Recombinant Expression Vector Research Articles

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H2O2)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H2O2 to establish a model of H2O2 injury to cells. H2O2 was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H2O2-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H2O2-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins' expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H2O2 treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H2O2-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H2O2-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H2O2-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H2O2-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H2O2 injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H2O2-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.

Toxoplasma gondii: Enzyme-linked immunosorbent assay based on a recombinant multi-epitope peptide for distinguishing recent from past infection in human sera

Enzyme-linked immunosorbent assays (ELISAs) based on a recombinant multi-epitope peptide (rMEP) were used in an attempt to differentiate pregnant women with Toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). The recombinant expression vector pET-32c-MEP encoding MEP constructed previously was expressed in Escherichia coli and the rMEP was purified as a bioactive fusion protein. The IgG-ELISA and IgM-ELISA based on the purified rMEP were developed, and used to detect IgG and IgM antibodies against Toxoplasma gondii in human sera. Immunoblot assays showed that the purified rMEP could be strongly recognized by IgM antibodies in the pooled sera from women with acute profiles, and by IgG antibodies in the pooled sera from women with chronic profiles. ELISA results also proved that the reactivities of IgG and IgM antibodies differed significantly in sera from women with acute and chronic profiles. Compared with two commercial ELISA tests for seradiagnosis of toxoplasmosis, the total concordance (including positive and negative sera) of this rMEP-based assay was 93.2% and 95.7% for the detection of IgG and IgM antibodies, respectively. Our study suggests that the rMEP protein could be used as the diagnostic antigen to differentiate recent from past infections in human toxoplasmosis.

Characterization of T-cell Receptors Directed Against HLA-A*01-restricted and C*07-restricted Epitopes of MAGE-A3 and MAGE-A12

The ability of T cells that have been genetically engineered to express T-cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical trials. These TCRs have primarily targeted HLA-A*0201-restricted TCRs, as approximately 50% of whites, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of the patient population respectively. Therefore, 2 TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07 and 2 TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor-reactive T-cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the 2 MAGE-A3-reactive TCRs and one of the 2 MAGE-A12-reactive TCRs were superior to the additional TCRs in conferring transduced peripheral blood mononuclear cells with the capacity to recognize a broad array of antigen and MHC-positive target cells. These results provide support for the use of these TCRs in cancer adoptive immunotherapy trials.

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

BackgroundLactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.ResultsBest transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.ConclusionsTransformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

Overexpression of oxidored-nitro domain containing protein 1 inhibits human nasopharyngeal carcinoma and cervical cancer cell proliferation and induces apoptosis: Involvement of mitochondrial apoptotic pathways

Oxidored-nitro domain containing protein1 (NOR1) is a novel member of the nitroreductase family that was first isolated as a tumor suppressor gene from human nasopharyngeal carcinoma (NPC). However, the role of NOR1 gene dysfunction in human cancers has not been addressed. We analyzed the expression of NOR1 in various human cancer and benign tissue specimens and found significant downregulation in nine types of cancer compared with corresponding non-tumor tissues. The recombinant expression vector pCDNA3.1-myc-his-NOR1 was constructed and transfected into human NPC 6-10B nasopharyngeal cancer and HeLa cervical cancer (CCA) cells. We found that stable NOR1 overexpression resulted in suppression of 6-10B and HeLa cell proliferation and led to S phase cell cycle arrest. In addition, NOR1 upregulation enhanced apoptosis in pCDNA3.1-myc-his-NOR1 stably transfected cells, and it also altered the expression of proteins involved in the mitochondria-dependent apoptotic pathway. Furthermore, we also found that the NOR1 protein is a cytoplasmic protein that is partially localized in the mitochondria and endoplasmic reticulum. Therefore, NOR1 is an important tumor suppressor gene associated with NPC and CCA and may play antitumor roles by inhibiting proliferation, preventing colony formation, and promoting the apoptosis of tumor cells via the mitochondrial-dependent apoptotic pathway. However, the precise mechanism behind the NOR1 antitumor effects needs to be investigated further.

