CN112501190A

CN112501190A - Recombinant plasmid, whitening agent, preparation method and related application thereof

Info

Publication number: CN112501190A
Application number: CN202110150753.0A
Authority: CN
Inventors: 郭衡远; 张露; 杨婷婷
Original assignee: First Affiliated Hospital of Guangdong Pharmaceutical University
Current assignee: First Affiliated Hospital of Guangdong Pharmaceutical University
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-03-16

Abstract

One or more embodiments of the present disclosure provide a recombinant plasmid, a whitening agent, a preparation method and related applications. The recombinant plasmid comprises a pTyr promoter, an egfp gene, a T2A gene and a TCS gene on a basic plasmid; the pTyr promoter, egfp gene, T2A gene and TCS gene are located between the pTyr promoter and the terminator of the base plasmid. The preparation method comprises the following steps: carrying out enzyme digestion on the basic plasmid, carrying out PCR amplification and recombining to obtain a first recombinant plasmid pcDNA3.1+ egfp-T2A; enzyme-cutting the recombinant plasmid and inserting a TCS sequence to obtain a second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS; and amplifying the Tyr promoter fragment by PCR, carrying out enzyme digestion on the CMV promoter in the second recombinant plasmid, and carrying out recombination to obtain the recombinant plasmid. The recombinant plasmid can specifically induce the 'natural' death of melanocytes without damaging normal cells, and has excellent targeting property.

Description

Recombinant plasmid, whitening agent, preparation method and related application thereof

Technical Field

One or more embodiments of the present disclosure relate to the field of genetic engineering technologies, and in particular, to a recombinant plasmid, a whitening agent, a preparation method thereof, and a related application thereof.

Background

The whitening agents on the market at present mainly comprise a copper ion complexing agent and an oxidation reaction inhibitor. The copper ion complexing agent is mostly cytotoxic and causes more cell damage. The oxidation reaction inhibitor has problems such as unstable drug effect.

Disclosure of Invention

In view of the above, one or more embodiments of the present disclosure are directed to a recombinant plasmid, a whitening agent, a preparation method and related applications thereof, which are used to solve the problems of the prior art.

In view of the above objects, one or more embodiments of the present specification provide a recombinant plasmid including a pTyr promoter, an egfp gene, a T2A gene and a TCS gene on a base plasmid; the egfp gene, the T2A gene and the TCS gene are positioned between the pTyr promoter and the terminator of the basic plasmid; the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1; the nucleotide sequence of the egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown in SEQ ID NO. 3.

In one embodiment, the base plasmid is pcDNA3.1+, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO. 4.

One or more embodiments of the present specification also provide a method of preparing a recombinant plasmid, including:

enzyme digestion basic plasmid, PCR amplification pcDNA3.1+ egfp and recombination obtain the first recombinant plasmid pcDNA3.1+ egfp-T2A; wherein, the nucleotide sequence of egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown as SEQ ID NO. 3;

carrying out enzyme digestion on the first recombinant plasmid pcDNA3.1+ egfp-T2A, and inserting a TCS sequence into the obtained enzyme digestion site to obtain a second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS;

carrying out PCR amplification on a Tyr promoter fragment, carrying out enzyme digestion on a CMV promoter in the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS and carrying out recombination to obtain a recombinant plasmid pTyr + egfp-T2A-TCS; wherein, the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1.

In one or more embodiments of the present disclosure, the step of obtaining the first recombinant plasmid pcDNA3.1+ egfp-T2A specifically comprises:

using EcoRV-HF enzyme to enzyme-cut pcDNA3.1+ plasmid to obtain a first enzyme-cut fragment;

using pcDNA3.1+ egfp as a template and adopting first primers shown as SEQ ID NO. 5 and SEQ ID NO. 6 to carry out PCR amplification to obtain a first amplified fragment;

the first digested fragment and the first amplified fragment were ligated, and the ligation product was transformed into Trans10 competent and applied to an LB plate containing AMP, resulting in the first recombinant plasmid pcDNA3.1+ egfp-T2A.

In one or more embodiments of the present disclosure, the step of enzymatically digesting the first recombinant plasmid pcDNA3.1+ egfp-T2A specifically comprises:

the first recombinant plasmid pcDNA3.1+ egfp-T2A was digested with EcoRV-HF enzyme.

In one or more embodiments of the present disclosure, the step of obtaining the recombinant plasmid pTyr + egfp-T2A-TCS specifically comprises:

the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS is digested by MluI enzyme and NheI enzyme to obtain a second digested fragment;

b16 cell DNA is taken as a template, and a second primer shown as SEQ ID NO. 7 and SEQ ID NO. 8 is adopted to obtain a second amplified fragment through PCR amplification;

and (3) connecting the second enzyme digestion fragment and the second amplification fragment by using a kit, converting the connection product into a Trans10 competence, and coating the competence on an LB plate containing 100ug/mL AMP to obtain the recombinant plasmid pTyr + egfp-T2A-TCS.

