CN110229849B

CN110229849B - Time-air-conditioning control type uPA gene expression non-viral vector and preparation method and application thereof

Info

Publication number: CN110229849B
Application number: CN201910468269.5A
Authority: CN
Inventors: 谌平; 陈国创; 赵静; 张炳照
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2021-09-17
Anticipated expiration: 2039-05-31
Also published as: CN110229849A

Abstract

The invention provides a time-space regulation type uPA gene expression non-viral vector, wherein the time-space regulation type uPA gene expression non-viral vector comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box comprises: the gene comprises a liver-specific promoter, a uPA mutant gene and an unstable structural domain, wherein the uPA mutant gene is a uPA gene inserted with an endoplasmic reticulum retention signal gene. The regulation system of the space-time regulation type uPA gene expression non-viral vector is tighter and simpler; can control the uPA gene to be expressed in a specific space at a specific time, so that other organs are prevented from being subjected to toxicity risk.

Description

Time-air-conditioning control type uPA gene expression non-viral vector and preparation method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a space-time regulation type uPA gene expression non-viral vector and a preparation method and application thereof.

Background

Hepatitis B Virus (HBV) infection has strict species specificity and only infects livers of a few primates, such as humans, gorillas, tree shrews and the like. The common experimental animal models such as mice, rats and the like are not infected with HBV. Due to the lack of stable and reliable HBV infection animal models, the research on HBV infection/pathogenic mechanism and antiviral strategy is restricted. Establishing a humanized chimera liver mouse model is an effective way to overcome the bottleneck. The liver has a strong regenerative potential. The basic principle of the humanized chimera liver model is as follows: by expressing hepatotoxic genes in the liver of a host, death of host hepatocytes is artificially induced, proliferation of transplanted human hepatocytes is stimulated, and the human hepatocytes are replaced, so that a humanized chimeric liver is formed. In fact, the key core of the humanized chimeric liver is to design a suitable hepatotoxic gene expression vector. Based on the difference of hepatotoxicity genes, 3 human-mouse chimera liver models are mainly developed internationally. The first is albumin-uPA (urokinase plasminogen activator) -SCID (severe combined immunodeficiency) transgenic mouse, abbreviated uPA transgenic mouse. The second is FRG mouse, i.e., Fah (fumarylacetoacetate hydrolase)/Rag 2/Il2rg triple gene knock-out mouse. The third is AFC8 mouse, FKBP (FK506 binding domain), FK506 binding domain Caspase 8 (Caspase-8) fusion protein transgenic mouse. Of the three, uPA transgenic mice have high levels of human hepatocyte replacement (up to over 90%) that are difficult for the other two to reach.

uPA, a urokinase-type plasminogen activator, belongs to the family of serine proteolytic enzymes, catalyzes the conversion of plasminogen to plasmin, initiating the fibrinolysis and extracellular matrix proteolytic cascade. Generally speaking, uPA is mainly involved in the processes of cell differentiation, migration, tissue reconstruction, extracellular matrix degradation, tumor infiltration and metastasis under physiological and pathological conditions. In the existing construction of uPA transgenic mice, because uncontrolled over-expression of uPA can cause severe bleeding and organ damage, the death rate of newborn mice is high, and the breeding is difficult. The uPA can cause hepatocyte death and promote liver regeneration, and the uPA gene is still considered as the most ideal hepatotoxic gene for constructing a chimera liver model on the premise that the toxic and side effects can be effectively controlled.

At present, an improved method aiming at a uPA gene regulation and control expression system is a Tet-on tetracycline switch regulation and control uPA expression system based on AAV viral vectors. The Tet-on-uPA comprises two parts: constitutively expressed or liver-specifically expressed rtTA (reverse tetracycline-controlled trans-activator, tetracycline-controlled trans-transcriptional activator) and uPA expression vector (TRE-uPA) under the control of tetracycline-responsive element (TRE). The Tet-on-uPA can effectively control the expression of uPA in a characteristic time through tetracycline, and is superior to the original Alb-uPA.

The Tet-on regulation system consists of two parts and is relatively complex. Therefore, when the method is used for constructing the Tet transgenic animal, two transgenic animal strains of rtTA and TRE-uPA are firstly constructed and then hybridized, so that the method cannot be completed in one step, and the whole process is very complex, time-consuming and labor-consuming. In addition, although the expression of uPA is temporally regulated by tetracycline, uPA is a secretory protein, which is secreted to the outside of cells after being expressed in hepatocytes and then enters the circulatory system to be transported to other organs of the body, the spatial distribution of uPA is not controlled, and the risk of causing toxicity of other organs is still remained. Meanwhile, AAV virus vectors used by Tet-on-uPA in the prior art are complex to prepare and inconvenient to use.

Disclosure of Invention

The invention aims to provide a space-time regulation type uPA gene expression non-viral vector, and aims to solve the problem that the expression system of the uPA gene in the prior art cannot simultaneously regulate and control the induction time and space of the uPA gene expression system.

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

a time-space regulation type uPA gene expression non-viral vector comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box comprises: the gene comprises a liver-specific promoter, a uPA mutant gene and an unstable structural domain, wherein the uPA mutant gene is a uPA gene inserted with an endoplasmic reticulum retention signal gene;

and a method for preparing a space-time regulation type uPA gene expression non-viral vector, which comprises the following steps:

synthesizing DNA fragments of a liver-specific promoter, a uPA mutant gene and an unstable structural domain;

inserting the DNA fragment into a multiple cloning site of a vector to obtain the time-air-conditioning control type uPA gene expression non-viral vector, wherein the time-air-conditioning control type uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, and the time-space control uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted.

And an application of the space-time regulation type uPA gene expression non-viral vector in preparing a humanized chimera liver model.

Compared with the prior art, the time-space regulation type uPA gene expression non-viral vector comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain. The non-viral vector for expressing the air-conditioning control type uPA gene contains a liver specific promoter, so that the downstream gene can be controlled to perform specific expression in liver cells during uPA gene expression; the uPA mutant gene is inserted, and the uPA mutant gene is the uPA gene inserted with the endoplasmic reticulum retention signal gene, so that the distribution of the uPA gene can be strictly controlled, and the position for regulating and controlling the distribution of the uPA gene is limited in liver cells, so that the distribution space position of the uPA mutant gene is strictly regulated and controlled; a labile domain is inserted, when trimethoprim or trimethoprim derivatives are not added, the fusion protein of the labile domain and the uPA mutant gene is unstable and can be rapidly degraded in liver cells; when the trimethoprim or trimethoprim derivatives are used for carrying out induction expression on the time-space regulation uPA gene expression box, the fusion protein of an unstable structural domain and a uPA mutant gene can be stabilized and accumulated in liver cells, so that the uPA gene can be stably regulated, the time for stably expressing the fusion protein of the unstable structural domain and the uPA mutant gene is controlled by controlling the time for adding an inducer trimethoprim or trimethoprim derivatives, so that the fusion protein is induced and expressed in a specific time, the expressed uPA mutant protein is strictly limited in the liver cells, cannot be secreted to the outside of cells and cannot enter a circulatory system to influence other organs of the whole body, and the regulation system of the time-space regulation type uPA gene expression non-viral vector is tighter and simpler; can control the uPA gene to be expressed in a specific space at a specific time, so that other organs are prevented from being subjected to toxicity risk.

The preparation method of the space-time regulation type uPA gene expression non-viral vector is a non-viral vector, is simpler and more convenient to operate and use, and is simple, convenient and safe to operate, convenient to use, high in success rate and wide in applicability.

The time-space regulation type uPA gene expression non-viral vector is simple, convenient and safe in preparation method, simple and convenient in operation and use, high in success rate and wide in applicability in the application process of preparing the humanized chimera liver model.

Drawings

Figure 1 is a pmc. besxp map provided by an embodiment of the present invention.