The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro

Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

Construction of a recombinant shRNA expression vector targeting EZH2 gene and its inhibitory effect on colon adenocarcinoma SW480 cells

To construct a recombinant short hairpin RNA (shRNA) expression vector targeting EZH2 gene, and to determine its effect on the proliferation of colon adenocarcinoma SW480 cells. The DNA sequence with short hairpin structure was designed according to the EZH2 cDNA sequence and cloned into PGFP-V-RS vector to construct a recombinant expression vector silencing EZH2 gene. After identification, the shRNA-expressing vector was then transfected into SW480 cells. RT-PCR and Western blot were used to detect the inhibitory effect at both mRNA and protein levels. MTT was used to detect cell viability due to the alteration of EZH2 gene activity. At 48 h after transfection, the expression of EZH2 mRNA in the gene silencing group and negative control group were 0.339 ± 0.013 and 1.968 ± 0.072, respectively. The expression of EZH2 protein in the gene silencing group and negative control group were 0.229 ± 0.008 and 1.168 ± 0.053, respectively. The expression of EZH2 in the gene silencing group was significantly lower than that in the negative control group (P < 0.01, P < 0.05). At 48 and 72 h after transfection, the inhibition rate of cell growth in the gene silencing group was 30.7% and 25.9%, respectively, indicating that the cell growth was significantly inhibited in comparison with that in the blank control group (P < 0.05). A recombinant shRNA expression vector targeting EZH2 gene has been successfully constructed in this study, with a significant inhibitory effect on the proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.

THE ROLES OF PRESENILIN-2 GENE IN THE APOPTOSIS OF MYOCARDIAL CELL

ObjectivesPresenilin (PS) gene is a novel gene family of causative gene related with familial Alzheimer's disease. They have been detected not only in the brain, but also in some peripheral...

Overexpression of mouse TTF‐2 gene causes cleft palate

In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2 or FOXE1) result in Bamforth syndrome. Bamforth syndrome is characterized by agenesis, cleft palate, spiky hair and choanal atresia. TTF-2 null mice (TTF-2−/−) also exhibit cleft palate, suggesting its involvement in the palatogenesis. However, the molecular pathology and genetic regulation by TTF2 remain largely unknown. In the present study, the recombinant expression vector pBROAD3-TTF-2 containing the promoter of the mouse ROSA26 gene was created to form the structural gene of mouse TTF-2 and was microinjected into the male pronuclei of fertilized ova. Sequence analysis confirmed that the TTF-2 transgenic mouse model was established successfully. The transgenic mice displayed a phenotype of cleft palate. In addition, we found that TTF-2 was highly expressed in the medial edge epithelium (MEE) from the embryonic day 12.5 (E12.5) to E14.5 in TTF-2 transgenic mice. These observations suggest that overexpression of TTF-2 during palatogenesis may contribute to formation of cleft palate.

Construction of a plasmid for expression of rat platelet-derived growth factor C and its effects on proliferation, migration and adhesion of endothelial progenitor cells

Endothelial progenitor cells (EPCs) play a key role in restoring endothelial function and enhancing angiogenesis. Platelet-derived growth factor C (PDGF-C) is a newly discovered member of the PDGF family that binds to the PDGFR-α homodimer and the PDGFR-α/β heterodimer. Currently, the biological effects of PDGF-C on EPCs proliferation, migration and adhesion are not well understood. In this study, the full-length coding sequence (CDS) region for the PDGF-C gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified PDGF-C product was digested and inserted into the pMD 19-T simple vector and then subcloned into a pIRES2-EGFP plasmid to construct the pIRES2-EGFP-PDGF-C eukaryotic expression vector. After it was transfected to EPCs, the expression of PDGF-C protein in EPCs was verified by Western blotting analysis. Finally, we investigated the effects of PDGF-C protein overexpression on EPCs proliferation, migration and adhesion. In conclusion, we constructed a recombinant eukaryotic expression vector containing the complete CDS region of PDGF-C and expressed the full-length and functional PDGF-C protein successfully. Furthermore, PDGF-C promoted EPCs proliferation, migration and adhesion. This offers promise for the development of new therapeutic strategies for improving neovascularization and repair of blood vessel endothelium in patients with ischemic heart disease or peripheral arterial occlusive disease.