One or more embodiments of the present disclosure further provide a recombinant plasmid as described in any one of the above or a recombinant plasmid prepared by the method for preparing a recombinant plasmid as described in any one of the above, and an application of the recombinant plasmid in preparing a whitening agent.

One or more embodiments of the present disclosure also provide a whitening agent, including the recombinant plasmid as described in any previous item or the recombinant plasmid prepared by the preparation method of the recombinant plasmid as described in any previous item, and a pharmaceutically acceptable carrier.

In one or more embodiments of the present description, the carrier is selected from liposomes.

One or more embodiments of the present disclosure also provide a method of preparing a whitening agent, including:

mixing a liposome agent with the concentration of 80-120 mg/mL and a recombinant plasmid with the concentration of 5-9 mg/mL in equal volume to obtain a whitening agent; wherein the liposome agent comprises the following substances in percentage by mass: 50% of GDL liposome, 15% of CH liposome, 23% of POE-10 liposome and 12% of DOTAP liposome; the recombinant plasmid is prepared by the recombinant plasmid or the preparation method of the recombinant plasmid.

As can be seen from the above description, the recombinant plasmid, the whitening agent, the preparation method thereof and the related application are provided in one or more embodiments of the present disclosure. The recombinant plasmid comprises a pTyr promoter, an egfp gene, a T2A gene and a TCS gene on a basic plasmid; the pTyr promoter, the egfp gene, the T2A gene and the TCS gene are located between the pTyr promoter and the terminator of the base plasmid; the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1; the nucleotide sequence of the egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown in SEQ ID NO. 3. The recombinant plasmid can specifically induce the 'natural' death of melanoma cells without damaging normal cells, and has excellent targeting property. Meanwhile, the recombinant plasmid can be wrapped by liposome to prepare a whitening agent, can be directly smeared on skin when in use, does not need to use an injection, and has excellent convenience and safety.

Drawings

In order to more clearly illustrate one or more embodiments or prior art solutions of the present specification, the drawings that are needed in the description of the embodiments or prior art will be briefly described below, and it is obvious that the drawings in the following description are only one or more embodiments of the present specification, and that other drawings may be obtained by those skilled in the art without inventive effort from these drawings.

FIG. 1 is a schematic diagram of pcDNA3.1+ base plasmid in example 1 of this specification;

FIG. 2 is a schematic diagram of the recombinant plasmid pTyr + egfp-T2A-TCS in examples 1, 2,3 and 4 of the present specification;

FIG. 3 is a schematic flow chart of a method for preparing the recombinant plasmid pTyr + egfp-T2A-TCS in examples 1, 2,3 and 4 of the present specification;

FIG. 4 is a diagram showing the fluorescence expression state of B16 cells transfected by the recombinant plasmid pTyr + egfp-T2A-TCS in example 2 of the present specification;

FIG. 5 is a diagram showing the fluorescence expression state of TC-1 cells transfected by the recombinant plasmid pTyr + egfp-T2A-TCS in example 2 of the present specification;

FIG. 6 is a graph showing the results of apoptosis and necrosis of TC-1 cells of a blank control in flow cytometry in example 3 of the present specification;

FIG. 7 is a graph showing the results of apoptosis and necrosis of TC-1 cells transfected by the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS detected by flow cytometry in example 3 of the present specification;

FIG. 8 is a graph showing the results of apoptosis and necrosis of TC-1 cells transfected by the recombinant plasmid pTyr + egfp-T2A-TCS detected by flow cytometry in example 3 of the present specification;

FIG. 9 is a graph showing the results of detecting apoptosis and necrosis of blank control B16 cells by a flow cytometer in example 3 of this specification;

FIG. 10 is a graph showing the results of apoptosis and necrosis of B16 cells transfected with the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS, which was detected by flow cytometry in example 3 of the present specification;

FIG. 11 is a graph showing the results of apoptosis and necrosis of B16 cells transfected by the recombinant plasmid pTyr + egfp-T2A-TCS, which was detected by flow cytometry in example 3 of the present specification.

Detailed Description

For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.

It is to be noted that unless otherwise defined, technical or scientific terms used in one or more embodiments of the present specification should have the ordinary meaning as understood by those of ordinary skill in the art to which this disclosure belongs. The use of "first," "second," and similar terms in one or more embodiments of the specification is not intended to indicate any order, quantity, or importance, but rather is used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.

