Abstract:Silk fibroin is a natural macromolecular fibroin. It has broad prospects in tissue engineering application due to its good physical and mechanical properties and good biocompatibility. This paper reviews its chemistry structure, property, the usage as matrix of tissue-engineering scaffold using electrospinning technology, and the influence on growth, proliferation and function of vascular endothelial cell, smooth muscle cell, keratinocyte and fibroblast. It also addresses the advantages and disadvantages of silk fibroin applied in tissue engineering study like artificial vascular, skin, bone stent etc. The potential applications on esophageal tissue engineering and regenerative medicine were discussed.

Abstract:Marine can be considered as a rather unexplored source of biological material. Production of algal oligosaccharides by using valuable enzymes from marine origin has become an important way to utilize marine resources. As one of algal tool enzymes, the use of alginate lyases has been focused mainly on development and application of alginate oligosaccharides with bioactive function in recent years. In this paper, we reviewed the research of alginate lyases over the past decade in several aspects, including their origin, diversity, substrate specification, mode of action, structure and catalysis mechanism, assay of enzyme activity, enzyme characterization, as well as our own experience on this subject. At the end of the review, the application prospects of alginate lyases are presented.

Abstract:Antibacterial peptides are a family of host-defense peptides most of which are gene-encoded and produced by living organisms of all types. Antibacterial peptides are small molecular proteins with broad antimicrobial spectrum against bacteria, viruses, fungi and sometimes even as anticancer peptide. SMAP-29, a cathelicidin-like peptide derived from sheep myeloid, line α-helical Structure, exerts a powerful broad antimicrobial activity against different pathogens including Gram-positive and Gram-negative bacteria, fungi, viruses, parasites, spirochaetes, chlamydia and antiendotoxin activity, and particular antibacterial mechanism, rapidly to permeabilize membranes of susceptible organisms. This paper summarizes the lately research progress of SMAP-29 and Variants including the characteristics of structure, structure-activity relationships, mode of action, diverse biological functions, gene recombinant and expression. We put emphasis on the necessity of molecular design, and primary and secondary structure-based modification, to provides a strong foundation for further drug development and design of SMAP-29.

Abstract:Based on previous bioinformational analysis results, two Aspergillus niger lipase (ANL) mutants, ANL-Ser84Gly and ANL-Asp99Pro were constructed to screen ANL mutants with oil-water interface independence. ANL-Ser84Gly still displayed a pronounced interfacial activation, while ANL-Asp99Pro displayed no interfacial activation. The specific activity of ANL-Ser84Gly towards p-nitrophenyl palmitate (-myristate, -laurate and -decanoate) decreased by 29.8% (53.1, 60.1 and 77.1, respectively) than that of ANL, while the specific activity of ANL-Asp99Pro towards p-nitrophenyl palmitate increased by 2.2-fold. The mutation in the hinge region at both sides of the lid domain also destabilized various secondary structure factors of ANL-S84G and ANL-D99P, which resulted in a substantial decrease in thermostability. The achievement to construct oil-water interface-independent ANL mutants would help to further understand lipase interfacial activation mechanism.

Abstract:In plants, proteins with A20/AN1 zinc finger domain are involved in stress responses, named as "Stress Associated Protein" (SAP) gene family. Based on Expressed Sequence Tag (EST) sequences information in Badila Saccharum officinarum mature related cDNA library, we cloned an SAP gene from sugarcane full length cDNA library, named ShSAP1 (GenBank: Accession No. HM991960). To characterize ShSAP1, we analyzed its genome structure and expression pattern. Southern blot analysis showed ShSAP1 was present as one or two copy in the genome of Badila. Comparison of ShSAP1 1 008 bp full length cDNA with a genomic frangment (2 241 bp) generated by PCR amplification and sequencing, revealed the presence of two introns (202 bp and 1 052 bp) located in the 5'UTR region. Semiquantitative RT-PCR analysis found ShSAP1 expressed in leaves, roots and stalk in mature sugarcane. Compared with immature stems, ShSAP1 expressed higher in mature stalk. ShSAP1 was induced by different types of treatments, such as salt (200 mmol/L NaCl), drought (10% PEG 6 000), GA3 (200 mg/L), ABA (100 μmol/L) and ET (1 mmol/L) during sugarcane seedling stage. These results indicated that ShSAP1 may function in sugarcane maturation and abiotic stress response processes.

Abstract:In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.

Abstract: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.

Abstract:Clinical application of staphylococcal enterotoxin C2 (SEC2) was restricted during the cure of malignant tumor due to its side-effects. The aim of this study was to obtain SEC2 mutant, preserving the important functional sites responsible for the T-cell stimulatory activities but removing the sites responsible for emetic activity, through truncation of SEC2. It would efficiently solve the question of SEC2 side-effect. According to the results of methyl thiazol tetrazolium (MTT) assay in vitro, novel truncated SEC2 mutant (NSM) efficiently stimulated T-cell proliferation and inhibited the growth of such tumor cells as human colorectal cancer cells (Cx-1) and human breast cancer cells (MCF-7) in vitro. Activities of T cell stimulating and anti-tumor of NSM were similar to those of SEC2. According to results of animal experiments, the mutant no longer induced emetic response even if the dose was a 10-fold excess of the amount of SEC2 required. And also, NSM obviously inhibited the tumor growth in tumor-bearing mice. Therefore, we obtained novel truncated staphylococcal enterotoxin C2 mutant, which could efficiently inhibit the growth of tumor cells. It will become novel anti-tumor agents with the lowest side-effects and best treatment effects in clinic.

Abstract:Kv2.1 channel currents in pancreatic b-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 mmol/L and 1.96 mmol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.

