CN107287241B

CN107287241B - Expression vector for promoting expression of 3 β -HSD gene and construction method and application thereof

Info

Publication number: CN107287241B
Application number: CN201710405615.6A
Authority: CN
Inventors: 徐新云; 王利; 毛侃琅; 彭鹏
Original assignee: Center Of Diseases Prevention & Control Shenzhen City
Current assignee: Center Of Diseases Prevention & Control Shenzhen City
Priority date: 2017-06-01
Filing date: 2017-06-01
Publication date: 2020-03-27
Anticipated expiration: 2037-06-01
Also published as: CN107287241A

Abstract

The expression vector comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, wherein the target gene expression fragment is inserted between an EcoRI enzyme cutting site and a NotI enzyme cutting site in the positive direction.

Description

Expression vector for promoting expression of 3 β -HSD gene and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an expression vector for promoting expression of a 3 β -HSD gene, a construction method and application thereof.

Background

Steroid hormones play an important role in the differentiation, development, growth and exertion of physiological functions of most vertebrate cells, the production of steroid hormones depends on the provision of raw cholesterol and the catalysis of a range of steroid-producing enzymes, among which 3 β -super steroid deaminase (3 β -HSD) plays an important role in the biosynthesis of glucocorticoids, mineralocorticoids and sex hormones.

In the traditional 3 β -HSD gene expression technology, for example, the regulation of rat testicular interstitial cell 3 β -HSD gene expression is realized by using gonadotropin releasing hormone agonist through ERKMAPK pathway by suviet al, and regulation of hCG on 3 β -HSD expression in Leydig cell of adult mouse testis is researched by thaumatin and the like.

The researches are all that 3 β -HSD gene expression is regulated and controlled by applying an indirect means, and an expression vector which can be transfected into a human cell and can continuously, stably and efficiently express the 3 β -HSD gene in the human cell is lacked at present.

Disclosure of Invention

Based on the above, it is necessary to provide an expression vector capable of expressing 3 β -HSD gene in human cells continuously, stably and efficiently, and a construction method and application thereof.

An expression vector for promoting expression of a 3 β -HSD gene comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of a lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro, wherein the multiple cloning site comprises an EcoRI enzyme cutting site and a NotI enzyme cutting site, the target gene expression fragment contains a 3 β -HSD gene coding sequence for expressing 3 β -HSD, the 5 'end of the target gene expression fragment is provided with an EcoRI enzyme cutting site cohesive tail end, the 3' end of the target gene expression fragment is provided with a NotI enzyme cutting site cohesive tail end, and the target gene expression fragment is positively inserted between the EcoRI enzyme cutting site and the NotI enzyme cutting site of the multiple cloning site sequence.

In one embodiment, the nucleotide sequence of the coding sequence of 3 β -HSD gene is set forth in SEQ ID No. 1.

In one embodiment, the 3 'end of the cohesive end of the EcoRI cleavage site is directly linked to the 5' end of the coding sequence of the 3 β -HSD gene, the 5 'end of the cohesive end of the NotI cleavage site is directly linked to the 3' end of the coding sequence of the 3 β -HSD gene, the base sequence of the cohesive end of the EcoRI cleavage site is 5 '-aattc-3', and the base sequence of the cohesive end of the NotI cleavage site is 5 '-ggccgc-3'.

The method for constructing the expression vector for promoting the expression of the 3 β -HSD gene comprises the following steps:

taking a 3 β -HSD gene coding sequence as an amplification template, adding an upstream primer and a downstream primer, and then carrying out PCR amplification to obtain a PCR amplification product, wherein the upstream primer comprises a sequence designed aiming at the 5 'end of the 3 β -HSD gene coding sequence and a sequence designed aiming at an EcoRI enzyme cutting site, and the downstream primer comprises a sequence designed aiming at the 3' end of the 3 β -HSD gene coding sequence and a sequence designed aiming at a NotI enzyme cutting site;

carrying out A tail adding reaction on the PCR amplification product to enable two ends of the PCR amplification product to be connected with an A base, and connecting the PCR amplification product subjected to the A tail adding reaction to a T vector to obtain a recombinant T vector;

carrying out double enzyme digestion on the recombinant T vector by using restriction enzymes EcoRI and NotI to obtain a target gene expression fragment; and

and connecting the target gene expression fragment to a lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro subjected to double enzyme digestion treatment by restriction enzyme EcoRI and restriction enzyme NotI to obtain the expression vector for promoting the expression of the 3 β -HSD gene.

In one embodiment, the nucleotide sequence of the coding sequence of the 3 β -HSD gene is shown as SEQ ID No.1, the base sequence of the upstream primer is shown as SEQ ID No.2, and the base sequence of the downstream primer is shown as SEQ ID No. 3.

A lentivirus containing a 3 β -HSD gene is prepared by the following method:

transfecting the expression vector for promoting the expression of the 3 β -HSD gene into 293FT cells, and

and amplifying and expressing the 293FT cells after transfection to obtain the lentivirus containing the 3 β -HSD gene.

The application of a composition in preparing an anti-tumor medicament comprises the expression vector for promoting the expression of the 3 β -HSD gene.

In one embodiment, the composition further comprises a plasticizer.

In one embodiment, the plasticizer is di (2-ethylhexyl) phthalate.

In one embodiment, the composition is used for preparing a medicament for resisting breast cancer.

