Recombinant Expression Plasmid Research Articles

Construction of a recombinant eukaryotic vector of Importin 8 and its expression in HeLa cells

Objective To clone the coding sequence of Importin 8 (IPO8) cDNA from HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein (GFP)-IPO8 fusion protein expression in HeLa cells.Methods The total RNA was extracted from HeLa cells and the full length cDNA of IPO8 was amplified using reverse transcription-polymerase chain reaction (RT-PCR),and cloned into green fluorescence protein vector phosphorylated enhanced green fluorescent protein (pEGFP)-C1 to construct recombinant expression plasmid pEGFP-IPO8.After recombinant plasmids were transfected into HeLa cells,the expression of IPO8 was observed by fluorescent microscope.Results DNA sequence analysis showed that the amplified rat IPO8 gene was in concordance with that published on Gene Bank.The results of enzyme digestion and sequence analysis demonstrated that the recombinant plasmid pEGFP-IPO8 was constructed successfully.Fluorescence microscopy revealed the fusion protein GFP-IPO8 was mainly located in the nucleus of HeLa cells.Conclusion The IPO8 gene was cloned successfully and the recombinant plasmid were constructed successfully.The fusion protein GFP-IPO8 was demonstrated to be mainly located in the nucleus of HeLa cells,which provides a basis for biological functional study of IPO8 gene. Key words: Importin 8 gene; Green fluorescent protein; Fusion protein; Eukaryotic vector

Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis

Lacticin Q is a 53-amino acid Class II bacteriocin produced by Lactococcus lactis QU 5. It shows antibacterial activity comparable to that of nisin A in terms of both spectrum and intensity. Moreover, it remains stable at alkaline pH values, while nisin A was inactivated. It may possibly be employed as an alternative to or in combination with nisin A. The objective of this study was to express lacticin Q extracellularly with Small ubiquitin-related modifier (SUMO) fusion technology in Bacillus subtilis. Secretory SUMO-lacticin Q fusion protein was efficiently produced in B. subtilis WB600 transformed with the recombinant expression plasmid and accounted for 19% of the culture supernatant proteins. Fusion SUMO-lacticin Q was purified by nickel nitrilotriacetate (Ni–NTA) affinity chromatography and digested with SUMO protease to release lacticin Q. Lacticin Q was further purified by Ni–NTA chromatography to yield about 2.5mg of lacticin Q with more than 93% purity from 1L of supernatant of fermentation culture. An activity assay indicated that the recombinant bacteriocin exhibited excellent antimicrobial activity against indicator strains. The results obtained suggest that the secretory lacticin Q was efficiently expressed using SUMO fusion technology in B. subtilis. The expression and purification system could promote the application of lacticin Q in food and medicine.

Recombinant attenuated Salmonella typhimurium carrying a plasmid co-expressing ENDO-VEGI151 and survivin siRNA inhibits the growth of breast cancer in vivo

The aim of this study was to investigate the antitumor effect of a plasmid co-expressing ENDO-VEGI151 and survivin siRNA on breast cancer in nude mice, and to explore the feasibility of attenuated Salmonella typhimurium (S. typhimurium) as a delivery vector for cancer gene therapy in vivo. Three recombinant expression plasmids pENDO‑VEGI151 (pEV), pSurvivin-siRNA (psi-survivin) and co-expressing plasmid pENDO-VEGI151/survivin‑siRNA (pEV/si-survivin), were transferred into the attenuated S. typhimurium strain SL7207, respectively. MDA-MB-231 cells were infected with these recombinants in vitro, and the expression of ENDO-VEGI151 and survivin was detected. In order to detect S. typhimurium distribution and gene delivery efficiency in vivo, the plasmid pEGFP-N1 which encodes green fluorescent protein was transferred into SL7207, and the recombinant known as SL-pEGFP was orally administered to tumor-bearing nude mice. The gene transfer efficiency, distribution and survival time of the SL-pEGFP in vivo were evaluated by detection of GFP fluorescence. SL-pEGFP not only infected the cancer cells effectively, but also allowed the survival and expression of specific genes mainly in the xenografts of nude mice. To further identify the anticancer effects of these recombinants in vivo, mice burdened with xenografts were randomly divided into 6 groups, which were subjected to intragastric administration of vehicle, SL7207, SL-pcDNA3.1, SL-pEV, SL-psi-survivin and SL-pEV/si-survivin, respectively. Eight weeks after implantation, tumor size, weight, inhibition rate, intratumoral microvessel density (MVD), apoptotic index (AI), ENDO‑VEGI151 and survivin expression were evaluated. Compared with the SL-pEV or SL-psi-survivin-treated groups, the growth of tumors was significantly reduced in the SL-pEV/si-survivin group with an inhibition rate of 90.28 vs. 69.12 and 65.61%, respectively. MVD and the expression of survivin were decreased significantly in the SL-pEV/si-survivin-treated group, while AI increased significantly in the SL-pEV/si-survivin-treated group. These results indicated that attenuated S. typhimurium carrying the dual function plasmid pEV/si-survivin cannot only be specifically enriched in the tumor tissue, but also showed a synergistic antitumor effect in vivo.

