Construction of a restriction-endonuclease-Eam1105I-generated T-vector for high-throughput cloning and expression.

A novel T-vector was constructed that could be used for direct cloning and expression of PCR-amplified cDNA. The technique was based on the insertion into the parent vector of two endonuclease-Eam1105I restriction sequences spaced by an expression cassette of the full-length beta-galactosidase, which helped to improve cloning efficiency and to minimize the non-recombinant background of the T-vector when used to clone PCR products. Moreover, this method took advantage of the reconstitution of the rarest restriction sequence of MssI to enable directional cloning. These advantages make the T-vector suitable for high-throughput expression and analysis.

[Effects of various iodin-nutritional on activity of T4 5'-and 5-deiodinase in rat brain].

OBJECTIVE: To investigate the changing of T4 5'-and 5-deiodinase within rat brain under various iodin-nutritional states. METHODS: Animal model of iodine-deficiency rat was performed and the rats were divided into 4 groups by the intake of iodine-nutrition, and then killed at an age of 20 days. The thyroid hormones level in serum was measured by ELISA and the activity of T(4) 5'-and 5-deiodinase within brain was analyzed. RESULTS: In less-iodine (LI) group,TT4 and FT4 were accounting for 3.5% of the neutral-iodine (NI) group's, and FT3 was 174.0% of NI group's (P < 0.05). In NI group,TT4 and FT4 were 114.5% and 127.7% of NI group's (P < 0.05). In high-iodine (HI) group, TT4 and FT4 were 61.86% and 62.0% of NI group's, and FT3 was 184.9% of NI group's (P < 0.05). In LI group, the activity of T4 5'-deiodinase tissue of per gram (1.95 +/- 0.32) ngT3.microgT4(-1).h was significantly higher than that of NI group (P < 0.05), and the activity of 5-deiodinase (1.38 +/- 0.21) ngrT3.microg T4(-1).h(-1) is significantly less than that of NI group (1.59 +/- 0.23) (P < 0.05). In HI group the activity of T4 5'-and 5-deiodinase tissue of per gram (1.12 +/- 0.19 and 1.73 +/- 0.36) ngrT3.microgT4(-1).h(-1)was significantly less than that of NI group (P < 0.05). CONCLUSION: The activity of T4 5'-deiodinase in iodine deficiency heightens and that in iodine excess is debased, the activity of T4 5-deiodinase in iodine deficiency and in iodine excess is debased.

[Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli].

GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl beta-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.

pEGFP-T, a novel T-vector for the direct, unidirectional cloning and analysis of PCR-amplified promoters.

A novel T-vector, designated as pEGFP-T, was constructed which could be used for direct and unidirectional cloning and analysis of promoters. The method involves minimizing the non-recombinant background of the T-vector when used to clone PCR products, and maintaining only the forward orientation of the PCR products prior to transformation. The usefulness of this vector is demonstrated in cloning and analyzing promoters.