ABSTRACT
Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).
Subject(s)
Escherichia coli , Genetics , Metabolism , Escherichia coli Proteins , Genetics , Metabolism , Plasmids , Genetics , Metabolism , Shikimic Acid , MetabolismABSTRACT
The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.
Subject(s)
Humans , Enzyme Inhibitors , Chemistry , Metabolism , Fusarium , Chemistry , Metabolism , Hepacivirus , Genetics , Hepatitis C , Virology , Magnetic Resonance Spectroscopy , Viral Nonstructural Proteins , MetabolismABSTRACT
AIM@#A quantitative ELISA kit for the detection of human secreted CD306/LAIR-2 was developed using monoclonal and polyclonal antibodies which were raised against a highly purified recombinant human secreted CD306/LAIR-2.@*METHODS@#Anti-human secreted CD306/LAIR-2 monoclonal and polyclonal antibodies were raised by immunizing mouse or rabbit with recombinant human secreted CD306/LAIR-2. The monoclonal antibody was purified by protein G affinity, whereas the polyclonal antibody was purified by protein A affinity. The best match pair of antibodies were found and used to develop a double antibody sandwich ELISA kit for the detection of human secreted CD306/LAIR-2 in human samples.@*RESULTS@#A human secreted CD306/LAIR-2 ELISA kit was formulated with highly purified recombinant human secreted CD306/LAIR-2, highly specific monoclonal and polyclonal antibodies. This kit realized the quantitative measurement of recombinant human CD306/LAIR-2 and natural CD306/LAIR-2 in human serum samples.@*CONCLUSIONS@#The developed human secreted CD306/LAIR-2 ELISA kit is a reliable quantitation immunoassay kit.
Subject(s)
Animals , Humans , Mice , Rabbits , Antibodies, Monoclonal , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , Mice, Inbred BALB C , Receptors, Immunologic , Blood , Allergy and Immunology , Recombinant ProteinsABSTRACT
<p><b>OBJECTIVE</b>To study neonatal Streptococcus agalactiae (GBS) infection in The Children's Hospital of Gansu Province through Polymerase Chain Reaction(PCR) Array.</p><p><b>METHOD</b>After obtaining the informed consent from parents or guardians, blood samples of 286 neonates were collected and studied in The Children's Hospital of Gansu Province from June 2011 to January 2012. DNA of the selected samples was extracted through the method of 5% Chelex-100 + 0.5% NP40 solution. Twenty-five genes were ultimately selected and then 25 pairs of primers were designed respectively through primer-BLAST tool of NCBI database.For every primer, PCR conditions were optimized through the identified GBS, and 25 pairs of primers were arrayed as to be used to study neonatal GBS infection.</p><p><b>RESULT</b>The results of PCR Array showed that the 14 samples were detected positive, accounting for 4.90% of all the selected specimens. As for neonatal GBS infection, the positive rate was 4.55% within 7 days after birth and 5.19% in those older than 7 days. The positive rate of 53 preterm infants was 5.66%. The follow-up survey showed that none of the cases died.</p><p><b>CONCLUSION</b>In the Children's Hospital of Gansu Province neonatal GBS infection rate was 4.90%, which is similar to the previous domestic reports, but is lower than the reports from Europe and the United States.Studies have shown that the gene expression related to immune evasion has a higher frequency. The present study suggests that the strategy of GBS immune adaptation may play an important role in neonatal GBS infection.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , Bacterial Proteins , Genetics , China , Epidemiology , DNA Primers , DNA, Bacterial , Genetics , Gene Amplification , Hospitals, Pediatric , Infant, Newborn, Diseases , Diagnosis , Epidemiology , Microbiology , Polymerase Chain Reaction , Sensitivity and Specificity , Streptococcal Infections , Diagnosis , Epidemiology , Microbiology , Streptococcus agalactiae , GeneticsABSTRACT
Objective To explore the clinical significance of monocyte chemoattractant protein-1(MCP-1)in children with Henoch-Schonlein purpura nephritis(HSPN).At the same time compare the association between serum and urine MCP-1,to investigate the impact of the both on them in children with HSPN.Methods Serum and urine MCP-1 were measured by enzyme linked immunosorbent assay in 50 children with Henoch-Schonlein purpura(HSP)(25 cases of them patients with renal injures),and 25 healthy children,the changes of serum and urine MCP-1 were compared;at the same time serum urea nitrogen,creatinine,urinary albumin,urine N-acetyl-D-glucosaminidase(NAG),urine ?2-MG,24 hours urinary levels of protein were investigated in children with HSPN by analyzing the correlation between these indicators and serum and urine MCP-1;urine MCP-1 in HSPN group were measured in recovery period,and were compared with urine MCP-1 in HSP group and HSPN group in acute period.Results 1.The expressions of urine MCP-1 was significantly higher in HSPN group than those in HSP group and healthy controls(P0.05).2.Urine MCP-1 levels were associated with proteinuria in children with HSPN,but serum MCP-1 levels had nothing to do with HSPN.3.There was a close correlation between urine MCP-1 expression and urinary albumin,urine NAG,urine ?2-MG and 24 hours urinary levels of protein,but the expression of urine MCP-1 levels were not correlated with the serum urea nitrogen and creatinine.4.There was statistical significance in urine MCP-1 in acute and recovery periods with HSPN group(P
ABSTRACT
?-lactamase inhibitor research is popular for its potential on ?-lactam antibiotics resistant strain.A ?-lactamase binding peptide SIPIS04-01 was obtained by the yeast two-hybid system.In vitro assay showed that it can inhibit the ?-lactamase activity.In order to improve the expression level of the recombinant peptide,a two-copy expression plasmid pYG563 was constructed by random orientation tandem repeat method after codon modification,the two-copy plasmid was successfully expressed and the product was increased by 48.4% than that of one-copy plasmid.Purified peptide showed inhibitory activity against TEM-1 ?-lactamase in vitro and the inhhibitory constant Ki was measured.
