小鼠β防御素4体外对人呼吸道合胞病毒抑制作用研究

Research on efficient inhibition of human respiratory syncytial virus with murine β-defensin 4 in vitro

目的观察小鼠β防御素4体外对人呼吸道合胞病毒的抑制作用。方法将小鼠β防御素4基因克隆到pET32a载体中并导入大肠埃希菌表达,获得的小鼠β防御素4融合蛋白,通过镍亲和层析法纯化和离子交换层析法复性,分别以半数组织感染剂量法和荧光定量PCR法分别测定小鼠β防御素4和阳性对照利巴韦林对人呼吸道合胞病毒的半数抑制浓度,以噻唑蓝比色法检测小鼠β防御素4和利巴韦林对细胞的半数毒性浓度,依据半数毒性浓度与半数抑制浓度的比值计算小鼠β防御素4和利巴韦林对人呼吸道合胞病毒的治疗指数(TI)。同时检测不同浓度小鼠β防御素4对人呼吸道合胞病毒感染细胞的保护程度。结果成功构建pET32a-Trx-His-β-D-4重组质粒,转化BL21后诱导表达融合小鼠β防御素4蛋白。以半数组织感染剂量法和荧光定量PCR法分别测定纯化的小鼠β防御素4对人呼吸道合胞病毒的TI值分别为189和152,而利巴韦林的TI值分别为87和85,且小鼠β防御素4对人呼吸道合胞病毒的抑制作用呈量效关系。结论体外实验表明,融合小鼠β防御素4能高效抑制人呼吸道合胞病毒感染,其效果优于利巴韦林。

Objective To investigate the inhibition of human respiratory syncytial virus with murineβdefensin 4 in vitro. Methods cDNA of murineβdefensin 4 was cloned into pET32 aand expressed in Escherichia coli BL21.A murineβdefensin 4-thioredoxin fusion protein was obtained and an His-tag was attached.This protein with enterokinase recognizing sites was expressed.After purification with nickel affinity chromatography and ion exchange chromatography,murineβdefensin 4 was recovered from the fusion protein and its effect against human respiratory syncytial virus was evaluated with TCID50 and Q-RT-PCR assays in a HEp-2 cell line.In addition,the IC50 values for murineβdefensin 4 and ribavirin（positive control）with respect to human respiratory syncytial virus and the TC50 values murineβdefensin 4 and ribavirin in HEp-2 cells were calibrated with a TCID50 assay/Q-RT-PCR assay and MTT assay in HEp-2 cells.The therapeutic index（TI）was then calculated.Various concentrations of murineβdefensin 4 were used to detect its effect against infection with human respiratory syncytial virus in HEp-2 cells. Results The TIs（IC50/TC50）of murineβdefensin 4 and ribavirin with respect to human respiratory syncytial virus,which were separately determined from the TCID50 assay and Q-RT-PCR assay,were 189 and 152 for murineβdefensin 4,and 87 and 85 for Ribavirin（the former was deduced from the result of TCID50 assay,and the latter was deduced from the result of Q-RT-PCR assay）.An assay of action against human respiratory syncytial virus revealed a dose-dependent effect for murineβdefensin 4 and ribavirin. Conclusion Murineβdefensin 4efficiently inhibits infection with human respiratory syncytial virus in an in vitro assay.