Expert consensus on microbiome sequencing and analysis / 生物工程学报

In the past ten years, the research and application of microbiome has continued to increase. The microbiome has gradually become the research focus in the fields of life science, environmental science, and medicine. Meanwhile, many countries and organizations around the world are launching their own microbiome projects and conducting a multi-faceted layout, striving to gain a strategic position in this promising field. In addition, whether it is scientific research or industrial applications, there has been a climax of research and a wave of investment and financing, accordingly, products and services related to the microbiome are constantly emerging. However, due to the rapid development of microbiome sequencing and analysis related technologies and methods, the research and application from various countries have not yet unified on the standards of technology, programs, and data. Domestic industry participants also have insufficient understanding of the microbiome. New methods, technologies, and theories have not yet been fully accepted and used. In addition, some of the existing standards and guidelines are too general with poor practicality. This not only causes obstacles in the integration of scientific research data and waste of resources, but also gives related companies unfair competition opportunity. More importantly, China still lacks national standards related to the microbiome, and the national microbiome project is still in the process of preparation. In this context, the experts and practitioners of the microbiome worked together and developed the consensus of experts. It can not only guide domestic scientific research and industrial institutions to regulate the production, learning and research of the microbiome, the application can also provide reference technical basis for the relevant national functional departments, protect the scale and standardized corporate company's interests, strengthen industry self-discipline, avoid unregulated enterprises from disrupting the market, and ultimately promote the benign development of microbiome-related industries.

The induction, purification, whole genome sequencing and comparative genome analysis of a novel staphylococcus haemolyticus bacteriophage IME-SA4 / 中国医师杂志

Objective To induce a novel temperate bacteriophage from staphylococcus haemolyticus strain SA98,observe the morphology and size,complete the whole genome sequencing,analyse the structure of genome and evolutionary relationship.Methods The mitomycin C was used to induce the temperate phage from staphylococcushaemolyticus strain SA98,the induced phage was observed by transmission electron microscopy after be concentrated and purified.The genome DNA was extracted and high through sequenced.The feature of whole genome and evolutionary relationship was analyzed.Results A temperate phage IME-SA4 was successfully induced from staphylococcus haemolyticus strain SA98.Transmission electron microscopy analysis indicated that IME-SA4 had an isometric head and a non-contractile long tail.The whole genome of IME-SA4 was long as 41 843 bp,and the whole genome blast result indicated IME-SA4 shared only 13％ homology with most related strain phiRS7.Conclusions A novel staphylococcus haemolyticus temperate phage with low homology with other staphylococcusphages was successfully induced from staphylococcus haemolyticus strain SA98.The research of its morphology,whole genome sequencing and comparative genome analysis make the foundation for further study of staphylococcus phages' properties and practical application.

Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence / 生物工程学报

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.

Expression of anti-avian influenza virus H5N1 secretory IgA in Chinese hamster ovary cells / 生物工程学报

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.

Rapid genetic characterization of a novel Enterobacteria phage and determination of its host recognizing genes / 生物工程学报

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.

Close Relationship between the 2009 H1N1 Virus and South Dakota AIV Strains / 中国病毒学·英文版

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.

Construction of anti-H5N1 virus chimeric igA antibody gene and its expression in CHO cells / 生物工程学报

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.

Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis / 生物工程学报

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.

Research Progress of Small Non-coding RNA in Bacteria / 微生物学通报

Small non-coding RNA (sRNA) is a kind of newly discovered 50 nt~500 nt small RNAs that do not encode proteins. To date, more than 150 sRNA have been found in bacteria. The small RNA acting by base-pairing with target mRNAs, resulting in post-transcriptionally regulating gene expression, is important regulators in the bacterial response to stress, virulence and metabolism. At present, researches of sRNA mainly based on bioinformatical prediction and molecular biological experiments. The sRNA that obtained through these methods needs confirmation in laboratory, and then study of its functions through a variety of experimental methods.