Ashayeri-Panah Mitra, Eftekhar Fereshteh, Kazemi Bahram, Joseph Joan
Department of Microbiology, Faculty of Biological Sciences and Technology, Shahid Beheshti University, Tehran, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Microbiol. 2017 Apr;9(2):64-73.
Rv1733c is a latency antigen from , a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in for purification.
Chemically synthesized coding sequence was cloned in pET-23a(+) followed by transforming BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting.
Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K HPO , 17 mM KH PO and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient's serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in which was successfuly purified.
We believe that the purified Rv1733c recombinant protein from might be a good candidate for vaccine production against tuberculosis.
Rv1733c是来自结核分枝杆菌的一种潜伏抗原，可能是一种具有混杂T细胞和B细胞表位的整合膜蛋白，这使其成为抗结核潜在疫苗候选物。本研究旨在克隆并优化重组Rv1733c在大肠杆菌中的表达以进行纯化。
化学合成的编码序列克隆至pET-23a(+)中，随后转化大肠杆菌BL21(DE3)细胞。为评估优化表达的诱导条件，采用实验的析因设计，使用四种不同培养基以及异丙基-β-D-硫代半乳糖苷（IPTG）浓度的四个水平和诱导持续时间。然后使用His标签纯化试剂盒纯化重组蛋白，并通过蛋白质印迹法进行检测。
重组Rv1733c（> 24 kDa）在大肠杆菌细胞的细胞质中表达并积累。培养基组成对重组蛋白产量影响最为显著（P = 0.000）。在37°C孵育10小时后，于 terrific肉汤培养基（含有1.2%胰蛋白胨、2.4%酵母提取物、72 mM KH₂PO₄、17 mM KH₂PO₄和0.4%甘油）中，0.4 mM IPTG存在的情况下，重组Rv1733c产量最高。在此条件下，表达水平约为0.5 g/L培养基。纯化的Rv1733c通过抗多组氨酸抗体和一名结核病患者的血清进行检测。诱导条件的系统优化使我们在大肠杆菌中获得了高产量的重组多组氨酸标签的Rv1733c，且成功进行了纯化。
我们认为从大肠杆菌中纯化得到的Rv1733c重组蛋白可能是抗结核疫苗生产的良好候选物。