As described in the background section, current whitening agents mainly include two types, i.e., a copper ion complexing agent and an oxidation reaction inhibitor. The copper ion complexing agent is mainly focused on inhibiting tyrosinase. The oxidation reaction inhibitor is mainly focused on inhibiting oxidation reaction and promoting melanin metabolism.

Applicants have discovered in the course of practicing the present disclosure that copper ion complexing agents tend to suffer from cytotoxicity and instability. The oxidation reaction inhibitor has the problems of unstable drug effect, damage to skin barrier and the like. The trichosanthin recombinant plasmid can specifically induce the 'natural' death of melanocytes without damaging normal cells, and has stronger targeting property.

Hereinafter, the technical means of the present disclosure will be described in further detail with reference to specific examples.

Some of the plasmids, enzymes and the like involved in the present invention are commercially available. For example, pcDNA3.1+ plasmid is available from Youbao, and the plasmid map is shown in FIG. 1. TCS is commercially available from the phylum cristatum. A general DNA product purification kit can be purchased from TIANGEN. Ligation kits are available from Beyotime. Trans10 cells were purchased from TransGen.

Referring to fig. 2, the present invention provides a recombinant plasmid, which includes ptyr (phosphotyrosyl) phosphotyrosine promoter, egfp (enhanced Green Fluorescent protein) enhanced Green Fluorescent protein gene, T2A gene and tcs (trichosantin) trichosanthin gene on a base plasmid. The pTyr promoter, the egfp gene, the T2A gene and the TCS gene are located between the pTyr promoter and the terminator of the base plasmid; the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1; the nucleotide sequence of the egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown in SEQ ID NO. 3. Wherein, the nucleotide sequence of TCS is shown in SEQ ID NO. 9.

In one or more embodiments of the present disclosure, the base plasmid is pcdna3.1+, which is a eukaryotic plasmid. The nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 4. It should be noted that the terminator of the base plasmid pcDNA3.1+ is the terminator of the recombinant plasmid.

Based on the same inventive concept, one or more embodiments of the present specification further provide a method for preparing a recombinant plasmid corresponding to any of the recombinant plasmids described above.

Referring to FIG. 3, the present disclosure also provides a method for preparing a recombinant plasmid, comprising:

s100, carrying out enzyme digestion on a basic plasmid, carrying out PCR amplification on pcDNA3.1+ egfp, and recombining to obtain a first recombinant plasmid pcDNA3.1+ egfp-T2A; wherein, the nucleotide sequence of egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown as SEQ ID NO. 3;

s200, carrying out enzyme digestion on the first recombinant plasmid pcDNA3.1+ egfp-T2A, and inserting a TCS sequence into the obtained enzyme digestion site to obtain a second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS;

s300, carrying out PCR amplification on a Tyr promoter fragment, carrying out enzyme digestion on a CMV promoter in the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS, and carrying out recombination to obtain a recombinant plasmid pTyr + egfp-T2A-TCS; wherein, the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1.

In one or more embodiments of the present disclosure, in the step S100, the step of obtaining the first recombinant plasmid pcdna3.1+ egfp-T2A specifically includes:

the pcDNA3.1+ vector was digested with EcoRV-HF enzyme to obtain the first digested fragment.

Referring to Table 1, a first amplified fragment was obtained by PCR amplification using pcDNA3.1+ egfp as a template and the first primers shown in SEQ ID NO:5 and SEQ ID NO: 6.

TABLE 1 nucleotide sequences

And (3) connecting the first enzyme cutting fragment and the first amplification fragment, converting the connection product into a Trans10 competence, and coating the competence to an LB plate containing 100ug/mL AMP to obtain the first recombinant plasmid pcDNA3.1+ egfp-T2A.

Wherein the pcDNA3.1+ vector is a eukaryotic plasmid. The EcoRV-HF enzyme is a NEB restriction enzyme that cleaves the Ampr ampicillin resistance gene, producing ligatable blunt ends. After enzyme digestion, gel recovery is carried out by adopting a common DNA product purification kit, and a first enzyme digestion fragment can be obtained. A common DNA product purification kit may be TIANGEN.

Wherein, when PCR amplification is carried out by using pcDNA3.1+ egfp as a template, a target fragment is amplified by Takara PrimerSTAR MAX DNA polymerase high fidelity polymerase. The steps and conditions of PCR amplification were: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10 s; annealing at 57 ℃ for 15s and extension at 72 ℃ for 1 min. The number of cycles was 35. Additional extension was included, at a temperature of 72 ℃ for a duration of 7 min. After PCR amplification, gel recovery is carried out by using a common DNA product purification kit, and a first amplified fragment can be obtained. Wherein the DNA product purification kit may be TIANGEN. By the PCR amplification, the egfp-T2A fragment can be obtained quickly and accurately.

