the constructed plasmid into the cell-free extract, wherein the reporter protein is green fluorescent protein GFP, the addition amount of the plasmid is 300ng, and subpackaging the cell-free system to form tcCFPS (temperature control cell-free protein synthesis system).

The reaction was started at different temperatures, respectively. As shown in FIG. 5, the GFP levels of the pcI plasmids were higher than 30 ℃ at 37 ℃ and the constructed pcI plasmids were able to function as high-temperature activators for protein synthesis in the tcCFPS (temperature-controlled cell-free protein synthesis system) in BS extract.

Example 4: the influence of the redox characteristics of the cell-free protein synthesis system on the folding of the translated protein further verifies the temperature control expression effect of the pcI plasmid under different redox conditions.

Glutathione (GSH), the most abundant multifunctional antioxidant in the cell, has been demonstrated to be a natural scavenger of free radical detoxification and active electrophiles. In addition, glutathione disulfide (GSSG) is an endogenous peptide, the oxidized form of GSH. The two redox regulators were added to the pcI plasmid containing tcCFPS in different ratios to obtain different redox conditions. The reaction was started at different temperatures, respectively.

As shown in FIG. 6, the reaction system with excessive addition of GSH solution has no significant difference in GFP expression of pcI plasmid at both 30 ℃ and 37 ℃, while the temperature control effect of GFP protein expression is better when GSSG solution is added to tcCFPS in excess. When the molar ratio of GSSG to GSH is 2, 3, 4, i.e. the ratio is 2:1-4:1, the temperature control effect of GFP protein expression is more prominent, especially when the ratio is 3:1, the temperature control induced expression fold is the highest, and is 3.40. This is because different GSSG/GSH ratios change the redox potential of the cell free system, affecting the formation of protein disulfide bonds, while the partial oxidation (GSSG) conditions lead to the best temperature control in tcCFPS.

Example 5: temperature control effect on cell-free systems by expressing phage lambda repressor cI in cell extracts

This experiment was conducted using extracts containing the cI repressor protein to test for p.DELTA.cI, and E.coli BL21Star (DE3) extract expressing the cI (cI content 7.2ug/mL) repressor protein was mixed with the original cell extract not containing the cI repressor protein in various ratios and added to a cell-free system with a GSSG: GSH ratio of 3:1 together with the plasmid p.DELTA.cI, under the same conditions as in example 3. When mixed cell extracts of different volume fractions of the cI-BS cell extracts were observed, the expression levels and ratios of GFP mean fluorescence at 37 ℃ and 30 ℃ were observed, and the results are shown in FIG. 7, and when different amounts of cI-BS cell extracts were added, the temperature control effect was achieved in the cell-free system, indicating that exogenously expressed cI could impart p.DELTA.cI temperature control capability, especially the best temperature control effect when the volume fraction of the cI-containing extract was 0.67, at a cI protein concentration of 4.8 ug/mL.

Example 6: tcCFPS expression kinetics study

In order to further explore the mechanism of the increase of the transcription and protein expression level of the tcCFPS, a tcCFPS system based on the extract of Escherichia coli BL21Star (DE3), pcI plasmid and 3:1 GSSG: GSH ratio was used as an observation object, and the expression kinetics of the tcCFPS system were researched by combining the detection of mRNA level at different temperatures and different time points for 8 hours.

Transcription Process of the-tcCFPS System

And monitoring the transcription process of the tcCFPS system by adopting a real-time quantitative PCR detection system (qPCR). The mRNA level transcribed by the cI gene (referred to as cI-mRNA herein) was observed at different temperatures (30 ℃ C., 37 ℃ C.), and as a result, as shown in FIG. 8, under the conditions of 30 ℃ C. and 37 ℃ C., the mRNA level transcribed by the cI gene rapidly increased in the first 2 hours, then decreased, and finally reached a steady level, and the degradation rate of mRNA and the transcription rate of DNA reached equilibrium, whereas in the steady level stage, the cI-mRNA at 30 ℃ C. was significantly higher than 37 ℃ C., indicating that the amount of the cI gene that could exert the inhibitory effect at 37 ℃ C. was much lower than 30 ℃.

