CN115141846B

CN115141846B - Double-promoter plasmid and construction method and application thereof

Info

Publication number: CN115141846B
Application number: CN202210620366.3A
Authority: CN
Inventors: 刘滨磊; 蔡林康
Original assignee: Wuhan Binhui Biotech Co ltd
Current assignee: Wuhan Binhui Biotech Co ltd
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2023-03-10
Anticipated expiration: 2042-06-02
Also published as: CN115141846A

Abstract

The application discloses a dual-promoter plasmid and a construction method and application thereof. The plasmid comprises: eukaryotic CMV promoter, ribosome binding site, T7RNA polymerase gene, T7 promoter, poly adenine nucleotide sequence, bovine growth hormone polyadenylation signal sequence and target gene. After the plasmid vector enters a tumor cell, the CMV promoter starts the transcription of the T7RNA polymerase gene, the T7RNA polymerase is translated to generate T7RNA polymerase, the T7RNA polymerase recognizes the T7 promoter on the plasmid, and the transcription target gene is translated into protein. The plasmid vector can obtain the target protein with higher expression quantity by accurately combining with the target gene and loading into an expression system.

Description

Double-promoter plasmid and construction method and application thereof

Technical Field

The application relates to the technical field of plasmid construction, in particular to a dual-promoter plasmid, a construction method and application thereof, and more particularly relates to a method for inhibiting tumor growth by expressing a cytokine in a constructed dual-promoter plasmid tumor.

Background

Vector construction is one of the commonly used means for molecular biology research. Mainly comprises the reconstruction of the existing carrier Multiple Cloning Sites (MCS) and the reconstruction of the existing carrier promoters, enhancers, screening markers and other functional elements. In order to deliver a DNA molecule into a recipient cell, a vehicle must be found which can enter the cell and replicate as it is after loading with foreign DNA fragments. The carrier is preferably plasmid (plasmid), and in genetic engineering, artificially constructed plasmid is usually used as the carrier, and the carrier construction is to construct plasmid containing exogenous DNA.

In the prior art, when recombinant immune protein is prepared by genetic engineering, relevant immune protein is obtained by expressing strong promoters such as CMV promoter (cytomegalovirus promoter) in plasmids in an eukaryotic expression system (such as tumor cells), and the method has the problems of low expression level, low content of the obtained recombinant protein, high cost and the like.

Therefore, it is highly desirable to construct a plasmid vector that can obtain a higher expression level of a target egg by precisely binding a target gene and loading the plasmid vector into an expression system.

Disclosure of Invention

In view of the above technical problems, the present application contemplates providing a continuous dual promoter plasmid, which optimizes the expression structure of genes, i.e., constructs a recombinant plasmid structure different from the prior art, so that it can express target proteins in higher amounts in an expression system.

Combining the above concepts, the present application provides a plasmid vector with a dual promoter and a brand new structure, after the plasmid vector enters a tumor cell, a cytomegalovirus (hereinafter referred to as "CMV") promoter starts T7RNA polymerase gene transcription, the T7RNA polymerase is generated by translation, the T7RNA polymerase recognizes the T7 promoter on the plasmid, the transcription target Gene (GOI) is translated into protein, and since the T7RNA polymerase has an amplification effect on the T7 promoter transcription, the plasmid can generate a larger amount of target protein after entering the tumor cell.

In a first aspect, the present application discloses a plasmid comprising:

a CMV promoter; the CMV promoter sequence is shown as SEQ ID NO:1 is shown in the specification;

a ribosome binding site (IRES); the sequence of the IRES is shown in SEQ ID NO:2 is shown in the specification;

a T7RNA polymerase gene; the sequence of the T7RNA polymerase gene is shown as SEQ ID NO:3 is shown in the figure;

a T7 promoter; the sequence of the T7 promoter is shown as SEQ ID NO:4 is shown in the specification;

a poly-adenine nucleotide (pA) sequence; the pA sequence is an RNA sequence formed by connecting a plurality of adenine As;

a bovine growth hormone polyadenylation signal (BGHpA) sequence; the BGHpA sequence is shown as SEQ ID NO:5 is shown in the specification; and

gene of interest (GOI).

Further, in the constructed plasmid, the T7RNA polymerase gene sequence is located downstream of the eukaryotic CMV promoter, the BGHpA sequence is located downstream of the T7RNA polymerase gene, the T7 promoter is located downstream of the BGHpA sequence, the IRES sequence is located downstream of the T7 promoter, and the gene of interest (GOI) is located downstream of the IRES.

