Naguib Mohamed M, Mohamed Mohamed R, M Ali Mohamed A, Karim Amr M
Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.
Indian J Pathol Microbiol. 2019 Jul-Sep;62(3):391-398. doi: 10.4103/IJPM.IJPM_774_18.
Hepatitis C virus (HCV) represents a serious worldwide healthcare problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt, and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds against NS3/4A protease from HCV genotype 4a.
Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into β-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by coexpression of β-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of β-galactosidase was colorimetrically estimated in the cell lysate using orthonitro phenyl β-D-galactopyanoside (ONPG) as a substrate.
We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/nonfunctional β-galactosidase enzyme.
丙型肝炎病毒（HCV）是全球范围内严重的医疗保健问题。由于HCV具有快速突变的特性，能够使病毒逃避中和抗体，因此尚未开发出针对HCV的保护性疫苗。对HCV的了解最初因无法在细胞培养中实现病毒复制而受到阻碍。鉴于其在病毒多聚蛋白加工和免疫逃逸中的重要作用，HCV NS3/4A蛋白酶是抗病毒化疗的主要靶点。我们旨在建立一种基于体内细胞的检测系统，用于监测来自埃及和中东地区主要基因型HCV 4a型的NS3/4A蛋白酶的活性。此外，所开发的系统用于评估一系列计算机设计的化学合成化合物对HCV 4a型NS3/4A蛋白酶的抑制效力。
将NS3/4A蛋白酶的天然及突变切割位点框内克隆到TA克隆载体的β-半乳糖苷酶基因中。通过在细菌细胞中共表达含有蛋白酶切割位点的β-半乳糖苷酶与NS3/4A蛋白酶构建体来评估HCV NS3/4A的靶标特异性。使用邻硝基苯基β-D-吡喃半乳糖苷（ONPG）作为底物，比色法测定细胞裂解物中β-半乳糖苷酶的活性。
我们成功开发了一种基于细菌细胞蓝/白筛选的高效细胞检测系统，该系统能够表达功能性/非功能性β-半乳糖苷酶。
