Construction Of Expression Plasmid Research Articles

Truncated SSX Protein Suppresses Synovial Sarcoma Cell Proliferation by Inhibiting the Localization of SS18-SSX Fusion Protein

Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2;q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.

Construction of Y376C-FGFR4 eukaryotic expression plasmid and its biological activity in HEK293 cell

Jing Huang1, Liuxin Feng1, Hongli An2, and Tao Zhang1* School of Pharmaceutical Sciences, Xi’an Jiaotong University, Xi’an 710061, China Center for Translational Medicine, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an 710061, China These authors are equal first author. *Correspondence address. Tel: þ86-29-82656788; Fax: þ86-29-82655451; E-mail: taozhang@mail.xjtu.edu.cn

Molecular Cloning and Expression of Bovine Viral Diarrhea Virus Nonstructural Protein 3 in Escherichia coli

animals. Objectives: The aim of this study was to synthesize a plasmid construct for high-level expression of NS3 with more solubility in Escherichia coli. Materials and Methods: A segment of BVDV genome encoding the NS3 protein was amplified using RT-PCR and cloned into pMAL-c2X expression vector, under the control of the lac promoter. After sequencing of the amplified gene, the recombinant protein was expressed in E. coli strain BL21 and analyzed by SDS-PAGE and western blotting. Results: The strong promoter of pMAL-c2X vector allowed a high level expression of NS3 as a maltose binding protein-NS3 (MBP-NS3) fusion protein. Expression of the expected fusion protein was confirmed by electrophoresis on SDS-PAGE and immunoblotting, using a BVDV positive bovine serum. Conclusions: Based on our results, it appears that this plasmid construct may be suitable for the production of NS3 recombinant antigen to develop BVDV laboratory diagnostic assays.

Expression and immune characteristics analysis of TcpA protein of Vibrio cholerae

Vibrio cholerae is a causative agent of mucous-sloughing disease and bacterial enteritis in fish,with high morbidity and mortality.Toxin-coregulated pilus(TCP) is a major virulence factor of V.cholerae.As a major structural protein of TCP,toxin-coregulated pilin A(TcpA) is potential candidate for diagnostic antigen and vac-cine development.In this study,the tcpA gene was amplified by PCR from genomic DNA of the Y1 strain of Vi-brio cholerae isolated from diseased grass carp(Ctenopharyngodon idellus).The tcpA gene was cloned into a pMD18-T vector and sequenced.The tcpA gene fragment containing the open reading frame(ORF) was then sub-cloned into pGEX-4T-1 to construct expression plasmid pGEX-4T-1-tcpA.The recombinant fusion protein(rGST-TcpA) was expressed by IPTG induction in E.coli for subsequent immunological characterization.Se-quence analysis revealed that the ORF of the tcpA gene from the Y1 strain(GenBank accession no.EU649677) is 657 bp and encodes a protein of 224 amino acids.The gene is 99.6%–99.7% identical at the nucleotide level and 98.7%–99.1% identical at the protein level to seven tcpA sequences in GenBank,which suggested that the TcpA protein is considerably conserved.SDS-PAGE analysis showed that the 47.0 kD rGST-TcpA fusion protein was mainly expressed in inclusion bodies.Western blotting demonstrated that rGST-TcpA could react specifically with mouse antisera raised against the pilus protein of strain Y1.The purified rGST-TcpA was used to immunize grass carp(Ctenopharyngodon idellus).The titer of the antisera was 1:16,as determined by a double immunodiffusion test,and it could significantly inhibit adherence of Y1 strain to HEp-2 cells in vitro.A relative percentage survival(RPS) of 73.33% was found in immunized grass carp(Ctenopharyngodon idellus) suffering from Y1 strain chal-lenge at day 25 after immunization.This study indicated that the rTcpA protein possess the same immunogenicity,immunoreactivity,and protective efficacy as the natural pilus protein of V.cholerae.

Key regulatory elements of a strong constitutive promoter, P GCW14 , from Pichia pastoris

The promoter of the Pichia pastoris gene GCW14 is strong and constitutive when glycerol is the available carbon source. To identify the cis-acting elements of this promoter (P GCW14), we constructed expression plasmids where the enhanced green fluorescent protein gene was fused to a series of mutants of P GCW14. We identified one negative (-114 to -94) and three positive regulatory regions (-426 to -152, -134 to -114, -94 to -77). The TATA box of P GCW14 was located at -48. One negative and four positive regulatory sites were identified combining error-prone PCR and directed mutation. The mutated promoter, M+20, with an increased promoter activity, was then used to express the gene for lipase B from Candida antarctica.

Presence of Transcription Factor OCT4 Limits Interferon-tau Expression during the Pre-attachment Period in Sheep.

Interferon-tau (IFNT) is thought to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. We and others have observed that OCT4 expression persists in the trophectoderm of ruminants; thus, both CDX2 and OCT4 coexist during the early stages of conceptus development. The aim of this study was to examine the effect of CDX2 and OCT4 on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG-3 cells were cotransfected with an ovine IFNT (-654-bp)-luciferase reporter (-654-IFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with Cdx2, Ets2 and Jun increased transcription of -654-IFNT-Luc by about 12-fold compared with transfection of the construct alone. When cells were initially transfected with Oct4 (0 h) followed by transfection with Cdx2, Ets2 and/or Jun 24 h later, the expression of -654-IFNT-Luc was reduced to control levels. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Thus, when combined with the other transcription factors, OCT4 exhibited little inhibitory activity towards CDX2. An inhibitor of the transcriptional coactivator CREB binding protein (CREBBP), 12S E1A, reduced CDX2/ETS2/JUN stimulated -654-IFNT-Luc expression by about 40%, indicating that the formation of an appropriate transcription factor complex is required for maximum expression. In conclusion, the presence of OCT4 may initially minimize IFNT expression; however, as elongation proceeds, the increasing expression of CDX2 and formation of the transcription complex leads to greatly increased IFNT expression, resulting in pregnancy establishment in ruminants.

Evaluation of the immunogenicity and the protective efficacy in mice of a DNA vaccine encoding SP41 from Brucella melitensis

Brucella melitensis is a facultative intracellular Gram-negative bacterial pathogen that may enter the host via ingestion or inhalation, or through conjunctiva or skin abrasions. Some Brucella spp surface proteins (SPs) play an important role in bacterial adhesion and invasion and thus represent targets for the host immune system. Brucella spp surface protein with apparent molecular mass of 41 kDa interacts selectively with HeLa cells. To evaluate the role of SP41 (41 kDa) as a DNA vaccine against Brucella spp., pCISP41, a plasmid construct for protein expression in mammalian cells, was established. Exogenous SP41 was detected in pCISP41-transfected Vero cell line by immune blotting using specific polyclonal antibody. The protective role of pCISP41 against B. melitensis 16M in mice was evaluated by measuring B and T cell responses in comparison to those achieved with attenuated B. melitensis Rev. 1 vaccine. BALB/c mice injected with pCISP41 were able to develop SP41-specific serum immunoglobulin G (IgG) antibodies. In addition, splenocytes from DNA-SP41-vaccinated mice elicited a T-cell-proliferative response and also induced gamma interferon (IFN-γ) production, but not interleukin-5 (IL-5), suggesting the induction of a T-helper-1-dominated immune response. Vaccination with attenuated B. melitensis Rev.1 strain induced better protection levels than DNA vaccination with SP41 against B. melitensis 16M in mice. Such responses play an important role against intracellular infecting agents such as Brucella spp. Altogether, our data suggest that SP41 may represent a promising candidate for DNA vaccination against brucellosis, but more investigation to increase its protective efficacy should be done.

Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3.

Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3.

In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene

BackgroundTransmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined.ResultsFour theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency.ConclusionsOur results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.

A natural mutation of the hepatitis B virus X gene affects cell cycle progression and apoptosis in Huh7 cells

Hepatocellular carcinoma (HCC), one of the most prevalent cancers, and HCC-associated diseases are caused by the hepatitis B virus (HBV). Viral sequences often contain mutations in the basal core promoter (BCP), which overlaps with the open reading frame encoding HBV X protein (HBx). HBx protein plays a very important role in the development of HCC. Influence of naturally-occurring mutation of HBx (BCP1) on cell cycle progression was investigated in the Huh7 HCC line. BCP1 mutation was cloned from clinical samples into recombinant green fluorescent protein-fusion expression plasmid constructs that were then transfected into Huh7 cells for either transient expression studies or isolation of stable transfectants, along with wild-type HBx and empty vector controls. The mutant protein was more effective than wild-type in reducing expression of cyclindependent kinase (CDK) inhibitor p27kip1, promoting cell cycle progression, inhibiting production of cleaved poly ADP ribose polymerase (PARP) (a marker for apoptosis), and down-regulating the multifunctional cytokine transforming growth factor-β (TGF-β). Since TGF-controls the cell cycle and apoptosis, the effect of BCP1 mutation on the enhanced activity of HBx to promote cell cycle progression and to inhibit apoptosis in Huh7 cells may result from down-regulation of TGF-β.