The first amplified fragment was 832bp in length.

When the first enzyme-digested fragment and the first amplified fragment are connected, the first amplified fragment is inserted into the first enzyme-digested fragment in one step by a homologous recombination method. Homologous recombination occurs only between the first amplified fragment and the end of the first cleaved fragment, and the resulting ligation product does not contain additional base sequences. The method is simple to operate, and the first amplified fragment can be rapidly and directionally inserted into the first enzyme-digested fragment by only one-step reaction. Ligation can be performed by using a ligation Kit (Seamless Cloning Kit). The reaction can be carried out rapidly at 50 ℃ for about 15 min.

When the ligation product is transformed into Trans10 cell competence, 8-12 uL of the ligation product can be transformed into Trans10 cell competence and plated on LB (Luria-Bertani) -containing medium plate with AMP (adenine ribonucleotide) concentration of 80-120 ug/mL. The first recombinant plasmid pcDNA3.1+ egfp-T2A was obtained by sequence screening. The conversion reaction may be carried out at 50 ℃ for about 40 minutes.

In one or more embodiments of the present disclosure, in the step S200, the step of enzymatically digesting the first recombinant plasmid pcdna3.1+ egfp-T2A specifically includes: the first recombinant plasmid pcDNA3.1+ egfp-T2A was digested with EcoRV-HF enzyme. Among them, EcoRV-HF enzyme is a NEB restriction enzyme capable of cleaving Ampr ampicillin resistance gene to generate ligatable blunt ends. After the enzyme digestion, gel recovery was performed using a common DNA product purification kit.

The TCS sequence can be shown as SEQ ID NO 9. The TCS sequence can be inserted into the obtained cleavage site by the same method as in step S100 and by the same ligation Kit (Seamless Cloning Kit). The reaction conditions are also the same as in the above S100, and are not described in detail here.

In one or more embodiments of the present disclosure, in the step S300, the step of obtaining the third recombinant plasmid pTyr + egfp-T2A-TCS specifically includes:

And performing double enzyme digestion by using MluI enzyme and NheI enzyme, and performing gel recovery by using a common DNA product purification kit to obtain the second enzyme digestion fragment. A common DNA product purification kit may be TIANGEN.

Among them, when PCR amplification was carried out using B16 cell DNA as a template, the target fragment was amplified by Takara PrimerSTAR MAX DNA polymerase high fidelity polymerase. The steps and conditions of PCR amplification were: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10 s; annealing at 57 ℃ for 15s and extension at 72 ℃ for 1 min. The number of cycles was 35. Additional extension was included, at a temperature of 72 ℃ for a duration of 7 min. After PCR amplification, gel recovery is carried out by using a common DNA product purification kit, and a first amplified fragment can be obtained. Wherein the DNA product purification kit may be TIANGEN. By the PCR amplification, the pTyr promoter fragment can be obtained quickly and accurately.

When the second enzyme-cleaved fragment and the second amplified fragment are connected, the second amplified fragment is inserted into the second enzyme-cleaved fragment in one step by a homologous recombination method. Homologous recombination occurs only between the second amplified fragment and the end of the second digested fragment, and the resulting ligation product does not contain additional base sequences. The method is simple to operate, and the second amplified fragment can be rapidly and directionally inserted into the second enzyme-digested fragment by only one-step reaction. Ligation can be performed by using a ligation Kit (Seamless Cloning Kit). The reaction can be carried out rapidly at 50 ℃ for about 15 min.

When the ligation product is transformed into Trans10 cell competence, 8-12 uL of the ligation product can be transformed into Trans10 cell competence and plated on LB (Luria-Bertani) -containing medium plate with AMP (adenine ribonucleotide) concentration of 80-120 ug/mL. Through sequence screening, the recombinant plasmid pTyr + egfp-T2A-TCS is obtained. The conversion reaction may be carried out at 50 ℃ for about 40 minutes.

Based on the same inventive concept, one or more embodiments of the present specification further provide a whitening agent corresponding to the recombinant plasmid of any of the above embodiments.

The embodiment of the invention also provides a whitening agent, which comprises the recombinant plasmid or the recombinant plasmid prepared by the preparation method of the recombinant plasmid and a pharmaceutically acceptable carrier. The whitening agent can be introduced into melanocyte, and can rapidly kill melanocyte through TCS gene expression.

It should be noted that a pharmaceutically acceptable carrier refers to one or more solid or liquid fillers, diluents, or encapsulating substances. The pharmaceutically acceptable carrier includes various solutions, diluents, solvents, dispersants, liposomes, emulsions, sugar coatings, antibacterial agents, antifungal agents, etc., and other carriers suitable for the application of the recombinant plasmid of the present invention. Injectable carriers include water, physiological saline, balanced salt solution, buffer, dextrose solution, glycerol, and the like. At the same time, as biologically neutral carriers, the pharmacological components employed may contain non-toxic auxiliary substances including wetting or emulsifying agents, preservatives, pH buffering agents, sodium acetate, monolaurate and the like.

