Jihong Yang

Identification and characterization of a chitin deacetylase from a metagenomic library of deep-sea sediments of the Arctic Ocean.

BACKGROUND The chemical and biological compositions of deep-sea sediments are interesting because of the underexplored diversity when it comes to bioprospecting. The special geographical location and climates make Arctic Ocean a unique ocean area containing an abundance of microbial resources. METHODS A metagenomic library was constructed based on the deep-sea sediments of Arctic Ocean. Part of insertion fragments of this library were sequenced. A chitin deacetylase gene, cdaYJ, was identified and characterized. RESULTS A metagenomic library with 2750 clones was obtained and ten clones were sequenced. Results revealed several interesting genes, including a chitin deacetylase coding sequence, cdaYJ. The CdaYJ is homologous to some known chitin deacetylases and contains conserved chitin deacetylase active sites. CdaYJ protein exhibits a long N-terminal and a relative short C-terminal. Phylogenetic analysis revealed that CdaYJ showed highest homology to CDAs from Alphaproteobacteria. The cdaYJ gene was subcloned into the pET-28a vector and the recombinant CdaYJ (rCdaYJ) was expressed in Escherichia coli BL21 (DE3). rCdaYJ showed a molecular weight of 43kDa, and exhibited deacetylation activity by using p-nitroacetanilide as substrate. The optimal pH and temperature of rCdaYJ were tested as pH7.4 and 28°C, respectively. CONCLUSIONS The construction of metagenomic library of the Arctic deep-sea sediments provides us an opportunity to look into the microbial communities and exploiting valuable gene resources. A chitin deacetylase CdaYJ was identified from the library. It showed highest deacetylation activity under slight alkaline and low temperature conditions. CdaYJ might be a candidate chitin deacetylase that possesses industrial and pharmaceutical potentials.

Structure and Function of CW Domain Containing Proteins.

The CW domain is a zinc binding domain, composed of approximately 50- 60 amino acid residues with four conserved cysteine (C) and two to four conserved tryptophan (W) residues. The members of the superfamily of CW domain containing proteins, comprised of 12 different eukaryotic nuclear protein families, are extensively expressed in vertebrates, vertebrate-infecting parasites and higher plants, where they are often involved in chromatin remodeling, methylation recognition, epigenetic regulation and early embryonic development. Since the first CW domain structure was determined 5 years ago, structures of five CW domains have been solved so far. In this review, we will discuss these recent advances in understanding the identification, definition, structure, and functions of the CW domain containing proteins.

Enhanced liver functions of HepG2 cells in the alginate/xyloglucan scaffold

A scaffold provides a framework and initial support for the cells to attach, proliferate and differentiate, and form an extracellular matrix (ECM) in tissue engineering. Here, xyloglucan (XG) was used as a new synthetic ECM for HepG2 cell attachment in alginate capsules. The effects of XG on HepG2 cells on adherent behavior, albumin secretion, ammonia elimination, cell proliferation and gene expression of Connexin 32 and epithelial-cadherin were investigated. Xyloglucan could also promote the HepG2 cell–matrix interactions and the cell clusters formation of HepG2 cells in three dimensional scaffold, thus enhance the liver-specific functions in the three-dimensional space.

Development of Neutralizing Monoclonal Antibodies Against VP4 of Rotavirus CC0812-1

The G10P[15] rotavirus CC0812-1 isolated from a diarrheal woman in Wuhan, China, in 2008 is phylogenetically close to the Lanzhou lamb rotavirus (LLR) of a monovalent human rotavirus vaccine produced by the Lanzhou Institute of Biological Products, China, and rotavirus Lamb-NT. This rotavirus can be used as the backbone of the attenuated rotavirus reassortant as a rotavirus vaccine candidate. In this study, rotavirus CC0812-1 was purified from the culture supernatant of CC0812-1-infected MA104 cells and used as antigen to immunize BALB/c mice. Four hybridoma clones were developed secreting antibodies that reacted with CC0812-1, designated as 1B1, 1B8, 1F11, and 1G10, respectively. Western blot analysis indicated that the four monoclonal antibodies (MAbs) were all specific for VP4 of rotavirus CC0812-1. Isotyping revealed that MAbs 1B1, 1B8, and 1G10 belonged to the IgM class, while MAb 1F11 belonged to the IgG1 subclass. A neutralization test demonstrated that the four MAbs all had the capacity to neutralize rotavirus CC0812-1. The neutralizing titers of the BALB/c mice ascites were 1:2048, 1:1024, 1:512, and 1:512 for MAbs 1B1, 1B8, 1F11, and 1G10, respectively.