FIG. 2 is a flow chart of the time-controlled air-conditioning type uPA gene expression non-viral vector prepared by the seamless cloning method according to the embodiment of the present invention.

FIG. 3 is a map of a mini-circle DNA mother plasmid (pMC. ApoE-muPA-DD) for expressing the time-controlled uPA gene provided by the embodiment of the invention.

FIG. 4 is a spatiotemporal regulatory uPA gene expression cassette including a liver-specific promoter, a uPA mutant gene and an unstable domain, provided in an embodiment of the invention.

FIG. 5 is a spatiotemporal regulation uPA gene expression cassette including liver-specific promoter, uPA mutant gene and unstable domain, polyA element provided by the embodiment of the present invention.

FIG. 6 is a temporal and spatial regulatory uPA gene expression non-viral micro-ring DNA (ApoE-muPA-DD MC) map provided by the embodiment of the invention.

FIG. 7 is a schematic diagram of a DD-GFP expression vector and a schematic diagram of a GFP-DD expression vector provided in the examples of the present invention.

FIG. 8 shows the results of expression control of a DD-GFP expression vector and a GFP-DD expression vector provided in the examples of the present invention.

FIG. 9 shows the relationship between the induced concentration of trimethoprim and the expression level of a target protein, which is provided in the examples of the present invention.

FIG. 10 is a graph showing the results of Western Blot detection of ApoE-muPA-DD vector expression in hepatocytes, according to the present invention.

FIG. 11 shows the result of the lysis of the ApoE-muPA-DD transfected liver cell line on fibrinogen agar plate after induction with TMP.

Detailed Description

In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the present invention, the "domain" refers to a region having a specific structure and an independent function in a biological macromolecule, and particularly refers to such a region in a protein. A domain is a structural hierarchy between secondary and tertiary structures, and is the basic structural unit of the tertiary structure of a protein, as well as a functional unit of a protein. In multi-domain proteins, different domains are often associated with different functions.

In the present invention, the "unstable Domain" refers to Destabilizing Domain, which is a small-sized protein Domain of full human origin, which can ensure the stability of fusion payload protein, can be easily added to cells or gene therapy products to turn on or off related functions by conformational change, and is a Destabilizing Domain (DD) technology developed by Obsidian therapeutics and Celgene, usa in cooperation, and can be used to control the expression of two immune regulators, IL12 and CD 40L.

In the invention, the 'uPA', namely urokinase-type plasminogen activator, belongs to a serine proteolytic enzyme family, and the uPA can catalyze plasminogen to be converted into plasmin so as to start a fibrinolysis cascade reaction and an extracellular matrix protein lysis reaction.

In the present invention, the "signal for retention in the endoplasmic reticulum" refers to a tetrapeptide sequence at the carboxy terminus of the structural and functional proteins of the endoplasmic reticulum: Lys-Asp-Glu-Leu-COO-, i.e., the KDEL signal sequence. This sequence has corresponding receptors on the membrane of the Golgi apparatus, and once it enters the Golgi apparatus it is bound by the receptors on the Golgi apparatus to form reflux vesicles which are transported back to the endoplasmic reticulum, so the sequence is called the endoplasmic reticulum retention signal.

In the present invention, the "promoter" refers to a nucleic acid sequence which is usually present upstream (5' -end) of a target nucleic acid sequence and is capable of activating RNA polymerase so that it can bind to a template DNA precisely and has specificity for transcription initiation.

In the present invention, the "liver-specific promoter" means that the promoter is capable of selectively activating transcription in stem cells or in cell lines derived from stem cells. In the present invention, liver-specific promoters refer to those promoters that are generally more active in the liver than in any other body tissue. In general, liver-specific promoters are much more active in the liver than other tissues. Thus, liver-specific promoters allow the active expression of a gene in that liver and prevent the expression of a gene in other cells or tissues. The purpose of inserting the "liver-specific promoter" is to control the specific expression of a downstream gene of interest in hepatocytes.

In the present invention, the "vector" refers to a structure into which a foreign DNA can be inserted and which can replicate in a recipient cell in genetic engineering studies.

In the present invention, the "multiple cloning site" refers to an artificially synthesized DNA fragment contained in a vector, which contains a plurality of single cleavage sites and is an insertion site of foreign DNA.

The embodiment of the invention provides a time-space regulation type uPA gene expression non-viral vector, wherein the time-space regulation type uPA gene expression non-viral vector comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box comprises: the gene comprises a liver-specific promoter, a uPA mutant gene and an unstable structural domain, wherein the uPA mutant gene is a uPA gene inserted with an endoplasmic reticulum retention signal gene.

Specifically, the time-air-conditioning control type uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, wherein the time-space control uPA gene expression box is a base sequence inserted into a multi-cloning site of a vector and is used for expressing uPA genes in cells. Specifically, the space-time regulation uPA gene expression cassette comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain.

An unstable structural domain is inserted into the space-time regulation uPA gene expression box, the fusion protein of the unstable structural domain and the uPA mutant gene is unstable, and the fusion protein can be rapidly degraded in liver cells under the condition that no specific small molecular ligand exists; the time for stably expressing the fusion protein of the unstable structure domain and the uPA mutant gene can be controlled by controlling the time for adding the inducer trimethoprim or trimethoprim derivatives, so that the fusion protein can be induced and expressed within specific time, and the uPA gene expression can be regulated and controlled by the non-viral vector for expressing the uPA gene in time.

Preferably, the unstable domain is linked to a liver-specific promoter and a uPA mutant gene, in a preferred embodiment of the invention, the unstable domain can be linked to the C-terminal or N-terminal of the uPA mutant gene, the unstable domain is linked to the uPA mutant gene to form a fusion protein, and the formed fusion protein is unstable and can be rapidly degraded in cells in the absence of a specific small molecule ligand trimethoprim or a trimethoprim derivative; if the specific small molecular ligand trimethoprim or trimethoprim derivative is added for induction expression, the fusion protein formed by connecting the unstable structural domain and the uPA mutant gene is more stable and can be accumulated in cells. In the specific embodiment of the invention, the unstable domain is fused at the C-terminal of the uPA mutant gene, which can strictly limit the retention of the target protein in hepatocytes, and the target protein cannot be transported to the outside of cells, and further cannot enter the circulatory system to affect other organs of the whole body.

In a specific embodiment of the present invention, the base Sequence of the unstable domain is as follows (Sequence No.1 in the Sequence listing):

5’-ATGATCTCTCTGATTGCCGCTCTGGCCGTGGACTACGTGATCGGGATGGAAAACGCTATGCCATGGAATCTGCCCGCCGATCTGGCTTGGTTCAAGAGGAACACCCTGAACAAGCCAGTGATCATGGGCAGACACACTTGGGAGTCCATTGGCCGGCCCCTGCCTGGACGCAAGAACATCATTCTGAGCTCCCAGCCCTCTACCGACGACAGGGTGACATGGGTGAAAAGTGTGGACGAAGCCATTGCCGCTTGCGGAGATGTGCCCGAGATCATGGTCATCGGCGGAGGGAGAGTGATCGAGCAGTTCCTGCCTAAGGCCCAGAAACTGTACCTGACTCACATTGACGCTGAGGTGGAAGGGGACACCCATTTTCCTGATTATGAGCCAGACGATTGGGAAAGCGTGTTCTCCGAGTTTCACGACGCCGATGCTCAGAATTCTCATAGTTATTGCTTTGAGATCCTGGAAAGGAGA-3’。

specifically, the space-time regulation uPA gene expression cassette further comprises a uPA mutant gene, wherein the uPA mutant gene is the uPA gene inserted with an endoplasmic reticulum retention signal gene.