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1

BackgroundCancer is both a systemic and a genetic disease. The pathogenesis of cancer might be related to dampened immunity. Host immunity recognizes nascent malignant cells – a process referred to as immune surveillance. Augmenting immune surveillance and suppressing immune escape are crucial in tumor immunotherapy.MethodsA recombinant plasmid capable of co-expressing granulocyte-macrophage colony- stimulating factor (GM-SCF), interleukin-21 (IL-21), and retinoic acid early transcription factor-1 (Rae-1) was constructed, and its effects determined in a mouse model of subcutaneous liver cancer. Serum specimens were assayed for IL-2 and INF-γ by ELISA. Liver cancer specimens were isolated for Rae-1 expression by RT-PCR and Western blot, and splenocytes were analyzed by flow cytometry.ResultsThe recombinant plasmid inhibited the growth of liver cancer and prolonged survival of tumor-loaded mice. Activation of host immunity might have contributed to this effect by promoting increased numbers and cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) following expression of GM-SCF, IL-21, and Rae-1. By contrast, the frequency of regulatory T cells was decreased, Consequently, activated CTL and NK cells enhanced their secretion of INF-γ, which promoted cytotoxicity of NK cells and CTL. Moreover, active CTL showed dramatic secretion of IL-2, which stimulates CTL. The recombinant expression plasmid also augmented Rae-1 expression by liver cancer cells. Rae-1 receptor expressing CTL and NK cells removed liver cancer.ConclusionsThe recombinant expression plasmid inhibited liver cancer by a mechanism that involved activation of cell-mediated immunity and Rae-1 in liver cancer.

Cloning and Transcription Function Analysis of Cotton Transcription Factor GhGT 3 0 Gene

Trihelix transcription factors play an important role in the regulation of plant growth and development, as well as in their response to many kinds of abiotic stress. According to expressed sequence tag (EST), full-length cDNA sequence of trihelix transcription factor was cloned from upland cotton (Gossypium hirsutum L.) using the methods of rapid-amplification of cDNA ends (RACE) and RT-PCR. Sequence analysis showed that the full-length of GhGT30 (GenBank accession No. JQ013098) was 2210 bp, containing a 2025 bp open reading frame which encoded a protein of 675 amino acids with predicted molecular weight of 76.26 kD and a isoelectric point of 6.21. SMART analysis showed GhGT30 with each trihelix domain at N-terminal and C-terminal, belonging to GT-2-type factors. Amino acid sequence alignment revealed that N-terminal of GhGT30 shared high degree of identity with other higher plant GT proteins. The phylogenetic tree showed that GhGT30 was located at the same branch with GmGT-2B. Transient expression of recombinant plasmid GhGT30/PBI221-GFP in Arabidopsis protoplasts showed that GhGT30 was located in cell nuclei. Real-time quantitative PCR (qPCR) showed that GhGT30 was expressed in a higher level in flower, fiber (12 DPA) than in root, stem leaf, and ovule (0 DPA). The gene was differentially respondes to various abiotic stresses (dehydration, high salinity, and low temperature) and ABA, indicating that it may play important roles in response of cotton plant to abiotic stresses.

Expression and purification of GST-FHL2 fusion protein

Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.

