Genome-wide analysis and expression profiling under heat and drought treatments of HSP70 gene family in soybean (Glycine max L.).

Heat shock proteins (HSPs) perform a fundamental role in protecting plants against abiotic stresses. Previous studies have made great efforts in the functional analysis of individual family members, but there has not yet been an overall analysis or expression profiling of the HSP70 gene family in soybeans (Glycine max L.). In this study, an investigation of the soybean genome revealed 61 putative HSP70 genes, which were evaluated. These genes were classified into eight sub-families, denoted I-VIII, based on a phylogenetic analysis. In each sub-family, the constituent parts of the gene structure and motif were relatively conserved. These GmHSP70 genes were distributed unequally on 17 of the 20 chromosomes. The analysis of the expression profiles showed that 53 of the 61 GmHSP70 genes were differentially expressed across the 14 tissues. However, most of the GmHSP70s were differentially expressed in a tissue-specific expression pattern. Furthermore, the expression of some of the duplicate genes was partially redundant, while others showed functional diversity. The quantitative real-time PCR (qRT-PCR) analysis of the 61 soybean HSP70 genes confirmed their stress-inducible expression patterns under both drought and heat stress. These findings provide a thorough overview of the evolution and modification of the GmHSP70 gene family, which will help to determine the functional characteristics of the HSP70 genes in soybean growth and development.

Genetic diversity of parvovirus isolates from dogs and wild animals in China.

We isolated three new parvovirus variants in China. The isolate from a blue fox was related to feline parvovirus, but possessed a mutation of VP2 residue A300P. Isolates from a raccoon dog and a masked civet were antigenically similar to canine parvovirus-2a but had a substitution of VP2 residue G300S.

[Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

AIM: To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. METHODS: The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. RESULTS: The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. CONCLUSION: The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

Characterization of monoclonal antibodies against goat interleukin-18 and their application in the measurement of goat interleukin-18 in LPS-stimulated peripheral blood mononuclear cells by sandwich ELISA.

In order to develop a specific assay for the measurement of goat IL-18 level, two stable hybridoma cell lines were established which secreted IgG1 monoclonal antibodies (mAbs) against goat IL-18. Specific binding of two mAbs named 2E8 and 4C4 to recombinant goat IL-18 expressed in Escherichia coli was demonstrated in an ELISA and Western blotting. Results also showed that mAbs 2E8 and 4C4 bound to distinct epitopes in the ELISA additivity test. These two mAbs were applied in IFA analysis for the detection of goat IL-18 expressed in 293FT cells and in the sandwich ELISA for the measurement of goat IL-18 levels in LPS-stimulated PBMC. Results from this study demonstrated that mAbs against goat IL-18 recognize bovine and human IL-18 and could be used to measure IL-18 levels in different inflammations or immune responses in future studies.

A tetranuclear Cu(II)-based 2D aggregate with an unprecedented tetradentate mu(4)-N1,N3,N7,N9-adeninate nucleobase.

A unique tetranuclear Cu(II)-based 2D aggregate containing 26-membered Cu(II)ade macrocycles, [Cu(4)(DMF)(6)(mu(4)-ade)(2)(mu(2)-Cl)(2)Cl(4)](n) (1; Hade = adenine, DMF = N,N-dimethylformamide), was isolated by the reaction of CuCl(2) and Hade in a mixed DMF-methanol medium and structurally characterized by X-ray crystallography, elemental analysis, Fourier transform infrared, fluorescence spectroscopy, and thermogravimetry-differential thermal analysis techniques. Unexpectedly, the adeninate in 1 represents an unprecedented tetradentate mu(4)-N1,N3,N7,N9-bridging mode, which significantly contributes to both the aggregate of four Cu(II) cores within the subunit and the extension of the 2D covalent framework. Additionally, 1 displays the intense Hade-based fluorescence emission in solution at room temperature.

catena-Poly[[[bis-(1,10-phenanthroline-κN,N')manganese(II)]-µ-9,10-dioxo-anthracene-1,5-disulfonato-κO:O] tetra-hydrate].

The title complex, {[Mn(C(14)H(6)O(8)S(2))(C(12)H(8)N(2))(2)]·4H(2)O}(n), exhibits a chain-like polymeric structure with 9,10-dioxo-anthracene-1,5-disulfonate anions bridging Mn(II) atoms in a bis-monodentate mode. The unique Mn(II) atom is located on a crystallographic centre of inversion. Four N atoms from two chelating 1,10-phenanthroline ligands and two sulfonate O atoms from two symmetry-related 9,10-dioxoanthracene-1,5-disulfonate anions give rise to a slightly distorted octa-hedral coordination environment around the Mn(II) centre. The centroid of the central ring of the anthraquinone ligand represents another crystallographic centre of inversion. In the crystal structure, inter-ligand π-π stacking [centroid-to-centroid distances 3.532 (1) and 3.497 (3) Å] and inter-molecular O-H⋯O hydrogen-bonding inter-actions assemble the chains into a three-dimensional supra-molecular network.

[Artificial chaperone-assisted refolding of recombined chicken Interleukin-18 gene in E.coli].

AIM: To study the technique of boosting the renaturation yield of rChIL-18 by using artificial molecular chaperone composed of cetyl trimethyl ammonium bromide (CTAB) and beta-cyclodextrin(beta-CD). METHODS: The recombinant plasmid of mChIL-18 prokaryotic expression was transformed into E.coli BL21 (DE3) strain and then induced by IPTG at 37 degrees Celsius. The recombinant mChIL-18 was expressed efficiently in inclusion bodies in E.coli. After crushed and washed, the inclusion bodies were thoroughly denatured with 6 mol/L of guanidine hydrochloride, and then the artificial molecular chaperone was used to promote protein refolding. After the rehabilitation of products was purified by bag filter, its activity was detected by lymphocyte proliferation assays. RESULTS: The SDS-PAGE analysis indicated the expressed ChIL-18 protein had molecular weight of 44000. The expressed product existed in the form of inclusion body.Two protein bands of Mr 44000 and 26000 appeared on SDS-PAGE gel. The percentage of renaturation was 42.54 with artificial molecular chaperone.The results of MTT assay showed the expression of ChIL-18 protein in E.coli BL21 (DE3) greatly induced the proliferation of chicken T lymphocytes. CONCLUSION: The artificial chaperone technique can obviously boost the renaturation yield of rChIL-18. The purified and expressed product of fusion chicken Interleukin-18 gene in E.coli have relativity high bioactivity.

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens.

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.

Cloning and expression of goat interleukin-18 gene.

We isolated and sequenced a 480 bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pET32a (+) vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle. Fusional expression with pET32a (+) of gIL-18 of about 38kD was obtained by SDS-PAGE analysis after induction by IPTG in the E. Coli BL21 expression system. The recombinant protein can induce IFN-gamma production in PBMC. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in Peripheral blood mononuclear cells (PBMCs) treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.

Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system.

In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.