Recombinant Plasmid Vector Research Articles

Naringin Regulates the Growth and Apoptosis of Ovarian Cancer Cells via the TGF-β Signaling Pathway: A Prospective Laboratory Based Study

Background: Traditional Chinese medicine (TCM) exhibits anti-tumor capabilities. This study explored the anti-tumor activity of Naringin in ovarian cancer and the signaling mechanism. Methods: The current investigation comprised the administration of various concentrations of Naringin, paclitaxel, and cisplatin to SKOV3 cells. Following the therapy, the protein expression levels for (TGF-β) and its downstream (Snail1)/(SMAD2) were measured in SKOV3 cells. Finally, small interfering RNA (siRNA) targeting TGF-β was synthesized, and the recombinant plasmid vector overexpressing TGF-β was constructed to detect the mRNA and protein expression of Snail1/SMAD2. The results were verified in animal models. Results: In this study, the expression of TGF-β varied significantly with varying Naringin concentrations. The expression of the downstream Snail1/SMAD2 was also affected. With increasing Naringin concentration, the expression of Snail1/SMAD2 decreased gradually in cells. Moreover, overexpressing TGF-β increased the expression of Snail1/SMAD2 and vice versa. In addition, Naringin further decreased the expression of Snail1/SMAD2 produced by transforming growth factor-β (TGF-β). Consistent outcomes were achieved when TGF-β agonists and inhibitors were employed alongside the inclusion of Naringin in animal models. Conclusions: The results of the present study demonstrated that Naringin suppressed the proliferation of ovarian cancer cells in a manner that depended on the dosage and also triggered programmed cell death (apoptosis) in ovarian cancer cells by activating the TGF-β mediated signaling pathway.

Construction of a Stable Expression System Based on the Endogenous hbpB/hbpC Toxin-Antitoxin System of Halomonas bluephagenesis.

Halomonas bluephagenesis is a halophilic bacterium capable of efficiently producing polyhydroxyalkanoates and other valuable chemicals through high salinity open fermentation, offering an appealing platform for next-generation industrial biotechnology. Various techniques have been developed to engineer Halomonas bluephagenesis, each with its inherent shortcomings. Genome editing methods often entail complex and time-consuming processes, while flexible expression systems relying on plasmids necessitate the use of antibiotics. In this study, we developed a stable recombinant plasmid vector, pHbPBC, based on a novel hbpB/hbpC toxin-antitoxin system found within the endogenous plasmid of Halomonas bluephagenesis. Remarkably, pHbPBC exhibited exceptional stability during 7 days of continuous subculture, eliminating the need for antibiotics or other selection pressures. This stability even rivaled genomic integration, all while achieving higher levels of heterologous expression. Our research introduces a novel approach for genetically modifying and harnessing nonmodel halophilic bacteria, contributing to the advancement of next-generation industrial biotechnology.

Multiplexed long-read plasmid validation and analysis using OnRamp.

Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

Connexin 43 overexpression induces lung cancer angiogenesis in vitro following phosphorylation at Ser279 in its C-terminus.

Blocking angiogenesis can inhibit tumor growth and metastasis. However, the mechanism underlying regulation of lung cancer angiogenesis remains unclear. The gap junction protein connexin 43 (Cx43) is implicated in angiogenesis. The aim of the present study was to determine the role of Cx43 in angiogenesis in vitro and its signaling pathways. Human pulmonary microvascular endothelial cells were transfected with Cx43-targeting siRNA or Cx43-overexpressing recombinant plasmid vector. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine Cx43, zonula occludens-1 (ZO-1), E-cadherin, β-catenin, von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression levels, respectively. Tyr265, Ser279, Ser368, and Ser373 phosphorylation levels in the C-terminus of Cx43 and intracellular and membranal Cx43 contents were determined using western blotting. Additionally, immunofluorescence, tube formation, Cell Counting Kit-8, and Transwell migration assays were performed. The results revealed that compared with that in the control samples, Cx43, ZO-1, E-cadherin, β-catenin, vWF, and PAI-1 mRNA and protein expression were significantly increased in the Cx43 overexpression group and significantly decreased in the Cx43-knockdown group. Moreover, the phosphorylation level of Ser279 as well as cell proliferation and migration rates were markedly increased in the Cx43 overexpression group, and tube formation revealed that the potential of angiogenesis was also increased. Conversely, in the Cx43-knockdown group, the phosphorylation level of Ser279 and cell proliferation and migration rates were reduced, and the potential of angiogenesis was greatly impaired. Under Cx43 overexpression, membranal Cx43 content was significantly increased, whereas under Cx43 knockdown, it was significantly reduced. Therefore, Cx43 overexpression could induce pulmonary angiogenesis in vitro by promoting cell proliferation and migration and activating ZO-1, E-cadherin, β-catenin, vWF, and PAI-1. This may be achieved by promoting phosphorylation and activation of the intracellular signal site Ser279 at the C-terminus of Cx43.

