前半胱天冬酶-1及活性位点突变真核表达载体的构建、表达及生物活性鉴定

发布时间：2018-01-12 17:46

本文关键词：前半胱天冬酶-1及活性位点突变真核表达载体的构建、表达及生物活性鉴定　出处：《军事医学》2013年02期 　论文类型：期刊论文

【摘要】：目的构建和表达前半胱天冬酶(procaspase)-1及其活性位点C285A突变真核表达载体,并检测其生物学活性。方法从人胚肾细胞系HEK293中提取总RNA,逆转录后,PCR获得前半胱天冬酶-1全长DNA,并通过重组PCR获得其酶活性位点突变片段,酶切连接到载体pcDNA3-Flag中,经鉴定将阳性克隆测序;脂质体转染两种载体到HEK293中表达,用免疫印迹检测目的蛋白的表达,并用酶联免疫吸附方法检测在病原菌毒力蛋白刺激下,前半胱天冬酶-1及C285A对IL-1β分泌的影响。结果构建的2种真核表达载体能够在HEK293中很好表达,当前半胱天冬酶-1过表达时,病原菌毒力蛋白可以刺激IL-1β的分泌,而C285A酶活性位点突变之后,IL-1β的分泌明显降低。结论构建重组的pcDNA3-Flag-procaspase-1具有生物学活性,而C285A突变导致前半胱天冬酶1丧失切割底物的功能,且使IL-1β的分泌明显降低。
[Abstract]:Objective to construct and express the eukaryotic expression vector of procaspase1 and its active site C285A. Methods Total RNAs were extracted from human embryonic kidney cell line HEK293, and the first cysteinase-1 full-length DNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The mutant fragment of the enzyme activity site was obtained by recombinant PCR and ligated into the vector pcDNA3-Flag. The positive clones were sequenced. The expression of the target protein was detected by Western blotting, and the virulence protein stimulated by pathogenic bacteria was detected by enzyme-linked immunosorbent assay (Elisa). Results the two eukaryotic expression vectors were well expressed in HEK293, and the current overexpression of C285A and C285A on the secretion of IL-1 尾 was observed. Virulence protein of pathogenic bacteria can stimulate the secretion of IL-1 尾, but after C285A enzyme activity site mutation. Conclusion the recombinant pcDNA3-Flag-procaspase-1 has biological activity. The mutation of C285A resulted in the loss of the substrates function and the decrease of the secretion of IL-1 尾.
【作者单位】：安徽大学生命科学学院;军事医学科学院生物工程研究所;
【基金】：国家自然科学基金资助项目(31170029)
【分类号】：R341;Q55
【正文快照】： 半胱天冬酶(cysteinyl aspartate-specific protea-ses,Caspase)家族是一组存在于细胞质中,活性位点均含半胱氨酸残基,能够特异性地切割靶蛋白的天冬氨酸残基后肽键的蛋白酶。目前发现的哺乳动物细胞caspase家族成员共有15种,其中caspase-1和14主要介导细胞因子的炎症反应[1]

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