Preferably, the ER retention signal gene may be added to at least one of the C-terminus and the N-terminus of the uPA gene, and the ER retention signal gene may be selected from any one of the C-terminus, the N-terminus, and the C-terminus and the N-terminus of the uPA gene. Specifically, the endoplasmic reticulum retention signal gene is inserted into the uPA gene, so that the distribution of the uPA gene can be strictly controlled, and the position for regulating and controlling the distribution of the uPA gene is only limited in liver cells, so that the distribution space position of the uPA mutant gene is strictly regulated and controlled.

In a specific embodiment of the invention, the endoplasmic reticulum retention signal gene comprises a C-terminal endoplasmic reticulum retention signal gene and an N-terminal endoplasmic reticulum retention signal gene;

specifically, the amino acid Sequence of the C-terminal endoplasmic reticulum retention signal is as follows (Sequence No.2 in the Sequence listing), and the auxiliary amino acid Sequence containing a signal promoting the binding of KDEL to a KDEL receptor located on the Golgi is the "EEDTSE" and "KDEL" signal sequences (underlined):

EEDTSEKDEL。

the C-terminal endoplasmic reticulum retention signal base Sequence is as follows (as Sequence No.3 in the Sequence table):

5’-GAAGAAGATACCTCTGAAAAAGATGAGCTC-3’。

specifically, the amino acid Sequence of the N-terminal retention signal is as follows (Sequence No.4 in the Sequence table): wherein the RR (Arg-Arg) retention signal is "MHRRRSRSCREDQKP" (amino acids 1-15); the lip33 spacer peptide is "VIDDQRDLISNNEQLPMLGRRPGAPESKCSR" (amino acids 16-46); the transmembrane sequence is "GALYTGFSILVTLLLAGQATTAYFL" (amino acids 47-71):

MHRRRSRSCREDQKPVIDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFL。

the base Sequence of the N-terminal retention signal is as follows (as Sequence No.5 in the Sequence table): 5'-ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTG-3' are provided.

In the specific embodiment of the invention, the uPA mutant gene is a uPA gene with a C-terminal endoplasmic reticulum retention signal gene added at the C-terminal and an N-terminal endoplasmic reticulum retention signal gene added at the N-terminal, and the base sequences thereof are as follows (as Sequence No.6 in the Sequence table):

5’-ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGTTCCATCGAACTGTGACTGTCTAAATGGAGGAACATGTGTGTCCAACAAGTACTTCTCCAACATTCACTGGTGCAACTGCCCAAAGAAATTCGGAGGGCAGCACTGTGAAATAGATAAGTCAAAAACCTGCTATGAGGGGAATGGTCACTTTTACCGAGGAAAGGCCAGCACTGACACCATGGGCCGGCCCTGCCTGCCCTGGAACTCTGCCACTGTCCTTCAGCAAACGTACCATGCCCACAGATCTGATGCTCTTCAGCTGGGCCTGGGGAAACATAATTACTGCAGGAACCCAGACAACCGGAGGCGACCCTGGTGCTATGTGCAGGTGGGCCTAAAGCCGCTTGTCCAAGAGTGCATGGTGCATGACTGCGCAGATGGAAAAAAGCCCTCCTCTCCTCCAGAAGAATTAAAATTTCAGTGTGGCCAAAAGACTCTGAGGCCCCGCTTTAAGATTATTGGGGGAGAATTCACCACCATCGAGAACCAGCCCTGGTTTGCGGCCATCTACAGGAGGCACCGGGGGGGCTCTGTCACCTACGTGTGTGGAGGCAGCCTCATCAGCCCTTGCTGGGTGATCAGCGCCACACACTGCTTCATTGATTACCCAAAGAAGGAGGACTACATCGTCTACCTGGGTCGCTCAAGGCTTAACTCCAACACGCAAGGGGAGATGAAGTTTGAGGTGGAAAACCTCATCCTACACAAGGACTACAGCGCTGACACGCTTGCTCACCACAATGACATTGCCTTGCTGAAGATCCGTTCCAAGGAGGGCAGGTGTGCGCAGCCATCCCGGACTATACAGACCATCTGCCTGCCCTCGATGTATAACGATCCCCAGTTTGGCACAAGCTGTGAGATCACTGGCTTTGGAAAAGAGAATTCTACCGACTATCTCTATCCGGAGCAGCTGAAAATGACTGTTGTGAAGCTGATTTCCCACCGGGAGTGTCAGCAGCCCCACTACTACGGCTCTGAAGTCACCACCAAAATGCTGTGTGCTGCTGACCCACAGTGGAAAACAGATTCCTGCCAGGGAGACTCAGGGGGACCCCTCGTCTGTTCCCTCCAAGGCCGCATGACTTTGACTGGAATTGTGAGCTGGGGCCGTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAGTCACACCAAGGAAGAGAATGGCCTGGCCCTCGAAGAAGATACCTCTGAAAAAGATGAGCTC-3’。

specifically, the space-time regulation uPA gene expression cassette comprises a liver-specific promoter.

Preferably, the liver-specific promoter is selected from the group consisting of apolipoprotein E promoter, albumin promoter, phosphoenolpyruvate carboxykinase promoter, alpha-I-antitrypsin promoter, thyroid hormone-binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, IGF-II promoter, factor VIII promoter, HBV basic core protein promoter, HBV pre-s 2 protein promoter, thyroxine-binding globulin promoter, HCR-Ap0CII hybrid promoter, HCR-hAAT hybrid promoter, AAT promoter in combination with enhancer elements of mouse albumin gene, low density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyltransferase promoter, apolipoprotein H promoter, transferrin promoter, thyroxine-carrying protein promoter, and the like, Any one of promoters of alpha-fibrinogen and beta-fibrinogen, alpha-I-antichymotrypsin promoter, alpha-2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoter, complement C3 activator promoter, hemopexin promoter and alpha-I-acidic glycoprotein promoter.

In the specific embodiment of the invention, the liver-specific promoter is apolipoprotein E promoter, and the apolipoprotein E promoter is selected mainly for controlling the downstream target gene to perform specific expression in liver cells. The base Sequence of the apolipoprotein E promoter is as follows (as Sequence No.7 in a Sequence table):

5’-TAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCTCGACGGTATCGAT-3’。

preferably, the space-time regulation uPA gene expression cassette also comprises a polyA element, and the addition of the polyA element mainly prevents newly synthesized mRNA from being degraded, so that the space-time regulation uPA gene expression cassette has very important significance on the stability of the mRNA. The poly A is bovine growth hormone poly A (bpA for short), and the signal Sequence is as follows (as shown in Sequence No.8 in the Sequence table):

5’-CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG-3’。

in a preferred embodiment of the present invention, a full-length DNA fragment (ApoE-muPA-DD) is synthesized in the order of apolipoprotein E promoter, uPA mutant gene and unstable domain. The obtained full-length DNA fragment of the ApoE-muPA-DD comprises an apolipoprotein E promoter, and the promoter is selected as a hybrid promoter and can strongly control the downstream target gene to perform specific expression in the liver cell; the uPA mutant genes are sequentially connected, wherein the uPA mutant genes are uPA genes inserted with endoplasmic reticulum retention signal genes, the endoplasmic reticulum retention signal genes are added at the C end and the N end of the uPA genes, and the endoplasmic reticulum retention signal genes are added at the C end and the N end of the uPA genes, so that the stability of the distribution of the uPA genes can be enhanced, the distribution of the uPA genes can be strictly controlled, the distribution positions of the uPA genes are limited in liver cells, and the distribution space positions of the uPA mutant genes are strictly controlled; furthermore, the unstable domain is connected to the C-terminal of the uPA mutant gene in sequence, which can strictly limit the target protein to be only distributed in the liver cells within a specific time when the specific small molecule is induced, and the target protein can not be transported to the outside of the cells and can not enter the circulatory system to affect other organs of the whole body. The base sequence of the full-length DNA fragment of the ApoE-muPA-DD is as follows: (Sequence No.9 in the Sequence table):

