猪TNF-α高效表达及其多克隆抗体的制备

Porcine TNF-α high level expression and preparation of its polyclonal antibody

【目的】明确猪源肿瘤坏死因子α(TNF-α)多克隆抗体的特异性和反应性,并探索猪瘟病毒(CSFV)感染对PK-15细胞分泌TNF-α的影响,为揭示CSFV的致病机理打下基础。【方法】提取CSFV病料基因组RNA,通过RT-PCR扩增TNF-α基因,构建原核表达载体pGEX-4T-1-TNF-α并转化BL21感受态细胞诱导表达融合蛋白,经纯化和浓缩后免疫SPF级昆明小鼠制备TNF-α多克隆抗体;同时构建真核表达载体pcDNA3.0-TNF-α,分别转染HEK-293T细胞和PK-15细胞表达融合蛋白,通过Western blotting、ELISA和间接免疫荧光分析等方法检测TNF-α多克隆抗体效价、反应性及特异性。【结果】以原核表达载体pGEX-4T-1-TNF-α转化BL21感受态细胞,经IPTG诱导后能表达出约43 kD的融合蛋白,且主要以包涵体形式进行表达。以真核表达载体pcDNA3.0-TNF-α转染HEK-293T细胞可表达出25 kD的融合蛋白,主要在细胞质中表达,且均匀分布。制备获得的TNF-α多克隆抗体能与转染HEK-293T细胞表达的融合蛋白TNF-α及PK-15细胞的内源蛋白TNF-α发生良好反应,即具有较好的反应特异性,其抗体效价高达1∶8000。CSFV能刺激PK-15细胞分泌蛋白TNF-α上调表达,且TNF-α与其下游因子(TRAF1)的表达变化趋势基本一致,即二者间存在一定关联性。【结论】制备获得的TNF-α蛋白抗体具有效价高、反应性好及特异性强的特点,可用于检测CSFV感染后真核细胞中过表达的TNF-α水平。TNF-α可刺激TRAF1产生,参与TRAF1相关信号通路而发挥其生物学功能,CSFV感染PK-15细胞后TNF-α和TRAF1的表达变化趋势基本一致,说明CSFV能刺激TNF-α和TRAF1信号通路,使机体产生炎症反应。

【Objective】To determine the specificity and reactivity of porcine tumor necrosisfactor α(TNF-α)polyclonal antibody,and the effects of CSFV infection on TNF-α secretion of PK-15 cells were investigated,to lay a foundation for revealing the pathogenic mechanism of classical swine fever virus(CSFV).【Method】The TNF-α gene was amplified by RT-PCR using RNA template extracted from CSFV infected PK-15 cells,to construct prokaryotic expression vector pGEX-4 T-1-TNF-α and transform BL21 competent cells to induce the expression of fusion protein.Purified and concentrated SPF level Kunming mice were immunized with TNF-α polyclonal antibody.At the same time,eukaryotic expression vector pcDNA3.0-TNF-α was constructed and transfected into HEK-293 T cell and PK-15 cell to express TNF-α.Titer,reactivity and specificity of TNF-α polyclonal antibody was detected by Western blotting,ELISA andindirect immunofluorescence methods.【Result】Prokaryotic expression vector pGEX-4 T-1-TNF-α transformed BL21 competent cells could express about 43 kD fusion protein induced by IPTG,and it was mainly expressed in the form of inclusion body.The eukaryotic expression vector pcDNA3.0-TNF-α transfected HEK-293 T cells could express 25 kD fusion protein,which was mainly expressed in cytoplasm and evenly distributed.The prepared TNF-α polyclonal antibody could react well with the fusion protein TNF-α expressed in HEK-293 T cells and the endogenous protein TNF-α in PK-15 cells,and had a good reaction specificity and the antibody titer was as high as 1∶8000.CSFV could up-regulate the expression of TNF-α secreted by PK-15 cells.The expression trend of TNF-α and its downstream factor(TRAF1)was basically consistent,therewas certain correlation between them.【Conclusion】The TNF-α protein antibody has the characteristics of high effective valence,good reactivity and strong specificity,it can be used to detect the overexpression of TNF-α level in eukaryotic cells after CSFV infection.TNF-α can stimulate TRAF1 production and participate in TRAF1-related signaling pathway to play its biological function.The expression of TNF-α and TRAF1 is similar after CSFV infected PK-15 cells.These results suggest that CSFV can stimulate TNF-α and TRAF1 signaling pathways,and leads to the inflammatory reaction.