Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.
Cloning, Molecular ; DNA, Complementary ; Flavonoids ; biosynthesis ; Intramolecular Lyases ; genetics ; Plants, Medicinal ; enzymology ; genetics ; Rhus ; enzymology ; genetics

To construct soluble TNF related apoptosis inducing ligand (TRAIL) expression system and investigate the effect of the expression product on tumor cell. It may provide valuable information for research into the immune system of the finless porpoise. The full-length cDNA of TRAIL (designated fTRAIL) was cloned from the total RNA of the finless porpoises blood using RT-PCR techniques and then the extracellular soluble fragments of fTRAIL (designated fsTRAIL) was ligated into pET43.1a. Recombinant soluble fTRAIL (pET43.1a-fsTRAIL) fused with Nus-his tag was efficiently expressed in Escherichia coli BL21 (DE3) and the Nus-His-fsTRAIL protein was purified. The expression of Nus-His-fsTRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fsTRAIL protein on Jurkat and HeLa cells were etected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay, TrypanBlue and Flow Cytometry analysis. The expression system pET43.1a-fsTRAIL was constructed and Nus-His-fsTRAIL protein was expressed successfully. In vitro, the Nus-His-fsTRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner. The Nus-His-fsTRAIL protein has anti-tumor activity against Jurkat and HeLa cells in vitro.
Animals ; Apoptosis ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; HeLa Cells ; Humans ; Jurkat Cells ; Porpoises ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis

ObjectiveTo construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells.

METHODSFull-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy.

RESULTSThe construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells.

CONCLUSIONSConclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.

Animals ; Baculoviridae ; Blotting, Western ; DNA, Complementary ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; Mice ; Plasmids ; Polymerase Chain Reaction ; Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Sf9 Cells ; Transfection

Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.
Arabidopsis ; genetics ; Arabidopsis Proteins ; biosynthesis ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Pichia ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Thiolester Hydrolases ; biosynthesis ; Transformation, Genetic

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.
Amino Acid Sequence ; Carbon-Carbon Double Bond Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; Paclitaxel ; biosynthesis ; Phylogeny ; Plant Proteins ; genetics ; Taxus ; enzymology ; genetics

UNLABELLED: Abstract OBJECTIVE: To measure the expression of CK18 and CK19 in the cells from peripheral blood and tumor tissue of the nasopharyngeal carcinoma patients,to test whether CK 18 and CK 19 could be biomarkers of nasopharyngeal carcinoma fordiagnosis. METHOD: The mRNA was extracted from the blood and carcinoma tissue of nasopharyngeal carcinoma and was reversed transcription to cDNA. The 3 pairs primers were designed for RT-PCR and the fold value was calculated to evaluated expression by ΔCT. RESULT: There are no statistical differences between the CK18 and CK19 gene expression and the gender, age and metastasis in tumor tissue of 45 nasopharyngeal carcinoma patients (P>0. 05). There are significant differences among 3 pathological stages and 2 genes expressed increase as the grade malignancy (P<0. 05). The detecting of the 2 genes expression from blood cells shows that CK18 and CK19 had a high positive ratio 64% and 75% respectively. Meanwhile this method showed a same detection characteristic in tumor and blood, the positive.rate of CK18 and CK19 genes in metastasis is higher than non-metastasis. The results showed CK18 has a high specificity and CK19 has a high sensitivity for prognosis and all relapsed cases are associated with the expression of CK18 and CK19. CONCLUSION CK18 and CK19 may be used as biomarkers of nasopharyngeal carcinoma for diagnosis.
Biomarkers, Tumor ; Carcinoma ; DNA, Complementary ; Gene Expression ; Humans ; Keratin-18 ; biosynthesis ; Keratin-19 ; biosynthesis ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; diagnosis ; metabolism ; pathology ; Neoplasm Metastasis ; Prognosis ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli).

METHODSHuman GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate.

RESULTSThe correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively.

CONCLUSIONThe expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Glutathione S-Transferase pi ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Azulenes ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Open Reading Frames ; Polyisoprenyl Phosphates ; Recombinant Proteins ; biosynthesis ; Sesquiterpenes ; metabolism ; Sesquiterpenes, Guaiane ; Thymelaeaceae ; enzymology ; genetics

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection