Supervisor(s): Chinese Academy of Sciences Sponsor(s): Institute of Microbiology, Chinese Academy of Sciences(CAS); Chinese Society for Microbiology CN：11-1998/Q
Founded in 1985,Chinese Journal of Biotechnology is the official journal of the Institute of Microbiology, the Chinese Academy of Sciences and the Chinese Society for Microbiology. It is an international,peer-reviewed journal that publishes original papers and reviews on all aspects of Biotechnology, such as genetic engineering,cell engineering, enzyme engineering, biochemical engineering, and so on.The journal is indexed/abstracted in various important citation resources such as Chemistry Abstracts,MEDLINE/PubMed,AJ OF vINITI, JST, Scopus, Center for Agriculture and Bioscience International, EMBASE, CNKI,CBM,Chinese Science and Technological Periodical(CD).
We isolated and identified the symbiotic and adnascent microorganisms from an unidentified sponge collected from 10-meter-deep seawater of the Xisha Islands in China. A total of 16 strains were obtained and identified. Through bacteriostatic activity assay, one of the strains, Dermacoccus sp. X4, was found to effectively inhibit the growth of Staphylococcus aureus. Subsequently, its secondary metabolites were purified by silica gel partition, octadecylsilane (ODS) reverse phase, Sephadex TMLH-20 size exclusion, and C18 reverse phase chromatography. Using liquid chromatography, mass spectrometry, and nuclear magnetic resonance, three of the purified compounds were structurally characterized to be one 3-(4-hydroxybenzyl) hexahydropyrrolo [1, 2-a]pyrazine-1, 4-dione and two indole acid glycerides. This is the first report about indole acid glyceride isolated from microbial secondary metabolites, enriching marine drug candidate resources.
Effective expression of pIFN-α in recombinant Pichia pastoris was conducted in a 5 L fermentor. Ethanol accumulation during the late glycerol feeding period inhibited heterologous protein expression. Comparative transcriptome analysis was thus performed to compare the gene transcription profiles of Pichia pastoris KM71H in high and low ethanol concentration environments. The results showed that during the glycerol cultivation stage, 545 genes (265 up-regulated and 280 down-regulated) were differentially expressed with ethanol stress. These genes were mainly involved in protein synthesis, energy metabolism, cell cycle and peroxisome metabolism. During the methanol induction stage, 294 genes (171 up-regulated and 123 down-regulated) were differentially expressed, which were mainly related to methanol metabolism, amino acid metabolism and protein synthesis. Ethanol stress increased protein misfolding and reduced structural integrity of ribosome and mitochondria during cultivation stage, and led to the failure of endoplasmic reticulum stress removal and damaged amino acid metabolism during induction stage in Pichia pastoris.
To analyze the immunogenicity and protective ability of recombinant IgG-binding protein (EAG) of Streptococcus equi subspecies equi and to evaluate its value when used as equine vaccine antigen, EAG gene was amplified by PCR and inserted into pET-28a vector. The EAG recombinant proteins were expressed and purified to immune mice. The serum antibody and challenge protection were tested. The purified recombinant protein of EAG was 26 kDa, and the protein reacted specifically with positive serum of Streptococcus equi subspecies equi. The mice antibody level for EAG immunization group was 1:8 100. The immunological protection result showed that the protection rate of the EAG recombinant protein was 90%. The results suggested that the EAG protein has good immunogenicity and immunological protection, and it can effectively increase the humoral immune response and immunological protection of mice.
The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with micro RNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar larvae, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3′UTR of Bmlin-41 of 1 434 bp in length was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3′UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.
Listeria monocytogenes (LM) is an excellent tumor vaccine vector. In this study, recombinant LM expressing human papillomavirus type 16 (HPV16) E7 protein, a vaccine candidate was constructed and the biological characteristics were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 hly promoter-signal sequence, and introduced into the LM4 genome. The recombinants, constructed as LM4△hly::E7, were plasmid- and antibiotic-resistant gene-free. LM4△hly::E7 could express and secrete a 66 kDa E7-LLO fusion protein, which had immunological activities. Proliferation of LM4△hly::E7 in RAW264.7 macrophages was observed under confocal laser scanning microscope. Specific anti-E7 antibodies could be detected by ELISA in mice immunized with LM4△hly::E7. The LD50 of LM4△hly::E7 was 3.863 × 109 CFU in C57BL/6 mice with intraperitoneal immunization, which was 104 times higher than that of wild type LM4. No obvious pathological changes were detected in the immunized mice. These results suggest that LM4△hly::E7 expressing E7-LLO fusion protein may be a safe and promising vector-vaccine candidate for cervical cancer, and may also be important biological materials for anti-tumor research.
We aimed at analyzing the structure of extracellular polysaccharide A from Grifola frondosa (EXGFP-A) and testing its immunological activity. Structural analysis showed that EXGFP-A contained α-D-glucoside bond and pyranose ring. GC analysis revealed that EXGFP-A was mainly composed of rhamnose, arabinose, xylose, mannose, glucose, galactose, with the molar ratio of 0.28:0.31:0.30:0.06:7.98:0.61. The results of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicated when EXGFP-A was at a concentration of 80 μg/mL and treatment time of 48 h, RAW264.7 cells proliferation index reached a maximum of 137.5%. Meanwhile, the AO staining showed that EXGFP-A activated RAW264.7 cells and improved the level of intracellular nucleic acid metabolism. In addition, in a certain range of concentration, EXGFP-A was able to increase the release of NO in RAW264.7 cells, and upregulate the mRNA expression of immunological factors TNF-α, IL-1β, IL-6, IL-12, IFN-γ and iNOS of RAW264.7 cells. Our results confirm that EXGFP-A had immunomodulatory activity. Our findings provided scientific basis for the structural analysis and application of Grifola frondosa polysaccharide.
We isolated and enriched mixed microorganisms SWA1 from landfill cover soils supplemented with trichloroethylene (TCE). The microbial mixture could degrade TCE effectively under aerobic conditions. Then, we investigated the effect of copper ion (0 to 15 μmol/L) on TCE biodegradation. Results showed that the maximum TCE degradation speed was 29.60 nmol/min with 95.75% degradation when copper ion was at 0.03 μmol/L. In addition, genes encoding key enzymes during biodegradation were analyzed by Real-time quantitative reverse transcription PCR (RT-qPCR). The relative expression abundance of pmoA gene (4.22E-03) and mmoX gene (9.30E-06) was the highest when copper ion was at 0.03 μmol/L. Finally, we also used MiSeq pyrosequencing to investigate the diversity of microbial community. Methylocystaceae that can co-metabolic degrade TCE were the dominant microorganisms; other microorganisms with the function of direct oxidation of TCE were also included in SWA1 and the microbial diversity decreased significantly along with increasing of copper ion concentration. Based on the above results, variation of copper ion concentration affected the composition of SWA1 and degradation mechanism of TCE. The degradation mechanism of TCE included co-metabolism degradation of methanotrophs and oxidation metabolism directly at copper ion of 0.03 μmol/L. When copper ion at 5 μmol/L (biodegradation was 84.75%), the degradation mechanism of TCE included direct-degradation and co-metabolism degradation of methanotrophs and microorganisms containing phenol hydroxylase. Therefore, biodegradation of TCE by microorganisms was a complicated process, the degradation mechanism included co-metabolism degradation of methanotrophs and bio-oxidation of non-methanotrophs.
