Biological Toxicity Effects of Soil Pollution Caused by Galvanized Wastewater Based on Vibrio Qinghaiensis sp.-Q67 / 法医学杂志

Objective To establish a rapid diagnosis method for the biological toxicity of soil, accurately and rapidly evaluate the toxicity of contaminated sites and identify the dominant pollutants. Methods Take the soil pollution of a galvanized factory as an example, while the metal concentration level was analyzed and detected, a rapid biological toxicity detection method based on the acute toxicity test of luminescent bacteria （Vibrio qinghaiensis sp.-Q67） was established, and the dominant pollutants were identified by stepwise multiple regression. Results The pollutants came from wastewater and metal plating fragments directly discharged from the manufacturing line of the factory. The concentration of those pollutants was correlated with the acute toxicity of Vibrio qinghaiensis sp.-Q67. The dominant pollutants in the study were zinc （Zn）, aluminum （Al） and copper （Cu）. Conclusion The luminescent bacteria toxicity test method based on Vibrio qinghaiensis sp.-Q67 can conveniently and rapidly assess the degree of toxic damage of polluted soil and identify the dominant pollutants and can be applied to the acute toxicity evaluation of polluted soil.

Analysis of the Relationship Between Era and YggG in E.coli by Double-promoter Expression Vector pDH2-YggG-P_(tac)-Era / 中国生物工程杂志

yggG, a Era-binding protein gene, was isolated and cloned from the E.coli genomic DNA library. Previous studies indicated that the product of yggG gene, YggG294(amino acids 1-294), strongly inhibited the growth of host bacteria and caused the death of bacteria cells. To elucidate whether Era is related to the death of bacterial cells expressed YggG294,A double promoter expression vector that can express YggG294 and Era proteins controllably in cells was constructed. Using this vector to express YggG294 and Era protein in the same E.coli cells, then analyzed the relation between YggG294 and Era. The results showed that the ratio of Era proteins to total proteins increased with the increase of induction time in E.coli cells without YggG294 expression and with little YggG294 expression;the ratio of Era proteins to total proteins seemed to be a constant level in E.coli cells overexpressing YggG294;but we could not detect any Era hydrolyzate in E.coli cells overexpressed YggG294 could not be detected. The results also showed that pre-expression of Era protein did not produce any effect on the growth inhibition of E.coli cells caused by YggG294. These results indicate that YggG294 can not hydrolyze Era protein in E.coli cells, and that YggG-Era interaction is not associated with the death of bacteria expressed YggG294. It is thus reasonable to draw a conclusion that Era is not associated with the growth inhibition of E.coli cells caused by YggG294. YggG294 inhibits the growth of bacteria by other way.

Antitumor immune responses induced by gene transfer of 4-1BBL into hepatocellular carcinoma Hepa1-6 in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To study the cytotoxic activity against tumor cells and cytokines production of spleen cells induced in vitro by murine 4-1BBL gene transfected Hepa1-6.</p><p><b>METHODS</b>The eukaryotic expression vector pCDNA3.1(+)-m4-1BBL was transfected into murine hepatocellular carcinoma cell line Hepa1-6 by Liposomes. Then the transfected cells were selected in medium containing G418 (400 - 800 micro g/ml) and termed as Hepa1-6-m4-1BBL. The TCV-m4-1BBL was obtained by treating them with mitomycin (MMC). Cocultivation TCV with syngeneic murine spleen cells, then the lymphocytes were tested for cytotoxic activity against Hepa1-6-wt cells and the supernatants were harvested for detecting the cytokines (IL-2, TNF-alpha and GM-CSF).</p><p><b>RESULTS</b>Hepa1-6-m4-1BBL cells expressed 4-1BBL protein with highest cell surface level. The 4-1BBL mRNA could still be detected in the cells when cultured 48 h after treated with MMC (80 mg/L). Comparing with TCV-Hepa1-6, the tumor cell vaccine derived from Hepa1-6-m4-1BBL (TCV-m4-1BBL) could induce a more efficient cytotoxic activity of syngeneic murine lymphocyte against its parental tumor cell Hepa1-6 (P < 0.05), but not against non-parental tumor cell H22 and NIH3T3. Higher levels of IL-2, TNF-alpha and GM-CSF were released by the splencytes after stimulated by TCV-m4-1BBL.</p><p><b>CONCLUSIONS</b>These results suggest the expression of m4-1BBL by tumor cells is effective in inducing antitumor immune responses.</p>

Construction and biological activity study of human osteoprotegerin expressing adenoviral system / 生物工程学报

Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.

Expression of human osteoprotegerin gene in E. Coli and bioactivity analysis of expression product / 中华外科杂志

<p><b>OBJECTIVE</b>To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro.</p><p><b>METHODS</b>Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products.</p><p><b>RESULTS</b>The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L.</p><p><b>CONCLUSIONS</b>Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.</p>

The OPG/RANKL/RANK system and bone resorptive disease / 生物工程学报

The OPG/RANKL/RANK system plays an important role in osteoclastogenesis and represents a great progress in bone biology. RANKL, which expresses on the surface of osteoblast/stromal cells and activated T cells, binds to RANK on the osteoclastic precursors or mature osteoclasts, and promotes osteoclastogenesis and bone resorption. While osteoprotegerin (OPG), which is expressed by osteoblasts/stromal cells, strongly inhibits bone resorption by binding to its ligand RANKL and thereby blocks the interaction between BANKL and RANK. A number of cytokines and hormones exert their effects on bone metabolism by regulating the OPG/RANKL ratio in the bone marrow microenvironment. RANK is also expressed on mammary epithelial cells and RANKL expression in these cells is induced by pregnancy hormones, RANKL and RANK are essential for the formation of the lactating mammary gland and the transmission of maternal calcium to neonates in mammalian species. Modulation of these systems provides a unique opportunity to develop novel therapeutics to inhibit bone loss in osteoporosis, rheumatoid arthritis, and bone metastasis of cancer. Further research should be focused on the cooperation of OPG/RANKL/RANK system with other signal pathways and the interactions among bone remodeling, immune system and endocrinology system. Currently, the development of OPG analogues or compounds which may stimulate OPG expression is becoming an attractive industry which may be profitable to both patients and manufacturers.

High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding / 生物工程学报

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.

Strategies of functional analysis of new genes / 生物工程学报

Functional analysis of new genes is playing a central role in postgenomic era. Here we reviewed several main strategies including bioinformatics, gene transduction, antisense technology, certain gene silence induced by RNA interference (RNAi), transgene and gene knockout and artificial chromosome transduction.