Objective To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot test. Results The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH Ⅰ and Hind Ⅲ), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2×103 and 30.3×103 respectively. The results of Westernblot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.
Objective To observe the effects of δ-opioid receptor agonists D-Ala2-D-Leu5-enkephali （DADLE） on hepatocyte apoptosis and expressions of bcl-2 and caspase-3 in septic rat, and to investigate the possible mechanism by which DADLE protects the liver in sepsis. Methods Sepsis was reproduced in rats by cecum ligation and puncture (CLP). Fifty-four SD rats (either male or female) were randomly divided into CLP group (n=18), DADLE group (n=18) and sham operation (SO) group (n=18). The rats were respectively killed at different time (2 h, 4 h and 6 h after operation). Hepatocyte apoptosis was detected by TdT-mediated dUTP Nick End Labeling (TUNEL). The expressions of bcl-2 and caspase-3 protein were detected by immunohistochemistry. And the changes of pathology in hepatic tissue were detected by light microscope. Results The hepatic pathological lesion of rats in CLP group was obviously serious compared with SO group, while it was obviously improved in DADLE group. The apoptosis index of rat hepatocytes in CLP group significantly increased compared with SO group, and further it was prominent at 4 h （P＜0.01). The apoptosis index of rat hepatocytes at each time of DADLE group was significantly decreased compared with CLP group （P＜0.01）. Expression of caspase-3 protein in liver tissues of CLP group significantly increased compared with SO group （P＜0.01）, while the expression of bcl-2 protein significantly decreased （P＜0.05）. Expression of caspase-3 protein in liver tissues of DADLE group significantly decreased compared with the CLP group （P＜0.01）, while the expression of bcl-2 protein significantly increased （P＜0.05）. There was positive correlation between expression of caspase-3 in liver tissues and apoptosis index of hepatocyte (r=0.83, P＜0.01) and negative correlation between expression of bcl-2 in liver tissues and apoptosis index of hepatocyte (r=-0.65, P＜0.01). Conclusions The findings indicate that δ-opioid receptor agonists DADLE can obviously improve hepatic pathological changes of septic rats. And its protective mechanism contains down regulation of caspase-3 expression, upregulation of bcl-2 expression and thus the apoptosis of hepatocyte is repressed.