Construction of an Expression Plasmid pEGFP-N1-boTLR2 for Full-length Bovine TLR2 and Its Expression in HEK293 Cells

[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.

Inhibitory Effects of Neferine on Na_v1.5 Channels Expressed in HEK293 Cells

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Construction of recombinant plasmid pEGFP-C2-L539fs/47 and its expression in HEK293 cells

Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.

Construction of eukaryotic expression vector of humanarresten gene and its secreted expression in HEK 293 cells

The eukaryotic expression vector of human arresten gene was constructed and its secretive expression in human embryonic kidney(HEK 293)cells was detected.Human arresten gene was amplified from recombinant plasmid pGEM-Arr by polymerase chain reaction(PCR),and then digested with restriction endonucleases BamH I and EcoR I.The target fragment was inserted into the BamH I and EcoR I restriction sites of eukaryotic expression vector pSecTag2 to construct pST-AT.Restric-tion analysis and DNA sequencing indicated that the arresten gene was successfully inserted into pSecTag2.The recombinant plasmid was subsequently transfected into HEK 293 cells with LipofectAMINETM2000 Reagent,and the expression of the target gene was detected.Reverse transcription PCR(RT-PCR)revealed that the mRNA of the target gene was transcribed in the transfected HEK 293 cells.Western blot analysis verified that the recombinant protein in supernatants was correct.The supernatants of transfected cells were prepared,and 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide(MTT)assay was carried out to assess their effects on the proliferation of human umbilical vein endothelial cells,which showed that the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells in vitro.These results provided a solid foundation to explore the usage of arresten in tumor anti-angiogenic gene therapy.

Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of factor Ⅸ gene and expression in HEK-293 cells

Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using

Electrophysiology of hyperpolarization-activated cyclic nucleotidegated cation channel 2 and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 expressed in HEK293 cells

The action potential of pacemaker cells in the sino-atrial node forms the automatic rhythm of the heart. The automatic depolarization in phase 4 is me DaSlS of the automaticity in pacemaker cells. Many currents are included in phase 4, such as calcium current, TTX-sensitive sodium current, sustained inward current （Isi）, decay of delayed rectifier potassium current, etc. Funny current （If） has long been recognized as important in this phase and is activated at hyperpolarized potentials during cell diastole and in turn activates other currents to form automatic depolarization.

Cellular Oxidative Damage of HEK293T Cells Induced by Combination of CdCl_2 and Nano-TiO_2

This study investigated the conjoined cellular oxidative damage of human embryo kidney 293T（HEK293T） cells induced by cadmium chloride（CdCl2） and nanometer titanium dioxide（nano-TiO2）.RT-PCR technique was used to detect the expressions of Heme oxygenase-1（HO-1） and 8-oxoguanine DNA glycosylase（OGG1）.The activities of superoxide dismutase（SOD） and catalase enzyme（CAT） and concentrations of reactive oxygen species（ROS） and maldondialdehyde（MDA） were measured by different approaches.The results showed that CdCl2 and nano-TiO2 at a low concen-tration of 0.75 total toxic unit（TU） exerted an additive effects on HO-1 gene expression,CAT activities and MDA concentrations.When the total TU was increased to 1 or 1.25 TU,the interaction was syner-getic.Moreover,the mixture with high proportion of CdCl2 produced an additive effect on the OGG1 gene expression,and the interaction was changed to be synergetic when the concentration of CdCl2 was lower than or equal to that of nano-TiO2.Synergetic effects of CdCl2 and nano-TiO2 on cellular oxida-tive damage of HEK293T cells were found as indicated by the changes in the SOD activities and ROS concentrations.It was concluded that CdCl2 and nano-TiO2 exerts synergistic effects on the cellular oxidative damage of HEK293T cells,and the sensitivity of these indicators of oxidative damage varies with the proportion of CdCl2 and nano-TiO2 in the mixture.

Protective Effect of Reduced Glutathione C_(60) Derivative against Hydrogen Peroxide-induced Apoptosis in HEK 293T Cells

Hydrogen peroxide（H_2O_2） and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene（C_（60）） is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione（GSH） is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative（C_（60）-GSH） was successfully synthesized, and its beneficial roles in protecting against H_2O_2-induced oxidative stress and apoptosis in cultured HEK 293 T cells were investigated. Fourier Transform infrared spectroscopy and 1H nuclear magnetic resonance were used to confirm the chemical structure of C_（60）-GSH. Our results demonstrated that C_（60）-GSH prevented the reactive oxygen species（ROS）-mediated cell damage. Additionally, C_（60）-GSH pretreatment significantly attenuated H_2O_2-induced superoxide dismutase（SOD） consumption and malondialdehyde（MDA） elevation. Furthermore, C_（60）-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H_2O_2 stimulation in HEK 293 T cells. Importantly, these protective effects of C_（60）-GSH were superior to those of GSH. In conclusion, these results suggested that C_（60）-GSH has potential to protect against H_2O_2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.

The Function of SeMNPV IAP3 in Mammalian Cells

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus （SeMNPV）, amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I/Barn H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.

Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

Transient Expression of BVDV N^(pro) Gene in vitro and Distribution of Fusion Protein in Cells

The gene encoding nonstructural protein Npro of BVDV was amplified by PCR,then was inserted into pEGFP-C1 vector.The 293 cells were transfected in vitro with recombinant plasmid by liposome.Npro-EGFP fusion protein was viewed directly with fluorensce microscope and mRNA expression of Npro was detected by reverse transcription-polymerase chain reaction（RT-PCR）.Results showed that recombinant plasmid was confirmed to be constructed correctly by PCR,restriction enzyme digestion and DNA sequencing;meanwhile,the gene carried was expressed in 293 cells.The fusion protein had properties of both the two Npro and enhanced green fluorescent proteins（EGFP） and distributed in the cytoplasm.This research lays a foundation for further researches which explored Npro gene function in the process of viral replication and synthesis.

Enhancement of Virus Replication in An Influenza A Virus NS1-Expresssing 293 Cell Line

The nonstructural protein 1 （NS1） of influenza A virus, which is absent from the viral particle, but highly expressed in infected cells, strongly antagonizes the interferon （IFN）-mediated antiviral response. We engineered an NS1-expressing 293 （293-NS1） cell line with no response to IFN stimulation. Compared with the parental 293 cells,

Downregulation of Serum PTEN Expression in Mercury-Exposed Population and PI3K/AKT Pathway-Induced Inflammation

Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation. Hg-exposed cell models and PTEN lowexpression models were established in vitro using 293T cells. PTEN gene expression was assessed using qRT-PCR, and Western blotting was used to measure PTEN, AKT, and PI3K protein levels. IL-6 expression was determined by ELISA.Results Combined findings from gene expression microarray analysis, bioinformatics, and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group. In the Hg-exposed cell model(25 and 10 μmol/L), a significant decrease in PTEN expression was observed, accompanied by a significant increase in PI3K, AKT, and IL-6 expression.Similarly, a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K, AKT, and IL-6 levels.Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene, activates the PI3K/AKT regulatory pathway, and increases the expression of inflammatory factors, ultimately resulting in kidney inflammation.

Construction of recombinant human nerve growth factor beta adenovirus and evaluation of its function An in vitro and in vivo study

Exogenous delivery of nerve growth factor （NGF） promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-β （hNGF-β） DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia CoIL The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-β adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells （primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts） compared with non-infected cells. Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.

Cytotoxicity assessment of a gold nanoparticle-chitosan nanocomposite as an efficient support for cell immobilization:comparison with chitosan hydrogel and chitosan-gelatin

Cell-based biosensors have become a research hotspot in the biosensors and bioelectronics fields. The main feature of cell-based biosensors is immobilization of living cells on the surface of transducers. Different types of polymers which are used as scaffolds for cell growth should be biocompatible and should have reac- tive functional groups for further attachment of biomolecules. In this work, cell attachment and proliferation on chitosan hydrogel, chitosan-gelatin and gold nanoparticle-chitosan nanocomposite membranes was stud- ied. Characterization of the membranes was performed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Cytotoxicity assessment on HEK293 cells was carried out for all mem- branes using the MTT assay. Cell morphology and viability were assessed to evaluate cell attachment and pro- liferation. Regarding cell studies, the findings revealed that none of the membranes induced cytotoxic effects. However, the data showed that gold nanoparticle nanocomposite membranes improved HEK293 attachment and adhesion more than other membranes, indicating that it provides an effective surface for immobilizing cells for sensing applications.

Expression of fluorescent tagged recombinant erythroferrone protein

Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE(ERFE_eGFP) protein was expressed in human embryonic kidney(HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein(approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.

Heterologous expression and cell membrane localization of dinoflagellate opsins (rhodopsin proteins) in mammalian cells

Rhodopsins are now found in all domains of life,and are classified into two large groups:type II,found in animals and type I found in microbes including Bacteria,Archaea,and Eukarya.While type II rhodopsin functions in many photodependent signaling processes including vision,type 1 among others contains rhodopsins that function as a light-driven proton pump to convert light into ATP as in proteobacteria(named proteorhodopsin).Proteorhodopsin homologs have been documented in dinoflagellates,but their subcellular localizations and functions are still poorly understood.Even though sequence analyses suggest that it is a membrane protein,experimental evidence that dinoflagellate rhodopsins are localized on the plasma membrane or endomembranes is still lacking.As no robust dinoflagellate gene transformation tool was available,we used HEK 293T cells to construct a mammalian expression system for two dinoflagellate rhodopsin genes.The success of expressing these genes in the system shows that this mammalian cell type is suitable for expressing dinoflagellate genes.Immunofluorescence of the expressed protein locates these dinoflagellate rhodopsins on the cell membrane.This result indicates that the protein codons and membrane targeting signal of the dinoflagellate genes are compatible with the mammalian cells,and the proteins'subcellular localization is consistent with proton pump rhodopsins.

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis.In addition,SR-AI modulates macrophage activation through cell signaling.However,investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors.Therefore,we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling.On 293T cells,SR-AI could respond to E.coli DH5α,leading to NF-κB activation and IL-8 production.However,this requires E.coli DH5αto be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion.Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5αand blocks DH5αstimulation of SR-AI signaling.Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b.By mutagenesis,The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α.These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.