Construction of a recombinant eukaryotic expression vector containing PHD3 gene and its expression in HepG2 cells

Prolyl hydroxylase domain 3 (PHD3) is a hypoxia inducible factor-α (HIFα) regulator; it degrades HIFα in the presence of oxygen. Recently, there have been an increasing number of studies about the role of PHD3 in proliferation and apoptosis of cancer cells. However, most of the evidence for the role of PHD3 is observational, and little is known of the molecular mechanism. In our current study, we constructed a recombinant eukaryotic expression vector containing the PHD3 gene and detected its biological activity in human hepatoma cell line (HepG2 cells). We successfully constructed a recombinant pcDNA 3.1(+)-PHD3 plasmid; the results showed that PHD3 overexpression could inhibit the proliferation of HepG2 cells and induce apoptosis by activating caspase-3 activity. Our study has provided preliminary materials and data for further investigation of the effect of PHD3 on HepG2 cells.

Package and identification of replication deficient recombinant adenovirus expression vector of channelrhodopsin-2

Background Channelrhodopsin-2 (ChR2)is a cation channel isolated from the eyespot of Chlamydomonas algae and has been used to control neuron activity.The light stimulation is a more precise fashion whether space or time than that of electrical,magnetic and ultrasound stimulation. Objective This study was to construct a replication deficient recombinant adenovirus cxpression vector of ChR2 and to determine its function.Methods Human embryo kidney 293 (HEK293) cell line was cultured and passaged in DF12 medium containing 10％ fetal bovine serum(FBS).The ChR2 gene was cloned at the downstream of cytomegalovirus(CMV)promoter of the adenoviral shuttle plasmid pSB291 in sense direction,and the resultant recombinant plasmid pSB291-hChR2- GFP was transfected into HEK293 cell together with plasmid pBHG lox ( deltaE1,3 ) containing adenoviral genome,then small amounts replication deficient recombinant adenovirus expression vector of ChR2 (Ad-ChR2) was obtained.Through amplification gradient centrifugation and dialysis,pure Ad-ChR2 was obtained.Visual cortex cells derived from 4 1-day-old clean Long Evans rats were primary cultured with serum-free culture media and infected by AdChR2.When expressing green fluorescencc,those cells received the stimulated of blue light with 460 nm.Patch clamp technique was applied to record an action potential. Results After purification and concentration,the titer of AdhCHR2 reached 7.9×1010 PFU/ml.Twenty-four hours after transfect of Ad-ChR2,HEK293 cell membrane showed the green fluorescence for the recombinant plasmid with green fluorescence protein under the inversed fluorescence microscope.The HEK293 cells change their shape from flat to round 13 days after transfected.The primary cultured visual cortex cells exhibited the green fluorescence 3-5 days after infected by Ad-ChR2.The action potentials evoked by blue light stimulation were recorded with patch clamp on those cells expressing green fluorescence. Conclusions Ad-ChR2 expressing vector is constructed successfully in this study.It is verified that Ad-ChR2 expressing vector can infect visual cortex cells with visual function.This result is very important for visual plasticity study. Key words: Channelrhodopsin-2; Adenovirus; Patch clamp

Prevalence of internal parasites in sheep/goats and effective economic de-worming plan at upland Balochistan,Pakistan