Furthermore, in the process of constructing the plasmid, a T7 promoter for prokaryotic expression is derived from a T7 bacteriophage and is a commonly used strong promoter for an escherichia coli expression system, the promoter can have specific reaction on T7RNA polymerase, and the transcription after recognition is very active;

the CMV promoter is used as an enhancer/promoter element, the element is derived from a virus genome, the CMV promoter is a most powerful promoter for promoting the expression of eukaryotic genes, and has a strong transcription promotion effect in a plurality of eukaryotic cells, and the promotion efficiency is higher;

an IRES capable of mediating a translation initiation process independent of the end, which IRES can express two or more genes under the same promoter;

thus, by inserting an IRES sequence upstream of the gene of interest (GOI) mRNA which directs transcription from the T7 RNAP/promoter is translated in eukaryotic cells, the CMV promoter in the recombinant vector initiates transcription of the T7RNA polymerase gene, the resulting T7RNA polymerase is translated, the T7 promoter on the plasmid is recognized, and the gene of interest (GOI) is transcribed and translated into protein.

Further, a bovine growth hormone polyadenylation signal (BGHpA) sequence serves as a terminator sequence for transcription of the T7RNA polymerase gene.

In a second aspect, the present application discloses a method for constructing the aforementioned plasmid, the method comprising the steps of:

synthesizing a gene of interest (GOI);

constructing a first recombinant eukaryotic expression plasmid containing a target Gene (GOI);

recombining a CMV promoter, a ribosome binding site (IRES), a T7RNA polymerase gene, a poly-adenine nucleotide (pA) sequence and a bovine growth hormone polyadenylation signal (BGHpA) sequence to a first recombinant eukaryotic expression plasmid to obtain a second recombinant eukaryotic expression plasmid;

and screening and verifying the second recombinant eukaryotic expression plasmid to obtain the plasmid.

Further, the method for synthesizing the gene of interest (GOI) includes at least one of a chemical synthesis method and a PCR amplification method.

In a third aspect, the embodiment of the present application discloses an application of the aforementioned plasmid in tumor drugs.

In a fourth aspect, the present application discloses the use of the aforementioned plasmid in the preparation of an mRNA medicament.

In a fifth aspect, the present application discloses a drug, wherein the active ingredients of the drug comprise the aforementioned plasmid and an adjuvant for drug combination.

Further, the plasmid contains a gene of interest (GOI) which is the mGM-CSF gene.

In a sixth aspect, the present application discloses the use of the aforementioned plasmid in tumor cells.

In a seventh aspect, the present application discloses the use of the aforementioned drugs in tumor cells.

In an eighth aspect, the present embodiments disclose a recombinant cell comprising the aforementioned plasmid.

In a ninth aspect, the present application discloses a genetically engineered bacterium, which includes the recombinant cell of the eighth aspect.

Compared with the prior art, the application has at least the following beneficial effects:

the application relates to a dual-promoter plasmid, a construction method and application thereof, wherein the plasmid comprises: a eukaryotic CMV promoter, a ribosome binding site (IRES), a T7RNA polymerase gene, a T7 promoter, a poly adenine nucleotide (pA) sequence, a bovine growth hormone polyadenylation signal (BGHpA) sequence, and a gene of interest (GOI). After the plasmid vector enters a tumor cell, the CMV promoter starts the transcription and translation of the T7RNA polymerase gene to generate T7RNA polymerase, and the T7RNA polymerase recognizes the T7 promoter on the plasmid, and the transcription target Gene (GOI) is translated into protein. The plasmid vector is accurately combined with a target gene and loaded into an expression system to obtain a target protein with higher expression quantity.

Drawings

FIG. 1 is a schematic diagram of a recombinant plasmid expression structure provided in the present application.

Fig. 2 is a graph of mean trend of tumor volumes of mice provided in the examples of the present application.

FIG. 3 is a graph showing the change in mean tumor volume on day 21 in mice, which is provided in the examples of the present application.

FIG. 4 is a graph of fluorescence signals of mice observed by in vivo imaging provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

In the present application, the term "gene" refers to a nucleic acid fragment that expresses a particular protein or functional RNA molecule and that may comprise regulatory sequences preceding (5 'non-coding regions) and following (3' non-coding regions) the coding sequence. "elements" means (upstream promoter elements) including the CAAT box and the GC box, which are usually located around-70 bp, and upstream elements further from the transcription start point.