The expression vector for promoting the expression of the 3 β -HSD gene comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, wherein the multiple cloning site sequence comprises an EcoRI restriction site and a NotI restriction site, the target gene expression fragment contains a 3 β -HSD gene coding sequence for expressing 3 β -HSD, the 5 'end of the target gene expression fragment is provided with an EcoRI restriction site cohesive end, the 3' end is connected with a NotI restriction site cohesive end, the target gene expression fragment is positively inserted between the EcoRI restriction site and the NotI restriction site of the multiple cloning site sequence, the inventor conducts a large amount of research on vector selection, a target gene insertion site, a construction method and other aspects, unexpectedly discovers that the target gene expression fragment containing the 3 β -HSD gene coding sequence is inserted between the EcoRI restriction site and the NotI restriction site of a lentiviral vector pLVX-mCGGrsGrK-PGK-expression vector, the expression vector and the expression vector are capable of increasing the expression of the tumor cell expression of the tumor-transfected human tumor receptor-transfected by the human MCHSD gene expression vector pLVX-ZsGrrC, the expression vector is expected to increase the expression of the tumor-transfected tumor cell expression vector, the tumor-expressing the tumor cell expressing the tumor-expressing the tumor cell expressing the tumor.

Drawings

FIG. 1 is a flowchart of a method for constructing an expression vector for promoting expression of 3 β -HSD gene according to one embodiment;

FIG. 2 is an agarose gel electrophoresis of the PCR amplification product of the coding sequence of the 3 β -HSD gene in example 1;

FIG. 3 is a map of the pLVX-mCMV-ZsGreen-PGK-Puro backbone vector used in example 1;

FIG. 4 is a graph showing the results of fluorescent quantitative PCR detection of mRNA expression levels of 3 β -HSD in MCF-7 cells and MCF-7 cells transfected with an expression vector promoting expression of 3 β -HSD gene in example 4;

FIG. 5 is a graph showing the results of Western blot detection of the expression levels of 3 β -HSD proteins in two groups of cells in example 5;

FIG. 6 is a graph showing the results of quantitative analysis of the expression level of 3 β -HSD protein by grayscale using Image J software on the Image in FIG. 5;

FIG. 7 is a graph showing the results of detecting the mRNA expression level of the apoptosis gene Bax of two groups of breast cancer cells MCF-7 under the conditions of different concentrations of DEHP by fluorescent quantitative PCR in example 6;

FIG. 8 is a graph showing the results of detecting the mRNA expression level of the apoptosis gene Caspase-3 of two groups of breast cancer cells MCF-7 under the conditions of DEHP at different concentrations by fluorescent quantitative PCR in example 6;

FIG. 9 is a graph showing the results of detecting the mRNA expression levels of the apoptosis gene Caspase-8 of two sets of breast cancer cells MCF-7 under the conditions of DEHP at different concentrations by fluorescent quantitative PCR in example 6;

FIG. 10 is a graph showing the results of the fluorescent quantitative PCR assay for the expression level of mRNA of the estrogen receptor gene ER α in two groups of breast cancer cells MCF-7 under the conditions of different concentrations of DEHP in example 6;

FIG. 11 is a graph showing the results of the fluorescent quantitative PCR assay for the expression level of mRNA of the estrogen receptor gene ER β in two groups of breast cancer cells MCF-7 under the conditions of different concentrations of DEHP in example 6;

FIG. 12 is a graph showing the results of the fluorescent quantitative PCR assay for the mRNA expression level of the DNA damage gene hOGG1 in two groups of breast cancer cells MCF-7 under the condition of varying concentrations of DEHP in example 6;

FIG. 13 is a graph showing the results of the fluorescent quantitative PCR assay for the mRNA expression level of the DNA damage gene MTH1 in two groups of breast cancer cells MCF-7 under the conditions of different concentrations of DEHP in example 6.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The expression vector for promoting the expression of the 3 β -HSD gene comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, wherein the multiple cloning site comprises an EcoRI enzyme cutting site and a NotI enzyme cutting site, the target gene expression fragment contains a 3 β -HSD gene coding sequence for expressing the 3 β -HSD, the 5 'end of the target gene expression fragment is provided with an EcoRI enzyme cutting site cohesive end, the 3' end of the target gene expression fragment is provided with a NotI enzyme cutting site cohesive end, and the target gene expression fragment is positively inserted between the EcoRI enzyme cutting site and the NotI enzyme cutting site of the multiple cloning site sequence.

In particular, the resistance gene sequence is used to screen for transformed expression vectors.

In one embodiment, the resistance gene sequence is selected from at least one of an ampicillin resistance gene sequence and a kanamycin resistance gene sequence.

Specifically, the sequence of the multiple cloning site includes multiple restriction sites, and besides an EcoRI restriction site and a NotI restriction site, the sequence of the multiple cloning site may also include an HpaI restriction site, a SfiI restriction site, an SbfI restriction site, and the like.

In particular, promoter (Promoters) sequences are used to control the initiation time and extent of expression of genes in expression vectors.

In one embodiment, the 3 'end of the cohesive end of the EcoRI cleavage site is directly linked to the 5' end of the coding sequence of the 3 β -HSD gene, and the 5 'end of the cohesive end of the NotI cleavage site is directly linked to the 3' end of the coding sequence of the 3 β -HSD gene, further, the base sequence of the cohesive end of the EcoRI cleavage site is 5 '-aattc-3', and the base sequence of the cohesive end of the NotI cleavage site is 5 '-ggccgc-3'.

Specifically, the target gene expression fragment contains a 3 β -HSD gene coding sequence for expressing 3 β -HSD, and the 3 β -HSD gene coding sequence is obtained by screening from an NCBI database.