5’-TAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCTCGACGGTATCGATAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGTTCCATCGAACTGTGACTGTCTAAATGGAGGAACATGTGTGTCCAACAAGTACTTCTCCAACATTCACTGGTGCAACTGCCCAAAGAAATTCGGAGGGCAGCACTGTGAAATAGATAAGTCAAAAACCTGCTATGAGGGGAATGGTCACTTTTACCGAGGAAAGGCCAGCACTGACACCATGGGCCGGCCCTGCCTGCCCTGGAACTCTGCCACTGTCCTTCAGCAAACGTACCATGCCCACAGATCTGATGCTCTTCAGCTGGGCCTGGGGAAACATAATTACTGCAGGAACCCAGACAACCGGAGGCGACCCTGGTGCTATGTGCAGGTGGGCCTAAAGCCGCTTGTCCAAGAGTGCATGGTGCATGACTGCGCAGATGGAAAAAAGCCCTCCTCTCCTCCAGAAGAATTAAAATTTCAGTGTGGCCAAAAGACTCTGAGGCCCCGCTTTAAGATTATTGGGGGAGAATTCACCACCATCGAGAACCAGCCCTGGTTTGCGGCCATCTACAGGAGGCACCGGGGGGGCTCTGTCACCTACGTGTGTGGAGGCAGCCTCATCAGCCCTTGCTGGGTGATCAGCGCCACACACTGCTTCATTGATTACCCAAAGAAGGAGGACTACATCGTCTACCTGGGTCGCTCAAGGCTTAACTCCAACACGCAAGGGGAGATGAAGTTTGAGGTGGAAAACCTCATCCTACACAAGGACTACAGCGCTGACACGCTTGCTCACCACAATGACATTGCCTTGCTGAAGATCCGTTCCAAGGAGGGCAGGTGTGCGCAGCCATCCCGGACTATACAGACCATCTGCCTGCCCTCGATGTATAACGATCCCCAGTTTGGCACAAGCTGTGAGATCACTGGCTTTGGAAAAGAGAATTCTACCGACTATCTCTATCCGGAGCAGCTGAAAATGACTGTTGTGAAGCTGATTTCCCACCGGGAGTGTCAGCAGCCCCACTACTACGGCTCTGAAGTCACCACCAAAATGCTGTGTGCTGCTGACCCACAGTGGAAAACAGATTCCTGCCAGGGAGACTCAGGGGGACCCCTCGTCTGTTCCCTCCAAGGCCGCATGACTTTGACTGGAATTGTGAGCTGGGGCCGTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAGTCACACCAAGGAAGAGAATGGCCTGGCCCTCGAAGAAGATACCTCTGAAAAAGATGAGCTCCACCGGTCGATGATCTCTCTGATTGCCGCTCTGGCCGTGGACTACGTGATCGGGATGGAAAACGCTATGCCATGGAATCTGCCCGCCGATCTGGCTTGGTTCAAGAGGAACACCCTGAACAAGCCAGTGATCATGGGCAGACACACTTGGGAGTCCATTGGCCGGCCCCTGCCTGGACGCAAGAACATCATTCTGAGCTCCCAGCCCTCTACCGACGACAGGGTGACATGGGTGAAAAGTGTGGACGAAGCCATTGCCGCTTGCGGAGATGTGCCCGAGATCATGGTCATCGGCGGAGGGAGAGTGATCGAGCAGTTCCTGCCTAAGGCCCAGAAACTGTACCTGACTCACATTGACGCTGAGGTGGAAGGGGACACCCATTTTCCTGATTATGAGCCAGACGATTGGGAAAGCGTGTTCTCCGAGTTTCACGACGCCGATGCTCAGAATTCTCATAGTTATTGCTTTGAGATCCTGGAAAGGAGATAA-3’。

in a preferred embodiment of the present invention, a full-length DNA fragment (ApoE-muPA-DD-polyA) was sequentially synthesized in the order of apolipoprotein E promoter, uPA mutant gene and unstable domain, poly A element as shown in FIG. 5.

The time-space regulation type uPA gene expression non-viral vector provided by the embodiment of the invention comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain. The non-viral vector for expressing the air-conditioning control type uPA gene contains a liver specific promoter, so that the downstream gene can be controlled to perform specific expression in liver cells during uPA gene expression; the uPA mutant gene is inserted, and the uPA mutant gene is the uPA gene inserted with the endoplasmic reticulum retention signal gene, so that the distribution of the uPA gene can be strictly controlled, and the position for regulating and controlling the distribution of the uPA gene is limited in liver cells, so that the distribution space position of the uPA mutant gene is strictly regulated and controlled; a labile domain is inserted, when trimethoprim or trimethoprim derivatives are not added, the fusion protein of the labile domain and the uPA mutant gene is unstable and can be rapidly degraded in liver cells; when the trimethoprim or trimethoprim derivatives are used for carrying out induction expression on the time-space regulation uPA gene expression box, the fusion protein of an unstable structural domain and a uPA mutant gene can be stabilized and accumulated in liver cells, so that the uPA gene can be stably regulated, the time for stably expressing the fusion protein of the unstable structural domain and the uPA mutant gene is controlled by controlling the time for adding an inducer, namely trimethoprim or trimethoprim derivatives, so that the fusion protein is induced and expressed in a specific time, the fusion protein is strictly limited to be distributed in the liver cells after induction expression, the fusion protein cannot be transported to the outside of cells and cannot enter a circulatory system to influence other organs of the whole body, and the regulation system of the time-space regulation type uPA gene expression non-viral vector is tighter and simpler; can control the uPA gene to be expressed in a specific space at a specific time, so that other organs are prevented from being subjected to toxicity risk.

Correspondingly, the embodiment of the invention also provides a preparation method of the space-time regulation type uPA gene expression non-viral vector, which comprises the following steps:

s01, synthesizing a liver specific promoter, a uPA mutant gene and a DNA fragment of an unstable structural domain;

s02, inserting the DNA fragments into multiple cloning sites of a vector to obtain the time-air-conditioning control type uPA gene expression non-viral vector, wherein the time-air-conditioning control type uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, and the time-space control uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted.

Specifically, in the above step S01, DNA fragments of the liver-specific promoter, uPA mutant gene and unstable domain were synthesized. Preferably, the DNA fragment (ApoE-muPA-DD) is synthesized in the order of apolipoprotein E promoter, uPA mutant gene and unstable domain; the obtained full-length DNA fragment of the ApoE-muPA-DD comprises an apolipoprotein E promoter, and the promoter is selected as a hybrid promoter and can strongly control the downstream target gene to perform specific expression in the liver cell; the uPA mutant genes are sequentially connected, wherein the uPA mutant genes are uPA genes inserted with endoplasmic reticulum retention signal genes, the endoplasmic reticulum retention signal genes are added at the C end and the N end of the uPA genes, and the endoplasmic reticulum retention signal genes are added at the C end and the N end of the uPA genes, so that the stability of the distribution of the uPA genes can be enhanced, the distribution of the uPA genes can be strictly controlled, the distribution positions of the uPA genes are limited in liver cells, and the distribution space positions of the uPA mutant genes are strictly controlled; the unstable structure domain is connected to the C end of the uPA mutant gene in sequence, so that the target protein can be strictly limited to be only distributed in liver cells, can not be distributed to the outside of cells and can not enter a circulatory system to influence other organs of the whole body within a specific time when the specific small molecule is induced. The base Sequence of the full-length DNA fragment of the ApoE-muPA-DD is shown as Sequence No.9 in a Sequence table.

Specifically, in step S02, the DNA fragment is inserted into a multiple cloning site of a vector to obtain the temporal air conditioning control type uPA gene expression non-viral vector, where the temporal air conditioning control type uPA gene expression non-viral vector includes a temporal and spatial control uPA gene expression cassette, and the temporal and spatial control uPA gene expression cassette includes: a liver-specific promoter, a uPA mutant gene, and an unstable domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted.

