利用昆虫细胞表达猪RANKL蛋白及其生物学活性分析

Expression of porcine RANKL protein using insect cells and analysis of its biological activity

将猪RANKL基因克隆至杆状病毒供体质粒pFastBacHTA中,经酶切鉴定及测序筛选出阳性重组供体载体pFastBacHTA-RANKL,将其转化至含有杆状病毒穿梭载体Bacmid的DH10Bac感受态细胞,构建杆状病毒表达载体,获得重组转座子Bacmid-RANKL,在脂质体介导下转染sf9昆虫细胞,获得含RANKL基因的重组杆状病毒。经SDS-PAGE分析、Western-blot分析以及IFA试验,证明RANKL基因在sf9细胞中成功表达。利用表达的RANKL蛋白刺激IPEC-J2细胞,采用qPCR方法检测处理后细胞中M细胞相关标志基因的表达。结果显示,与阴性对照组相比,猪RANKL处理后的细胞中M细胞特异性的标记基因CK-18、Marcksl1、Sgene1、Spib、CCL20、UBD、NCF4、TRAF6表达量都显著上调。这表明本研究表达的猪RANKL具有良好的生物学活性,可成功诱导IPEC-J2细胞向M细胞分化。本研究结果为后续进一步研究猪肠道M细胞的分化,及研制增强猪肠道黏膜免疫应答的M细胞靶向疫苗打下了良好的物质基础。

The porcine RANKL gene was cloned into the baculovirus donor plasmid pFastBacHTA,and the positive recombinant donor vector pFastbachTA-RANKL was identified by restriction enzyme digestion and sequencing.The recombinant vector pFastbachTA-RANKL was transformed into DH10Bac competent cells containing Bacmid to construct baculovirus expression vector.The recombinant baculovirus containing porcine RANKL gene were obtained via sf9 insect cells transfected by recombinant transposon Bacmid-RANKL using Liposome.The identification results of SDS-PAGE,Western-blot and IFA showed that RANKL protein was expressed successfully by recombinant baculovirus in sf9 insect cells.IPEC-J2 cells were treated with expressed RANKL protein,and M cell-associated gene were tested by PCR assay.The results showed that the expression of M cell-specific marker genes CK-18,Marcks11,Sgene1,Spib,CCL20,UBD,NCF4,and TRAF6 were significantly up-regulated in cells treated with porcine RANKL,indicating the expressed porcine RANKL protein has biological activity and could induce IPEC-J2 cells differentiation towards M cells.In conclusion,the successfully expressed porcine RANKL protein provides the maRANterial basis for further study of M cell differentiation in porcine intestinal tract and development of M cell targeted vaccines.