Khademi Farzad, Yousefi-Avarvand Arshid, Derakhshan Mohammad, Meshkat Zahra, Tafaghodi Mohsen, Ghazvini Kiarash, Aryan Ehsan, Sankian Mojtaba
Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
Rep Biochem Mol Biol. 2017 Oct;6(1):15-21.
The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of in a prokaryotic system.
An gene construct was synthesized and ligated into a pGH plasmid, TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting.
The gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein.
An HspX/EsxS fusion protein was expressed in and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.
本研究的目的是在原核系统中克隆、表达和纯化一种新型多结构域融合蛋白。
合成一个基因构建体并将其连接到pGH质粒中，转化TOP10细胞，然后纯化载体。用相同的酶消化含有构建体的载体和pET-21b(+)质粒，并将构建体连接到pET-21b(+)中。通过菌落PCR和测序确认克隆的准确性。用pET-21b(+)/hspX/esxS表达载体转化BL21细胞并评估蛋白表达。最后，在镍-亚氨基二乙酸（Ni-IDA）柱上纯化表达的融合蛋白，并通过SDS-PAGE和蛋白质印迹法进行验证。
将基因构建体插入pET-21b(+)中，并用异丙基-β-D-硫代半乳糖苷（IPTG）在BL21细胞中诱导重组蛋白表达。测试了不同浓度的IPTG以确定表达诱导的最佳浓度。重组蛋白在不溶性包涵体中表达。使用3摩尔盐酸胍溶解不溶性蛋白。
在中表达了一种HspX/EsxS融合蛋白并纯化了重组蛋白。经过免疫学分析，HspX/EsxS融合蛋白可能是未来临床试验研究中的一种抗结核疫苗候选物。