In the present application, "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different naturally occurring promoters, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause gene expression in most cell types, in most cases, are commonly referred to as "constitutive promoters".

In this application, the term "expression" refers to the stable accumulation of coding RNA (mRNA) or functional RNA transcribed and derived from a gene, and may also refer to the translation of mRNA into a polypeptide or protein.

In the present application, the term "messenger RNA (mRNA)" refers to RNA that is without introns and that can be translated into protein by a cell.

In the present application, the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in stable inheritance in the gene. The transformed nucleic acid may be in the form of a plasmid retained in the host cell, or some of the transformed nucleic acid may be integrated into the host cell genome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

In the present application, the terms "plasmid" and "vector" refer to an extrachromosomal element that typically carries genes that are not part of the central metabolism of the cell, and is often in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences (linear or circular) of single-or double-stranded DNA or RNA, derived from any source, in which multiple nucleotide sequences have been joined or recombined into a unique construct which is capable of introducing a promoter fragment and a DNA sequence of a selected gene product into a cell along with the corresponding 3' terminal untranslated sequence.

Construction of plasmids

The synthesis of the gene of interest (GOI) here employs PCR amplification.

This example details the construction of recombinant plasmids using mGM-CSF as the target Gene (GOI), whose sequence is shown in SEQ ID NO: and 6, respectively.

1. Designing and synthesizing a PCR amplification primer pair, wherein the nucleotide sequence of the primer pair is as follows:

p1: as shown in SEQ ID NO:7 is shown in the specification;

p2: as shown in SEQ ID NO: shown in fig. 8.

PCR amplification

The PCR amplification reaction system is shown in Table 1:

TABLE 1

Reagent	Amount of the composition used
		TaKaRa Taq Version 2.0	25μl
Template DNA	1μl
		P1（10μM）	1μl
P2（10μM）	1μl
		ddH ₂ O	Mixing the solution to 50. Mu.l

The PCR amplification procedure is shown in table 2:

TABLE 2

Step (ii) of	Temperature of	Time
			1	98℃	5min
2	98℃	10sec
			3	55℃	30sec
4	72℃	35sec
				Repeating the steps for 2 to 34 cycles
5	72℃	5min

3. Constructing a first recombinant eukaryotic expression plasmid containing a gene of interest (GOI) of mGM-CSF.

And respectively carrying out double enzyme digestion on the PCR amplification product and the original plasmid, carrying out electrophoretic detection, and recovering the mGM-CSF sequence and the linearized plasmid DNA after enzyme digestion.

In the enzyme digestion process, a 50-microliter target fragment enzyme digestion system is designed as follows:

10×H buffer ，7μl；

enzymes, 1. Mu.l each (10U/. Mu.l);

35. Mu.l of PCR amplification product

Adding up the double distilled water to 50 mu l;

after mixing well, the mixture was digested at 37 ℃ (for 4 hours for PCR amplification).

The 20. Mu.l vector digestion system was designed as follows:

10×M buffer ，7μl；

enzymes, 1. Mu.l each (10U/. Mu.l);

plasmid (1 ug/. Mu.l) 2. Mu.l

Adding double distilled water to 20 μ l;

after mixing well, the mixture was digested at 37 ℃ (for PCR amplification product, digestion time 4 h).

The recovered mGM-CSF sequence and linearized plasmid DNA were ligated using T4 DNase.

For ligation, a 20. Mu.l ligation system was designed as follows:

2×Quick Ligation Buffer，10μl；

linear plasmid DNA,200ng;

87.2ng of the digested mGM-CSF sequence fragment;

t4 ligase, 1. Mu.l (350U/. Mu.l);

adding double distilled water to 20 μ l;

ligation was carried out overnight at 16 ℃.

4. Recombining a CMV promoter, a ribosome binding site (IRES), a T7RNA polymerase gene, a poly adenine nucleotide (pA) sequence and a bovine growth hormone polyadenylation signal (BGHpA) sequence to a first recombinant eukaryotic expression plasmid to obtain a second recombinant eukaryotic expression plasmid; the expression structure of the obtained second recombinant eukaryotic expression plasmid is shown in figure 1.

5. And (4) transforming and screening.

E.coliDH5. Alpha. Competent cells were prepared by the calcium chloride method, recombinant plasmids were transformed into E.coliDH 5. Alpha. Cells, respectively, and plated on LB plates containing 100. Mu.g/mL ampicillin (Amp), and positive colonies were selected and cultured in LB plates containing 100. Mu.g/mL ampicillin. The strains which have high in vitro expression quantity of the recombinant plasmids and are identified correctly by the restriction enzyme are screened out and stored below 70 ℃ for amplification culture.