Impact of the biological function on epithelial ovarian cancer with ITIH4 gene expression down-regulating in vitro

To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer. The four pairs ITIH4 gene siRNA interference fragments (ITIH4-546, ITIH4-795, ITIH4-917 and ITIH4-1568) were designed respectively, and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently. Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment. The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells. The stably transfected cells- pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418). The experiment was divided into three groups, namely ITIH4-917 transfection group, the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group), and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group). Fluorescence quantitative reverse transcription (RT) PCR and western blot were used to detect the ITIH4 mRNA and protein expression. The cell proliferation, the cell cycle, colony formation of cells, cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT), flow cytometry, colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)], respectively. The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells, the relative copy number was only 0.26 ± 0.15. Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ± 0.10, it significantly lower than that in empty vector group (1.87 ± 0.12, P = 0.008) and negative control group (1.58 ± 0.21, P = 0.032); Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells, empty vector group and negative control group were 0.51, 1.64 and 1.74, respectively, there were statistically significant differences (0.51 vs. 1.64, P = 0.012; 0.51 vs. 1.74, P = 0.014). MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group, and there were statistically significant differences among them (P = 0.001). The S + G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2%, which was significantly higher than that in the empty vector group or negative control group (26.3% and 31.3%, respectively, all P < 0.05). The colony formation rate (55.7 ± 0.7)% in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ± 0.9)% (P = 0.037) and negative control group (31.4 ± 0.3)% (P = 0.043). Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18, whicht was significantly higher than that in the negative control group or empty vector (0.30 ± 0.03, P = 0.031;0.25 ± 0.03, P = 0.028, respectively). Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ± 0.34) was higher than that in the control cells (1.05 ± 0.68) and empty vector group (1.14 ± 0.08), while there were not significant difference (P > 0.05). It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.

Construction and expression of a recombinant eukaryotic expression plasmid containing the preS1-preS2-S genes of hepatitis B virus and the granulocyte-macrophage colony stimulating factor gene: A study of its immunomodulatory effects

A total of 10-20% of the population remains unresponsive or weakly responsive to hepatitis B vaccine, which is composed of hepatitis B surface antigen HBsAg (S protein). Therefore, it is necessary to develop a hepatitis B vaccine with a better penetrating and responsive rate. In the present study, a plasmid pVAX1-L-GM was constructed and its immunomodulatory effect of as hepatitis B virus (HBV) DNA vaccine was analyzed through the immunization of BALB/c mice. Immune responses were measured after immunization by anti-HBsAg, proliferation of splenocytes, the number of CD4+ and CD8+ molecules, CTL cytotoxicity, cytokines of IFN-γ and IL-2 secretion assays. Following the immunization, mice in the pVAX1-L-GM group produced antibody 2 weeks earlier compared to the control plasmid pVAX1 and pVAX1HBsAg groups and antibody levels showed significant differences. Enhanced HBsAg-specific splenocyte proliferation as well as specific cytotoxic activities of splenic CTLs were also detected. Furthermore, pVAX1-L-GM plasmid increased the number of CD4+ and CD8+ molecules on the surface of the spleen T cell and the level of IFN-γ, IL-2 secretion. pVAX1-L-GM induced a specific immune response in mice and enhanced the immune effect. Thus, a foundation was laid for developing immunogenicity of a better prevention and treatment of HBV via a hepatitis B vaccine.

An improved method of xylose utilization by recombinant Saccharomyces cerevisiae

The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

Toxoplasma gondii: Bioinformatics analysis, cloning and expression of a novel protein TgIMP1

Toxoplasma gondii is an obligate intracellular protozoan parasite, infecting a large variety of animals and human beings. In recent years, the study of DNA vaccine against T. gondii has made a great progress; however, few vaccines have completely controlled toxoplasmosis. Thus people started to look for more effective antigenic proteins. Here we report a novel T. gondii protein termed immune mapped protein 1 (TgIMP1). We used multiple bioinformatics approaches to predict the physical and chemical characters, signal peptide, transmembrane domain, epitope, topological structure and function of the protein, and we theoretically determined that the TgIMP1 has multiple epitopes, and with immunogenicity, suggesting that the TgIMP1 may be a vaccine candidate against toxoplasmosis. Then the gene coding TgIMP1 was obtained by PCR and connected with cloning vector. Recombinant plasmid was identified by PCR, double digestion and sequencing analysis. Then the TgIMP1 gene was directly inserted into the eukaryotic expression vector pBudCE4.1, so that the recombinant eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed. After identification by PCR and restriction enzyme digestion, the recombinant plasmid pBudCE4.1-TgIMP1 was transfected into cells of HFF, and then identified by RT-PCR. The results showed that the eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed and was transfected to the HFF cells successfully.

Effect of shRNA-mediated silencing of CTGF and TIMP-1 on mRNA expression of CTGF, TIMP-1, and PC I and secretion of extracellular matrix in rat hepatic stellate cells

To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.

Reactive Center Loop Moiety Is Essential for the Maspin Activity on Cellular Invasion and Ubiquitin‐Proteasome Level

Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.

