ABSTRACT
Genes encoding thaumatin-like protein () are frequently found in fungal genomes. However, information ongenes inis still limited. In this study, threegenes were cloned from. The full-length coding sequence of,andwere 768, 759 and 561 bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that,andwere clustered with sequences fromand, respectively. The expression patterns of the threegenes were higher inwithinfection than in the sclerotia without. Furthermore, over-expression of three PuTLPs were carried out inBL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that,andinmay be involved in the defense response toinfections.
ABSTRACT
Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.
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Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.
ABSTRACT
<p><b>OBJECTIVE</b>To isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.</p><p><b>METHOD</b>Fungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.</p><p><b>RESULT</b>Ninety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.</p><p><b>CONCLUSION</b>Endophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.</p>
Subject(s)
Anti-Infective Agents , Metabolism , Pharmacology , Aspergillus fumigatus , Bacillus subtilis , Base Sequence , Biodiversity , Candida albicans , China , Cryptococcus neoformans , DNA, Fungal , Chemistry , DNA, Ribosomal Spacer , Chemistry , Genetics , Dendrobium , Microbiology , Physiology , Endophytes , Classification , Genetics , Physiology , Escherichia coli , Fungi , Classification , Genetics , Physiology , Microbial Sensitivity Tests , Molecular Sequence Data , Phylogeny , Plant Roots , Microbiology , Physiology , Plant Stems , Microbiology , Physiology , Sequence Alignment , Sequence Analysis, DNA , Staphylococcus aureusABSTRACT
The oral administration of bioactive macromolecular drugs such as proteins, peptides and nucleic acids represents unprecedented challenges from the drug delivery point of view. One key consideration is how to overcome the gastrointestinal tract absorption barrier. Recent studies suggest that microfold cell (M cell), a kind of specialized antigen-sampling epithelial cell which is characterized by a high endocytic rate and low degradation ability, may play an important role in macromolecule oral absorption. The development of an in vitro M cell coculture system and its modified models greatly advanced the study of M cells and the development of oral delivery system for macromolecular drugs. The special structure, function and formation characteristics, and biomarkers of M cell are summarized in this review. The applications of in vitro M cell models in developing oral delivery system ofbioactive macromolecular drugs are discussed.
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0.05),but enhanced the expression of CD80,CD86 significantly(P