Effects of hepatitis C virus core protein on cell cycle of HepG2 cells

AIM: To investigate the molecular mechanism of hepatitis C virus (HCV) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D1 and pRb/p130 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV-core-1b was constructed. The cDNA of HCV core protein was subcloned into pBabe-Flag-puro vector to generate pBabe-Flag-HCV-core-1b. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus-containing supernatant collected from the cell culture was used to infect HepG2 cells and subsequently the cell line that stably expressed the core protein of HCV was obtained.The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/p130 was examined by Western blotting. RESULTS: The pBabe-Flag-HCV-core-1b vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type 1b core protein impaired the cell cycle progression in G₀/G₁ phase and significantly reduced the levels of cyclin D1 and pRb/p130 in the cells. CONCLUSION: A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed, and a HepG2 cell line which stably expressed the core protein of HCV is also established. HCV gene type 1b core protein inhibits the cell cycle possibly through down-regulation of cyclin D1 and pRb/p130 proteins in the cells.     

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

Identification of domain of protein kinase R interacting with hepatitis C virus core protein

AIM: To observe if hepatitis C virus (HCV) core protein (CP) influences the expression level of protein kinase R (PKR) and to map the direct interaction domain between PKR and CP.METHODS: The expression levels of PKR in Huh-7,Huh-7 transfected with CP plasmid and replicon Huh-7 harboring selecting full length of HCV genome were studied.HCV structure and non-structure proteins in replicon Huh-7 with interferon (IFN) stimulation were compared.Co-immunoprecipitation and glutathione S-transferase (GST) binding assay were done between PKR and CP.RESULTS: PKR expression level in replicon Huh-7 was higher than that in Huh-7 and Huh-7 transfected with CP expression plasmid.PKR was increased but structure and non-structure proteins in replicon Huh-7 were decreased after treated with IFN.The N-terminal 1-180 amino acid of PKR was the key binding site to CP.CONCLUSION: CP directly binds to N-terminal 1-180 amino acid of PKR and leads to constitutive expression of PKR,which interferes signal transfer mediated by PKR.The interaction between CP and PKR might be a novel model of virus protein-cell protein interaction,which might play an important role in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.     

Ligation of the HCV 5’ NCR,C, E1, E2/NS1 sequences andits cloning into pLNSX

AIM: To construct retrovirus recombinant HCV/pLNSX (pLHC), which is used to investigate new ways to control HCV infection and gene therapy. METHODS and RESULTS: Primers with their sequences homologous to multiple strains of HCV had been designed, synthesized and used in RT-PCR to amplify 5 fragments from 4 regions, 5’ NCR, C, E1 and E2/NS1. The products had been cloned, restrictively cut aside, and overlappingly amplified to ligate a consecutive sequence of 2547 bp in length that contains the whole HCV 5’ NCR and all the structural protein coding regions. The sequence was then inserted into the vector pGEM-3Zf (+) yielding a recombinant pHC2547, and into the retrovirus pLNSX yielding a pLHC. Both plasmid DNA were analyzed by PCR, Southern blot, and enzymatic cut. CONCLUSION: Retrovirus recombinant HCV/pLNSX (pLHC) constructed successfully is useful to study both the regulation of intracellular HCV gene expression and the pathogenesis of HCV infection, as well as the molecular foundation of related transgene animals and gene therapy.     

Advances in experimental research in HCV cell model and anti-HCV effect of As2O3

Hepatitis C virus (HCV) is a human pathogen responsible for liver diseases including acute and chronic hepatitis and hepatocellular carcinoma. However, the high prevalence, the absence of antiviral drugs and vaccines for prevention and treatment are the difficult medical problems. Lacking appropriate culture method and small animal model have severely limited investigation of the HCV infection mechanism and the development of the therapeutic strategy. Recently, the in vitro culture system develops rapidly, and provides a powerful tool for HCV related research. Despite the well known toxicity of the chemical, arsenic trioxide (As₂O₃) is effective for treating the patients with refractory or relapsed acute promyelocytic leukemia and many other cancers. Interestingly, As₂O₃ shows the ability of inhibiting HCV RNA replication and infection. This review describes the different types of in vitro HCV models developed, and many studies of the potent effect of As₂O₃ against HCV and its associated molecular mechanisms.     