In one embodiment, the 3 β -HSD gene coding sequence is designed according to the mRNA sequence of 3 β -HSD, specifically GenBank NM-001328615 of the selected NCBI database, and the nucleotide sequence of the 3 β -HSD gene coding sequence is shown in SEQ ID No. 1.

In a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, a 3 β -HSD gene coding sequence exists in a double-chain form, the 5 'end of the 3 β -HSD gene coding sequence is connected with an EcoRI enzyme cutting site cohesive end, the EcoRI enzyme cutting site cohesive end comprises a base sequence which is cut by EcoRI enzyme, for example, the 3' end of the aattc.3 β -HSD gene coding sequence is connected with a NotI enzyme cutting site cohesive end, the NotI enzyme cutting site end comprises a base sequence which is cut by NotI enzyme, for example tcgag, and the EcoRI enzyme cutting site cohesive end are arranged so that the 3 β -HSD gene coding sequence can be smoothly inserted into the lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro.

Furthermore, the cohesive end of the EcoRI enzyme cutting site and the cohesive end of the NotI enzyme cutting site also contain protective sequences, so that the enzyme cutting efficiency is improved.

The inventor carries out a great deal of research on the aspects of vector selection, target gene insertion sites, construction methods and the like, and unexpectedly discovers that a target gene expression fragment containing a 3 β -HSD gene coding sequence is positively inserted between an EcoRI enzyme cutting site and a NotI enzyme cutting site of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro to successfully construct an expression vector capable of specifically promoting 3 β -HSD gene expression.

Further, the expression vector can also increase the toxicity of the plasticizer to tumor cells. Experimental results show that the expression levels of the apoptosis gene, the estrogen receptor gene and the DNA oxidative damage gene of the human breast cancer cell MCF-7 transfected with the expression vector are higher than those of the non-transfected human breast cancer cell MCF-7, the expression vector can accelerate the apoptosis of tumor cells and increase the damage effect of a plasticizer on the tumor cells, and the expression vector is expected to be applied to anti-tumor drugs and provides a thought for tumor treatment.

Referring to fig. 1, the method for constructing the expression vector for promoting expression of the 3 β -HSD gene according to an embodiment includes the following steps S110 to S140.

S110, taking the coding sequence of the 3 β -HSD gene as an amplification template, adding an upstream primer and a downstream primer, and carrying out PCR amplification to obtain a PCR amplification product, wherein the upstream primer comprises a sequence designed aiming at the 5 'end of the coding sequence of the 3 β -HSD gene and a sequence designed aiming at an EcoRI enzyme cutting site, and the downstream primer comprises a sequence designed aiming at the 3' end of the coding sequence of the 3 β -HSD gene and a sequence designed aiming at a NotI enzyme cutting site.

In one embodiment, the 3 β -HSD gene coding sequence is designed according to the mRNA sequence of 3 β -HSD, specifically GenBank NM-001328615 of NCBI database is screened, the nucleotide sequence of the 3 β -HSD gene coding sequence is designed as shown in SEQ ID No.1, and the designed 3 β -HSD gene coding sequence can be obtained by means of gene synthesis.

The upstream primer comprises a sequence designed aiming at the 5 'end of the coding sequence of the 3 β -HSD gene and a sequence designed aiming at an EcoRI enzyme cutting site, the downstream primer comprises a sequence designed aiming at the 3' end of the coding sequence of the 3 β -HSD gene and a sequence designed aiming at a NotI enzyme cutting site, and the coding sequence of the 3 β -HSD gene is provided with the EcoRI enzyme cutting site and the NotI enzyme cutting site through the amplification of the upstream primer and the downstream primer.

Specifically, the upstream primer comprises a sequence for amplifying an EcoRI enzyme cutting site and a protection sequence, and the downstream primer comprises a sequence for amplifying a NotI enzyme cutting site and a protection sequence, so that a PCR amplification product obtained by PCR amplification has the EcoRI enzyme cutting site and the NotI enzyme cutting site. The upstream and downstream primers have no primer dimer and have small annealing temperature difference.

In one embodiment, the base sequence of the upstream primer is shown as SEQ ID No.2, and the base sequence of the downstream primer is shown as SEQ ID No. 3.

Specifically, a reaction system is configured according to the instructions of Premix Prime 3 β -HSD HS to carry out PCR, the annealing temperature is 62 ℃, agarose gel electrophoresis identification is carried out after PCR amplification is finished, and then a PCR product of about 1000bp is recovered by a DNA gel recovery kit to obtain a PCR amplification product.

And S120, carrying out A tail adding reaction on the PCR amplification product obtained in the S110, connecting A bases at two ends of the PCR amplification product, and connecting the PCR amplification product subjected to the A tail adding reaction to a T vector to obtain a recombinant T vector.

Specifically, the A-tail reaction is carried out on the PCR amplification product by using ExTaq enzyme, referring to the using instruction of the ExTaq enzyme, A bases are respectively added to two ends of the PCR amplification product under the condition of the ExTaq enzyme to form the PCR amplification product with the A-tail, so that the PCR amplification product can be smoothly connected into a T carrier.

Specifically, referring to the instruction manual of T4DNA ligase, PCR amplification products after adding A base at both ends are connected to a T vector to form a recombinant T vector.

The method specifically comprises the steps of transforming the recombinant T vector into Escherichia coli competent bacteria, screening to obtain positive monoclonals, culturing the positive monoclonals, and extracting to obtain a large amount of recombinant T vectors, wherein the Escherichia coli competent bacteria are JM107 competent bacteria or DH5 α competent bacteria and the like.

