Farnesyl diphosphate synthase (FPS) is one of the key rate-limiting enzymes in the sesquiterpene metabolic pathways. In this study, the open reading frame (ORF) of FPS was cloned by PCR based on the transcript sequence of AsFPS1 from the Aquilaria sinensis transcriptome database and sequenced. Total RNA was extracted from the root, stem and leaves of three-year-old A. sinensis, and from healthy and wounded A. sinensis calli, and then reverse-transcribed into single-stranded cDNA as a template for real-time PCR, to detect the expression specificity of AsFPSI in different tissues and its expression profile in responding to different treatments. The result showed that the full length of AsFPS1 was 1 342 bp with the ORF 1 029 bp, encoding 342 amino acids. Tissue expression analysis indicated that AsFPS1 was mainly expressed in root and stem, and was lower in leaves. Inducible-experiments showed that the genes was induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 12 h, respectively. The full-length cDNA clone of AsFPSI and its expression patterns analysis will provide a foundation for follow-up study on its biological function and agarwood sesquiterpene biosynthesis mechanism.
DNA, Complementary ; genetics ; Geranyltranstransferase ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Thymelaeaceae ; enzymology ; genetics ; metabolism

OBJECTIVEExpress a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.

METHODSWe express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.

RESULTSThe plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.

CONCLUSIONSkod-ssb may in future be used to enhance DNA and cDNA amplification.

Archaeal Proteins ; genetics ; isolation & purification ; metabolism ; Chromatography, Affinity ; DNA, Bacterial ; genetics ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Thermococcus ; genetics ; metabolism

AIMTo clone and analyse the coding cDNA sequence of alpha1, beta2 and gamma2 subunit of GABAA receptor in American king Pigeon.

METHODSWithdrew total RNA from the American king pigeon brain, reverse transcribing general primers to acquire a gene set cDNA. Designing specific primers of three subunit mRNA of the GABAA receptor, by RT-PCR respectively expanded the conservative gene of al subunit, beta2 subunit and gamma2 subunit of GABAA receptor, and carried on clone, plastid identification and the sequence measurese of three genes.

RESULTSThe experiment on sequence measures has succeeded that sequence analysis indicated that lengths of the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor was respectively 899 bp, 597 bp and 563 bp, homology on reference sequence was respectively 94.99%, 94.64% and 96.28%.

CONCLUSIONHomology is high on the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor of brain tissue of pigeon and chicken but there is a discriminating characteristic in different kinds of animals.

Animals ; Brain ; metabolism ; Cloning, Molecular ; Columbidae ; DNA, Complementary ; genetics ; RNA, Messenger ; genetics ; Receptors, GABA-A ; classification ; genetics ; Sequence Analysis, DNA

Human Sodium/Iodide symporter gene cDNA was amplified from thyroid tissue of the patient suffering from Graves disease by RT-PCR, and T/A cloned into pGEM-TEasy-NIS for sequencing, subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene, and then forwarded to homologous recombinant in the bacteria BJ5183 that already contained AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the 293 cells to obtain recombinant adenovirus. The results showed that the recombinant AdNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 2. 5 - 3 x 10(9) efu/ml. So, the recombinant adenovirus vector carrying hNIS was successfully constructed, thus providing a basis for researches on 131I therapy in nonthyroid carcinoma.
Adenoviridae ; genetics ; metabolism ; DNA, Complementary ; genetics ; Genetic Vectors ; genetics ; metabolism ; Graves Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Symporters ; biosynthesis ; genetics

Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02), and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the alpha-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut+ carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction, the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7 u/mg.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Superoxide Dismutase ; biosynthesis ; genetics

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

OBJECTIVETo investigate whether introducing promoterless DNA containing the cDNA sequence of green fluorescent protein (GFP) induces gene-specific silencing in human pancreatic cancer cell line harboring genomically integrated GFP gene.

METHODSUsing G418 selection and fluorescent separation we established a highly purified monoclonal pancreatic cancer cell line, recombinant PANC-1, which had a steady level of GFP expression. GFP cDNA was amplified by PCR from plasmid pEGFP-C1 and ligated to the promoterless plasmid PUC19. PUC-GFP was then transfected into monoclone cells along or cotransfected with pEGFP-C1 to panc-1 cells. Each had PUC plasmid transfected group as their control to eliminate possibility of plasmid toxicity and GFP small interferent RNA (siGFP) transfected group as positive control. Western blot, flow cytometry and phase contrast fluorescence microscopy were used to detect the changes of GFP expression.