Preferably, the vector is selected from any one of prokaryotic plasmid vector and eukaryotic plasmid vector. In a particular embodiment of the invention, the vector is preferably pmc. Map information of the vector pMC. BESXP is shown in the attached figure 1 of the specification, the full length of the vector is 4082bp, and attB and attP recombination sites are contained.

In the preferred embodiment of the present invention, the appropriate genetically engineered bacteria are selected for vector transformation according to the selected vector. The genetically engineered bacteria can be selected from various Escherichia coli strains, such as ZYCY10P3S2T, DH5 alpha, TOP10, JM109, etc. In the embodiment of the invention, the genetically engineered bacterium is preferably used in ZYCY10P3S 2T.

Preferably, the pmc. besxp vector is selected for use with the ZYCY10P3S2T engineered bacterium, and under the influence of Φ C31 recombinase expressed by ZYCY10P3S2T, the pmc. besxp vector spontaneously recombines to form two small circular DNA molecules: one is minicircle DNA (MC) only containing a target gene expression frame and 36bp attR sites, and the other is a small loop consisting of plasmid skeleton DNA (containing a plasmid DNA replication origin, an antibiotic resistance gene, I-SceI 32 enzyme cutting sites and the like). Wherein, I-SceI 32 refers to 32 tandem repeated I-SceI enzyme cutting sites, so that a small ring consisting of plasmid framework DNA and a parent plasmid (pMC. BESXP) can be recognized and degraded by I-SceI endonuclease self-expressed by the engineering bacteria in the thalli of the engineering bacteria ZYCY10P3S 2T. Finally, only one kind of circular DNA molecule of the micro-circular DNA is left in the engineering bacteria ZYCY10P3S 2T. That is, the micro-loop DNA can be generated only when the pMC. BESXP vector is used in combination with ZYCY10P3S2T engineering bacteria. Neither other vectors nor the engineered strain ZYCY10P3S2T nor the vector pMC. BESXP nor other E.coli strains (commonly used such as DH 5. alpha., TOP10, JM109, etc.) are capable of producing minicircle DNA. The formed micro-ring DNA molecule only contains a target gene expression frame and 36bp attR sites, so the micro-ring DNA molecule has the following advantages: firstly, MC molecules are small and are more than half smaller than common plasmids; thus, the host cell can be easily accessed, and the transfection efficiency in vivo is higher. Secondly, due to the removal of gene silencing effect (gene silencing effect) caused by the DNA component of the plasmid skeleton, MC can be stably expressed in vivo for a long time; in addition, MC does not contain resistance antibiotic resistance genes, avoiding the concerns of resistance gene spreading and therefore has high safety. Moreover, because MC does not contain the bacterially derived plasmid backbone DNA component, immune responses of the body to bacterially derived DNA components are avoided.

Preferably, in the step of inserting the DNA fragment into the multiple cloning site of the vector, either seamless cloning or specific enzyme ligation may be used, and the insertion of the DNA fragment into the multiple cloning site of the vector is mainly performed.

Taking the ApoE-muPA-DD fragment as an example, the above DNA fragment is inserted into the multiple cloning site of the vector pmc. besxp by a seamless cloning method, which includes the following steps (as shown in fig. 2):

s201, carrying out double enzyme digestion linearization on the vector pMC. BESXP by using restriction enzymes SpeI and SalI to obtain a linearized vector;

s202, using the synthesized DNA fragment as a template, and performing PCR amplification by using overlapping primers priFOR and priREV to obtain a PCR product;

s203, mixing the linearized vector and the PCR product, and adding In-Fusion recombinase to perform Fusion reaction to obtain the time-air-conditioning control type uPA gene expression micro-ring DNA mother plasmid (pMC.

Specifically, in step S201, the double enzyme digestion linearization system is as follows:

the conditions of the double enzyme digestion linearization are 37 ℃ and 4 hours.

In the above step S202, the specific base sequences of the overlapping primers priFOR and priREV are as follows:

priFOR (Sequence No.11 in Sequence listing):

5’-CCCGGGCGCGACTAGTTAGGCTCAGAGGCACACAGG-3’；

priREV (Sequence No.10 in Sequence listing)

5’-GCCCCCATGGGTCGACTTATCTCCTTTCCAGGATCTCAAAGCAATAACT-3’；

The PCR amplification system is as follows:

KOD-Plus high fidelity DNA polymerase (TOYOBO) 1. mu.l.

The PCR amplification reaction conditions are as follows:

25 cycles.

In the above step S203, the linearized vector and the PCR product are mixed, and an In-Fusion recombinase is added to perform a Fusion reaction,

the fusion reaction system is as follows:

the reaction conditions were as follows:

the reaction was carried out at 50 ℃ for 15 min.

The time-air-conditioning controlled-type uPA gene expression micro-ring DNA mother plasmid (pMC.ApoE-muPA-DD) is obtained through the reaction, the map of the time-air-conditioning controlled-type uPA gene expression micro-ring DNA mother plasmid (pMC.ApoE-muPA-DD) is shown in figure 3, and the size of the micro-ring DNA mother plasmid is 7 kb. The air-conditioning control type uPA gene expression micro-ring DNA mother plasmid comprises a time-space control uPA gene expression box (as shown in figure 4), and the time-space control uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted.

The preparation method of the time-air-conditioning controlled type uPA gene expression micro-ring DNA mother plasmid provided by the embodiment of the invention is a non-viral vector, is simpler and more convenient to operate and use, and is simple, convenient and safe, convenient to operate and use, high in success rate and wide in applicability.

Correspondingly, the embodiment of the invention also provides an application of a space-time regulation type uPA gene expression non-viral vector or a preparation method of the space-time regulation type uPA gene expression non-viral vector.

Preferably, the appropriate genetically engineered bacteria are selected for vector transformation according to the selected vector. The genetically engineered bacteria can be selected from various Escherichia coli strains, such as ZYCY10P3S2T, DH5 alpha, TOP10, JM109, etc. In the embodiment of the invention, the genetically engineered bacterium is preferably used in ZYCY10P3S 2T. The transformation steps of transforming the air-conditioning control type uPA gene expression micro-ring DNA mother plasmid (pMC. ApoE-muPA-DD) into the genetically engineered bacterium ZYCY10P3S2T are as follows:

G01. thawing the genetically engineered bacteria ZYCY10P3S2T on ice to liquid state;

G02. adding 1-2 μ L (less than or equal to 50ng) of the time-air-conditioning control type uPA gene expression non-viral vector into the gene engineering bacteria ZYCY10P3S2T thawed to be liquid, uniformly mixing to obtain a first mixture, and placing on ice for 30-40 minutes;

G03. placing the first mixture in a water bath at 40-42 ℃ for heat shock for 80-90s, rapidly placing on ice for 1-2min, adding 180-;

G04. and spreading 5-10 μ L of the second mixture on LB solid plate medium containing kanamycin antibiotic, and culturing at 37 deg.C for 12-14h, wherein the final concentration of kanamycin antibiotic is 0.1%.

And (3) transforming the time-air-conditioning controlled uPA gene expression non-viral vector into a genetically engineered bacterium ZYCY10P3S2T, culturing at 37 ℃ for 12-14h to obtain a recombinant, and performing monoclonal sequencing verification on the recombinant.

The positive clones obtained by the screening were inoculated into TB medium containing kanamycin (final concentration: 0.1%), subjected to shake culture at 37 ℃ for 12-16h, then added with an arabinose-containing induction medium, subjected to shake culture at 32 ℃ for 8h, and finally subjected to separation and purification with a Plasmid DNA purification Kit (QIAGEN EndoFree Plasmid Mega Kit, Qiagen, Germany) to obtain a spatio-temporally regulated uPA gene expression non-viral micro-loop DNA (ApoE-muPA-DD MC) having a map of FIG. 5 and a size of 3 kb.

