[Responses of stoichiometric characteristics of rhizosphere soil to the degradation of alpine meadow.] / é«˜å¯’è‰ç”¸æ ¹é™ åœŸå£¤åŒ–å­¦è®¡é‡ç‰¹å¾å¯¹è‰åœ°é€€åŒ–çš„å“åº”.

This study aimed to understand the stoichiometric characteristics of carbon (C), nitrogen (N) and phosphorus (P) and soil nutrients in rhizosphere and non-rhizosphere soils and to obtain information on the status of soil and microbial nutrient limitation in degraded alpine meadow. We collected soil samples from rhizosphere (0-2 mm) of dominant plant species and non-rhizosphere (0-10 cm) of the alpine meadow with four different degraded degrees in the Qilian Mountains. We measured the concentration of C, N and P and extractable C, N, P (Ext-C, Ext-N, Ext-P), the activity and proportion of extracellular enzymes (ß-1, 4-glucosidase, ß-1, 4-N-acetylglucosaminidase, leucine aminopeptidase and acid phosphatase) involved in C, N, P cycles, as well as soil microbial biomass (MBC, MBN, MBP). The results showed that nutrient concentrations in the rhizosphere of dominant species was higher than that in non-rhizosphere. With the increases of degradation degree, soil C:N:P changed significantly, and resulted in a serious imbalance of C:N and severe N limitation. In the degraded alpine meadows, the ratio of log-transformed rhizosphere C-, N- and P-extracellular enzymes deviated from the 1:1:1 of global ecosystem, indicating that nutrient supply was mainly restricted by N and followed by P. The contents of soil total nutrients in degraded alpine meadow was relatively high, but the contents of soil available nutrients were low, which would hinder plant growth.

Oral administration activity determination of recombinant osteoprotegerin from silkworm larvae.

Osteoprotegerin (OPG) regulates the formation of osteoclasts and is involved in the regulation of bone resorption and remodeling. To investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant osteoprotegerin as a oral administration drug, the rh-OPG was expressed in the larvae of silkworm through the silkworm baculovirus expression system, and was orally administered to mice. Compared with the control, oral administration of rh-OPG was effective to decrease serum calcium concentration in normal mice, and block the bone loss induced by the loss of estrogen in ovariectomized mice. These results indicated that oral administration of rh-OPG expressed in silkworm larvae had the proper bioactivity.

[Expression and activity determination of TNFR domain of osteoprotegerin in E.coli and corresponding antibody preparation].

Osteoprotegerin (OPG) plays an important role in the regulation of bone resorption and remodeling. The TNFR domain of OPG, which is involved in the inhibition of formation and activity of osteoclasts, was amplified by PCR and inserted into multiple cloning site of PET-28a. The recombinant plasmid was transferred into E.coli BL21 to express recombinant protein. It was found that expressed product existed in the form of inclusion body. The inclusion body was solubilized, renatured and purified by affinity chromatography. Polyclonal antibodies with high specificity were obtained from the serum of rabbit immunized with purified recombinant protein. Mice were used to determine the hypocalcemic effect of the recombinant protein. Results showed that the recombinant protein expressed in E.coli had the proper bioactivity.

Expression and identification of mutated osteoprotegerin in culture cells and larvae of silkworm.

The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.