Wenhua Ren

Nanjing Normal University, Nanjing, Jiangsu Sheng, China

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Publications (8)19.79 Total impact

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    ABSTRACT: Antimicrobial peptide scolopin 1 (AMP-scolopin 1) is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans. It has broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We first report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1. The fusion protein expressed in a soluble form was purified to a purity of 95% by Ni-IDA chromatography. After the SUMO-scolopin 1 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was reapplied to a Ni-IDA. The recombinant scolopin1 had similar antimicrobial properties to the synthetic scolopin 1. Thus, we successfully established a system for purifying peptide of centipede, which could be used for further research.
    Protein Expression and Purification 10/2013; · 1.43 Impact Factor
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    ABSTRACT: In this study, we isolated the cDNA of a gamma-interferon-inducible lysosomal thiol reductase (GILT), which is critical for innate immune regulation, from the Yangtze finless porpoise (FpGILT). This gene encoded a protein with 244 amino acids and a predicted molecular weight of 28 kDa. The amino acid sequence of FpGILT includes an active-site CXXC motif, a GILT signature sequence, CQHGX2ECX2NX4C, and three N-linked glycosylation sites. Phylogenetic analysis showed that FpGILT and other GILT family members were derived from a common ancestor and finless porpoises are closely related to artiodactyla. Recombinant protein (FpsGILT) was then efficiently expressed and purified, and thiol reductase activity assays suggested that FpGILT catalyses disulfide bond reduction. These findings provide a basis for understanding the characteristics of immunity in the finless porpoise and other aquatic mammals.
    Developmental and comparative immunology 06/2013; · 3.29 Impact Factor
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    ABSTRACT: The Yangtze finless porpoise (Neophocaena phocaenoides asiaorientalis) is listed on the First Order of Protected Animals in China and was identified as an endangered species by the International Union for Conservation of Nature and Natural Resources (IUCN) in 2011. A proliferation inducing ligand (APRIL), belonging to the tumour necrosis factor (TNF) family, is critical for immune regulation. In this study, we identified a finless porpoise APRIL cDNA (fAPRIL) by RACE (rapid amplification of cDNA ends) strategies, from the Yangtze finless porpoise (fAPRIL). This gene encodes 247 amino acids containing a predicted transmembrane domain and a TNF domain, and phylogenetic analysis of the APRIL sequence indicated that finless porpoises are closely related to Artiodactyla. In vitro, soluble fAPRIL (fsAPRIL) not only promoted the survival/proliferation of the mouse spleen lymphocytes, but also bound specifically to the surface of the B cells. The results of this study contribute to our understanding of the immune mechanisms in the finless porpoise and other aquatic mammals.
    International immunopharmacology 04/2013; · 2.21 Impact Factor
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    ABSTRACT: The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species. Research into the immune system of the finless porpoise is essential to the protection of this species, but, to date, no genes coding for proteins from the tumor necrosis factor family (TNF family) have yet been reported from finless porpoises. The TNF B cell activating factor (BAFF) is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. It acts through its three receptors, BAFF-R, BCMA, and TACI. In the present study, the full-length cDNA of BAFF (designated NpBAFF) from the finless porpoise was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. To our knowledge, this is the first report of any BAFF gene being cloned from an aquatic mammal. The full-length cDNA of NpBAFF consists of 1502 bases including an 852 bp open reading frame encoding 283 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known BAFF homologues. Sequence comparison indicated that the amino acid sequence of NpBAFF was very similar to its bovine (87.68%), porcine (76.33%), hircine (87.68%) and canine (82.19%) counterparts. The predicted three-dimensional (3D) structure of the NpsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its human counterpart. Phylogenetic analysis indicated that NpBAFF showed a notable homology with Artiodactyla BAFFs. The SUMO-NpsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that NpsBAFF could bind to its receptors on B cells. In vitro, MTT assays indicated that SUMO-NpsBAFF could promote the survival or proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that NpBAFF plays an important role in the survival or proliferation of B cells and has functional cross-reactivity among cetaceans and other mammals. The present findings may provide valuable information for research into the immune system of the finless porpoise.
    Gene 05/2012; 504(1):13-21. · 2.20 Impact Factor