Preclinical Safety Evaluation of a Recombinant Plasmid Vector Encoding Mature Human Neutrophil Peptide-1 by Repeated Local Administrations in Nonhuman Primates.

In our previous studies, a novel gene therapy approach was developed based on a plasmid vector pSecTag2B in which recombinant HNP1 gene was regulated under a cytomegalovirus promoter to encode a mature human neutrophil peptide-1 (HNP1) form. We showed for the first time in various tumor models, including human cancer xenografts, that overexpression of HNP1 in the tumor milieu by intratumoral pSecTag-HNP1 (pHNP1) administration efficiently attenuated in vivo tumor progression, mediated host immune responses to tumors, and produced a synergistic effect when combined with chemotherapeutics. In this study, a preclinical safety investigation of HNP1 gene therapy was conducted in nonhuman primates. Eleven cynomolgus monkeys were divided into three groups of three to four animals each and received either repeated s.c. injections of pHNP1/cationic liposome complexes at a low (0.625 mg/kg) or a high (2.5 mg/kg) dose or glucose as control. Significant HNP1 in vivo accumulation was detected after consecutive administrations. All primates reached the end of the study with good body conditions. Injection site inflammation was the only obvious toxic reaction during observation period. In addition, elevation of monocyte/macrophage and neutrophil as well as decline of lymphocyte were detected in the peripheral blood of pHNP1-treated primates. These alterations were partially alleviated at the end of observation period. Besides, dose-related histopathological changes of the immune organs were observed at necropsy, including a minimal thymic lymphocyte decrease and a minimal-to-mild lymph node erythrocyte increase, but which cannot be excluded from HNP1-induced immune reactions. Together, these data support future clinical studies of pHNP1-based local gene delivery in tumor patients.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Protection of BALB/c mice against pathogenic Brucella abortus and Brucella melitensis by vaccination with recombinant Omp16.

Objective(s):Prevention of the globally spread zoonotic infection, brucellosis which affects an extensive range of hosts is still challenging researchers. There are no approved vaccines for the prevention of human disease and those used for animal brucellosis have adverse properties, which limit their application. We investigated the immunological and protective effects of recombinant 16 kDa outer membrane protein of Brucella abortus (Omp16) which introduced a new candidate for brucellosis subunit vaccine. Materials and Methods: Brucella Omp16 gene was cloned in pET-23a and expressed in Escherichia coli BL21 (DE3). Recombinant Omp16 (rOmp16) was purified using nickel resin and confirmed by Western blot analysis. BALB/c mice were immunized with rOmp16, afterward, specific serum antibodies and cytokine responses were evaluated. Protection of immunized mice against pathogenic B. abortus 544 and B. melitensis 16M was evaluated by the intraperitoneal bacterial challenge. Results:Sequencing results of the recombinant plasmid vector along with Western blotting confirmed the cloning procedure. Recognition of rOmp16 by specific IgG from serum samples of infected cases suggests the stimulation of immune response to this protein. Significant total serum IgG along with remarkable IgG1 and IgG2a response to the protein was recorded. A significant increase in IFN-γ, and IL-4 levels were observed from splenocyte cultures of immunized mice which were stimulated with rOmp16 suggesting the development of T-lymphocyte mediated immunity against the recombinant antigen. Conclusion:The intraperitoneal challenge with B. abortus 544 and B. melitensis 16M confirmed that rOmp16 is able to elicit efficient protective immune responses in the animal host.

Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females.

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/104 cells in SA and 3.02 ( ± 1.86 [SD]) copy/104 cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/104 cells and 4.09 ( ± 4.26 [SD]) copy/104 cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.

