ABSTRACT
Background Studies showed that activation of microglia-derived nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the neurodegenerative diseases and neural cell death in central nervous system.The effect of NADPH on cone degeneration have been determined in rd rats,its role in rod degeneration is relatively less studied.Objective This study was to study the expression of NADPH oxidase in the retinal degenerative process in rd mice and further explore its role in the photoreceptor degeneration.Methods rd Mice at postnatal day 8 (P8),P10,P12,P14,P16 and P18 were collected.The mice were sacrificed,and retinal sections,RNAs and proteins were prepared in above-mentioned time points.The expressions of the gp91 phox,a major subunit of NADPH oxidase,in transcript level and protein level in the retinas were semi-quantitatively detected by real-time PCR and Western blot respectively.Expression of gp91phox was localized in the rd retinas as ageing by immunohistochemstry,and the co-expression of gp91phox with CD11b,a specific marker of microglial cells,was assayed by immunofluorescent double labeling.The C57BL/6N mice were served as controls.The use and care of the animals complied with the Guideline of ARVO.Results Real-time PCR showed that gp91phox mRNA was not expressed in the retinas of C57BL/6N mice.Gp91phox mRNA was found to have less expressed in retinas of P8 rd mice.With aging,the expression level of gp91phox mRNA (gp91phox mRNA/β-actin) in rd mouse retinas was gradually increased with the highest level in P14 mice(1.136±0.370).A significant difference was seen in the gp91 phox mRNA expression among various groups of mice (F=17.81,P =0.00),and gp91phox mRNA expression was significantly elevated in P10,P12,P14,P16 and P18 rd mice compared with P8 rd mice(all at P＜0.05).The expression level of gp91phox protein (A value) in the retinas presented with a similar trend in the rd mice,with a significant difference among the various ages of rd mice and C57BL/6N mice (F =354.00,P＜0.01).The expression level of gp91 phox protein was increased in the rd mice in comparison with the C57BL/6N mice (all at P＜0.05).Immunochemistry revealed that the positive response cells for gp91phox increased in the inner layers of retinas in P10 rd mice and peaked in P14 mice.Immunofluorescent double labeling exhibited that gp91phox were seen to present a co-expression with CD11b,showing an orange fluorescence.Conclusions Expression of NADPH oxidase in the rctinas in the rd mice up-regulates and is parallels to the microglial activation and photoreceptor degeneration,suggesting that NADPH oxidase plays a role in the retinal dystrophy associated with microglial activation.
ABSTRACT
Objective To express the recombinant mouse histidyl-tRNA synthetase (HARS) and maltose binding protein (MBP) gene in Escherichia coli and obtain the purified protein which possesses antigen specificity.Methods Total RNA was extracted from the myocytes of C57BL/6 mouse and reversely transcripted to cDNA.The gene of N-terminal origin of 591 base pairs was amplified,then cloned into pMALc-5e vector.The recombinant plasmid was transformed into Rosetta-gami B,then IPTG was used to induce the expression of HARS-MBP.Fusion protein was purified by affinity chromatograph.The molecular weight (MW) of HARS-MBP was roughly determined by SDS-PAGE.The antigen specificity was identified by Western blotting using anti-Jo-1 serum from patients,commercial anti-HARS and anti-MBP antibodies.Results The recombinant HARS-MBP protein gene was efficiently expressed in Escherichia coli,and the MW was consistent with predicted MW of 66 000.The fusion protein was specifically combined with its antibody.Conclusion The HARS-MBP fusion protein could be efficiently and steadily synthesized in Escherichia coli,which shows satisfactory antigen specificity and provides the key requirement for making a deep study of HARS in the pathogenesis of idiopathic inflammatory myopathy(IIM) and animal modeling of IIM.
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Objective: To develop a therapeutic adjuvantfree protein vaccine against HPV16 which is closely related to cervical cancer of China. Method: First the E6/E7 gene by PCR technology from HPV16z virus strain was isolated in the highrisk cervical cancer area of Shanxi province of China in1990s, and again got the gene segment of Hsp65 from BCG by the same method, mutated the transforming codes in sequences of HPV 16 E6/E7 genes and thus constructed the expression vector pET28aHsp65E6/E7, expressed the Hsp65E6/E7 fusion protein in E.coli BL21(DE3) strain and researched optimal protein purification procedures. Results: The expression vector pET28aHsp65E6/E7 was constructed successfully and E6/E7 gene was mutated correctly. Hsp65E6/E7 fusion protein was renatured and purified on the affinity chromatography column simultaneously. The protein purity achieved 95% after the anionic exchange chromatograph purification. conclusions: This research laid a foundation for further functional study of the therapeutic adjuvantfree protein vaccine——Hsp65E6/E7.
ABSTRACT
Objective: To study the biological effects of the HPV16Z-Hsp65-E6/E7 fusion protein vaccine on the tumor associated with HPV16 infection. Methods: We tested the cellular immune responsive intensity to the vaccine by the lymphocyte proliferation and CTL response, and studied the therapeutic effect of the vaccine on mouse TC-1 cell transplanted cancer in vivo and the influence on mouse lifetime. Results: The spleen lymphocytes from the C57BL/6 mouse immunized by the Hsp65-E6/E7 vaccine could proliferate obviously in the presence of the protein and TC-1 tumor cell could be lysed specifically by immune activated lymphocytes in vitro. This animal therapeutic experiment in vivo showed that the vaccine suppressed the growth of TC-1 cell transplanted tumor remarkably. Conclusion: The recombined vaccine can induce specific cellular immune response in vitro and suppress HPV16 positive TC-1 tumor cell growth obviously in vivo.