In one or more embodiments of the present description, the carrier is selected from liposomes. The whitening agent can be directly applied on skin in a smearing mode by selecting a mode of wrapping recombinant plasmids by liposome, and can directly transfect the recombinant plasmids into skin cells and mediate in-vivo transient expression. When in use, the whitening agent is directly smeared on the skin, the use is convenient, and the toxic and side effects caused by systemic administration are avoided.

Based on the same inventive concept, one or more embodiments of the present specification further provide a method for preparing a whitening agent, corresponding to the recombinant plasmid of any of the above embodiments.

The embodiment of the invention provides a preparation method of a whitening agent, which comprises the following steps:

and (3) mixing the liposome agent with the concentration of 80-120 mg/mL and the recombinant plasmid with the concentration of 5-9 mg/mL in equal volume, and incubating at room temperature to obtain the whitening agent.

In one or more embodiments of the present disclosure, the liposome agent comprises the following substances by mass: 50% of GDL (polyoxyethylene-9-dodecyl ether) liposomes, 15% of CH cholesterol, 23% of POE-10 liposomes and 12% of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine) liposomes. The liposome preparation contains alpha-tocopherol, and the alpha-tocopherol accounts for 1% of the total liposome amount in the liposome preparation. The recombinant plasmid is the recombinant plasmid or is prepared by the preparation method of the recombinant plasmid.

In one or more embodiments of the present disclosure, the liposomal agent is present in the form of a suspension. Can be prepared by respectively weighing 50g of GDL liposome, 15g of CH, 23g of POE-10 liposome and 12g of DOTAP liposome; mixing and heating at 70 ℃ and centrifuging; dissolving the filter residue in 70 deg.C water bath; and mixing with water to obtain liposome in the form of suspension.

In one or more embodiments of the present disclosure, prior to mixing the liposome agent and the recombinant plasmid, the method further comprises sonicating the liposome suspension for 20 min. The incubation time may be 45 min.

The technical solution of the present invention will be further described with reference to the following embodiments.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

EXAMPLE 1 preparation of recombinant plasmid pTyr + egfp-T2A-TCS

a. The pcDNA3.1+ plasmid was digested with EcoRV-HF (NEB) and recovered from the gel using a common DNA product purification kit (TIANGEN). The target fragment was amplified by Takara PrimerSTAR MAX DNA polymerase high fidelity polymerase using the first primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 with pcDNA3.1+ egfp as a template, and then recovered by gel using a general DNA product purification kit (TIANGEN). The digested fragment and the amplified fragment were ligated by using a Seamless Cloning Kit (Beyotime) Kit, 10uL of the ligation product was aspirated to transform Trans10 (TransGen) competence, the resulting product was spread on an LB plate of 100ug/mL AMP, and the next day was sequenced, and a colony containing a plasmid with the correct sequence was selected, thereby finally obtaining the plasmid pcDNA3.1+ egfp-T2A.

b. The pcDNA3.1+ egfp-T2A is digested by EcoRV-HF (NEB), and then the Trichosanthin (TCS) sequence is biosynthesized by the engine family and inserted into the site of the digestion, thus obtaining the plasmid pcDNA3.1+ egfp-T2A-TCS.

c. pcDNA3.1+ egfp-T2A-TCS was digested with MluI and NheI (NEB) and gel recovered using a common DNA product purification kit (TIANGEN). Using the second primers shown in SEQ ID NO:7 and SEQ ID NO:8, the target fragment (pre-denatured 98 ℃ C. for 30s, 98 ℃ C. for 10s, 57 ℃ C. for 15s, 72 ℃ C. for 1min, for 35 cycles, and additionally extended 72 ℃ C. for 7 min) was amplified by Takara PrimerSTAR MAX DNA polymerase using B16 cell DNA as a template, and gel recovery was performed using a general DNA product purification kit (TIANGEN). The digested fragment and the amplified fragment were ligated by using a Seamless Cloning Kit (Beyotime) Kit, 10uL of the ligation product was aspirated to transform Trans10 (TransGen) competence, the resulting product was spread on an LB plate of 100ug/mL AMP, and the next day was sequenced, and a colony containing a plasmid with the correct sequence was selected, thereby finally obtaining the plasmid pTyr + egfp-T2A-TCS.

Meanwhile, in order to verify the effect of the recombinant plasmid pTyr + egfp-T2A-TCS specifically inducing melanocyte death, the following examples 2 and 3 were performed.

EXAMPLE 2 transfection of the recombinant plasmid pTyr + egfp-T2A-TCS into TC-1 cells (lung epithelial cells) and B16 cells (melanoma cells), respectively

a. Cell culture (six well plate): TC-1 cells and B16 cells (about 50 million cells per well) were seeded into six-well plates and cultured the day before transfection to achieve a cell density of about 70-80% the next day.

b. Six-well plates with TC-1 cells and B16 cells were replaced with 2ml of fresh medium (complete medium with serum and antibiotics) per well, respectively, prior to transfection.

c. Transfection: a clean sterile centrifuge tube is taken, 125ul of DMEM culture solution (either high-sugar DMEM or low-sugar DMEM) or Opti-MEM granules without antibiotics and serum are respectively added into cells of each hole in a six-hole plate to be transfected, 2.5ug of recombinant plasmid pTyr + egfp-T2A-TCS is added, and the mixture is lightly blown and uniformly mixed by a gun. Then 4. mu.l of Lipo 8000-transfectant was added thereto, and the mixture was gently blown with a gun and mixed. After the preparation is finished, the product is stable within 6 hours at room temperature. Each group of 3 multiple wells.

d. And (3) detection: transient cells were screened 24h after transfection using 400ug/mL G418 (Geneticin), and the expression of fluorescence in both cells was observed 48h later under a fluorescence microscope.

Example 3 transfection of the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS and the recombinant plasmid pTyr + egfp-T2A-TCS into TC-1 and B16 cells, respectively

c. Transfection: a clean sterile centrifuge tube is taken, 125ul of DMEM culture solution (either high-sugar DMEM or low-sugar DMEM) or Opti-MEM Medium which does not contain antibiotics and serum is added into cells of each hole in a six-hole plate to be transfected, 2.5ug of second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS and recombinant plasmid pTyr + egfp-T2A-TCS are added into the cells to be transfected to serve as a treatment group, and the cells are lightly blown and uniformly mixed by a gun. Then, 4. mu.l of Lipo 8000-antibody transfection reagent was added, and the mixture was gently blown with a gun and mixed. After the preparation is finished, the product is stable within 6 hours at room temperature. The control group was a group to which only transfection reagent was added, and the treatment group and the control group were each 3 duplicate wells.

d. And (3) detection: and detecting the necrotic and apoptotic conditions of the cells by a flow cytometer 24 hours after transfection.

1) The cell culture fluid is sucked out into a proper centrifugal tube, adherent cells are washed once by PBS, and a proper amount of pancreatin cell digestive juice (which can contain EDTA) is added to digest the cells. And (4) incubating at room temperature until the adherent cells can be blown down by gentle blowing, and sucking the digestive juice of the pancreatin cells. Excessive digestion of pancreatin is to be avoided.

2) Adding the cell culture solution collected in the step A, slightly mixing, transferring into a centrifugal tube, centrifuging for 5 minutes at 1000g, discarding supernatant, collecting cells, gently resuspending the cells with PBS and counting.

3) About 10-100 million cells per sample were collected in a 1.5ml centrifuge tube and the supernatant discarded by centrifugation. The cell pellet was resuspended in 0.8-1 ml of cell staining buffer.

4) Add 5 microliters of Hoechst staining solution.

5) Add 5 μ l PI staining solution.

6) Mix well and incubate for 20-30 minutes at ice bath or 4 ℃.

7) Red and blue fluorescence was detected with a flow cytometer.

Analysis of results

1. Results of specificity of the recombinant plasmid pTyr + egfp-T2A-TCS for B16 cells

The results are shown in FIGS. 4 and 5. As can be seen, the recombinant plasmid pTyr + egfp-T2A-TCS showed fluorescent expression only in B16 cells and no fluorescent expression in TC-1 cells after 48h of transfection. As can be seen, the recombinant plasmid pTyr + egfp-T2A-TCS has excellent specificity for melanoma cells.

2. Effect of the second recombinant plasmid pcDNA3.1+ egfp-T2A-TCS and the recombinant plasmid pTyr + egfp-T2A-TCS on apoptosis of TC-1 and B16 cells

The results are shown in tables 2 and 3, and FIGS. 6 to 11. It can be seen that the apoptosis rate of B16 cells was significantly higher than that of TC-1 cells in each treatment group. Compared with the control group, the apoptosis rate of the TC-1 cells in the recombinant plasmid pTyr + egfp-T2A-TCS treatment group is only improved by 6.9 percent, while the apoptosis rate of the TC-1 cells in the recombinant plasmid pcDNA3.1+ egfp-T2A-TCS treatment group is improved by 19.4 percent. As can be seen, the recombinant plasmid pTyr + egfp-T2A-TCS hardly induced the apoptosis of TC-1 cells. Compared with the control group, the apoptosis rate of the B16 cell in the recombinant plasmid pTyr + egfp-T2A-TCS treatment group is increased by 22.2%, while the apoptosis rate of the TC-1 cell in the recombinant plasmid pcDNA3.1+ egfp-T2A-TCS treatment group is only increased by 19.4%. Therefore, the recombinant plasmid pcDNA3.1+ egfp-T2A-TCS has no specificity, while the recombinant plasmid pTyr + egfp-T2A-TCS can specifically induce B16 cell apoptosis.

