结核分枝杆菌Rv3295蛋白的单克隆抗体制备及抗原表位鉴定
发布时间：2019-02-22 18:10
【摘要】：结核分枝杆菌作为结核病的病原微生物,很早就引起了人们的关注,并对其进行过大量的研究。结核分枝杆菌内存在着数量众多的转录调控因子,组成庞大的基因调控网络及次级调控网络,调控着不同基因在不同生理条件下的表达水平,使菌体能够适应环境变化而维持正常代谢,从而可能成为药物设计的靶标。结核分枝杆菌中转录调控因子的作用目前研究的还不是很清楚,其中Rv3295蛋白隶属于TetR转录调控家族,TetR家族参与了细菌的新陈代谢,小分子物质的运输,抗生素的产生等多种生理过程,具有重要调控作用。本研究以结核分枝杆菌H37Rv标准株基因组DNA为模板,通过PCR扩增出683 bp的rv3295基因,经限制性内切酶NdeΙ和XhoΙ双酶切后,克隆至表达载体pET-22b中,构建pET22b-rv3295重组质粒,并将测序正确的阳性重组质粒转化到大肠杆菌BL21(DE3)感受态细胞中。该重组蛋白在终浓度1 mmol/L IPTG于30℃条件下诱导并获得了可溶性表达。经SDS-PAGE电泳分析呈现出了一个分子量为25 ku的蛋白条带,与预期蛋白大小相符。并通过亲和层析纯化,获得较高纯度的Rv3295蛋白,然后经Western-Blot分析验证,结果表明成功表达了带有His标签的重组蛋白。以该重组蛋白为免疫原,免疫BALB/C小鼠,利用细胞融合、克隆和筛选等技术,获得1株抗Rv3295重组蛋白的单克隆抗体Rv3295-1F7。采用Western blot验证抗体的特异性,通过亚型鉴定试剂盒鉴定抗体亚型,结果显示该抗体重链为IgG1,轻链为κ链,可用于后期对Rv3295蛋白功能及应用研究。应用噬菌体随机肽库对Rv3295-1F7单抗进行3轮生物淘选,得到25株阳性噬菌体。测序结果表明:6个噬菌体序列为LTIHMDNHGPHR,2个噬菌体序列为SWFGATGVGPHR。这分别与结核分枝杆菌Rv3295蛋白的氨基酸序列18-29位和107-119位氨基酸有一定的相似度。噬菌体竞争抑制试验与ELISA方法证实,筛选出的噬菌体多肽的空间构像和天然抗原表位相似,可用于检测结核分枝杆菌及相关表位疫苗的研究。
[Abstract]:As the pathogenic microorganism of tuberculosis, Mycobacterium tuberculosis has attracted people's attention for a long time and has been studied extensively. There are a large number of transcription regulators in Mycobacterium tuberculosis, which form a large gene regulatory network and secondary regulatory network, and regulate the expression level of different genes under different physiological conditions. Bacteria can adapt to environmental changes and maintain normal metabolism, which may become the target of drug design. The role of transcriptional regulators in Mycobacterium tuberculosis is not well understood. The Rv3295 protein belongs to the TetR transcriptional regulatory family, and the TetR family is involved in the metabolism of bacteria and the transport of small molecules. The production of antibiotics and other physiological processes play an important regulatory role. In this study, the genomic DNA of Mycobacterium tuberculosis (H37Rv) standard strain was used as template, and the rv3295 gene of 683 bp was amplified by PCR. The rv3295 gene was digested by restriction endonuclease Nde I and Xho I, and cloned into the expression vector pET-22b to construct the pET22b-rv3295 recombinant plasmid. The positive recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The recombinant protein was induced and expressed at 30 鈩，
本文编号：2428476
[Abstract]:As the pathogenic microorganism of tuberculosis, Mycobacterium tuberculosis has attracted people's attention for a long time and has been studied extensively. There are a large number of transcription regulators in Mycobacterium tuberculosis, which form a large gene regulatory network and secondary regulatory network, and regulate the expression level of different genes under different physiological conditions. Bacteria can adapt to environmental changes and maintain normal metabolism, which may become the target of drug design. The role of transcriptional regulators in Mycobacterium tuberculosis is not well understood. The Rv3295 protein belongs to the TetR transcriptional regulatory family, and the TetR family is involved in the metabolism of bacteria and the transport of small molecules. The production of antibiotics and other physiological processes play an important regulatory role. In this study, the genomic DNA of Mycobacterium tuberculosis (H37Rv) standard strain was used as template, and the rv3295 gene of 683 bp was amplified by PCR. The rv3295 gene was digested by restriction endonuclease Nde I and Xho I, and cloned into the expression vector pET-22b to construct the pET22b-rv3295 recombinant plasmid. The positive recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The recombinant protein was induced and expressed at 30 鈩，
本文编号：2428476
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