Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, Pakistan.
Virol J. 2011 Jul 26;8:364. doi: 10.1186/1743-422X-8-364.
Pakistan is facing a threat from hepatitis C infection which is increasing at an alarming rate throughout the country. More specific and sensitive screening assays are needed to timely and correctly diagnose this infection.
After RNA extraction from specimen (HCV-3a), cDNA was synthesized that was used to amplify full length core gene of HCV 3a. After verification through PCR, DNA sequencing and BLAST, a properly oriented positive recombinant plasmid for core gene was digested with proper restriction enzymes to release the target gene which was then inserted downstream of GST encoding DNA in the same open reading frame at proper restriction sites in multiple cloning site of pGEX4t2 expression vector. Recombinant expression vector for each gene was transformed in E. coli BL21 (DE3) and induced with IPTG for recombinant fusion protein production that was then purified through affinity chromatography. Western blot and Enzyme Linked Immunosorbant Assay (ELISA) were used to detect immuno-reactivity of the recombinant protein.
The HCV core antigen produced in prokaryotic expression system was reactive and used to develop a screening assay. After validating the positivity (100%) and negativity (100%) of in-house anti-HCV screening assay through a standardized panel of 200 HCV positive and 200 HCV negative sera, a group of 120 serum specimens of suspected HCV infection were subjected to comparative analysis of our method with commercially available assay. The comparison confirmed that our method is more specific than the commercially available assays for HCV strains circulating in this specific geographical region of the world and could thus be used for HCV screening in Pakistan.
In this study, we devised a screening assay after successful PCR amplification, isolation, sequencing, expression and purification of core antigen of HCV genotype 3a. Our developed screening assay is more sensitive, specific and reproducible than the commercially available screening assays in Pakistan.
巴基斯坦正面临丙型肝炎感染的威胁，该国的感染率正在以惊人的速度上升。需要更特异和敏感的筛查方法来及时、正确地诊断这种感染。
从标本（HCV-3a）中提取 RNA 后，合成 cDNA，用于扩增 HCV 3a 全长核心基因。通过 PCR、DNA 测序和 BLAST 验证后，用适当的限制酶消化定向正确的核心基因阳性重组质粒，释放目的基因，然后将其插入 pGEX4t2 表达载体的多克隆位点中的 GST 编码 DNA 的下游，在适当的限制位点上处于相同的开放阅读框。将每个基因的重组表达载体转化到 E. coli BL21（DE3）中，并诱导 IPTG 产生重组融合蛋白，然后通过亲和层析进行纯化。用 Western blot 和酶联免疫吸附试验（ELISA）检测重组蛋白的免疫反应性。
在原核表达系统中产生的 HCV 核心抗原具有反应性，并用于开发一种筛查方法。通过对 200 份 HCV 阳性和 200 份 HCV 阴性血清的标准化面板验证了内部 HCV 筛查试验的阳性（100%）和阴性（100%），然后对 120 份疑似 HCV 感染的血清标本进行了比较分析，该方法与市售检测方法的比较。比较证实，我们的方法比市售的在该地区流行的 HCV 株更特异，因此可用于巴基斯坦的 HCV 筛查。
在这项研究中，我们在成功地进行了 PCR 扩增、分离、测序、表达和纯化 HCV 基因型 3a 的核心抗原后，设计了一种筛查方法。与巴基斯坦市售的筛查方法相比，我们开发的筛查方法更敏感、特异和可重复。
