To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.

METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.

RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.

CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.

Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVE: To analyze the expression and location of coding protein of UBAP1 gene and to understand the relationship between the expression pattern of the protein and cell carcinogenesis. METHODS: Bioinformatics was used to analyze the protein character to provide an available clue of subsequent research. The codon frame cDNA was amplified by PCR, and subcloned into enhance green fluorescence protein (EGFP) of pEGFP-C2. The recombinant plasmid was transfected into HNE1 cells. The expression of coding protein was observed by fluorescence microscopy. RESULTS: The expressed GFP-fusion protein generated striking green fluorescence in the cytoplasm in HNE1 cells. EGFP/UBAP1 was expressed and existed mainly in the nuclear, especially accumulated on the nuclear envelope. CONCLUSION The expression difference in HNE1 might be related to the carcinogenesis of NPC.
Base Sequence ; Carrier Proteins ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; metabolism ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Bacterial Proteins ; biosynthesis ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; D-Amino-Acid Oxidase ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics ; Yeasts ; enzymology ; genetics

Omics docking study for polygenic inheritance tumors has become an important strategy in oncology research. This review focuses on the conceptions and technologies of omics, and puts forward the central contents and omics docking for polygenic inheritance tumor to reveal the role of molecular changes at different stages of polygenic inheritance tumor at multidisciplinary and multilayer level. It is a new strategy to explore the mechanism of tumor carcinogenesis, and to regulate the network, key molecules, and drug target by combined biology effects.
Carrier Proteins ; biosynthesis ; genetics ; Genomics ; methods ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Membrane Proteins ; biosynthesis ; genetics ; Multifactorial Inheritance ; genetics ; Neoplasms ; genetics ; metabolism ; Phosphoproteins ; biosynthesis ; genetics ; Proteomics ; methods ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo study the expression and localization of ATP50 by construction of ATP50-pEYFP-N1 in primary cultured mouse Leydig cells.

METHODSPrimary cultured mouse Leydig cells were confirmed by 3B-HSD staining. ATP50 was cloned into pEYFP-N1 between Bam HI and Eco RI sites. Cell-transfection and living-cell fluorescence imaging microscopy were employed to investigate the sub-cellular localization of YFP-ATP50 in TM3 mouse Leydig cells.

RESULTSATP50 green fluorescent protein was well co-localized with red fluorescence mitochondrion marker-Mitotracker in TM3 mouse Leydig cells.

CONCLUSIONATP50 was expressed in primary cultured mouse Leydig cells. The fluorescent expression vector of ATP50 was constructed successfully and YFP-ATP50 was located in mitochondria in TM3 mouse Leydig cells, which provided a useful clue for further research on the steroidogenesis dysfunction in aging males.

Adenosine Triphosphatases ; biosynthesis ; genetics ; Animals ; Carrier Proteins ; biosynthesis ; genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Leydig Cells ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Mitochondria ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Transfection

OBJECTIVETo construct the eukaryotic expression vector of HBEBP1 gene and express HBEBP1 recombinant protein in yeast.

METHODSPCR was performed to amplify the gene of HBEBP1 from the cDNA template origining from HepG2, and the gene was cloned into pGEM-T vector. After sequencing, the correct DNA fragment was cut from pGEM-T-HBEBP1 and inserted into yeast expression plasmid pGBKT7. The reconstructed plasmid pGBKT7-HBEBP1 was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium (SD/-Trp/Kana). The yeast protein was isolated and analyzed with SDS-PAGE and Western Blot.

RESULTSThe eukaryotic expressive vector was constructed successfully. The results of Western Blot showed HBEBP1 protein was existed within yeast cells and the molecular weight of it was about 33 x 10(3).

CONCLUSIONSThe successful expression of HBEBP1 protein in yeast cells lay the foundation for studying biological function of HBEBP1.

Blotting, Western ; Carrier Proteins ; genetics ; Hepatitis B e Antigens ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Saccharomyces cerevisiae ; genetics

OBJECTIVETo express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step.

METHODSThe hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot.

RESULTSThe expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification.

CONCLUSIONThe D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.

Bacterial Proteins ; genetics ; Blotting, Western ; Carrier Proteins ; genetics ; Escherichia coli ; genetics ; Haemophilus influenzae type b ; genetics ; Immunoglobulin D ; genetics ; Lipoproteins ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; Solubility

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.
Adaptor Proteins, Signal Transducing ; Carbachol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokines ; biosynthesis ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Muscarinic M3 ; agonists ; physiology ; Signal Transduction

OBJECTIVETo investigate the expression of vitamin D3 upregulated protein-1 (VDUP1) in peripheral eosinophils at different stages of asthma and its correlation with clinical manifestations of asthma.

METHODSFourteen normal volunteers and 51 mild to moderate asthma patients, including 16 untreated patients with asthma attack and 35 with asthma remission by continuously corticosterone inhalation. The symptom severity and pulmonary function indices were evaluated and induced sputum eosinophil counts and blood eosinophil count measured. VDUP1 and beta-actin gene fragments were amplified simultaneously by RT-PCR from the total RNA of peripheral eosinophils, and 10 microl of the RT-PCR product underwent agarose gel electrophoresis and the VDUP1/beta-actin ratio was obtained by Gel-Pro software.

RESULTSVDUP1/beta-actin ratio significantly decreased in untreated patients with asthma attacks in comparison with normal volunteers (0.314+/-0.242 vs 0.532 +/-0.279) but not in patients with asthma remission (0.612+/-0.381). In the former patients, a positive correlation of VDUP1/beta-actin ratio was found with FEV1.0% (r=0.587, P=0.046) and %PEF (r=0.563, P=0.033), whereas an inverse one observed with sputum eosinophil count (r=-0.436, P=0.049).

CONCLUSIONVDUP1 expression in the eosinophils correlates to eosinophil activation and may influence the disease severity of asthma patients.

Actins ; biosynthesis ; genetics ; Asthma ; blood ; metabolism ; Carrier Proteins ; biosynthesis ; genetics ; Eosinophils ; metabolism ; Humans ; Maximal Expiratory Flow-Volume Curves ; Peak Expiratory Flow Rate ; Respiratory Function Tests ; Thioredoxins ; biosynthesis ; genetics