Screening,identifying and sequencing of human single-chain variable fragment specific to hepatitis B virus core protein

AIM:To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence.METHODS:The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli.HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced.RESULTS:The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli.HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION:Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.     

青苹果竹芋的组培快繁技术

青苹果竹芋(Calathca rotundi fla CV.Fasciata)又称圆叶竹芋,属于竹芋科肖竹芋属多年生常绿草本.青苹果竹芋是近年来从国外引进的室内高档观叶植物品种,曾在2005年1月广州花博园举行的"中国首届盆栽花卉交易会"上,以其叶片圆润,质感好,观赏价值高,适宜于室内盆栽观赏等特点,一举获得"金花奖",令人十分注目.     

Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay

AIM: To develop a real-time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.METHODS: The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold serially diluted primary viral stock was used for plaque assay and DNA extraction.Bacmid (baculovirus plasmid) was 10-fold serially diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve.We also find that the“vg/mL”titer value is generally about 10 times than“pfu/mL”titer of the same recombinant virus stock.CONCLUSION: A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the“vg/mL”titer of virus.The method is rapid and quantitative over a wide range of virus titers.     

Cloning of human sperm protein(SP17) and expression in escherichia coli DH5α

AIM: To obtain GST fusion protein of hSP17 gene and construct the recombinant plasmidfor expression in E.coli.METHODS:Total fragment of hS P17 cDNA gene were amplified by RT-PCR,then subcoloned into p GEX-3b to generate recombinant hS P17/pGEX.Right orientation of insert are identified by restricted enzyme digestion.Transform the correct recombinant plasmid into the E.coli DH5a.The expression of fusion proteins hS P17-GST were induced by adding isopropylthiogalactoside(IPTG).RESUL TS and CONCLUSION:The recombinant plasmid hS P17/pGEX-3b could express effectively in E.coli and a high level of fusion protein hsp17-GST with the predicted molecular weight was detected.     

Phenotypic transformation of myoblast to osteoblast induced by introduction of bone morphogenetic protein-4 coding sequence into C2C12 cells

AIM: To study the effect of introduced bone morphogenetic protein-4 (BMP-4) gene on the differentiation of C2C12 cells. METHODS: BMP-4 gene was constructed into pCI-neo plasmid, which was then introduced into C2C12 cells. Cells transfected with pCI-neo-BMP-4 recombinant plasmid or pCI-neo plasmid alone and non-transfected cells were cultured in the same condition and studied. RESULTS: It was demonstrated that cells with/without pCI-neo plasmid showed no expression of BMP-4 both in their proliferating and differentiating stages. Cells transfected with pCI-neo-BMP-4 showed abundant BMP-4 expression both in their proliferating and differentiating stages. The undifferentiated C2C12 cells with/without pCI-neo plasmid displayed the characteristic stellate morphology, and fused with each other to form characteristic thin, elongated multinucleated myotubes. In contrast, the cells transfected with pCI-neo-BMP-4, subject to both proliferation or differentiation stages did not form myotubes, instead they formed osteoblast-like morphology with high level expression of alkaline phosphatase (ALP) in the cells and also secreted osteocalcin in their culture medium. CONCLUSION: The introduction of BMP-4 gene into C2C12 cells could convert their committed myogenic differentiation pathway into that of osteoblast lineage, and the conversion of differentiation pathway is heritable.     

Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

AIM: To explore the mechanism of Coriaria sinica Maxim’s extract (CSME) promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage, based on the signaling pathways, such as transforming growth factor-β₁ (TGF-β₁) regulated by integrin-linked kinase (ILK) and ILK regulated by PI3K/AKT. METHODS: Female SD rats (n=180; 180~200 g) were randomly divided into 6 groups: normal control (NC) group, vaseline (VL) group, silver sulfadiazine (SS) group and low-, medium- and high-dose of CSME (CSME-L, CSME-M and CSME-H) groups, with 30 rats in each group. Except for the rats in NC group, VL, SS, and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II° burn model was made on their waist-back in the condition of chloral hydrate anesthesia. To calculate the healing rate (HR), 10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change, detecting the expression of related proteins by Western blot and RT-qPCR, and checking the collagen shrinkage (SK) by fibroblast culture at the 7th, 14th, and 21st days. RESULTS: The expression of ILK, fibronectin (FN), TGF-β₁, α-smooth muscle actin (α-SMA) and integrin-β₁ (ITG-β₁) at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner, but weaker than that in VL group and SS group at the 21st day (P<0.05). Meanwhile, the protein and mRNA expression of collagen type I (Col I) in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day, but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). However, the protein and mRNA expression of collagen type III (Col III) in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day, but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h. SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner, but lower than that in VL group and SS group at 96 h (P<0.05). CONCLUSION: CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages. The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β₁ regulated by ILK and ILK regulated by PI3K/AKT. It may also be related to the ratio of Col I and Col III expression.     

Preparation of m1AChR-G11 fusion protein and m4AChR-G16 fusion protein and effects of various ligands on them

AIM:To prepare m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein in Baculovirus-Sf9 cell system and detect the effects of various muscarinic ligands on the interaction between m₁AChR and G₁₁ and m₄AChR and G₁₆, and screen different kinds of ligands specific for m₁ and m₄. METHODS:To prepare fused DNA of m₁AChR-G₁₁and m₄AChR-G₁₆ in two PCR, then expressed in Sf9 cells and detect the pharmacological function of m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein by QNB and GTPγS binding experiments; To expore the way of the activation of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein by various ligands includingcetylcholine (ACh), Pilocarpine (Pilo), 4-hydroxy-2-butynyl-1-trimethylammonium-m-chloro-carbanilatechloride (McN-A-343), tetrandrine, pirenzepine (PZ), alcuronium, atropine, R-(+)-hyoscyamine and gallamine by displacement by GDP on GTPγS binding experiments. RESULTS:The expression levels of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein were (45.39±2.62) nmol·g^-1 protein, (47.04±1.58) nmol·g^-1 protein. The affinity of GDP to G₁₁ and G₁₆ partner changed in the presence of different muscarinic ligands. CONCLUSION: The m₁AChR-G₁₁ and m₄AChR-G16 showed the pharmacological specificity to m₁ and m₄ receptor and the efficient signaling of the two partners. Ligands of m₁AChR and m₄AchR mediated different signal transduction by changing the affinity of G₁₁/G₁₆ and GDP. So m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein can be taken as a tool to screen ligands specific for m₁AChR and m₄AChR.     

Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

结球甘蓝雌蕊调控转录因子SPT 基因的克隆 与序列分析

以结球甘蓝E1 为材料,提取花蕾总RNA,反转录cDNA。根据拟南芥SPT 基因设计引物,
采用同源克隆的方法从中克隆SPT 基因序列1 085 bp,开放阅读框1 062 bp。通过cDNA 推导得到的氨
基酸序列分析表明,BoSPT 编码353 个氨基酸残基,预测分子量为37.67 kD,pI 为6.83。经过EcoRⅠ和
KpnⅠ限制酶双酶切后,构建原核表达质粒pET43.1a-BoSPT 转化表达菌株E. coli Rosetta（ DE3）,通过
SDS-PAGE 检测该蛋白的表达。经Smart-embl 预测其具有bHLH 家族结构域,位于序列第173～221 位氨
基酸残基处。进化树表明结球甘蓝BoSPT 与拟南芥AtSPT 和筷子芥AlSPT 的亲缘关系较近。BoSPT 基因
的原核表达得到纯化的融合蛋白。     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