In a specific embodiment of the invention, the

Specifically, the screened space-time regulation type uPA gene expression non-viral micro-ring DNA (ApoE-muPA-DD MC) is used for transfecting the hepatocyte and carrying out induced expression, and the specific operation steps are as follows:

D01. seeding density per well on 6-well plates 1 × 10⁶Inoculating the cells with the individual cells;

D02. at 37 ℃ 5% CO₂Culturing the cells in a DMEM (containing 10% fetal calf serum) culture medium for 24 hours to obtain cells to be transfected;

D03. transferring the ApoE-muPA-DD MC plasmid into the cell to be transfected by using a transfection reagent to obtain a recombinant cell, and incubating for 24 hours;

D04. adding trimethoprim into the recombinant cell culture medium until the final concentration of trimethoprim is 10 mu M, and then inducing for 24 hours; the uPA gene can be detected.

In the application process of the space-time regulation type uPA gene expression non-viral vector in preparing the humanized chimera liver model, the preparation method is simple, convenient and safe, the operation and the use are simpler and more convenient, the success rate is high, and the applicability is wide.

The following examples are provided to further illustrate the non-viral vector expressing uPA gene under controlled conditions of air-conditioning and the preparation method thereof.

The vector pMC. BESXP and the strain E. coli ZYCY10P3S2T used in the embodiment of the invention are all commercial products, and the used reagents are all commercial products; the primers and DNA sequences used were synthesized by Shanghai Invitrogen corporation.

The LB culture medium used in the examples of the present invention was configured as follows:

LB culture medium: 10g/L Tryptone (Tryptone); 5g/L of yeast powder (Yeastextract); NaCl10 g/L; pH7.0

The primers used in the examples of the present invention are shown in table 1:

TABLE 1 primer sequence Listing

Example 1

Determining a DNA fragment that synthesizes a liver-specific promoter, a uPA mutant gene and an unstable domain, comprising the steps of:

(1) determining the position of the unstable structure domain, taking a fluorescent group GFP as an indicator group, constructing a GFP-DD expression vector, and comparing the influence of the unstable structure domain at different positions on the transgenic regulation. The method comprises the following specific steps:

placing a DD structure domain at the N end (DD-GFP) or the C end (GFP-DD) of a GFP fluorescence indicator gene respectively, and constructing a DD-GFP expression vector and a GFP-DD expression vector respectively (figure 6);

and transfecting 293T cells with the DD-GFP expression vector and the GFP-DD expression vector respectively, adding a trimethoprim inducer for induction, and observing the cells in a fluorescence microscope.

And (4) analyzing results: as analyzed by FIG. 7, the expression levels of both DD-GFP and GFP-DD were equivalent when induced by 10. mu.M TMP; in the absence of TMP, the GFP-DD signal is very weak, and the DD-GFP still has relatively more protein residues; the expression of GFP by the DD placed at the C-terminal was found to be more tightly regulated.

Further, inducing GFP-DD transfected 293T cells with trimethoprim at different concentrations (0. mu.M, 0.01. mu.M, 1. mu.M, 10. mu.M) gave the results shown in FIG. 8, where the higher the concentration of trimethoprim, the higher the amount of the target protein, and the positive correlation between the amount of the target protein and the concentration of TMP was observed.

(2) Sequentially synthesizing a DNA fragment (ApoE-muPA-DD) according to the sequence of the apolipoprotein E promoter, the uPA mutant gene and the unstable structural domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted, and the endoplasmic reticulum retention signal gene is added to the C-terminal and N-terminal of the uPA gene. The base Sequence of the synthesized DNA is shown as Sequence No.9 in the Sequence table.

Example 2

The DNA fragment is inserted into a vector pMC. BESXP through seamless cloning, and the specific steps are as follows:

(1) preparing a linearized vector, and carrying out double enzyme digestion linearization on the vector pMC. BESXP by using restriction enzymes SpeI and SalI to obtain a linearized vector;

specifically, the double-enzyme digestion linearization system is as follows:

(2) Overlapping PCR was performed. Using the synthesized DNA fragment as a template, and carrying out PCR amplification by using overlapping primers priFOR and priREV to obtain a PCR product;

specifically, the PCR amplification system is as follows:

KOD-Plus high fidelity DNA polymerase (TOYOBO) 1. mu.l

The PCR amplification reaction conditions are as follows:

25 cycles

(3) Seamless clonal joining was performed. Mixing the linearized vector and the PCR product, and adding In-Fusion recombinase to perform PCR reaction to obtain the time-air-conditioning-controlled uPA gene expression non-viral vector (pMC. ApoE-muPA-DD);

specifically, the linearized vector and the PCR product are mixed, In-Fusion recombinase is added for Fusion reaction,

the fusion reaction system is as follows:

the reaction conditions were as follows:

50℃，15min

and obtaining the time-air-conditioning controlled uPA gene expression non-viral vector (pMC. ApoE-muPA-DD) through the reaction. The time-air-conditioning control type uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, and the time-space control uPA gene expression box comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted.

Example 3

The time-controlled air-conditioning type uPA gene expression non-viral vector is transformed into a genetic engineering bacterium ZYCY10P3S2T to prepare a micro-ring, and the specific transformation steps are as follows:

(1) thawing the genetically engineered bacteria ZYCY10P3S2T on ice to liquid state;

(2) adding 1 mu L (less than or equal to 50ng) of the time-space regulation type uPA gene expression non-viral vector into the gene engineering bacteria ZYCY10P3S2T thawed to be liquid, uniformly mixing to obtain a first mixture, and placing on ice for 30 minutes;

(3) placing the first mixture in 42 ℃ water bath for heat shock for 90s, rapidly placing on ice for 2min, then adding 200 mu L of LB liquid culture medium without antibiotics, uniformly mixing, and placing at 37 ℃ and 180-year 200rpm for culturing for 40min to obtain a second mixture;

(4) mu.L of the second mixture was applied to LB solid plate medium containing kanamycin antibiotic at a final concentration of 0.1% and cultured at 37 ℃ for 12 hours.

And (3) transforming the time-air-conditioning control type uPA gene expression non-viral vector into a genetic engineering bacterium ZYCY10P3S2T, culturing at 37 ℃ for 12-14h to obtain a recombinant, and then carrying out positive screening on the recombinant to carry out monoclonal sequencing verification.

And (3) separating and purifying the positive clones obtained by screening by using a plasmid DNA purification kit to obtain the space-time regulation type uPA gene expression non-viral micro-ring DNA (ApoE-muPA-DD MC).

Example 4

And transfecting the hepatocyte by using the screened space-time regulation type uPA gene expression non-viral micro-ring DNA (ApoE-muPA-DD MC) and performing induced expression.

The method comprises the following specific steps:

(1) seeding density per well on 6-well plates 1 × 10⁶Inoculating the cells with the individual cells;

(2) culturing the cells to be transfected for 24 hours in a DMEM (containing 10% fetal calf serum) culture medium at 37 ℃ under the condition of 5% CO 2;

(3) transferring the ApoE-muPA-DD MC plasmid into the cells to be transfected by using a transfection reagent Lipofectamine 2000 to obtain recombinant cells, and incubating for 24 hours;

(4) adding trimethoprim into the recombinant cell culture medium until the final concentration of trimethoprim is 10 mu M, and then inducing for 24 hours; the uPA gene can be detected.

