Advances in the research and applications of orange fluorescent protein / 生物工程学报

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Pathogenicity of white-spot syndrome virus in Macrobrachium nipponensis via different infection routes / 生物工程学报

Macrobrachium nipponensis is delicious and has high economic value, but its susceptibility to white-spot syndrome virus (WSSV) is unknown. Susceptibility, morbidity, and multiplication of WSSV in M. nipponense were studied by epidemiological survey, infection experiment and qPCR. M. nipponense was the natural host of WSSV, and the natural carrying rate was about 8.33%. M. nipponense could be infected with WSSV via oral administration, muscle injection and immersion, and the cumulative infection rate of 10 d exposure was 100%, and the cumulative mortality rates were 100%, 75% and 0%, respectively. The infection of WSSV is fast by muscle injection. The virus content after 5 day's injection is 1 000 times higher than that of the first day of infection, and the mortality rate reached 100% after 8 days. The median lethal dose (LD₅₀) measured as the mortality of infected M. nipponense via injection indicated the LD₅₀ in the concentration of WSSV of 2.71×10⁵ virions/μL. In shrimp farming, M. nipponense can be infected by ingesting WSSV infected shrimp or dead shrimp, and also by soaking in WSSV-containing water and thus become a vector, consequently affecting the spread and pathogenicity of WSSV.

Effects of light quality on cell growth and psbA promoter of engineered Synechococcus sp. PCC7002 / 生物工程学报

Light quality can regulate both psbA genes and vector promoter psbA of the engineered Synechococcus. Through light regulation, we tried to improve yield of the recombinant protein for vp28 gene-expressed Synechococcus sp. PCC7002. To drive photon-capturing efficiently, three limiting factors (irradiance, temperature and pH) were optimized by measuring net photosynthesis. High cell density cultures were performed with variant ratios of white, red and blue light in a 5-L photo-bioreactor. Yields of biomass, expressions of vp28 and transcription levels of psbA were compared. High ratio blue light-induced vp28 transcription had tripled and the relative accumulation of VP28 protein was doubled. The relative expressions of psbAII and psbAIII had positive correlations with higher ratio of blue light, not the red light. With high ratio red light inducing, dry biomass reached 1.5 g/L in three days. Therefore, we speculated that red light accelerated biomass accumulation of the transgenic strain and blue light promoted transcription for PpsbA and psbA. These results provided useful information for mass production of cyanobacteria and its secondary metabolites.

Scale-up preparation of phycoerythrin from Porphyra haitanensis / 生物工程学报

We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using "swelling and smash" method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280 > 3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.

Photodynamic effect of two kinds of phycobiliproteins on human liver cancer cell line SMMC-7721 in vitro / 生物工程学报

We studied the effect of photodynamic therapy with phycobiliproteins on human liver cancer cells in vitro. With 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), we used two phycobiliproteins, R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) prepared from Porphyra yezoensis, to determine the killing rates and apoptosis rates of human liver cancer cells (SMMC-7721) mediated by laser. When the concentration of R-PE was 120 mg/L, the survival rate of human liver cancer cells was 27% after treated by Argon laser with 100 J/cm2 doses, while the survival rate in the control group (without adding R-PE) was 65%. When the C-PC concentration was 120 mg/L, the survival cell rate was 47% after treated by He-Ne laser with 35 J/cm2 dose, while the survival rate in the control group (without adding C-PC) was 70%. After handled only with these two kinds of phycobiliproteins for 72 h, the growth of cancer cells presented significant inhibition. The maximal inhibition rates reached up to 31% with R-PE (120 mg/L concentration) and 27% with C-PC (250 mg/L concentration) respectively. After irradiated by laser for 8 h, the maximal cell apoptosis rates were 31.54% with R-PE and 32.54% with C-PC, respectively. It indicated that R-PE and C-PC extracted from Porphyra yezoensis could develop to new photosensitizers for cancer photodynamic therapy.

Reform on the examination of molecular biology / 中华医学教育探索杂志

To analyse and compare the advantages and disadvantages of various kinds of examinations in teaching molecular biology,and detail the process and effect of thesis proseminar domained examination.