Expression of GAP-43 mRNA and protein following brachial plexus avulsion injury

AIM:To investigate the expression of GAP-43 mRNA and protein of the motor neurons in spinal cord following the brachial plexus avulsion injury. METHODS:In the present study, three kinds of models of brachial plexus avulsion injury were made: right C₇ anterior root avulsion (group A), C₇ anterior root avulsion with right C₅-T₁ posterior roots breaking (group B), right C₇ anterior root avulsion with hemi-transect between C₅ and C₆ segment of spinal cord (group C). The expression of GAP-43 mRNA in anterior horn of spinal cord was detected at 14 days after operation by SYBR green quantification RT-PCR technique. The amounts of GAP-43 positive neurons in spinal cord were detected at 1, 3, 7 and 14 days after operation by immunohistochemistry technique. RESULTS:In control group, the expression of GAP-43 mRNA was very low in anterior horn. By 14 days after operation, the expression of GAP-43 mRNA was evidently up-regulated compared with control group. GAP-43 positive neuron was observed in control group at 1st day and 3rd day after operation. GAP-43 positive neurons appeared at 7th day and peaked at 14th day after operation. The expression of GAP-43 mRNA and protein were maximum in group C, group B was the lowest. CONCLUSION:The expression of GAP-43 mRNA and GAP-43 protein were up-regulated following brachial plexus injury. The expression of GAP-43 protein results from the recombination of proteins. GAP-43 is closely related to the axon regeneration and functional reconstruction.     

Regulation of DHCR24 expression and its progress with HCV infection

3β-hydroxysterol △²⁴-reductase (DHCR24) catalyzes the conversion of desmosterol to cholesterol and in turn affects lipid metabolism. DHCR24 is a multifunctional protein with enzymatic activity, antioxidant stress, scavenging of reactive oxygen species (ROS) and anti-apoptotic effects. Hepatitis C virus (HCV) infection results in severe oxidative stress which is correlated closely with fatty liver, liver cirrhosis, liver cancer and a series of liver diseases. Recent studies have shown that DHCR24 expression is regulated by various factors such as sterols, hormones and growth factors, and the expression of DHCR24 is significantly increased after HCV infection. In-depth study of the function and regulation of DHCR24 and its relationship with disease can be help to further discover and understand the pathogenesis, and contribute to early diagnosis and treatment of disease. This review summarizes the regulation of DHCR24 expression and its progress with HCV infection.     

Erythropoietin inhibits eryptosis induced by reactive oxygen species

AIM: To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H₂O₂),and to explore its related mechanism. METHODS: The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group, the culture medium was PBS), H₂O₂ group (H group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L) and EPO group (E group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L and EPO at final concentration of 2×10⁴ U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion concentration ([Ca²⁺]_i) were also analyzed by flow cytometry. RESULTS: The eryptosis in C group was increased as the incubating time extended. The eryptosis in H group was higher than that in C group (P<0.01), while that in E group was lower than that in H group (P<0.01). Meanwhile, ROS production and[Ca²⁺]_i were higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION: EPO inhibits eryptosis induced by H₂O₂ and its mechanism may be related to antioxidant effect and change of[Ca²⁺]_i.     

Effect of calcitonin gene-related peptide on proliferation of vascular smooth muscle cells

AIM: To elucidate the effect of calcitonin gene-related peptide (CCRP) in the therapy of atherosclerosis.METHODS:Effect of CGRP on cell cycle kinetics of cultured vascular smooth muscle cells(HA-VSMC) was investigated by flow cytometry. The expression of cyclins D₁ and E required for initiation of S phase were also studied by immunochemistry method. RESULT: CGRP was shown to arrest VSMC in the G₀/G₁ phase of cell cycle and reduced expression of cyclins D₁ and E. CONCLUSION:CGRP inhibits proliferation of HA-VSMC by arresting cells in G₁ phase via limiting accumulation of cyclin D₁ and E. It might play a role in the therapy of atherosclerosis.