A nitrite reductase gene related to somatic embryogenesis was first cloned from Gossypium hirsutum. The cDNA sequence of the gene, named GhNiR, is 2,257 bp in length, with 254 bp of the 5’ untranslated region and 236 bp of the 3’ untranslated region. The open reading frame is 1,767 bp in length, encoding a deduced amino acid sequence of 588 residues with a molecular weight of 65.722 kDa and an isoelectric point of 7.07. Semi-quantitative RT-PCR analysis showed that the expression level of GhNiR was higher in embryogenic calli and somatic embryoids than in nonembryogenic calli among different somatic embryogenesis stages, and that the level of GhNiR mRNA was also higher in the cultivar with higher somatic embryogenesis ability. The catalytic GhNiR was verified by transformation in E. coli BL21 (DE3) strain with the recombinant expression vector pET-28A-GhNiR. NiR activity assay showed that the crude GhNiR protein had obvious activity to NaNO2 substrate.

In vivo transient expression for the functional analysis of polydnaviral genes

Transient expression of a foreign gene in an organism is useful to determine its physiological function. This study introduces an efficient expression technique in the insect system using a recombinant eukaryotic expression vector. A recombinant construct expressing an enhanced green fluorescence protein (EGFP) gene under an immediately early promoter was injected into the larval hemocoel of Spodoptera exigua along with a cell transfection reagent. The expression of EGFP occurred earlier, and persisted for longer period with increasing injection dose. However, there was significant variation in expression efficiency among different cell transfection reagents. In addition, the transfection efficiency measured by RT-PCR varied among tissues with high expression of EGFP in hemocytes and fat body, but not in epidermis, gut, and nerve tissues. Two functional genes (CpBV15α and CpBV15β) derived from a polydnavirus were inserted into the eukaryotic expression vector and injected into S. exigua larvae. Expression levels in hemocytes and fat body were measured by RT-PCR and immunofluorescence assay. Both mRNAs and proteins were detected in the two tissues, in which expression signals depended on the amount of injected DNA. These immunosuppressive factors significantly inhibited hemocyte behavior, such as hemocyte-spreading, nodule formation, and phagocytosis. These results demonstrate the use of in vivo transient expression of polydnaviral genes for direct analysis of biological function in the host insect.

DNA Vaccines Co-Expressing Containing Hsp70 and GP5/M Protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Display Enhanced Immunogenicity

Construction of Eukaryotic recombinant expression Vector pCI-ORF5-ORF6/ pCI-ORF5-ORF6B-Hsp70 Containing Hsp70 and PRRSV GP5/M (encoded by ORF5 and ORF6 genes), and to study its immune effect. After ifentified by enzyme analysis and nucleotide sequencing test,the repression vector plasmid was transfected into COS-7 cells. The transient expression protein was detected by indirect immunofluorescence assay (IFA) and Western-blotting. The immunogenicities of this DNA vaccine constructs were firstly investigated in a mouse moder. IFN-γ, IL-4 of cytokine, and the spleen T-lymphocyte subgroup quantity (CD4+/CD8+) were detected. The result shows that the recombinant plasmid pCI-ORF5-ORF6-Hsp70 could induce higher response of cellular immune responses and specific hummune responses in mouse, providing the clinical basic data and theoretical basis for success of the DNA vaccine development.

Expression, renaturation and biological activity of recombinant conotoxin GeXIVAWT

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Conotoxin GeXIVAWT (CTX-GeXIVAWT) is a 28-amino acid peptide containing five cysteines isolated from the venom of Conus generalis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. The codes of novel conotoxin gene GeXIVAWT were optimized and generated two pairs of primers by chemical synthesis for construction of expression vector. Recombinant expression vector pET22b(+)-GeXIVAWT fused with pelB leader and His-tag was successfully expressed as an insoluble body in Escherichia coli BL21(DE3) cells. Recombinant conotoxin GeXIVAWT (rCTX-GeXIVAWT) was obtained by dissolving the insoluble bodies and purifying with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The rCTX-GeXIVAWT renatured in vitro could inhibited the growth of Sf9 cell with biological activity assay. This expression system may prove valuable for future structure-function studies of conotoxins.