Application of recombinant plasmid

The target Gene (GOI) contained in the constructed plasmid is mGM-CSF gene, wherein the mGM-CSF gene expresses human macrophage colony stimulating factor and stimulates dendritic cells to mature.

In this example, recombinant plasmids in which the target Gene (GOI) is mGM-CSF gene and Green Fluorescent Protein (GFP) gene were constructed by the aforementioned method, wherein the carrier plasmid is pT7AMPce, and the constructed recombinant plasmids are pT7AMPcemGM-CSF and pT7AMPce GFP, respectively; the anti-tumor effect of pT7AMPce mGM-CSF applied to the tumor is verified by taking a mouse colon cancer model as an experimental object.

First, a mouse model of CT26 colon cancer was constructed.

Mice were injected subcutaneously with 100. Mu.L of a solution containing 1X 10 ⁶ CT26 cell suspension. When the tumor volume of the tumor-bearing mouse reaches 50 to 80 mm ³ All mice were randomized into 7 groups.

Next, a recombinant plasmid grouping scheme was designed as shown in Table 3.

TABLE 3

IS buffer IS a solvent control containing no plasmid.

As shown in the above table, the pT7AMPce GFP group was used as a plasmid control group; the IS Buffer group IS a non-plasmid blank control group.

Next, the volume of the mice reached 1500mm ³ Mice were then euthanized, tumor volumes were measured twice weekly, and results of tumor volume in mice were analyzed statistically using Student-t-test.

As a result: as shown in figures 2 to 3, the antitumor effect of the pT7AMPce mGM-CSF plasmid group is remarkably superior to that of the pT7AMPce GFP group through statistical analysis by comparing the change of the tumor volume of the mice.

Application of recombinant plasmid in preparation of mRNA (messenger ribonucleic acid) medicament

In the embodiment of the application, the recombinant plasmid can be used for preparing mRNA medicaments.

Firstly, constructing a recombinant plasmid by the plasmid construction method; selecting different target Genes (GOI) to construct in recombinant plasmid according to the type of mRNA medicine to be prepared, such as preparing an anti-tumor cell mRNA medicine, and adopting D-type envelope protein gene gD of an oncolytic virus, i.e. II-type herpes simplex virus (HSV 2) ^ED Constructing a recombinant plasmid for a target gene;

in the second step, the recombinant plasmid is subjected to In Vitro Transformation (IVT) to convert the DNA strand into mRNA, and in this step, RNA polymerase (RNA polymerase) transcribes the DNA into mRNA.

Third, the delivery system is loaded. This step is carried out by encapsulating the mRNA in a lipid carrier (LNP), suspending the lipid in an alcohol solution, contacting the mRNA and encapsulating it, the two substances being attracted by the opposite charge.

And fourthly, checking and filling to obtain the mRNA medicine.

In the examples of the present application, the delivery system for encapsulating mRNA is a lipid carrier (LNP) commonly used or published at present, and can be obtained by those skilled in the art by applying conventional technical means, which are not described herein.

Mouse intramuscular injection plasmid for observing fluorescence expression

Firstly, constructing a recombinant plasmid pT7AMPCe-fluc with a target Gene (GOI) being a firefly fluorescent protein (fluc) gene by the method;

secondly, 7-week-old female balb/c mice are selected as experimental mice, wherein the left thigh muscle and the right thigh muscle of the experimental group of mice are respectively injected with 30 mu g of pT7AMPCe-fluc recombinant plasmid, and the control group of mice are injected with equal doses of IS Buffer solvent. After 48h, the mouse is injected with fluorescein substrate of 15mg/ml in the abdominal cavity, and the mouse fluorescence signal is observed by the living body imaging of the small animal.

As a result: as shown in fig. 4: the result shows that the pT7AMPCe-fluc recombinant plasmid expresses firefly fluorescent protease in muscle cells; wherein the mouse No. 1 is a control group mouse, and the mouse No. 2 is injected with 30 mu g of pT7AMPCe-fluc recombinant plasmid.

