Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell tr...Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.展开更多
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
文摘Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.
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。