Combinational biosynthesis and characterization of a fluorescent 82β-phycocyanin of Spirulina platensis

To biosynthesize fluorescent Spirulina platensis (Sp) beta-phycocyanin (PC) in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 (S6) proteome. A highly homologous gene, slr2049, was obtained from the S6 genome. Sites 82 and 153 in beta-phycocyanin of Sp were modified by site-directed mutagenesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21: (i) pCDF-cpcB (C153A)-slr2049-sll0583-ho1-pcyA; and (ii) pCDF-cpcB (C82I)-slr2049-sll0583-ho1-pcyA. Lyases encoded by the genes slr2049 and sll0583 catalyzed the linking of Sp 82 beta-PC to phycocyanobilin (PCB), and fluorescent CpcB (C153A)-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca)

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.

Construction of a novel vector expressing the fusion suicide gene yCDglyTK and hTERT-shRNA and its antitumor effects

This study aimed to construct a novel recombinant expression vector, pcDNA3.1(-)hTERT-shRNA/yCDglyTK. Its bioactivity and antitumor effects were investigated in the SGC7901 human gastric cancer cell line. Interfering RNA (RNAi) targeting human telomerase reverse transcriptase (hTERT) was applied to construct the pYr1.1-hTERT-shRNA vector. The shRNA expression cassette (including U6 promoter) was subcloned into the pcDNA3.1(-) CV-yCDglyTK vector to build a new vector, pcDNA3.1(-) hTERT-shRNA/yCDglyTK, which was identified by restriction enzyme digestion and gene sequencing. All the plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expression of yCDglyTK and hTERT was detected by immunofluorescence, real-time PCR and western blot analysis. MTT assays were applied to measure the cytotoxic effect of the plasmids with 5-fluorocytosine (5-FC). Cell apoptosis was detected by flow cytometry. Restriction enzyme digestion and gene sequencing confirmed that the recombinant vector pcDNA3.1(-)hTERT-shRNA/yCDglyTK had been successfully constructed. Immunofluorescence, real-time PCR and western blot analysis showed that yCDglyTK was expressed, and that hTERT expression was inhibited in cells transfected with the recombinant vector. The cells transfected with the recombinant vector were the most sensitive to 5-FC and the apoptosis rates of the cells were also increased. The pcDNA3.1(-)hTERTshRNA/yCDglyTK vector was constructed successfully; it was confirmed that targeting hTERT through RNAi could synergize with suicide gene therapy.

Construction of A eukaryotic expression vector carrying the iNOS gene and its effect on A549 lung cancer cells

背景与目的iNOS与NO介导的抗肿瘤效应有关。本研究旨在构建pVAX-iNOS载体并转染A549肺癌细胞，检测其基因的表达并初步探讨iNOS基因表达增高后对A549肺癌细胞的抗肿瘤作用。方法应用RT-PCR方法扩增人iNOS编码序列的CDS片段，构建pVAX-iNOS载体后转染肺癌A549细胞，通过RT-PCR和Western blot方法检测目的基因的表达；采用MTT法、Hoechst 3235染色和划痕实验分别检测iNOS高表达在体外对肺癌A549细胞增殖、凋亡和迁移作用的影响。结果真核表达质粒载体pVAX-iNOS构建成功，iNOS蛋白在转染后的A549细胞中表达升高。pVAX-iNOS转染A549肺癌细胞后能明显诱导细胞发生凋亡并抑制肿瘤细胞的生长和迁移。结论本研究成功构建pVAX-iNOS真核表达质粒，高表达iNOS能明显抑制A549细胞的增殖、迁移并促进细胞发生凋亡。本研究有望为临床治疗肺癌提供一个新的有效策略。