Abstract:Human bocavirus (HBoV) is a recently discovered parvovirus, which is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in human. In the present study, we screened 941 nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections from October 9, 2007 to March 20, 2009 in the Children’s Hospital of Hubei Province. Our results showed that 33 of 941 samples (3.51%) were detected positive for HBoV. To obtain a full-length HBoV clone, three segments which covered the nearly full-length genome were amplified by PCR from HBoV positive samples separately and cloned into pBluescript SKⅡvector, and the resulting plasmid was designated as pWHL-1 (GenBank Acession No. GU139423). We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter, respectively. Our data demonstrated that the HBoV promoter exhibited very high activity in all mammalian cells tested by fluorescent microscopy observation of the EGFP and luciferase activity assay and its strength was 4?5 fold higher compared to that of the CMV promoter. This work provided an excellent tool for further study of the mechanism of transcription and expression of the viral genome.

Abstract:In Candida albicans, adaptation to environmental pH is relevant to its pathogenicity. Calcium signaling pathway involves in many stress responses and often accompany with Ca2+ fluctuation. We constructed CCH1 and MID1 mutant strains and studied their effect on calcium influx and further investigated the regulation by Crz1p transcription factor. We used PCR-directed gene disruption to construct cch1Δ/Δ and mid1Δ/Δ null mutant. By using a flow cytometry-based method we monitored the free cytosolic Ca2+ levels under alkaline stress. Moreover, we constructed pPHO89-LacZ plasmids and by β-Galactosidase assays, we analyzed the changes of LacZ activities after gene disruption. The results showed that alkaline stress induced calcium burst reduced obviously in cch1Δ/Δ and mid1Δ/Δ mutant strains, also for LacZ activities, and fully abolished in crz1Δ/Δ mutant strain. Finally, by realtime PCR, we confirmed the regulation role of Crz1p in CCH1 and MID1 genes but in a calcineurin independent way. Studies on the effect of calcium pathway on response to alkaline stress will provide an important theoretical basis for Candida albicans infection-oriented treatment and new drug targets.

Abstract:A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORF5/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted. Conclusions: double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

Abstract:We established a stable Chinese hamster ovary (CHO-S) cell line for recombinant human VEGF165-expressing. We co-transfected GS-expression vector and rhVEGF165 expression plasmid into CHO-S cells, and selected the highest VEGF165- expressing clone as the working cell line to express VEGF165 protein. After 7-day fed-batch culture in a 5 L bioreactor and 3 steps chromatographic purification, we got the rhVEGF165 protein for series of binding and biological activity examination. The production was over 50 mg/L. The purified rhVEGF165 protein was functionally active with a half-maximal Human Umbilical Vein Endothelial Cells (HUVEC) growth-enhancing effect concentration of 1.94 ng/mL. It was slightly better than commercially available Escherichia coli expressing rhVEGF165. So we expressed successfully rhVEGF165 protein in high-level and obtained the fully active rhVEGF165 protein in large quantity.

Abstract:To produce high-affinity monoclonal antibodies against pesticide imidacloprid, we synthesized the haptens 1-[(6-Carboxylethylthio-3-pyridinyl) methyl] -N-nitro-imidazolidinimine (named as H1) and 1-[(6-Chloro-3-pyridinyl) methyl]-3-carboxylpropyl-N-nitro-2-imidazolidinimine (termed as H2). And then the haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) for immunogen (H1-BSA) and coating antigen (H2-OVA) respectively by NHS ester method. BALB/c mice were immunized with H1-BSA conjugate. We obtained two hybridoma cell lines 2F11/A9 and 2G6/G12 secreting antibody specific for imidacloprid from the conventional hybridoma technology. The result showed that the subtypes of obtained monoclonal antibodies were IgG3 and IgG1, respectively, and the titers of ascites were up to 1:128 000. The indirect competitive ELISA indicated the IC50 values of 5.3 and 28.3 ng/mL with detection limits of 1.1 ng/mL and 7.7 ng/mL, respectively. Two monoclonal antibodies had no apparent cross reactivity with six analogous compounds. Thus, two prepared monoclonal antibodies had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of imidacloprid residue and laid a solid foundation for research and development of products for immunoassay.

Abstract:Nowadays, SUMO fusion system is important for recombinant protein production in Escherichia coli, yet a few aspects remain to be improved, including the efficacy for vector construction and protein solubility. In this study, we found the SUMO gene Smt3 (Sm) of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning, and the gene coding regions of SUMOs in most species had a s70-dependent prokaryotic promoter embedded, through the prediction via the BPROM program developed by Softberry. By combining the characters of Sm promoter activity and the Stu I site (added at the 3’-terminal of Sm), and introducing a His-tag and a hyper-acidic solubility-enhancing tag, we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm’-LacZα fusion gene. Experimentally started from these vectors, several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis. The results manifest a few of expectable advantages such as rapid vector construction, highly soluble protein expression and feasible co-expression of correlated proteins. Conclusively, our optimized SUMO fusion technology herein could confer a large potential in E. coli protein expression system, and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.

Abstract:In order to construct a eukaryotic expression vector of bovine c-myc gene, the coding sequence (CDS) of c-myc gene was amplified from bovine primordial genital ridges by RT-PCR. The CDS was subcloned into pMD19-T vector, and then inserted into vector pIRES2-AcGFP1-Nuc. After confirmed by restriction enzyme digestion and sequencing, the recombined plasmid was transfected into skin fibroblast cells. RT-PCR and Western Blotting were used to detect the expression of c-myc mRNA and protein, respectively. The results show that the complete CDS of c-myc gene was cloned from fetal bovine primordial genital ridges. The eukaryotic expression vector of bovine c-myc gene was constructed and efficiently expressed in the skin fibroblast cells. The present study will lay a good foundation for further study of c-myc gene function and bovine induced pluripotent stem cells from somatic cells by defined factors.