ABSTRACT
The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.
Subject(s)
Corydalis/classification , DNA Barcoding, Taxonomic/methods , DNA, Plant/genetics , DNA, Ribosomal Spacer/genetics , Papaveraceae/classification , Base Sequence , China , Corydalis/chemistry , Corydalis/genetics , DNA, Plant/chemistry , DNA, Ribosomal Spacer/chemistry , Molecular Sequence Data , Nucleic Acid Conformation , Papaveraceae/chemistry , Papaveraceae/genetics , Phylogeny , Plants, Medicinal/chemistry , Plants, Medicinal/classification , Plants, Medicinal/geneticsABSTRACT
Ni2+ binding peptides were selected from phage random dodecapeptide library by metal affinity chromatography. After four rounds of biopanning, phage amplification and DNA sequencing, a group of peptide sequences were obtained. GenBank blast found no homogenous sequences, Clustal W analysis showed no motifs but they were really riched in histidines and contained di- or more histidines(his). Affinity assays of selected metal-binding phages for various metal-charged NTA resins and the experiments of E. coli suppression and detoxification gave positive results for Ni2+ binding peptides: strong affinities for Ni2+ were found for Ni2+ binding peptide displayed phages, as well as for other metals (Cu2+, Co2+, Zn2+, Cr2+, Cd2+); affinities of the binding peptides for Cu2+, Ni2+, Co2+ and Zn2+ were much higher than that of Cd2+ and Cr2+; in addition, Ni2+ binding peptides displayed phages had effects on E. coli as to enhance the tolerance and detoxification of E. coli for heavy metals when exposed to Ni2+ and Cd2+. The interactions of meal binding peptides for heavy metals were also disclosed by microscopic observation. The research offered great values for the study of the interaction between metals and peptides, as well as in other areas such as heavy metal bioremediation.
Subject(s)
Carrier Proteins/metabolism , Nickel/metabolism , Peptide Library , Peptides/metabolism , Animals , Biodegradation, Environmental , Chromatography, Affinity , MiceABSTRACT
Nickel (Ni) performs its biological or toxic functions in nickel-protein coordination form. Novel Ni-binding peptides were isolated from a random dodecapeptide library displayed on the flagella of Escherichia coli against immobilized ions. On the basis of isolated sequences rich in histidine residues, two secondary libraries were constructed respectively. By consequent selection, more Ni-chelating peptides were identified and the consensus motif RHXHR (where X was always H) was deduced. The result suggested that not only histidine, but also arginine, play an important role in Ni-binding. Furthermore, two selected clones (1035 and 2022) were chosen for further identification. They exhibited similar relative binding affinity, which was about nine times that of the original library derived clones and statistically much more significant than the positive control with polyhistidine insert. Free nickel ions could almost completely inhibit the binding of the clones 1035 and 2022 to immobilized nickel, implicating that the peptides were able to chelate nickel ions. These studies reveal that bacterial surface displayed peptide libraries may have promising future potential for the development of metal bioadsorbents. Furthermore, novel Ni-binding peptides may provide lead molecules for Ni-chelation and applications thereof.
Subject(s)
Flagella/chemistry , Nickel/metabolism , Peptide Library , Peptides/chemistry , Amino Acid Sequence , Base Sequence , Binding, Competitive , DNA Primers , Escherichia coli/chemistry , Peptides/metabolismABSTRACT
Bacterially-displayed peptide libraries have been widely used as an alternative to phage-displayed peptide libraries in screening epitopes or mimotopes of antibodies. Using a protective monoclonal antibody (mAb) 3B9 against hepatitis B virus (HBV) preS protein as target, mimotopes were successfully screened from a FliTrx random peptide library. To monitor the enrichment ratios of each round and to isolate higher affinity clones from the library, a modified procedure was performed in which the titer of eluted bacteria from an antibody-coated well (P value) was compared with that from a non-coated well (N value). After sufficient enrichment of the library, bacterial colonies were randomly picked and identified further by the monoclonal bacterial P/N value assay and Western blotting analysis. Immunization of mice with the selected bacterially-displayed mimotopes, including the enriched populations without clone identification, elicited strong specific immune responses against the recombinant preS protein. The present study provides a potentially rapid and effective strategy for the development of engineered live bacterial vaccines without the need for information about the aetiological agents or their antigens.
Subject(s)
Epitopes/immunology , Hepatitis B Surface Antigens/immunology , Hepatitis B virus/immunology , Peptide Library , Protein Precursors/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/analysis , Antibodies, Viral/biosynthesis , Blotting, Western , Cloning, Molecular , DNA/genetics , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Female , Mice , Mice, Inbred BALB CABSTRACT
OBJECTIVE: To establish an improved procedure for isolation and identification of epitopes from a random peptide library displayed on the bacterial surface. METHODS: Epitopes were screened from FliTrx random peptide library by a monoclonal antibody 3B9 against HBV-PreS2 protein. The enrichment was monitored in each round. Higher affinity clones were obtained by increasing the washing strength and randomly selected for sequencing and Western blot analysis. RESULTS: Clones specifically binding to antibody were enriched in each round. Ten sequences were obtained from sixteen sequenced clones, seven of them contained the common motif RXRGXY with high homogeneity to 135-140 amio acids in HBV-PreS protein and have positive results in Western blot analysis. The other three sequences have no typical motif RXRGXY and showed different Western blot results. CONCLUSIONS: It's easy and quick to drive epitopes from a random peptide library displayed on the bacterial surface.