Influence of introduction of interleukin-2 and interleukin-12 into experimental marker DNA-vaccine

Aim. To determine the effects of the combined administration of recombinant expression vectors containing genes of murine interleukin-2 and interleukin-12 on humoral immune response, elicited by the experimental marker DNA-vaccine against classical swine fever. Methods. Expression of chimeric proteins in vitro and in vivo was determined by western-blot analysis. The antibodies specific to target antigens in blood serum have been detected by ELISA. Results. A series of recombinant plasmid vectors containing the genes of murine interleukin-2 and chimeric murine interleukin-12 have been developed. It has been shown that target chimeric proteins were expressed from the developed vectors both in vitro in HEK293 and in vivo in murine muscle tissue. The use of combined administration of murine interleukin-2 and interleukin-12 genes resulted in significant enhancement of titer of the anti-E2 and anti-β-galactosidase IgG, induced by vaccination with experimental marker DNA-vaccine against CSF, and model DNA-vaccine respectively. Conclusions. The data obtained show that the introduction of recombinant expressing vectors containing genes of murine interleukin-2 and interleukin-12 into vaccine preparations enhances humoral immune response elicited by the experimental marker DNA-vaccine against CSF and modelDNA-vaccine. Keywords: DNA-vaccine, humoral immune response, interleukin-2, interleukin-12, classical swine fever.

Shiga-like toxin I exerts specific and potent anti-tumour efficacy against gastric cancer cell proliferation when driven by tumour-preferential Frizzled-7 promoter.

Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.

Construction of a Bioluminescent Labelling Plasmid Vector for Bifidobacteria.

Bifidobacterium is recognized as one of the most beneficial microorganisms in our gut. Many researches on bifidobacteria have been done to understand their roles in the gut. The objective of the present study was to develop a bioluminescent labelling plasmid vector for bifidobacteria to facilitate their visualization in vitro, in situ, and in vivo. A plasmid replicon (2.0 kb) of plasmid pFI2576 previously identified from B. longum FI10564 was amplified by PCR and cloned into pUC19 plasmid vector (2.68 kb). The cloned replicon was subcloned into pTG262 (luc+) recombinant plasmid vector (7.4 kb) where a luciferase gene (luc+) from pLuc2 (8.5 kb), an Escherichia coli and lactobacilli shuttle vector, was inserted into pTG262 plasmid vector. The final recombinant DNA, pTG262::pFI2576 rep (luc+), was transferred into a B. catenulatum strain. This recombinant strain showed 3,024 relative luminescence units at OD600 value of 0.352. Thus, this recombinant plasmid construct can be broadly used for labelling bifidobacteria.

In vitro study on reversal of ovarian cancer cell resistance to cisplatin by naringin via the nuclear factor-κB signaling pathway.

The aim of the present study was to investigate the mechanism of action by which naringin reverses the resistance of ovarian cancer cells to cisplatin. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting assays were used to detect the effects of different concentrations of naringin on the expressions of nuclear factor (NF)-κB and P-glycoprotein (P-gp) in the SKOV3/CDDP cell line. Small interfering RNA (siRNA) targeting NF-κB was designed and synthesized to silence NF-κB, and recombinant plasmid vectors overexpressing NF-κB were constructed to transfect cells. RT-qPCR and western blotting assays were subsequently performed to detect the effects of NF-κB on the expression of P-gp at the mRNA and protein levels. Naringin was added to the NF-κB-overexpressing SKOV3/CDDP cells and cultured for 48 h, followed by the detection of the expression of P-gp. RT-PCR and western blotting results demonstrated that the gene and protein expressions of NF-κB and P-gp were significantly decreased in a dose-dependent manner by naringin treatment (P<0.05). In cells overexpressing NF-κB, P-gp expression was significantly elevated (P<0.05), and the expression of P-gp was significantly decreased when NF-κB was silenced (P<0.05). Treatment with naringin was able to significantly ameliorate the NF-κB-induced overexpression of P-gp (P<0.05). These results indicate that naringin is able to inhibit the expression of NF-κB and P-gp in SKOV3/CDDP cells. Such an inhibitory effect may increase gradually with concentration, and is associated with blockade of the NF-κB signaling pathway. This pathway may represent one of the mechanisms of action by which Naringin reverses resistance to platinum-based agents in ovarian cancer cells.