Western Blot is used for detecting the expression of the ApoE-uPA-DD carrier in the liver cells, and the detection steps are as follows:

(1) preparing a protein sample: collecting cells expressed by the ApoE-uPA-DD carrier, using cell lysate (RIPA lysate, Bycnanthes) to perform lysis, and further processing to obtain a protein sample;

(2) electrophoresis: adding a proper amount of loading buffer solution into the protein sample, heating with boiling water for 3-5 min to denature the protein, cooling, loading the protein into SDS-PAGE gel loading hole, and performing 80-100V electrophoresis for 1 hour

(3) Film transfer: proteins were transferred from SDS-PAGE gel to PVDF membrane using a wet-type transfer apparatus (Bio-Rad, USA) at 300mA for 1 hour

(4) And (3) sealing: after cleaning the PVDF membrane, adding Western Blot sealing solution for sealing;

(5) primary antibody incubation: diluted uPA antibody (Abcam, UK) was added and incubated for 1 hour at room temperature

(6) And (3) secondary antibody incubation: adding diluted horseradish peroxidase (HRP) -labeled secondary antibody, and incubating at room temperature for 1 hr

(7) Color development: proteins were detected using ECL chemiluminescence reagent (Cell Signaling, usa).

As shown in FIG. 9, the detection results of the Control (blank Control) and the ApoE-uPA group (positive Control) were not changed as shown in FIG. 9; after the ApoE-uPA-DD carrier transfects the liver cell line for 24 hours, TMP is added for induction (10 mu M), the uPA protein amount in the cell is gradually increased along with the accumulation of time, and when the ApoE-uPA-DD is added with TMP for induction for 17 or 24 hours (Lane 4), a remarkable target protein band is detected by Western Blot; when the ApoE-uPA-DD is induced without adding TMP, only very weak bands exist at each time point, the uPA protein level is very low, and the situation that after the unstable domain DD is inserted, the trimethoprim is not added for induction, and the fusion protein of the uPA and the DD can be rapidly degraded; when trimethoprim is added for induction, the fusion protein of uPA and DD can be expressed, and the amount of distributed intracellular uPA protein gradually increases along with the accumulation of time along with the increase of the induction time of trimethoprim.

The method for monitoring the activity of the uPA protein by using a fibrinogen plate method comprises the following specific operation steps:

(1) preparing 1% agar with PBS, heating to melt, cooling to 40-50 deg.C, adding fibrinogen 5mg and thrombin 60U into 15mL melted agar

(2) Pouring into a culture dish, spreading a plate (thickness about 5 mm)

(3) After solidification, a glass tube with a diameter of 4mm was used to perforate the agar

(4) A uPA protein sample (cell lysate) or uPA standard (Sigma, USA) to be tested is added into the wells, and incubated at 37 ℃ for 4 hours. The uPA activity can be judged according to the size of the cracking ring.

As can be seen from FIG. 10, significant lysis circles were generated by transfecting the liver cell line with ApoE-muPA-DD, inducing with TMP, and adding cell lysate to the fibrinogen agar plate, in FIG. 10, A is a positive control (uPA protein, Sigma); b is blank control; c is a cell lysate; the results indicate that muPA retains urokinase activity.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> advanced hospital

<120> space-time regulation type uPA gene expression non-viral vector and preparation method thereof

<130> 2019.5.23

<160> 11

<170> PatentIn version 3.3

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atgatctctc tgattgccgc tctggccgtg gactacgtga tcgggatgga aaacgctatg 60

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atcatgggca gacacacttg ggagtccatt ggccggcccc tgcctggacg caagaacatc 180

attctgagct cccagccctc taccgacgac agggtgacat gggtgaaaag tgtggacgaa 240

gccattgccg cttgcggaga tgtgcccgag atcatggtca tcggcggagg gagagtgatc 300

gagcagttcc tgcctaaggc ccagaaactg tacctgactc acattgacgc tgaggtggaa 360

ggggacaccc attttcctga ttatgagcca gacgattggg aaagcgtgtt ctccgagttt 420

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Glu Glu Asp Thr Ser Glu Lys Asp Glu Leu

