Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide / 生物工程学报

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.

Apoptosis and the expressions of caspase-3 and programmed cell death 5 protein in perihematoma tissue after intracerebral hemorrhage in rats / 国际脑血管病杂志

Objective To observe apoptosis and its relationship between the expression of caspase-3 and programmed cell death 5 (PDCD5) protein in perihematoma tissue after intracerebral hemorrhage in rats and to investigate the injury mechanism after intracerebral hemorrhage. Methods A total of 54 male Wistar rats were randomly divided into sham operation and intracerebral hemorrhage groups, and the latter were redivided into 3,6, 12 hours, days 1,2,3, 5, and 7 subgroups (n = 6 in each group). A model of intracerebral hemorrhage was induced by injecting 50 μL autologous tail artery blood into the caudate nucleus. Apoptosis was detected by TUNEL method. The expressions of Caspase-3 and PDCD5 was observed by immunohistochemistry. Results The apoptotic cells were found in perihematoma tissue of rats at 3 hours,they reached the peak at day 2 to day 3 and reduced gradually after 3 days. Caspose-3 and PDCD5 positive cells were found in perihematoma tissue of rats at 3 hours, they reached peak at day 1 to day 2 and reduced gradually after 3 days. The numbers of Caspase-3 (r =0. 971, P ＜0. 01 ) and PDCD5 (r = -0. 334, P ＜0. 01 ) positive cells were positively correlated with those of apoptotic cells in perihematoma tissue after intracerebral hemorrhage in rats. Conclusions The perihematoma tissue of intracerebral hemorrhage in rats existed apoptosis, and it was consistent with the expressions of Caspase-3 and PDCD5. Caspase-3 and PDCD5 may promote apoptosis in perihematoma tissue after intracerebral hemorrhage.