ABSTRACT
TTI gene coding for Tsetse thrombin inhibitor was modified with E.coli bias codon and expressed in Escherichia coli with high efficiency.Recombinant protein was purified to more than 98% purity.Assay for enzyme activity determination was set up.The result showed that the fusion protein exhibited inhibiting activity for thrombin.Inhibitory rate of purified TTI was 73% when concentration of thrombin and substrate was 10U/ml and 250?mol/L respectively.Inhibition pattern was determined as competitive with Ki at 35?mol/L.
ABSTRACT
A novel gene,located between dnrX and drrB in the genome of daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482,was cloned and named as dauW.The full sequence of dauW was submitted to GenBank(Accession No.EF523565).Blast result indicated that it showed high homology with dnrW in GenBank.The exact function of dauW is as yet unknown despite the possibility that it might belong to a family of FAD-dependent oxidoreductases on the basis of conserved domain analysis.dauW was cloned into expression plasmids pET-28a(+)and pET-32a(+),respectively,and was successfully expressed in E.coli DE3 after induction with IPTG.The preliminary results of the expression of dauW suggested that it might be involved in the self resistance in Streptomyces coeruleorubidus due to the increased resistance to daunorubicin in the E.coli host.
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To improve the growth enhancement activity of Vitreoscilla hemoglobin(VHb), Vitreoscilla hemoglobin gene(vgb) was mutated by error-prone PCR and then reconstituted by DNA shuffling. The shuffling library was constructed by inserting the shuffled genes into the downstream of vgb natural promoter and transforming them into E.coli DH5?. Mutated active VHb proteins were first screened in test tubes according to host cell pellets color and then in shake flasks according to host pellets wet weight .One active mutant protein, VHb′042506, was obtained after second screening. It could increased the host wet weight by 31.25% and 58.75% than that of the control which bearing natural VHb under microaerobic and extremely microaerobic conditions, respectively. Sequencing and alignment results showed that 11 nucleotides were mutated, thus resulted in 4 amino acids changes occurred in this mutant protein. CO difference spectrum test also indicated that it had higher specific absorption.
ABSTRACT
TDP-4-ketohexulose reductase, encoded by dnmV, is important in daunorubicin biosynthesis. To obtain a daunorubicin block mutant, double cross-over plasmid pYG817 was constructed by inserting apramycin resistant gene and amplified dnmV together with upstream dnmU into vector pUC18. dnmV was successfully disrupted after transformation of daunorubicin-producing strain SIPI-1482 by pYG817. Daunorubicin was absent from metabolites of the resulting transformant, and its biosynthesis can be reconstituted by introducing dnmV expression plasmid into the disruptant, although the yield is lower than wild-type SIPI-1482, according to HPLC analysis. This mutant can be a good candidate for production of anthracycline such as epi-daunorubicin by introducing exogenous gene into the host.
ABSTRACT
A new sisomicin resistance gene sisR was cloned from sisomicin-producing Micromonospora inyoensis. The sisR fragment was obtained by PCR amplification. The primer pairs were designed based on grm gene sequence from gentamicin-producing Micromonospora purpurea. The template DNA was isolated from Micromonospora inyoensis. A series of different DNA fragments were amplified by PCR, which were sub-cloned to vector pUC19 for further identification. It was found that five specific transformants containing target DNA fragments could resist high concentrations of sisomicin (over 1000 microg/mL sisomicin). One of them designated as sisR, was then sequenced and the alignment among sisR and other related genes showed that sisR gene differs from any known genes. It was concluded that sisR gene is a sequence that has not been reported so far.
Subject(s)
Anti-Bacterial Agents , Pharmacology , Bacterial Proteins , Genetics , Base Sequence , Cloning, Molecular , Drug Resistance, Microbial , Genetics , Genes, Bacterial , Micromonospora , Genetics , Molecular Sequence Data , Sisomicin , PharmacologyABSTRACT
Objective To observe the changes of serum ferritin (SF)in neonates with mechanical ventilation and its clinical significance in the ventilation-induced lung injury (VILI). Methods The study was carried out in 36 neonates with mechanical ventilation and 31 neonates without mechanical ventilation in neonate intensive care unit(NICU).SF level in venous blood was measured on 1,24,48,72 hours after mechanical ventilation and 24 hours after mechanical ventilation removal by radioimmunoassay (RIA ).SF level of non-mechanical ventilation group were determined at the same time. Results SF levels in mechanical ventilation groups were significantly higher than those of control group (P
ABSTRACT
Canrenone is an important intermediate for the synthesis of eplerenone,a cardiovascular drug.C_ 11 ?-hydroxylation of canrenone is the key reaction,which can be done by microbial transformation.Rhizopus sp.SIPI-0602,kept in our lab,could high selectively transform canrenone to a compound named SIPI-11.By determining and analyzing the MS,UV,NMR etc.spectra of compound SIPI-11,its chemical structure was elucidated to be 11?-hydroxycanrenone.The study on flask transformation technology showed that the transformation ratio exceeded 90% when the substrate concentration was not more than 6g/L.
ABSTRACT
A promoter-trap vector pGBT14 for selecting promoters of fungus gene was constructed with E. coli-yeast shuttling plasmid pGBT9. Using this vector, a0. 5-2. 0kb chromosomal DNA library of Cepholosporium acremonium was constructed, and twenty four DNA fragments with promoter function in Saccharomyces oerevisiae Y153 were selected from this DNA library. And the promoter function of these DNA fragments was analyzed.