The killing effect of NY-ESO-1-sensitzed dendritic cells-induced cellular toxic lymphocyte on human retinoblastoma cell

Background Cell immunologic therapy for retinoblastoma （RB）is becoming a hot research topic. Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors. However,its effect on RB has not been investigated. Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes （CTL）induced by the cancer-testis antigen, NY-ESO-l-sensitized dendritic cells （DCs）, on human RB. Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI. Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1. The expression of NY-ESO-1 protein in human RB cells strain, HXO-RB44,was detected by immunofluoreseence and Western blot. Monoeytes wereisolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 x 107/ml. DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4 （ rhIL-4 ）. The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes. The resultant CTL were used as effector ceils. The growth of the CTL was detected by MTT assay. The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay. Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-lwas conforms with the sequence of the NY-ESO-1 gene. The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot. DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs. These DCs had high expression of surface molecules such as HLA-DR （42. 1% ） , CD80 （54.2%） , CD83 （39.7%） and CD86 （94.8%）. The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells. CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs, as shown by MTT asssay（ P〈0.05 ）. The anti-tumor activity was highest when the ratio of effector to target was 75 ： 1 （ P〈0. 05 ）. Conclusions DCs transfected by the recombinant plasmid pDC316/ NY-ESO-1 can induce the proliferation of allogenic CTLs, which showed a specific anti-tunmr effect against HXORB44 cells. These results present a new type of immunotherapy for the treatment of RB. Key words: Retinoblastoma; Ddendritic cell; Cytotoxic T lymphocyte; NY-ESO-1

Transforming Growth Factor beta1 Gene Variation Leu10Pro Affects Secretion and Function in Hepatic Cells

Our previous work revealed transforming growth factor beta1 (TGFβ1) gene polymorphisms are associated with susceptibility to hepatocellular carcinoma and liver cirrhosis. However, no further study of functional substitution in hepatic cells has yet been reported. This study was designed to uncover the functional mechanisms of TGFβ1 gene polymorphisms in the pathogenesis of liver diseases. Two recombinant TGFβ1 expression plasmids containing TGFβ1 codon 10 Leu/Pro variation were constructed with CMV promoter and transfected into human hepatoma cell lines (HepG2 and SMMU 7721), hepatic stellate cells (LX-2), and immortalized hepatocytes (L02). The secretion capacities of TGFβ1 protein in the transfected cells were determined by ELISA. Apoptosis, proliferative activity, and expression of CD 105, CD83, and CD80 were also measured by use of flow cytometry. The ELISA results showed that cells transfected with CMV-Pro10 were more capable of TGFβ1 secretion than those transfected with CMV-Leu10. Functionally, CMV-Pro10 was more apoptosis-protective and induced more proliferation than CMV-Leu10 in transfected hepatic cells. Pro10 up-regulated expression of CD105 and down-regulated expression of CD83. TGFβ1 gene Leu10Pro variation in signal peptide has significant effects on TGFβ1 secretion and functions in hepatic cells.

Biological function explore of protein CCL15 in HCC cell lines

To explain biological function of protein CCL15 in HCC cell lines. The different expression level of CCL15 among HCC cell lines was validated by RT-PCR and Western blot. The expression recombinant plasmid of siRNA-CCL15 was constructed successfully and transfected into high metastasis cell lines HCCML3 to observe the alteration of biological function of HCCML3. The overexpression of CCL15 in high metastasis HCC cell lines was confirmed by validation tests. After transfected with siRNA-CCL15, the average amounts of invaded cells in cell invasion assay were 657.9 (HCCML3) and 148.4(HCCML3-siCCL15) (t=19.34, P less than 0.05). And in the scratch assay, the migrating distance were (0.35+/-0.02) mm (HCCML3) and (0.82+/-0.03)mm (HCCML3-siCCL15) (t=15.67, P less than 0.05). The expression of MMP-9 in HCCML3 was higher than HCCML3-siCCL15 through Western blot. Some biological properties (migration, invasion, MMP-9) of HCCML3 transfected with siRNA-CCL15 were decreased. The results suggest CCL15 might play an important role in HCC cell invasion and metastasis through two paths of MMPs regulation and invasion potential strengthening.

Experimental Research of RB94 Gene Transfection Into Retinoblastoma Cells Using Ultrasound-Targeted Microbubble Destruction

The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His+, Mut+) with multiple hly copies. One transformant resistant to 4.0 mg/ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol.

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.

Preparation and identification of a recombinant hepatitis C virus (HCV) based on sindbis virus vector

To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector. The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA). The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2. The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.