RESULTSThe data showed that (1) PUC-GFP inhibited the GFP expression in monoclonal cell line in a dosage dependent manner. The inhibitory effect of 3 microg PUC-GFP did not show significant difference with siGFP. (2) The significant repression appeared on the fourth day after transfecting monoclonal cells with 3 microg PUC-GFP. By the end of the sixth day, GFP expression in PUC-GFP group and siGFP group remained at a low level. (3) Cotransfecting PUC-GFP with pEGFP-C1 plasmids into PANC-1 cells showed a decreased transfection efficiency when compared with transfecting pEGFP-C1 alone. Higher PUC-GFP vs pEGFP-C1 corresponded with lower transfection efficency. (4) When adding new pEGFP-C1 plasmid to cells after inhibition appeared, the GFP expression recovered.

CONCLUSIONTransfection of the promoterless DNA fragment containing full-length cDNA effectively induces a gene-specific silencing in mammalian cells.

Cell Line, Tumor ; DNA, Complementary ; genetics ; DNA, Recombinant ; genetics ; Gene Silencing ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Transfection

NHL proteins, which play important roles in regulation of cell proliferation and differentiation, have been extensively studied on mammals. Here, we cloned a member of NHL protein family namely BmBrat in silkworm. The full-length cDNA sequence of BmB rat was obtained by means of the rapid amplification of cDNA ends (RACE), including 3 614 bp. The ORF is 2 580 bp long, encoding a protein with 859 amino acid residues. The molecular weight is 94.3 kDa and the isoeledtric point (pI) is 6.65. The BmBrat expression profile was detected by RT-PCR at L5D3 larval stage, and it was expressed in all tissues, including silk gland, midgut, fat body and malpighian tubule. However, it was highly expressed in ovary and head. The expression profile was also detected at different stage of embryo development, and reached a peak at the 4th and 5th days of the embryonic period. Anti-BmBrat polyclonal antibody was generated f6llowing prokaryotic expression, protein purification and mice immunization, which is highly specific and effective for recognizing BmBrat protein through Western blotting and immunofluorescence staining. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. This study provides a foundation for further research of the biological function of BmBrat gene.
Animals ; Bombyx ; Cloning, Molecular ; DNA, Complementary ; Insect Proteins ; genetics ; metabolism ; Larva ; Mice

OBJECTIVETo investigate the splice variants of the calpain 3 gene existing in human skeletal muscle tissue and white blood cells, and to explore the feasibility of gene diagnosis using CAPN3 mRNA extracted from peripheral leukocytes.

METHODSTotal RNA was extracted from peripheral blood and skeletal muscle tissue in healthy individuals. CAPN3 cDNAs were determined by reverse transcriptase polymerase chain reaction and DNA sequencing. CAPN3 cDNAs from peripheral leukocytes were compared with sequences obtained from skeletal muscle tissue.

RESULTSRT-PCR and DNA sequencing showed that the CAPN3 cDNAs comprised 24 exons in human skeletal muscle tissue, while the number of exons was 23 in white blood cells. Exon 15 was spliced out in human white blood cells.

CONCLUSIONSplice variants exist in human skeletal muscle tissue and white blood cells. Gene diagnosis may omit the mutations of exon 15 using mRNA extracted from peripheral leukocytes. These findings suggest that mutation analysis of the CAPN3 cDNA should use skeletal muscle tissue as materials instead of peripheral blood.

Calpain ; genetics ; DNA Mutational Analysis ; DNA, Complementary ; genetics ; Exons ; genetics ; Humans ; Leukocytes ; metabolism ; Muscle Proteins ; genetics ; Muscle, Skeletal ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B.

METHODSThe total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector.

RESULTSThe titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.

CONCLUSIONA high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

DNA, Complementary ; genetics ; Gene Library ; Hepatitis B, Chronic ; genetics ; Humans ; Liver ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA ; analysis ; genetics