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    ABSTRACT: A proliferation-inducing ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the full-length cDNA of APRIL (designated bAPRIL) from bat was cloned using RT-PCR and its biological activities have been characterized. The open reading frame (ORF) of this cDNA consists of 753 bases, encoding a protein of 250 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known APRIL homologs. Real-time quantitative PCR (qPCR) analysis indicated that bAPRIL mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsAPRIL was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that bsAPRIL could bind to its receptors on B cells. In vitro, MTT assays indicated that bsAPRIL could promote the survival/proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that bsAPRIL plays an important role in the survival and proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.
    Gene 02/2012; 498(2):196-202. · 2.20 Impact Factor
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    ABSTRACT: B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat (Vespertilio superans Thomas) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of bBAFF consists of 986 bases including an 873 bp open reading frame encoding 290 amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the bsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO (Small Ubiquitin-like Modifier)-bsBAFF was efficiently expressed in Escherichia coliBL21 (DE3) and confirmed by SDS-PAGE and Western blotting analysis. Laser scanning confocal microscopy analysis showed that bsBAFF could bind to its receptors on B cells. In vitro, the MTT assays indicated that SUMO-bsBAFF was not only able to promote survival/proliferation of bat lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These findings indicate that bsBAFF plays an important role in the survival/proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.
    International immunopharmacology 02/2012; 12(2):433-40. · 2.21 Impact Factor
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    ABSTRACT: B-cell activating factor (BAFF), a member of the TNF family, is critical to the survival, proliferation, maturation, and differentiation of B-cells. In the present study, a CpBAFF was amplified from the white-spotted catshark (Chiloscyllium plagiosum) using RT-PCR and RACE (rapid amplification of cDNA end) techniques. To our knowledge, this is the first report of any BAFF gene being cloned from a cartilaginous fish. The open reading frame (ORF) of CpBAFF cDNA consists of 819 bases encoding a protein of 272 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other identified BAFF homologues. Sequence alignment showed that CpBAFF shares 37-57% identity with BAFF amino acid sequences reported in other vertebrates. Three-dimensional structure modeling analysis revealed a soluble mature portion of CpBAFF (CpsBAFF) with a long D-E loop specific to the BAFF gene, which has not been found in other reported TNF proteins. Phylogenetic reconstruction showed that CpBAFF is most closely related to other fish BAFFs and clusters with BAFF genes from higher vertebrates (reptiles, birds, and mammals). Real-time quantitative RT-PCR demonstrated that CpBAFF mRNA expression was high in the spleen but moderate in the kidney and branchia. Recombinant CpsBAFF fused to NusA-His(6)-tag was efficiently expressed in Escherichia coli BL21 (DE3), and a molecular weight of approximately 83 kDa was determined using SDS-PAGE and Western blotting. In vitro MTT assay indicated that the purified pET43.1a (+)-CpsBAFF protein can co-stimulate the proliferation of mammalian B-cells with anti-IgM in a dose-dependent manner. The present findings not only present novel information that may be relevant to shark immunity but also provide some new insights into the origins and evolution of immunity in all vertebrates.
    Fish &amp Shellfish Immunology 09/2011; 31(6):1088-96. · 2.96 Impact Factor
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    ABSTRACT: In this study, an IL-8 homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlIL-8). The open reading frame (ORF) of XlIL-8 consists of 312 bases encoding a protein of 103 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in South African clawed frog IL-8. By quantitative real-time PCR, mRNA transcript of XlIL-8 was detectable in all the examined tissues with higher level in spleen and kidney. The temporal expression of XlIL-8 mRNA in the monocytes was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at about 6h post-stimulation. Recombinant soluble XlIL-8 (XlsIL-8) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The fusing protein SUMO-XlsIL-8 was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. Chemotaxis assays showed that lymphocytes but not monocytes could be recruited toward SUMO-XlsIL-8 or XlsIL-8 protein in a dose-dependent manner in vitro. The present study may be useful for understanding the anti-bacteria immunity in amphibian and gives the potential to use the recombinant proteins to manipulate the immune response.
    Developmental and comparative immunology 04/2011; 35(11):1159-65. · 3.29 Impact Factor