Effects of interleukin-34 expressed by human bone marrow derived mesenchymal stem cells on THP-1 cells

To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.

Long non-coding RNA HOTAIR enhances radioresistance in MDA-MB231 breast cancer cells.

The aim of the present study was to investigate the radiosensitizing effects of homeobox (HOX) transcript antisense RNA (HOTAIR) long non-coding RNA on breast cancer tumor cells and examine the underlying mechanisms. Recombinant plasmid vectors containing HOTAIR gene were constructed and MDA-MB231 cells were transfected with these plasmids using liposomes. The cells were treated with radiation and cell apoptosis, proliferation, and double-stranded DNA breaks were examined. HOXD10, phosphorylated AKT (p-AKT) and p-BAD expression levels were measured using western blot analysis. The results showed a higher expression of HOTAIR in advanced tumor cells. HOTAIR efficiently enhanced radioresistance in MDA-MB231 breast cancer cells and accelerated proliferation through the Akt pathway by targeting HOXD10. In conclusion, the findings demonstrated that HOTAIR gene is a valid therapeutic target for the reversal of radiotherapy resistance in breast cancer.

Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth.

Frizzled-7 protein plays a significant role in the formation of several malignant tumors. Up regulation of the Frizzled-7 in cancer cell lines is associated with nuclear accumulation of wild-type β-catenin from the Wnt/β-catenin pathway which is frequently activated in tumors. To analyze activity of the Frizzled-7 promoter in tumor cells, we constructed two recombinant plasmid vectors in which the Frizzled-7 promoter was used to drive the expression of green fluorescent protein (GFP) and Shiga-like toxin I (Stx1) (pFZD7-GFP/Stx1) genes. The Frizzled-7 protein was found to be expressed in the cancer cell lines but not in the normal cell lines. The GFP expression was restricted to the cancer cell lines and xenografts in the BALB/C mice but not to normal cell lines. Moreover, cell proliferation and tumor growth decreased significantly after transfection with the pFZD7-Stx1. Results from this study will help determine a highly effective strategy for gene therapy of tumors.

Infection by Cx43 adenovirus increased chemotherapy sensitivity in human gastric cancer BGC-823 cells: not involving in induction of cell apoptosis

Background and objectiveThere is a lower basal expression of Connexin43 (Cx43) in human gastric cancer BGC-823 cells. In the present study, BGC-823 cells were transfected with recombinant Cx43 adenovirus plasmid vector, and we explored the influences of Cx43 expression on cell proliferation, chemo-sensitivity, colony forming ability, invasion ability and apoptosis. Moreover, we also determined the expression of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase3 and caspase 9). MethodsMTT assay was performed to determine the proliferation of BGC-823 cells before and after Cx43 transfection. The influences of Cx43 infection on sensitivity of chemotherapy (including Doxorubicin, fluorouracil, oxaliplatin) were detected by MTT assay. Expression levels of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase-3 and caspase-9) in BGC-823 cells were determined by Western blotting analysis before and after the infection with Cx43 adenovirus. MDR expression was determined by RT-PCR before and after Cx43 infection. Invasive ability was detected by invasion chamber. Influence of Cx43 adenovirus infection on apoptosis of BGC-823 cells was determined by flow cytometry. ResultsAfter infection by Cx43 adenovirus, colony forming rate and invasive ability of BGC-823 cells were decreased. Flow cytometry results revealed that cell apoptosis were insignificantly increased. The data of MTT assay revealed that infection with Cx43 adenovirus, cell proliferation ability decreased and sensitivity to chemotherapy drugs (including doxorubicin, fluorouracil, oxaliplatin) increased. Results of Western blotting analysis revealed that increasing expression levels of Cx43, decreasing expression levels of Pgp, and insignificant changes of bcl-2, bax, caspase3 and caspase 9 were detected. RT-PCR revealed the expression of MDR1 gene, the gene encoding Pgp, decreased significantly (p<0.05). ConclusionThe human gastric cancer BGC-823 cells were infected with Cx43-IRES2-EGFP recombinant adenovirus vector. Colony formation, invasive ability and cell proliferation all decreased, whereas chemo-sensitivity increased in Cx43 infected BGC-823 cells. The increasing Cx43 expression was accompanied by decreasing Pgp expression and MDR1 m RNA levels. However, apoptosis-related proteins (bcl-2, bax, caspase3 and caspase 9) and cell apoptosis increased insignificantly. All results demonstrated that Cx43 may be negatively regulated the development, invasion and metastasis of gastric cancers, however, it had no obvious relationship with tumor cell apoptosis.