Meanwhile, the GFP gene transcription level (herein, referred to as GFP-mRNA) was observed, and as a result, as shown in FIG. 9, although the GFP gene transcription mRNA level (herein, referred to as GFP-mRNA) was slightly lower than 30 ℃ at 37 ℃ in the previous hour, the GFP-mRNA at 37 ℃ rapidly decreased to a lower level and reached equilibrium after one hour, indicating that the inhibition of the pR-pL-manipulated promoter by the cI protein resulted in a lower GFP-mRNA at 30 ℃ and that the expression of the cI protein decreased more slowly at 37 ℃ indicating that the cI protein was inactivated and that the RNA polymerase transcribed the GFP gene to have a higher transcription level at 37 ℃ and above.

Example 7: expression of plasmids in Artificial cells

The artificial cells imitating the structure and functional characteristics of living cells can be used for further researching the physical principle of life and promoting the development of cell engineering and biomedicine. The development of cell-free systems has greatly facilitated the development of artificial cells. The synthesis of proteins and enzymes in artificial cells has been successfully demonstrated. In the present invention, tcCFPS is compartmentalized into artificial cells that can respond to thermal signals. The artificial cell with the function of starting protein synthesis at high temperature can be used as a potential carrier for drug synthesis and delivery, and can allow a specific part to synthesize and release therapeutic protein when abnormal thermal reaction such as fever occurs in vivo.

Preparation of lipid solutions

A water-in-oil (w/o) emulsion transfer method was used to create an artificial cell chamber containing a cell-free temperature control system. Lipid solutions were used to prepare liposomes using a modified version of the protocol proposed by previous studies.

mu.L of POPC chloroform solution (100mg/mL) and 15. mu.L of cholesterol chloroform solution (100mg/mL) were dissolved in 450. mu.L of liquid paraffin and 1. mu.L of Liss Rhod (lissamine rhomadine B sulfonyl chloride) was added to make the lipids red. To evaporate the chloroform sufficiently, the solution was heated at 80 ℃ for 1 hour to obtain a lipid solution.

Preparing an external and an internal solution containing tcCFPS.

The inner solution consisted of cell-free reagent, 40 mg of glucose was added, prepared in a vial, and placed on ice to prevent the reaction from occurring. The outer solution is prepared by adopting the same components and concentration as the inner solution, no plasmid is added, and glucose with the same molar concentration is added into the outer solution. The concentrations of the inner and outer solutions should be kept consistent. If the concentration of the external solution is low, leakage of the internal small molecular weight components from the liposome to the outside may result.

Preparation of liposomes

100 μ L of lipid solution was added to 200 μ L of external solution (LS-OS) and incubated on ice for 30 minutes to form an oil-water interface. mu.L of the inner solution was added to 150. mu.L of lipid solution (LS-IS), rotated for 1 minute, and then incubated on ice for 20 minutes. The LS-IS solution was slowly added to the LS-OS solution and centrifuged at 4000g for 15 minutes at 4 ℃. The lower liposome suspension was transferred to another 1.5ml LEppendorf tube, mixed with 200. mu.L of fresh external solution, and centrifuged at 4000g at 4 ℃ for 10 minutes. The supernatant was removed and the liposome particles were suspended in 20-50. mu.L of external liquid. Liposomes encapsulating the previously constructed tcCFPS system solution were obtained as shown in figure 10.

Example 8

The effect of protein production was examined by flow cytometry (BD LSRFortessa SORP) analysis of reactions in liposomes encapsulating previously constructed solutions of the tcCFPS system at 30 ℃ and 37 ℃. In 19 cases of flow cytometry, a single fluorescent signal of GFP was detected. A total of 60000 data samples were obtained for the measurements. GFP was excited at 488nm and the emission was detected with a 550 + -30 nm band pass filter. From the flow cytometry results, it was shown that the tcCFPS encapsulated liposomes were responsive to temperature control as shown in fig. 11, the liposomes reacted at 37 ℃ to produce more fluorescent protein than at 30 ℃ and the percentage of the liposome population in the high fluorescence band was higher.