In summary, the present application relates to a dual promoter plasmid, a construction method and applications thereof, wherein the plasmid comprises: eukaryotic CMV promoter, ribosome binding site (IRES), T7RNA polymerase gene, T7 promoter, poly adenine nucleotide (pA) sequence, bovine growth hormone polyadenylation signal (BGHpA) sequence, and gene of interest (GOI). After the plasmid vector enters a tumor cell, the CMV promoter starts the transcription of a T7RNA polymerase gene and translates the generated T7RNA polymerase, and the T7RNA polymerase recognizes the T7 promoter on the plasmid and transcribes a target Gene (GOI) and translates the target gene into protein. The plasmid vector is accurately combined with a target gene and loaded into an expression system, so that the expression quantity of the target immune protein is higher. According to the method, mGM-CSF gene is used as a target Gene (GOI) to construct recombinant plasmid, and the recombinant plasmid is used for anti-tumor treatment, so that the growth rate of tumor cells can be obviously inhibited, and the treatment effect is good.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Sequence listing

<120> double-promoter plasmid and construction method and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 655

<212> DNA

<213> CMV promoter (CMV promoter)

<400> 1

cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc 60

aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120

aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180

tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg 240

gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300

cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360

tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420

gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480

cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540

taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600

aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaatt 655

<210> 2

<211> 585

<212> DNA

<213> IRES

<400> 2

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccaca 585

<210> 3

<211> 2652

<212> DNA

<213> T7RNA polymerase gene (T7 RNA polymerase gene)

<400> 3

atggacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60

ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120

catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180

gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500

tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860

ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

<210> 4

<211> 18

<212> DNA

<213> T7 promoter (T7 promoter)

<400> 4

taatacgact cactatag 18

<210> 5

<211> 232

<212> DNA

<213> BGHpA

<400> 5

ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 60

cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 120

atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 180

ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg ga 232

<210> 6

<211> 426

<212> DNA

<213> mGM-CSF gene (mGM-CSF gene)

<400> 6

atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc agcacccacc 60

cgctcaccca tcactgtcac ccggccttgg aagcatgtag aggccatcaa agaagccctg 120

aacctcctgg atgacatgcc tgtcacgttg aatgaagagg tagaagtcgt ctctaacgag 180

ttctccttca agaagctaac atgtgtgcag acccgcctga agatattcga gcagggtcta 240

cggggcaatt tcaccaaact caagggcgcc ttgaacatga cagccagcta ctaccagaca 300

tactgccccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggatttc 360

atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accaggccaa 420

aaatga 426

<210> 7

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 7

tggccacact cgagagatct atgtggctgc agaatttact tttcctgggc 50

<210> 8

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 8

tttttttttt tttttagatc ttcatttttg gcctggtttt ttgcattcaa agggg 55

Claims

1. A plasmid, wherein the plasmid comprises the following elements linked in a specific order:

a T7RNA polymerase gene downstream of a eukaryotic CMV promoter; the sequence of the T7RNA polymerase gene is shown as SEQ ID NO:3 is shown in the specification;

a bovine growth hormone polyadenylation signal sequence located downstream of the T7RNA polymerase gene; the bovine growth hormone polyadenylation signal sequence is shown as SEQ ID NO:5 is shown in the specification;

a T7 promoter located downstream of the bovine growth hormone polyadenylation signal sequence; the sequence of the T7 promoter is shown as SEQ ID NO:4 is shown in the specification;

a ribosome binding site downstream of the T7 promoter; the sequence of the ribosome binding site is shown as SEQ ID NO:2 is shown in the specification;

a gene of interest, mGM-CSF, located downstream of the ribosome binding site; the nucleotide sequence of the target gene mGM-CSF is shown as SEQ ID NO:6 is shown in the specification; and

a poly-adenine nucleotide sequence; the poly-adenine nucleotide sequence is an RNA sequence formed by connecting a plurality of adenine As.

2. The method for constructing the plasmid of claim 1, which comprises the steps of:

synthesizing a target gene mGM-CSF;

constructing a first recombinant eukaryotic expression plasmid containing a target gene mGM-CSF;

recombining a CMV promoter, a ribosome binding site, a T7RNA polymerase gene, a poly-adenine nucleotide sequence and a bovine growth hormone polyadenylation signal sequence to a first recombinant eukaryotic expression plasmid according to the connection sequence of each element of the plasmid in claim 1 to obtain a second recombinant eukaryotic expression plasmid;

3. The use of the plasmid of claim 1 in the preparation of an anti-tumor medicament.

4. Use of the plasmid of claim 1 for the preparation of a mRNA medicament.

5. A medicament, the active ingredient of which comprises the plasmid of claim 1 and an adjuvant for pharmaceutical composition.

6. A recombinant cell comprising the plasmid of claim 1.