Be Aware of Immunogenic But not Protective Antigens: The Actinobacillus pleuropneumoniae PalA as an Example

BACKGROUND Identification of immunogenic antigens is an important step for the vaccine improvement. Previous studies indicated that Actinobacillus pleuropneumoniae PalA is homologous to a Haemophilus influenzae protective antigen Hi-PAL (P6) protein. However, PalA protein adversely affects the Apx toxinbased subunit vaccine. The role of PalA in the inactivated vaccine is not known, and the mechanism involved in the PalA-mediated interference has not been investigated. OBJECTIVES The main objective of this study was to investigate the possible impacts of PalA on the protective immunity of A. pleuropneumoniae inactivated vaccine. METHODS Coding sequence of the mature peptide of PalA was amplified from A. pleuropneumoniae SLW01, and inserted into the prokaryotic expression plasmid pGEX-KG, so as to generate the recombinant PalA (rPalA) protein. The immunogenicity of rPalA was verified in rabbits. For the protection assay, mice were assigned into 4 groups, and were immunized with TSB, rPalA, bacterin (Bac) and bacterin + rPalA (BacPal), respectively. Humoral immune response was evaluated before each immunization and before challenge. Two weeks after three immunizations, mice were infected with virulent A. pleuropneumoniae 4074. The clinical signs, survival rates and lung bacteria loads were determined. Then a passive protection assay was performed using pooled sera from the active immunization assay. RESULTS rPalA was produced in E. coli and was confirmed to be immune-reactive. rPalA is able to elicit a strong humoral immune response in rabbit. Besides, polyclonal antibodies against rPalA is able to recognize the natural PalA in the outer membrane of A. pleuropneumoniae. The positive immunization assay showed that mice immunized with BacPal produced significantly less antibodies against Apx toxins, relative to that of animals immunized with Bac before challenge (P <0.01). After virulent challenge, all mice in the TSB and rPalA groups died within 48 hpi. The survival rates of the Bac and the BacPal groups were 100% and 75%, respectively. The average bacterial loads of the BacPal group was lower than that of the TSB and rPalA groups (P <0.01), but higher than that of the Bac group (P <0.01). The survival rates of mice received pooled anti-sera against TSB, rPalA, BacPal and Bac, were 0%, 0%, 37.5% and 100% after challenge, respectively. In addition, mice in the BacPal group showed moderate to severe lung damage, whereas mice in the Bac group showed relatively normal lung tissues during the histological examination. CONCLUSION Our results demonstrate that A. pleuropneumoniae PalA is an immunogenic but not protective antigen, the existence of PalA suppresses the production of protective antibodies, and thus reduces the protective immunity of inactivated vaccine. Therefore, it should be taken into consideration of these immunogenic but not protective proteins during the development of highly effective vaccines in future.

Crystal structure of human Intersectin-2L C2 domain.

Intersectin-2L (ITSN-2L) is a long isoform of ITSN family, which is a multimodule scaffolding protein functioning in membrane-associated molecular trafficking and signal transduction pathways. ITSN-2L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain, suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. But the role of C2 domain is obscure in this process. Here we report the crystal structure of human ITSN-2L C2 domain at 1.56Å resolution. The sequence and structural alignment of ITSN-2L C2 domain with other members of C2 domain protein family indicate its vital cellular roles in membrane trafficking, the generation of lipid-second messengers and activation of GTPases. Moreover, our data show the possible roles of ITSN-2L C2 domain in regulating the activity of Cdc42.

Outer Membrane Lipoprotein Lip40 Modulates Adherence, Colonization, and Virulence of Actinobacillus pleuropneumoniae

Bacterial lipoproteins are a set of membrane proteins with various functions; many of which are virulence factors of pathogenic bacteria. In the present study, we investigated the role of an outer membrane lipoprotein Lip40 in the pathogenesis of Actinobacillus pleuropneumoniae. A mutant strain (Δlip40) lacking Lip40 and a complemented strain (CΔlip40) were constructed. Δlip40 exhibited reduced adherence to the St. Jude porcine lung cells. The ability of the Δlip40 mutant to colonize the mouse lung tissues was significantly impaired compared to that of the wild type and complementation strains. Furthermore, an infection assay revealed that pigs infected with Δlip40 showed fewer clinical signs and lung lesions, indicating that Lip40 contributed to the development of porcine pleuropneumonia. Collectively, our data suggest that Lip40 is involved in the virulence of A. pleuropneumoniae.

Monoclonal Antibodies Directed Against VP7 Protein of Human Group B Rotavirus

The aim of this study was to prepare and identify a monoclonal antibody that binds the viral proteins 7 (VP7 protein) of human group B rotavirus (GBRV) and to describe its immunologic characterization. Human group B rotavirus vp7 gene was successfully ligated into pGEX-KG vector and transformed into Escherichia coli TOP10 cells. The glutathione S-transferases (GST)-fusion protein GST-VP7 was induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and immediately purified to immunize BALB/c mice. Splenocytes were then prepared from the immunized mouse and fused with SP2/0 myeloma cell line. In the end we obtained one positive hybridoma cell line stably secreting monoclonal antibody against GST-VP7 protein by indirect enzyme-linked immunosorbent assay (ELISA) and limiting dilution. The production of the monoclonal antibody against GBRV will benefit the further study of GBRVs structures and functions and also lay a solid foundation for the research of disease prevention, clinical diagnosis, and treatment.

Monoclonal Antibodies Against Actinobacillus pleuropneumoniae TonB2 Protein Expressed in Escherichia coli

TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified. The rTonB2 was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Four clones of TonB2-specific MAb secretion hybridomas--2F2, 2G8, 3D2, and 6F10--were selected. The MAbs 2F2, 3D2, and 6F10 were classified as IgG1 isotype and 2G8 was of IgG2a isotype. Western blot and ELISA results indicated that MAbs had specific binding activity to rTonB2. The MAbs generated here will be used for further functional analyses of the TonB2 protein.