Example 9

The liposomes after the reaction described above were observed by confocal microscopy (Zeiss LSM 710). Prior to measurement, the vesicles were diluted to the appropriate concentration with dilution buffer (100mM HEPES-KOH pH 7.6,24mM magnesium acetate, 280mM potassium glutamate, 1.5mM DTT,200mM glucose). An x 63 objective lens for microscopic observation. Fluorescence images of GFP were obtained through corresponding filters and showed that vesicles were typically between 5-20 μm in size, capturing some clear fluorescent vesicles at 30 ℃ and 37 ℃, as shown in figure 12. FIGS. 12A and 12B show the fluorescence emitted from the light-emitting part of the Liss Rhod stained on the liposome membrane, and it can be seen that the liposome morphology was normal at both 30 ℃ and 37 ℃, and FIGS. 12C and 12D also include a GFP light-emitting part, and the fluorescence emitted from GFP observed at an excitation wavelength of 488nm showed that the expression level of GFP in the liposome was very high at 37 ℃ and the expression effect of the target protein was very good, simulating an artificial cell expressing strong fluorescence.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the specific embodiments and applications described above, which are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Qinghua university

<120> construction method of temperature-controlled cell-free reaction system, plasmid used in the method and use

<130> PE01969

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 1

cctatagtga gtcgtatta 19

<210> 2

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 2

gctagcataa taccaaggac tgagctagct gtaaa 35

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 3

ttgactattt tacctctggc ggtgataa 28

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> description of human procedures: artificially synthesized sequences