The above results indicate that recombinant plasmid pTyr + egfp-T2A-TCS can specifically induce B16 apoptosis.

TABLE 2 detection of apoptosis of B16 cells 24h after transfection by flow cytometry

TABLE 3 detection of apoptosis of TC-1 cells 24h after transfection by flow cytometry

Example 4 preparation of whitening agent

a. A nonionic/cationic liposome agent (containing GDL (50%), CH (15%), POE-10 (23%) and DOTAP (12%), mixed in a system containing α -tocopherol (1% of total lipid) was used. 100g of total lipids were weighed out in a certain proportion, mixed and heated to 70 ℃ and dissolved in a sterile polystyrene centrifuge tube. The dissolved lipids were filtered through a 0.22 μ M filter, the filtrate was dissolved in a 70 ℃ water bath and drawn into one sterile syringe, and sterile double distilled water preheated to 65 ℃ was added to the other sterile syringe. The two syringes were connected by a sterile three-tube plunger, the aqueous phase was slowly pushed into the lipid phase syringe, then the water-oil mixture was rapidly mixed back and forth, cooled to room temperature under tap water, and the liposomes were stored at 4 ℃ until use, with a lipid concentration of 100mg/mL in the suspension.

b. Before the experiment in the animal body, the liposome suspension is subjected to ultrasonic treatment for 20min at room temperature, then the recombinant plasmid pTyr + egfp-T2A-TCS aqueous solution (7 mg/mL) with the same volume is added, and after mixing, the incubation is carried out for 45min at room temperature, so as to obtain the whitening agent capable of being smeared on the skin of the mouse.

It should be noted that the above description describes certain embodiments of the present disclosure. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some embodiments, multitasking and parallel processing may also be possible or may be advantageous.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the spirit of the present disclosure, features from the above embodiments or from different embodiments may also be combined, steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present description as described above, which are not provided in detail for the sake of brevity.

While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of these embodiments will be apparent to those of ordinary skill in the art in light of the foregoing description.

It is intended that the one or more embodiments of the present specification embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements, and the like that may be made without departing from the spirit and principles of one or more embodiments of the present disclosure are intended to be included within the scope of the present disclosure.

Sequence listing

<110> Guangdong university of pharmacy affiliated first Hospital

<120> recombinant plasmid, whitening agent, preparation method and related application thereof

<130> FI200965-ND

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 423

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttttgacaaa atgagttcta taaatgttat tgtctactta tgatctctaa atacaacagg 60

cttgtattca gaatctagat gtttcatgac ctttattcat aagagatgat gtattcttga 120

tactacttct catttgcaaa ttccaattat tattaatttc atatcaatta gaataatata 180

tcttccttca atttagttac ctcactatgg gctatgtaca aactccaaga aaaagttagt 240

catgtgcttt gcagaagata aaagcttagt gtaaaacagg ctgagagtat ttgatgtaag 300

aaggggagtg gttatatagg tcttagccaa aacatgtgat agtcactcca ggggttgctg 360

gaaaagaagt ctgtgacact cattaaccta ttggtgcaga ttttgtatga tctaaaggag 420

aaa 423

<210> 2

<211> 717

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

<210> 3

<211> 63

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggaagcggag agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggc 60

