【摘要】：結核分枝桿菌作為結核病的病原微生物,很早就引起了人們的關注,并對其進行過大量的研究。結核分枝桿菌內存在著數量眾多的轉錄調控因子,組成龐大的基因調控網絡及次級調控網絡,調控著不同基因在不同生理條件下的表達水平,使菌體能夠適應環(huán)境變化而維持正常代謝,從而可能成為藥物設計的靶標。結核分枝桿菌中轉錄調控因子的作用目前研究的還不是很清楚,其中Rv3295蛋白隸屬于TetR轉錄調控家族,TetR家族參與了細菌的新陳代謝,小分子物質的運輸,抗生素的產生等多種生理過程,具有重要調控作用。本研究以結核分枝桿菌H37Rv標準株基因組DNA為模板,通過PCR擴增出683 bp的rv3295基因,經限制性內切酶NdeΙ和XhoΙ雙酶切后,克隆至表達載體pET-22b中,構建pET22b-rv3295重組質粒,并將測序正確的陽性重組質粒轉化到大腸桿菌BL21(DE3)感受態(tài)細胞中。該重組蛋白在終濃度1 mmol/L IPTG于30℃條件下誘導并獲得了可溶性表達。經SDS-PAGE電泳分析呈現出了一個分子量為25 ku的蛋白條帶,與預期蛋白大小相符。并通過親和層析純化,獲得較高純度的Rv3295蛋白,然后經Western-Blot分析驗證,結果表明成功表達了帶有His標簽的重組蛋白。以該重組蛋白為免疫原,免疫BALB/C小鼠,利用細胞融合、克隆和篩選等技術,獲得1株抗Rv3295重組蛋白的單克隆抗體Rv3295-1F7。采用Western blot驗證抗體的特異性,通過亞型鑒定試劑盒鑒定抗體亞型,結果顯示該抗體重鏈為IgG1,輕鏈為κ鏈,可用于后期對Rv3295蛋白功能及應用研究。應用噬菌體隨機肽庫對Rv3295-1F7單抗進行3輪生物淘選,得到25株陽性噬菌體。測序結果表明:6個噬菌體序列為LTIHMDNHGPHR,2個噬菌體序列為SWFGATGVGPHR。這分別與結核分枝桿菌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。噬菌體競爭抑制試驗與ELISA方法證實,篩選出的噬菌體多肽的空間構像和天然抗原表位相似,可用于檢測結核分枝桿菌及相關表位疫苗的研究。
[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，

本文編號：2428476