Safarnezhad Tameshkel Fahimeh, Rahimi Pooneh, Khataminejad Mohammad Reza
Department of Biology, Islamic Azad University, Tonekabon branch, Tonekabon, IR Iran.
Department of Hepatitis and AIDS, Pasteur Institute of Iran.
Iran J Microbiol. 2015 Aug;7(4):221-5.
Hepatitis C virus (HCV) chronically infects around 200 million people worldwide and frequently causes liver cirrhosis and hepatocellular carcinoma. Rapid detection of this virus results in decreasing the distance between infection and initiation the anti-viral treatment, and may prevent most of the undesirable consequences. The new detected HCV protein "Core+1" made from the ribosomal frame shift in Core region is an important candidate for diagnostic tools. This study was conducted to design a recombinant Bacmid plasmid expressing the HCV 1a Core+1 sequence in the Baculovirus expression system for further diagnostic applications.
The HCV Core +1 gene was amplified by PCR using the pcDNA-HAF recombinant vector that contained the Core+1 sequence from HCV genotype 1a as a template, and the specific primers with 2 restriction sites for Nco I and Xba I restriction enzymes. The PCR product was cloned in XbaI/NcoI restriction sites of the linearized pFastBac-HTB vector and evaluated by using those restriction enzymes and sequencing. Then the recombinant pFastBac-HTB vector was transformed in DH10Bac and the result was screened and confirmed by X-Gal discrimination and PCR.
The HCV 1a Core+1 was successfully amplified and the PCR product was confirmed by using the related restriction enzymes and sequencing. Cloning of pFastBac vector with the purified PCR product of HCV Core+1 was confirmed. Finally, the recombinant Bacmid was successfully transformed in DH10Bac.
The recombinant Bac-Core+1 expression vector is considered as an important tool to transfect the sf9 cell line and expression the Core+1 protein.
丙型肝炎病毒（HCV）在全球约2亿人中长期感染，并经常导致肝硬化和肝细胞癌。快速检测这种病毒可缩短感染与开始抗病毒治疗之间的时间间隔，并可能预防大多数不良后果。新检测到的由核心区域核糖体移码产生的HCV蛋白“Core+1”是诊断工具的重要候选物。本研究旨在设计一种重组杆粒质粒，在杆状病毒表达系统中表达HCV 1a Core+1序列，用于进一步的诊断应用。
以含有HCV 1a基因型Core+1序列的pcDNA-HAF重组载体为模板，通过PCR扩增HCV Core +1基因，并使用带有Nco I和Xba I两种限制性内切酶的2个限制性位点的特异性引物。将PCR产物克隆到线性化的pFastBac-HTB载体的XbaI/NcoI限制性位点，并使用这些限制性内切酶和测序进行评估。然后将重组pFastBac-HTB载体转化到DH10Bac中，并通过X-Gal鉴别和PCR筛选并确认结果。
成功扩增出HCV 1a Core+1，PCR产物经相关限制性内切酶和测序确认。用纯化的HCV Core+1 PCR产物克隆pFastBac载体得到确认。最后，重组杆粒成功转化到DH10Bac中。
重组Bac-Core+1表达载体被认为是转染sf9细胞系并表达Core+1蛋白的重要工具。
