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Construction of eukaryotic expression vector pTAR-GET-
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DNTβRⅡand its expression in breast cancer COS-7 cells
Yue Zhao1* , Jinyue Hu2* , Rongguo Li1, Jian Song1, Dongwei Zhang1
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Medical University, China
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China
The Department of General Surgery, The Second Affiliated Hospital of Harbin
The Department of Breast and Thyroid Surgery, The Third Hospital of Zhengzhou,
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Abstract
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Objective: To synthesize the gene of human dominant-negative transforming growth
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factor beta receptor (DNTβRⅡ) by means of oligo chemic synthesis and PCR
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amplification, construct pTAR-GET-DNTβRⅡ eukaryotic expression vector and
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investigate whether its expression in breast cancer COS-7 cells.
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Methods: The DNTβRⅡ gene fragment was inserted into pTAR-GET to conduct a
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eukaryotic plasmid, then sequenced and identified by restrictive endonuclease
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digestion. The eukaryotic expression vector pTAR-GET-DNTβRⅡ was constructed
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and then transfected into COS-7 cells, to evaluate its expression by RT-PCR method.
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Results: The eukaryotic expression vector pTAR-GET-DNTβRⅡ was successfully
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constructed and could be
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cells.
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Conclusion: Full length gene of DNTβRⅡ can be synthesized by means of oligo
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chemic synthesis and PCR amplification. The construction of eukaryotic expression
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vector pTAR-GET-DNTβRⅡ and its successful expression in breast cancer COS-7
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cells, may provide the basis for further study of breast cancer anti-tumor
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immunotherapy by adoptive immune cell therapy.
instantaneously transfected and finally expressed in COS-7
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Key words: DNTβRⅡ; breast cancer cells; transfection; eukaryotic expression
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Introduction
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Breast cancer is immunogenic, and infiltrating immune cells in primary breast
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tumors convey important clinical prognostic and predictive information [1]. To date,
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surgery, chemotherapy, radiotherapy and endocrine therapy have generated small
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improvements in clinical outcomes, but breast cancer can recur and metastasize. The
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immune system can play a dual role in breast cancer, both promoting tumorigenesis
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through inflammatory pathways that also suppress adaptive immunity and thereby
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preventing tumor formation through active immune surveillance. Thus, adoptive
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immune cell therapy provides a new rationale way in the treatment of breast cancer.
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Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that regulates
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cellular responses, such as proliferation, growth, and tumorigenesis [2]. TGF-β can
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function as both tumor suppressor and promoter. In benign epithelia and many
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early-stage tumors, TGFβ is a potent inducer of growth arrest. However, TGF-β
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promotes cell motility, invasion, and metastasis in advanced tumors [3]. The tumor
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microenvironment is characterized by immediate fencing the tumor cells with the
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non-epithelial components of the area. This plays a vital role in cancer etiology
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through the interactions with tumor cells (Reference???). TGF-β is a potent tumor
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suppressor that has a negative impact on surrounding host immune cells in the tumor
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microenvironment [4,5]. Therefore, adoptive immune cell therapy must consider the
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immunosuppression of TGF-β in tumor microenvironment. Based on various
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prospective and retrospective studies, blocking of TGF-β signaling pathway can
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obviously enhance the antitumor effect of adoptive T-cells [6,7]. TGF-β starts the
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downstream signaling pathways should first combined with TβRⅡ in cell membrane,
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the transfection of DNTβRⅡ can competitive inhibition of normal TβRII , blocking of
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TGF-β signaling pathways, as a result, the construction of eukaryotic expression
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vector is a basis for further study[8]. In our study, we construct eukaryotic expression
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vector pTAR-GET-DNTβRⅡ and express it successfully in breast cancer COS-7 cells,
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and may provide basis for further study of breast cancer anti-tumor immunotherapy
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by adoptive immune cell therapy.
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Materials and methods
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Cell lines and Materials
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Human breast cancer COS-7 cells were obtained from the neurobiology teaching
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and research section of Harbin Medical University, Place???. TGF-β1 enzyme-linked
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immune detection kits was purchased from Rapidbio company (USA). Eukaryotic
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plasmid vector pTAR-GET was synthesized by Settlebio Company. COS-7 cells were
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cultured in DMEM medium supplemented with 10 % FBS and antibiotics. Cells were
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maintained at 37°C in a humidified 5% CO2 in air atmosphere.
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Design and synthesis of DNTβRⅡ
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Tthe gene of human dominant-negative transforming growth factor beta receptor
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(DNTβRⅡ) was synthesized by means of oligo chemic synthesis and PCR
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amplification,
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5’-ATGCTTCTCGAGATGGGTCGGGGGCT-3’;
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5’-CTGAATTCCTACTGCCGGTTAACGCTGA-3’. According to the expression
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requirements of pTAR-GET vector , the upstream primer initiation codon is ATG (at
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the beginning), and the downstream primer termination codon is TGA (at the 3’ end of
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primer). XhoI and EcorI restriction enzyme sites were annexed at the end of the
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sequences, respectively and synthesize the objective gene oligo by Gene2 Oliga
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software. Eventually, transforming growth factor beta receptor (DNTβRII) was
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amplified by polymerase chain reaction (PCR).
using
Forward
and
Reverse
primer:
primer:
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Construction of eukaryotic expression vector pTAR-GET-DNTβRⅡ
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A 591 bp-long fragment, DNTβRII was amplified by PCR using the primer-pairs
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given above. and the target sequence was sub-cloned into the corresponding
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restriction site on eukaryotic expression vector pTAR-GET. The sequence of the
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plasmid was confirmed by DNA sequencing analysis.
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Transfection of COS-7 cells
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COS-7 cells were cultured in RPMI-1640 medium supplemented with 10% FCS,
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in an incubator at 37˚C, with 5% CO2 and saturated humidity. One day prior to
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transfection, 2x106 cells were inoculated in a 6-well plate with 2 ml medium per well.
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When cells reached 70-80% confluence, transfections were performed with eukaryotic
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expression vector pTAR-GET-DNTβRⅡ, following the Lipofectamine 2000
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manufacturer's instructions. Cells were collected 48 h following transfection.
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Detection of eukaryotic expression vector pTAR-GET-DNTβRⅡ in COS-7 cells
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RT-PCR was used to analyze the DNTβRⅡ expression in COS-7. Briefly, total
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RNA was extracted from transfected COS-7 cells using TRIzol reagent, according to
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the manufacturer's instructions. cDNA was subsequently synthesized from the total
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RNA. PCR amplifications were performed with denaturation at 94˚C for 2 min,
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followed by 30 cycles of denaturation at 94˚C for 40 sec, annealing at 54˚C for 60 sec
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and 72˚C for 50 sec, followed by a final 10 min extension at 75˚C. PCR products were
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separated on 1% agarose gel by electrophoresis and visualized by ethidium bromide
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staining.
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Result
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Synthesize the DNTβRⅡ gene
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To synthesize the gene of human dominant-negative transforming growth factor
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beta receptor (DNTβRⅡ) by means of oligo chemic synthesis and PCR amplification.
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The PCR products were subsequently analyzed by agarose gel electrophoresis. At
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about 591 bp have seen a DNA stripe which the size match with the expected purpose
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gene (Figure 1).
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Identification and sequencing of recombinant plasmid
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The amplicon was ligated with the pTAR-GET plasmid using T4 DNA Ligase and
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then transformed into E. coli JM109 competent cells. Colony PCR was done for
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screening the bacteria harboring the pTAR-GET-DNTβRⅡ, followed by custom
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sequencing the target from Shanghai United Gene Company. Finally, we get four
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recombinant plasmid which connecting direction and reading code are correct were
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used for our study.
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Expression of the recombinant plasmid pTAR-GET-DNTβRⅡ in COS-7 cells
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To determine whether or not the recombinant plasmid pTAR-GET-DNTβRⅡ was
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expressed in COS-7 cells,
the later wase transiently transfected with
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pTAR-GET-DNTβRⅡ and its expression was detected using RT-PCR analysis.
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amplicon of expected size of ~591 bp was detected (Figure 2). The normal COS-7
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cells group and blank group without the stripes, the result showed that the RT-PCR
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products come from the recombinant plasmid transcription of mRNA, suggesting that
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the recombinant plasmid pTAR-GET-DNTβRⅡcan be expressed in breast cancer
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COS-7 cells.