In this embodiment, the recombinant T vector is transformed into JM107 competent bacteria, spread on a resistant medium, picked up from positive monoclonal and cultured in small amounts and sequenced for identification. And then, carrying out amplification culture on the bacterial liquid with the correct sequencing, and extracting the recombinant T vector in the bacteria.

And S130, carrying out double enzyme digestion on the recombinant T vector obtained in the S120 by using restriction enzymes EcoRI and NotI to obtain a target gene expression fragment.

Specifically, selecting a recombinant T vector with correct sequencing, carrying out NotI enzyme digestion, recovering an enzyme digestion product by using a nucleic acid coprecipitation agent, carrying out enzyme digestion by using EcoRI, carrying out electrophoretic identification, and recovering the enzyme digestion product by using a DNA gel recovery kit to obtain a target gene expression fragment. The 5 'end of the target gene fragment is provided with an EcoRI enzyme cutting site cohesive end, and the 3' end of the target gene fragment is provided with a NotI enzyme cutting site cohesive end.

And S140, connecting the target gene expression fragment obtained in the S130 to the lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro subjected to double enzyme cutting treatment by restriction enzyme EcoRI and restriction enzyme NotI to obtain the expression vector for promoting the expression of the 3 β -HSD gene.

The lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro is subjected to double enzyme digestion treatment by restriction enzyme EcoRI and restriction enzyme NotI, and a gap is opened, so that a target gene expression fragment is smoothly inserted between an EcoRI enzyme digestion site and a NotI enzyme digestion site of the lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro, and the expression vector for promoting the expression of the 3 β -HSD gene is obtained.

The lentivirus containing the 3 β -HSD gene is prepared by transfecting the expression vector promoting the expression of the 3 β -HSD gene into 293FT cells, and amplifying and expressing the 293FT cells after transfection to obtain the lentivirus containing the 3 β -HSD gene.

Specifically, after the transfected 293FT cells are cultured for 36-60 h, a cell supernatant culture medium is collected and filtered by a filter membrane to obtain a lentivirus supernatant containing the 3 β -HSD gene, the lentivirus containing the 3 β -HSD gene is obtained, and the virus titer is determined by using a Lenti-X GoStix gold-labeled kit and then the lentivirus titer is stored at-80 ℃.

The construction method of the expression vector for promoting the expression of the 3 β -HSD gene is simple to operate, a large amount of research and research are carried out on aspects of vector selection, target gene insertion sites, construction methods and the like, and a target gene expression fragment containing a 3 β -HSD gene coding sequence is positively inserted between an EcoRI enzyme cutting site and a NotI enzyme cutting site of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro to successfully construct the expression vector capable of specifically promoting the expression of the 3 β -HSD gene.

Also provided herein is the use of a composition of an embodiment comprising the above expression vector for promoting expression of 3 β -HSD gene in the preparation of a medicament for the treatment of tumors.

In particular, the composition can be a vaccine applied to prevent tumor, and can also be a drug applied to treat tumor and even cancer which are already diagnosed.

Furthermore, the composition is used for preparing a medicine for resisting breast cancer.

In one embodiment, the composition further comprises a plasticizer.

Specifically, the plasticizer is di (2-ethylhexyl) phthalate (DEHP).

Further, the concentration of the plasticizer in the composition is 0.05mmol/L or more, preferably 0.80mmol/L or more.

It is understood that pharmaceutical adjuvants such as starch, sodium stearate, etc. may also be included in the composition.

In one embodiment, the composition is used in an anti-tumor drug for promoting expression of apoptosis genes Bax, Caspase-3 or Caspase-8. Preferably, the composition is applied to an anti-tumor drug for promoting apoptosis gene Bax.

In one embodiment, the composition is applied to an anti-tumor drug for promoting the expression of the estrogen receptor gene ER α or ER β.

In one embodiment, the composition is used in an anti-tumor drug for promoting the expression of DNA oxidative damage gene hOGG1 or MTH 1.

Furthermore, the composition is applied to antitumor drugs which simultaneously promote the expression of apoptosis genes, estrogen receptor genes and DNA oxidative damage genes.

Furthermore, the composition formed by the plasticizer and the expression vector for promoting the expression of the 3 β -HSD gene is applied to the antitumor drugs for simultaneously promoting the expression of the apoptosis gene, the estrogen receptor gene and the DNA oxidative damage gene.

Experimental results show that after the expression vector for promoting the expression of the 3 β -HSD gene is transfected into a breast cancer cell line MCF-7 cell, the expression level of the 3 β -HSD expressed by the MCF-7 is 104.5 times that of a control group, and the expression quantity has obvious difference.

In addition, even under the condition that the concentration of the plasticizer is 0mmol/L, namely the plasticizer does not exist, the expression levels of the apoptosis gene Bax, Caspase-3 or Caspase-8, the estrogen receptor gene ER α, ER β and the DNA oxidative damage gene hOGG1 of the human breast cancer cell MCF-7 transfected by the expression vector are higher than those of the non-transfected human breast cancer cell MCF-7, which shows that the expression vector for promoting the expression of the 3 β -HSD gene has toxicity to the tumor cells and has the damage effect on the tumor cells, and is expected to be applied to antitumor drugs.

The following is a detailed description of the embodiments.

In the following examples, unless otherwise specified, the experimental procedures without specifying the specific conditions are usually carried out according to conventional conditions, for example, the conditions described in the molecular cloning's Experimental guidelines [ M ] (Beijing: scientific Press, 1992) by Sammbruke, EF Friech, T Mannich, et al (translated by Kindong goose, Rimeng maple, et al) or the procedures recommended by the manufacturers of the kits. The reagents used in the examples are all commercially available.