<400> 4

ttgacataaa taccactggc ggtgatact 29

<210> 5

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 5

tcacacagga aag 13

<210> 6

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 6

atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg tcgccttaaa 60

gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt cgcagacaag 120

atggggatgg ggcagtcagg cgttggtgcc ttatttaatg gcatcaatgc attaaatgct 180

tataacgccg catcgcttac aagaattctc aaagttagcg ttgaagaatt tagcccttca 240

atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt 300

gagtatgagt accctgtttt ttctcatgtt caggcaggga tgctctcacc tgagcttaga 360

acctttacca aaggtggtgc ggaaaggtgg gtaagcacaa ccaaaaaagc cagtgattct 420

gcattctggc ttgaggttga aggtaattcc atgacagcac caacaggctc caagccaagc 480

tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc aggtgatttc 540

tgcatagcca gactcggggg tggtgagttt accttcaaga aactgatcag ggatagcggt 600

caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa tgagagttgt 660

tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg ctga 714

<210> 7

<211> 5033

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequences

<400> 7

gaattcgcgg ccgcttctag agtttacagc tagctcagtc cttggtatta tgctagctcc 60

tagggtcaca caggaaagta ccatatgagc acaaaaaaga aaccattaac acaagagcag 120

cttgaggacg cacgtcgcct taaagcaatt tatgaaaaaa agaaaaatga acttggctta 180

tcccaggaat ctgtcgcaga caagatgggg atggggcagt caggcgttgg tgccttattt 240

aatggcatca atgcattaaa tgcttataac gccgcatcgc ttacaagaat tctcaaagtt 300

agcgttgaag aatttagccc ttcaatcgcc agagaaatct acgagatgta tgaagcggtt 360

agtatgcagc cgtcacttag aagtgagtat gagtaccctg ttttttctca tgttcaggca 420

gggatgctct cacctgagct tagaaccttt accaaaggtg gtgcggaaag gtgggtaagc 480

acaaccaaaa aagccagtga ttctgcattc tggcttgagg ttgaaggtaa ttccatgaca 540

gcaccaacag gctccaagcc aagctttcct gacggaatgt taattctcgt tgaccctgag 600

caggctgttg agccaggtga tttctgcata gccagactcg ggggtggtga gtttaccttc 660

aagaaactga tcagggatag cggtcaggtg tttttacaac cactaaaccc acagtaccca 720

atgatcccat gcaatgagag ttgttccgtt gtggggaaag ttatcgctag tcagtggcct 780

gaagagacgt ttggctgaaa atttagctaa acacacatga attttcagat gtgttttatc 840

cgggtactag agccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 900

gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg 960

gtgggccttt ctgcgtttat atactagaga tgccggccac gatgcgtccg gcgtagagga 1020

tcgagtatca ccgcaaggga taaatatcta acaccgtgcg tgttgactat tttacctctg 1080

gcggtgataa tggttgcatg tactaaggag gttgtatgga acaacgcata accctgaaag 1140

attatgcaat gcgctttggg caaaccaaga cagctaaaag atctctcacc taccaaacaa 1200

tgcccccctg caaaaaataa attcatataa aaaacataca gataaccatc tgcggtgata 1260

aattatctct ggcggtgttg acataaatac cactggcggt gatactgagc acatcagcag 1320

gacgcactga ccaccatgaa ggtgacgctc ttaaaaatta agccctgaag aagggcagca 1380

ttcaaagcag aaggctttgg ggtgtgtgat acgaaacgaa gcattggtta aaaattaagg 1440

agggaattat gcgtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat 1500

tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa 1560

catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc 1620

caacacttgt cactactttc ggttatggtg ttcaatgctt tgcgagatac ccagatcata 1680

tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta 1740

tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata 1800

cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg 1860

gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa 1920

agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac 1980

tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca 2040

accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca 2100

tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca 2160

aataataata ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 2220

tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct 2280

tcgggtgggc ctttctgcgt ttatatacta gtagcggccg ctgcagtccg gcaaaaaaac 2340

gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac ttcgcgttat 2400

gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2460

agctcactca aaggcggtaa tctcgagtcc cgtcaagtca gcgtaatgct ctgccagtgt 2520

tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat 2580

ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 2640

gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 2700

actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 2760

gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct 2820

ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 2880

aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 2940

ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 3000

atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 3060

gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 3120

ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 3180

ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 3240

attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 3300

tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata 3360

acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt 3420

ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat 3480

cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc 3540

gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac 3600

cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca 3660

acaccttctt cacgaggcag acctcagcgc tagcggagtg tatactggct tactatgttg 3720

gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt 3780

gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg 3840

ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga 3900

agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat 3960

aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac 4020

ccgacaggac tataaagata ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg 4080

ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca 4140

cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc 4200

cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa 4260

agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt 4320

gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc 4380

aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg 4440

caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga 4500

agatcatctt attaaggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4560

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 4620

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 4680

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 4740

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 4800

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 4860

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctattc catggtgcca 4920

cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 4980

aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt ctg 5033

<210> 8

<211> 3993

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized sequence

<400> 8

gaattcgcgg ccgcttctag agttgactat tttacctctg gcggtgataa tggttgcatg 60

tactaaggag gttgtatgga acaacgcata accctgaaag attatgcaat gcgctttggg 120

caaaccaaga cagctaaaag atctctcacc taccaaacaa tgcccccctg caaaaaataa 180

attcatataa aaaacataca gataaccatc tgcggtgata aattatctct ggcggtgttg 240

acataaatac cactggcggt gatactgagc acatcagcag gacgcactga ccaccatgaa 300

ggtgacgctc ttaaaaatta agccctgaag aagggcagca ttcaaagcag aaggctttgg 360

ggtgtgtgat acgaaacgaa gcattggtta aaaattaagg agggaattat gcgtaaagga 420

gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg 480

cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 540

aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc 600

ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc 660

aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg 720

aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag 780

ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac 840

tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac 900

ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa 960

aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa 1020

tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta 1080

acagctgctg ggattacaca tggcatggat gaactataca aataataata ctagagccag 1140

gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 1200

gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt 1260

ttatatacta gtagcggccg ctgcagtccg gcaaaaaaac gggcaaggtg tcaccaccct 1320

gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc tcgctcactg 1380

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 1440

tctcgagtcc cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat taaccaattc 1500

tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc 1560

aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 1620

ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca 1680

acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 1740

gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg 1800

ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga 1860

ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat 1920

cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg 1980

atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc 2040

atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca 2100

gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag 2160

aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc 2220

gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg 2280

cggcctcgag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt 2340

tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca 2400

tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa 2460

ggatcagatc acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa 2520

aatcaccaac tggtccacct acaacaaagc tctcatcaac cgtggctccc tcactttctg 2580

gctggatgat ggggcgattc aggcctggta tgagtcagca acaccttctt cacgaggcag 2640

acctcagcgc tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt 2700

gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat 2760

acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc 2820

gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta 2880

acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa 2940

gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata 3000

ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc 3060

ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg 3120

gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg 3180

cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact 3240

ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag 3300

gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca 3360

aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt 3420

cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaaggggt 3480

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3540

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3600

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3660

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3720

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3780

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3840

caactttatc cgcctccatc cagtctattc catggtgcca cctgacgtct aagaaaccat 3900

tattatcatg acattaacct ataaaaatag gcgtatcacg aggcagaatt tcagataaaa 3960

aaaatcctta gctttcgcta aggatgattt ctg 3993

Claims (10)

1. A plasmid, comprising:

a gene encoding a temperature sensitive element;

a manipulation unit that is located downstream of the gene encoding a temperature sensitive element and is capable of being regulated by the temperature sensitive element;

a foreign gene expressing a protein of interest located downstream of the manipulation unit.

2. The plasmid according to claim 1, wherein the manipulation unit that can be regulated by the temperature sensitive element means that when the temperature is increased to a given temperature, the temperature sensitive element is inactivated so that the manipulation unit initiates expression of the target protein; and the temperature sensitive element inhibits the manipulation unit from initiating the expression of the target protein when the temperature is lower than a given temperature, preferably 37-40 ℃.

3. The plasmid according to claim 1 or 2, wherein the temperature-sensitive element and the operator are used in combination, and the temperature-sensitive element and the operator are selected from any one of bacteriophage lambda-repressed cI-pR-pL promoter, TlpA protein-TlpA promoter, TetR thermo-sensitive variant-tet operator of E.coli repressor protein, LacI (Ts) -lacO site + LacI promoter of E.coli repressor protein, or E.coli heat shock protein-pHSP promoter, preferably the temperature-sensitive element and the operator are selected from bacteriophage lambda-repressed cI-pR-pL promoter.

4. The plasmid according to any one of claims 1 to 3, wherein the initial plasmid used for constructing the plasmid is selected from any one of the pSB3K3 plasmid, pET series plasmid, pGEX series plasmid, pMAL series plasmid, pQE-1 plasmid, pQE-60 plasmid, pGS-21a plasmid, pCDFDuet-1 plasmid, pRSFDuet-1 plasmid or pBluescriptII series plasmid, preferably pSB3K3 plasmid.

5. The plasmid according to any one of claims 1 to 4, wherein the target protein is selected from any one or more of a fluorescent protein, a vaccine protein, an antibody protein, a biocatalytic enzyme, a membrane protein, a polypeptide, a cytokine protein, a hormone protein, and a complement protein.

6. The plasmid according to claim 5, wherein the fluorescent protein is any one or more selected from the group consisting of a red fluorescent protein, a green fluorescent protein, an orange fluorescent protein and a yellow fluorescent protein.

7. Use of a plasmid according to any one of claims 1-6 in a cell-free reaction system for the synthesis of a protein of interest.

8. A liposomal drug delivery system comprising:

the plasmid of any one of claims 1-4, wherein the protein of interest is a pharmaceutical protein;

Cell-free extracts of prokaryotic or eukaryotic cells; and temperature sensitive proteins.

9. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract;

-mixing the cell extract with a plasmid comprising a gene encoding a temperature sensitive element and a manipulation unit capable of being regulated by the temperature sensitive element located downstream of the gene encoding the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell-free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the protein of interest in a reaction system at a given temperature.

10. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract mixture of a cell extract comprising a temperature sensitive element and a cell extract not comprising a temperature sensitive element;

-mixing the cell extract mixture with a plasmid comprising a manipulation unit capable of being manipulated by the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature-controlled cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature controlled cell-free reaction system;

-expressing the target protein in a reaction system at a given temperature.

Images