An
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Discussion
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The immune system can play a dual role in breast cancer, both promoting
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tumorigenesis through inflammatory pathways that also suppress adaptive immunity
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and preventing tumor formation through active immune surveillance. Despite multiple
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approaches to therapy and prevention, cancer remains a major cause of death
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worldwide. Conventional therapies targeting dividing cells, using chemotherapy or
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radiation therapy, also affect normal cells and often fail in preventing the metastatic
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spread of the disease. Therefore, immunotherapy is an alternative modality of
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treatment that attempts to harness the specificity of the immune system to target tumor
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propagation without harming normal cells [9]. However, passive immunotherapeutic
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strategies with adoptive cell transfer (ACT) involve ex vivo stimulation of
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tumor-reactive T cells that are then transferred back to the patient [10].
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TGF-β is critically important for mammary morphogenesis and secretory function
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through specific regulation of epithelial proliferation, apoptosis, and extracellular
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matrix. In the normal mammary gland, TGF-β controls tissue homeostasis by inhibiting
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cell cycle progression, inducing differentiation and apoptosis, and maintaining
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genomic integrity [11]. In breast cancer, higher levels of TGF-β are often detected in
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tumors when compared to corresponding normal mammary gland tissue, and it
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appears even higher in the most advanced stages of tumor progression [12, 13]. In
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humans, three isoforms of TGF-β have been described, TGF-β1, TGF-β2 and TGF-β3.
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The signaling of these isoforms is comparable but the expression levels differ across
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tissues [14]. Canonical TGF-β signaling pathway is activated upon binding of TGF-β
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family members to type II TGF-β receptor (TβRII). In our study, we synthesize the
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gene of human dominant-negative transforming growth factor beta receptor
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(DNTβRⅡ) by PCR amplification, and construction of pTAR-GET-DNTβRⅡ
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eukaryotic expression vector. DNTβRⅡ does not contain Ser/Thr kinase domain in it,
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so it also can combine with TβRII, but it can't start the downstream signaling
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pathways [8,15]. In addition, peptide chain which express a large number of
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DNTβRⅡ transfer to the cell membrane, competitive inhibition of normal TβRII,
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blocking of TGF-β signaling pathways in tumor microenvironment and without
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affecting the normal tissue TGF-β signaling pathway.
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In our study, we synthesize the gene of human dominant-negative transforming
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growth factor beta receptor (DNTβRⅡ) by means of oligo chemic synthesis and PCR
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amplification, and successfully construction of eukaryotic expression vector
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pTAR-GET-DNTβRⅡ. XhoI and EcorI restriction enzyme site were imported on the
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end of sequence respectively and synthesize the objective gene oligo Gene2 Oliga
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software, and DNTβRII was amplified by PCR. In addition, we connected between
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the purified purpose gene fragments and pTAR - GET plasmid under the T4DNA
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ligase, connect the product transform the E.coliJM109 competent cell. Colony PCR
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method used for screening the bacteria which may contain pTAR-GET-DNTβRⅡ.
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Finally, we get four recombinant plasmid which connecting direction and reading
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code are correct were used to transfected with pTAR-GET-DNTβRⅡ and express it in
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breast cancer COS-7 cells. The results were analyzed by RT-PCR showed that
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recombinant plasmid pTAR-GET-DNTβRⅡ express in breast cancer COS-7 cells. In
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conclusion, the construction of eukaryotic expression vector pTAR-GET-DNTβRⅡ
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and express it successfully in breast cancer COS-7 cells, may provide basis for further
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study in both blocking the TGF-β signaling pathway and breast cancer anti-tumor
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immunotherapy.
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Acknowlegments
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This work was supported by the Ministry of Education of the People’s Republic of
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China (grants 20122307120034), Heilongjiang Province of Science and Technology
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(grants
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2012RFQS064).
D201273),
and
Harbin
Science
and
Technology
Bureau
(grants
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Reference
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Figure 1
Amplification of DNTβRII gene
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1.DNA marker; 2.blank group; 3.control group; 4. normal COS-7 cell; 5.plasmid compared
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Figure 2
Expression of the recombinant plasmid pTAR-GET-DNTβRⅡ in
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COS-7 cells.
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