The materials used in the examples were restriction enzymes EcoRI, NotI (Fermentas USA), T4DNA ligase (Fermentas USA), DNA gel recovery Kit RNeasy Mini Kit (QIAGEN, Germany), Prime script RT reagent Kit and SYBR Primescript RT-PCR Kit (TAKARA, China), plasmid miniprep and endotoxin-free plasmid miniprep Kit (OMEGA bio-tek, USA), J.coli 107 competence (Tiangen Bio Inc.), eukaryotic expression vector pLVX-mCUV-ZSgreen-PGLC-Puro (pLVX vector, Biovector), packaging helper plasmid PLVX-GFP and the corresponding lentiviral packaging Kit (Clonteche, Clontech), 293FT cells for lentiviral packaging (US Itrogen), RPMI-1640-E culture medium and 10% fetal calf serum (Gibco Cassia), heat goat packaging Kit (Clontech, USA), anti-rabbit DNA library DNA polymerase (MCH-7), anti-DNA polymerase chain reaction primers, anti-DNA polymerase chain reaction (SAGE-DNA polymerase chain reaction products) (RG, DNA polymerase chain reaction products, DNA recovery Kit, plasmid.

Example 1

Construction of expression vector for promoting expression of 3 β -HSD Gene

(1)3 β -HSD target gene expression fragment primer design.

A3 β -HSD gene coding sequence is designed according to NCBI database GenBank NM _001328615, a 3 β -HSD gene coding sequence (a cDNA sequence of 3 β -HSD) with a nucleotide sequence shown as SEQ ID No.1 is obtained, Oligo7 is used for analyzing the coding sequence, an upstream primer and a downstream primer are searched (no primer dimer is required as far as possible and the annealing temperature difference is small), then a protective base and an EcoR I enzyme cutting site sequence are added to the 5 'end of the upstream primer, a protective base and a Not I enzyme cutting site sequence are added to the 5' end of the downstream primer, the designed primer sequence is shown in Table 1, and the designed PCR primer is synthesized by Shanghai Bioengineering technology service Limited.

TABLE 1 PCR amplification primers for the coding sequence of 3 β -HSD gene

Upstream primer	5’-GGAATTCCATGACGGGCTGGAGCTG-3’(SEQ ID No.2)
		Downstream primer	5’-CGCGGCCGCCTGAGTCTTGGACTTCAG-3’(SEQ ID No.3)

(2) Amplification of 3 β -HSD gene coding sequence (cDNA) and construction of recombinant T vector thereof

Preparing a reaction system according to instructions of Premix Prime 3 β -HSD HS to perform PCR, carrying out agarose gel electrophoresis identification after the PCR is finished at an annealing temperature of 62 ℃, obtaining a result shown in figure 2, then recovering a PCR product of about 1000bp by using a DNA gel recovery kit to obtain a PCR amplification product, carrying out A tail reaction by using ExTaq enzyme, connecting the A tail product with a T carrier according to the instructions of T4DNA ligase, transforming JM107 competent bacteria, carrying out conventional screening identification, selecting a small amount of cultured bacterial liquid of PCR identification positive clones to carry out sequencing identification, carrying out amplification culture on the bacterial liquid with correct sequencing, and extracting a recombinant T carrier in the bacteria.

(3) Expression vector for promoting expression of 3 β -HSD gene and identification

Selecting a recombinant T vector with correct sequencing, carrying out NotI enzyme digestion, recovering an enzyme digestion product by using a nucleic acid coprecipitation agent, carrying out EcoRI enzyme digestion, carrying out electrophoretic identification, and then recovering the enzyme digestion product by using a DNA gel kit to obtain a target gene expression fragment, connecting the target gene expression fragment with a pLVX-mCMV-ZsGreen-PGK-Puro lentiviral vector subjected to the same treatment, referring to a spectrogram of a pLVX-mCMV-ZsGreen-PGK-Puro skeleton vector in figure 3, further converting the connected vector into JM107 competent bacteria, carrying out PCR identification on the bacteria selected by small-scale culture, carrying out sequencing identification on the bacteria, carrying out amplification culture on the bacteria solution in which the cDNA sequence of the 3 β -HSD gene is correctly inserted by using a plasmid sequencing result, extracting a recombinant plasmid by using an endotoxin extraction kit, and obtaining an expression vector (pLVX-3 β -HSD) for specifically promoting the expression of the 3 β -HSD gene.

Example 2

Preparation of lentivirus containing 3 β -HSD Gene

293FT cells were cultured, and cells in good growth state were inoculated into six wells 10 per well⁶For each cell, 2. mu.g of the pLVX-3 β -HSD recombinant vector extracted in example 1 was transfected into 293FT cells using a lentivirus packaging helper kit, cultured at 37 ℃ for 48 hours, then the supernatant medium was filtered through a 0.45. mu.M filter, the virus supernatant was collected, the virus titer was determined using a Lenti-X GoStix colloidal gold kit, and then stored at-80 ℃.

Example 3

Lentivirally transduced human breast cancer MCF-7 cells

The virus supernatant obtained in example 2 was diluted 1:1 with RPMI-1640 complete medium and Polybrene was added to a final concentration of 6. mu.g/mL-10. mu.g/mL for further use. Will be 3X 10⁵MCF-7 cells were inoculated into a T25 cell culture flask, the confluency of the cells reached 50% after 18 hours of culture, the original complete medium in the flask was removed, washed twice with PBS and the complete medium was added to the above lentivirus-containing RPMI-1640. Transfection for 24h to remove lentivirusAdding normal RPMI-1640 complete medium into RPMI-1640 complete medium, culturing for 24h, selecting cells with puromycin of 1 μ g/mL, changing the medium 1 time every 2 days for 7 days, and selecting to obtain MCF-7 cell strain (pLVX-3 β -HSD cell strain) with high expression of 3 β -HSD gene.

Example 4

Fluorescent quantitative PCR detection of 3 β -HSD gene expression

PCR primers were designed using primer design software Oligo 7.0 based on GAPDH (GenBank NM-002046.5) and 3 β -HSD (GenBank NM-001328615) gene mRNA sequences, and the primer sequences are shown in Table 2.

TABLE 2 PCR primers for GAPDH and 3 β -HSD genes

MCF-7 cells and MCF-7 cell line (pLVX-3 β -HSD cell line) highly expressing the 3 β -HSD gene prepared in example 3 were inoculated into a 6-well plate, respectively, and when the cell density reached 80% to 90%, total RNA of each group of cells was extracted using RNeasy Mini Kit, and mRNA was reverse-transcribed into cDNA using PrimeScript RT reagent Kit under conditions of 37 ℃ for 15min, 85 ℃ for 5s, 4 ℃ and infinity, after the reverse transcription was completed, 90. mu.L of RNase Free dH2O was added to dilute the cDNA, and the cDNA was stored at-20 ℃ for later use.

Taking 1 microliter of cDNA of each group of cells as a template, respectively adding primers in a table 2, detecting the relative expression quantity of 3 β -HSD by using GAPDH as an internal reference and real-time fluorescence quantitative PCR, setting reaction conditions of 95 ℃ 30s, 1 cycle, 54 ℃ 30s, 40 cycles, 95 ℃ 5s, 60 ℃ 1min and 95 ℃ 15s, and detecting the relative expression quantity of the 3 β -HSD gene of each group of cells by using SYBR Primescript RT-PCR Kit, wherein the result is shown in figure 4. the result shows that the expression level of the 3 β -HSD gene of a pLVX-3 β -HSD cell strain is improved by 104.5 times compared with that of a normal MCF-7 cell, and has very obvious difference (p is less than 0.01), which indicates that the cDNA sequence of the 3 β -HSD gene provided by the invention is successfully inserted into a pLVX-mCCV-ZSgreen-PGLC-PURO slow virus vector, and can specifically, continuously, efficiently and stably promote the expression of the 3-HSD gene 3 β -HSD gene.

Example 5

Western blot detection of 3 β -HSD protein expression level

Normal MCF-7 cells and pLVX-3 β -HSD cell line prepared in example 3 were inoculated into T25 cell culture flasks (1X 10), respectively⁶Removing the culture medium after the confluence of the cells reaches 90% after culturing for about 24 hours, removing the culture medium, absorbing the cell culture solution, washing the cells with cold PBS for 3 times, adding cell lysate to extract total cell proteins, denaturing at 100 ℃ for 8min, quantifying the proteins by a bicinchoninic acid method (BCA method), carrying out 10% SDS-PACE electrophoretic separation on the total proteins, then electrically transferring the proteins onto a PVDF membrane, sealing 5% skimmed milk powder at room temperature for 2h, respectively cutting off the parts containing GAPDH (37kD) and 3- β -HSD protein (42kD) on the membrane according to Marker and molecular weight, adding corresponding primary antibody, incubating on ice overnight, washing the membrane with TBST buffer for 3 times, adding corresponding secondary antibody for 3 times each 10min, incubating for 1h at room temperature, washing the membrane with TBST buffer for 3 times, adding Western blot chemiluminescence reagent light for 10min each time, exposing the membrane to a cold imaging system, and obtaining an image with software for quantitative analysis, wherein the difference of the expression level of the HSD protein is shown in a pLVX 3- β -32-HSD statistical comparison graph, and the difference of the expression level of the HSD is shown in a statistical analysis software shown in figure 5<0.01), the result shows that the pLVX-3 β -HSD cell strain is successfully constructed, and the 3 β -HSD protein can be continuously, efficiently and stably highly expressed.

Example 6

Effect of expression vector promoting expression of 3 β -HSD Gene on expression amounts of mRNA of apoptosis Gene, Estrogen receptor Gene and DNA Oxidation injury Gene

MCF-7 cells and pLVX-3 β -HSD cell strains (MCF-7 cells with high expression of 3 β -HSD gene) prepared in example 3 are respectively inoculated into a 6-well plate, DEHP contamination is carried out when the cell fusion degree reaches 80%, the final concentrations of DEHP are respectively 0.05, 0.1, 0.2, 0.4 and 0.8mmol/L, 5 thousandth DMSO is used as a control, after the cell contamination is finished for 24h, the changes of the expression levels of the Bax, Caspase-3, Caspase-8, ER α, ER β, hOG 1 and MTH1 genes in each group of cells are respectively determined by a method for detecting the expression level of the 3 β -HSD gene by fluorescent quantitative PCR in example 4, and specifically, the changes of the expression levels of the Bax, Caspase-3, Caspase-8, ER α, ER β, hOG 1 and MTH1 genes are extracted by an RNeasy Mini KitTotal RNA of group cells, reverse transcription of mRNA to cDNA using primescrrip RT reagent Kit, reverse transcription conditions: 15min at 37 ℃; 5s at 85 ℃; storing at 4 ℃. After the reverse transcription was complete, 90. mu.L of RNase Free dH was added₂The cDNA was diluted O and stored at-20 ℃ for later use in assays.

