ABSTRACT
ObjectiveProteomics was used to investigate the protein differences between porcine cardiac blood（PCB） and porcine blood（PB） from Menghe medical school and to compare the effects of both on the microglial inflammation of Salviae Miltiorrhizae Radix et Rhizoma（DS）. MethodNanoliquid chromatography-quadrupole orbitrap mass spectrometry（nLC-MS/MS） and bioinformatics were utilized to compare the proteomic differences of PCB and PB in simulated gastrointestinal digestion. Furthermore， Western blot was used to verify the contents of some shared proteins and differential proteins identified in PCB and PB. In addition， BV2 neuroinflammation model constructed by corticosterone（CORT） and lipopolysaccharide（LPS） was applied to detect the intervention effects of PCB and PB on the levels of tumor necrosis factor（TNF）-α and interleukin（IL）-6 in BV2 inflammatory cells of DS. ResultA total of 69 common proteins and 68 differential proteins were identified in PCB and PB， among which the common proteins included transferrin（Tf） with brain-targeting effect， and the differential proteins in the two were 41 and 27， respectively. Western blot validation showed that the difference in the content of the same protein Tf between PCB and PB was not statistically significant， while the difference in the contents of the specific proteins of creatine kinase M and heart-type fatty acid binding protein（H-FABP） were statistically significant（P<0.05）. Moreover， in vitro experimental studies revealed that compared with the same concentration of DS group， in addition to the 100 mg·L-1 PB-DS group， PCB-DS and PB-DS groups could significantly inhibit the levels of TNF-α and IL-6 in BV2 inflammatory cells（P<0.05， P<0.01）， and PCB-DS group had more significant anti-inflammatory effect than PB-DS group with the same concentration（P<0.05， P<0.01）. ConclusionBoth of PCB and PB can enhance the inhibitory effect of DS on the release of inflammatory factors， thus playing a neuroprotective role， and PCB promotes DS inhibition more significantly， which may be due to the existence of the two involved in energy metabolism-related differential proteins， which can lay a foundation for revealing the scientific connotation of the processing of PCB-DS and PB-DS.
ABSTRACT
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .