猪TRIM32分子的克隆、表达及其功能分析

Cloning,Expression and Functional Analysis of Porcine TRIM32

TRIM32作为TRIM家族中的一员,在宿主先天性免疫防御中起着重要的作用。然而,现今对猪TRIM32分子的研究鲜有报道。首先,利用RT-PCR从猪肺中扩增获得了猪TRIM32分子,并将其克隆入真核表达载体p3xFlag-CMV-14,构建重组真核表达载体pFlag-porTRIM32。随后,在Marc145细胞上利用过表达猪TRIM32,鉴定其在PRRSV感染中的作用。此外,为了进一步验证其在PRRSV感染中的作用,在沉默Marc145细胞中内源性TRIM32表达的情况下,检测了PRRSV的感染情况。结果显示:成功构建了pFlag-porTRIM32真核表达载体;Western blot的结果显示,Flag抗体能识别瞬时表达的融合蛋白Flag-porTRIM32;在Marc145细胞上过表达猪TRIM32分子,可显著地抑制PRRSV的复制;相反,在Marc145细胞上干扰TRIM32的表达,可明显增加PRRSV的复制。因此,我们的研究结果初步揭示了猪TRIM32分子具有抗PRRSV感染的作用,为进一步探究猪TRIM32抗HP-PRRSV感染的机制奠定基础。

TRIM32 is a member of TRIM family and plays an important role in host immune defense.However,it is little known about the role of porcine TRIM32 in innate antiviral response.In the present study,we first cloned the porcine TRIM32 gene from pig lung by using RT-PCR,and the recombinant plasmid pFlag-porTRIM32 was constructed by inserting the porcine TRIM32 gene into p3xFlag-CMV-14 vector.Subsequently,effect of porcine TRIM32 on PRRSV infection was determined in Marc145 cell line by the gain-of-function analysis through over expression of pFlag-porTRIM32.Moreover,we further set up the loss-of function analysis through knockdown expression of endogenous TRIM32 in Marc145 cell line using RNA interference.Our results demonstrated that the recombinant plasmid pFlag-porTRIM32 was successfully constructed and anti-Flag antibody recognized recombinant protein Flag-porTRIM32 transiently expressed in 293T cell line in Western blotting.Overexpression of porcine TRIM32 in Marc145 cells inhibited PRRSV infection in comparison to infected-vector-group.In contrast,knock-down expression of endogenous TRIM32 in Marc145 cell line increased virus titters compared with those in infected-NC group.Thus,the results of this study not only revealed that porcine TRIM32 had a role in anti-PRRSV infection but also provided a basis for understanding insights into effect of TRIM32 on anti-PRRSV infection.