ccg 63

<210> 4

<211> 6765

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtttttgac 240

aaaatgagtt ctataaatgt tattgtctac ttatgatctc taaatacaac aggcttgtat 300

tcagaatcta gatgtttcat gacctttatt cataagagat gatgtattct tgatactact 360

tctcatttgc aaattccaat tattattaat ttcatatcaa ttagaataat atatcttcct 420

tcaatttagt tacctcacta tgggctatgt acaaactcca agaaaaagtt agtcatgtgc 480

tttgcagaag ataaaagctt agtgtaaaac aggctgagag tatttgatgt aagaagggga 540

gtggttatat aggtcttagc caaaacatgt gatagtcact ccaggggttg ctggaaaaga 600

agtctgtgac actcattaac ctattggtgc agattttgta tgatctaaag gagaaagcta 660

gcgtttaaac ttaagcttgg taccgagctc ggatccacta gtccagtgtg gtggaattct 720

gcagatgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 780

gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 840

gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 900

ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 960

gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 1020

cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 1080

ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1140

atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1200

aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1260

gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1320

cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1380

gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1440

ctgtacaagg gaagcggaga gggcagggga agtcttctaa catgcgggga cgtggaggaa 1500

aatcccggcc cgatgatcag attcctggtg ctgagcctgc tgatcctgac cctgttcctg 1560

accacccccg ctgtggaagg agacgtgtct ttcagactga gcggagccac cagcagcagc 1620

tacggagtgt tcatcagcaa cctgagaaag gccctgccca acgaacgcaa actgtacgac 1680

atcccactgc tgagaagctc cctgcccgga tcacagagat acgccctgat ccacctgacc 1740

aactacgccg acgaaaccat cagcgtggcc atcgacgtga caaacgtgta catcatgggc 1800

taccgggccg gcgatacctc ttactttttc aacgaggcct ccgccaccga agccgctaaa 1860

tacgtgttca aggacgccat gagaaaagtg accctgccat actctggcaa ctacgagcgc 1920

ctgcagaccg ctgctggaaa gatcagagag aacatccccc tgggcctgcc cgctctggat 1980

tctgctatca ccaccctgtt ctactataac gccaacagcg ccgcctccgc cctgatggtg 2040

ctgatccaga gtacaagcga ggccgccaga tacaagttca tcgaacagca gatcggcaag 2100

agagtggaca aaaccttcct gcccagcctg gccatcatca gcctggaaaa cagctggtcc 2160

gccctgagca aacagattca gatcgccagc accaacaacg gccagttcga gagccccgtg 2220

gtgctgatta acgcccagaa ccagagagtg accatcacca acgtggacgc cggcgtggtg 2280

acatcaaaca tcgccctgct gatccagcac agtggcggcc gctcgagtct agagggcccg 2340

tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 2400

cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 2460

atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 2520

ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 2580

gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 2640

cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2700

ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2760

ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2820

tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2880

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2940

tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 3000

ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 3060

attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 3120

cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3180

ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3240

gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 3300

tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt 3360

ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc 3420

ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga 3480

acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 3540

ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 3600

gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga 3660

ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 3720

tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 3780

gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 3840

gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 3900

agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 3960

ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga 4020

tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 4080

ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 4140

ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 4200

ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 4260

cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca 4320

cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 4380

gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 4440

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 4500

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 4560

tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 4620

tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 4680

aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 4740

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 4800

gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 4860

cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 4920

tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 4980

aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5040

catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5100

caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5160

ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 5220

aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5280

gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5340

cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5400

ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 5460

tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 5520

tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc 5580

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5640

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5700

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5760

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5820

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5880

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5940

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 6000

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6060

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 6120

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6180

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6240

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6300

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6360

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6420

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6480

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6540

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6600

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6660

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6720

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc 6765

<210> 5

<211> 48

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccagtgtggt ggaattctgc agatgccacc atggtgagca agggcgag 48

<210> 6

<211> 106

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cggccgccac tgtgctggat atcgggccgg gattttcctc cacgtccccg catgttagaa 60

gacttcccct gccctctccg cttcccttgt acagctcgtc catgcc 106

<210> 7

<211> 70

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgatgtacgg gccagatata cgcgtttttg acaaaatgag ttctataaat gttattgtct 60

acttatgatc 70

<210> 8

<211> 62

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

accaagctta agtttaaacg ctagctttct cctttagatc atacaaaatc tgcaccaata 60

gg 62

Claims

1. A recombinant plasmid comprising a pTyr promoter, egfp gene, T2A gene and TCS gene on a base plasmid; the egfp gene, the T2A gene and the TCS gene are located between the pTyr promoter and the terminator of the base plasmid; the nucleotide sequence of the pTyr promoter is shown as SEQ ID NO. 1; the nucleotide sequence of the egfp gene is shown as SEQ ID NO. 2; the nucleotide sequence of the T2A gene is shown in SEQ ID NO. 3.

2. The recombinant plasmid of claim 1, wherein the base plasmid is pcDNA3.1+, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO. 4.

3. A method for preparing a recombinant plasmid, comprising:

4. The method for preparing recombinant plasmid of claim 3, wherein the step of obtaining the first recombinant plasmid pcDNA3.1+ egfp-T2A specifically comprises:

5. The method for preparing the recombinant plasmid according to claim 3, wherein the step of enzymatically digesting the first recombinant plasmid pcDNA3.1+ egfp-T2A specifically comprises:

6. The method for preparing the recombinant plasmid according to claim 3, wherein the step of obtaining the recombinant plasmid pTyr + egfp-T2A-TCS specifically comprises: