池蝶蚌CyPA的应激表达及对Hela细胞生长的影响

THE EXPRESSION OF CYCLOPHILIN A(Cy PA) IN HYRIOPSIS SCHLEGELII IN RESPONSE TO PATHOGENS AND ITS EFFECT ON THE GROWTH OF HELA CELLS

研究分析了池蝶蚌(Hyriopsis schlegelii)亲环蛋白A(Cyclophilin A,Cy PA)诱导的应激反应和对Hela细胞生长的影响。采用嗜水气单胞杆菌诱导刺激池蝶蚌,并利用Real-time PCR技术对其肝脏、性腺和血淋巴中Cy PA基因m RNA的表达量进行分析,应激实验表明,在嗜水气单胞杆菌刺激后血淋巴、肝脏和性腺中的Cy PA在诱导4h出现一个表达峰,8h后开始下降,说明池蝶蚌Hs Cy PA基因参与免疫防御应答反应;利用p ET-32a(+)表达载体,根据Hs Cy PA基因c DNA的序列,构建了含有完整开放阅读框(ORF)的表达载体并在大肠杆菌BL21(DE3)中进行原核表达,优化诱导条件后,通过亲和层析获得了含量较高的上清可溶性蛋白;采用MTT法,将原核表达的重组Cy PA蛋白稀释到相应(50、100、200、400和800 ng/m L)浓度,刺激Hela细胞系后发现,重组池蝶蚌Cy PA蛋白能促进Hela细胞生长,刺激浓度为100 ng/m L时,达到最大增殖率18.5%。结果初步表明,池蝶蚌Cy PA是一个既参与免疫应激又对细胞的生长发育具有影响的功能广泛的蛋白质。

In this study we explored the expression of Hyriopsis schlegelii Cyclophilin A in response to infection and the effect of this protein on the growth of Hela cells. The m RNA level of this gene in the haemocytes, liver and gonad was measured with Real-time PCR after the Aeromonas hydrophila challenge. It was observed that the transcription of Hs Cy PA in haemocytes, gonad and liver was significantly up-regulated at 4h after the challenge and then decreased at 8h. These results suggested that Hs Cy PA might be involved in the immune response. The complete ORF of Hs Cy PA gene was subcloned into p ET-32a（＋） prokaryotic expression vector, and then expressed in E. coli BL21（DE3）. After the optimization of induction conditions, the soluble fusion protein was purified with Ni-NTA affinity chromatography. To test the effect of this recombinant protein on Hela cells, we diluted it into certain concentrations（50, 100, 200, 400, 800 ng/m L） and applied them into Hela cell strains through MTT assay. It was revealed that the recombinant Hs Cy PA could promote the proliferation of Hela cells, and the proliferation rate reached the maximum（18.5%） when the Hs Cy PA concentration was 100 ng/m L. These preliminary results indicated that Cyclophilin A could be a multi-functional protein which might participate in immune responses and cell growth regulation.