Taking 1 mu L of cDNA of each group of cells as a template, respectively adding primers in the table 3, detecting the relative expression quantity of 3 β -HSD by using GAPDH as an internal reference and real-time fluorescence quantitative PCR (polymerase chain reaction) under the set reaction conditions of 95 ℃ for 30s, 1 cycle, 54 ℃ for 30s, 40 cycle, 95 ℃ for 5s, 60 ℃ for 1min and 95 ℃ for 15s, and detecting the relative expression quantity of Bax, Caspase-3, Caspase-8, ER α, ER β, hOGG1 and MTH1R genes of each group of cells by using SYBR Primescript RT-PCR Kit, wherein the sequences of PCR primers of Bax, Caspase-3, Caspase-8, ER α, ER β, hOGG1 and MTH1 are shown in the table 3.

TABLE 3 PCR primers for Bax, Caspase-3, Caspase-8, ER α, ER β, hOGG1, MTH1

The experimental results are as follows:

(1) effect of expression vector promoting expression of 3 β -HSD Gene on expression level of apoptosis Gene

Under the condition of different concentrations of DEHP, the expression levels of apoptosis genes Bax, Caspase-3 and Caspase-8 of two groups of human breast cancer cells MCF-7 are respectively shown in figure 7, figure 8 and figure 9, wherein MCF-7-cells represent a normal MCF-7 cell control group, 3 β -HSD overexpression MCF-7-cells represent an experimental group transfected with an expression vector promoting the expression of 3 β -HSD genes, and the expression levels of the genes under different conditions are calculated by taking the expression level of the normal MCF-7 cells with the final concentration of the DEHP of 0mmol/L as reference 1.

As can be seen from FIG. 7, after the plasticizer DEHP is infected for 24h, the expression level of the apoptosis gene Bax of the breast cancer cell MCF-7 transfected with the expression vector for promoting the expression of the 3 β -HSD gene is obviously higher than that of the control group, the expression level of the apoptosis gene Bax of the 3 β -HSD high expression cell is respectively increased to 24h from the low dose group to the high dose group, the expression level of the apoptosis gene Bax of the plasticizer DEHP is obviously higher than that of the control group, and is respectively increased by 75% -126% from the low dose group to the high dose group, the difference has statistical significance (P <0.01) — especially when the final concentration of DEHP is 0.8mmol/L, the Bax expression level is 2.2 times of that of the normal MCF-7 cell, and the difference of the expression amount is.

As can be seen from FIG. 8, the expression level of the apoptosis gene Caspase-3 of the breast cancer cell MCF-7 transfected with the expression vector promoting the expression of the 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, the expression level is respectively increased by 23% -83% from the low dose group to the high dose group, and the difference has statistical significance (P < 0.01).

As can be seen from FIG. 9, the expression level of the apoptosis gene Caspase-8 of the breast cancer cell MCF-7 transfected with the expression vector promoting the expression of the 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, the expression level of the apoptosis gene Caspase-8 is respectively increased by 39% -125% from the low dose group to the high dose group, the difference has statistical significance (P < 0.01). particularly, when the final concentration of the DEHP is 0.8mmol/L, the expression level of Caspase-8 is 2.3 times that of the normal MCF-7 cell, and the difference of the expression amount is obvious.

(2) Influence of expression vector for promoting expression of 3 β -HSD gene on expression level of estrogen receptor gene

The expression levels of the estrogen receptor genes ER α and ER β of two groups of human breast cancer cells MCF-7 under different concentrations of DEHP are shown in FIG. 10 and FIG. 11, respectively.

As can be seen from FIG. 10, the expression level of estrogen ER α gene of breast cancer cells MCF-7 transfected with expression vector promoting expression of 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, and the difference is statistically significant (P <0.01) because the expression level is increased by 25% -167% from the low dose group to the high dose group.

As can be seen from FIG. 11, the expression level of estrogen ER β gene of breast cancer cells MCF-7 transfected with expression vector promoting expression of 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, and the difference is statistically significant (P <0.01) when the expression level is respectively increased by 49% -245% from the low dose group to the high dose group.

(3) Effect of expression vector promoting expression of 3 β -HSD Gene on DNA Damage Gene expression level

The expression levels of hOGG1 and MTH1 of the DNA damage genes hOGG1 and MTH1 of two groups of human breast cancer cells MCF-7 under different concentrations of DEHP are shown in FIG. 12 and FIG. 13, respectively.

As can be seen from FIG. 12, the expression level of hOGG1 gene of breast cancer cells MCF-7 transfected with expression vector promoting expression of 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, and the difference is statistically significant (P <0.01) when the gene is increased by 52% -223% from the low dose group to the high dose group.

As can be seen from FIG. 13, the expression level of MTH1 gene of the breast cancer cell MCF-7 transfected with the expression vector promoting the expression of the 3 β -HSD gene is obviously higher than that of the control group after the plasticizer DEHP is infected for 24h, and the difference is statistically significant (P <0.01) when the expression level is increased by 20% -150% from the low dose group to the high dose group.

The above results show that the expression level of the apoptosis gene, estrogen receptor gene and DNA oxidative damage gene of the human breast cancer cell MCF-7 transfected with the expression vector is higher than that of the non-transfected human breast cancer cell MCF-7, the expression vector can accelerate the apoptosis of the tumor cell and increase the damage effect of plasticizer on the tumor cell, in addition, even under the condition that the concentration of the plasticizer is 0mmol/L, namely the plasticizer does not exist, the expression levels of the apoptosis gene Bax, Caspase-3 or Caspase-8, the estrogen receptor gene ER α, ER β and the DNA oxidative damage gene hOGG1 of the human breast cancer cell MCF-7 transfected with the expression vector are higher than that of the non-transfected human breast cancer cell MCF-7, and the expression vector for promoting the expression of the 3 β -HSD gene is toxic to the tumor cell and has the damage effect on the tumor cell, so that the expression vector is expected to be applied to anti-tumor drugs.

The above-mentioned embodiments only express one or several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Shenzhen disease prevention and control center

<120> expression vector for promoting expression of 3 β -HSD gene, and construction method and application thereof

<160>21

<170>PatentIn version 3.3

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<213>3 β -HSD gene coding sequence

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cacaccgcct gtatcattga tgtcttcggt gtcactcaca gagagtctat catgaatgtc 300

aatgtgaaag gtacccagct cctgttagag gcctgtgtcc aagctagtgt gccagtcttc 360

atctacacca gtagcataga ggtagccggg cccaactcct acaaggaaat catccagaat 420

ggccatgaag aagagcctct ggaaaacaca tggcccgctc catacccaca cagcaaaaag 480

cttgctgaga aggctgtact ggcggctaac gggtggaatc tgaaaaacgg cggcaccctg 540

tacacttgtg ccttacgacc catgtatatc tatggggaag gaagccgatt cctttctgct 600

agtataaacg aggccctgaa caacaatggg atcctgtcaa gtgttggaaa gttctccact 660

gttaacccag tctatgttgg caatgtggcc tgggcccaca ttctggcctt gagggccctg 720

caggacccca agaaggcccc aagcatccga ggacagttct actatatctc agatgacacg 780

cctcaccaaa gctatgataa ccttaattac accctgagca aagagttcgg cctccgcctt 840

gattccagat ggagctttcc tttatccctg atgtattgga ttggcttcct gctggaaata 900

gtgagcttcc tactcaggcc aatttacacc tatcgaccgc ccttcaaccg ccacatagtc 960

acattgtcaa atagcgtatt caccttctct tataagaagg ctcagcgaga tctggcgtat 1020

aagccactct acagctggga ggaagccaag cagaaaacgg tggagtgggt tggttccctt 1080

gtggaccggc acaaggagac cctgaagtcc aagactcag 1119

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cgcggccgcc tgagtcttgg acttcag 27

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gccagcaaac tggtgctcaa 20

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atgtccagcc catgatggtt c 21

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gactctggaa tatccctgga caaca 25

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gtggactccc acttccaaga 20

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cgatgttgtt gttggaggaa 20

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Claims

1. The use of a composition for the manufacture of an anti-tumor medicament, wherein the anti-tumor medicament is an anti-breast cancer medicament, the composition comprising:

the expression vector for promoting the expression of the 3 β -HSD gene is a lentiviral expression vector, the expression vector for promoting the expression of the 3 β -HSD gene comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of a lentiviral vector pLVX-mCMV-ZsGreen-PGK-Puro, wherein the multiple cloning site comprises an EcoRI enzyme cutting site and a NotI enzyme cutting site, the target gene expression fragment contains a 3 β -HSD gene coding sequence for expressing the 3 β -HSD, the 5 'end of the target gene expression fragment has an EcoRI enzyme cutting site cohesive end, the 3' end of the target gene expression fragment has a NotI enzyme cutting site cohesive end, the target gene expression fragment is positively inserted between the multiple cloning site sequence and the NotI enzyme cutting site, and the nucleotide sequence of the 3 β -HSD gene coding sequence is shown in SEQ ID No. 1;

or, the lentivirus containing the 3 β -HSD gene is prepared by transfecting an expression vector promoting the expression of the 3 β -HSD gene into 293FT cells, amplifying and expressing the 293FT cells after transfection to obtain the lentivirus containing the 3 β -HSD gene, wherein the expression vector promoting the expression of the 3 β -HSD gene comprises a basic sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and a target gene expression fragment of the lentivirus vector pLVX-mCMV-ZsGreen-PGK-Puro, the multiple cloning site comprises an EcoRI digestion site and a NotI digestion site, the target gene expression fragment contains a 3-mCMV-ZsGreen-PGK-Puro gene for expressing the 3-387 coding sequence, the 5 ' end of the target gene expression fragment has an EcoRI digestion site cohesive end, the 3 ' end of the target gene expression fragment has a NotI digestion site cohesive end, the target gene expression fragment is inserted between the coding sequence of the multiple cloning site and the nucleotide sequence of the coding sequence β ' end of the target gene expression fragment, and the NotI digestion site of the target gene expression fragment is shown in nucleotide sequence β.

2. The use of claim 1, wherein the 3 'end of the cohesive end of the EcoRI cleavage site is directly linked to the 5' end of the coding sequence of the 3 β -HSD gene, the 5 'end of the cohesive end of the NotI cleavage site is directly linked to the 3' end of the coding sequence of the 3 β -HSD gene, the base sequence of the cohesive end of the EcoRI cleavage site is 5 '-aattc-3', and the base sequence of the cohesive end of the NotI cleavage site is 5 '-ggccgc-3'.

3. The use according to claim 1 or 2, wherein the expression vector for promoting expression of 3 β -HSD gene is constructed by the following construction method:

4. The use according to claim 3, wherein the base sequence of the upstream primer is shown as SEQ ID No.2, and the base sequence of the downstream primer is shown as SEQ ID No. 3.