ABSTRACT
Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.
Subject(s)
Adenosine Triphosphate , Chemistry , Chromatography, Affinity , Escherichia coli , Luminescent Measurements , Methods , Lysostaphin , Chemistry , Recombinant Proteins , Chemistry , Staphylococcus aureusABSTRACT
Secretory IgA (SIgA) antibodies in external secretions play an important role in mucosal immune response. Polymeric SIgA was advantageous over monomeric IgA (mIgA) and IgG in several aspects. To express secretory IgA antibody against H5N1 virus, we constructed the secretory component and immunoglobulin J expressing plasmids and co-transfected the plasmids into the Chinese hamster ovary cells (CHO) stably expressing immunoglobulin A. Then we used Zeocin to select the positive clone cells, monoclonal cells stably secreting SIgA was screened through fold dilution method at last. The SIgA antibody secreted from the CHO cells was confirmed by Western blotting, which demonstrated that we had got the complete SIgA molecular. The successful expression of this polymeric anti-H5N1 SIgA in CHO cells will contribute to the production of recombinant SIgA as a preventive agent for infectious disease control.
Subject(s)
Animals , Cricetinae , Antibodies, Viral , Genetics , CHO Cells , Cloning, Molecular , Cricetulus , Genetic Vectors , Immunoglobulin A , Allergy and Immunology , Immunoglobulin A, Secretory , Genetics , Allergy and Immunology , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and ImmunologyABSTRACT
We isolated a novel Enterobacteria phage IME08 from hospital sewage, then confirmed it was a double-stranded DNA phage by digesting its genetic material with DNase I, RNase A and several restriction endonucleases respectively. BLAST results of random fragments generated by a random PCR cloning method revealed that it belonged to T4-like virus. We subsequently determined the host recognizing genes (g37 and g38) sequence with a PCR-based "genome jumping" protocol based on highly conserved region at 5' terminus of g37 from four other T4-like Bacteriophages (T4, JS98, T2 and K3). These molecular biological methods enabled us to readily characterize the bacteriophage and efficiently determine the sequence of the genes of interest based on very limited conserved sequence information.
Subject(s)
Bacteriophage T4 , Genetics , Cloning, Molecular , DNA, Viral , Genetics , Escherichia coli , Genetics , Virology , Genome, Viral , Genetics , Host Specificity , Genetics , Polymerase Chain Reaction , MethodsABSTRACT
Although previous publications suggest the 2009 pandemic influenza A(H1N1)virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.
ABSTRACT
To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes.
Subject(s)
Amino Acid Sequence , Base Sequence , DNA , Genetics , DNA Restriction Enzymes , Genetics , DNA-Directed DNA Polymerase , Genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Methods , Plasmids , Receptors, Polymeric Immunoglobulin , GeneticsABSTRACT
Abstract: To express human-mouse chimeric IgA antibody directed against H5N1 virus, an anti-H5N1 chimeric IgA antibody gene was constructed by joining the light and heavy chain variable region genes and the corresponding signal peptide coding sequences of the anti-H5N1 mouse monoclonal antibody H5N1-HA with the coding sequences of the constant region of the human IgA2 heavy chain and Kappa chain respectively. Then the full-length chimeric light and heavy chain expressing plasmids pEF-IGHA9 and pEF-IGK9 were constructed and transfected into the CHO/dhfr cells. The chimeric IgA antibody expression was confirmed by ELISA, SDS-PAGE and Western blotting. The successful expression of this anti-H5N1 chimeric IgA may help to provide a stand for developing passive immunological agents for H5N1 virus infection prophylaxis.