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<213> Artificial Synthesis

<400> 4

Met His Arg Arg Arg Ser Arg Ser Cys Arg Glu Asp Gln Lys Pro Val

1 5 10 15

Ile Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met

20 25 30

Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala

35 40 45

Leu Tyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln

50 55 60

Ala Thr Thr Ala Tyr Phe Leu

65 70

<210> 5

<211> 213

<212> DNA

<213> Artificial Synthesis

<400> 5

atgcacagga ggagaagcag gagctgtcgg gaagatcaga agccagtcat cgatgatcag 60

cgcgacctta tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg 120

gagagcaagt gcagccgcgg agccctgtac acaggctttt ccatcctggt gactctgctc 180

ctcgctggcc aggccaccac cgcctacttc ctg 213

<210> 6

<211> 1470

<212> DNA

<213> Artificial Synthesis

<400> 6

atgcacagga ggagaagcag gagctgtcgg gaagatcaga agccagtcat cgatgatcag 60

cgcgacctta tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg 120

gagagcaagt gcagccgcgg agccctgtac acaggctttt ccatcctggt gactctgctc 180

ctcgctggcc aggccaccac cgcctacttc ctgtaccagc agcaggttcc atcgaactgt 240

gactgtctaa atggaggaac atgtgtgtcc aacaagtact tctccaacat tcactggtgc 300

aactgcccaa agaaattcgg agggcagcac tgtgaaatag ataagtcaaa aacctgctat 360

gaggggaatg gtcactttta ccgaggaaag gccagcactg acaccatggg ccggccctgc 420

ctgccctgga actctgccac tgtccttcag caaacgtacc atgcccacag atctgatgct 480

cttcagctgg gcctggggaa acataattac tgcaggaacc cagacaaccg gaggcgaccc 540

tggtgctatg tgcaggtggg cctaaagccg cttgtccaag agtgcatggt gcatgactgc 600

gcagatggaa aaaagccctc ctctcctcca gaagaattaa aatttcagtg tggccaaaag 660

actctgaggc cccgctttaa gattattggg ggagaattca ccaccatcga gaaccagccc 720

tggtttgcgg ccatctacag gaggcaccgg gggggctctg tcacctacgt gtgtggaggc 780

agcctcatca gcccttgctg ggtgatcagc gccacacact gcttcattga ttacccaaag 840

aaggaggact acatcgtcta cctgggtcgc tcaaggctta actccaacac gcaaggggag 900

atgaagtttg aggtggaaaa cctcatccta cacaaggact acagcgctga cacgcttgct 960

caccacaatg acattgcctt gctgaagatc cgttccaagg agggcaggtg tgcgcagcca 1020

tcccggacta tacagaccat ctgcctgccc tcgatgtata acgatcccca gtttggcaca 1080

agctgtgaga tcactggctt tggaaaagag aattctaccg actatctcta tccggagcag 1140

ctgaaaatga ctgttgtgaa gctgatttcc caccgggagt gtcagcagcc ccactactac 1200

ggctctgaag tcaccaccaa aatgctgtgt gctgctgacc cacagtggaa aacagattcc 1260

tgccagggag actcaggggg acccctcgtc tgttccctcc aaggccgcat gactttgact 1320

ggaattgtga gctggggccg tggatgtgcc ctgaaggaca agccaggcgt ctacacgaga 1380

gtctcacact tcttaccctg gatccgcagt cacaccaagg aagagaatgg cctggccctc 1440

gaagaagata cctctgaaaa agatgagctc 1470

<210> 7

<211> 762

<212> DNA

<213> Artificial Synthesis

<400> 7

taggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60

cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120

ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180

ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240

ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300

tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360

ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420

cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480

tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540

ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600

ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660

aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720

cagtgaatga tccccctgat ctgcggcctc gacggtatcg at 762

<210> 8

<211> 225

<212> DNA

<213> Artificial Synthesis

<400> 8

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225

<210> 9

<211> 2756

<212> DNA

<213> Artificial Synthesis

<400> 9

taggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60

cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120

ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180

ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240

ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300

tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360

ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420

cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480

tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540

ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600

ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660

aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720

cagtgaatga tccccctgat ctgcggcctc gacggtatcg ataagcttgg cattccggta 780

ctgttggtaa agccaccatg cacaggagga gaagcaggag ctgtcgggaa gatcagaagc 840

cagtcatcga tgatcagcgc gaccttatct ccaacaatga gcaactgccc atgctgggcc 900

ggcgccctgg ggccccggag agcaagtgca gccgcggagc cctgtacaca ggcttttcca 960

tcctggtgac tctgctcctc gctggccagg ccaccaccgc ctacttcctg taccagcagc 1020

aggttccatc gaactgtgac tgtctaaatg gaggaacatg tgtgtccaac aagtacttct 1080

ccaacattca ctggtgcaac tgcccaaaga aattcggagg gcagcactgt gaaatagata 1140

agtcaaaaac ctgctatgag gggaatggtc acttttaccg aggaaaggcc agcactgaca 1200

ccatgggccg gccctgcctg ccctggaact ctgccactgt ccttcagcaa acgtaccatg 1260

cccacagatc tgatgctctt cagctgggcc tggggaaaca taattactgc aggaacccag 1320

acaaccggag gcgaccctgg tgctatgtgc aggtgggcct aaagccgctt gtccaagagt 1380

gcatggtgca tgactgcgca gatggaaaaa agccctcctc tcctccagaa gaattaaaat 1440

ttcagtgtgg ccaaaagact ctgaggcccc gctttaagat tattggggga gaattcacca 1500

ccatcgagaa ccagccctgg tttgcggcca tctacaggag gcaccggggg ggctctgtca 1560

cctacgtgtg tggaggcagc ctcatcagcc cttgctgggt gatcagcgcc acacactgct 1620

tcattgatta cccaaagaag gaggactaca tcgtctacct gggtcgctca aggcttaact 1680

ccaacacgca aggggagatg aagtttgagg tggaaaacct catcctacac aaggactaca 1740

gcgctgacac gcttgctcac cacaatgaca ttgccttgct gaagatccgt tccaaggagg 1800

gcaggtgtgc gcagccatcc cggactatac agaccatctg cctgccctcg atgtataacg 1860

atccccagtt tggcacaagc tgtgagatca ctggctttgg aaaagagaat tctaccgact 1920

atctctatcc ggagcagctg aaaatgactg ttgtgaagct gatttcccac cgggagtgtc 1980

agcagcccca ctactacggc tctgaagtca ccaccaaaat gctgtgtgct gctgacccac 2040

agtggaaaac agattcctgc cagggagact cagggggacc cctcgtctgt tccctccaag 2100

gccgcatgac tttgactgga attgtgagct ggggccgtgg atgtgccctg aaggacaagc 2160

caggcgtcta cacgagagtc tcacacttct taccctggat ccgcagtcac accaaggaag 2220

agaatggcct ggccctcgaa gaagatacct ctgaaaaaga tgagctccac cggtcgatga 2280

tctctctgat tgccgctctg gccgtggact acgtgatcgg gatggaaaac gctatgccat 2340

ggaatctgcc cgccgatctg gcttggttca agaggaacac cctgaacaag ccagtgatca 2400

tgggcagaca cacttgggag tccattggcc ggcccctgcc tggacgcaag aacatcattc 2460

tgagctccca gccctctacc gacgacaggg tgacatgggt gaaaagtgtg gacgaagcca 2520

ttgccgcttg cggagatgtg cccgagatca tggtcatcgg cggagggaga gtgatcgagc 2580

agttcctgcc taaggcccag aaactgtacc tgactcacat tgacgctgag gtggaagggg 2640

acacccattt tcctgattat gagccagacg attgggaaag cgtgttctcc gagtttcacg 2700

acgccgatgc tcagaattct catagttatt gctttgagat cctggaaagg agataa 2756

<210> 10

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 10

cccgggcgcg actagttagg ctcagaggca cacagg 36

<210> 11

<211> 49

<212> DNA

<213> Artificial Synthesis

<400> 11

gcccccatgg gtcgacttat ctcctttcca ggatctcaaa gcaataact 49

Claims

1. A time-space regulation type uPA gene expression non-viral vector is characterized in that the time-space regulation type uPA gene expression non-viral vector comprises a time-space regulation type uPA gene expression box, and the time-space regulation type uPA gene expression box sequentially comprises: the uPA mutant gene is a uPA gene inserted with an endoplasmic reticulum retention signal gene, wherein the uPA mutant gene is a uPA gene added with a C-terminal endoplasmic reticulum retention signal gene at the C end and a uPA gene added with an N-terminal endoplasmic reticulum retention signal gene at the N end, the base Sequence of the uPA mutant gene is shown as Sequence No.6, the base Sequence of the C-terminal endoplasmic reticulum retention signal is shown as Sequence No.3, the base Sequence of the N-terminal endoplasmic reticulum retention signal is shown as Sequence No.5, and the base Sequence of the unstable structural domain is shown as Sequence No. 1.

2. The spatiotemporal regulated uPA gene expression nonviral vector of claim 1, wherein the liver specific promoter is selected from the group consisting of apolipoprotein E promoter, albumin promoter, phosphoenolpyruvate carboxykinase promoter, alpha-1-antitrypsin promoter, thyroid hormone binding globulin promoter, alpha-fetoprotein promoter, alcohol dehydrogenase promoter, IGF-II promoter, factor VIII promoter, HBV basal core protein promoter, HBV pre-s 2 protein promoter, thyroxine-binding globulin promoter, HCR-Ap0CII hybrid promoter, HCR-hAAT hybrid promoter, AAT promoter bound to enhancer elements of mouse albumin gene, low density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyltransferase promoter, and the like, Any one of apolipoprotein H promoter, transferrin promoter, transthyretin promoter, promoters of alpha-fibrinogen and beta-fibrinogen, alpha-1-antichymotrypsin promoter, alpha-2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoter, promoter of complement C3 activator, hemopexin promoter and alpha-1-acidic glycoprotein promoter.

3. A method for preparing a space-time regulation type uPA gene expression non-viral vector comprises the following steps:

inserting the DNA fragments into multiple cloning sites of a vector to obtain the time-air-conditioning control type uPA gene expression non-viral vector, wherein the time-air-conditioning control type uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, and the time-space control uPA gene expression box sequentially comprises: a liver-specific promoter, a uPA mutant gene, and an unstable domain; the uPA mutant gene is a uPA gene inserted with an endoplasmic reticulum retention signal gene; wherein the uPA mutant gene is a uPA gene with a C-terminal endoplasmic reticulum retention signal gene added at the C end and an N-terminal endoplasmic reticulum retention signal gene added at the N end, the base Sequence of the uPA mutant gene is shown as Sequence No.6, the base Sequence of the C-terminal endoplasmic reticulum retention signal is shown as Sequence No.3, the base Sequence of the N-terminal endoplasmic reticulum retention signal is shown as Sequence No.5, and the base Sequence of the unstable structural domain is shown as Sequence No. 1.

4. The method for producing a spatiotemporal regulated uPA gene expression non-viral vector according to claim 3,

sequentially synthesizing DNA fragments according to the sequence of the apolipoprotein E promoter, the uPA mutant gene and the unstable structural domain;

inserting the DNA fragment into a multiple cloning site of a vector pMC. BESXP to obtain the time-air-conditioning controlled uPA gene expression non-viral vector, wherein the time-air-conditioning controlled uPA gene expression non-viral vector comprises a time-space control uPA gene expression box, and the time-space control uPA gene expression box sequentially comprises: apolipoprotein E promoter, uPA mutant gene and labile domain; wherein the uPA mutant gene is a uPA gene into which an endoplasmic reticulum retention signal gene is inserted, and the endoplasmic reticulum retention signal gene is added to the C-terminal and N-terminal of the uPA gene.

5. The method for producing a spatiotemporal regulated uPA gene expression non-viral vector according to claim 4,

the genetic engineering bacteria for transforming the time-air-conditioning control type uPA gene expression non-viral vector is ZYCY10P3S 2T.

6. Use of the spatiotemporal regulated uPA gene expression non-viral vector of claim 1 in the preparation of a humanized chimera liver model.