目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3'UTR(107)和3CD(1)-3'UTR(107),并克隆入p M D19-T。利用双酶切、T4...目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3'UTR(107)和3CD(1)-3'UTR(107),并克隆入p M D19-T。利用双酶切、T4连接酶将两种重组3CD-3'UTR分别置换入本室构建并保存的EV71的SDLY107株的感染性c DNA克隆p M D19-T-107,得到重组p M D19-T-107后,将其线性化并转染入横纹肌肉瘤(RD)细胞,盲传得到重组病毒107(1-3C)和107(1-3CD)。对收获病毒进行鉴定。结果构建了重组EV71全长c DNA克隆;RNA转染并盲传至第3代,36 h后RD细胞出现细胞病变(CPE),48 h出现明显的CPE,成功拯救重组病毒SDLY107(1-3C)和SDLY107(1-3CD);重组病毒产生的CPE更接近SDLY107。结论成功构建了重组EV71反向遗传系统,拯救了重组病毒SDLY107(1-3C)和SDLY107(1-3CD),为进一步研究3C与3D蛋白在EV71致病机制中的作用提供基础。展开更多
AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver dis...AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hep展开更多
文摘目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3'UTR(107)和3CD(1)-3'UTR(107),并克隆入p M D19-T。利用双酶切、T4连接酶将两种重组3CD-3'UTR分别置换入本室构建并保存的EV71的SDLY107株的感染性c DNA克隆p M D19-T-107,得到重组p M D19-T-107后,将其线性化并转染入横纹肌肉瘤(RD)细胞,盲传得到重组病毒107(1-3C)和107(1-3CD)。对收获病毒进行鉴定。结果构建了重组EV71全长c DNA克隆;RNA转染并盲传至第3代,36 h后RD细胞出现细胞病变(CPE),48 h出现明显的CPE,成功拯救重组病毒SDLY107(1-3C)和SDLY107(1-3CD);重组病毒产生的CPE更接近SDLY107。结论成功构建了重组EV71反向遗传系统,拯救了重组病毒SDLY107(1-3C)和SDLY107(1-3CD),为进一步研究3C与3D蛋白在EV71致病机制中的作用提供基础。
基金the National Natural Science Foundation of China,No.39970719,30170919
文摘AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hep