Cloning and expression analysis of histocompatibility complexⅠα antigen (MHCⅠα)from humphead snapper Lutjanus sanguineus

In the present study,full-length cDNA sequences of histocompatibility complexⅠα antigen( MHC Ⅰα) was cloned by rapid amplification of cDNA ends technique( RACE) from humphead snapper( Lutjanus sanguineus) . Full-length cDNA sequence of MHC Ⅰ α is 1 369 bp,encoding 354 amino acids. BLAST analysis revealed that the amino acids sequence of MHCⅠα shared high identity( 84% ) w ith other MHCⅠ. Phylogenetic tree w as constructed by the Neighbor-Joining method,and the results suggested that MHC Ⅰα of humphead snapper shared the closest genetic relationship w ith the MHC Ⅰof Epinephelus coioides. The results of fluorescent real-time quantitative RT-PCR show ed that the expression of MHCⅠα mRNA could be detected in head kidney,and the maximum expression appeared in 6 - 15 h post infection. MHC Ⅰα w as subcloned into pET32a( + ) to construct expression plasmids pET32-MHCⅠα. SDS-PAGE and Western blot analysis indicated that the recombinant proteins w ere successfully expressed in Escherichia coli BL21 ( DE3) . Then the recombinant proteins were purified and the antiserum was obtained by immunizing rabbits w ith the purified recombinant proteins emulsified w ith adjuvant. ELISA analysis show ed that the titer of the antiserum prepared in this study w as 1∶ 40 000. The results of the Western blot revealed that specific antigenantibody reaction occurred betw een the antiserum and the recombinant proteins.

Construction and Identification of Eukaryotic Expression Plasmids of Caspase-3 and RNA Interference Plasmids of Rats

Objective To construct the eukaryotic expression plasmids and RNA interference plasmids of Caspase-3 in SD rats, and to investigate their inhibitory effects on Caspase-3. Methods The cDNA was obtained from the endplate cartilage cells of SD rats, and the full-length fragment of Caspases-3 was constructed, about 834 bp. The target segment was cloned into the expression vector pEGFP-N1 and the recombinant plasmid pEGFP-N1-Caspases-3 was obtained. Four pairs of pre-microRNA (pre-miRNA) sequences were designed according to Caspases-3 gene, and pre-miRNA was cloned into the expression vector pcDNA6. 2, while non-related interference plasmid was constructed. pcDNA6. 2-Caspases-3-1, pcDNA6. 2-Caspases-3-2, pcDNA6. 2-Caspases-3-3, pcDNA6. 2-Caspases-3-4 and pcDNA6. 2-miRNC were constructed successfully, and they were co-transfected into 293T cells with pEGFP-N1-Caspases-3 (7:1 ). The inhibition of Caspases-3 expression was analyzed by qPCR 48 h after transfection. Results pEGFP-N1-Caspases-3 and RNA interference plasmids were successfully constructed and confirmed by colony PCR, Western blotting and sequence analysis, qPCR results showed that in the intervention group of pcDNA6. 2-Caspase-3-2 and pcDNA6. 2-Caspases-3-3, Caspases-3 expression levels were increased as compared with the control group (33％ and 8％, respectively). But in the intervention group of pcDNA6.2-Caspase-3-1 and pcDN6.2-Caspases-3-4, Caspases-3 expression levels were significantly reduced as compared with the control group,and the inhibition efficiency reached 24％ and 67％ (P ＜ 0. 05 ). Conclusion Caspases-3 eukaryotic expression vector and the RNA interference plasmids were constructed successfully, and it was verified that pcDNA6. 2-Caspase-3-4 could specifically inhibit the Caspases-3 expression at the cellular level. Key words: RNAi; Caspase-3 ; Plasmids

Construction of eukaryotic expression plasmid of hTGF-β3 and its inducing effect on differentiation of precartilaginous stem cells into chondroblasts

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

Effective inhibition of foot-and-mouth disease virus (FMDV) replication in vitro by vector-delivered microRNAs targeting the 3D gene.

BackgroundFoot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined.ResultsFour pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV.ConclusionOur results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

Construction of Gadd45a expression plasmid and its expression in human T cells.

To construct Gadd45a expression plasmid and induce its expression in human T cells. Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells, and cloned into the pcDNA3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4(+)T cells using electroporation, and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot. Human Gadd45a expression plasmid was constructed successfully. Gadd45a was overexpressed both in Jurkat cells and normal human CD4+T cells after these cells were transfected with pcDNA3.1-Gadd45a. The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45a in the epigenetic mechanism.

NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.

Construction of eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 WT or its variants

To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its variants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells. The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+). pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs (W39R, R264C, W39R-R264C). pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells. The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing. pcDNA3.1(+)-CYP19(W39R)-GFP, pcDNA3.1(+)-CYP19(R264C)-GFP, pcDNA3.1(+)- CYP19(W39R-R264C)-GFP plasmids were confirmed by DNA sequencing. The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP. The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully, which lays the foundation for the research of biological activities of CYP19 variant allozymes.

Effect of Human RyR2 CPVT Mutations on Interaction with Calmodulin

The ryanodine receptor (RyR) is a member of a family of intracellular calcium release channels that regulate calcium efflux from intracellular stores. The RyR2 isoform is most abundant in the heart and plays a key role in cardiac muscle excitation-contraction coupling. Clusters of mutations associated with the inherited arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), have been found in specific regions throughout RyR2, a large protein of ∼5000 amino acids. Many of these CPVT mutations (total >100) are thought to occur in significant functional domains and result in the dysregulation of RyR channel function.One such region of RyR2 is believed to comprise a calmodulin (CaM) interaction site and two EF hand motifs. The calcium-sensitive binding of CaM has been shown to regulate the opening of RyR. Hence, examining the RyR2 interaction with CaM and the potential effects of CPVT mutations on this binding may help reveal mutation-dependent mechanisms of channel dysfunction.We have prepared bacterial expression plasmid constructs containing the wild-type human RyR2 CaM-interacting domain and introduced a series of CPVT mutations that have previously been identified to occur within this region. Expression and purification of the corresponding recombinant fusion proteins has enabled calcium-dependent binding of CaM to be determined with all these constructs. Examining the distinct functional role of calcium concentration on CaM binding kinetics and further comparative structural analyses of the wild-type and mutant RyR2 domains may help reveal the specific effect(s) that CaM-mediated regulation may have in mediating CPVT-linked arrhythmogenesis.

Construction and identification of small hairpin RNA expression plasmid targeting opticin gene in rat

Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene. Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Non-transfected NPE cells were set as control group. The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Results The opticin mRNA expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were decreased compared with the control group (F = 10. 239, P = 0. 000);the inhibitory rate were 85.7% ,62. 87% ,54.87% and 48.77% respectively. The opticin protein expression of the shRNA-1,shRNA-2, shRNA-3, shRNA-4 group were also decreased compared with the control group (F=17.870, P= 0.000);the inhibitory rate were 78.7%, 34.6%, 31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells. Key words: RNA, Small Interfering; Gene transfer techniques; OPTC; Animal experimentation

Effect of PEP-1-heme oxygenase-1 fusion protein transduction on hypoxia-reoxygenation injury in rat H9c2 cells

Objective To investigate the effect of PEP-1-heme oxygenase-1 (PEP-1-HO-1) fusion protein transduction on hypoxia-reoxygenation (H/R) injury in rat H9c2 cells. Methods After construction of the prokaryotic expression plasmid pET15b-PEP-1-hHO-1 containing the human heme oxygenase-1 gene, it was then transformed to make PEP-1-HO-1 fusion protein express. The H9c2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum and randomly divided into 4 groups (n = 4 each): control group (group C), H/R group, low-concentration fusion protein group (group L-HO), and high-concentration fusion protein group (group H-HO). The cells were exposed to 22 h of hypoxia followed by 8 h of reoxygenation. PEP-1-HO-1 fusion protein was added to the culture medium with a final concentration of 1.0 μ mol/L (group L-HO) or 2.0 μmol/L (group H-HO) before hypoxia. The cells and supernatant of the culture medium were collected after reoxygenation to determine the activity of lactate dehydrogenase (LDH) in the supernatant and the content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in the cells. Results The SOD activity was significantly lower, while the MDA content and LDH activity were significantly higher in group H/R, L-HO and H-HO than in group C (P ＜0.05). The SOD activity was significantly higher, while MDA content and LDH activity were significantly lower in group L-HO and H-HO than in group H/R, and in group H-HO than in group L-HO ( P ＜ 0.05). Conclusion PEP-1-HO-1 fusion protein transdution can protect H9c2 cells against H/R injury in rats. Key words: Recombinant fusion proteins; Transduction, genetic; Heme oxygenase-1; Myocytes,cardiac; Cell hypoxia; Oxygen