Cloning of Dense Granular (GRA) 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids

Background: Serological assay based on dense granular (GRA) proteins of Toxoplasma gondii (T. gondii) is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production.Materials and Methods: Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites’ DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing.Results: Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white (recombinant) and blue (non-recombinant) colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures.Conclusion: The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein.

Effect of microRNA-10b silencing on expression of peroxisome proliferator activated receptor-a in human pancreatic cancer cell line

Objective To observe the effect of silencing microRNA-10b on the expression of peroxisome proliferator activated receptor-α(PPAR-α) in human pancreatic cancer cell line AsPC-1.Methods The recombinant eukaryotic expression plasmid vector with miR-10b antisense sequence was transfected into AsPC-1 cells with lipofectamine,and the efficiency of the instant expression was confirmed by real-time quantitative polymerase chain reaction (Real-time PCR) and fluorescence microscope.The difference of the proliferation of the cells was measured by proliferation assay,and the expression of PPAR-α was detected by Western blotting.Results The expression of miR-10b in the antisense groups was decreased to 14％ as compared with the control group (P ＜ 0.05).The cell proliferation in the antisense groups was decreased to 36％ as compared with the control group (P ＜ 0.05).The expression of PPAR-α protein in the antisense groups was 1 230.5 ± 110.4,and that in the control groups was 2 398.1 ± 156.3 and 2 603.7 ± 227.5 (P ＜0.05).The apoptosis ratio in the antisense group was 19.4％,and that in the control groups was 10.1％ and 11.5 ％ (P ＜ 0.05).Conclusion The silencing of miR-10b can inhibit the expression of PPAR-α and the cell proliferation,and promote the apoptosis of the ASPC-1 cells. Key words: Pancreatic cancer; MicroRNA-10b; Peroxisome proliferator activated receptor-α

Overexpression of the PLAP-1 gene inhibits the differentiation of BMSCs into osteoblast-like cells.

Periodontal ligament-associated protein-1 (PLAP-1) is a newly discovered member of the extracellular matrix family of proteins known as proteoglycans and is a negative regulator that plays a crucial role in the homeostasis of periodontal tissues. It can protect the periodontal ligament from excessive osteogenesis. However, the molecular mechanisms of PLAP-1 during osteogenic differentiation and osteogenesis remain unclear. In this study, we constructed a PLAP-1 recombinant retroviral plasmid vector named pBABE-hygro-PLAP-1. We transfected this plasmid into rat bone marrow stromal cells (rBMSCs) to obtain a stable cell line with overexpression of PLAP-1 to verify whether PLAP-1 also acts as an inhibitory factor in rBMSCs during bone mineralization. A rBMSC line stably overexpressing PLAP-1 was established successfully as determined by the mRNA levels of PLAP-1, which were measured by real time-qPCR (RT-qPCR), and protein expression, which was measured by immunocytochemistry and western blot analysis. At the same time, a Cell Counting Kit-8 assay did not reveal any statistically significant changes in the transfected cells (P > 0.05). Then, mineral-inducing cultures were performed, and mineralized nodules were observed at weeks 2, 3 and 4 under a microscope. Alizarin Red (Sigma) staining was performed at 4 week to illustrate calcium accumulation. The mineralized nodules in the PLAP-1-transfected rBMSC group were fewer than those in the control groups. The time span of the formation of the mineralized nodules was prolonged. Meanwhile, osteogenic genes were also detected in the mineral-inducing cells by RT-qPCR. An RT-qPCR analysis demonstrated that the levels of the osteoblast markers of rBMSCs that were transfected with pBABE-hygro-PLAP-1, including Runx2, Osterix, alkaline phosphatase, bone sialoprotein and osteocalcin, were lower than those in the non-transfected rBMSCs and rBMSCs that were transfected with empty vector (P < 0.01). These results suggest that PLAP-1 has an inhibitory function in rBMSCs when they differentiate into osteoblast-like cells.